Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 3th to the 15th of May, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted testing guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Principles of method if other than guideline:
The purpose or this oral toxicity (range-finding) study was to assess the toxicological profile of the substance when administered daily to rats by oral gavage, according to OECD401, for a period of 5 days. This study should provide a rational basis for the dose selection for a 28-day oral toxicity (gavage) study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Environmental fate/Ecotoxicological/Toxicological studies:
Storage: Room temperature, 20-24 °C, in the dark
Stability: pure, for 5 years; in solvent > 24 hours in water, DMSO, DMF, stable for at least 2 hours in aqueous medium.
Evidence of chemical instability at pH 6: none
Safety precaution: routine hygienic procedures were sufficient to assure personnel health and safety.
Toxicological studies:
Preparation: On the day of the experiment, the test article was suspended in Carboxymethylcellulose (1 %).
The vehicle was chosen to its nontoxicity for the animals. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM
Acclimatization: May 3 to May 9, 1990
Observation: May 10 to 14, 1990
Source: BRL, Biological Research Laboratories Ltd, Wolferstrasse 4, CH-4414 Fullinsdorf
Total number of animals 9 males, 9 females
Age at start of treatment males: 6 weeks
Body weight at start pretest/acclimatization 158-171 g males, 167- 178 g females
Identification: By unique cage number and corresponding color coded spots on the tail.
Randomization:Computer-generated random algorithm at time of delivery.
Acclimatization: Seven days under test conditions, after veterinary examination.

HUSBANDRY
Room No.: 136
Standard Laboratory Conditions:
Air-conditioned with 10-15 air changes per hour, and hourly monitored environment with temperature 22±3 °C, relative humidity 40-70 %, 12 hours artificial fluorescent light/12 hours dark, music/light period.
Accommodation: the rats were housed individually in Makrolon type-3 cages with standard softwood bedding, (“Lignocel”, Schill AG, CH-4132 Muttenz).
Diet: pelleted standard Kliba 343, Batch 68/90 rat maintenance diet (‘Kliba’, Klingentalmuehle AG, CH-4303 Kaiseraugst) available ad libUtum.
Analysis for contaminants performed.
Water: community tap water from Itingen, available ad libitum. Analyses for contaminants performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The animals received a single dose of the test article on a mg/kg body weight base by oral gavage.
Frequency: daily, for 5 days
Dose volume: 10 mL/kg body weight
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg (control) Group 2: 200 mg/kg Group 3: 1000 mg/kg
Basis:

No. of animals per sex per dose:
3 males
3 females
for every group
Total
9 males
9 females

Examinations

Observations and examinations performed and frequency:
OBSERVATION
Mortality: daily
Body Weights: the body weight of each animal was recorded during acclimatization and on days 1, 3 and 5 of treatment period using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to a computer system.
Clinical Signs: daily. A description of any abnotmality was recorded and the subsequent progress was monitored.
Food consumption: the food consumption was recorded during acclimatization and on days 1, 3 and 5 of the treatment period using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to a computer system.
Opthalmoscopic examination: Ophthalmoscopic examinations were performed on all animals at the end of the treatment period. Ten minutes after theapplication of a mydriatic solution (Dispersa AG, CH—8400 Winterthur) the cornea, lens, anterior chamber, vitreous body and ocular fundus
of both eyes were examined under dimmed light using a Heine-Biftcal Ophthalmoscope miroflex type (Eisenhut Vet AG, CH-4123 Allschwil).
Sacrifice and pathology:
PATHOLOGY
Organ weights
The following organ weights were recorded on the scheduled date or necropsy: Heart, Liver, Kidneys, Adrenal glands, Spleen, Testes, Ovaries.
Necropsy: All animals were necropsied by experienced prosectors, and descriptions of all macroscopic abnormalities were recorded. All animals werefasted for 19 to 21 hours before necropsy, but water was provided. Immediately before necropsy, all fasted individuals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the organs and tissues weighed as well as those with gross lesions were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution for possible further histopathological examination.
Other examinations:
The food consumtion is calculated per cage and per food consumption interval. It expresses the average food consumption per animal and per day over the food consumption interval.
Formula: FC: C/AD

FC Food consumption (in g of food per animal and per day).

C Measured food consumption per animal over the consumption interval (in g of food).

AD Total of consumption days during the consumption interval.

The relative food consumption is calculated according to the following formula:

RFC: FC (in gram)/BW(i) (in gram) x 1000

Unit: Gram food per kg body weight and day

FC = Food consumption per animal and per day (gram)

BW(i) = Most ideal body weight = body weight (of the corresponding rat individual) recorded on the day most close to the middle of the food consumption interval. In cases of equal “closeness” of two body weight records the earlier one is chosen.
Statistics:
The computer generated values which appear on the tables represent the rounded off results of the raw data values or of calculations which used the exact raw data values. Group means were calculated according to the definition of any mean value using the individual values per animal or per cage and the number of ani mals or the number of cages. Medians were calculated instead of mean values in case of discrete data like scores and counts.
The following data were recorded online: mortality, clinical signs , food consumption, body weight, organ weights, macroscopic findings. As an exception, the clinical finding of discolored faeces and slight 7 diarrhea in group 3 on test day 5 was only recorded on data sheets.
The following data were recorded on data sheets (except for mortality) and transcribed for compilation and analysis:
Viability, mortality, clinical signs, ophthalmoscopfc examinations, terminal body weights at necropsy.
Daily evaluation: The individual data represent the maximum scores observed per animal and per day.The summary data show for each treatment group the median of the individual maximum scores, together with a code representing the percentage o animals affected.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No death occurred prior to the scheduled date of termination. All animals were killed at necropsy on the day after termination of administration.
Mortality:
mortality observed, treatment-related
Description (incidence):
No death occurred prior to the scheduled date of termination. All animals were killed at necropsy on the day after termination of administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All males of group 3 lost weight during the entire treatment period. This was consistent with the body weights statistically significantly lower than their controls during the latter half of the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see below
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see below
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Ophtalmoscopic examination of each rat individual at the end of the treatment period did not reveal any abnormalities.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Details on results:
All males of the high dose group (1000 mg/kg) showed slightly ruffled fur as well as slight and/or moderate emaciation from test day 3 or 4. One out of three females of this group showed slightly ruffled rur on test days 3 and 4. In addition to these findings, all males and females of the high dose group showed slight diarrhea and yellowish brown discoloration or the faeces on test day 5.

No macroscopical findings were noted on control group 1 and on the females of group 2 (200 mg/kg). One out of three males of group 2 showed pelvic dilation in its right kidney. The main findings in both sexes of group 3 (1000 mg/kg) were reddish, red or dark red discoloration of the mucosa of several gastro- intestinal organs. In addition to this, yellowish discoloration of the mucosa of the forestomach was found in two females of group 3 and in one of these 0 also a gray white focus (diameter 5 mm) in the spleen.

Gross pathology
The kidney weights and to a lesser degree the Testes weights of male rats of group 3 were statistically significantly lower than their controls. The Testes weights of group 2 (200 mg/kg) were also slightly lower than control, but at a statistically non-significant level. However, such differences did not apply tothe corresponding organ to body weight ratios, probably because the average O terminal body weights of the males were negatively dose-related. The Adrenal glands to body weight ratio was higher in male rats of group 3 than in their controls, and the Spleen weights and Spleen to body weight ratios higher in females of group 2 than in control females. However, the Spleen weights and Spleen to body weight ratios of male rats of group 3 were slightly lower than the corresponding controls at a statistically non-significant level.

Food consumption:
Males of the high-dose group 3 (1000 mg/kg) ate statistically significantly less than the respective controls throughout the treatment period. They ate less and less towards the end of the treatment. During test days 1 to 3, the food consumption rates of females of this group was slightly lower than the corresponding controls at a statistically non-significant level. Females of group 2 (200 mg/kg) ate statistically significantly more than their controls during test days 3 to 5. This was explained by two out of three females of this group eating more than their respective controls.

Relative food consumption
All male rats of group 3 and all females of this group showed Relative Food Consumption significantly lower than their controls throughout the treatment period and on test days 1 to 3 respectively. Relative Food consumption of females of group 2 (200 mg/kg) was statistically significantly higher than the respective control during the days 3 to 5. This was consistent with a high absolute food consumption of two females of this group.


Effect levels

Dose descriptor:
NOAEL
Remarks:
LD0
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
LD0>=1000 mg/Kg bw
According to the results observed, it is recommended to select the following dose groups for the subacute 28 day oral (gavage) study:
Group 1-0 mg/kg body weight (control)
Group 2-50 mg/kg body weight (low dose)
Group 3-200 mg/kg body weight (mid dose)
Group 4-800 mg/kg body weight (high dose)
Executive summary:

A pre-arange test on the substance is available. This study should provide a rational basis for the dose selection for a 28 day oral toxicity (gavage) study.

Material and methods

In the oral toxicity (range-finding) study the substance was administered daily to rats by oral gavage, for a period of 5 days. The study was comprised of three groups, each containing three male and three female rats.

The study should provide a rational basis to propose dose groups for a 28-day oral toxicity study.

The following dose levels were administered:

Group 1: 0 mg/kg body weight (control)

Group 2: 200 mg/kg body weight

Group 3: 1000 mg/kg body weight

Observations

MORTALITY

No death occurred prior to the scheduled date or termination.

CLINICAL SIGNS

All males of the high dose group (1000 mg/kg) showed slightly rurfled fur as well as slight and/or moderate emaciation from test day 3 or 4. One out of three females or this group showed slightly ruffled fur on test days 3 and 4. In addition to these findings, all males and females or the high dose group showed slight diarrhea and yellowish brown discoloration of the faeces on test day 5.

FOOD CONSUMPTION

Males of the high-dose group 3 (1000 mg/kg) ate statistically significantly less than the respective controls throughout the treatment period. During test days 1 to 3, the food consumption rates of females of this group was slightly lower than the corresponding controls at a statistically non-significant level.

Females of group 2 (200 mg/kg) ate statistically significantly more than their controls during test days 3 to 5.

RELATIVE FOOD CONSUMPTION

Relative Food Consumption of males of group 3 (1000 mg/kg) was statistically significantly lower than their controls throughout the treatment period.

Relative Food Consumption of females of group 3 was statistically si9nificantly lower than their controls on test days 1 to 3 and statistically significantly higher than controls for female rats of group 2 (200 mg/kg) during test days 3 to 5.

BODY WEIGHTS

From test day 3 the body weights of males of group 3 (1000 mg/kg) were statistically significantly lower than their controls. No other statistically significant differences from control occurred.

OPHTHALMOSCOPIC EXAMINATION

No findings were observed during the study.

MACROSCOPIC FINDINGS

The only findings considered to be treatment related were reddish, red, dark red or yellowish discoloration of the mucosa of several gastro-intestinal organs in high dose individuals (group 3, 1000 mg/kg). However, these findings may have been the result of staining by the test article, rather than an expression of toxicity.

ORGAN WEIGHTS AND ORGAN WEIGHT RATIOS

The Kidney weights were statistically significantly lower than their controls in males of group 3 (1000 mg/kg). Testes weights were also lower than their controls in this group and there was a similar trend in males of group 2 (2000 mg/kg) at a statistically non significant level. The Adrenal glands to body weight ratio was higher in male rats of group 3 than in their controls and the Spleen weights and Spleen to body weight ratios higher in females of group 2 than in control females.

Results

LD0 male/female (5 d): >= 1000 mg/Kg bw