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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:
from December 6, 1977 to June 1st, 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study with detailed information, fulfilling the scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report Date:
1978

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
a white granular solid substance was received from the principal on 30th September 1977. It was stored at room temperature.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals
Fifty male and fifty female weanling SPF albino rats (Wistar derived) were obtained from the Central Institute for the Breeding of Laboratory Animals (CPB-TNO), Zeist, The Netherlands, and kept for one week on stock diet. Thereafter they were divided according to body weight into five groups of ten male and ten female rats each, and fed the diets.
The animals were housed conventionally in screen-bottom cages (five to a cage) in a well-ventilated room at a temperature of 24 ± 1°C and a relative humidity of about 50%. Diets and tap water were given ad libitum during the whole experimental period of 13 weeks.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test article was thoroughly mixed into stock diet by means of a mechanical blender(Stephan) at levels of 0, 0.075, 0.1, 0.15 and 1%. The diets were freshly prepared once a fortnight and stored in a room at ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stock diet used is analysed for nutrients and contaminants at regular intervals.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.075, 0.1, 0.15 and 1% stock diet
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
This study was carried out in addition to a previous experiment(De Rijke and Willems, CIVO report no. R 5228, 1977). Only those parameters showing an effect in the previous experiment were examined in this study.

Positive control:
no data

Examinations

Observations and examinations performed and frequency:
The experiment was started on 6th December 1977. Individual body weights were recorded weekly. The food intake of each group was measured during the first four weeks and in week 11 and 12.
Sacrifice and pathology:
After 13 weeks the rats were anaesthetized by ether inhalation and blood was collected via puncture of the aorta abdominalis in heparinized plastic: tubes.
After centrifugation of the blood samples for 15 minutes at 2000 rpm, using Sure—Sep® from General Diagnostics for good separation, plasma was obtained. In these plasma samples the activity of alkaline phosphatase (ALP-EC 3.1.3,1) was determined by the method of Bessey et al. (J. Biol. Chem. (1946) 364, 321) using a Technicon AutoAualyxer.
The animals were examined macroscopically for pathological changes after having been bled to death. The liver, thyroid and kidneys were weighed. Samples of these organs were fixed in a 4 % neutralized formaldehyde solution and processed into haematoxylin- eosin stained paraffin sections for microscopical examination.
Other examinations:
no data
Statistics:
no data

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth rate of male rats fed 1 % of the test substance was statistically significantly lower than in controls throughout the experimental period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption and food efficiency figures in males of the highest dose group were decreased
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The food consumption and food efficiency figures in males of the highest dose group were decreased
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A distinctly increased ALP-activity was observed at 1 % in both sexes.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increased relative liver weights were observed only at 1 % in both sexes.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopical examination minimal, though clearly treatment- related, changes were observed in the livers of males and females of the highest dose group only.
Histopathological findings: neoplastic:
not examined
Details on results:
General condition, growth and food consuraption:
During the experiment, no abnormalities of condition or behaviour vere observed and none of the rats diet.
The mean body weights are presented, Growth rate of male rats fed 1 % of the test substance was statistically significantly lower than in controls throughout the experimental period.
The mean body weights of all other groups (males and females) were comparable to those of their controls.
The food consumption and food efficiency figures in males of the highest dose group were decreased.
Biochemical blood values:
The mean activities of alkaline phosphatase (ALP) in plasma are given. A distinctly increased ALP-activity was observed at 1 % in both sexes.
Organ weights
At 0.15 % relative liver weights were slightly higher than those of controls. Statistically significantly increased relative liver weights were observed only at 1 % in both sexes.
The decrease in kidney weight at the 0.1 % level was not found at higher levels and is therefore considered to be a fortuitous finding rather than an effect of the test compound. Moreover this finding was not observed in the previous study.
Pathology:
Gross examination at autopsy did not reveal any abnormalities which could be attributed to the feeding of the test compound.
Upon microscopical examination minimal, though clearly treatment- related, changes were observed in the livers of males and females of the highest dose group only. These changes consisted of swollen periportal hepatocytes containing too homogeneous, eosinophilic cytoplasm.
In the kidneys of male rats fed 0.1, 0.15 and 1 % test article the incidence and degree of normally occurring proteinaceous droplets in tubular epithelial cells was slightly higher than in control animals.
In view of comparable findings in the previous experiment it seems justified to ascribe this shift in incidence and degree to the treatment, although the incidence was not significantly different from that found in control kidneys, when tested according to the Fisher !s exact: probability test.
Only minor inter individual differences were found in morphological activation of the thyroid gland. The degree and incidence of this activation in controls and. the various test groups was comparable.
All other lesions were about equally distributed or a few animals only. Moreover, they are common findings in the strain of rats used and may therefore not be ascribed to the treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
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

Conclusions:
Since in a previous study a slight increase in liver weight was observed at 0.15 % and higher, a more conservative no-effect level, on the basis of all information available, is 0.1%,which is approximately equivalent with an intake of 50mg/kg body weight/day.
Executive summary:

The study was conducted based on the results given in a previous study(De Rijke and Willems, CIVO report no. R5228 1977). The test material was fed at levels of 0, 0.075, 0.1, 0.15 and1% in stock diet to groups of ten males and nen females each.

Observations were made of general appearance and behaviour, growth, food intake and efficiency, and alkaline phosphatase activity in blood plasina. At the end of the experimental period all rats were killed and examined grossly.Liver, kidneys and thyroid of all animals were weighed and samples were taken for histopathological examination.

Mean body weight, food intake and food efficiency were decreased at 1% in males,Alkaline phosphatase activity in blood plasma was increased at I % in both sexes. Relative liver weights were statistically significantly increased in both sexes at 1% only. Histopathological examination revealed minimal, though clearly treatment related,changes in the liver of animals at the I % level.

From the results of the present study it was concluded that 0.15 % was a no-effect level, which is approximately equivalent with a daily intake of 75 mg /kg body weight.