Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1967

Materials and methods

Principles of method if other than guideline:
Male and female rats were dosed in food for 90 days. Examination of hematological and biochemical parameters along with appearance, weight and functioning of organs was done.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: weanling rats
- Housing: 4/cage
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
Doses: 0.0 (control), 0.5, 1.0 and 2.0 % in diet
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
In food, ad libitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 other: % in diet
Dose / conc.:
1 other: % in diet
Dose / conc.:
2 other: % in diet
No. of animals per sex per dose:
4 sex/dose
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
Body weight and food intake: weekly examination

Terminal haematological examination involved the determination of total erythrocyte count, haemoglobin and methaemoglobin concentrations, haematocrit value and total and differential leucocyte counts. Erythrocytes were examined for the presence of reticulocytes and Heinz bodies.

Liver and kidney function tests: terminal examination

Blood parameters: levels of blood urea nitrogen and activities of serum glutamic-oxaloacetic and glutamic-pyruvic transaminases.

Urinalysis: examination of colour, pH, protein, reducing substances, bile salts, blood and microscopic constituents and activity of glutamic-oxaloacetic transaminase. A concentration test involved measuring the volume and specific gravity of the urine
excreted during a 6-hr period of water deprivation and during a 4-hr period commencing 16 hr after a water load of 25 ml/kg.

Palatability test: pairs of male rats were allowed free access to stock diet and to diet containing either 0.5 or 2 % of test substance. The consumption of both diets was recorded over a period of 8 days.


Sacrifice and pathology:
At autopsy: gross appearance of organs and weights of brain, heart, liver, spleen, kidneys, adrenals and gonads. Paraffin wax sections of these organs and a wide range of other organs were stained with haematoxylin and eosin for histological examination.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control male rat died on day 68 but autolysis prevented a diagnosis of the cause of death.
All other animals remained healthy throughout the study.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In the palatability test, animals showed slight preference for control diet, as compared with the diet at 2 %, but no difference was seen between 0 and 0.5 % diets.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological indices were not affected by dietary levels of up to 1 %.
At 2 % level, and especially in females, a slightly depressed red cell count was associated with decreases in haemoglobin concentration and haematocrit value.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated activities of transaminase enzymes occurred starting at the 2 % dietary level.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Elevated organ weights were randomly distributed throughout varoius groups and were not dose-related.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination revealed slight fatty liver and bronchopneumonia (the incidence of both these findings was randomly distributed in the various groups), myocarditis in a few females at the two highest levelst.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mammary adenofibroma in one female on 0.5 % group.
Other effects:
not specified
Details on results:
Effects were only seen at the 2 % level and consisted of a slight normochromic, normocytic anaemia and some elevation of the serum transaminase levels. These effects which were more pronounced in females could not be correlated with any other findings.
None of the histological findings observed can be attributed to treatment. Slight fatty liver is frequently found in our colony of rats and group differences in the incidence of this lesion were not significant. Myocarditis has been encountered in other experiments in both test and control animals and is of the spontaneous type. No significance can be assigned to the single mammary tumour in a female of the 0.5 % group since there were no signs of a similar lesion at higher levels and also because this lesion is frequently encountered in the strain of rat used.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Remarks on result:
other: 1 % in diet
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Remarks on result:
other: 2 % in diet

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL = 500 mg/kg bw/d in rats upon 90 days exposure.
Executive summary:

Method

Groups of 4 rats/sex were treated with test substance at 0 (control), 0.5, 1 and 2 % in diet for 90 days. Weekly examinations of body weight and food intake as well as terminal examination of haematological parameters, serum chemistry, urine and organs weight were performed. In addition histological examination was done.

Results

The following was reported: no mortality, except for one in the control group; no effect on growth rate and food consumption; no treatment related histological changes; effect on blood parameters (depressed red cell counts, statistically significant decreased haemoglobin concentrantion and decreased haematocrit value) mainly in females of the 2 % group; increased transaminase levels, possibly indicative of liver injury, in the 2 % group.

On these bases, NOAEL of 500 mg/kg bw/d (1 % dose group) was identified.