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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1974
Report Date:
1974

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no data on test material purity, no neurotoxicological examination
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bred on own premises
- Weight at study initiation: Mean of males 197 g and females 159 g
- Housing: Rats were caged in groups of 5
- Diet: Commercial diet, ad libitum (Dakes Special Diet with added Vitamine E) to which the compound was added
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 10

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Freshly each week
- Mixing appropriate amounts with (Type of food): Powdered diet was mixed with dry extract of malt (Diamalt C) in the ratio of 85 % food to 15 % malt extract by weight. A premix of powdered diet and compound was prepared for each dose level. The premix was then thoroughly mixed with the bulk diet to produce the required concentrations of the test substance. 15 % Water was added and the mixture passed through a mincer. The pellets so produced were dried for approximately 12 hours at a temperature not exceeding 45°C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
8 g pulverised rat diet containing the test substance were extracted for 16 hours with benzene. The quantitative determination of the test compound in the food-extract was realised by spectrophotometry. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the concentrations found in the diets and the nominal values.
Duration of treatment / exposure:
91 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 000 ppm
Remarks:
corresponding to 311 mg/kg bw
Dose / conc.:
10 000 ppm
Remarks:
corresponding to 694.5 mg/kg bw
Dose / conc.:
20 000 ppm
Remarks:
corresponding to 1297.3 mg/kg bw
No. of animals per sex per dose:
15, additional 5 males and females of the control group and the 20000 ppm dose group for the recovery group.
Control animals:
yes, plain diet
Details on study design:
- Post-exposure recovery period in satellite groups: 4 weeks with each 5 males and females from the control group and the 20000 mg/kg bw group

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each dose group determined and mean weekly diet consumption calculated: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes
- Time schedule for examinations: Pretest (all animals), during weeks 5, 9, and 13 (10 each sex of the control group and the 20000 ppm dose group), and in week 17 all animals of the recovery group.
- Dose groups that were examined: See above

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In weeks 5, 9, and 13 and in the recovery period (week 17)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals of the control group and the 20000 ppm dose group and 5 males and females of the 5000 and 10000 ppm dose group.
- Parameters checked: Haemoglobin (Hb), Erythrocytes, Haematocrit, Inclusion bodies, Thrombocytes, Leucocytes (total count and differential count), Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In weeks 5, 9, and 13 and in the recovery period (week 17)
- Animals fasted: No data
- How many animals: All animals of the control group and the 20000 ppm dose group and 5 males and females of the 5000 and 10000 ppm dose group.
- Parameters checked: Glucose, Urea, Serum Glutamic Oxaloacetic Transaminase (S.G.O.T.), Serum Glutamic Pyruvic Transaminase (S.G.P.T.), Serum Alkaline Phosphatase (S.A.P.)

URINALYSIS: Yes
- Time schedule for collection of urine: In weeks 5, 9, and 13 and in the recovery period (week 17)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: All animals of the control group and the 20000 ppm dose group and 5 males and females of the 5000 and 10000 ppm dose group.
- Parameters checked: pH, Specific Gravity, Protein, Glucose, Bilirubin, Ketones, Blood, Urine Sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (organ weights: Adrenals, Brains, Hearts, Kidneys, Livers, Gonads)
HISTOPATHOLOGY: Yes (Adrenals, Aorta, Bone marrow, Brain, Colon, Eye (+ optic nerve), Gonads, Gross lesions, Heart, Kidneys, Lymph nodes (axillary & mesenteric), Mammary gland, Muscle, Pancreas, Peripheral nerve (sciatic) Pituitary, Prostate, Small intestine, Spleen, Spinal cord, Stomach, Thymus, Thyroids, Urinary bladder, Uterus
Only for the control group and the high dose group histopathological examination was conducted.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical symptoms were recorded.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gains were within normal limits and, in treated groups, comparable to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was within normal limits and, in treated groups, comparable to controls. The dose levels of 5000, 10000, and 20000 ppm were equivalent to an average daily intake of 311, 694.5 and 1297.3 mg/kg bw.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination did not reveal any eye changes caused by treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
No significant differences were seen between the groups in respect of haematology parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant differences were seen between the groups in respect of clinical chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant differences were seen between the groups in respect of urine analysis parameters.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights (both absolute and relative to brain) were comparable in all groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No changes referable to the administration of the test substance seen were seen at autopsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Thirty rats (15 males and 15 females) of the 20000 ppm dose group and of the control group, respectively, were examined in detail. The lungs of several rats in both groups showed evidence of mild chronic murine pneumonia and in many livers small fat droplets were seen in individual parenchymal cells in Sudan stained frozen sections. Spleens invariably showed moderate haemosiderosis and extra medullary haematopoiesis and nematode parasites were frequently observed in the lumen of the large intestine. Changes referable to the test substance were not found, as there was no significant difference in the incidence or severity of any of the above changes between test and control animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
RECOVERY EXPERIMENT
No deaths and no clinical symptoms were recorded. Laboratory parameters were all within normal limits. Body weight gains and food consumption were within normal limits. The apparent fall in body weights of male animals in group 1 (control)
during the recovery period is due to the fact that the five animals had a lower average starting weight than the remainder of that group. No treatment induced changes were seen at autopsy. In view of the negative findings in group 4 (20000 p.p.m.) of the main study, no histopathological examination was carried out in group 5 (20000 p.p.m.).

Effect levels

Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
1 297.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The test compound administered in the diet to rats at a level of 20000 ppm (equivalent to an average daily intake of 1297.5 mg/kg) for 91 days did not cause any adverse effect.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results presented, the NOAEL was set at 1297.3 mg/kg body weight in rats, which was the highest concentration tested in this study.
Executive summary:

The test article was administered in the diet to rats at levels of 0, 5000, 10,000 and 20,000 p.p.m. (equivalent to an average daily intake of 0, 311, 594.5 and 1297.3 mg/kg body weight), for not less than 91 days. No deaths occurred and no clinical symptoms were recorded. Body weight gains and food consumption were within normal limits and, in treated groups, comparable to controls.

No important differences were seen between groups in respect of haematology, clinical chemistry and urine analysis parameters.

Ophthalmic examination did not reveal any eye changes caused by treatment. Neither at autopsy nor on histopathological examination were any changes referable to the administration of the test item seen. Organ weights (both absolute and relative) were comparable in all groups. In the recovery group, no deaths and no clinical symptoms were recorded. Laboratory parameters were all within normal limits. Body weight gains anc food consumption were within normal limits. The apparent fall in body weights of male animals in group 1 (control) during the recovery period was due to the fact that the five animals had a lower average starting weight than the remainder of that group. No treatment induced changes were seen at autopsy. In view of the negative findings in group 4 (20,000 p.p.m.) of

the main study, no histopathological examination was carried out in group 5 (20,000 p.p.m.). Based on these findings, a NOAEL was set at 1297.3 mg/kg body weight in rats, which was the highest concentration tested in this study.