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Description of key information

In a 28day palatabiltiy study with limited examinations a NOAEL was derived at 1010 mg/kg body weight for male rats and 987 mg/kg body weight for female rats (highest concentration tested). In a 90 days subchronic feeding study with rats performed similar to OECD guideline 408, a NOAEL was set at 1297.3 mg/kg body weight in the rat (highest concentration tested). In a second 90day feeding study with dogs a NOAEL was set at 818 mg/kg body weight in the dog (highest concentration tested). No specific systemic toxicity was observed in neither study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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
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
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.
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.).
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.
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 297.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Subacute study

Rodents

The palatability of the test substance was determined in a subacute study in rats. The study was conducted similar to the OECD-Guideline 407 with limited examinations. 10 male and 10 female Tif: RAIf (SPF) rats per dose group were daily fed with food containing the test compound in dose levels of 1000, 3000, and 10000 ppm (equivalent to 97, 302, and 1010 mg/kg body weight for males and 91, 277, and 987 mg/kg body weight for females; calculation based on body weight and food consumption for 28 days. The control animals (10 each sex) were fed plain diets. During exposure the rats were observed for clinical signs and examined concerning body weight development, food consumption, and food efficiency. At the end of the study gross pathology was conducted. No deaths occurred during the course of the study, and no clinical signs of local or systemic toxicity were observed. The mean body weight of all treated males and females of the test substance groups was similar to that of the respective control group. The mean food consumption of all treated male and female test groups was similar to that of the respective control group. The food efficiency in the test groups was similar to that of the control group in both male and female animals. Macroscopical examination revealed no evidence of treatment-related effects. Based on these findings, a NOEL was set at 1010 mg/kg body weight for male rats and 987 mg/kg body weight for female rats, which was the highest concentration tested in this study (Ciba-Geigy, 1983).

 

Subchronic studies

Rodents 

The subchronic oral toxicity of the test substance was determined in rats. The study was conducted similar to the OECD-Guideline 408. 15 male and 15 female Sprague Dawley rats per dose group were daily fed with food containing the test compound in dose levels of 5000, 10000, and 20000 ppm (equivalent to 311, 694.5, and 1297.3 mg/kg body weight; calculation based on body weight and food consumption) for 91 days. The control animals (15 each sex) were fed plain diets. Additional a satellite group of 5 animals each sex of the control group and the highest dose level group were kept for a recovery period of 4 weeks. During the study period the rats were observed for clinical signs and examined concerning body weight development, food consumption, haematology, clinical chemistry, and urinalysis. Furthermore, ophthalmic examination was carried out. At the end of the study gross pathology and histopathology were conducted. No deaths occurred and no clinical symptoms were recorded. The body weight gains and food consumption were within normal limits and, in treated groups, comparable to controls. The ophthalmic examination did not reveal any eye changes caused by treatment and no important differences were seen between the groups in respect of haematology, clinical chemistry and urine analysis parameters. The organ weights (both absolute and relative to brain) were comparable in all groups and no changes referable to the administration of the test substance seen were seen at autopsy. Thirty rats (15 males and 15 females) of the 20000 ppm dose group and of the control group, respectively, were examined histopathologically 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 instained frozen sections. Spleens invariably showed moderate haemosiderosis and extra medullary haemopoiesis and nematode parasites were frequently observed in the lumen of the large intestine. Changes referable to the test substance were not found. During the recovery period no deaths and no clinical symptoms were recorded. The laboratory parameters, body weight gains, and food consumption were all within normal limits. In view of the negative findings in the 20000 ppm does group of the main study, no histopathological examination was carried out after the recovery period. 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 (Geigy Pharmaceuticals, 1974).

 

Non-rodents 

The subchronic oral toxicity of the test substance was determined in dogs. The study was conducted similar to the OECD-Guideline 409. Male and female beagle dogs were daily fed a diet containing the test substance at dose levels of 7500, 15000, and 30000 ppm for 90 days (equivalent to 237, 410, and 818 mg/kg body weight; calculation based on body weight and food consumption; 4 male and female dogs in the low and mid dose as well as 5 males and 5 females in the high does group) The control animals (5 animals each sex) were fed plain diets. Additionally, a satellite group of 1 animal each sex of the control group and the highest dose level group were kept for a recovery period of 4 weeks. During the study period the dogs were observed for clinical signs and examined concerning body weight development, food consumption, haematology, clinical chemistry, and urinalysis. Furthermore, ophthalmic examination was carried out. At the end of the study gross pathology and histopathology were examined. No deaths occurred and no treatment induced symptoms were recorded. Treatment with the test substance produced no adverse effect on body weight gains and food consumption. No effects were observed at ophthalmic examination. Erythrocyte parameters in the 30000 ppm dose group were slightly below control levels throughout the trial but as a similar situation was present in pretest samples no significance was attached to this finding. At week 13 one animal each in the 7500 and 15000 ppm dose groups and two animals in the 30000 ppm dose group had an erythrocyte sedimentation rate slightly above normal. No significant changes were recorded in clinical chemistry or urine analysis. At autopsy no changes caused by treatment were seen, variation in organ weights were within normal limits. The histopathological examination did not reveal any changes caused by the administration of the test compound. In the majority of animals minute foci of calcification were seen in the medullary region of the kidneys. These foci are very common in beagle dogs. In many cases spleens were very congested and the architecture was difficult to visualise. This condition was caused by the use of pentabarbitone for the purpose of euthanasia. During the recovery period one control animal had an erythrocyte sedimentation rate slightly aboue normal levels. Other laboratory parameters were within acceptable limits. No changes referable to treatment were seen at autopsy or on histopathological examination. A mild tracheitis was present in 3out of 4 animals (two of which were controls).

Based on these findings, a NOAEL was set at 818 mg/kg body weight in dogs, which was the highest concentration tested in this study (Geigy Pharmaceuticals, 1974).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data is reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for oral repeated dose toxicity is not warranted.