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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (performed prior to GLP). Due to read-across from CAS 2440-22-4 reliability was set to 2.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1975
Report Date:
1975
Reference Type:
other: Amendment to report
Title:
Unnamed
Year:
1975
Report Date:
1975
Reference Type:
other: Letter by CRO in response to questions by US FDA
Title:
Unnamed
Year:
1984
Report Date:
1984
Reference Type:
other: Addendum to report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
no
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): TK 10047
- Physical state: pale yellow powder
- Analytical purity: not specified
- Lot/batch No.: 200983K
Specific details on test material used for the study:
CAS 2440-22-4

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CFY rats (hysterectomy-derived strain of Spague-Dawley origin)
- Source: Carworth Europe, England
- Age at study initiation: 25 ± 1 days old
- Weight at study initiation: individual body weights are reported
- Fasting period before study: not reported
- Housing: the rats were housed five to a cage (unless the number was reduced by mortality), in suspended metal cages fitted with wire-mesh floors
- Diet: powdered laboratory rat food, Spratt's Laboratory Animals Diet No. 2, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- A premix containing 20000 ppm was prepared each week, and from this the different dietary concentrations were obtained by direct dilution with further quantities of diet. Homogeneity was achieved by mixing for ten minutes in a rotary double-cone blender; all diets were stored until use in heat-sealed, opaque polythene bags.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the diet fed to the rats have been analysed for their content by spectrophotometry. The estimated relative reproducibility of this method is ± 10% or better. Within the limits of error of sampling technique and of the analytical method, there was good correspondence between the concentrations found in the diets and the nominal values .

Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
4 - 6 mg/kg bw ; 14 - 17 mg/kg bw; 47 - 58 mg/kg bw; 142 - 169 mg/kg bw for males and females
Basis:
actual ingested
Dose / conc.:
100 ppm
Dose / conc.:
300 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
3 000 ppm
No. of animals per sex per dose:
50
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: daily
- Cage side observations checked: all signs of ill-health and toxicity, together with any behavioural changes, detailed descriptions were made of any skin lesions, cataracts or palpable growths, together with the progression or regression of such lesions.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly for first 12 weeks, and at 4 week intervals thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: Yes, but only for weeks 0 - 24


WATER CONSUMPTION: Yes
- Time schedule for examinations: assessed in the first instance by inspection of the water bottles


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, and after 13, 26, 52, 75 and 102 weeks
- Dose groups that were examined: control and group 5 (3000 ppm)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13, 26, 52, 78 and 102 weeks of treatment (control and group 5), during week 103 blood was withdrawn from control and group 4 (1000 ppm)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex from control group, group 5 (3000 ppm) and group 4 (1000 ppm)
- Parameters examined: packed cell volume (PCV, % red cells), haemoglobin (Hb), red cell count (RBC), mean corpuscular haemoglobin concentration (MCHC) and mean cell volume (MCV), total white cell count (WBC), differential count (neutrophils, lymphocytes, eosinophils, basophils, monocytes)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13, 26, 52, 78 and 102 weeks of treatment (control and group 5), during week 103 blood was withdrawn from control and group 4 (1000 ppm) for examination of urea nitrogen (males only), serum alkaline phosphatase (SAP) and serum glutamic-pyruvic transaminase (SGPT)
- Animals fasted: No data
- How many animals: 10 animals/sex from control, group 5 (3000 ppm) and group 4 (1000 ppm)
- Parameters examined: plasma urea nitrogen, plasma glucose, serum alkaline phosphatase (SAP) and serum-glutamic-pyruvic transaminase (SGPT)


URINALYSIS: Yes
- Time schedule for collection of urine: After 13, 26, 52, 78 and 103 weeks of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: pH, specific gravity (SG), protein, reducing substances, glucose, ketones, bile pigments, urobilin, blood pigments, microscopy of spun deposit (1000 rev/min for 10 min): epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, casts, abnormal constituents, sperm
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
(superficial tissues (including urinogenital orifices and tail, each pinna, orbit and external auditory meatus), mammary tracts, cervical subcutaneous structures, nares, buccal cavity, tongue, pharynx and auditory region, brain, pituitary gland and cranial nerves, subcutaneous tissues (including regional lymph nodes, mammary and salivary glands), abdominal viscera, urinary bladder, mucosae of stomach and caecum, thoracic viscera, thymus, heart, oesophagus, intestines, uterus, lungs, liver, kidneys, gonads, adrenals, thyroids, intra-abdominal lymph nodes and accessory reproductive organs)

ORGAN WEIGHTS: adrenals, brain, heart, kidneys, liver, testes, pituitary, spleen, thyroid

HISTOPATHOLOGY: Yes
(all macroscopically observed lesions suggestive of neoplasia, for every animal; all macroscopically abnormal tissues from decedents in an attempt to ascertain cause of death; 10 rats of each sex from control and 3000 ppm groups killed at 104 weeks). The following organs were fixed and preserved: adrenals, aorta, brain (cerebrum,cerebellum and pons), caecum, colon, duodenum, eyes, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, skin, spinal cord, stomach (cardiac, fundus and pylorus), testes, thymus, thyroid, urinary bladder, uterus, spleen

All nodules, tissue masses and otherwise macroscopically abnormal tissues were routinely preserved and processed along with samples of adjacent tissue where appropriate.
Statistics:
Student's 't' test was employed to assess the significance of intergroup differences where the data suggested a treatment related response. Differences in tumour incidence were compared by a 2 x 2 contingency test used as a one tailed test, computing the exact probability of the observed tumour distribution occurring or one which is more extreme.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
An inferior body weight gain for male animals recieving 3000 ppm was recorded during the last 52 weeks of treatment in comparison with the body weight gain for control animals (p<0.05). Bodyweight gains among other groups were not changed by treatement.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first 52 weeks of the study, food intake of treated rats was comparable with that of the controls. Thereafter, the quantity of food consumed by rats treated with 3000 ppm was lower than that of the controls, although only the difference for the food intake of females during weeks 53 to 80 attained a level of significance. Similarly, during weeks 53 to 80, the intake of females receiving 1000 ppm was significantly lower than that of the controls but as this group consumed a similar quantity of food to that of the controls during the remainder of the study, the difference cannot be unequivocally related to treatment. Food intake of rats treated with 100 or 300 ppm and males treated with 1000 ppm remained comparable with that of the controls throughout the study.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Although at week 13, a statistically significant reduction in red blood cell count (RBC), with the consequent elevation of the mean cell volume (MCV) was observed in male rats receiving 3000 ppm, the values were within the 'normal' range for the strain of rat employed. The differences were, therefore, not considered to be of biological significance.
At week 52, female rats receiving 3000 ppm showed a significant decrease in mean cell volume, and a small increase in the total white blood cell count. The values recorded were, however, within the 'normal' range for the strain of rat employed. No intergroup differences were recorded in male rats receiving 3000 ppm.
At week 78, male and female rats receiving 3000 ppm showed a small decrease in the mean haemoglobin value which in females associated with a reduction in the mean cell haemoglobin content (MCHC). The values recorded were, however, within the 'normal' range for the strain of rats employed.
At week 102 values for male rats receiving 3000 ppm showed a small to moderate statistically significant decrease in mean packed cell volume (PCV), haemoglobin value and red blood cell count (RBC), with evidence of marked anaemia in 3 rats. In view of these findings, the investigation was extended at week 103 to cover male rats receiving 1000 ppm. This investigation showed the erythrocytic parameters in this group were comparable with the control values with the exception of 1 rat which was anaemic with an elevated white cell count related to the haemorrhagic ulcer on its hind limb. As abnormal blood values are commonly seen in a proportion of aged rats, and since no treatment related effects had been recorded on haematological parameters, these findings were not considered to be of toxicological significance. No other intergroup differences were recorded.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Among rats treated with 3000 ppm, there was a marginal reduction in urea nitrogen recorded among females at week 13, and a marginal increase among males at week 26, females at week 78 and males at week 102. With the exception of the values recorded for males treated with 3000 ppm, all values for urea nitrogen recorded during the study were within the 'normal' limits for the strain of rat employed. Among males treated with 3000 ppm, 3 of the 10 rats sampled had values above the normally accepted range for CFY rats. As a small incidence of elevated urea nitrogen values are frequently recorded in aged rats, this finding was not considered to be of toxicological significance. At week 103, urea nitrogen values were determined for males treated with 1000 ppm. This investigation revealed values for this group which were similar to those of the controls.
No significance is attached to the apparent elevation of serum alkaline phosphatase levels recorded at weeks 102 or 103 in males treated with 3000 or 1000 ppm, as the values obtained were within the 'normal' range for rats of the strain employed and the apparent effect was not dosage related.
At the 102 week investigation, 3 females treated with 3000 ppm had markedly elevated SGPT values. Consequently, this parameter was investigated in female rats treated with 1000 ppm. Although the values obtained were in the upper range of normality, only one rat had a markedly elevated value. Subsequent histopathological examination of livers from rats treated with 3000 ppm revealed no evidence of a treatment-related hepatotoxic effect.
Urinalysis findings:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A marginally lower survival rate was recorded during the final 26 weeks of treatment among males receiving 3000 ppm although the difference from the control male group survival rate did not attain a level of significance. Survival rates among other treated rats was similar to that of the controls .
Details on results:
FOOD EFFICIENCY
No treatment related disturbance of efficiency of food utilization was recorded during the period of fastest growth (the first 24 weeks of treatment). Food utilization was not determined for weeks 53 to 80 during which reduced food consumption was observed for 1000 and 3000 ppm treated animals.



ORGAN WEIGHTS
A small increase in relative adrenal weights was recorded among rats treated with 1000 ppm. As the difference from control values was of a small order of magnitude and was not dosage related in degree, this finding was considered to be fortuitous. Although heart weight in some groups attained a level of significance either in the absolute or relative weights, in the case of males treated with 3000 ppm, this reflected the intergroup disparity in bodyweight and in treated females this was a reflection of the lower than normal heart weight recorded for the control group. Other small differences in weights of organs from treated rats were attributable to intergroup disparity in bodyweight and were considered to be of no toxicological significance.
Organ weight analysis performed on rats killed after 104 weeks of treatment revealed slightly heavier thyroid/parathyroid weights among treated animals, which only attained a level of statistical significance among males treated with 1000 ppm and females treated with 1000 or 3000 ppm when related to bodyweight, and among females treated with 100, 1000 or 3000 ppm when related to brainweight.
After statistical re-evaluation of significance levels in comparison with control using Williams' test:, for the males, no significant treatment effects were detected. For females significant increases in both absolute kidney (p < 0.05) and thyroid weights (p < 0.01) were found in both 1000 and 3000 ppm treatment groups. Although statistically significant, there is no dose-response relationship. As the values obtained for treated rats were within the normal range for CFY rats, the differences were considered to have arisen fortuitously.

GROSS PATHOLOGY
The macroscopic pathology among rats dying during the course of the study showed no relation to treatment. Macroscopic examination of rats killed after 104 weeks of treatment showed pathology which was common to animals from control and treated groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology of decedents
Lungs: Chronic respiratory disease, characterized by congestion, peribronchiolar and perivascular lymphoid aggregations or hyperplasia, aggregates of alveolar macrophages and granulomatous foci, was common to a number of rats from control and treated groups.
Liver: Changes in the liver, common to some rats from control and treated groups, consisted of varying degrees of hepatocyte vacuole formation, aggregates of periportal and parenchymal chronic inflammatory cells, necrosis, haemorrhage and focal areas of bile duct hyperplasia. A hyperplastic nodule was observed in the liver of one male rat (1000 ppm).
Kidney: Varying degrees of progressive glomerulonephrosis was seen in a proportion of rats from both control and treated groups, with occasional animals showing a minor degree of dystrophic mineralization.
Cardiovascular system: Myocarditis and fibrosis was observed in 2 male rats (1000 ppm) and myocarditis in 1 male rat (3000 ppm). An auricular thrombus in 2 male rats (1000 ppm) and mineralization in the heart of 3 male rats of the 100 ppm, 1000 ppm and 3000 ppm groups. A proportion of animals from control and treated groups showed mineralization of the aortic median, and periarteritic changes with or without aneurysm formation. These changes are commonly seen in ageing rats of this strain and were considered to be of no toxicological significance.
Mammary gland: A proportion of female rats from both control and treated groups showed hyperplasia of the mammary tissue. Ducts and acini were increased in number with evidence of hypersecretion and galactocele formation in some instances.
Adrenal gland: Areas of adrenal haemorrhage were observed in a proportion of female rats from both control and treated groups.
Parathyroid gland: Hyperplasia of the parathyroid gland frequently associated with varying degrees of progressive glomerulonephrosis was observed in occasional rats .
The changes listed above are those commonly seen among rats of the strain employed and were, therefore, not considered to be related to treatment.

Histopathology of rats killed after 104 weeks
Lungs: A proportion of animals from each group showed changes indicative of chronic respiratory disease. The morphological features included perivascular and peribronchiolar lymphoid hyperplasia, hyperplasia of the bronchus-associated lymphoid tissue, peribronchiolar chronic inflammatory cell aggregates and occasional subpleural granulomatous foci. Such changes are characteristic of the chronic respiratory disease frequently seen in laboratory rodents and were considered to be without significance.
Liver: No group-related changes were seen in any of the animals examined. A variety of non-specific features were seen in animals from all groups. These included focal areas of hepatocytic vacuolation, aggregates of periportal and parenchymal chronic inflammatory cells, subcapsular areas of hepatocytic necrosis and focal areas of bile duct hyperplasia. All these features are commonly seen in the livers of ageing laboratory rodents . Multiple
areas of nodular regeneration were also seen in a single female animal (3000 ppm).
Kidneys: A number of animals from each group showed a varying degree of progressive glomerulonephrosis. This degenerative condition is very common in ageing laboratory rats and as neither the incidence or the severity of the change was group-related these findings are of no importance in the context of the experiment as a whole. Occasional animals also showed a minor degree of dystrophic mineralisation or interstitial nephritis. In one female rat (Control) a small area of hyperplasia of the pelvic epithelium was identified in close relationship to a lipoma.
Cardiovascular system: A small proportion of animals from each group showed histological evidance of myocarditis. The features were interstitial chronic inflammatory cell aggregations and focal myocytolysis with or without associated fibrosis. Myocarditis is a common disease of laboratory rats and as the Incidence was not group-related the findings are of no experimental importance. A small number of animals from each group showed evidence of periarteritis with or without aneurysm formation. Amongst the areas affected were testis, intestinal mesentery and pancreas This is the usual pattern of involvement in the ageing laboratory rodent; the changes were not group-related and were therefore not considered to be related to treatment.
Mammary gland: A proportion of female animals from both the control and treated groups showed hyperplasia of mammary tissue. Ducts and acini were increased in numbers and there was often evidence of hypersecretion, sometimes with galactocele formation. The changes were not group-related.
Adrenal gland: Areas of adrenal haemorrhage were identified in many animals. Female animals were affected more often than male animals but there was no relationship to treatment. One male rat (Control) showed multiple small areas of cystic degeneration; one male rat (3000 ppm) showed multiple areas of cytoplasmic vacuolation. Both these changes are regarded as unimportant incidental findings.
Parathyroid gland: 2 male rats (3000 ppm) showed hyperplasia of the parathyroid gland. In both instances this was associated with a moderate or marked degree of progressive glomerulonephrosis. It is assumed that these parathyroid changes are entirely secondary to the primary renal disease.

No morphological abnormalities or variations from normal were seen in any of the tissues examined which were considered to be due to the administration of the test substance.

HISTOPATHOLOGY: NEOPLASTIC
Lymphoreticular system: The low incidence of tumours of the lymphoreticular system showed no evidence of a treatment-related effect.
Liver: No evidence of hepatocarcinogenicity was recorded.
Liver cell tumours were recorded in 3 male rats (Control, 1000 ppm and 3000 ppm) and a fibrosarcoma was observed in the liver of one female rat (300 ppm).
Kidney: A lipoma in close assocation with hyperplasia of the renal pelvic epithelium was observed in one female rat (Control) and an undifferentiated sarcoma in the kidney of another female rat (1000 ppm).
Reproductive system: Tumours of the reproductive systems showed no evidence of a treatment-related increase over the spontaneous incidence.
One interstitial cell adenoma of the testis was recorded in one male rat (1000 ppm) and a fibrosarcoma of the prostate with metastatic deposits in the lungs in another male rat (300 ppm). A fibrosarcoma was recorded in the uterus of one female rat (100 ppm) and in the vagina of another female (1000 ppm). An adenocarcinoma of the ovary was recorded in a third female rat (1000 ppm) and a moderately differentiated fibrosarcoma with metastatic deposits in the lungs was seen in the ovary of a fourth rat (1000 ppm). Neither the incidence nor the multiplicity of mammary tissue tumours, consisting of fibroadenomas, adenomas, adenocarcinomas and a carcinoma, showed any evidence ot a treatment-related increase.
Endocrine glands: The distribution of tumours of the endocrine glands showed no treatment-related disturbance. The incidence recorded was within the normal limits previously recorded within our laboratories for the CFY rat.
Subcutaneous tissues: There was no evidence of a treatment-related effect on the incidence of connective tissue tumours found in subcutaneous locations.
Miscellaneous tumours: The individual tumour types and number of tumours fall within the control range for the strain of rat employed.
Total tumour incidence: There were slightly more tumour-bearing rats among females receiving the test substance at 300 or 1000 ppm, killed after 104 weeks of treatment, compared to controls, although these differences did not attain a level of statistical significance. The incidence of tumour-bearing rats in other treated groups was comparable with that of the controls.

The administration of test substance was not associated with any evidence of an effect on the spontaneous tumour incidence of the CFY rat.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOEL
Effect level:
> 47 - < 58 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
> 142 - < 169 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the above findings it was concluded that 1000 ppm (47-58 mg/kg bodyweight/day) was the no-effect level. The marginally lower bodyweight shown by some males on 3000 ppm and the slightly lower food consumption of some females of the same dose recorded during the second year of the study is difficult to assess. Therefore, the highest dose of 3000 ppm (142- 169 mg/kg bodyweight/day) was considered to be the NOAEL in this study.