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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
Humidity variations reached at times > 80 %. Highest dose too low (58-147 mg/kg/day) to elicit minimal toxicity. Detailed clinical observation not specified and functional observations are missing. Animals not fasted before blood withdraw.)
GLP compliance:
not specified
Remarks:
no data (Statement of compliance is missing, but GLP compliance is mentioned on page 5 paragraph 4.1.7 of the report)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: organic
- Physical state: solid, white amorphous powder
- Storage condition of test material: room temperature (21°C) protected from moisture

Test animals

Species:
rat
Strain:
other: CD strain (a hysterectomy-derived strain of remote Sprague-Dawley origin)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Ltd., Margate, UK
- Age at study initiation: 4 weeks
- Weight at study initiation: 60 - 80 g
- Housing: groups of animals in suspended polypropylene cages (Pattern RCl from North Kent Plastics L t d . , measuring 52 x 35 X 18 cm), with stainless steel mesh floors and lids.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40 - 80 , sometimes > 80 %
- Air changes (per hr): 15
- 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:
The test article was incorporated into the powdered diet to provide the concentrations indicated. The latter were held constant throughout the treatment period. A pre-mix was prepared each week for each group and from this the dietary concentrations were obtained by direct dilution with further quantities of diet.Homogeneity was achieved by mixing for 20 minutes in a horizontal, screw-type mixer (Gardner type SOL 286M, maximum capacity 45 kg), which was electrically grounded (= earthed).

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: All diets were stored until use in sealed light-proof polythene bags and stored at room temperature.A complete powdered rodent diet produced by the manufacturer by pulverisation of fat-coated pellets (Spratts Laboratory Animal Diet No. 2) was available to the animals prior to and during the first 20 weeks of treatment. From Week 21, the diet supplied by the manufacturer was no longer coated with fat prior to the pellets being ground. This was an expanded autoclaved diet supplied in a discardable outer paper sack with a sealed inner sterilisable polythene bag. It contained no added antibiotic or other chemotherapeutic or prophylactic agent. It was monitored for chlorinated hydrocarbon, aflatoxin and polychlorinated biphenyl contaminants at intervals. Each batch of diet was analysed by the manufacturer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A 200 g aliquot of each test diet was taken each week, sealed into polythene sachets and stored at room temperature pending possible future analysis as required by Section 58 195 of the Good Laboratory Practice regulations (GLPs). Before treatment commenced and during Weeks 13, 26, 53, 71, 78, 86, 94 and 104, a further 200 g aliquot from each test diet and 5 g of the test compound were sent to the Sponsor for determination of the levels of active ingredient.
Duration of treatment / exposure:
105 weeks
Frequency of treatment:
continous
Doses / concentrationsopen allclose all
Dose / conc.:
250 ppm
Remarks:
corresponding to 7 - 18 mg/kg bw/day
Dose / conc.:
750 ppm
Remarks:
corresponding to 20 – 55 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
corresponding to 58 - 147 mg/kg bw/day
No. of animals per sex per dose:
70
Control animals:
yes, plain diet
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily- Cage side observations: morbidity and mortality. Regular examination by palpation was performed for the purpose of detecting superficial tumours.

DETAILED CLINICAL OBSERVATIONS: No dataBODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on the day that treatment commenced, at weekly intervals for Weeks1 - 13, then at Week 16 and at monthly intervals during the remainder of the treatment period, with additional weighings during Weeks 26 and 79.

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/week: Yes
- Compound intake calculated as mg/kg bodyweight/day, calculated, for Weeks 6, 13, 26, 52, 78 and 104, using the formula:Achieved dosage (mg/kg/day) = [concentration of test substance (ppm) -x weekly food intake (g)]/[end of week bodyweight (g) x 7]

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment commenced, both eyes of each rat were examined The eyes of all rats in each of Groups 1 (control) and 4 (2000 ppm) were similarly examined after treatment had continued for 13, 26, 53, 80 and 103 weeks.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 12, 25, 52, 78, and 103 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: no data
- How many animals: 10 animals of each sex from each group
- Parameters checked: haemoglobin concentration (Hb); erythrocyte count (RBC); leukocyte count (WBC), total; leukocyte count (WBC), differential; packed cell volume (PCV); platelet count; prothrombin time (PT); reticulocyte count.
From the erythrocytic data, the mean cell haemoglobin concentration [(Hb x 100) / PCV] and mean cell volume [(PCV x 10) / RBC] were calculated for each sample. Additional blood samples (for the examination of prothrombin times) were withdrawn from ten females of each group during Week,15, owing to the large number of dotted samples obtained at Week 13. Reticulocytes, Howell-Jolly bodies and Heinz bodies were stained and counted (if present) after 52 weeks for male rats and for all animals after 78 weeks of treatment. After 103 weeks of treatment any animals showing abnormalities of erythrocyte characteristics were examined for reticulocyte count. These parameters were also examined in respect of rats dying after the 85th week of treatment. For purposes of calculating group mean values for reticulocytes, values of < 2 were taken as 1.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 12, 25 (without anticoagulant), 52, 78, and 103 (with anticoagulant) weeks
- Animals fasted: no data
- How many animals: 10 animals of each sex from each group
- Parameters checked: urea; glucose; total protein; electrophoretic protein fractions; alkaline phosphatase activity (SAP or AP); alanine amino-transferase activity (ALT); aspartate amino-transferase activity (AST); cholesterol; Sodium (Na) and Potassium (K); Chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: after 12 (females) or 13 (males), 26 (males) or 27 (females), 52, 78, and 103 (males) or 104 (females) weeks
- Metabolism cages used for collection of urine: no data
- Animals fasted: no data
- Parameters checked: pH; specific gravity (SG); reducing substances; glucose; protein; ketones; bile pigments; urobilin; haemoglobin.
Microscopy: the sediment from centrifugation at 3400 r.p.m. for five minutes was examined for epithelial cells, polymorph and mononuclear leucocytes, red blood cells, casts or other abnormalities
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
All animals, including those which died or were killed in extremis, were subjected to a detailed necropsy involving opening of the cranial, thoracic and abdominal cavities.
Upon gross inspection, the external features were palpated and particular attention was directed toward the mammary tracts, the lymph nodes and the subcutaneous tissues, and compared with any relevant comments on the clinical history report.
The external auditory meatus, the nares, the buccal cavity and the tongue were inspected. The eyes, complete with optic nerve and relevant adnexa, were removed. The cranial cap was lifted and the brain dissected free of meninges. The pituitary was freed from the sella turcica and fixed separately. Any abnormality of meninges, brain, pituitary or cranial nerves was recorded.
Subcutaneous structures were then examined after reflection of the ventral skin from a mid line incision. Thoracic and abdominal viscera were examined before and after removal and note made of any abnormal position, morphology or interactions. The urinary bladder was slightly inflated with fixative and the urethra ligated. After removal, this was viewed by transmitted light. The mucosa was examined at the time of embedding. The entire intestinal tract was re-examined after removal. The stomach was opened along its greater curvature and rinsed in isotonic saline, prior to fixation . The caecum was similarly treated. The lungs were removed, weighed and all pleural surfaces were examined. They were then slightly inflated with fixative via the trachea, prior to immersion in fixative. After weighing, both kidneys and the liver were repeatedly sectioned at intervals of a few millimeters, and the cut surfaces were examined. The spinal cord was fixed within the cervical vertebrae.

HISTOPATHOLOGY: Yes.
Samples of the following tissues, taken from each rat, were preserved in 10% buffered formol saline (except for eyes, which were placed in Davidson's fixative): adrenal glands; colon; aortic arch; duodenum; bone; epididymides; brain; eyes and optic nerves; caecum (one sectioned initially); Harderian gland; heart; ileum; jejunum; kidneys; liver (at least two lobes); lungs; lymph nodes (cervical mesenteric); mammary gland (posterior and anterior); oesophagus; ovaries; pancreas; pituitary gland; prostate gland; salivary gland (submaxillary); sciatic nerve; seminal vesicles; skeletal muscle; skin; spinal cord (in situ) (cervical and thoracic); spleen; sternum; stomach (cardia, fundus and pylorus); testes; thymus; thyroid and parathyroid glands; tissue masses, suspected tumours and adjacent lymph nodes; tongue; trachea; urinary bladder; uterus (including cervix).
In addition, samples of any other tissues showing macroscopic abnormality were preserved in fixative. Blood smears were made, but on inspection these were found to be of insufficient quality for detailed examination.

The weights of the following organs were recorded: adrenal; glands; kidneys; brain; liver; heart; lungs; testes.
The ratios of organ weight to bodyweight were calculated for animals killed at termination.
Statistics:
The significance of any inter-group differences in blood composition or growth performance was assessed by Student's t-test, using a pooled variance. The probability of mortality and palpable mass distributions arising by chance was calculated by constructing 2 x 2 contingency tables and deriving Chi2 values therefrom.The sign test was applied to group mean food consumption data.All of these tests were applied as two-tailed tests.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were significantly fewer (P < 0.05) male rats bearing one or more subcutaneous masses in the high dosage group than there were in the control group. In other respects the appearance and behavior of rats treated with the test material was indistinguishable from that of controls.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality among treated animals was slightly higher than it was in the control group, but only among females receiving 750 ppm was statistical significance (P < 0.05) attached to this observation. Because of the lack of dosage-relation and the fact that the enhanced mortality was apparent only in the terminal stages of the study, the difference from the control value was considered fortuitous and unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly increased mean bodyweight gain was recorded during the first year of treatment for males receiving 2000 ppm and for all three treated female subgroups, but only in the case of females receiving 2000 ppm was the overall increase statistically significant (P < 0.01). During the second year of the study there were no significant variations in bodyweight change.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no marked inter-group differences in food intake. Group mean values for males receiving 2000 ppm were consistently and significantly (P < 0.001) slightly higher than the corresponding control values, but the total increase in consumption during the study was less than 4% of the control intake.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no remarkable inter-group differences during the first 13 weeks of treatment. Between Weeks 14 and 26, however, slightly increased utilization efficiency was apparent in all three treated female subgroups and in males receiving 2000 ppm, reflecting the slightly enhanced weight gains of these animals during that period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual assessment of water intake did not reveal any inter-group differences and quantitative measurements were not instituted.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The lesions discovered at these examinations were those commonly found in rats of this strain and there was no evidence of a response to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 12 and 25 weeks, the platelet counts of treated females were significantly lower (P < 0.05 - P < 0.001) than those of control females. After 52 weeks, however, values for treated females were higher than the control values (significantly so in the case of females receiving 750 ppm, P < 0.05). On most occasions the values recorded were within the normal range and there was no evidence for dosage-relationship. There were no notable inter-group disparities in platelet values for male rats.
A number of inter-group differences in other parameters attained levels of statistical significance but there were no persistent trends suggestive of a response to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A number of inter-group differences between treated and control animals reached a level of statistical significance. There were, however, no marked changes in treated rats and no persistent trends, although treated females, but not males, showed a tendency to higher plasma α2-globulin levels (except after 103 weeks).
Urinalysis findings:
no effects observed
Description (incidence and severity):
These data provided no evidence of a response to treatment.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant inter-group differences in the absolute or bodyweight-relative organ weights recorded for rats sacrificed after 105 weeks of treatment. A number of grossly abnormal organ weights were excluded from the calculation of group mean values and from statistical analysis. Among these were a number of adrenal weights for females, predominantly controls, which were excluded because of unilateral enlargement. No importance is attached to the uneven distribution between the groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A range of changes were present in all animals examined; the lesions seen were those commonly found in rats of the strain used. A number of subcutaneous tissue masses was present in all treatment groups. In the female rats which received 750 ppm and were killed at termination, a slight increase in the incidence of subcutaneous tissue masses was present.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was a wide range of non-neoplastic changes which were of a type and frequency commonly present in CD rats at this Laboratory. These included myocarditis, almost exclusively in the males, and sub-acute epicarditis of the heart; peribronchiolar and perivascular lymphoid hyperplasia and fibrino-purulent bronchopneumonia of the lungs; slight or moderate haemosiderosis and extramedullary haemopoiesis of the spleen; geriatric nephropathy, fine vacuolation of epithelial cells and slight hydronephrosis of the kidneys; peri thymic inflammation; focal acinar fibrosis of the pancreas and cortical pallor and hyperplasia of the adrenal glands. A wide range of other occasional lesions were observed. These lesions, listed above, were not considered to be treatment-related.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were three animals with malignant lymphoma and four other animals with sarcomas of the skin and subcutis. There were five other tumours, two of which occurred in one animal (No. 552♀). These tumours listed above were of a type and frequency common in CD rats at this Laboratory and not considered to be treatment-related.
Details on results:
HISTOPATHOLOGY ASSESSMENT OF RATS DYING OR KILLED IN EXTREMIS BETWEEN WEEK 53 AND TERMINATION
NON-NEOPLASTIC CHANGES
There was a wide range of banal degenerative and inflammatory changes present, similar to those considered usual in CD rats of this age and not associated with the test material. These included cortical haemorrhagic degeneration in the adrenals; status spongiosus in the brain; chronic myocarditis; geriatric nephropathy, cortical cysts and hydronephrosis; centriacinar hepatocytic fatty change/vacuolation, chronic inflammation, congestion and biliary hyperplasia in the liver; slight peribronchiolar lymphoid hyperplasia, perivascular lymphocytic infiltration, congestion, fibrous pleurisy and aggregations of distended macrophages in the lungs; hyperplasia, secretory activity and galactoceles in the mammary gland; pancreatic acinar degeneration and fibrosis of islet tissue; parathyroid hyperplasia; periarteritis; prostatitis; reduction of secretion in the lumina of the salivary glands; reactive hyperplasia and haemosiderosis in the spleen; ulceration of the non-glandular region, hyperplasia of the epithelium, oedema, mineralisation and distension of the crypt glands of the stomach; testicular atrophy and hyaline change in the endometrial stroma, hydrometra and endometrial polyps in the uterus. There were occasional changes that attained some statistical significance but were not considered to be associated with treatment with the test substance. In two instances, this was attributed to chance,. For example, there-was an increase in tubular,epithelial fatty change in the highest dosage group males and an increase in slight glomerular fatty change in the intermediate dosage group males. In two other instances a significant variation in incidence could be attributed to an unusually high and low incidence in the control groups. For example, there was a significant decrease in the incidence of chronic myocarditis in the lowest dosage group males. Mammary secretory activity in intermediate dosage group males was significantly increased.

NEOPLASTIC CHANGES
There was a wide range of neoplasms present which were not related to treatment with the test article. These included benign skin and subcutis epithelial tumours; benign fibroepithelial tumours of the mammary gland; islet cell adenomas of the pancreas; pituitary adenomas; thyroid parafollicular cell adenomas; phaeochromocytomas of the adrenal medulla; benign and malignant skin and subcutis mesenchymal tumours and malignant lymphomas.

HISTOPATHOLOGY ASSESSMENT OF RATS KILLED AT THE TERMINATION OF THE STUDY
NON-NEOPLASTIC CHANGES
There was a wide range of banal degenerative and inflammatory changes present, similar in type and incidence to those considered usual in CD rats at Life Science Research and not associated with treatment with the test article. These included adrenal cortical haemorrhagic degeneration; parasitic worms in the lumen of the colon; subepithelial lymphocytic infiltration in the pharyngeal region of the head; chronic myocarditis; geriatric nephropathy; hydronephrosis, cortical cysts and calculus formation in the renal pelvis of the kidneys; periacinar and centriacinar hepatocytic vacuolations, biliary hyperplasia, clear cell foci, chronic inflammation and distended sinusoids in the liver; slight peribronchial lymphoid hyperplasia, slight perivascular lymphocyti infiltration, medial calcification in the pulmonary artery, macrophage infiltration, fibrous pleurisy and congestion in the lungs; mammary hyperplasia, secretory activity and galactoceles; pancreatic acinar degeneration; periarteritis; sciatic nerve fibre degeneration; reduction in colloid contents in the seminal vesicles; nerve fibre degeneration in nerve roots and cauda equina of the spinal cord; siderosis of the spleen; testicular atrophy; regression/involution of the thymus; parafollicular cell hyperplasia in the thyroids and endometrial polyps and cystic endometrial hyperplasia in the uterus.
There were occasional changes that attained some statistical significance but were not related to treatment. These were due to high or low incidence in the control groups or due to a chance effect. These included a zero incidence of marked geriatric nephropathy in highest dosage group males and a higher incidence of moderate geriatric nephropathy in intermediate dosage group females. There was a low incidence of slight centriacinar hepatocytic vacuolation in the intermediate dosage group females. The reduction in colloid content in the seminal vesicles was increased in intermediate dosage group males. Statistically significant variations in incidence were seen in the liver and spinal cord. There was a zero incidence of nerve fibre degeneration in the cauda equina of the spinal cord in the highest dosage group males. Biliary hyperplasia in the liver also had a zero incidence in the highest dosage group males.

NEOPLASTIC CHANGES
There was a range of neoplasms present which were not related to treatment with the test substance. These included benign skin and subcutis epithelial tumours; mammary gland benign fibro-epithelial tumours; pituitary adenomas; thyroid parafollicular cell adenomas; phaeochromocytomas of the adrenal medulla; interstitial cell adenomas of the testes and skin and subcutis benign mesenchymal tumours. There was an increase in the incidence of pituitary adenomas in the intermediate dosage group males and highest dosage group females which are regarded as chance effects.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: corresponds to 58 - 147 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Assays of experimental diets

Concentrations of the test substance in diet samples collected at intervals during the study were determined; these were in tolerable agreement with the desired concentrations.

Achieved dosage

The reduction in achieved dosage as the study progressed reflected the declining ratio between food intake and bodyweight. The range of achieved dosages was:

Level (ppm): 250; 750; 2000

Dosage (mg/kg/day): 7 - 18; 20 – 55; 58 - 147

Applicant's summary and conclusion