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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Ro 1525 corresponds to Colipa A 155
Batch number : Ro-RN-6567-083
SAT 0 10232 and SAT 000344

Test animals

Species:
rat
Strain:
other: 130 Wistar and 10 Sprague Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague Dawley rats in high dose and control group for inter-strain comparison

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 w
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15, 60 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 Wistar rats per sex per dose.
In mid-dose, high-dose and control group additionally 5 animals per sex per dose for evaluation of 4 weeks recovery period.
In high-dose and control group additionally 5 male Sprague Dawley rats for evaluation of inter-strain differences.
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
Clinical signs, post-dose observations and neurotoxicity assessment
All clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily before dosing, immediately after, and approximately 1 and 2 hours after dosing.
Once before commencement of treatment and once a week therafter each animal was subjected to a detailed clinical exarmination, which included an evaluation of neurotoxicity. Animals were examined in an open arena for a period of three minutes. Observed parameters, described by an
evaluation scale, are indicated below:
Removal (from cage), Handling reactivity, Lachrymation, Palpebral closure, Salivation, Piloerection, Rearing, Mobility impairment, Arousal (animal activity), Vocalisation, Stereotypies, Unusual respiratory pattern, Bizarre behaviour, Urination, Defecation, Clonic movements, Tonic movements, Gait
Once during week 12/13 of treatment and once during week 4 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, audio and proprioceptive stimuli) and assessment of grip strength were also performed.

Motor activity assessment (MA)
The MA of the first 5 males and 5 fernales was measured once during week 12/13 of treatment and week 4 of recovery by an automated activity recording device (Mini Opto-Varimex, Columbus International Cop.). Measurements were performed using a computer generated random order.

Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon to look for dead or moribund animals. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Body weight
All animals were weighed on the day of allocation to treatment groups, on the day that treatment comenced, weekly therafter and just prior to necropsy.

Food consumption
The weight of food consumed by each cage of rats was recorded weekly following allocation and the group mean daily intake per rat calculated.

Ophthalmoscopy
Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment, by means of both an ophthalmoscope and a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic, Merck, Sharp and Dohme, Rome, Italy). The eyes of control and high-dose animals were re-examined during week 12/13 of treatment. During the examination particular attention was paid to:
Anterior chamber, Conjunctivae and eyelids, Cornea and sclera, Iris, lens, Posterior segment - vitreous humour, Ocular fundus

Clinical pathology investigations
Once during week 13 of treatment period, samples of blood were withdrawn under light ether anaesthesia from the retro-orbital sinus of all surviving male and female animals, under conditions of food and water deprivation. Once during week 13 and again during week 4 of the recovery period individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 ml/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. Blood samples were collected into tubes containing EDTA anticoagulant for haematological investigations, heparin anticoagulant for biochemical tests and citrate anticoagulant for coagulation tests. Blood samples were collected and analysed in the same order, a computer-generated random cage order being used. The estimations performed on blood samples are listed below:
Haematology
Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count (not performed as no signs of anaemia wae present), Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count (Differential leucocyte count, Neutrophils, Lymphocytes, Eosinophils, Basophil, Monocytes, Large unstained cells), Abnormalities of the blood film, Platelets, Prothrombin time
Clinical chemistry
Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Urea, Creatinine, Glucose, Albumin, Total bilirubin, Total cholesterol, Total protein, Sodium, Potassium, Calcium, Chloride
Urinalysis
Appearance, Volume, Specific gravity, pH, Protein, Total reducing substances, Glucose, Ketones, Bilirubin, Urobilinogen, Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
Epithelial cells, Polymorphonuclear leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components
Sacrifice and pathology:
Euthanasia
All animals were killed by carbon dioxide narcosis at the end of the scheduled treatment period and were subjected to necropsy supervised by a pathologist, as detaiied below.

Necropsy procedure
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surfaces and orifices). Changes were noted and the requisite Organs weighed (see following section).

Organ weights
Adrenal glands, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Liver, Uterus, Kidneys.
The ratios of Organ weight to body weight was calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed below were fixed and preserved in 10% buffered formolsaline (except eyes, which were fixed in Davidson's fluid; testes and epididymides which were fixed in Bouin's solution. These were preserved in 70% ethyl alcohol).
Abnomalities, Adrenal glands, Aorta, Bone marrow (from sternum), Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum (including Peyer's patches), Jejunurn, Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes - cervical, Lymph nodes - mesenteric, Mammary gland, Oesophagus, Ovaries, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus, Vagina

Histopathological examination
Tissues listed above were fixed and preserved. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. The examination was carried out as detailed below
a) Tissues specified above from all animals in the control and high dose groups.
b) Kidneys from all animals.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated fiom the actual values in the computer without rounding off.
Microscopic observations were tested for statistical significance using the non-parametric Kolmogorov-Smimov test.

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral toxicity of RO 1525 when given by daily adrninistration to rats at dosages of 5, 15 and 60 mg/kg/day has been investigated over a period of 13 weeks and possible recovery from any treatment-related changes over a 4 week recovery period. No changes of toxicological importance were observed in any of the in-life parameters evaluated. The only exceptions were slight variations observed in urine parameters, which disappeared after the recovery period. In addition, the relative weight of the kidney appeared slightly increased at the end of the treatment period. Discolouration of the urine was also observed for the duration of the treatrnent period. The observed changes suggest a toxic effect of the test item to the kidneys, evident at the high-dose level. This evidence is supported by the histopathological examination of this organ, which appears to be damaged (tubular degeneration) by the presence of the test item (pigmentation) at the mid- and high-dose levels. This change, considered to be treatment related, does not appear to be reversible following a 4 week recovery period.
On the basis of these results, a toxic effect of the test item to the kidney, mainly demonstrated by the histopathological examination of this organ, is evident at the high-dose level in both strains of rats (Wistar and Sprague Dawley). Some Wistar rats treated with 15 mg/kg/day showed similar changes, but the incidence of the changes was not statistically significant compared with controls. At the dose level of 5 mg/kg/day there was no evidence
of any toxic effect. Therefore, 5 mg/kg/day is the No Observed Effect Level (NOEL) for the test item under the described test conditions.
Executive summary:

The oral toxicity of RO 1525 when given by daily administration to rats, has been investigated over a period of 13 consecutive weeks and recovery from any potential treatment-related effects over a period of 4 consecutive weeks.

Three groups, each of 10 male and 10 female rats of the Wistar strain, received the test item daily by oral gavage at dosages of 5, 15 and 60 mg/kg/day for a minimum of 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (distilled water) and acted as a control. Five additional animals for each sex were included in the high, mid and control groups for recovery assessment. In addition, the control and high dose groups included 5 male Sprague Dawley rats for inter-strain comparison. One of these satellite groups was treated at 60 mgkg and the second one with the vehicle alone (distilled water).

Mortality

One female from the low-dose and 2 males from the high-dose groups were found dead during the study. In addition, 1 control female and 2 males from the mid-dose group died on days 87 and 88, respectively due to technical problems during the bleeding procedure.

Daily post-dose observations and detailed clinical signs

No significant daily post-dose observations were noted during the study. Detailed clinical signs with neurotoxicity assessment did not show any signs which could be clearly related to the treatment with the test item. Skin/fur staining of different regions of the body surfaces and dark staining in the litter tray were observed in all treated animals.

Motor activity and sensory reaction to stimuli

Neurotoxicity tests and rneasurements taken at the end of treatrnent did not show changes clearly attributable to the test item.

Body weight

Body weight was not affected by treatment.

Food consumption

No change of toxicological relevance was observed in food consumption.

Ophthalmoscopy

No findings were seen at the ophthalmic examination performed at the end of the study.

Haematology

No changes of toxicogical significance were observed for haematological parameters.

Clinical chemistry

A slight increase in total protein, aspartate aminotransferase and albumin haematic levels, stastically significant in high-dose males (main groups), was noted at the end of treatment. These changes and all the other occasional statistically significant changes observed were

slight and within historical control values and, therefore, were considered of no toxicological relevance.

Urinalysis

An increased number of epithelial cells and leukocytes, with a dose related trend, was noted in all treated groups, when compared to controls, at the end of treatment. The presence of ketones, proteins and bilirubin, particularly evident in the high-dose groups (main and satellite groups), was also seen at the end of the treatment period. They were no longer present at the end of the recovery period. The urine volurne was also significantly increased in the treated males when compared to controls (main and satellite groups). This change was still present in the mid-dose males only at the end of the recovery period.

No toxicological significance was attributed to the other statistically significant changes observed.

Organ weights

A slight, but statistically significant, increase in relative kidneys weight was seen at the end of the treatment period in the high-dose males (main and satellite groups) and in relative spleen weights in the high-dose males of the main group. This change disappeared at the end of the recovery period.

Macroscopic observations

No treatment related macroscopic changes were detected in the animals killed at termination or in those found dead during the treatment period or sacrificed after the recovery period.

Microscopic observations

Two distinct, apparently unrelated, patterns of pathological change were observed in the kidneys in this study.

There was a clearly spontaneous set of changes seen in both control and treated Sprague Dawley rats, but not in any Wistar rats. These changes have been grouped under the term "nephropathy".

The most important feature of this nephropathy was cast formation or thickened basement

membrane and tubular basophilia. The changes were seen in individual nephrons and/or groups of nephrons. The glomerular capsules of some affected nephrons were dilated and often contained proteinaceous material. This type of spontaneous pathology has long been recognised in the Sprague Dawley rat (Peter CP, Burek JD, van Zwieten MJ, Spontaneous Nephropathies in Rats. Toxicol Pathol 1986; 14(1):91-100).

In addition, the high dose animals of both strains showed a distinct renal pathology when compared to the relevant controls. This pathology was mainly characterised by cytoplasmatic brown pigmentation in groups of proximal tubules, located in the mid-cortical region. These tubules were lined by slightly basophilic cells with vesicular nuclei and marginal chromatin.

Condensed cells were also observed in the affected tubules. The condensed cells were shed into the tubular lurnen and showed shrinkage of cell volume, condensed nuclei, and intensely eosinophilic cytoplasm, suggesting apoptosis. Interstitial inflammatory reactions were also detected around the involved tubules. These changes were still present at the end of the recovery period.

This renal pathology could be considered a degenerative condition due to the deposition (brown pigmentation) and/or interaction of the test item or its metabolites with tubular cells.

Pigmented macrophages were observed in the mesenteric lymph nodes of some rats in the high dose groups of both strains. Such a finding was also detected in the spleen in most high dose Wistar females and Sprague Dawley males. This aspect was considered to be an expression of deposition of the test item.

The kidneys of some mid-dose females at the end of the treatment period and some mid-dose males at the end of the recovery period showed slight cortical tubular pigmentation associated with interstitial chronic inflammation. These changes were not statistically significant.

No treatrnent related changes were observed in the low dose animals.

Conclusion

On the basis of these results, a toxic effect of the test item to the kidney, rnainly demonstrated by the histopathohgical. examination of this organ, is evident at the high dose level in both strains of rats (Wistar and Sprague Dawley). Some Wistar rats treated with 15 mg/kg/day showed similar changes, but the incidence of the changes was not statistically significant compared with controls. At the dose level of 5 mg/kg/day there was no evidence of any toxic effect. Therefore, 5 mg/kg/day is the No Observed Effect Level (NOEL) for the test itern under the described test conditions.