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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The in life phase of the study was undertaken between 4 November 1992 and 3 December 1992.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
45W81 was administered to groups of 10 males and 10 females for at least 28 consecutive days by gavage, at dose levels of 200,600 or 2000mg/kg/day. At the end of the dosing period all animals were killed and necropsied.
During this study, animals were assessed for clinical signs, body weight changes and food and water consumption. Ophthalmoscopy was carried out prior to, and towards the end of the dosing period. Blood and urine samples were taken at a selected timepoint for clinical pathology and urinalysis. At the end of the dosing period, animals were killed and subjected to extensive macro- and microscopic examination, including evaluation of organ weights.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-bromo-6-(4-methylbenzoyl)pyridine
EC Number:
618-079-0
Cas Number:
87848-95-1
Molecular formula:
C13H10BrNO
IUPAC Name:
2-bromo-6-(4-methylbenzoyl)pyridine
Specific details on test material used for the study:
45W81 (2-bromo-6-(4-toluoyl)-pyridine), batch BX C67

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rationale
Exposure to 45W81 by the oral route is a potential hazard that requires evaluation.
The doses are based upon the results from a previous 11 day dose range finding study (Wellcome Report No: BPAT 91/0119). The no observable effect dose in that study was the high dose of 2000mg/kg/day, hence the same dose levels were used.
The Wistar rat is a readily available rodent species acceptable to regulatory authorities with a wide range of background data, and with documented susceptibility to a large number of toxic substances.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
40 male and 40 female Wistar (VAF) rats, approximately 5 weeks of age in the weight range 115--lSOg, were supplied by Charles River (U.K.) Ltd., Manston, Kent. The animals were housed in Room 14, Building 88, Beckenham, under controlled environmental conditions (room temperature 22°C ± 2°C, photoperiod light phase 0700-1900h). The animals were allowed to acclimatise for approximately 2 ,veeks prior to the start of dosing. The animals were allocated, 10 males and 10 females per group, following a routine randomisation procedure, and housed 5 animals of the same sex to a cage.
Each animal was identified by a unique six figure number tattooed on the tail and a within-cage system of ear-marking.
Pelleted diet (RMl(E) SQC, Batch Nos. 8153 and 8247, Special Diet Services Ltd., Manea, Cambs.) and water (Thames Water Authority) were provided ad libitum.
There were no contaminants seen in the analytical data for food or water which were considered to prejudice the integrity of the study.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Doses were administered orally by stomach tube at a volume to bodyweight ratio of 10 mL/kg, once daily for a minimum of 28 consecutive days. At the end of the 28 day dosing period, necropsies took 2 days to complete. Dosing was continued as necessary until the day prior to the scheduled sacrifice for each animal.
Vehicle:
methylcellulose
Details on oral exposure:
Formulation
Dosing suspensions were prepared once weekly by the Drug Formulation Laboratory, Division of Drug Safety Evaluation, and stored in the dark at 4°C.
45W81 was suspended in 1 % methyl cellulose (Celacol; Sigma Chemical Company Ltd., Lot No. 30H--0239) to give a final concentration of 200mg/rnl.
Further dosing suspensions of 60 and 20mg/rnl were prepared in a similar manner by dilution in 1 % aqueous Celacol.

Experimental Design
Group 1 - Vehicle - 0 mg/kg (0 mg/L)
Group 2 - low dose - 200 mg/kg (20 mg/L)
Group 3 - mid dose - 600 mg/kg (60 mg/L)
Group 4 - high dose - 2000 mg/kg (200 mg/L)

Doses were administered orally by stomach tube at a volume to bodyweight ratio of 10mL/kg, once daily for a minimum of 28 consecutive days. At the end of the 28 day dosing period, necropsies took 2 days to complete. Dosing was continued as necessary until the day prior to the scheduled sacrifice for each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the dosing formulations was performed by the Analytical Development Laboratories, Dartford. The results of the assays indicated satisfactory agreement with the labelled strength.
Duration of treatment / exposure:
10 mL/kg, once daily for a minimum of 28 consecutive days.
Frequency of treatment:
10 mL/kg, once daily for a minimum of 28 consecutive days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10 Male and 10 Female per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Observations
Clinical Signs
All animals were observed for signs of ill health from Day -4 prior to dosing, daily for the first 7 days of dosing and then at weekly intervals as no signs persisted.
Bodyweight
Individual body weights were recorded on Days -5, 1, 8, 15, 22 and 28.

Food Consumption
The quantity of food consumed per cage was recorded on Days-4, 2, 9, 16, 23 and 29.

Water Consumption
The quantity of water consumed per cage was recorded on Days-4, 2, 9, 16, 23 and 29.

Ophthalmoscopy
Examination of the eyes was carried out using an ophthalmoscope after dilatation of the pupils with 1 % Tropicamide sterile ophthalmic solution (Mydriacyl, Alcon Laboratories U.K. Ltd.). All animals from Groups 1 to 4 were examined prior to the start of dosing (Day -5). Only animals in Groups 1 and 4 were examined towards the end of the treatment period (Day 23).

Clinical Pathology
Blood samples were collected from all animals via the vena cava prior to necropsy. Approximately 5mL of blood was taken and dispensed into tubes containing either EDTA anticoagulant (haematology), heparin anticoagulant (clinical chemistry), or citrate (coagulation). The following parameters were measured:

Haematology
Red cell count (RBC)
Haemoglobin (HB)
Packed cell volume (PCV)
Mean cell volume (MCV)
Mean cell haemoglobin (MCH)
Mean cell Hb concentration (MCHC)
Platelets (PLT)
Reticulocyte count (RETC)
Qualitative examination of blood film
Prothrombin time (PT)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (NEUT)
Lymphocyt~ (LYMP)
Monocytes (MONO)
Eosinophils (EOSN)
Basophils (BASO)
Nucleated red blood cells (NRBC)
Activated partial thromboplastin time (APTT)

Clinical Chemistry
Glucose (GLU)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (AST)
Alanine aminotransferase (ALT)
Total bilirubin (TBIL)
Urea (UREA)
Creatinine (CRE)
Sodium (NA)
Potassium (K)
Total protein (TP)
Albumin (ALB)
Globulin (GLOB)
Cholesterol (CHOL)
Calcium (CA)
Inorganic phosphate (PHOS)
Lactate dehydrogenase (LDH)
Creatine kinase (CK)
Gamma glutamyl transpeptidase (GGT)

Urinalysis
On Day 23 following dosing, the animals (5 males and 5 females, the first cage of either sex from each group) were placed individually into metabolism cages without food and water and urine collected over a 5 hour period.
The individual samples were analysed for:
Volume (UVOL)
Osmolality (UOSM)
Hydrogen ion cone. (PH)
Microscopy of spun sediment
Dipstick tests for:
Glucose (UGLU)
Bilirubin (UBIL)
Ketone (UKET)
Blood (UBLD)
Protein (UPRO)
Urobilinogen (UUBG)
Sacrifice and pathology:
Terminal Procedures
All animals were killed at the end of the treatment period on Days 29 and 30.

Euthanasia
All animals were killed by overdose of barbiturate (i.p. sodium pentobarbitone - Sagatal, May & Baker, Dagenham) followed by exsanguination via the vena cava.

Necropsy
All animals were subjected to a full necropsy examination with the following tissues removed, weighed (*) and placed into fixative following Standard Operating Procedures.

*Adrenals
Bone marrow smear (Methanol)
*Brain
Caecum
Cervical lymph node
Colon
Duodenum
Eyes/Harderian glands (Davidson's)
Femur and femoro/tibial joint
*Heart
Ileum
Jejunum
*Kidneys
*Liver
Lungs and mainstem bronchi
Mesenteric lymph node
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin/ mammary gland
Spinal cord
*Spleen
Sternum
Stomach
Submandibular salivary gland
Tail
*Testes/Epididymides (Bouins)
Thoracic aorta
Thymus
Thyroids/ parathyroids
Tongue
Trachea
Urinary bladder
Uterus
Vagina/ cervix

All tissues were fixed in neutral buffered formalin except where indicated.

Histopathology
Samples of all tissues taken into fixative were routinely processed (excluding the tail). Haematoxylin and Eosin stained sections were prepared from all animals in Groups 1 and 4, from any animal dying before the end of the study (Group 3 male - 923256), and all gross abnormalities for histopathological examination by light microscopy.
Statistics:
Statistical Methods
Results were analysed separately by gender (and day as appropriate). No comparison of male and female results was carried out. No comparisons across sacrifice dates (day 29 versus day 30) were carried out. No 'repeated measures' or covariance analysis were carried out. Cage effects were not allowed for, or estimated.
All comparisons were inter-group. With the exception of bodyweight, parameters were only assessed once on each individual at sacrifice (urinalysis - at day 24). Changes from baseline measures could not be assessed in this study.
The data was not transformed before analysis.
Analyses of variance were employed to detect any differences between the groups of animals globally. Graphs of residuals versus fitted values were used to qualitatively and subjectively check the need for. transformation of the data. Animals giving standardized residuals (= 'studentized residuals' in SAS) of >2 or <-2 were noted. Dunnett's multiple comparison test was used to compare the treated groups to the control.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A few observations were made which were considered not to be treatmentrelated.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One group 3 male was found dead on Day 28.
The one unscheduled death (923256) was attributable to mis-dosing.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects on the individual or group mean values for body weight were seen.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the average food consumption per animal per cage.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the average water consumption per animal per cage.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
All findings were within the normal range for rats of this age and strain. There was no evidence of any ophthalmological finding attributable to the administration of the test compound.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects of 45W81 on the haematological parameters measured.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no effect of 45W81 on the clinical chemistry parameters measured.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Female animals showed a dose-related increase in urine volume (statistically significant for group 4 females).
The statistically significant parameter values and the dose-related increase in urine volume of the females, is thought not to be of toxicological significance.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects on the individual or group mean values for absolute or relative organ weights were noted for animals killed at the end of the dosing period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no evidence of treatment-related effects with 45W81.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no findings at necropsy that can be attributable to 45W81.
A variety of incidental findings was encountered in rats from all dose groups. The findings were consistent with spontaneous changes commonly encountered in young rats kept under laboratory conditions.
The one unscheduled death (923256) was attributable to mis-dosing.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There was no evidence of any microscopic findings that can be attributable to 45W81.

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
There was no evidence of any adverse effects attributable to 45W81. The statistically significant parameter values and the dose-related increase in urine volume of the females, is thought not to be of toxicological significance.
The no observable effect level of 45W81 in the Wistar rat in this study is 2000mg/kg/ day.