Harmonization of animal clinical pathology testing in toxicity and safety studies. The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing.

Ten scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in regulated toxicity and safety studies. For repeated-dose studies in rodent species, clinical pathology testing is necessary at study termination. Interim study testing may not be necessary in long-duration studies provided that it has been done in short-duration studies using dose levels not substantially lower than those used in the long-duration studies. For repeated-dose studies in nonrodent species, clinical pathology testing is recommended at study termination and at least once at an earlier interval. For studies of 2 to 6 weeks in duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals. If a study contains recovery groups, clinical pathology testing at study termination is recommended. The core hematology tests recommended are total leukocyte (white blood cell) count, absolute differential leukocyte count, erythrocyte (red blood cell) count, evaluation of red blood cell morphology, platelet (thrombocyte) count, hemoglobin concentration, hematocrit (or packed cell volume), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In the absence of automated reticulocyte counting capabilities, blood smears from each animal should be prepared for reticulocyte counts. Bone marrow cytology slides should be prepared from each animal at termination. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis. The core clinical chemistry tests recommended are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. For hepatocellular evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, glutamate dehydrogenase, or total bile acids. For hepatobiliary evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alkaline phosphatase, gamma glutamyltransferase, 5' -nucleotidase, total bilirubin, or total bile acids. Urinalysis should be conducted at least once during a study. For routine urinalysis, an overnight collection (approximately 16 hr) is recommended. It is recommended that the core tests should include an assessment of urine appearance (color and turbidity), volume, specific gravity or osmolality, pH, and either the quantitative or semiquantitative determination of total protein and glucose. For carcinogenicity studies, only blood smears should be made from unscheduled sacrifices (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia.

Assessment of drug exposure in rat dietary studies.

1. The objective of this study was to justify the evaluation of exposure of animals to chemical substances on the basis of only three blood samples taken during a 24-h period, but still with acceptable accuracy. 2. Fischer rats were fed a diet mixed with either paracetamol, 100 mg.kg-1 (short half-life compound), antipyrine, 100 mg.kg-1 (medium half-life compound), or phenylbutazone, 50 mg kg-1 (long half-life compound) for 3 weeks. It had been shown in a preliminary study that these compounds when administered at these dose levels did not influence feeding behaviour. At the end of 3 weeks, five rats were sampled every 3 h beginning and ending at 19.00 h (45 rats in total) and plasma concentrations were measured using h.p.l.c. 3. The area under the curve over 24 h (AUC24), calculated using all nine concentrations was considered to be the true AUC24. Subsequently, estimates of this parameter were made using different combinations of concentrations at three or even two selected time points. 4. For each compound, the highest concentration occurred at 07.00 h. It was shown that using the concentrations at 07.00, 10.00 and 16.00 h the estimate of the AUC24 was within 15% of the true value. 5. In comparison with a gavage study in the same rat (strain and age), bioavailability was lower in the diet study with relative bioavailabilities of 27, 22 and 61% for paracetamol, antipyrine and phenylbutazone, respectively. 6. In conclusion, drug exposure as expressed by AUC24 and Cmax can be accurately determined in rat studies using compound administration in the diet by measuring concentrations at three selected time points for compounds with elimination half-lives ranging from about 1 to 5 h.