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Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies*
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
29, 198–201 (1996)
0022
Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies* KURT WEINGAND,**,1 GEOFF BROWN,2 ROBERT HALL,3 DAI DAVIES,4 KENT GOSSETT,3 DOUG NEPTUN,1 TREVOR WANER,5 TOSHIAKI MATSUZAWA,6 PAUL SALEMINK,7 WILHELM FROELKE,8 JEAN-PIERRE PROVOST,9 GIANNI DAL NEGRO,10 JOHN BATCHELOR,2 MAMORU NOMURA,6 HORST GROETSCH,8 ALPHONS BOINK,7 JON KIMBALL,1 DAVID WOODMAN,4 MALCOLM YORK,4 EVA FABIANSON-JOHNSON,5 MICHEL LUPART9, AND ELSA MELLONI10 The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing consisting of delegates from 1American Association for Clinical Chemistry’s Division of Animal Clinical Chemistry (United States), 2Association for Comparative Haematology (United Kingdom), 3 American Society for Veterinary Clinical Pathology (United States), 4Animal Clinical Chemistry Association (United Kingdom), 5 International Society for Animal Clinical Biochemistry (multinational), 6Japanese Pharmaceutical Manufacturer’s Association (Japan), 7Dutch Association for Comparative Haematology (Netherlands), 8Arbeitsgruppe Klinische Chemie bei Laboratoriumstieren der Deutschen Gesellschaft fur Klinische Chemie E.V. (Germany), 9Association des Biologistes Cliniciens pour Animaux de Laboratoire (France), and 10Gruppo di Ematologia ed Ematochimica Applicate alla Tossicologia (Italy) Received December 19, 1994; accepted August 29, 1995
Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. (1996) WEINGAND, K., BROWN, G., HALL, R., DAVIES, D., GOSSETT, K., NEPTUN, D., WANER, T., MATSUZAWA, T., SALEMINK, P., FROELKE, W., PROVOST, J-P, DAL NEGRO, G., BATCHELOR, J., NOMURA, M., GROETSCH, H., BOINK, A., KIMBALL, J., WOODMAN, D., YORK, M., FABIANSON-JOHNSON, E., LUPART, M., AND MELLONI, E. Fundam. Appl. Toxicol. 29, 198–201. 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 * Author affiliations: The Procter & Gamble Company, Cincinnati, Ohio (K.W.); Huntingdon Research Centre Ltd., Huntingdon-Cambs, England (G.B.); Corning Hazleton, Madison, Wisconsin (R.H.); Zeneca Pharmaceuticals, Macclesfield-Cheshire, England (D.D.); SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania (K.G.); Novel Pharmaceutical, Inc., Research Triangle Park, North Carolina (D.N.); Life Science Research Israel, Ness Ziona, Isreal (T.W.); Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan (T.M.); Organon International B.V., Oss, Netherlands (P.S.); Boehringer Ingelheim KG, Ingelheim-Rhein, Germany (W.F.); Pfizer, Amboise, France (J.P.P.); Glaxo Italia, Verona, Italy (G.D.N.); Sanofi-Winthrop, Alnwick-Northumberland, England (J.B.); Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan (M.N.); Hoechst AG, Frankfurt, Germany (H.G.); Rijksinstituut Voor Volksgezondheid en Milieuhygiene, Bilthoven, Netherlands (A.B.); R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey (J.K.); SmithKline Beecham, Welwyn-Herts, England (D.W.); Glaxo Wellcome, Ware-Hertfordshire, England (M.Y.); Kabi Pharmacia, Helsingborg, Sweden (E.F.J.); Servier, Gidy, France (M.L.); Boehringer Mannheim Italia, Monza, Italy (E.M.). ** To whom reprint requests and correspondence should be addressed at The Procter and Gamble Company, Health Care Research Center, P.O. Box 8006, Mason, OH 45040-8006.
0272-0590/96 $12.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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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
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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. q 1996 Society of Toxicology
Clinical pathology testing of laboratory animals in toxicity and safety studies is an important part of safety assessment for food additives, drugs, and chemicals. Some government regulatory agencies and professional standards organizations provide guidelines for clinical pathology testing in animal toxicity and safety studies. These testing guidelines vary considerably (Stitzel and Waner, 1992). Clinical pathology testing of laboratory animals is a distinct scientific specialty that requires qualified professionals for test selection, technical conduct, and data interpretation (Dooley, 1979) (Carakostas and Banerjee, 1990a). Clinical pathology tests often provide information not detected by direct examination of organs and tissues, e.g., evaluations of hepatic and renal function, oxygen carrying capacity of the blood, circulating cells of the immune system, hemostatic function, and systemic mineral and electrolyte homeostasis. This information is useful for determining the biological significance of findings in toxicity and safety studies conducted with laboratory animals and the safety testing to be conducted in clinical trials for development of new drugs and food additives. In an effort to improve clinical pathology testing guidelines for safety assessment, 10 scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in toxicity and safety studies. This document presents minimum recommendations for clinical pathology testing. Additional testing may be needed to better characterize any observed or expected toxic effects of a test material. These recommendations should be implemented to comply with all appropriate laws and regulations concerning animal welfare and husbandry. BLOOD SAMPLING
Hematologic and clinical chemistry tests are subject to influence by the conditions under which blood is collected, such as feeding (fasting), anesthesia, and the site of blood collection. For this reason, it is critical that the blood be collected in the same manner from concurrent control and treatment groups of animals. Feeding or fasting prior to blood sampling may have effects on animal health, behavior,
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blood sample quality, and carbohydrate and lipid metabolism; these effects vary among species. The decision to feed or fast before blood sample collection should be based on individual study objectives, the species used, and the biological activity of the test material. Blood samples should be collected in a manner that minimizes time-related biases. Blood samples from multiple animals should not be pooled. Predose clinical pathology testing is useful for general health screening of nonrodent species considered for placement in toxicity and safety studies (Leissing et al., 1985). Predose clinical pathology testing of rodents is not necessary because of the large number of animals per group in rodent studies and the homogeneity of the laboratory animal population. Additionally, predose blood sampling of young rodents is limited by small blood volume and may have adverse effects on animal health. Clinical pathology testing of mice is limited by blood sample volume. Because of this limitation, interim (in-life) study blood samples should not be collected routinely from mice. Blood samples from mice for clinical pathology testing are collected optimally at terminal euthanasia only. The frequency and timing of clinical pathology testing is dependent upon study duration, study objectives, the biological activity of the test material, and the species tested. The following are minimum recommendations that may be modified because of these factors. 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 because of the small number of animals per group and interanimal variability. For studies of 2 to 6 weeks’ duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals (Davies, 1992). If a study contains recovery groups, clinical pathology testing at study termination is recommended. For carcinogenicity studies, the recommendation is that blood smears be made only from unscheduled terminations (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia. Quantitative clinical laboratory testing during the latter half of a 104-week carcinogenicity, toxicity, or safety study involving rodents is considered to be generally inappropriate and is not recommended (Weingand et al., 1992). As rodents age, they become progressively more susceptible to naturally occurring diseases that affect clinical laboratory tests, resulting in greater test value variability that obscures meaningful interpretation of test material effects.
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The animals used for clinical pathology testing should be examined for morphologic pathology findings. Use of separate subgroups of rodents for clinical pathology testing is scientifically acceptable, but maximal information is obtained when clinical pathology findings can be associated with gross and histopathologic observations from the same animal.
creatine kinase, and lactate dehydrogenase activities and fractionation of serum/plasma proteins are not recommended for routine use in animal toxicity and safety studies (Weingand et al., 1992). Additional tests may be useful in the evaluation of a test material known or suspected of affecting a specific tissue or metabolic pathway.
HEMATOLOGY
URINALYSIS
The core hematology tests recommended for animal toxicity and safety studies are total leukocyte (white blood cell) count, absolute differential leukocyte count (Zawidzka, 1990), 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. Reticulocyte counts and bone marrow cytology are not routinely necessary as screening tests in toxicity and safety studies but may be indicated when the test material has an effect on the hematopoietic system. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis for animal toxicity and safety studies (Vargaftig et al., 1979; Theus and Zbinden, 1984; Godsafe and Singleton, 1992). CLINICAL CHEMISTRY
The core clinical chemistry tests recommended for animal toxicity and safety studies are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. There are a variety of clinical chemistry tests for evaluation of liver health and function appropriate for use with laboratory animals (Thompson et al., 1984, 1987; Hoffman et al., 1989; Carakostas et al., 1990; Leonard et al., 1984; Woodman, 1988). 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. Ornithine decarboxylase, ornithine carbamoyltransferase,
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Uroanalytic tests are often inaccurate due to artifactual changes induced in vitro by the suboptimal conditions under which timed urine samples are commonly collected from animals in metabolic cages (Osborne and Stevens, 1981; Evans and Parsons, 1986; Ragan, 1989; Taylor and Neal, 1982). Urine sample contamination should be minimized, e.g., food, drinking water, feces, vomit, blood. Pooling of urine samples from multiple animals is not recommended. Urinalyses are subject to influence by the conditions of urine collection, such as feeding, fasting, and sample time. For this reason, it is recommended that urine be collected in the same manner from concurrent control and treatment groups. Urinalysis should be conducted at least once during a study. It is optimal to conduct and interpret uroanalytic tests with concurrent clinical laboratory tests. Urine sample quality can be maintained by collecting samples into a cooled container or one containing a suitable preservative. 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. Microscopic examination of urine sediment and urinary mineral and electrolyte excretion are not recommended as routine screening tests in nonclinical studies (Weingand et al., 1992). Additional tests with appropriate urine collection procedures may be necessary for evaluating test materials suspected of causing renal toxicity (Stonard, 1990; Stonard et al., 1987; Kluwe, 1981; Fettman, 1987; Bovee, 1986). STATISTICAL ANALYSIS
Appropriate statistical methods should be used to analyze clinical pathology data (Gad and Weil, 1989). Regardless of the outcome of statistical analysis, scientific interpretation is necessary for the ultimate determination of test material treatment effects. Statistical significance alone should not be used to infer toxicological or biological relevance of clinical pathology findings. Additionally, the absence of statistical significance should not preclude the possibility that test material treatment effects exist. The concurrent control data
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are more appropriate than historical reference ranges for comparison with test material treatment groups. CONCLUSIONS
This paper represents the consensus of a joint international scientific committee and provides minimum clinical pathology testing recommendations for animal toxicity and safety studies based on current technical and scientific information. It is hoped that these recommendations will serve as the basis for international harmonization of clinical pathology testing guidelines developed by government regulatory agencies and professional standards organizations. ACKNOWLEDGMENTS Nemi Jain and Michael Carakostas served as impartial observers at the meetings of this joint scientific committee. Walter Loeb served as a scribe.
REFERENCES Bovee, K. C. (1986). Renal function and laboratory evaluation. Toxicol. Pathol. 14, 26–36. Carakostas, M. C., and Banerjee, A. K. (1990a). Interpreting rodent clinical laboratory data in safety assessment studies: Biological and analytical components of variation. Fundam. Appl. Toxicol. 15, 744–753. Carakostas, M. C., Power, R. J., and Banerjee, A. K. (1990b). Serum 5* nucleotidase activity in rats: A method for automated analysis and criteria for interpretation. Vet. Clini. Pathol. 19, 109–113. Davies, D. T. (1992). Enzymology in preclinical safety evaluation. Toxicol. Pathol. 20(3), Part 2, 501–505. Dooley, J. F. (1979). The role of clinical chemistry in chemical and drug safety evaluation by use of laboratory animals. Clin. Chem. 25, 345– 347. Evans, G. O., and Parsons, C. E. (1986). Potential errors in the measurement of total protein in male rat urine using test strips. Lab. Anim. 20, 27– 31. Fettman, M. J. (1987). Evaluation of the usefulness of routine microscopy in canine urinalysis. J. Am. Vet. Med. Assoc. 190, 892–896. Gad, S. C., and Weil, C. S. (1989). Statistics for toxicologists. In Principles and Methods of Toxicology (A. W. Hayes Ed.), 2nd ed., pp. 435–483. Raven, New York. Godsafe, P. A., and Singleton, B. K. (1992). The use of the whole blood thrombotest time (511 ) as a routine monitor of vitamin K-dependent blood coagulation factor levels in the rat. Compar. Haematol. Int. 2, 51–55.
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Hoffman, W. E., Kramer, J., Main, A. R., and Torres, J. L. (1989). Clinical enzymology. In Clinical Chemistry of Laboratory Animals (W. F. Loeb and F. W. Quimby, Eds.), pp. 237–278. Pergamon, New York. Kluwe, W. M. (1981). Renal function tests as indicators of kidney injury in subacute toxicity studies. Toxicol. Appl. Pharmacol. 57, 414–424. Leissing, N., Izzo, R., and Sargent, H. (1985). Variance estimates and individuality ratios of 25 serum constituents in beagles. Clin. Chem. 31, 83–86. Leonard, T. B., Neptun, D. A., and Popp, J. A. (1984). Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver. Am. J. Pathol. 116, 262–269. Osborne, C. A., and Stevens, J. B. (1981). Handbook of Canine and Feline Urinalysis, pp. 57–120. Ralston Purina, St. Louis, MO. Ragan, H. A. (1989). Markers of renal function and injury. In The Clinical Chemistry of Laboratory Animals. (W. F. Loeb and F. W. Quimby, Eds.), pp. 321–343. Pergamon, New York. Stitzel, K. A., and Waner, T. (1992). Introduction. Toxicol. Pathol. 20(3), Part 2, 469. Stonard, M. D. (1990). Assessment of renal function and damage in animal species. J. Appl. Toxicol. 10, 267–274. Stonard, M. D., Gore, C. W., Oliver, G. J. A., and Smith, I. K. (1987). Urinary enzymes and protein patterns as indicators of injury to different regions of the kidney. Fundam. Appl. Toxicol. 9, 339–351. Taylor, D. M., and Neal, D. L. (1982). False negative hyperglucosuria teststrip reactions in laboratory mice. Lab. Anim. 16, 192–197. Theus, R., and Zbinden, G. (1984). Toxicological assessment of the hemostatic system, regulatory requirements, and industry practice. Regul. Toxicol. Pharmacol. 4, 74–95. Thompson, M. B., Blair, P. C., Morris, R. W., Neptun, D. A., Deyo, D. F., and Popp, J. A. (1987). Validation and application of a liquid chromatography/enzymatic assay for individual bile acids in the serum of rats. Clin. Chem. 33, 1856–1862. Thompson, M. B., Neptun, D. A., Garvey, L. K., and Popp, J. A. (1984). Effects of three hepatocarcinogens, alpha-naphthylisothiocyanate (ANIT), lithocholic acid (LCA) on serum bile acids in the rat. Proc. Am. Coll. Vet. Pathol. 79. Vargaftig, B. B., Conard, J., and Samama, M. (1979). Blood coagulation and platelet function. Pharmacol. Ther. 5, 225–227. Weingand, K., Bloom, J., Carakostas, M., Hall, R., Helfrich, M., Latimer, K., Levine, B., Neptun, D., Rebar, A., Stitzel, K., and Troup, C. (1992). Clinical pathology testing recommendations for nonclinical toxicity and safety studies. Toxicol. Pathol. 20(3), Part 2, 539–543. Woodman, D. D. (1988). Assessment of hepatic function and damage in animal species. J. Appl. Toxicol. 8, 249–254. Zawidzka, Z. Z. (1990). Hematologic evaluation. In Handbook of In Vivo Toxicity Testing (D. L. Arnold, H. C. Grice, and D. R. Krewski, Eds.), pp. 463–508. Academic Press, San Diego.
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29, 198–201 (1996)
0022
Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies* KURT WEINGAND,**,1 GEOFF BROWN,2 ROBERT HALL,3 DAI DAVIES,4 KENT GOSSETT,3 DOUG NEPTUN,1 TREVOR WANER,5 TOSHIAKI MATSUZAWA,6 PAUL SALEMINK,7 WILHELM FROELKE,8 JEAN-PIERRE PROVOST,9 GIANNI DAL NEGRO,10 JOHN BATCHELOR,2 MAMORU NOMURA,6 HORST GROETSCH,8 ALPHONS BOINK,7 JON KIMBALL,1 DAVID WOODMAN,4 MALCOLM YORK,4 EVA FABIANSON-JOHNSON,5 MICHEL LUPART9, AND ELSA MELLONI10 The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing consisting of delegates from 1American Association for Clinical Chemistry’s Division of Animal Clinical Chemistry (United States), 2Association for Comparative Haematology (United Kingdom), 3 American Society for Veterinary Clinical Pathology (United States), 4Animal Clinical Chemistry Association (United Kingdom), 5 International Society for Animal Clinical Biochemistry (multinational), 6Japanese Pharmaceutical Manufacturer’s Association (Japan), 7Dutch Association for Comparative Haematology (Netherlands), 8Arbeitsgruppe Klinische Chemie bei Laboratoriumstieren der Deutschen Gesellschaft fur Klinische Chemie E.V. (Germany), 9Association des Biologistes Cliniciens pour Animaux de Laboratoire (France), and 10Gruppo di Ematologia ed Ematochimica Applicate alla Tossicologia (Italy) Received December 19, 1994; accepted August 29, 1995
Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. (1996) WEINGAND, K., BROWN, G., HALL, R., DAVIES, D., GOSSETT, K., NEPTUN, D., WANER, T., MATSUZAWA, T., SALEMINK, P., FROELKE, W., PROVOST, J-P, DAL NEGRO, G., BATCHELOR, J., NOMURA, M., GROETSCH, H., BOINK, A., KIMBALL, J., WOODMAN, D., YORK, M., FABIANSON-JOHNSON, E., LUPART, M., AND MELLONI, E. Fundam. Appl. Toxicol. 29, 198–201. 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 * Author affiliations: The Procter & Gamble Company, Cincinnati, Ohio (K.W.); Huntingdon Research Centre Ltd., Huntingdon-Cambs, England (G.B.); Corning Hazleton, Madison, Wisconsin (R.H.); Zeneca Pharmaceuticals, Macclesfield-Cheshire, England (D.D.); SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania (K.G.); Novel Pharmaceutical, Inc., Research Triangle Park, North Carolina (D.N.); Life Science Research Israel, Ness Ziona, Isreal (T.W.); Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan (T.M.); Organon International B.V., Oss, Netherlands (P.S.); Boehringer Ingelheim KG, Ingelheim-Rhein, Germany (W.F.); Pfizer, Amboise, France (J.P.P.); Glaxo Italia, Verona, Italy (G.D.N.); Sanofi-Winthrop, Alnwick-Northumberland, England (J.B.); Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan (M.N.); Hoechst AG, Frankfurt, Germany (H.G.); Rijksinstituut Voor Volksgezondheid en Milieuhygiene, Bilthoven, Netherlands (A.B.); R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey (J.K.); SmithKline Beecham, Welwyn-Herts, England (D.W.); Glaxo Wellcome, Ware-Hertfordshire, England (M.Y.); Kabi Pharmacia, Helsingborg, Sweden (E.F.J.); Servier, Gidy, France (M.L.); Boehringer Mannheim Italia, Monza, Italy (E.M.). ** To whom reprint requests and correspondence should be addressed at The Procter and Gamble Company, Health Care Research Center, P.O. Box 8006, Mason, OH 45040-8006.
0272-0590/96 $12.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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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
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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. q 1996 Society of Toxicology
Clinical pathology testing of laboratory animals in toxicity and safety studies is an important part of safety assessment for food additives, drugs, and chemicals. Some government regulatory agencies and professional standards organizations provide guidelines for clinical pathology testing in animal toxicity and safety studies. These testing guidelines vary considerably (Stitzel and Waner, 1992). Clinical pathology testing of laboratory animals is a distinct scientific specialty that requires qualified professionals for test selection, technical conduct, and data interpretation (Dooley, 1979) (Carakostas and Banerjee, 1990a). Clinical pathology tests often provide information not detected by direct examination of organs and tissues, e.g., evaluations of hepatic and renal function, oxygen carrying capacity of the blood, circulating cells of the immune system, hemostatic function, and systemic mineral and electrolyte homeostasis. This information is useful for determining the biological significance of findings in toxicity and safety studies conducted with laboratory animals and the safety testing to be conducted in clinical trials for development of new drugs and food additives. In an effort to improve clinical pathology testing guidelines for safety assessment, 10 scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in toxicity and safety studies. This document presents minimum recommendations for clinical pathology testing. Additional testing may be needed to better characterize any observed or expected toxic effects of a test material. These recommendations should be implemented to comply with all appropriate laws and regulations concerning animal welfare and husbandry. BLOOD SAMPLING
Hematologic and clinical chemistry tests are subject to influence by the conditions under which blood is collected, such as feeding (fasting), anesthesia, and the site of blood collection. For this reason, it is critical that the blood be collected in the same manner from concurrent control and treatment groups of animals. Feeding or fasting prior to blood sampling may have effects on animal health, behavior,
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blood sample quality, and carbohydrate and lipid metabolism; these effects vary among species. The decision to feed or fast before blood sample collection should be based on individual study objectives, the species used, and the biological activity of the test material. Blood samples should be collected in a manner that minimizes time-related biases. Blood samples from multiple animals should not be pooled. Predose clinical pathology testing is useful for general health screening of nonrodent species considered for placement in toxicity and safety studies (Leissing et al., 1985). Predose clinical pathology testing of rodents is not necessary because of the large number of animals per group in rodent studies and the homogeneity of the laboratory animal population. Additionally, predose blood sampling of young rodents is limited by small blood volume and may have adverse effects on animal health. Clinical pathology testing of mice is limited by blood sample volume. Because of this limitation, interim (in-life) study blood samples should not be collected routinely from mice. Blood samples from mice for clinical pathology testing are collected optimally at terminal euthanasia only. The frequency and timing of clinical pathology testing is dependent upon study duration, study objectives, the biological activity of the test material, and the species tested. The following are minimum recommendations that may be modified because of these factors. 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 because of the small number of animals per group and interanimal variability. For studies of 2 to 6 weeks’ duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals (Davies, 1992). If a study contains recovery groups, clinical pathology testing at study termination is recommended. For carcinogenicity studies, the recommendation is that blood smears be made only from unscheduled terminations (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia. Quantitative clinical laboratory testing during the latter half of a 104-week carcinogenicity, toxicity, or safety study involving rodents is considered to be generally inappropriate and is not recommended (Weingand et al., 1992). As rodents age, they become progressively more susceptible to naturally occurring diseases that affect clinical laboratory tests, resulting in greater test value variability that obscures meaningful interpretation of test material effects.
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The animals used for clinical pathology testing should be examined for morphologic pathology findings. Use of separate subgroups of rodents for clinical pathology testing is scientifically acceptable, but maximal information is obtained when clinical pathology findings can be associated with gross and histopathologic observations from the same animal.
creatine kinase, and lactate dehydrogenase activities and fractionation of serum/plasma proteins are not recommended for routine use in animal toxicity and safety studies (Weingand et al., 1992). Additional tests may be useful in the evaluation of a test material known or suspected of affecting a specific tissue or metabolic pathway.
HEMATOLOGY
URINALYSIS
The core hematology tests recommended for animal toxicity and safety studies are total leukocyte (white blood cell) count, absolute differential leukocyte count (Zawidzka, 1990), 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. Reticulocyte counts and bone marrow cytology are not routinely necessary as screening tests in toxicity and safety studies but may be indicated when the test material has an effect on the hematopoietic system. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis for animal toxicity and safety studies (Vargaftig et al., 1979; Theus and Zbinden, 1984; Godsafe and Singleton, 1992). CLINICAL CHEMISTRY
The core clinical chemistry tests recommended for animal toxicity and safety studies are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. There are a variety of clinical chemistry tests for evaluation of liver health and function appropriate for use with laboratory animals (Thompson et al., 1984, 1987; Hoffman et al., 1989; Carakostas et al., 1990; Leonard et al., 1984; Woodman, 1988). 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. Ornithine decarboxylase, ornithine carbamoyltransferase,
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Uroanalytic tests are often inaccurate due to artifactual changes induced in vitro by the suboptimal conditions under which timed urine samples are commonly collected from animals in metabolic cages (Osborne and Stevens, 1981; Evans and Parsons, 1986; Ragan, 1989; Taylor and Neal, 1982). Urine sample contamination should be minimized, e.g., food, drinking water, feces, vomit, blood. Pooling of urine samples from multiple animals is not recommended. Urinalyses are subject to influence by the conditions of urine collection, such as feeding, fasting, and sample time. For this reason, it is recommended that urine be collected in the same manner from concurrent control and treatment groups. Urinalysis should be conducted at least once during a study. It is optimal to conduct and interpret uroanalytic tests with concurrent clinical laboratory tests. Urine sample quality can be maintained by collecting samples into a cooled container or one containing a suitable preservative. 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. Microscopic examination of urine sediment and urinary mineral and electrolyte excretion are not recommended as routine screening tests in nonclinical studies (Weingand et al., 1992). Additional tests with appropriate urine collection procedures may be necessary for evaluating test materials suspected of causing renal toxicity (Stonard, 1990; Stonard et al., 1987; Kluwe, 1981; Fettman, 1987; Bovee, 1986). STATISTICAL ANALYSIS
Appropriate statistical methods should be used to analyze clinical pathology data (Gad and Weil, 1989). Regardless of the outcome of statistical analysis, scientific interpretation is necessary for the ultimate determination of test material treatment effects. Statistical significance alone should not be used to infer toxicological or biological relevance of clinical pathology findings. Additionally, the absence of statistical significance should not preclude the possibility that test material treatment effects exist. The concurrent control data
ftoxas
AP: Fund Tox
HARMONIZED CLINPATH TESTING FOR SAFETY STUDIES
are more appropriate than historical reference ranges for comparison with test material treatment groups. CONCLUSIONS
This paper represents the consensus of a joint international scientific committee and provides minimum clinical pathology testing recommendations for animal toxicity and safety studies based on current technical and scientific information. It is hoped that these recommendations will serve as the basis for international harmonization of clinical pathology testing guidelines developed by government regulatory agencies and professional standards organizations. ACKNOWLEDGMENTS Nemi Jain and Michael Carakostas served as impartial observers at the meetings of this joint scientific committee. Walter Loeb served as a scribe.
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