Recommendations (best practices) are provided by the Society of Toxicologic Pathology's Adversity Working Group for making consistent interpretations of test article-related effects as adverse and assigning a no observed adverse effect level (NOAEL) in nonclinical toxicity studies. Adverse is a term indicating harm to the test animal, while nonadverse indicates lack of harm. Adverse findings in the study reports should be defined in relation to effects on the test species used and within the context of the given study. Test article-related effects should be described on their own merits, and decisions to consider them as adverse or nonadverse should be justified. Related effects may be discussed together; in particular, markers of toxicity that are not in and of themselves adverse ideally should be discussed in conjunction with the causal toxicity to determine adversity. Adverse findings should be identified in subreports (clinical data, pathology data, etc.) if sufficient information is available, and/or in the final study report as individual or grouped findings, but study NOAELs should be established at the level of the overall study report. Interpretations such as not biologically relevant or not toxicologically important should be avoided unless defined and supported by scientific rationale. Decisions defining adverse findings and the NOAEL in final study reports should combine the expertise of all contributing scientific disciplines. Where possible, use of NOAELs in data tables should be linked to explanatory text that places them in context. Ideally, in nonclinical summary documents, NOAELs from multiple studies are considered together in defining the most important adverse responses in the most sensitive species. These responses are then considered along with an understanding of their likely mechanisms, as well as other information such as variability in species sensitivity, comparative pathology, reversibility and progression, kinetics, and metabolism of the test substance to help assess human risk.

The process of drug discovery and development is highly complex, demanding extraordinary scientific acumen as well as an understanding of the regulatory milieu. Veterinary pathologists in industry find themselves at the vanguard of this process, helping companies to bring innovative new therapies to patients in need. In this chapter we focus on the role of the toxicologic pathologist in understanding both the nature of drug efficacy, as well as the pathogenic mechanisms of drug toxicity, and the way in which both perspectives play into decisions to either develop or halt development of a potentially promising drug candidate. We review the role of the toxicologic pathologist in discovering and developing small molecules as well as some of the extraordinary variety of biotherapeutic agents, with their inherent challenges and often unexplored potentialities. Other issues such as reversibility or delayed toxicity, imaging, GLP regulations, and the use of some advanced scientific tools used by toxicologic pathologists developing pharmaceutical agents are discussed. In progressive companies, as well as regulatory agencies, toxicologic pathologists bring their expertise to bear across the entire drug discovery and development pipeline to improve our understanding of the advantages and potential liabilities of new therapies.

CHARLOTTE KEENAN (CHAIR), SUSAN ELMORE, SABINE FRANCKE-CARROLL, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, USA Pfizer Inc., Groton, Connecticut 06340, USA National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Charles River, Frederick, Maryland 21701, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, Suffolk IP23 7PX, UK U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA

CHARLOTTE KEENAN, SUSAN ELMORE, SABINE FRANCKE-CARROLL, RAMON KEMP, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania, USA National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina, USA Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration, College Park, Maryland, USA Merck Research Laboratories, Riom, France Pfizer Inc., Groton, Connecticut, USA NIEHS, Research Triangle Park, North Carolina, USA Charles River, Frederick, Maryland, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, United Kingdom U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

KEN SCHAFER, DVM, PHD, DIPLOMATE, ACVP (CHAIR), SABINE FRANCKE-CARROLL, DVM, PHD, DAVID HUTTO, DVM, PHD, DIPLOMATE, ACVP, NATASHA NEEF, DVM, PHD, DIPLOMATE, ACVP, LEE SILVERMAN, DVM, PHD, DIPLOMATE, ACVP, JOHN VAHLE, DVM, PHD, DIPLOMATE, ACVP AND KATHARINE WHITNEY, DVM, PHD, DIPLOMATE, ACVP Vet Path Services, Inc. Greenfield, Indiana, USA Office for Food Additive Safety, Center for Food Safety and Applied Nutrition, US FDA, College Park, Maryland, USA Millennium Pharmaceuticals Cambridge, Massachusetts, USA Pfizer Gorton, Connecticut, USA Millennium Pharmaceuticals Cambridge, Massachusetts, USA Eli Lilly and Co. Indianapolis, Indiana, USA Abbott Laboratories Abbot Park, Illinoise, USA

Peroxisomal proliferator-activated receptor (PPAR)-alpha is a ligand-activated transcriptional factor that regulates genes involved in lipid metabolism and energy homeostasis. PPAR-alpha activators, including fibrates, have been used to treat dyslipidemia for several decades. In contrast to their known effects on lipids, the pharmacological consequences of PPAR-alpha activation on cardiac metabolism and function are not well understood. Therefore, we evaluated the role that PPAR-alpha receptors play in the heart. Our studies demonstrate that activation of PPAR-alpha receptors using a selective PPAR-alpha ligand results in cardiomyocyte necrosis in mice. Studies in PPAR-alpha-deficient mice demonstrated that cardiomyocyte necrosis is a consequence of the activation of PPAR-alpha receptors. Cardiac fatty acyl-CoA oxidase mRNA levels increased at doses in which cardiac damage was observed and temporally preceded cardiomyocyte degeneration, suggesting that peroxisomal beta-oxidation correlates with the appearance of microscopic injury and cardiac injury biomarkers. Increased myocardial oxidative stress was evident in mice treated with the PPAR-alpha agonists coinciding with increased peroxisomal biomarkers of fatty acid oxidation. These findings suggest that activation of PPAR-alpha leads to increased cardiac fatty acid oxidation and subsequent accumulation of oxidative stress intermediates resulting in cardiomyocyte necrosis.

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

The contribution of hyperplastic lesions in two-year rodent carcinogenicity studies to human hazard identification and risk assessment is a complex issue. Although hyperplasia is a common, spontaneous age-related change in many tissues, it can also be part of other pathologic conditions that in some cases represent a morphologic/biologic continuum leading to neoplasia, and therefore may be considered part of the “the weight of evidence” for carcinogenicity assessment (3, 4, 5). This paper provides a general perspective for toxicologists, pathologists, and other scientists involved in carcinogenicity hazard identification and risk assessment. There currently are no regulatory guidelines that discuss the relationship of non-neoplastic proliferative changes (i.e., regenerative hyperplasia, atypical hyperplasia, physiologic hyperplasia, etc.) to neoplastic processes in rodent carcinogenicity studies; however, some useful generalizations for interpreting hyperplastic lesions can be made. First and foremost, hyperplasia must be viewed within the context of the study. Factors that may contribute to an accurate assessment of the relevance of hyperplasia to coexisting neoplastic processes include a common cell of origin for hyperplastic and neoplastic processes, the presence or absence of a morphological continuum between hyperplasia and neoplasia within the study, histologic similarities of hyperplastic and neoplastic lesions, the incidence and severity of spontaneous chronic diseases that may influence development of hyperplastic and neoplastic lesions, incidences of hyperplasia and neoplasia, and other evidence for treatment-related toxicity. Furthermore, hyperplasia has different implications depending on the specific type(s) of morphologic changes (diffuse or focal, with or without cellular atypia), the biochemical mechanisms stimulating cellular proliferation, concurrent lesions, the organ/tissue sites involved, and the presence or absence of other neoplastic findings in the tissue with hyperplasia. The pathologist evaluating the study must determine whether nonneoplastic lesions are relevant to assessment of hyperplastic and neoplastic responses based on morphologic similarities, mechanistic data, and scientific knowledge. Information regarding the specific compound or class of compound in question, including expected pharmacologic effects and mechanisms of action, mutagenicity, chemical groups known to be associated with carcinogenicity, and characteristics of absorption, distribution, metabolism, and excretion should be considered. Data from other nonclinical studies with the same or a related compound may add to the weight of evidence. Obviously, consistent diagnostic terminology must be used to distinguish hyperplastic lesions relevant to carcinogenicity assessment. In this regard, it is noteworthy that there may be multiple terms currently in use that can be applied to hyperplastic lesions. Use of standardized nomenclature such as that endorsed by the Society of Toxicologic Pathology (1) and clear, cogent communication of the findings and their interpretations are essential to appropriate interpretation. It is important to determine if hyperplastic lesions are a direct effect of compound action, or alternatively are secondary to a primary degenerative, necrotic or apoptotic event leading to a reparative (regenerative) hyperplasia. Focal hyperplasia that appears morphologically similar to neoplasia in the same tissue without evidence of concurrent toxicity or tissue injury may be considered more indicative of a potential direct treatment-related neoplastic response. Hyperplasia and cancer in tissue with an inciting factor such as inflammation or degeneration/regeneration induced by the test agent may suggest that the carcinogenic process is secondary to chronic tissue injury. If hyperplasia can be clearly associated with tissue toxicity, then one can assume that exposures insufficient to cause the inflammation or degenerative/regenerative changes are unlikely to cause cancer. Even if the hyperplastic precursor lesions (whether in the same study or in earlier shorter term studies) are considered to be a primary effect of the test article, there are different implications depending on the type of change (i.e., diffuse or focal, with or without cellular atypia or dysplasia), the biochemical mechanism(s) stimulating cellular proliferation, associated concurrent lesions, the tissue involved, and the type of related neoplastic findings. For example, a chemical producing a marginal increase in transitional cell papilloma or carcinoma of urinary bladder at the high dose in a 2-year bioassay in rats, but also with multiple foci of transitional cell hyperplasia with cellular atypia in the high and mid dose groups, may have greater risk for carcinogenicity than a chemical with only a marginal increase in transitional cell neoplasms at the high dose. Thus, the appropriate perspective of pathologic changes is critical in assessing and communicating the risk. The role of hyperplastic lesions in the assessment of carcinogenicity should be interpreted very carefully since our understanding of the causes and progression of many chemically-induced neoplastic processes is limited. Although it is common to analyze incidences of benign and malignant neoplasms separately, the incidences of benign and malignant neoplasms arising from the same cell type are usually also combined for statistical analyses (2, 3). This is logical, since many benign and malignant neoplasms generally are considered irreversible with the potential for biological/morphological progression (3, 5). However, unlike benign and malignant neoplasms, hyperplastic lesions may not progress to neoplasia and may be reversible (3). Although some morphologic and biochemical criteria may

An 8-year-old female spayed Cocker Spaniel mix breed dog was presented with generalized erythroderma, scaling and alopecia. Radiographs of the thorax demonstrated a discrete lung mass which was aspirated using ultrasound guidance and cytological analysis revealed large abnormal lymphocytes. Similar cells were observed in the peripheral blood and in skin biopsies. The cells in the skin biopsies were epidermotropic, indicative of an uncommon cutaneous lymphoma termed cutaneous T cell lymphoma (CTCL), sometimes also called mycosis fungoides. Immunohistochemical staining of a skin biopsy was positive for the CD3 antigen demonstrating that the lymphocyte infiltrate was of a T-cell lineage. The presence of neoplastic lymphocytes in the epidermis and peripheral blood indicate that this is a rare variant of Cutaneous Epidermotropic Lymphoma (CEL) called Sézary syndrome based on nomenclature used in the human literature. An unusual feature of this dog, not seen in previous cases, was the presence of a discrete neoplastic lung mass.