One of the principal challenges facing a toxicologic pathologist is to determine and differentiate a true adverse effect from a nonadverse or an adaptive response. Recent publications from the Society of Toxicologic Pathology (STP) and the European STP provide guidance for determining and communicating adversity in nonclinical toxicology studies. In order to provide a forum to inform and engage in a discussion on this important topic, a continuing education (CE) course was held during the 2016 STP Annual meeting in San Diego, CA. The lectures at this course provided guidance on determining and communicating adversity using case studies involving both clinical pathology and anatomic pathology. In addition, one talk also focused on data quality, study design, and interpretation of artifacts that could hinder the determination of adversity. The CE course ended with a talk on understanding adversity in preclinical studies and engaging the regulatory agencies in the decision-making process. This manuscript is designed to provide brief summaries of all the talks in this well-received CE course.

Pathology peer review is one of those pesky perennial issues that continue to haunt industrial pathologists. Just when you think it has been nailed once and for all by the issuance of a ‘‘Best Practice’’ document from the Society of Toxicological Pathology (STP 1997; Crissman et al. 2004; Morton et al. 2010), it rises, phoenix like from the ashes, to consume us for another few months of letter writing and fervent hallway discussion. Nevertheless, as new generations join the professions of toxicology and pathology, it will likely continue to be a recurring theme and so maybe will merit continued discussion, in which we are happy to engage. The opinion article by Engelhardt, Hardisty, and Mann published in this issue has a noble goal, which we applaud, of trying to increase transparency and thus reduce anxiety among regulators about the ‘‘black box’’ of pathology peer review. But, we submit that the means they believe will accomplish this will fail, and indeed, we hope to convince you that it will actually create the opposite effect. Their proposal is simply to retain the interim tables created by the peer review pathologist (PRP), thus apparently demystifying the process of arriving at the final consensually agreed set of diagnoses. And where there is general agreement between the study pathologist (SP) and the PRP, or where disagreement centers around nontreatment-related findings, there is no problem, although what value this material may be is questionable. But when there are differences of opinion in treatment-related findings in the initial tables, it creates an issue and a potential set of dilemmas where none should exist. If an SP diagnoses hyperplasia and a PRP diagnoses adenoma, but the final tables say hyperplasia, what is a regulator supposed to think? Has there been undue influence on the PRP pressuring them to move to a less impactful diagnosis? Or, did the SP convince the PRP that he or she was overcalling the diagnosis in a legitimate educative process of coequal learning and intellectual give and take? What about when an inexperienced SP breaks out components of chronic progressive nephropathy (CPN) in a rat study resulting in tables showing an apparent increase in incidence of some components with dose, but a decrease of others with increasing dose. The experienced PRP convinces the SP that this is a purely serendipitous effect and that lumping the components together shows no treatment effect for CPN and an incidence that is not increased above the historical background rate for the rat strain. An inexperienced regulator may surmise that the PRP inappropriately influenced the SP to suppress some treatment-related renal findings. Fundamentally, there is no value to retention of the interim tables. Where there is disagreement between tables created by SP and PRP, one or both of these interim data will be wrong— mutually agreed as wrong by the SP and PRP—and the final tables will have the consensually agreed correct diagnosis. There is no support by either pathologist for a diagnosis in any interim table which they both have agreed is now incorrect, and therefore no point in reporting this wrong diagnosis. To be continually challenged to defend why they changed diagnoses *This is an opinion article submitted to the Regulatory Forum and does not constitute an official position of the Society of Toxicologic Pathology or the journal Toxicologic Pathology. The views expressed in this article are those of the authors and do not necessarily represent the policies, positions, or opinions of their respective agencies and organizations. The Regulatory Forum is designed to stimulate broad discussion of topics relevant to regulatory issues in toxicologic pathology. Readers of Toxicologic Pathology are encouraged to send their thoughts on these articles or ideas for new topics to regulatoryforum@toxpath.org. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Dr. Roy Kerlin, Global Research and Development, Pfizer Inc, Eastern Point Road, MS 8274-1210, Groton, CT 06340, USA; e-mail: roy.l.kerlin@pfizer.com. Abbreviations: CPN, chronic progressive nephropathy; PRP, peer-review pathologist; STP, Society of Toxicological Pathology; SP, study pathologist.

Weak peroxisome proliferator–activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal β-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased β-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000→1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle β-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart β-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. β-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle β-oxidation and resultant muscle injury in the rat.

Peroxisome proliferator-activated receptors (PPARs) represent therapeutic targets for the management of type 2 diabetes mellitus and dyslipidemia. Rodent carcinogenicity studies have revealed a link between gamma and dual gamma/alpha PPAR agonist treatment and the increased incidence of subcutaneous (SC) liposarcomas/fibrosarcomas or hemangiosarcomas, but very little has been reported for potent and selective PPAR alpha agonists. We present a mode of action framework for the development of SC mesenchymal tumors in rodents given PPAR agonists. (1) Tumor promotion results from pharmacologically mediated recruitment (proliferation and differentiation), thermogenesis and adipogenesis of stromovascular cells, and subsequent generation of oxidative free radicals. (2) Tumor initiation consists of chemotype-driven mitochondrial dysfunction causing uncontrolled oxidative stress and permanent DNA damage. Promotion is characterized by enhanced adipogenesis in the SC adipose tissue, where the baseline PPAR gamma expression and responsiveness to PPAR gamma ligands is the highest, and by thermogenesis through expression of the uncoupling protein 1 (UCP-1) and the PPAR gamma co-activator 1 alpha (PGC-1 alpha), two factors more highly expressed in brown versus white adipose tissue. Initiation is supported by the demonstration of mitochondrial uncoupling and OXPHOS Complexes dysfunction (Complexes III, IV and V) by compounds associated with increased incidences of sarcomas (muraglitazar and troglitazone), but not others lacking malignant tumor effects (pioglitazone, rosiglitazone).

Peroxisome proliferator-activated receptors (PPARs) represent therapeutic targets for the management of type 2 diabetes mellitus and dyslipidemia. Rodent carcinogenicity studies have revealed a link between γ and dual γ/α PPAR agonist treatment and the increased incidence of subcutaneous (SC) liposarcomas/fibrosarcomas or hemangiosarcomas, but very little has been reported for potent and selective PPARα agonists. We present a mode of action framework for the development of SC mesenchymal tumors in rodents given PPAR agonists. (1) Tumor promotion results from pharmacologically mediated recruitment (proliferation and differentiation), thermogenesis and adipogenesis of stromovascular cells, and subsequent generation of oxidative free radicals. (2) Tumor initiation consists of chemotype-driven mitochondrial dysfunction causing uncontrolled oxidative stress and permanent DNA damage. Promotion is characterized by enhanced adipogenesis in the SC adipose tissue, where the baseline PPARγ expression and responsiveness to PPARγ ligands is the highest, and by thermogenesis through expression of the uncoupling protein 1 (UCP-1) and the PPARγ co-activator 1 α (PGC-1α), two factors more highly expressed in brown versus white adipose tissue. Initiation is supported by the demonstration of mitochondrial uncoupling and OXPHOS Complexes dysfunction (Complexes III, IV and V) by compounds associated with increased incidences of sarcomas (muraglitazar and troglitazone), but not others lacking malignant tumor effects (pioglitazone, rosiglitazone).

Zoniporide, an inhibitor of the Na+-H+ exchanger-1, was administered by continuous intravenous infusion to rats and dogs for up to 1 month. In 1-month studies, histological and functional changes were observed in select portions of the peripheral nervous system; however, these findings were not detected in 2-week studies at similar or higher doses. In the 1-month rat study, there was dose-dependent, minimal, focal, or multifocal nerve fiber (axonal) degeneration in the spinal cord and/or sciatic nerve. In a follow-up rat study, findings included slowing of caudal nerve conduction velocity and axonal degeneration in the spinal cord (dorsal funiculus), dorsal roots, dorsal root ganglia (DRG), radial, sciatic, and tibial nerves. In the 1-month dog study, there was impairment of the patellar reflex and associated postural reaction changes, minimal to marked proximal nerve fiber degeneration in the DRG, and minimal nerve fiber degeneration in the dorsal roots and funiculi of the spinal cord. Minimal nerve fiber degeneration of equivocal significance was noted in various peripheral nerves. Taken together, these findings were consistent with a specific effect on peripheral sensory nerve fibers. These studies demonstrated that zoniporide produces clinical, electrophysiologic, and microscopic evidence of peripheral sensory axonopathy and establishes the importance of careful preclinical evaluation of neurological function.

This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.

Cystic degeneration/spongiosis hepatis in rats has been proposed to be a preneoplastic and/or neoplastic lesion by some authors, because of its proliferative properties and persistent increased cell turnover rate in stop experiments using hepatocarcinogens , and the assumption that it can develop into a sarcoma. The neoplastic potential of cystic degeneration is questioned in this review article. Cystic degeneration, which appears to derive from altered Ito cells, does not have neoplastic histomorphologi c characteristics, although it may be composed of cells with an increased mitotic index. In this regard, persistent proliferation is also seen with other nonneoplastic lesions. Arguments are presented to show that the induced, probably extremely rare sarcoma that was associated with cystic degeneration most likely derives from the very rare induced spherical Ito-cell aggregate with an unusually high cellular turnover rate in rats treated with hepatocarcinogens , and not from cystic degeneration. Also, in none of 12 referenced standard oncogenicity studies with chemically induced cystic degeneration was the lesion associated with mesenchymal (Ito-cell) tumors. Consequently, evidence is lacking that cystic degeneration in rats should be classifi ed as a preneoplastic or neoplastic lesion. The 12 oncogenicity studies in rats with induced cystic degeneration showed a marked sex predilection, with males more likely to develop either spontaneous or chemically induced lesions. In these 12 studies, cystic degeneration was more often associated with hepatocellular hypertrophy or hepatotoxicity, rather than hepatocarcinogenicity. Thus, it is concluded that hepatocarcinogens induce cystic degeneration, not because they are carcinogenic, but because they have other effects on the liver, and that cystic degeneration may be a secondary/reparative change. Cystic degeneration in fi sh parallels the situation in rats in many respects, yet the existence of the lesion in other species, including man, is not as well supported. Based on the data presented in this review, spontaneous and induced cystic degeneration in rats and fi sh is not a preneoplastic or neoplastic lesion and risk assessment for man can be based on no-effect levels and safety margins, as for other nonneoplasti c adverse effects that have no counterpart in man.

Cystic degeneration/spongiosis hepatis in rats has been proposed to be a preneoplastic and/or neoplastic lesion by some authors, because of its proliferative properties and persistent increased cell turnover rate in stop experiments using hepatocarcinogens, and the assumption that it can develop into a sarcoma. The neoplastic potential of cystic degeneration is questioned in this review article. Cystic degeneration, which appears to derive from altered Ito cells, does not have neoplastic histomorphologic characteristics, although it may be composed of cells with an increased mitotic index. In this regard, persistent proliferation is also seen with other nonneoplastic lesions. Arguments are presented to show that the induced, probably extremely rare sarcoma that was associated with cystic degeneration most likely derives from the very rare induced spherical Ito-cell aggregate with an unusually high cellular turnover rate in rats treated with hepatocarcinogens, and not from cystic degeneration. Also, in none of 12 referenced standard oncogenicity studies with chemically induced cystic degeneration was the lesion associated with mesenchymal (Ito-cell) tumors. Consequently, evidence is lacking that cystic degeneration in rats should be classified as a preneoplastic or neoplastic lesion.The 12 oncogenicity studies in rats with induced cystic degeneration showed a marked sex predilection, with males more likely to develop either spontaneous or chemically induced lesions. In these 12 studies, cystic degeneration was more often associated with hepatocellular hypertrophy or hepatotoxicity, rather than hepatocarcinogenicity. Thus, it is concluded that hepatocarcinogens induce cystic degeneration, not because they are carcinogenic, but because they have other effects on the liver, and that cystic degeneration may be a secondary/reparative change. Cystic degeneration in fish parallels the situation in rats in many respects, yet the existence of the lesion in other species, including man, is not as well supported. Based on the data presented in this review, spontaneous and induced cystic degeneration in rats and fish is not a preneoplastic or neoplastic lesion and risk assessment for man can be based on no-effect levels and safety margins, as for other nonneoplastic adverse effects that have no counterpart in man.

Most nonclinical safety studies performed to satisfy regulatory requirements include numerous anatomic and clinical pathology endpoints. Consequently, the traditional predominant role of the pathologist in the pharmaceutical industry has focused upon the interpretation of histologic changes in tissues and of clinicopathologic changes in blood from animals that had been exposed to new chemical entities. In addition to this conventional role, pathologists are increasingly being recognized for their expanded contributions to drug discovery and development. The medical training, specialty or residency training, and comparative medicine perspective place the industrial pathologist in a unique position to contribute broadly to the objectives of drug discovery and development programs. In addition, the technologies in the pathologist’s repertoire have broad application and utility to many areas of biomedical research in the pharmaceutical industry. Such objectives and areas of research include characterization of ! disease states; identification of modified disease states as a response to administration of potential therapies; characterization and quantification of toxic responses to drug administration ; delineation of mechanisms of toxicities and potential relevance to target species; and facilitation of multidisciplinary efforts to monitor for the clinical occurrence, progression, and reversibility of adverse events. The business and science of pharmaceutical research and development is increasingly complex, and the pressures associated with them create competitive advantages for companies that are able to deploy resources in novel and beneficial ways. Use of pathologists to support drug discovery represents one such approach.