Jeffery A. Engelhardt

Skeletal Changes in Rats Given Daily Subcutaneous Injections of Recombinant Human Parathyroid Hormone (1-34) for 2 Years and Relevance to Human Safety

Fischer 344 rats (60/sex/group) were given daily subcutaneou s injections of recombinant human parathyroi d hormone (PTH)(1-34) for 2 years at doses of 0, 5, 30, or 75 μg/kg. Treatment caused substantial increases in bone mass consistent with the known pharmacologic effects of once-daily administration. As determined by quantitative computed tomography (QCT) and histomorphometry, bone mass was markedly increased. Substantial new bone formation resulted in a large decrease in marrow space accompanied by altered bone architecture. Bone proliferative lesions were observed in all PTH(1-34)-treated groups. Osteosarcoma occurred in 3, 21, and 31 male rats and in 4, 12, and 23 female rats in the 5-, 30-, and 75- μg/kg treatment groups, respectively. Focal osteoblast hyperplasia, osteoma, and osteoblastoma were much less frequent. Although the specific cellular or molecular mechanisms responsible for the rat bone tumors have not been fully elucidated, the data suggest that these lesions resulted from the long duration of treatment and the exaggerated pharmacologic response of the rat skeleton to daily treatment with PTH(1-34). Important differences between the rat study and clinical use in adult humans suggest that the increased incidence of bone neoplasia in rats treated for 2 years is likely not predictive of an increased risk of bone cancer in skeletally mature adult humans being given PTH(1-34) for a limited period of time in the treatment of osteoporosis.

Six-Month Continuous Intraputamenal Infusion Toxicity Study of Recombinant Methionyl Human Glial Cell Line-Derived Neurotrophic Factor (r-metHuGDNF) in Rhesus Monkeys

Recombinant human glial cell line-derived neurotrophic factor (r-metHuGDNF) is a potent neuronal growth and survival factor that has been considered for clinical use in the treatment of Parkinson’s disease (PD). Here we present results of a 6-month toxicology study in rhesus monkeys conducted to support clinical evaluation of chronic intraputamenal infusion of r-metHuGDNF for PD. Monkeys (6–9/sex/group) were treated with 0 (vehicle), 15, 30, or 100 μg/day r-metHuGDNF by continuous unilateral intraputamenal infusion (150 μl/day flow rate) for 6 months; a subset of animals (2–3/sex/group) underwent a subsequent 3-month treatment-free recovery period. Notable observations included reduced food consumption and body weight at 100 μg/day and meningeal thickening underlying the medulla oblongata and/or overlying various spinal cord segments at 30 and 100 μg/day. In addition, multifocal cerebellar Purkinje cell loss (with associated atrophy of the molecular layer and, in some cases, granule cell loss) was observed in 4 monkeys in the 100-μg/day group. This cerebellar finding has not been observed in previous nonclinical studies evaluating r-metHuGDNF. The small number of affected animals precludes definitive conclusions regarding the pathogenesis of the cerebellar lesion, but the data support an association with r-metHuGDNF treatment.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Incidence of Spontaneous Neoplastic and Nonneoplastic Lesions in Charles River CD-1® Mice Varies with Breeding Origin

Three separate control lifetime studies were conducted with untreated Crl: CD-1 ®(ICR)BR mice using a total of 400 mice/sex maintained to 21 mo of age. Similar husbandry practices and environmental conditions were used for all 3 studies. It was noted after study initiation that the Charles River breeding facility of origin was different for each study. The aggregate range of survival and incidence of neoplasms for the combined studies was similar to that previously reported. However, these 3 groups of mice had prominent variation in survival and in the incidence of pulmonary adenomas and systemic amyloidosis in males and females and in the incidence of hepatocellular neoplasms in males. The present studies indicate that consistent procurement of test animals is an additional variable to be considered in the establishment of a valid database within a test facility when using an outbred mouse.

Cephaloridine-Induced Renal Pathological and Biochemical Changes in Female Rabbits and Isolated Proximal Tubules in Suspension

Cephaloridine (Cld) is a nephrotoxic cephalosporin antibiotic. The intracellular biochemical changes that occur leading to Cld-induced nephrotoxicity may involve lipid peroxidation and/or mitochondrial injury. The purpose of this report was to examine and correlate the biochemical changes induced by Cld in vivo and in vitro with the observed pathological changes in an attempt to understand better the mechanisms of βT-lactam-induced nephrotoxicity. Cld treatment (500 mg/kg sc) caused elevations in blood urea nitrogen and decreases in the accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) by renal cortical slices. Histopathological alterations, characterized by individual cell necrosis of tubular epithelial cells, were first seen 6 hr after treatment in the pars recta of the outer stripe of the medulla. Ultrastructural alterations involved the straight (S2 and S3) segments of the proximal tubules. Mitochondrial morphology was, for the most part, unaffected by Cld exposure. Cld did not cause any significant changes in tissue malondialdehyde (MDA) content in vivo at any of the time points examined, but it did cause a depletion of GSH to approximately 40% of control by 1 hr after dosing that recovered toward control by 6 hr. Significant changes were observed in renal ATP content beginning at 6 hr after treatment; however, this change mirrored the onset of histological evidence of necrosis. In isolated tubules in vitro, the onset of glutathione (GSH) depletion and MDA formation clearly preceded lactate dehydrogenase (LDH) leakage, whereas ATP depletion was a mirror image of cell death. These data demonstrate that isolated proximal tubules in vitro are a reasonable model for Cld nephrotoxicity in vivo. Cld-induced mitochondrial alterations leading to ATP depletion and cell injury were not observed in this study.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.

Scientific and Regulatory Policy Committee Points-to-consider Paper* Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.

Industry-contract research organization pathology interactions: a perspective of contract research organizations in producing the best quality pathology report.

This article provides observations on the features of sponsor–contract research organization communication that will achieve the best quality pathology report based on our collective experience. Information on the test article and any anticipated findings should be provided, and initial slide examination should be done with knowledge of treatment group (but may be followed by blinded review of target tissues to determine no-effect levels). Only a pathologist should write or revise the pathology report or the pathology section of the overall study report. To address concerns related to undue sponsor influence, comments by sponsors should be presented as suggestions rather than directives. Adversity should be defined for each finding by the study pathologist, but the no-observed adverse effect level should not be discussed in the pathology report. Board-certified pathologists are recommended, but are not essential. Sponsors that have a particular format or report preferences should make them known well in advance. Histologic processing “to glass” of protocol-specified tissues from all dosage groups is recommended for rapid evaluation of target tissues. Telepathology is beneficial in certain situations, but it is usually more efficient for the study pathologist and reviewing pathologist to be in the same physical location to review differences of opinion and reach a consensus.

Regulatory forum opinion piece*: pathology peer review--to lock or not to lock, is that really the question?

It seems that every few years concerns are raised about the practice of pathology peer review, even though peer review has been part of toxicologic pathology since the late 1970s. While the individual procedures have varied slightly, the overarching intent of pathology peer review has been to provide diagnostic consistency and accuracy of interpretation of the pathology data to the global regulatory authorities to aid in their decision making regarding the safety of biopharmaceuticals and chemicals. The intent of peer review is not what raises periodic cause for concern among regulators, but rather other technical aspects of the process that surface periodically. That is why we believe that worksheets should be retained even though this step is not ‘‘required’’ or espoused by Society of Toxicological Pathology (STP; Morton et al. 2010). Kerlin (2012) briefly touched on some of the perceived reasons behind issues with peer review in his Regulatory Forum Opinion Piece on pathology raw data. Often times the ‘‘documentation’’ of the peer review is regarded as pathologist’s ‘‘interim notes’’ and are discarded after the report is completed, as these ‘‘notes’’ are not deemed to be necessary for the reconstruction of the pathology evaluation. While this practice may be expeditious in completing the peer review and reduces retained documents, it can be perceived as a ‘‘black box’’ around the peer-review process and opens the study for criticisms relative to data integrity and manipulation of data. While pathologists have long stated that peer review is intended to ensure the quality of the pathology evaluation in safety studies (Ward et al. 1995; Boorman et al. 2010), it does no good if the process used creates doubt in the skeptical mind of compliance and regulatory authorities relying on the final evaluation and interpretation of the data. It has previously been suggested that the peer review worksheets could be included as an appendix to the pathology report or maintained in the study file (Ward et al. 1995). Long (1996) and Mann (1996) continued to stress the need for a well-documented peer review process to give regulatory reviewers greater confidence in the pathology evaluation. However, a recent commentary by McKay et al. (2010), based upon current practices in the United States, European Union, and Japan, restated the position that peer review worksheets are not raw data and do not need to be retained or submitted. The issue is not what constitutes raw data and what should be retained, but instead the issue is one of transparency in the process. The latest controversy has focused not on the raw data but on the integrity of the data generated by the study pathologist. This is not a new issue, as Peters (1996) has already highlighted the need to maintain peer review worksheets to add credibility to the process and to ensure that the recommended data changes as a result of the peer review were made. More recently, a series of Food and Drug Administration (FDA) compliance inspections and interactions with agency representatives both in the United States and in Europe have questioned the timing of when the database should be ‘‘fixed or locked.’’ The ‘‘influence’’ by the sponsor and/or the peer review pathologist on the study pathologist’s findings has raised concerns on the part of regulators regarding how to maintain the integrity of the database. This has led to a recommendation from compliance inspectors that pathology data be locked prior to the peer review in order to track changes made during or after the peer review. The concerns raised by the regulators distill to transparency of the peer-review process. Although STP has provided *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: Jeffery A. Engelhardt, EPL, Inc., 5407 Castillo de Rosas, Camarillo, CA 93012, USA; e-mail: jengelhardt@epl-inc.com. Abbreviations: FDA, Food and Drug Administration; QC, quality control; STP, Society of Toxicological Pathology.

Meeting Summary: Nonrodent Species in Toxicologic Pathology

Nonrodent species are widely used in safety testing of chemicals and pharmaceuticals to meet worldwide regulatory requirements. As such, evaluation of nonrodent species may comprise a large percentage of the toxicologic pathologist’s work effort. This year, the STP Annual Symposium focused on the species other than rodents that may be encountered in the day-to-day activities of the pathologist, namely primates, dogs, minipigs, and fish. The Symposium provided a venue for comparison among these models, where these species differ from rodent models, the cross-species relevance of nonrodents models, and the relative importance of observations made in these species relative to risk assessment. Spontaneous tissue alterations and intercurrent diseases that may confound the interpretation of toxicology studies were addressed. A major theme of each of the speakers was how unique features of a particular species or model system may interfere with interpretation or be misinterpreted as potential compound related alterations. A broad spectrum of expertise provided numerous stimulating, and sometimes challenging and provocative, presentations. Our keynote speaker, Dr. Bernard Schwetz, Senior Advisor for Science in the US FDA, spoke on what the future for toxicologic pathology can and should hold. He made it clear that toxicologic pathologists must be intimately engaged in the transition to implement new technologies. This is necessary to assure that the outcome reflects the knowledge and discipline that are the hallmarks of informed regulatory decisions and links molecular data to the interpretation in the broader context of the whole animal. We must all reflect on his comments that the future role of the toxicologic pathologist will, in part, be determined by how well and how rapidly we adapt as the field moves from “:::icities” to “::: omics.” He did note, though, that we must not lose sight of what we do today, as these activities remain highly valuable for appropriate conclusions to be drawn from toxicologic studies. The first session provided a general overview of fundamental issues related to the use of the dog in toxicologic pathology and began with a discussion of common spontaneous lesions and aging changes in the beagle. The session then moved through presentations on specific aspects on the use of the dog, including interpretation of clinical pathology data, intravascular infusion models, evaluation of effects on QT interval, pharmacokinetics and drug metabolism, diagnosis of vascular injury and myeolodysplasia, and a vivid technicolor presentation on evaluating CNS toxicity. This was followed by an introductory discussion on the minipig as an alternative model to the dog in drug and chemical safety assessment. Fish, especially the zebrafish model, have been proposed as an attractive alternative to mammalian systems for drug discovery and testing safety of chemicals. The second session presented a general overview of fish as model systems in toxicologic pathology. Discussions focused on the utilization of the various models for risk identification and assessment. After a presentation on GLP issues and special procedures, the rainbow trout, zebrafish, and medaka models were described and relevant examples on the utility of these models were presented. The third session was devoted to practical examples of the use of primates in toxicologic pathology and began with a review of common diseases in primates. It was emphasized that all species are not alike and that biological differences among the species must be considered when interpreting potential pathological alterations. The session then proceeded with presentations on simian retroviruses, interpretation of clinical pathology data, intravascular infusion models, studies on immune modulators and antisense compounds, and the use of cynomolgus monkeys in teratology evaluations. The marmoset was presented for its important utility in the development of biotechnology products, but with the caveat of its unique set of challenges for husbandry. The session ended with a panel discussion on the appropriate use of primates in toxicology studies. The last session was devoted to regulatory perspectives in relation to nonrodent models. Broader issues on animal use were addressed in 2 presentations and a panel discussion. Our organization may have an unrealized social role beyond our role of assuring safety of medicines and agrichemicals. Indeed, we have the opportunity to influence international regulations on acceptable study designs and endpoints. The panel discussion, which included members from industry, academia, and regulatory agencies, addressed other study design issues and the regulatory perception of data derived from toxicology studies using nonrodent species. In particular, does the age of animals confound the pathology evaluation and is there a need to evaluate the reproductive tract in young nonrodents? Finally, this year brought record attendance to the symposium to hear presentations on a new variety of topics. I want to thank my session chairs, Suzanne Botts, Dennis Hoover, Mac Law, and Stu Levin, for their efforts in coordinating the various speakers and for bringing my vision for this symposium to fruition.