George L. Foley

Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic–pituitary–adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article–related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.).

Nonproliferative and proliferative lesions of the rat and mouse female reproductive system.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the female reproductive tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. There is also a section on normal cyclical changes observed in the ovary, uterus, cervix and vagina to compare normal physiological changes with pathological lesions. A widely accepted and utilized international harmonization of nomenclature for female reproductive tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

STP Position Paper: Ovarian Follicular Counting in the Assessment of Rodent Reproductive Toxicity

Evaluation of the ovary is an important endpoint in tox-icological assessments because xenobiotics that cause lossof oogonia, oocytes, or supportive somatic cells may haveadverse effects on reproduction. To make an adequate histo-logic assessment of the ovary, knowledge of all morphologiccomponents and an understanding of the changes that occurduring the normal estrous cycle and aging are required. Athorough,qualitativelightmicroscopicexaminationbyatox-icologic pathologist will detect morphologic features associ-atedwithmostfunctionalalterations,includingchangesintherelative number or density of ovarian components. Develop-ment of consistent, reliable, and cost-effective quantitativemethods to evaluate ovarian toxicity has been challenging.Much effort has been spent developing adequate methods forquantification of small follicles. The publications by Bolonetal.(1997)andBuccietal.(1997)providethemostthoroughrecent comparisons of follicle-counting techniques in mice.These follicular-counting methods have been adopted and/ormodified for use as first-tier screening methods in some reg-ulatory guidelines for reproductive toxicity studies.

Intratubular spermatic granulomas of the canine efferent ductules.

Spermatic granulomas are inflammatory lesions that occur in the efferent ductule and epididymis of humans, goats, rats, and bulls. Three clinically normal dogs from a control group had histologic lesions of the initial segment of the caput epididymis consisting of intratubular spermatic granulomas. The granulomas were located within the efferent ductules, and the inflammatory response consisted primarily of aggregates of spermiophagic macrophages in the ductules. Sperm stasis of the affected ductules was evident by the dilation and accumulation of large numbers of spermatozoa in adjacent cross-sections of efferent ductules. Blind-ending efferent ductules were demonstrated by serially sectioning the initial segment of the epididymis. The intratubular granulomas did not completely occlude the outflow tract because spermatozoa were present in the cauda epididymis. While spermatic granulomas can be induced by trauma, infection, or toxins, spontaneous granulomas due to blind-ending ductules should be considered as a differential. Clinical history, location of the granuloma, and serial sectioning can help determine the most likely etiology in early cases.

Society of Toxicologic Pathology Position Paper on Best Practices on Recovery Studies The Role of the Anatomic Pathologist

This article reviews the regulatory guidelines that provide for the inclusion of recovery groups in toxicology studies, presents the challenges in the design and interpretation of nonclinical recovery studies, and summarizes the best practices for the role of an anatomic pathologist regarding toxicology studies with recovery groups. Evaluating the potential recovery of histopathologic findings induced by a biopharmaceutical requires the active participation of one or more anatomic pathologists. Their expertise is critical in risk assessment regarding the potential for recovery as well as providing scientific guidance in the design and evaluation of studies with recovery groups.

Sponsor-CRO Practices That Facilitate the Creation of a High-Quality Pathology Report A Pharmaceutical Sponsor’s Perspective

Contract research organizations (CROs) provide a multitude of valuable services and expertise to the pharmaceutical industry, government, and other organizations. One of those services is conducting nonclinical toxicology studies to assess the safety of chemical and biological entities intended as pharmaceuticals. An integral part of these studies is the generation and interpretation of pathology data. Both the CROs and the sponsors of these studies have a genuine interest in ensuring that the pathology data are accurate and that the reports are of high quality. The goal of this article is to provide the sponsors’ perspectives on practices that affect the conduct of preclinical studies and that specifically facilitate sound technical and scientific execution for pathology components of those studies, leading to the creation of pathology reports of high quality. This perspective will be limited to topics that affect the pathology contributor report of toxicology studies covered by Good Laboratory Practices (GLP) and will not encompass every possible aspect of the sponsor-CRO relationship. The sponsor-CRO relationship must be a collaborative and nonadversarial relationship that balances the perspectives and needs of both CROs (Gosselin et al. 2011) and sponsors. The success of this relationship and the generation of a pathology report of high quality will greatly depend on thorough communication at all stages of a toxicology study. The peer review process provides the opportunity for a comprehensive review of pathology data by a second pathologist to ensure that all test article– related target organs and findings are accurately and consistently identified, diagnosed, interpreted, and scored. For the purposes of this article, the study pathologist and the peer review pathologist are generally assumed to be employees of the CRO and sponsor, respectively. However, situations in which the study pathologist and/or the peer review pathologist are not employees of the CRO or the sponsor will require even closer communication between the CRO and the sponsor.

Light Microscopic Sciatic Nerve Changes in Control Beagle Dogs from Toxicity Studies

Although the dog is a common choice among nonrodent species in evaluation of compound safety for regulatory submission, information regarding the incidence of spontaneous or incidental microscopic changes in canine peripheral nerve is limited. A retrospective examination was performed of routine histologic preparations of sciatic nerve from eighty-one control dogs in toxicity studies ranging from ten days to three months in duration. Spontaneous background changes included digestion chambers, foci of vacuolation, nerve fibers circumscribed by proliferating Schwann cells (bands of Büngner), and small foci of myelin aggregation. The latter accounted for 91% of the microscopic changes and were noted in all sections examined. These changes were quantified, and the number per square millimeter of evaluable nerve tissue was determined for each slide. Densities of foci varied among the slides examined; no age- or sex-related trends were apparent. In addition, anatomic features of peripheral nerves including nodes of Ranvier, Schmidt-Lanterman incisures, Renaut bodies, and effects resulting from sectioning plane were noted. By demonstrating the range of effects observed within control animals, these observations provide a basis for recognition of possible compound-related effects in routine nerve preparations from dogs included in toxicity studies.

Microscopic Background Changes in Brains of Cynomolgus Monkeys

Brain sections from control cynomolgus monkeys (Macaca fascicularis) used in toxicology studies were evaluated retrospectively in order to better understand spontaneous background changes in this species. Hematoxylin and eosin–stained slides from 76 animals (38 males and 38 females) of 9 studies were examined. Eleven animals (9 males and 2 females) were each observed to have 1 to 3 findings within the brain sections examined, for a total of 19 findings. No findings were noted in the spinal cord. The most common finding was focal to multifocal perivascular infiltration of mononuclear cells, affecting the parenchyma, the meninges, or the choroid plexus. Additionally, focal gliosis was observed in 6 animals and a single focus of hemosiderin deposition (coincident with focal gliosis and mononuclear cell infiltrate) was noted in 1 animal. Most of the glial foci were composed of cells consistent with microglial cells, with or without admixed lymphocytes. All findings were of slight or minimal severity, lacked an apparent cause, and were considered incidental and of negligible biologic significance. An awareness of the spontaneous incidence of these background findings may facilitate the discernment of toxicologically relevant effects when these findings are observed.