A ‘Best Practices’ Approach to Neuropathologic Assessment in Developmental Neurotoxicity Testing—for Today

Abstract

A key trait of developmental neurotoxicants is their ability to cause structural lesions in the immature nervous system. Thus, neuropathologic assessment is an essential element of developmental neurotoxicity (DNT) studies that are designed to evaluate chemically-induced risk to neural substrates in young humans. The guidelines for conventional DNT assays have been established by regulatory agencies to provide a flexible scaffold for conducting such studies; recent experience has launched new efforts to update these recommendations. The present document was produced by an ad hoc subcommittee of the Society of Toxicologic Pathology (STP) tasked with examining conventional methods used in DNT neuropathology in order to define the 'best practices' for dealing with the diverse requirements of both national (EPA) and international (OECD) regulatory bodies. Recommendations (including citations for relevant neurobiological and technical references) address all aspects of the DNT neuropathology examination: study design; tissue fixation, collection, processing, and staining; qualitative and quantitative evaluation; statistical analysis; proper control materials; study documentation; and personnel training. If followed, these proposals will allow pathologists to meet the need for a sound risk assessment (balanced to address both regulatory issues and scientific considerations) in this field today while providing direction for the research needed to further refine DNT neuropathology 'best practices' in the future.

STP Position Paper

“…Sampling of specific lumbar spinal cord segments and their CNS/PNS interface is not feasible as each segment exists in the vertebral canal at a level increasingly cranial to the corresponding vertebral body, so that trimming at vertebrae L 4 -L 5 actually samples the sacral spinal cord and surrounding cauda equina in adults. The degree of cranial displacement of lumbar cord segments varies among species, so that segment L 4 -L 5 in adults actually occurs within vertebrae at L 1 -L 2 in rats (Bolon et al 2006); L 3 -L 4 in dogs (Fletcher 1993); L 1 -L 2 in cynomolgus monkeys ( Figure 1); and L 1 -L 2 in humans (FitzGerald, Gruener, and Mtui 2007).…”

“…However, substantial variation exists across regulatory agencies (Bolon et al 2011a) regarding preferred neuropathology practices for registering new compounds. The recommendations vary by species (non-rodents [e.g., dog, nonhuman primate vs. rodents; Krinke 1989;Morawietz 2004]); by age (e.g., developing rodents [U.S. Environmental Protection Agency {EPA} 1998b; Organisation for Economic Co-operation and Development {OECD} 2007;Garman et al 2001;Bolon et al 2006Bolon et al , 2011b vs. adult rodents [Broxup 1991;EPA 1998a;OECD, 1997]); by the type of study (general toxicity screen vs. dedicated neurotoxicity bioassay; Bolon et al 2011a); by the kind of industry (agrochemical firms vs. pharmaceutical companies, for which potential exposure levels and, therefore, risk-to-benefit assessments will vary); and according to whether the study was conducted by Good Laboratory Practices (GLP) standards to support product registration. Regulatory guidelines for conducting the neuropathology analysis of GLP-type general toxicity studies (i.e., screening or ''Tier I'' surveys) provide wide-ranging advice for designing experiments that will evaluate many disparate organs and systems.…”

Neuroanatomy-based Matrix-guided Trimming Protocol for the Rat Brain

“…Moreover, a landmarkguided trimming approach has been recently suggested for general toxicity studies by the Working Group on Nervous System Sampling established by the Society of Toxicologic Pathology (STP; Bolon et al 2013). In the past decade, several anatomical landmark-guided trimming methods for the rat brain have been published, both for developmental neurotoxicity testing (Bolon et al 2006) and for the adult rat brain in general toxicity studies (Jordan et al 2011;Rao et al 2011;Rao, Little, and Sills 2014). Even if differences exist between the levels of trimming between these methods, all protocols recommend the sampling of the brain through a variable number of coronal cuts identified by target anatomical landmarks recognizable mainly on the ventral aspect of the brain (Bolon et al 2006).…”

“…In the past decade, several anatomical landmark-guided trimming methods for the rat brain have been published, both for developmental neurotoxicity testing (Bolon et al 2006) and for the adult rat brain in general toxicity studies (Jordan et al 2011;Rao et al 2011;Rao, Little, and Sills 2014). Even if differences exist between the levels of trimming between these methods, all protocols recommend the sampling of the brain through a variable number of coronal cuts identified by target anatomical landmarks recognizable mainly on the ventral aspect of the brain (Bolon et al 2006). However, the application of a landmark-guided trimming can be timeconsuming, and the identification of anatomical landmarks requires a high level of training for the histology technical staff.…”

Fundamentals of Translational Neuroscience in Toxicologic Pathology

“…Thus, the typical morphologic lesions in DNT studies are treatment-related morphometric differences in subregions of the brain. Although, volumetric measures and cell counting methods (such as those utilized in optical or physical dissector stereology) or computer-based areal measures frequently may be used as targeted investigational tools, simple linear measures are employed by most laboratories for obtaining morphometric data in DNT studies on rats (Bolon et al 2006). The ability to take such linear measures accurately requires highly homologous brain sections, which in turn depends on adequate technician training and the accurate identification of specific neuroanatomic regions within unstained sections floating on a water bath.…”