"Neurotoxicology is the study of the adverse effects of chemical, biological, and certain physical agents on the nervous system and/or behavior during development and in maturity" (Harry, et al., 1998). Many common substances are neurotoxic, including lead, mercury, some pesticides, and ethanol.
Neurotoxicity testing is used to identify potential neurotoxic substances. Neurotoxicity is a major toxicity endpoint that must be evaluated for many regulatory applications. Sometimes neurotoxicity testing is considered as a component of target organ toxicity; the central nervous system (CNS) being one of the major target organ systems. In utero exposure to chemicals and drugs can also exert an adverse effect on the development of the nervous system, which is called developmental neurotoxicity (DNT).
Like other target organ toxicities, neurotoxicity can result from different types of exposure to a substance; the major routes of exposure are oral, dermal, or inhalation. Neurotoxicity may be observed after a single (acute) dose or after repeated (chronic) dosing.
The Animal Test(s)
Neurotoxicity testing for regulatory purposes is based on in vivo animal test methods. Four Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TGs) describe in vivo neurotoxicity studies. Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure, TG 418, involves a single oral dose to hens who are observed for 21 days. Primary observations include the hen's behavior, weight, and gross and microscopic pathology. Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study, TG 419, involves daily oral dosing of hens with an organophosphorous pesticide for 28 days followed by biochemical and histopathological assessments. Neurotoxicity Study in Rodents, TG 424, involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer). Primary observations include behavioral assessments and evaluation of nervous system histopathology.
The OECD adopted a new Test Guideline in 2007 for DNT testing. The Developmental Neurotoxicity Study, TG 426, evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. Offspring are evaluated for neurologic and behavioral abnormalities, and brain weights and neuropathology are assessed at different times through adulthood. An OECD expert group conducted a retrospective performance assessment of DNT testing in support of OECD TG 426, and concluded that TG 426 "represents the best available science for assessing the potential for DNT in human health risk assessment, and data generated with this protocol are relevant and reliable for the assessment of these end points" (Makris, et al., 2009).
The type of exposure (single or repeated dose) and the outcome (lethal or nonlethal; immediate or delayed effects) will result in different classifications for substances under the Globally Harmonized System (GHS). GHS classifications are determined "on the basis of the weight of all evidence available," including human exposures and animal studies. Neurotoxic effects sufficient for classification include significant functional changes in the central or peripheral nervous system, signs of CNS depression, effects on the senses (sight, hearing, smell), and damage to the brain observed at necropsy or microscopically. Human data are generally not available, but when they are they take precedence over animal test results. The GHS may permit the use of (Quantitative) Structure Activity Relationships [(Q)SAR] and expert judgment to fill data gaps for structural analogs.
An expert working group of the International Life Sciences Institute (ILSI) Risk Science Institute published a series of four reports in 2008 "to assess the lessons learned from the implementation of standardized tests for developmental neurotoxicity in experimental animals" (Fitzpatrick, et al., 2008). These reports covered the following topics: need for positive control studies (Crofton, et al., 2008); understanding variability in study data (Raffaele, et al., 2008); statistical issues and appropriate techniques (Holson, et al., 2008); and interpretation of DNT effects (Tyl, et al., 2008).
Problems cited with the current regulatory testing approach for neurotoxicity and DNT include: high cost, long duration, low throughput, and "not always sensitive enough to predict human neurotoxicity" (Bal-Price, et al., 2008a). Experts also claim that the animal tests in the current test guidelines "do not always generate the mechanistic data required for a scientifically based human risk assessment" (ECVAM, 2002).
Non-animal Alternative Methods
A 1998 review of in vitro methods developed for neurotoxicity testing explains the desirability of using a battery of in vitro tests that would capture the complexity of the nervous system, and the processes involved in neurotoxicity (Harry, et al., 1998). Since these cellular and mechanistic processes had not been fully identified, the authors noted the difficulty in designing such an in vitro test battery. They described a more appropriate use of in vitro models to elucidate toxicity mechanisms and to identify the target cells of neurotoxicants. They also pointed out that cellular models usually cannot distinguish between pharmacological actions and toxicity responses and that this level of discrimination is required for risk assessment.
A European Centre for the Validation of Alternative Methods (ECVAM) Working Group reviewed some of the many individual assays and test batteries that have been developed for neurotoxicity testing. The best approach was described as the development of mechanistically relevant alternative methods "that encompass the most important neurotoxic endpoints" to be used in test batteries as part of a tiered testing strategy (ECVAM, 2002). The first testing tier would distinguish neurotoxicant from cytotoxic chemicals, and the second tier would consist of mechanism-specific tests. It was proposed that a minimum battery might consist of methods for assessing blood-brain barrier function, basal cytotoxicity, and energy metabolism. A number of other test battery approaches were summarized in this article (ECVAM, 2002).
A review article by Harry and Tiffany-Castiglioni (2005) covered in vitro systems for neurotoxicity testing ranging from "single cell types to systems that preserve some aspects of tissue structure and function." These authors reviewed the current state of in vitro methods and their potential limitations as a reference for future studies. Potential limitations associated with existing in vitro methods for neurotoxicity testing were identified as: "relevant drug concentrations, factors that limit or alter drug accessibility to the nervous system, and the need for assays to reflect biologically meaningful end points" (Harry & Tiffany-Castiglioni, 2005). Recent symposia and workshops have also reviewed the state of alternative testing approaches for developmental neurotoxicity (Bal-Price, et al., 2008a; 2008b; CAAT, 2009a; 2009b; Coecke, et al., 2007; Lein, et al., 2007).
In vitro models for neurotoxicology studies and testing include the following types (Breier, et al. 2009; Coecke, et al., 2007; ECVAM, 2002; Harry & Tiffany-Castiglioni, 2005; Prieto, et al., 2005):
- Primary cells: neurons and glia (microglia, oligodendrocyte, astrocyte) from different brain regions
- Cell lines: neuroblastoma, astrocytoma, glioma, pheocromocytoma
- Brain slices: hippocampus
- Reaggregating neuronal and glial cell cultures
- Organotypic (3D) cultures; usually co-cultures (more than one type of cell)
- Neural stem (progenitor) cells: primary cell cultures and cell lines
The primary cell cultures and brain slices require the use of animals for obtaining the cells and tissues. Continuous cell lines, originally derived from human or animal tissues, typically can be propagated, frozen, and thawed and therefore maintained for research and testing purposes for many years.
As with any cell-based test system, determining the type of cell(s) to be used is critical. For example, a commonly used neuroblastoma cell line (Neuro-2a) was less sensitive to some neurotoxicants than cultured primary neurons (LePage, et al., 2005). The authors suggest caution in interpreting neurotoxicity data obtained from tests using this transformed cell line. Tamm, et al., (2006) reported that neural stem cells "are more sensitive than differentiated neurones or glia" to methlymercury exposure. Both neural progenitor cell lines and primary cultures have been utilized in published reports. Costa (2008) reported that primary cells are not always more sensitive than cell lines, and that differences may be due to different culture conditions, such as the presence of serum. Any cell system, whether a primary or transformed cell line, or neuroprogenitor cells, needs to be characterized and preferably compared to primary cells and/or the in vivo response for the specific toxicity endpoint being assessed.
In addition to defining the appropriate cellular models, predictive in vitro toxicity assays require that neurotoxicity specific assay endpoints be determined and used in appropriate models, and in a combination (test battery/test strategy) that provides an overall response predictive of the in vivo human outcome. Commonly used cellular endpoints that are not neural specific include cytotoxicity, proliferation, migration, differentiation, and apoptosis. Some endpoints that have been described as relevant to cell-based neurotoxicity assays include electrical activity, neurotransmitter release, and neurite outgrowth (Bal-Price, et al., 2008a; Radio & Mundy, 2008).
The roles of the blood-brain barrier (BBB), metabolism, and toxicokinetics need to be included in assessing the potential neurotoxicity of a substance. Recent workshops have discussed how the results from in vitro assays might be coupled with (Q)SAR studies, computational modeling, and other techniques to form an integrated testing strategy for the prediction of neurotoxicity (Bal-Price, et al., 2008a; 2008b).
Validation and Acceptance of Non-animal Alternative Methods
ICCVAM, ECVAM, and the OECD have not reviewed or validated any non-animal methods or alternative testing strategies for assessing neurotoxicity. Regulatory authorities have not accepted any non-animal methods or alternative testing strategies for neurotoxicity testing.