Neurotoxicity refers to an alteration caused by natural or manmade substances of the normal structure or function of the nervous system. 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 discussed as a component of target organ toxicity, the central nervous system (CNS) being one of the major target organ systems.
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 still conducted using animal tests. Three Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TGs) describe in vivo neurotoxicity studies. TG 418, Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure, 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. TG 419, Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study, involves daily oral dosing of hens with an organophosphorous pesticide for 28 days followed by biochemical and histopathological assessments. TG 424, Neurotoxicity Study in Rodents, involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer).
The OECD adopted a new Test Guideline in 2007 for developmental neurotoxicity testing (DNT). The guideline, Developmental Neurotoxicity Study (OECD TG 426), evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. Random offspring are observed for neurologic and behavioral abnormalities, and brain weights and neuropathology are assessed at different times through their adulthood.
The type of exposure (single or repeated does) and the outcome (lethal or nonlethal; immediate or delayed effects) will result in different classifications for substances under the Globally Harmonized System (GHS) (UNECE, 2004). 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.
Some experts 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), which is another incentive for the development of mechanism-based alternative methods.
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).
In vitro models for neurotoxicology studies and testing include the following types (ECVAM, 2002; Prieto, et al., 2005):
- Primary neuronal cell cultures
- Neuronal cell lines (neuroblastoma)
- strocyte primary cultures and cell lines
- Oligodendrocytes
- Primary microglia cell cultures
- Hippocampus brain slices
- Reaggregating neuron and glial cell cultures
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.
Validation and Acceptance of Non-animal Alternative Methods
ICCVAM, ECVAM, and the OECD have not reviewed or validated any non-animal methods for assessing neurotoxicity. There are no regulatory accepted non-animal methods for neurotoxicity testing.
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