Toxicology through alternative methods that reduce, refine and replace animal use (3R principle)

Abstract

While animal Reduction and Refinement are currently in the mind of experimental toxicologists and scientists in general, the Replacement still requires of validated alternative test methods to evaluate the human hazard and risks posed by drugs and chemicals. The US National Research Council released a report in 2007 for the Toxicity Testing in the 21st Century. This report strongly recommended the use of in vitro methods and predictive models as an alternative to in vivo animal testing by using and validating new tests based on human cells and cellular components. Cell response networks would reveal interconnected pathways composed of biochemical interactions of genes, proteins and small molecules. Eventually, these toxicity pathways, when sufficiently disturbed, would lead to adverse health effects. The step-by-step connection of the initial molecular event with the adverse effect on health is called the Adverse Outcome Route (AOP). The development of AOPs was promoted by the Organization for Economic Cooperation and Development (OECD) in 2012 and is becoming important to expand the use of mechanistic toxicological data for risk assessment and regulatory applications.

Acute systemic toxicity test designs are described in OECD test guidelines. At present, a significant animal Reduction and Refinement has been achieved in the oral toxicity test designs: the up-and-down procedure, the acute toxic class method, and the fixed dose procedure, which use five to nine animals while the old LD50 test used 30 or more animals per chemical. To obtain Replacement of animals for acute systemic toxicity, several regulatory agencies have begun to accept the use of the IC50 value of an in vitro baseline cytotoxicity test in a cell culture assay to determine an initial dose for the LD50 oral test.

However, the toxicity of chemicals that are organ-specific toxicants might not be properly recognized in a cell-based cytotoxicity test. This is specially relevant for neurotoxic agents. Of the more than 80 000 known chemicals, more than 1000 are neurotoxic when assayed in animals, and human neurotoxicity has been proved for 200 (mostly solvents, pesticides, industrial compounds and metals). In addition, several neurotoxicants are permanent organic pollutants and bioaccumulate in food chains, contaminating the food, posing a risk for human health after long exposure to low concentrations.

Neurotoxicity is usually manifested as alterations of behavior, and cognitive motor and learning-memory processes, which are often a consequence of alterations in GABAergic and glutamatergic neurotransmission, and of neurodegenerative processes. Neurotoxic events may underlie acute human toxicity that may not be predicted by using an in vitro test that exclusively relies on general cytotoxicity. Acute human toxicity correlating to adverse neuronal function is mainly a result of over-excitation or depression of the central or peripheral nervous system. It is expected that neurotoxic chemicals will produce their effect by different mechanisms. The major molecular mechanisms include GABAergic, glutamatergic and cholinergic neurotransmission. Neurodegenerative processes are generally a consequence of glutamate-mediated excitotoxicity and /or of cellular red-ox imbalance.

Primary cultured neurons allow for testing of chemicals acting on general (red-ox homeostasis, cytoskeleton) and specific (receptor-operated ion channels, neurotransmitter transport) neural endpoints, both in mature neurons and during development. We have been using primary cultures of cortical and cerebellar granule neurons, mainly constituted by GABAergic and cholinergic and glutamatergic neurons, respectively, for th