Can case study approaches speed implementation of the NRC report: "Toxicity Testing in the 21st Century: A Vision and a Strategy?"

Abstract

The 2007 report "Toxicity Testing in the 21st Century: A Vision and a Strategy" argued for a change in toxicity testing for environmental agents and discussed federal funding mechanisms that could be used to support this transformation within the USA. The new approach would test for in vitro perturbations of toxicity pathways using human cells with high-throughput testing platforms. The NRC report proposed a deliberate timeline, spanning about 20 years, to implement a wholesale replacement of current in-life toxicity test approaches focused on apical responses with in vitro assays. One approach to accelerating implementation is to focus on well-studied prototype compounds with known toxicity pathway targets. Through a series of carefully executed case studies with four or five pathway prototypes, the various steps required for implementation of an in vitro toxicity pathway approach to risk assessment could be developed and refined. In this article, we discuss alternative approaches for implementation and also outline advantages of a case study approach and the manner in which the case studies could be pursued using current methodologies. A case study approach would be complementary to recently proposed efforts to map the human toxome, while representing a significant extension toward more formal risk assessment compared to the profiling and prioritization approaches inherent in programs such as the EPA's ToxCast effort.

Figure 1. The toxicity pathway knowledge base can be developed using an unbiased approach, a case study approach, or a combined approach

The knowledge needed to develop a functioning in vitro test system will be acquired over time (left-to-right). This knowledge can be acquired by having a comprehensive test platform that is challenged by numerous toxicants, and over time the interconnections and important response pathways emerge (unbiased approach). In this “unbiased approach”, knowledge expands over time as pathway interactions are understood (reflected by the width of the triangle increasing and the shading darkening from left to right). Alternatively, using a case study approach, a specific pathway can be evaluated in detail. While the density of knowledge increases (the shade darkens with time), the knowledge area covered does not change (the width of the knowledge area remains constant). Combining these approaches may be the best way to take advantage of the strengths of each approach; however, projects to demonstrate the applicability of the new toxicity testing paradigm may be most rapidly developed using a case study approach.

Figure 2. A Schematic showing steps in a toxicity pathway-based risk assessment

Results from the panel of assays identify the pathway targets and generate a point of departure (POD) for the subsequent risk assessment as an in vitro concentration. Computational systems biology pathway (CSBP) modeling of circuitry and dynamics for the assay system indicates the expected shape of the dose response at lower doses, leading to a POD. The POD concentration is then converted to an exposure standard through techniques of reverse dosimetry implemented by pharmacokinetic modeling. This step takes advantages of in vitro-in vivo extrapolation (IVIVE).

Figure 3. Developing Pathway Assays

The steps shown here constitute the process of assuring that pathway assay are fit for purpose, i.e., for identifying activity in a specific pathway, assessing the structure and dynamics of the overall signaling network, and providing computational systems biology pathway models to assist dose-response modeling of assay results and low-dose extrapolations. The process of pathway development is fundamental to their use as part of a testing suite for risk assessment, as shown in Figure 2.

Figure 4. A schematic for a “developmental network” that controls receptor-mediated signaling

The network is controlled by several incoherent (IFFL) and coherent feed forward loops (cFFL) with nodes to either activate (arrows) or repress (blunt lines) gene expression through critical signaling nodes – the y and Z factors. The goal in network inference is to understand the circuitry of various toxicity pathways at this level of detail in order to describe exposures that are without appreciable effects, doses with activation of early portions of the pathway, and full activation. These dose dependencies are expected to coincide with areas of sub-threshold, adaptive and adverse perturbations as outlined in the NRC report (NRC, 2007). The proteins designated Z1, Z2, and Z3 would have non-monotonic dose response curves; Y1, Y2, Y3 and Y4 would have monotonically increasing, but sequentially delayed, responses. Based on similar networks previously described (Alon, 2007); the code can be obtained by contacting MEA (MAndersen@thehamner.ore).