Here, we explore this conceptual model as a framework for understanding adverse effects, and for planning on how we will incorporate new knowledge into defining adverse effects, in the context of toxicity testing in the 21st century.

Before addressing this important question, let us backfill and introduce the new paradigm of toxicity testing. The National Research Council (NRC, 2007) of the National Academies produced a report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy that provides a roadmap for the future of toxicity testing. This Forum article is one of a series over the past year that comments on the report and the follow-up paper “Toxicity Testing in the 21st Century: Bringing the Vision to Life” by Andersen and Krewski (2009). The basic proposal is to reorient testing to the molecular level rather than observing apical responses at the level of whole organisms. The NRC report’s vision identified a sequence of steps for evaluating toxicants, including (1) chemical characterization, (2) assessment of toxicity pathway responses and targeted testing, (3) dose-response and extrapolation modeling, (4) benchmarking to population and exposure data, and (5) decision making within risk contexts. Each of these steps will require new tools and thought processes that together create a coherent system of toxicity testing.

Lead toxicity is a great example of how such limitations have changed our perception of adversity over time. The ancient Romans knew that lead was toxic, but their adverse effects of concern were saturnine gout and colic. Up until the mid-20th century, lead-induced inhibition of hemoglobin synthesis leading to anemia was the most sensitive adverse effect identified for regulatory purposes. We now know that developmental neurotoxicity is an adverse effect of lead that occurs at low levels of exposure. Indeed, it took much of the 20th century to understand that lead toxicity occurs in children (Gibson, 2005) and produces persistent neurodevelopmental effects (Byers and Lord, 1943) with long-lasting consequences for behavior and intellectual function (Lanphear et al., 2005; Needleman, 1995). Only in the last quarter century has this new understanding of children as a susceptible subpopulation been translated into regulatory action that limits exposure of this vulnerable group. While toxicologists focused on verifying the importance of each apical end point or active failure in turn, the biological implications of the underlying molecular mimicry shared by lead, zinc, and calcium were underappreciated. This is just the type of situation that TFACS can address, finding the latent failures inherent to shared chemical structure and inadvertent biological pathway activation that predispose to an adverse outcome.

Carcinogenicity is clearly a complex adverse effect, so it is useful to examine how the in vitro Ames test approach has been employed to address it. In this regard, a report of the European Centre for the Validation of Alternative Methods workshop entitled “How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests” (Kirkland et al., 2007) is illuminating. In a gathering of more than 20 genotoxicity experts, this workshop identified a research agenda relevant not only to genotoxicity testing but also to the toxicity testing vision developed by the NRC report. The workshop participants recognized the very poor discrimination of noncarcinogens from carcinogens using in vitro genotoxicity tests and proposed an ambitious plan that was specific and lengthy, detailing areas for further research and development, including (1) culture medium composition and toxicant oxidation, (2) deficiencies in the cell lines, (3) lack of normal metabolism, (4) cytotoxicity as a confounder, and (5) the need for new cell systems, such as three-dimensional culture models. As a bottom line, the workshop participants concluded “… that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health … is needed” (Kirkland et al., 2007).

As an example of how better mechanistic insight can be informative, the incorporation of gene expression profiling into in vitro genotoxicity assessment provides increased discriminatory power regarding carcinogenicity (Staal et al., 2006). A comparison of the effects of polycyclic aromatic hydrocarbons (PAHs) on gene expression with other end points indicative of carcinogenicity (e.g., DNA-adduct formation, Ah-receptor binding) provides insight into how we can discriminate better between carcinogenic and noncarcinogenic compounds. PAHs were found to generally induce a compound-specific response on gene expression, but when responses were examined at the pathway level, carcinogenic and noncarcinogenic compounds could be successfully distinguished (Staal et al., 2006). Pathways likely to be activated by carcinogens include DNA repair and oxidative stress response pathways among others. Pathway responses at the gene or protein level provide better guidance because they can not only serve as a discriminatory tool but also reveal mechanistic information related to the carcinogenicity. Understanding the mechanism behind a positive hit will provide greater confidence in whether it is a true or false positive, a vast improvement over the traditional Ames test. This confidence will extend beyond genotoxicity testing, with the mechanistic insight derived from gene and protein expression profiling providing increased discriminatory power regarding adverse effects in the new toxicity testing vision.

We are now in a post-Virchow era, and defining an adverse effect purely by its apical manifestations is no longer enough. The toxicity testing of tomorrow will use new molecular techniques to generate enormous amounts of data describing fundamental subcellular responses to toxicants. A TFACS framework, yet to be designed, will provide a path forward to mining this data, generating a deep understanding of toxicity and adversity at the molecular level.

The NRC report provides some guidance for creation of a TFACS framework. Roughly speaking, one can imagine that the TFACS categories might correspond to the initial sequential steps of toxicity testing as proposed in the NRC report: chemical characterization, assessment of toxicity pathway responses and targeted testing, and dose-response and extrapolation modeling. The testing itself will identify response patterns consistent with vulnerabilities in each of these categories (latent failures) that when sufficiently aligned predispose to an adverse effect (active failure). Analysis of the response patterns of numerous toxicants within the TFACS framework will, over time, develop and refine a Taxonomy of Adverse Effects. This conceptual approach is illustrated in Figure 1.

Looking into the future, one imagines that the features that predispose to adversity within the TFACS categories (Fig. 2) are similar to those already associated with an adverse effect. At the level of the first TFACS category, chemical characterization, response patterns will be identified by evaluating quantitative structure-activity relationships, physical and chemical properties, environmental concentrations, and possible metabolites and toxic properties of the test chemicals. Vulnerabilities predisposing to an adverse effect will likely include a biologically reactive chemical or metabolic product of a chemical, predicted molecular interactions with critical cellular macromolecules, and chemicals that have a high probability of reaching and persisting in the environment.

Assessment of toxicity pathway responses, the next category within the TFACS framework, will provide another level of predictive power for active failures. Chemical exposure may result in activation of adaptive response pathways (e.g., heat shock protein response pathways) and/or pathways that produce adverse consequences (e.g., proapoptotic pathways). Activation of adaptive pathways, while protective, may be a red flag for eventual adverse outcomes should the pathway perturbation remain for a sufficient period of time or intensity. In the context of toxicity pathway alterations, adaptive responses to toxicant exposure will likely be characterized by reversible changes, a limited scope of toxicity pathway–induced alterations, and gradual changes in dose-response. On the other hand, latent failures predisposing to an adverse effect will likely be characterized by irreversible changes, a spreading of toxicity pathway responses, and abrupt dose-response transitions. As we develop this new framework, we will likely identify selected pathways of concern that may be more indicative of an active failure than other pathways. Therefore, the specific toxicity pathways affected and the biological relevance of the pathway alteration are important considerations when characterizing a response as adaptive versus adverse. In a previous forward-thinking report from the NRC entitled Scientific Frontiers in Developmental Toxicology and Risk Assessment (Committee on Developmental Toxicology, Board on Environmental Studies and Toxicology, National Research Council, 2000), signaling pathways important during development were characterized and the importance of understanding how molecular perturbations in these pathways can result in adverse outcomes was stressed. This report provides an excellent example of how adverse effects will be classified in the future based on a more complete understanding of toxicity pathways.

Within the third TFACS category, dose-response and extrapolation modeling, there are additional predisposing manifestations of adversity. The characterization and interpretation of the dose-dependent changes in protective and adverse toxicity pathways are at the core of the new toxicity testing paradigm. At some low dose, a pathway may begin to be disrupted by a toxicant exposure, but the pathway will continue to function due to a homeostatic response (an “adaptive” behavior). At a higher dose, the adaptive response is overwhelmed, and an adverse effect takes place. Dose-response modeling is critical to identifying adverse effects, especially since there can be dose-dependent transitions in the principal mechanism of toxicity (Slikker et al., 2004). As discussed earlier, an abrupt dose-response transition and a transition in mechanism of toxicity at different doses will likely be indicative of an adverse effect. Physiologically based pharmacokinetic models will be needed to determine if the doses that cause toxicity pathway alterations in vitro are comparable to the human blood/tissue concentrations that would result from environmental exposure levels. A response will be classified as adverse if the dose required to elicit the effect is environmentally relevant.

The development of the Taxonomy of Adverse Effects can only be accomplished through national and international collaboration among laboratories. This will require the establishment of standardized data collection and integration methods. Publicly available databases must be the norm, facilitating collaboration and comparison of data among laboratories and maximizing the utility of the resource. The large amounts of data that will be generated and stored in these databases must be analyzed to identify adverse and adaptive effects of chemical exposure. Therefore, development of standardized bioinformatic techniques for data analysis will also be necessary. These are just a few of the steps that will be essential to the creation of a robust and comprehensive Taxonomy of Adverse Effects.