The Environmental Protection Agency (EPA) has invested tremendously in its new toxicity-testing program, ToxCast, which aims to use in vitro high-throughput screening (HTS) assays to evaluate the effects of thousands of chemicals and prioritize them for further in vivo testing. Yet, many questions remain regarding the reliability and relevance of these assays. For example, are they providing accurate predictions about the effects of interest? Are the assays consistent over time and between laboratories? And, ultimately, do we have enough confidence in the results to use them as the basis for decision-making?
While EPA has begun to evaluate some of their assays, a recently published article in Environmental Health Perspectives reports specifically on the performance of ToxCast assays and related tools in detecting chemicals that promote adipogenesis. Such “obesogenic” chemicals interact with pathways involving the peroxisome proliferator activated receptor (PPARγ), among others, to alter normal lipid metabolism and contribute to abnormal weight gain. (Note: the term “obesogen” was coined by Bruce Blumberg, the senior author of this paper).
For the first part of this work, the researchers evaluated ToxCast results for one specific pathway in adipogenesis. Of the top 21 chemicals that were reported to bind to PPARγ in ToxCast Phase I, only 5 were actually found to activate PPARγ in their own laboratory.
Next, they examined the predictive power of multiple ToxCast assays representing various pathways related to adipogenesis. The researchers chose eight biologically relevant targets (including PPARγ) and generated a ToxPi (Toxicological Priority Index) graphic based on assay results for the chemicals (see figure from the paper, below). Each color represents a specific target evaluated by one or more assays, and larger slices correspond to higher relative activity in those assays. In this way, they could combine the results of multiple assays for each chemical and easily compare adipogenic potential.
How well do these ToxPis – created based on weighted results from ToxCast assays – predict actual PPARγ activation and overall adipogenic activity? The researchers found that only 2 out of 11 highest scoring ToxPi chemicals could activate PPARγ in their laboratory assays, and only 7 out of 17 top and medium scoring ToxPi chemicals were active in cell culture adipogenesis assays. In addition, 2 of the 7 chemicals that appeared negative in ToxPi actually promoted adipogenesis in culture.
EPA had previously recognized the potential for false positive and false negatives in the testing program and had begun to implement correction methods, such as z-score adjustments, in more recent ToxCast phases. Unfortunately, problems remained even after researchers considered these supposed improvements. While many false positives and false negatives were removed, the true positives were also eliminated.
These results are concerning, to say the least. Why are the ToxCast assays performing so poorly in predicting PPARγ activity and overall obesogenic potential? The researchers suggest several possible reasons, including 1) the fact that there are relatively few specific obesogenic assays that have been developed (especially compared to estrogen and androgen receptor assays), and 2) the inherent difficulties in using simple receptor binding tests to reflect the complexities of the endocrine system.
These issues must be resolved if we are to move forward with the goal of using these assays for prioritization and risk assessment. Last year, EPA announced (see here and here) that they would allow the use of a combination of ToxCast estrogen receptor assays to replace several existing tests in the Endocrine Disruptor Screening Program (EDSP). Clearly, however, other areas of the ToxCast program need additional refinement and validation before they can be used confidently for regulatory purposes.
While it is discouraging to read about these weaknesses in ToxCast, such external assessments are essential and will motivate important improvements. With more input from and collaboration with the scientific community, we can be hopeful that EPA’s ToxCast program will be able to fulfill its goal of efficiently evaluating thousands of chemicals and serving as the basis for decision making to protect public health.