On the Road to In Vitro Testing: Are We There Yet?

John Balbus, M.D., M.P.H., is Chief Health Scientist.

EPA’s recently released draft Nanotechnology Research Strategy (NRS) proposes a tiered testing system to evaluate human toxicity of nanomaterials.  It puts in vitro tests, or those done in test tubes and petri dishes as opposed to living animals, front and center.  EPA says the results of the first, in vitro tier will be used for guidance on “what health endpoints to monitor” and the second, in vivo tier will then help “identify those in vitro assays that correlate with in vivo nanomaterial toxicity or health effects.”

Wait a second.  If the in vivo testing is necessary in order to figure out what the in vitro testing results really mean, how can the agency use the in vitro testing results to figure out what health endpoints to monitor?  This cart and horse confusion is a serious matter.

The recent National Academy of Sciences (NAS) report, “Toxicity Testing in the 21st Century,” lays out a compelling vision of a toxicology future far less reliant on animal testing.  The prominence of in vitro testing in the EPA’s NRS is partly explained as a response to NAS recommendations.  But NAS emphasizes that there’s a decades-long road to get there, which will require a huge influx of research dollars that has yet to materialize.

A recent paper by Fischer and Chan highlights the current limitations of in vitro testing for nanomaterials.  The authors note that “the combined results from multiple studies of different cells in vitro cannot be assumed to capture the same behavior as the same cells arranged in situ in an organ.”  They also point out the difficulty of using cellular and acellular systems to model impacts on coordinated cell signaling pathways and the influence of transport mechanisms via blood, lymph and bile.  It’s even harder to imagine how in vitro tests can assess toxicity to complex biological processes like reproduction, development and immune response.  And yet nanoparticles, by virtue of their transport properties and protein coatings, may very well have subtle but serious effects on all these systems.

While the desire to push the science of in vitro testing is admirable, EPA has to be careful not to push so hard that it all topples over like a house of cards.  There’s no escaping the fact that a huge amount of scientific research will be required to identify and characterize the myriad pathways and mechanisms operating in mammals that may produce toxicity – let alone to develop assays for all them that use cultured cells or acellular systems, which is what would be needed to replace animal testing.

In vitro assays are already proving themselves valuable for elucidating mechanisms and explaining observed effects, and they serve as a critical supplement to in vivo testing.  For acute effects like corrosivity and eye damage, they are definitely preferred over in vivo tests, and that list should grow rapidly over the next few years.  But they can’t stand alone to predict many critical types of toxicity in humans, certainly not for the foreseeable future.

The best way for in vitro screening approaches to move forward is through parallel testing approaches using side-by-side in vitro and in vivo assays.  Only in this way can we develop the data needed to determine how predictive in vitro assays are of in vivo behavior, and to understand how and to what extent the two approaches can work in concert to deepen our understanding of nanomaterial behavior.

Done correctly, the development of rapid, inexpensive in vitro screening tests will facilitate far more comprehensive and rapid assessment of potential risks from nanomaterials; however, done poorly, not only will there be potential for missing critical toxic effects, but the realization of the dream of effective in vitro testing will be set back for many years.  In its haste to get to an answer, it’s not at all clear the EPA is prepared to do the hard work needed to get from here to there.