The Next Mile Marker on the Road to High Throughput In Vitro Screening?

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

A new paper by Shaw et al., published in May in the Proceedings of the National Academy of Sciences, “suggests a generalizable and scalable method for the systematic characterization and comparison of novel nanomaterials” using high throughput in vitro tests.  Does this mean that the National Academy of Sciences’ vision for toxicity testing in the 21st century – proposed for conventional chemicals – is already here for nanomaterials?  Not quite. 

The National Academy of Sciences (NAS), the Environmental Protection Agency, industry, environmental NGOs, and animal rights groups all look to high throughput in vitro testing to reduce animal testing while increasing the efficiency and lowering the cost of assessing chemicals and other materials for safety concerns.  But, as the NAS report acknowledges, achieving this shared vision will require a tremendous input of new research (and monetary resources) in order to refine and validate the next generation of toxicological test methods.  I discussed this in a previous blog post.

The Shaw et al. study makes a meaningful advance in the path towards in vitro screening.  The investigators used a variety of assays, cell types, and doses to test 50 different nanoparticles, thereby developing a multidimensional data profile for each.  They then applied statistical clustering techniques to examine whether differences in the structures of the nanoparticles, subtle or otherwise, changed their toxicity profiles.  For three nanoparticles that showed interesting differences in their profiles, the authors conducted short-term in vivo assays to seek to validate what they had seen in vitro.

The authors were able to demonstrate that using four different assays and a range of doses and cell types provided a robust set of data with which to categorize and distinguish the different nanoparticles.  The authors did not try to use the assays to predict specific health effects, but rather to create a classification system for previously untested nanoparticles and then use comparisons with nanoparticles that had already been thoroughly assessed by in vivo tests to make inferences about potential toxicity.

The three nanoparticles chosen for in vivo validation in this study are all intended for use as medical diagnostic agents, and hence have been widely tested in animals.  The study demonstrated that the nanoparticle that appeared to have the highest toxicity of the three in the in vitro assays also caused the greatest response in vivo.

While the Shaw et al. study has considerable strengths, a couple of critical limitations highlight the large gap that still needs to be bridged if in vitro screening is to be effective in protecting human and ecosystem health from risks arising from the wide array of nanomaterial applications under development.

First, all of the 50 nanomaterials tested were designed for intravenous injection as medical diagnostic agents.  Because of this, the nanoparticles were water-soluble, making them easier to use in in vitro systems.  Most nanoparticles intended for non-medical purposes are poorly soluble and therefore difficult to apply and dose accurately in the liquid medium found in cellular assays.  And while in vitro assays using cells grown and exposed to a nanoparticle in liquid media reasonably simulate an injection exposure pathway, making inferences about health effects from exposure via other routes of exposure that are more relevant to non-medical uses – inhalation, dermal contact and even ingestion – is more difficult.

Second, both the nanomaterials and the endpoints assessed were of limited relevance to evaluating the kinds of effects that could arise from consumer, environmental, or occupational exposures to nanoparticles.  Most were variants of iron oxides, which are not commonly found nanomaterials in consumer products.  In addition, the investigators were most concerned with acute reactions to the injected materials, and therefore used acute assays and cell types relevant to intravenous injection:  vascular cells, monocytes (a type of white blood cell), and hepatocytes.

The supporting in vivo test was also a short-term assay designed to look for changes in monocyte counts.  This test is most relevant to identifying immediate systemic inflammation, but would be very unlikely to detect perturbational changes that occur over longer time periods and could lead to chronic effects such cancer, neurodevelopmental toxicity, endocrine disruption, etc.  The latter types of effects are of greater concern for occupational, consumer, and environmental exposures.

The authors note that this approach is most valuable as an initial screen in the relatively rigorous evaluation process required by the Food and Drug Administration (FDA) for approval of pharmaceuticals.  They point to the ability to identify the most promising nanomaterials by comparing their profiles to those of materials that have already gained FDA approval – and therefore were already extensively tested.  Of course, any nanomaterials selected through this screening process would then themselves be put through an extensive set of safety tests.

This is a very different application of high throughput screening than, for example, would be needed to help evaluate a novel material for which a Premanufacturing Notice is submitted under the Toxic Substances Control Act, where there is no backup of rigorous safety testing to assure protection.  Such an approach may have value, however, in screening alternative nanoparticles as companies develop new products.

So while this study is a step in the right direction, there is still a lot more good science needed if high throughput in vitro screening tests are to become a reliable means to assess the safety of occupational, consumer, and environmental exposures to nanomaterials.

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