Many of the screening-level hazard data being collected and analyzed under ChAMP that pertain to human health are derived from traditional laboratory animal studies.  The National Academy of Sciences (NAS) recently offered a "new paradigm for toxicity testing" in its 2008 report Toxicity Testing in the 21^st Century: a Vision and a Strategy.  Can ChAMP hazard data be used to facilitate the development of new testing strategies? 

First, more about the new toxicity testing paradigm envisioned by NAS:  Instead of exposing whole animals to chemicals to examine their effects, which takes a lot of time, costs a lot of money and may not always be a good model for how humans can be affected, might we use human cells or tissues (e.g., liver, kidney) to determine how chemicals act biologically?  Where chemicals interact with the cells and alter (e.g., inhibit or over-stimulate) critical biochemical pathways, such perturbations may well represent early events or signals that can eventually lead to an adverse effect.

ToxCast

There is an EPA initiative called ToxCast that is exploring the use of high-throughput screening (HTS) test methods to predict hazard, characterize so-called "toxicity pathways," and prioritize among large numbers of chemicals.  According to the ToxCast website:  "In its first phase, ToxCast^TM is profiling over 300 well-characterized chemicals (primarily pesticides) in over 400 HTS endpoints."  The endpoints include tests that measure DNA and protein synthesis activities, multi-cell interactions and developmental assays in zebrafish.  These methods are among those being developed to realize the vision described in NAS' report. 

The results of the first set of ToxCast assays are still being validated – meaning that they are being analyzed to determine how similar the outcomes are to those of standard laboratory animal tests.  This is one important step in determining whether the ToxCast testing array can actually substitute for some standard laboratory animal testing.

But we would suggest there are already two timely and useful applications of the data that ToxCast can generate in aiding EPA's effort to evaluate hazard information under ChAMP.

Supporting alternatives analysis

The first idea would be to compare the relative capacity for chemicals in the same functional use class (e.g., solvent, chelator, fragrance) or chemical category (e.g., fatty nitrogen derived cationics) to effect biological changes detectable by the ToxCast HTS methods.  In doing so, we would be testing the hypothesis that chemicals that demonstrate less capacity for effecting changes in the HTS tests are more likely to have lower hazard profiles. 

As we described in a previous post, alternatives analysis is a tool that compares hazard assessments of chemicals that are used to perform similar functions, in order to identify those that have a lower hazard profile.  Using ChAMP hazard data and characterizations for this purpose would start by comparing the results of laboratory animal study data.  By running some of the ChAMP chemicals through the ToxCast HTS system and comparing the results of the HTS tests with the ChAMP hazard data, EPA could both support the identification of safer chemicals and test how well the HTS methods predict in vivo effects – a true win-win.

Validating (or refuting) chemical categories

About 80% of the chemicals sponsored under EPA's HPV Challenge Program are members of proposed chemical categories.  Within these categories, sponsors and EPA propose that hazard data for tested category members can be "read across" to untested members, as an alternative to direct testing of each chemical.

Grouping chemicals into a category starts with an hypothesis that distinct chemicals that show similarity or regularity in their physical-chemical properties and chemical structures actually possess similar or predictably regular patterns of biological activity.  Establishing a valid category requires that the hypothesis actually be demonstrated to be true, once the available data on physical-chemical properties, environmental fate and toxicity/ecotoxicity for the proposed category members are assembled.  Then, some degree of read-across among category members can be justified.

However, as our comments on the several ChAMP assessments we've analyzed in recent posts make clear (see here, here and here), both industry sponsors under the Challenge and EPA under ChAMP appear frequently to be over-relying on category approaches, lumping together chemicals that are insufficiently related or similar to warrant read-across.

HTS data could play a very useful role, therefore, in helping either to demonstrate – or to negate – an hypothesis that members of a proposed category of chemicals actually exhibit similar biological activity.  If category members in fact exhibit similar or regular patterns of activity across an array of different cellular and subcellular assays, then the case for grouping them for purposes of read-across would be much stronger than a case based solely on similarity or regularity in their physical-chemical properties and chemical structures.  Alternatively, if HTS data do not show such patterns, the category should not be utilized and either it should be broken up into smaller groupings or its putative members should be tested individually.

Both of these uses of ChAMP hazard data and characterizations side-by-side with HTS data developed under ToxCast – in supporting alternatives analysis and in category validation – would offer the added benefit of helping to advance the transition to the "new paradigm for toxicity testing."

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.

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.