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Animal-free skin allergy testing

With the recent approval of the human Cell Line Activation Test (h-CLAT) for skin sensitization (allergy), toxicologists now have a battery of methods that allows them to test for sensitization without using animals.mice

Testing a chemical substance for skin irritation or corrosion is pretty straight-forward: the substance is applied to a skin sample (there are many non-animal in vitro options) and damage will be seen relatively quickly. But to learn if the chemical has the potential to cause skin allergies, testing is more complicated. Skin sensitization is a two-stage process. In the first stage, a chemical exposure “primes” the immune system. Additional exposures then provoke an allergic response (inflammation, redness, itching, etc). Because of the biological complexity of the process, skin sensitization testing is usually conducted on mice or guinea pigs.

But mice and guinea pigs don’t always react the same way as humans would to potential skin allergens. To replace these animal tests with more human-relevant methods, toxicologists have long recommended developing a battery of in vitro tests that could be combined in an Integrated Testing Strategy – where each test method captures a different part of the skin sensitization process. The biological processes that underlie the skin allergy reaction are pretty well understood, and have been described (the Organisation on Economic Co-operation and Development (OECD) has published a description of this process – the Adverse Outcome Pathway leading to skin allergies). Two of the pieces of this strategy are already in place: the OECD approved the use of the Direct Peptide Reactivity Assay (DPRA) and the KeratinoSens test – each measuring a different step in the sensitization process. The DRPA assays measures whether a chemical can react with proteins in a way that causes the protein to become an allergen. The KeratinoSens assay measures activation of genes involved in the allergic reaction in skin cells (keratinocytes). The h-CLAT method completes the battery of tests: it detects biomarkers that indicate activation of immune (dendritic) cells in the skin.

Many countries require that chemicals used in manufacturing, agriculture, medicine, and cosmetics be tested for their potential to cause skin allergies in humans. Without approved in vitro options, REACH regulations in the EU alone would force industry to use hundreds of thousands of animals for skin sensitization testing. The animal-free, 3-test strategy is a “textbook” example of the mechanistic, pathway-based approach to chemical testing promoted by the Human Toxicology Project Consortium. Use of this strategy has the potential to greatly reduce the numbers of animals used for skin sensitization testing, while also reducing the cost and time it takes to produce human-relevant results.

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From the Wyss Institute press release: "These fluorescence confocal microscopy images show both a high magnification view (left) of a region of the human brain capillary endothelium within the endothelium lined tube (shown at lower magnification at right) that, in combination with surrounding human pericytes and astrocytes, comprise the blood-brain barrier (cell junctions linking adjacent endothelial cells are shown in magenta). " Credit: Wyss Institute at Harvard University

“Blood-brain barrier on-a-chip”

Scientists at the Wyss Institute have created a 3-dimensional in vitro model of the human blood-brain-barrier (BBB)-“on-a-chip.”  The device will make it possible for researchers to test drugs, chemicals, and disease factors that interact with the BBB, without using animals – and in a 3-dimensional environment that mimics that of the human BBB in vivo.

The BBB is a semi-permeable cellular structure that allows some nutrients and substances to enter the blood flow in the brain, and keeps other elements (such as bacteria and potential toxins) out. Because it is so effective, it can also prevent useful treatments from reaching targets in the brain. Researchers need to understand how and why certain substances can pass through the barrier, in part so they can design therapeutic drugs accordingly, and so they can design other substances to prevent neurotoxicity.

From the Wyss Institute press release: "These fluorescence confocal microscopy images show both a high magnification view (left) of a region of the human brain capillary endothelium within the endothelium lined tube (shown at lower magnification at right) that, in combination with surrounding human pericytes and astrocytes, comprise the blood-brain barrier (cell junctions linking adjacent endothelial cells are shown in magenta). " Credit: Wyss Institute at Harvard University

From the Wyss Institute press release: “These fluorescence confocal microscopy images show both a high magnification view (left) of a region of the human brain capillary endothelium within the endothelium lined tube (shown at lower magnification at right) that, in combination with surrounding human pericytes and astrocytes, comprise the blood-brain barrier (cell junctions linking adjacent endothelial cells are shown in magenta). ” Credit: Wyss Institute at Harvard University

To create the device, the Wyss Institute team carved a tiny channel in a polymer chip and filled it with a gel matrix containing human astrocytes, the cells that comprise the extra-tight “barrier” around blood vessels in the brain. Another channel was tunneled through this matrix and seeded with human pericyte cells (contractile cells which control the “gaps” through which substances can enter the neurological bloodstream) and then with human endothelial cells (the cells that line the interior of a blood vessel). The cells “self-assembled” into the same layers and connections they exhibit in blood vessels in vivo.

To test the model, the team introduced a protein known to cause inflammation – one that has been associated with a number of central nervous system diseases and disorders, including Alzheimer’s, multiple sclerosis, and stroke (among others). The in vitro BBB responded by producing protective proteins. The device can thus be used to study neuroinflammation, and to test new treatments.

Read more in the Wyss Institute’s press release.

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Educational Infographic produced by the Human Toxicology Project Consortium

THE FUTURE OF TOXICITY TESTINGHTP_infographic_FINAL_revised

A new infographic produced by the Human Toxicology Project Consortium shows in three sections how the future of toxicity testing promises a steady reduction in testing costs, increases in human relevance and confidence in safety assessments, and the eventual elimination of animal tests.

The first section provides a snapshot comparison of the current and future costs, efficiency and efficacy of toxicity testing, while the mid portion uses pesticide testing as a specific example of now, vs near-future, vs the optimal approach that, given the focus and resources necessary, will be envisioned within the decade.

The near-future and optimal approaches rely increasingly on our understanding of biology and using it to build a predictive systems biology platform that is comprised of an interrelated network of biological pathways. This platform is used to design and interpret tests that provide much more efficient and effective characterization of chemical activity that can be used to predict safe use of chemicals.

Finally, the results of this progression are captured in the summary graphic at the end – decreasing costs, animal use and time while human relevance and our confidence in safety decisions continue to improve.

As explained on our Project page, the Human Toxicology Project Consortium works on three areas critical for the successful, international implementation of a pathways-based approach to chemical safety testing: advancing the science, communicating the purpose and goals of pathway-based toxicology, and lobbying for funding and policy changes that will support pathway-based approaches in the US and around the world.

To advance our communication and education efforts, HTPC member organizations worked together to create this infographic, to quickly and effectively illustrate the differences between traditional animal-based toxicity testing and pathway-based testing in terms of predictive power, cost, and testing capacity.

Details on the numbers used in this comparison are available here (PDF).

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Credit: The Wyss Institute at Harvard University

Chipping away at the use of animals to predict human diseases

The Wyss Institute recently announced two new human cell-based inflammatory disease models built on its rapidly expanding “organ chip” platform. Both models could speed the development of treatments for these diseases, and further reduce the use of animals in testing.

Using the “gut-on-a-chip” device first introduced in 2012, Wyss scientists co-cultured human intestinal cells with normal and pathogenic intestinal microbes, producing an in vitro model (viable for up to two weeks) of intestinal inflammation and bacterial overgrowth. These two disease features are present in a number of human intestinal disorders (such as ulcerative colitis and Crohn’s disease). Until now, it has been difficult to reproduce these disorders in the lab in order to test treatments for them. The gut-on-a-chip device “could allow breakthrough insights into how the microbial communities that flourish inside our GI tracts contribute to human health and disease.” The image below, from the Wyss Institute press release, shows how the cells in this microenvironment even reproduce normal peristalsis, the contraction/relaxation cycle of the intestinal walls that moves digested food down the tract.
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The Wyss Institute also used its chip technology to create a human lung “small-airway-on-a-chip.” When the chips are lined with airway cells from patients suffering from such inflammatory disorders as chronic obstructive pulmonary disease (COPD) or asthma, the physiological features of the disease can be observed and tested in vitro. As noted in the Wyss Institute press release, “Demand for such opportunities is especially high since small airway inflammation cannot be adequately studied in human patients or animal models and, to date, there are no effective therapies that can stop or reverse the complex and widespread inflammation-driven processes.”

Watch a video demo of the small-airway-on-a-chip below:

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Supercomputers link proteins to drug side effects

Image from Lawrence Livermore National Laboratories: “LLNL researchers Monte LaBute (left) and Felice Lightstone (right) were part of a Lab team that recently published an article in PLOS ONE detailing the use of supercomputers to link proteins to drug side effects. Photo by Julie Russell/LLNL”

Supercomputers link proteins to drug side effects.

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Rod photoreceptors (in green) within a "mini retina" derived from human iPS cells in the lab.

3D “mini retina” from stem cells

Scientists at Johns Hopkins University School of Medicine have cultivated functional 3-dimensional human retinal tissue in vitro from induced human pluripotent stem cells.  And in a significant technical advance over previous cultured retina studies, the resulting tissue exhibits mature cell differentiation and organization, and is able to detect light.

From the JHU press release:

(Lead investigator M. Valeria Canto-Soler) says that the newly developed system gives them the ability to generate hundreds of mini-retinas at a time directly from a person affected by a particular retinal disease such as retinitis pigmentosa. This provides a unique biological system to study the cause of retinal diseases directly in human tissue, instead of relying on animal models.

The system, she says, also opens an array of possibilities for personalized medicine such as testing drugs to treat these diseases in a patient-specific way. In the long term, the potential is also there to replace diseased or dead retinal tissue with lab-grown material to restore vision.

The study appears in last week’s issue of Nature Communications.

(To learn more about stem cells – especially about their potential in toxicity testing – see this stem cell “primer” on AltTox.org.)

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