Crop duster (photo credit: Roger Smith/Creative Commons)

Advances in human-relevant alternatives for inhalation toxicity testing & screening

Many of the situations in which inhaled particles or substances can cause toxicity involve repeated exposures to the substance – e.g., cigarette smoking, use of inhaled medications, or on-the-job exposure to agricultural or industrial sprays and vapors.

To test for the effects of repeated inhalation exposures without using animals, scientists are developing increasingly sophisticated in vitro models of the human respiratory system. These in vitro alternatives use cultured respiratory cells and tissues that are kept alive in conditions designed to imitate the environment of the human airway. For repeat-exposure tests, it is necessary to keep these “reconstructed” cells and tissues alive and functional for extended periods of time.

Rats in inhalation exposure chamber

Rats in inhalation exposure chamber – a standard apparatus for testing inhalation toxicity on animals

Scientists with British American Tobacco (BAT) recently demonstrated that a commercially-available reconstructed human airway tissue called MucilAir™ would remain fully responsive for at least six months in their experimental conditions, making it one of a growing number of viable and more human-relevant alternatives to testing inhalation toxicity on animals.

(Image credit: British American Tobacco)

(Image credit: British American Tobacco; click to enlarge)

The scientists monitored such regular lung tissue activities as cilia beat frequency, mucous secretion, enzyme activity and gene expression, and found that the MucilAir tissue showed normal responses throughout the test period.

According to the company press release, BAT will use the tissue model to “compare the toxicological effect of repeated exposures to aerosols generated from conventional and next-generation tobacco and nicotine products.”

image of iPSC-derived branching mini-lung

Branching mini-lung. Credit: Nick Hannan, University of Cambridge

Cultured and engineered respiratory tissues with extended lifespans can also be used to test drugs intended to treat chronic lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and asthma. Scientists at Cambridge University recently created a “mini-lung” model of cystic fibrosis by reprogramming skin cells from patients whose CF was caused by a particular genetic mutation (one that affects 3 out of every 4 CF patients). The skin cells were reprogrammed into an induced pluripotent state, from which they can develop into any kind of cell in the body. The researchers then guided the cells to develop into the distal airway lung tissue most affected by diseases such as CF, certain lung cancers, and emphysema, and demonstrated that the tissue – which the team refers to as “mini-lungs” – was functional. Said lead author Nick Hannan: “We’re confident this process could be scaled up to enable us to screen tens of thousands of compounds and develop mini-lungs with other diseases such as lung cancer and idiopathic pulmonary fibrosis… This is far more practical, should provide more reliable data and is also more ethical than using large numbers of mice for such research.”

3D cell & tissue culture alternative toxicity testing
vascular photo

New non-animal methods for predicting cardiotoxicity

Cardiotoxicity is defined as damage or dysfunction occurring in the heart as a result of exposure to a chemical substance.  Cardiotoxicity is one of the major reasons given when the development of a drug fails or a drug is withdrawn from the market.  With the cost of bringing a new drug to market averaging $2.6 billion USD, there are pressing ethical and economic reasons to find a faster, more accurate way to predict cardiotoxicity.

Developments announced in the last few weeks are encouraging.  UC Berkeley researchers led by Professor Kevin Healy published a study of their functional “heart-on-a-chip:” a 3-dimensional network of adult stem cell-derived heart muscle cells linked together on a microfluidic platform that reproduces blood flow.  The cells beat normally, and responded appropriately to the effects of four well-described heart drugs (isoproterenol, E-4031, verapamil and metoprolol).  According to Healy, “Ultimately, these chips could replace the use of animals to screen drugs for safety and efficacy.” (See the open-access paper here.)

In addition, NC3Rs recently awarded its 2014 3Rs prize to Oliver Britton, a PhD student who created an innovative computational model of cardiac electrophysiology. Because the model incorporates within-species variations in heart properties (which are usually averaged in more simplistic models), it has the potential to more accurately identify potentially toxic drug compounds – allowing them to be pulled from development before animal studies begin.  Quoted in the NC3Rs press release, Professor Ian Kimber said of the model: “Mr Britton’s paper really stood out to the panel because of (its) potential as a replacement for early-stage animal tests in drug safety studies, across a broad range of disciplines. The model has also been developed into a piece of user-friendly software, encouraging uptake and use by industry, which could have an important impact on the reduction of animals in research.”

Human-relevant alternative models such as these have the potential to reduce the cost, time, and numbers of animals expended in drug development, while increasing human safety.

Watch video of the beating UC Berkeley “heart-on-a-chip”:

alternative toxicity testing computational toxicology drug discovery organs-on-chips
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A step closer to 3D-printing organs to replace animal testing

The inventors of a 3D bioprinter capable of printing human stem cells have now developed a synthetic DNA gel that allows them to print a three dimensional scaffold containing live cells. The process brings the Heriot-Watt University team closer to their ultimate aim of 3D-printing organs for transplant or drug-testing.

Read more about the invention in R&D Mag online.

This video from 2013 explains the team’s stem cell-printing process:

3D cell & tissue culture alternative toxicity testing
DNA molecule

EPA expanding use of non-animal methods


EPA’s Tox21 high-throughput chemical screening robot (image attribution)

Last month, the EPA invited stakeholder comments on its proposed new guidance for testing pesticides for acute oral, dermal, and inhalation toxicity, as well as skin and eye irritation and skin sensitization.  The draft guidance outlines a procedure for expanding the use of alternative test methods in pesticide testing.  If implemented, the guidance stands to improve chemical screening efficiency and data relevance while greatly reducing the agency’s use of animals.  As the EPA press release acknowledges, “With the rapid advances in science and continual development of new technologies… there is an increasing potential for the use of alternative methods in regulatory risk assessments.”  The draft guidance is available here:  Comments are due to Christopher Schlosser ( by March 10.

As the press release also notes, the draft guidance represents continued progress in the agency’s efforts to adopt recommendations in the National Academy of Science’s report, Toxicity Testing in the 21st Century. For more about these achievements, see these two recent EPA blog posts:

Exposing the Missing Link: Advancing Exposure Science to Rapidly Evaluate Chemicals

EPA: Taking Action on Toxics and Chemical Safety

alternative toxicity testing EPA

Advancing Species Extrapolation: EPA’s “Sequence Alignment to Predict Across Species Susceptibility” | Science

…SeqAPASS provides us with a fast, efficient screening tool. Using it, we can begin to extrapolate toxicity information from a few model organisms (like mice, rats, zebrafish, etc.) to thousands of other non-target species to evaluate potential chemical susceptibility.

SeqAPASS provides an example of how EPA Chemical Safety for Sustainability researchers are leading the effort to usher in a new generation of toxicology practices that aspire to reduce the number of animals used, decrease costs, and increase the efficiency of chemical toxicity testing. The 21st century chemical toxicity testing strategy incorporates these ideals and has given rise to adverse outcome pathway (AOP) development and rapid, high-throughput chemical screening programs such as EPA’s ToxCast program.

Read more on the EPA’s science blog: Advancing Species Extrapolation: EPA’s “Sequence Alignment to Predict Across Species Susceptibility” | Science.

alternative toxicity testing AOPs computational toxicology databases EPA ToxCast
microscopic image of lab-grown muscle tissue

Contracting human muscle tissue grown in a lab

Duke University scientists have grown functional human muscle tissue in the lab. The team grew the muscle tissue from “myogenic precursors” – cells that have developed beyond stem cells but are not yet muscle cells, and then tested the tissue’s contractile and other responses.

From the Duke University press release:

To see if the muscle could be used as a proxy for medical tests, Bursac and Madden studied its response to a variety of drugs, including statins used to lower cholesterol and clenbuterol, a drug known to be used off-label as a performance enhancer for athletes.

The effects of the drugs matched those seen in human patients. The statins had a dose-dependent response, causing abnormal fat accumulation at high concentrations. Clenbuterol showed a narrow beneficial window for increased contraction. Both of these effects have been documented in humans. Clenbuterol does not harm muscle tissue in rodents at those doses, showing the lab-grown muscle was giving a truly human response.

microscopic image of lab-grown muscle tissue

A microscopic view of lab-grown human muscle bundles stained to show patterns made by basic muscle units and their associated proteins (red), which are a hallmark of human muscle. Photo credit: Duke University; used with permission

Read more about the work and its implications for toxicity testing and disease modeling here. Watch video of the engineered muscle tissue below:

3D cell & tissue culture alternative toxicity testing
vascular photo

Pfizer and HemoShear partner to predict toxicity in early stages of drug discovery

Pfizer Inc. and HemoShear, a privately held biotechnology company, are collaborating to develop methods to predict drug-induced vascular injury (DIVI) – a complication that slows or stops the development of many promising new drug candidates.

As Hemoshear’s press release explains:

Drugs in development may cause DIVI, such as inflammation or vascular lesions, in animals during testing for drug safety and toxicity. When this occurs, significant program delays and additional costs are incurred to investigate and explain the underlying biology and determine whether the compound is safe to move forward to human testing, or to determine if another compound would be safer. In some cases, decisions are made to stop the program altogether in the absence of a clear understanding of the injury and whether an animal response translates to human response.

The collaboration will make use of Hemoshear’s computational models and “translational tissue systems” — species-specific multidimensional tissue structures that replicate circulation and other physiological dynamics. Using these tissue systems, Hemoshear is able to run comparative studies (human tissues versus other animal tissues) that show whether, and how, human and animal responses differ.

The Pfizer/HemoShear collaboration has the potential to prevent unnecessary additional animal testing, and to reduce the cost and time it takes to bring new medications to market.


Read more about HemoShear’s translational tissue systems here.

alternative toxicity testing computational toxicology drug discovery