Will Inhalation Toxicology Tests Go Animal-Free

A Charles River and MatTek project funded by the American Chemistry Council is seeking to validate a New Approach Methodology that would rely on 3D tissue models of human airways

Last month, Charles River embarked on a project that could dramatically change how companies evaluate the impact of chemicals and aerosols on airway passages. Working with its longtime partner MatTek, a pioneer in the development of 3D in vitro inhalation systems, and the nonprofit research company Battelle, Charles River is seeking to advance the development of an integrated approach that moves beyond the rat experiments that regulators require, and companies routinely do, but which experts concede are far from perfect.

The two-year project, funded by a US$1.3 million grant from the American Chemistry Council focuses on development of in vitro approaches using human 3D in vitro models of the upper lungs to assess direct toxicity of chemicals without the need to conduct inhalation studies in lab animals. The project isn't seeking to replace animal models completely, but it is leaning heavily on the potential of 3D tissue platforms that replicate human and animal airways, and the power of computational models, to build a bridge toward a more modern, efficient, and effective way of testing the thousands of chemicals used in pharmaceuticals, industrial and agricultural products and even cosmetics, on respiratory systems.

Jo Wallace, a Principal Research Scientist at Charles River who is co-leading the ACC-funded project along with Seyoum Ayehunie Chief Scientific Officer at MatTek, said her hope is that their findings will lead to a new alternative method accepted by the OECD and regulatory agencies, though she cautioned it will take time, money, and lots of data to achieve these goals. "There is going to have to be more grants, more research, more data, but we're getting more and more confident that these models will ultimately be able to predict the human outcomes as well as or better than the rodent tests."

3D human tissue models in toxicology studies are nothing new. The earliest models are well-established, well understood and carry huge amounts of historical data, says Wallace. Leading commercialized 3D cell culture models - like MatTek's EpiAirway™ and Epithelix's MucilAir™-are used routinely for safety and risk assessment, antiviral research and inflammation. Companies also use them to optimize compounds or formulations for delivery to the nose or airways.

Yet while companies do include some in vitro data in their regulatory filings it is really just an adjunct to the rat inhalation studies that agencies require for IND applications or, in the case of the EPA, registration actions. While rat inhalation studies are still the norm challenges remain due to the differences between rat and human anatomies. "A rodent is like a long sausage, and whatever it breathes in goes straight in," says Wallace. "In humans, however, the upper respiratory tract turns a corner, which is going to affect the flow of whatever you are breathing in. Also, the lung structures are very different between rodents and people, not to mention species differences in genes and gene expression." These anatomical and genetic differences have built a good case for developing new approach methods like the one Charles River is working on with its collaborators. The work is years in the making.

Decades of work-and progress-on 3D airway models

3D airway tissues models trace their origins to the 1980s when scientists, primarily from big science hubs in Boston and San Diego, began detailing how they were using techniques-like scaffolds and hydrogels-for culturing cells in a 3D environment. Among them were MIT chemical engineering professors Raymond Baddour and Robert Cohen, who invented a surface treatment technology that could make plastic surfaces adherent or non-adherent for living cells. This turned out to be uniquely valuable in 3D tissue cultures, and out of this research rose MatTek, one of the first companies to market 3D models to a small but growing number of academic, industry and government stakeholders.

In 1993, it launched its first commercial product, EpiDerm-a reconstructed model of the outer layers of human skin-to predict skin irritation from chemicals and formulations, and today the OECD recognizes the in vitro procedure as an animal replacement for skin irritation, corrosion, and phototoxicity studies of chemicals and formulations. In 1999, the EpiAirway™ respiratory tissue model went on the market followed by two other 3D respiratory models; EpiAlveolar™ (2013), constructed of tissue from the lower respiratory tract and EpiNasal™ (2022), from the nasal system. Among their many uses were during the COVID-19 pandemic when scientists used the models to establish infection and test potential drug candidates against SARS-CoV-2. These models are ideal for chemical safety and risk assessment, respiratory viral infection and antiviral research, drug delivery, inflammation, and fibrosis research.

The tissues, which are about 40-75 microns thick, function like human airways in an animal or a human and are grown in cup-like devices about a centimeter in diameter. EpiAirway, for instance, consists of highly differentiated primary tracheal and bronchial epithelial cells, mucous-producing goblet cells, functional tight junctions that prevent free movement of fluid across the airway barrier, and beating cilia. "In order to appropriately model or reproduce the in vivo tissue it needs to be fed the same way it gets nourished in vivo," says Mitch Klausner, one of the early leaders with MatTek and today its Executive Vice President. "So, we feed the tissue from the underside only, supplying nutrients through the membrane."

Technology certainly propelled the revolution in 3D models, but so did concerns about animal welfare. Ever since William Russell and Rex Burch released their seminal paper in 1959 proposing a new way of research that involved the replacement, reduction, and refinement of animal models - known today as the 3Rs of alternative methods for minimizing animal pain and distress in research-laboratories have searched for in vitro, in silico and in chemico models that could be applied to the myriad of preclinical studies done on new drugs and chemicals.

Charles River, a global contract research organization (CRO) that works largely in the preclinical space, was an early leader in evaluating the use of 3D models as a possible replacement model for predictive toxicology studies, including for the pesticide, chlorothalonil made by Syngenta. As part of the re-registration process for chlorothalonil, the value of performing longer term inhalation rodent bioassays for materials like this was questioned, and in raising it with the US Environmental Protection Agency Syngenta suggested an in vitro assay should be done instead to characterize the risk to human health from inhalation. Charles River performed the test for Syngenta using MucilAirTM , which is derived from primary human airway epithelial cells. The study was so successful that Syngenta incorporated this data into a New Approach Methodology, with particle size distributions, computational fluid dynamics, and worker breathing measurements to reach the expected human equivalent concentrations for the risk assessment. This was published as an OECD Case Study in 2022.

"Ultimately, the EPA accepted the new approach methodology (NAM) and gave a lot of recommendations and advice for future use of models," said Wallace. "But it was the first instance of a regulatory body actually going, "Yes, okay, we'll accept this. You don't have to do the repeat dose chlorothalonil test."

Today, Charles River routinely employs 3D human-derived reconstructed airway tissue models to identify, test and identify specific markers of toxicity associated with epithelial damage. These human tissue models have been used in support of its 3Rs program by informing the design of better animal inhalation toxicology tests or as replacement of the animal tests in clinical, occupational and consumer toxicology risk and hazard assessments as well as in pharmaceutical drug development for safety and efficacy.

Another New Approach Methodology for Inhalation Tox?

The current ACC-funded project could usher in another NAM for inhalation toxicology, but unlike the work with chlorothalonil, it will impact a wide range of aerosolized chemicals. "Our goal is to develop a prediction model that we hope will be accurate in predicting the rodent outcomes, and ultimately the human outcomes," said Wallace. "We are specifically looking at chemicals and aerosol-delivered chemicals. It doesn't cover pesticides, it doesn't cover pharmaceuticals, and it doesn't cover vapors, mist, and dust, which are the other inhaled exposure routes."

Mary McElroy, an Associate Director at Charles River and Co-investigator on the ACC grant, said the overall strategy of the project is to build on Wallace's and Ayenie's deep knowledge of in vitro systems while including experts in lung toxicology, inhalation exposure, pathology and modelling. "This ensures the most robust approach will be applied to achieve the grant objectives and therefore confidence in the data and interpretation," she said.

Using 10 chemicals and 5 aerosols with a lot of good in vivo and in vitro data behind them, Wallace's team will be comparing a very simple cell culture model with a complex 3D EpiAirway model in a four-hour single exposure test. "We are also going to compare interspecies-so looking at that same exposure using human EpiAirway and rat EpiAirway to see if we can see translational species differences. And then we are going to compare the acute exposure with a chronic 14-day scenario and compare a direct application-applying test chemicals with a pipette to the surface of the cells-with aerosol application using our Vitrocell^TM equipment. After that is all done, the data goes into the dosimetry model to try and extrapolate the in vivo outcomes from the in vitro data."

Vitrocell® VC 12/12 Quick Connect Docking Station

Klausner said the respiratory models it has been offering for 20-25 years have understandably been a learning curve for companies. It takes time, he acknowledges, for companies to implement new assays into their product development cycles. Because of this, it can take time to generate the data needed to prove the value of these in vitro methods over animal models, and it can take time for agencies to accept alternative models.

"Regulators are very conservative, that is their job," said Klausner. "You need to develop the assay, prove it works, do a validation study and present the results to regulators. This all can take 5-10 years. With that said, 5-10 years from now, I would hope there would be an OECD test guideline for this."

Ayehunie agrees, adding that the key to approval by regulatory bodies like the EPA or FDA will be a painstaking process. "Validation of the 3D tissue models for their accuracy, reproducibility, reliability must be done across different laboratories. There also needs to be improvement in transferability to enhance prediction of human responses and collaboration between stakeholders and regulatory bodies," he said. "This collaboration between CRL and MatTek is a step in the right direction to get there."

The main image at top of page depicts an EpiAirway™ 3D structure, which consists of organized Keratin 5+ basal cells, mucus producing goblet cells (MUC5AC), functional tight junctions (E-cadherin) and beating cilia. EpiAirwayFT also incorporates human fibroblasts (Vimentin) in an extracellular stromal matrix. Images from: https://www.mattek.com/products/epiairway/

Check out the Alternative Methods Advancement Project (AMAP) and learn more about the scientific and technological innovations Charles River is using to lead the industry into the next frontier of drug discovery and development.

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