Reimagining alternatives to animal testing

Around 348 B.C., Aristotle took a two-year trip to the eastern Aegean island of Lesbos to study animals in a lagoon. Along with observing the creatures in their natural habitat and surmising, among other things, that dolphins were not fish, he dissected smaller animals to try and understand their internal workings. When he cut open eels, abundant in the lagoon, he was puzzled to find no evidence of reproductive tissue and made the false assumption that they generated spontaneously from the mud.

Aristotle's dissections were some of the first documented experiments on animals. Initially a practice aimed at understanding anatomy, these experiments evolved as biology and medicine progressed. For example, the Roman physician Galen of Pergamon developed techniques for dissection and vivisection of animals, which informed his treatises on medicine that remained canonical until the 14th century, when the Renaissance began in Italy.

It wasn't until the late 1930s that rigorous animal testing became a standard part of the drug development process. A U.S. pharmaceutical company had created an elixir with a raspberry aroma that promised to work as an antibiotic. The solution contained diethylene glycol. Unbeknownst to the company's chief chemist, the chemical proved poisonous to humans, and over 100 people died after the elixir hit store shelves. The resulting outcry led to the passage of the 1938 Federal Food, Drug, and Cosmetic Act, which required that drugs be tested on animals before being marketed.

Without animal testing, many of the medicines and procedures we take for granted wouldn't exist today. Transplantation of skin, corneas, and internal organs became possible owing to knowledge acquired through experimentation on animals. And polio—the devastating, paralysis-causing virus that was once one of the most feared diseases in the world—has been nearly eradicated because of a vaccine that was developed through experiments on monkeys.

Today, animals continue to be widely used in the biomedical sciences. One paper published in Sage Journals found that 79.9 million animals were used in scientific procedures in 2015, an estimated 37% increase from 2005. There have also been unprecedented levels of funding in drug development in the last decade. However, the number of drugs failing to make it to market that have passed animal testing reached an all-time high of 95% in 2021, according to a review paper in the journal Nature. Thomas Hartung, a professor of toxicology and director of the Johns Hopkins Center for Alternatives to Animal Testing, wants better science—and more options.

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Sometimes the consequences of animal experiments can go beyond a couple of failed experiments. In France in 2016, six people were hospitalized and one man died during a clinical trial. The drug in question had been tested in mice, rats, dogs, and monkeys with dosages 400 times stronger than those given to the human volunteers, and no ill effects were recorded.

Hartung's research has found that there are cases where animal models may no longer be necessary. In a paper published in a 2018 edition of the journal Toxicological Sciences, he and his team found they were able to predict—using a computer model that combed through a massive chemical hazard database—whether a particular chemical would be toxic to humans in more cases than animal models could. "The publication was a turning point," says the German-born Hartung, who has led the center since 2009.

The finding effectively put the center at the heart of a revolution in toxicology to move away from decades-old animal tests to the use of artificial intelligence and organoid cultures, 3D tissue models grown from stem cells programmed to mimic a specific organ. In the not-so-distant future, Hartung hopes, this emerging and quickly evolving technology and science could render many animal tests a remnant of the past.

That 2018 paper was a breakthrough in the use of machine learning to approach toxicology. One of Hartung's PhD students had built a database that could be used to predict—better than animal models could—how toxic a certain chemical would be to humans. More than 10,000 chemicals and their properties, provided by the European Chemical Agency, were reviewed.

The structure of a chemical determines whether it would be toxic to humans. So, when researchers want to determine the toxicology of a chemical, they can look at those with a similar structure known to produce a negative reaction. Manually assessing each chemical on a case-by-case basis would be time consuming, limiting its usefulness. What the researchers at the center, known as CAAT, did was to automate and accelerate that process, using big data to examine potential human interactions such as acute oral and dermal toxicity, eye and skin irritation, and mutagenicity (ability to induce a genetic mutation).

Computational tools are just one of the ways Hartung and other researchers at CAAT are attempting to move away from animal testing. The lab where Hartung is based is filled with brains, and that's not a reference to the staff located there. Little clumps of brain tissue, barely visible to the human eye, are grown in incubators every week by the thousands. Referred to as organoids, these clumps of tissue can't think or feel, but they can be used to see how brain cells respond to stimuli in a lab setting.

Brain organoids are made from pluripotent stem cells, which can produce any cell or tissue a body may need. The cells are placed in a matrix that helps them connect with each other and form larger tissues. They're then added to an incubator and allowed to grow for eight weeks, at which point they are essentially miniaturized 3D versions of organs able to be used for testing. "Once you have mastered production, it is a very robust and reasonably cheap process," Hartung says.

By combining brain organoids with AI, Hartung hopes to develop what he calls organoid intelligence, a major step forward "to make brain cell cultures do what the [human] brain is supposed to." Although it is currently still science fiction, he says, the organoid intelligence project (running since January 2023) recently produced a technical paper describing how to build such a system.

Hartung moved to the United States in 2009 to take over from Alan Goldberg as director at CAAT. Founded in 1981 with a $1 million grant from the Cosmetic, Toiletry, and Fragrance Association, the center and its researchers for the next few decades worked to harness scientific advances, such as in vitro experiments using human cell lines, where once mice and rats were used. Advances in biostatistics and computer modeling of biological systems enabled researchers to construct experiments using only a fraction of the animals they would otherwise have needed.

When Hartung arrived, the center was "an information hub of six people" down by Baltimore's Inner Harbor. He promptly moved activities to the university, under the auspices of the Bloomberg School of Public Health, because he wanted to have a larger lab space and students to work with. Today, CAAT involves more than 30 researchers. His background, he says, has also helped bring some diversity to the center. "We have an unusual number of expats and people from all over the world," he says.

Alternatives to animal testing would get a real boost when the COVID-19 pandemic hit. Scientists were desperate for answers, and fast, so they turned to nonanimal models to understand the virus. Similarly, researchers had to dramatically cut down the time it took to develop a vaccine, typically in the range of five to 10 years. "I'm not saying that animal studies don't give us good answers, but they're expensive and lengthy, and they're not for something that you need answers quickly on," says Suzanne Fitzpatrick, a toxicologist at the FDA's Center for Food Safety and Applied Nutrition.

In the years since, there has been growing support for alternatives to animal testing. Maryland became the first state to require animal testing labs to contribute money to nonanimal research. Monica Bertagnolli, the director of the National Institutes of Health, announced in February that it would prioritize the development and use of combinatorial NAMs. NAMs refers to new approach methods, another term for alternatives to animal testing.

In January 2023, the FDA Modernization Act took things a step further, declaring that animal testing was no longer required as evidence before clinical trials.

CAAT recently announced that it would collaborate with the FDA's Center for Food Safety and Applied Nutrition to discuss and share the latest developments in the field of animal testing alternatives. "There's so many papers coming out now in this area, it's hard to keep up with the science," Fitzpatrick says. The collaboration, she says, makes it easier for scientists to keep up with advances while "we're still doing our regular jobs."

Soon, Fitzpatrick expects "more and more methods coming in that might be of use to the FDA" as nonanimal models mature. But, she cautions, "I don't think we're at the point where we're not going to have animal testing."

Of the $42 billion of funding the NIH awarded in 2020, 47% went to projects based on animal testing. But it should be pointed out that there are many laws, regulations, and policies that protect animals used in federally funded research. According to the National Institutes of Health, these protections include considering nonanimal alternatives to meet the scientific objectives and using the fewest subjects needed for thorough and repeatable results. They also outline standards that reflect a commitment to animal care.

In short, animals are still a vital part of science and public health. Reporting in Fast Company from earlier this year highlighted a shortage of long-tailed macaques during the COVID-19 pandemic. A panel assembled by the National Academies of Sciences, Engineering, and Medicine concluded that a lack of nonhuman primates in research would "severely limit the ability of National Institutes of Health–supported research programs to respond adequately to public health emergencies, as well as to carry out high-impact biomedical research."

The panel also said: "While no model, animal or otherwise, can fully mimic the complexities of the human body, there remain research questions that currently cannot be answered outside of the context of a living organism."

"In an animal, you have the systemic interaction of multiple organs, says Eva-Maria Dehne, a senior scientist at TissUse, a biotechnology company in Germany. "This is what you need to replace [animal models]." Her work focuses on organ-on-a-chip systems, small chips roughly the size of a computer memory stick. Organoids are added to chambers on the chips, lined with canals along which liquid can flow, mimicking blood vessels. Valves allow researchers to control the rate of flow.

Different chips can be connected so that a researcher could, for example, end up with a brain-heart-liver system. TissUse develops these organ-on-a-chip systems and sells them to researchers in the biomedical field.

Dehne, who was initially interested in the field owing to an ethical opposition to animal testing, has become more and more convinced by the scientific arguments to move away from the practice.

In making the case for nonanimal testing, Hartung thinks it's best to focus on arguments around efficiency. He adds that in his experience people tend not to respond positively to ethical arguments. When you highlight the efficiency of alternatives, that can open more doors. Data suggests that organ-on-a-chip systems could reduce research and development costs by 26%. "It is much more powerful than saying you have to protect these animals," he says.

Dehne worked with others on a brain-to-liver chip to test the blood-brain barrier permeation of the drugs atenolol and propranolol, the results of which were published in the journal Cells in 2022. Not only did the drugs match the results from human clinical trials, so, too, did the metabolites. In another paper, cosmetic ingredients were tested on a skin-liver-thyroid chip, with results predicting safe dosages within a fraction of current safety standards.

Researchers from Columbia University tested the cancer drug doxorubicin on a heart-liver-bone-skin chip, which matched the results found from clinical trials of the drug. Emulate, a spin­off from Harvard's Wyss Institute that is also developing organ-on-a-chip systems, works with top pharmaceutical companies, such as AstraZeneca, Johnson & Johnson, and Roche.

Hartung says the main message he wants to get across is that today there are simply fewer reasons—scientifically, economically, and ethically—to keep experimenting on animals to the same degree as we have done historically. In his view, "it is time to complement and then to replace the animal tests where we can do better," he says.

There are, however, still a lot of technological hurdles to cross before nonanimal testing can become more prevalent. Unless organoid intelligence, or something similar, comes to fruition, running experiments that involve a conscious response may always have to be done on animals. For example, if testing the effects of a pain relief drug, you need a conscious being.

Organ-on-a-chip systems are also quite complex, and that limits their usefulness. Chengpeng Chen, an assistant professor of analytical chemistry at the University of Maryland, Baltimore County, has experienced that firsthand. He remembers setting up a chip system, but when it came to adding the cells, they were either contaminated or hadn't grown properly, and so he had to discard the whole configuration. "It takes days to have a setup ready," he says. "Any mistake or any problem in any of the steps can mess up the whole setup."

Chen himself is running a lab focused on organ-on-a-chip systems. One of his aims is to try and make the use of these alternatives to animal testing easier. "A lab has to have very well-trained personnel to fabricate and maintain such organs-on-a-chip," Chen says. If we want the technology to be more widely used, then it must become easier to handle. Focusing on gains in efficiency in lieu of accessibility, while still useful, will mean the technology remains largely in academia, he says.

When Hartung went to Germany's University of Tübingen in the 1980s, animal testing was the norm. "I really needed a big glass of whiskey in the evening when I had done an experiment on mice and rats," he says. Feeling uneasy with the prospect of a career in a field where he had to keep testing on animals, Hartung managed to convince his mentor at the time to let him run experiments on cell cultures instead.

"I got a lot of feedback from some fellow scientists who told me: 'How can you waste your beautiful career with this alternative nonsense?'" he says. Nevertheless, Hartung continued with his research into cell cultures until, in 1996, he made a breakthrough when he designed an in vitro version of a pyrogen test—a test, traditionally done on rabbits, to find out whether a product is clean of bacterial contamination.

Ecstatic to have made a contribution that could save animal lives, Hartung was disappointed when the test was finally approved in 2006 alongside a host of others with the same function. Almost nobody seemed interested in adopting them. "The appetite by both the regulators and the regulated industry to make changes is often not very big," Hartung says.

One moment that really highlighted this resistance to change for Hartung was when he was on a panel discussing his pyrogen test. An employee from a big pharmaceutical company opposed the test "harshly," yet away from the spotlight, in the safety of a private conversation, the employee said he thought the test was good; it's just that his company had taken the stance to oppose it as it might impact their profits.

A 2022 paper published in the Journal of the Royal Society of Medicine noted that the beneficial effects of "tissue plasminogen activator for stroke had been well documented in animal models by 2001, but research using several thousand animals continued for several years afterward." An analysis published in BMJ Open in 2020 found that most stroke researchers recognized that animal models had not been successful in the field, yet they were reluctant to relinquish them. The analysis looked at opinion papers published in journals from 1979 to 2018 and found that only one author out of 80 had advocated for alternatives to animal testing.

Others, however, seem to be embracing alternatives. Last year, CAAT, together with TissUse, organized a conference in Berlin based on alternatives to animal testing. Hartung said that while they initially expected around 700–1,000 guests, the capacity of 1,300 was reached months before the conference occurred.

CAAT recently received $17 million for a seven-year project called IMPACT. With the money, Hartung and his team hope to further refine the alternatives to animal testing they've developed and create the Human Exposome Project, a database cataloging chemical exposures a person might face over their lifetime, along with potential harmful side effects.

Dehne thinks the community building around nonanimal models is great, and she is happy that more companies and researchers are entering the space. "There will never be one system that can answer all the questions, and therefore there will always be a need for different systems," she says.

Hartung has a similar view. "We're trying to form communities, … hundreds of people ultimately collaborating because they buy in" to the mission, he says. "It's more important that things are being done than who does them."

Fitzpatrick from the FDA says that alternatives to animal testing will likely help reduce the number of animals used but not necessarily fully replace them. She suggests that alternatives could be used to study a particular chemical, so researchers would have a better understanding of what to look for in animal tests, meaning fewer overall tests, and therefore fewer test subjects, would be necessary. "Our responsibility is to put safe and effective products on the market—not ending animal testing," she said. "So, we have to do that however we can."

Almost three decades after Hartung designed his pyrogen test, the European Union finally decided to outlaw the industry standard rabbit pyrogen test by 2026. "If you would have told me as a young postdoc how long this might take, I probably would have gone into another field," he says. Though the U.S. is still lagging in that regard, Hartung is enjoying the moment of having finally pushed through a replacement that he says could save up to 170,000 rabbits from unnecessary suffering every year.