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Researchers examine how the use of animal testing to identify endocrine-disrupting substances in the EU can be reduced. Although it is, in principle, possible to identify such substances without using animals, non-animal methods are still rarely applied.

A team from the MERLON research project, led by the DTU National Food Institute, has mapped the use of alternatives to animal testing – so-called New Approach Methodologies (NAMs) – in the identification of endocrine disruptors. This required a thorough review of the complex legal framework underlying the EU’s regulation of chemicals in everyday products, ranging from hair shampoo to food.

The conclusion is that alternative methods are almost never used to demonstrate the harmful effects of endocrine-disrupting substances, even though EU legislation allows for their use in this context. The research has been published in the scientific journal Regulatory Toxicology and Pharmacology.

According to the EU law, alternative methods may be used to demonstrate harmful effects of endocrine disruptors if they provide a ‘similar predictive capacity’ as animal tests. The problem is that we currently have only one alternative method that can actually be used for this purpose – namely ‘read-across’, where data from one substance is used to assess another, similar substance.”

Marie Louise Holmer, Special Consultant at the DTU National Food Institute, and one of the authors of the scientific article

“Read-across is ready to be used now, and we should apply it as broadly as possible. However, when it comes to other alternative methods, such as computer-based models and cell-based tests, substantial further development is still required.”

Better for both animals and humans

Alternative testing methods are, from an animal welfare perspective, clearly preferable. However, these methods are also much faster to carry out, which means that NAMs could significantly increase the efficiency of identifying endocrine disruptors in the EU.

“This is an area in which we are in urgent need of the support offered by digitalisation and new laboratory techniques. The WHO estimates that over 60,000 chemical substances are in global commerce. With today’s methods, we would not be able to test them all for the many harmful effects associated with endocrine disruptors – not even in 100 years,” says Marie Louise Holmer.

Endocrine disrupting substances pose risks to humans, animals and the environment, and can affect health in multiple ways. They may, for example, be carcinogenic, impair fertility, or interfere with the development of the brain and immune system. In the EU, substances can be regulated if testing confirms they are endocrinedisrupting – and such testing has so far primarily relied on animal experiments.

Animal testing has been widely debated for many years. Following a large public petition, the European Commission is developing a roadmap to outline how all animal testing for chemical safety assessments can eventually be phased out.

A balanced transition

The researchers advocate a balanced approach. They argue that there is a need both to develop and validate alternative methods and to explore how these, potentially in combination, can predict harmful effects to the same degree as animal tests. At the same time, animal testing should be refined and optimised to provide as much valuable information as possible.

“We must ensure that the alternatives are just as effective as animal testing before animal methods can be phased out completely. That is why we also emphasise the continued need for animal testing until alternatives are fully accepted and routinely implemented in chemical regulation,” says Marie Louise Holmer.

Dialogue to build common ground

One of the recommendations from the researchers is to bring together all stakeholders affected by the legislation in order to build consensus.

“We need, among other things, to determine when results from New Approach Methodologies – or combinations of New Approach Methodologies – are sufficiently robust and reliable to predict harmful effects and thus replace animal testing,” says Marie Louise Holmer.

Stakeholders could include national authorities (such as the Danish Environmental Protection Agency), relevant EU agencies, researchers, industry representatives, and NGOs.

Facts: Identifying endocrine-disrupting substances in the EU

Three criteria must be fulfilled to classify a substance as endocrine-disrupting:

1. It must cause harm (as assessed via animal tests or NAMs – the latter are currently seldom used for this purpose).
2. It must interfere with hormone systems (NAMs are already used here).
3. A link must be demonstrated between the hormonal disruption and the adverse effect.

Facts: What are NAMs?

NAMs include:

• In vitro tests – laboratory experiments carried out on cells or tissues outside a living organism.
• In silico models – computer-based models that predict the properties and effects of chemicals.
• Read-across – using data from one chemically similar substance to assess another.

LA JOLLA, CA—A new study, published recently in Nature Communications, offers the first-ever map of which parts of Chikungunya virus trigger the strongest response from the body’s T cells. 

With this map in hand, researchers are closer to developing Chikungunya vaccines or therapies that harness T cells to strike specific targets, or “epitopes,” to halt infection. The new study also offers important clues for understanding why many people experience chronic, severe joint pain for years after clearing the virus.

“Now we can see what T cells are seeing patients with chronic disease,” says LJI Assistant Professor Daniela Weiskopf, Ph.D., senior author of the new study.

This research comes as many mosquito-borne viruses, including Chikungunya, are moving into new areas of the globe.

“Historically, Chikungunya was considered an emerging virus. Now all of Latin America has been exposed,” says Weiskopf. “These mosquitoes are traveling further north, and we need to know what’s going on with this virus before it arrives in the United States.”

T cells jump into action

Chronic Chikungunya virus disease strikes between 30 to 60 percent of those infected—usually women—and causes severe joint pain. This debilitating joint pain can last for years following the initial viral infection. 

In a study out earlier this year, Weiskopf and her colleagues showed that these patients have a population of inflammatory CD4+ T cells that closely resembles the T cell signature of rheumatoid arthritis, an autoimmune disease.

“So many people, mostly women, have chronic disease following Chikungunya virus infection,” says Weiskopf. “This has an impact on the workforce and impacts the economy. And there’s no treatment.”

Weiskopf and her colleagues are working to understand why these CD4+ T cells linger and cause problems long after a person clears the virus. For this study, they investigated whether people who develop chronic disease produce T cells that naturally target a different set of epitopes on Chikungunya virus.

Would a different “flavor” of T cells be more likely to stay in the body after infection?

Weiskopf and her team used a “peptide pool” approach to assemble a map of key T cell epitopes on Chikungunya virus. The researchers broke up the virus into very small amino acid sequences, called peptides. Then they took T cells from people with chronic Chikungunya virus disease and exposed these cells to the pool of peptides.

By stimulating the T cells, the researchers discovered exactly which parts of the virus are most likely to be recognized by T cells. These “immunodominant” regions may prove to be good targets for future Chikungunya treatments.

Rimjhim Agarwal, a UC San Diego graduate student and member of the Weiskopf Lab, spearheaded experiments to learn more about these T cells. Agarwal received funding from The Tullie and Rickey Families SPARK Awards for Innovations in Immunology to take a closer look. 

For her project, funded through the generosity of the Rosemary Kraemer Raitt Foundation Trust, Agarwal  compared CD4+ T cells from people with chronic Chikungunya virus disease to people who cleared the virus quickly with no lasting symptoms.

Agarwal found that both patient groups had T cells that targeted the same viral epitopes. People who developed chronic disease did not recognize different proteins of the virus.

Now the question is—why do these T cells stick around to cause inflammation in some but not all people? Weiskopf and Agarwal are now looking at where Chikungunya virus might hide in the body to stimulate a long-term T cell response.

The LJI team also hopes to help other laboratories shed light on how to fight the virus. “Identifying the immunodominant T cell epitopes could seed new research into Chikungunya-specific T cell responses,” says Agarwal.

Additional authors of the study, “Identification of immunogenic and cross-reactive chikungunya virus epitopes for CD4+ T cells in chronic chikungunya disease,” included Calvin Ha, Fernanda H. Côrtes, Yeji Lee, Amparo Martínez-Pérez, Rosa Isela Gálvez, Izabella N. Castillo, Elizabeth J. Phillips, Simon A. Mallal, Angel Balmaseda, Eva Harris, Claudia M. Romero-Vivas, Lakshmanane Premkumar, Andrew K. Falconar, Alba Grifoni, and Alessandro Sette.

The research was supported by LJI’s Tullie and Rickey Families SPARK Awards for Innovations in Immunology, the National Institutes of Health (grants 75N93019C00065 and 75N93024C00056), La Jolla Institute for Immunology, Kyowa Kirin, Inc. (KKNA-Kyowa Kirin North America), and ARPA-H (grant 1AY1AX000039).

DOI: 10.1038/s41467-025-60862-7