EU-ToxRisk: How to avoid animal testing

Animal-free toxicity testing and risk assessment for the 21st century

Paradigm shift: Away from animal testing and towards a deeper understanding of how chemical substances work

Project EU-ToxRisk – a HORIZON project of the European Union

The number of test animals in Germany has been largely stable for years: tests were conducted in 2,825,066 animals in 2018. Animal studies are also necessary for examining the safety and toxicity of chemical substances. As the head of the Department of In-silico Toxicology at the Fraunhofer ITEM, Dr. Sylvia Escher is looking for alternatives to animal testing.

“At our institute, we work with several groups on new concepts for chemical risk assessment,” the chemist explains. She mentions the EU-ToxRisk project as one example. Cultures of human cells and organ sections are meant to offer alternatives to animal testing. A total of 39 partners from 13 countries are working together in the EU-ToxRisk project, which has been ongoing since 2016 and has a budget of more than 30 million euros. In addition to universities, research institutes and companies, regulatory authorities are also taking part in the project – an important factor in the success of the undertaking. Because of animal testing can only truly be replaced if the national and EU authorities approve the newly developed processes for determining toxicity.

Maus Tierversuche vermeiden EU-ToxRisk
© H. & HJ. Koch
Mice are the most frequently used animals in research. Since the decoding of the mouse genome in 2002, the scientific community has known that mouse genes are 98 percent the same as those of humans.

The objective is not only a more conservative alternative, but also one that is better.

The classic procedure is to expose animals to the substances to be tested. And the researchers observe: are there any inflammatory reactions? Are any organs damaged? Is there damage due to continuous long-term exposure to lower concentrations of a substance, for example in the amounts that could be breathed in on a daily basis? However, which mechanisms lead to the adverse changes observed usually remains unclear.

“As part of EU-ToxRisk, we investigate the cascade that a substance causes in the body,” Escher points out the advantages. It begins with the first molecular interactions of the substance (for instance, with receptors on the cell), transitions to a reaction of the cells and then leads to the effects on organs, culminating in changes in the organism as a whole.

Multiple working groups from Fraunhofer ITEM are involved in three of the nine case studies being conducted as part of EU-ToxRisk. Dr. Tanja Hansen, head of the Working Group on In-vitro Test Systems, is currently investigating the toxicology of volatile compounds using diketones as an example. Diacetyl – a chemical compound that is a natural component of butter and whose industrially produced version serves as butter flavor, for example for popcorn – is the best-known representative of this substance group.

Simulations with human tissue

What happens when people breathe in diacetyl? Can it be harmful to the lungs? To answer these questions, Sylvia Escher and Tanja Hansen use an apparatus that was developed at Fraunhofer ITEM: the P.R.I.T.® ExpoCube®, which enables them to simulate how volatile substances affect cells and tissue.

Human bronchial epithelial cells are cultivated on membranes at the air-liquid interface to simulate the situation in the lung. Using the P.R.I.T.® ExpoCube®, gaseous diacetyl is passed over the surface of the cells. Biochemical methods are then used to examine the effect on the cells. Thanks to comprehensive analyses of gene expression, the research team is able to recognize which genes the cells have activated and/or deactivated. They can then use this data to determine which signal pathways were activated within the cell. These could be signal pathways that lead to the production of messenger substances that cause inflammation, for instance.

The investigation is taken to the organ level in the next step. To this end, the researchers use living tissue sections cultured from human lungs that also have many of the lungs’ functions. As with the cell cultures, the lung sections are exposed to diacetyl in the P.R.I.T.® ExpoCube® and then intensely scrutinized.

To simulate the behaviors of diacetyl in the body, the project partners make use of complex calculation models, which known as “in silico methods.” These computer-aided models reproduce to a high degree how an inhaled substance is absorbed, distributed and excreted in an organism. “The in vitro and in silico data result in a precise picture of how diacetyl damages the lungs,” Escher reports. “They are consistent with the in vivo data from the tests carried out in animals.”

Using data from similar substances

The first step in avoiding animal testing with these alternative methods is the read-across approach. If you want to have a new chemical approved in accordance with this method, you seek out similar substances for which toxicological data from animal testing already exists. This data is then transferred to the new “sister” chemical as part of the read across. “In principle, this approach is already possible today. Practically, however, it has been difficult to demonstrate that two chemicals are so similar that they truly have the same toxicity,” Escher emphasizes. “Which is why the read-across approaches have only seldom been accepted by the regulatory authorities thus far.”

In the case studies, the project teams investigated groups of closely related substances and gathered comprehensive in vitro and in silico data to increase the level of acceptance. With these investigations, they were able to demonstrate that the methods are ideally suited to determine the toxicity of structurally related materials. In a detailed study, the cooperating partners presented how a read-across approach is made possible using data acquired without animal testing. “We sought out a close dialogue with the regulatory authorities from early on, continuously adapting the concept as we went along. In doing so, we have taken a major step forward towards acceptance,” says Escher.

The EU-ToxRisk project partners already have the next objective in their sights: they hope to establish toxicity testing without the use of animals even for substances for which the read-across approach is not possible. The project timeline runs through the end of 2021.


Text by Christine Broll, published in the Fraunhofer magazine 03/2020.


Sylvia Escher

Contact Press / Media

Dr. Sylvia Escher

Head of Department of In-silico Toxicology

Phone +49 511 5350-330