Eawag: Test with fish cells replaces animal experiments

A large number of chemicals are used in everyday products, in agriculture or in industry. Many of them also end up in the environment at some point. For these substances to be approved for the market, manufacturers must be able to prove in advance that they are harmless to humans and nature. This is done in toxicity tests in which living creatures are exposed to the effects of the chemicals. Thousands of fish die every year because they are used to test the effects on aquatic vertebrates.

Thanks to a test method developed by the Eawag Water Research Institute in recent years, this should soon be a thing of the past. This test, which is based on isolated rainbow trout gill cells, has now been approved by the Organization for Economic Co-operation and Development (OECD) as the latest guideline in the field of environmental toxicology. This paves the way for animal-free approval procedures.

Great interest of the economy

The so-called fish cell line test is the world's first alternative to live fish testing. The new OECD guideline allows companies to use the fish cell line test for product development and as part of dossiers for chemical approval. "There is great interest on the part of industry in non-animal tests," confirms Prof. Kristin Schirmer, Head of Department at Eawag, who, together with Melanie Fischer (also at Eawag), has taken the lead in this pioneering work. This is because, on the one hand, the demands on environmental risk assessment are growing, and, on the other, the number of new chemicals and products that need to be tested is constantly increasing. Moreover, fish cell line testing also conserves resources: miniaturization through the use of the cells makes it possible to save chemicals, water and time.

Kristin Schirmer assumes that the regulatory authorities, such as the ECHA in Helsinki or the US EPA in the USA, will increasingly accept the fish cell line test as an equivalent substitute to the regular fish test: "The OECD recommendation was the last important step from our side, after our method was already certified by ISO two years ago. This should mean that nothing more stands in the way of an animal-free approval procedure."

In this way, Eawag contributes to the further development of toxicology and makes a significant contribution to the reduction of animal testing. In 2019, ecotoxicological tests were carried out on almost 8,000 fish in Switzerland alone.

The first idea

This marks the end of a long and intensive period that required perseverance and a supportive environment. Schirmer already had the idea for this test method during her doctoral thesis in the mid-1990s: At the University of Waterloo (CAN), she worked at that time with exactly the same cell line on which the method is still based today. The cell line goes back to Schirmer's doctoral supervisor Niels C. Bols, who isolated the cells from the gills of rainbow trout for the first time and cultivated them in such a way that they can be multiplied as often as desired.

"We therefore chose gill cells because it is the gills that first come into contact with a chemical due to their large surface area in the fish," explains Schirmer. If the chemical damages the gill cells, it means that vital functions, such as oxygenation, no longer function for the fish. "So by observing how the gill cells are damaged by a chemical, we can predict how that chemical would affect a living fish," Schirmer says.

The acid test

The award of a project proposal, which Schirmer received from the European Chemical Industry Council (CEFIC) in 2006, marked the actual starting signal for the development of the now validated fish cell line test. Schirmer continued her research first as head of department at the Helmholtz Centre for Environmental Research in Leipzig and, from 2008, as head of department at Eawag. In 2009, engineer and laboratory technician Melanie Fischer joined the team.

"In a first step, we developed and optimized our method and compared the results with the regular tests on fish," says Schirmer. A study published in 2013 was thus able to prove for the first time that the new fish cell line test arrives at the same toxicity values for over 30 chemicals as the fish test. "After that, we had to show that the method also works in other laboratories," Schirmer says. Six laboratories from industry and academia participated in the international ring study conducted for this purpose in 2013-2014. The results they obtained using the Eawag method proved to be repeatable and comparable.

At destination

This study data laid the foundation for ISO certification in 2019 and the OECD guideline in 2021. "Certification by internationally supported organizations is very important, as it is the only way to ensure that the tests are carried out in a standardized manner worldwide and that the results are therefore also recognized," says Schirmer. From the submission to the OECD to the validation that has now taken place, a record short time of just two years elapsed, during which the method was once again revised in an elaborate process to harmonize the suggestions and opinions of experts from many different representative countries and organizations. The team was supported by the country representatives of Switzerland (Petra Kunz, Federal Office for the Environment FOEN) and Norway (Sjur Andersen, Norwegian Federal Environmental Agency and Adam Lillicrap, NIVA). Eszter Simon (also FOEN) supported the work as an expert.

Schirmer attributes the fact that the guideline could be adopted in such a short time to the interest and great support of all those involved: "It was an active discussion with the OECD countries, with industry, NGOs and the regulatory authorities." This is also confirmed by Leon van der Wal of the OECD: "The rapid adoption of this first in vitro method for ecotoxicity testing reflects the commitment of OECD countries to reducing animal testing. Moreover, this is due to the well-organized and transparent way in which the lead countries communicated, as well as the hard work of the method developers and the OECD experts who reviewed this method."

Looking back, Kristin Schirmer states, "Our learning curve was very steep. But the investment of time and energy was worth it: this success for non-animal alternatives in ecotoxicology is great and something that will stay."

Source: Eawag News

The fish cell line test briefly explained: www.eawag.ch/de/news-agenda/news-plattform/news/eawag-test-mit-fischzellen-ersetzt-tierversuche/

Research awarded prize

Eawag's work on non-animal test methods is of great importance. This is also recognized by the 3R Competence Center Switzerland (3RCC) so: The association aims to replace, reduce and improve animal experiments and presented Kristin Schirmer and Melanie Fischer with the 3Rs Award in 2019 for their research. The researchers were also awarded around 251,000 Swiss francs from the 3RCC in 2019. With this grant, they are working on replacing the fetal calf serum that is currently still required for cultivating the cells with a serum-free medium.

aQuaTox-Solutions

Due to the growing interest of companies, wastewater treatment plant operators and environmental agencies in using the test as a service, in 2017 Eawag spin-off aQuaTox-Solutions. It is the world's first provider of this and other fish cell line-based test methods. With the certification by the OECD, and the accompanying confirmation of the high robustness, transferability and comparability, the interest in this test method is expected to increase further.