Animal testing can be phased out thanks to new in vitro assays and simulations

Specialists discuss methods to replace the use of animals in laboratory tests, minimize pain and distress, and assure better results.

By Diego Freire

Agência FAPESP – Animal testing studies the effects of chemical substances on organisms. A search for alternatives to this type of testing, which has been driven by public opinion and by scientific and technological developments in toxicology, is already presenting results: Computer simulations of molecular interactions and new technologies for in vitro assays minimize the use of laboratory animals and point to a future without in vivo testing.

Efforts in this direction that have been made by institutions in Brazil and in other countries were presented at the workshop “Challenges and perspectives in research on alternatives to animal testing”, which was held at FAPESP’s headquarters in São Paulo on March 31. According to the specialists who took part, alternatives to animal testing must be developed and implemented to not only reduce the use of laboratory animals but also to reduce risks to people, as the results of in vivo experiments are often not sufficiently reliable given the differences between animals and humans.

“Animal testing has been performed for decades, but the results have never adequately reflected the effects of the substances tested when applied to the human organism,” said Thomas Hartung, director of the Center for Alternatives to Animal Testing at Johns Hopkins Bloomberg School of Public Health, Maryland, USA. “Progress is required in both the ethical and scientific dimensions. We need new technologies and new approaches to toxicology,” Hartung told Agência FAPESP.

In his workshop presentation, Hartung explained how in 1959 two British scientists, William Russell and Rex Burch, introduced the “three Rs” for humane alternative methods of experimentation: Reducing animal use in research, Refining the management of tests so that laboratory animals experience less pain and distress, and Replacing animals by developing experimental systems that reproduce physiological conditions without live models.

“The idea is to use new methods that enhance the reliability of experiments instead of impairing the quality of our research,” Hartung said. “For example, refining the procedures we use in order to minimize the pain and distress experienced by laboratory animals makes experiments more reliable by controlling the animals’ psychological alterations, which increase the experimental variability of the results.”

Maria José Soares Mendes Giannini, Pro-Rector for Research at São Paulo State University (UNESP) and coordinator of the workshop, stressed that the search for alternatives to animal testing is a prerequisite for the advancement of science.

“In addition to the ethical reasons that lie at the heart of the search for alternatives to in vivo tests and all of the pressure from public opinion to reduce and avoid the suffering of animals, the issue of scientific progress is equally urgent,” Giannini said. “Animal models are proven to be limited in that they don’t enable us to obtain answers of sufficient quality.” Giannini is also a member of FAPESP’s Board of Trustees.

“If highly advanced new medications such as immunobiological drugs are tested in an animal model, the responses that they produce cannot be compared with the ways in which we humans respond. Toxicity testing must keep pace with these developments and move beyond animal models,” Giannini said.

Organs-on-a-chip

During the workshop, Wagner Quintilio, a researcher at the Butantan Institute in São Paulo, Brazil, presented results of studies that used in vitro methods instead of laboratory animals to develop and control the quality of the immunobiological drugs that are produced by the institution.

For Chantra Eskes, President of the European Society of Toxicology In Vitro (ESTIV), mankind is fast approaching the point where animals will no longer be used in laboratory tests, above all thanks to the evolution of in vitro testing.

“Humanity is experiencing a great scientific revolution driven by the possibility of restoring cells to their initial state and using them to make tissues and organs for applications that test toxic substances on the cells of the patients themselves. Growing knowledge of the human genome, transcriptome and proteome is paving the way for the replacement of in vivo testing by more advanced in vitro testing,” Eskes said.

Silvya Maria-Engler, a professor at the University of São Paulo (USP), presented promising results by Brazilian researchers in the development of a three-dimensional model of human epidermis.

Based on primary human skin cells, the model has been used in place of animal testing in studies of cutaneous irritancy and corrosion, to evaluate the effectiveness of candidate molecules for anti-melanoma drugs, and in studies of skin diseases, among other types of research.

“This revolution, combined with new technologies for in vitro testing, overcomes the deficiencies of animal models by much more accurately mimicking the human organism,” said Eduardo Pagani, Drug Development Manager of the National Bioscience Laboratory (LNBio) in Brazil.

“In addition to cell cultures, we can grow tissues, which are groups of organized cells that can be cultivated in more complex dimensions, as part of a search for a closer correspondence with the normal morphology of human tissue,” Pagani explained, referring to organotypic culture, which combines different types of cells. In his presentation, Pagani described the concept of organs-on-a-chip. This technology, which is in the early stages of development by a few foreign institutions, uses stem cells to grow human organs that are integrated onto microchips, which are designed to mimic the functioning of live organs such as the heart or lungs. According to Pagani, LNBio is working with the National Network for Alternative Methods (RENAMA) to find partners that have the technology that will enable joint development of toxicity tests using organs-on-a-chip.

In silico testing

Another important front for the development of alternatives to animal testing is “in silico testing”, which uses computational tools to simulate the performance of products and devices.

“Computer-based testing involves searching a database of drugs that have been tested to look for similarities with the new drug in terms of toxicity, pharmacokinetic absorption, and even therapeutic effectiveness for specified conditions. The methodology aims basically to produce theoretical predictions by comparing a new molecule with those already tested,” Pagani said.

“At this time, in silico testing doesn’t eliminate the need for experimentation with animals, but it does reduce the number of substances that have to be tested in vivo.”

Raymond Tice, until recently the head of biomolecular screening at the US National Institute of Environmental Health Sciences (NEIHS), told the workshop about Toxicology in the 21st Century (Tox21), a federal program aimed at developing better toxicity assessment methods. The Tox21 high-throughput screening robotic system can be used to test whether a large number of chemical compounds have adverse effects on human health via specific cellular signaling pathways. The initiative represents a new vision of risk assessment that differs from traditional toxicology in characterizing toxicity and disease pathways.

Cláudia Vianna Maurer Morelli, a professor at the University of Campinas (UNICAMP) in São Paulo, and Giannini delivered presentations on alternatives to testing in mammals, including models that use the zebrafish (Danio rerio) and the greater wax moth (Galleria mellonella).

Regulation

The international scientific community has adopted several alternative methods that reduce and replace the use of animals in toxicity testing. Parameters vary between countries. In 2003, for example, the European Union banned the sale of cosmetics if any of their ingredients or if the end product had been tested in animals, unless no alternative methods had been validated.

“Brazil is working hard to keep up with what’s happening in the US and Europe,” Giannini said. “We’re making important progress but we must accelerate and advance more.”

Last year, at the request of Brazil’s Animal Experimentation Control Council (CONCEA), the National Public Health Surveillance Agency (ANVISA) licensed 17 internationally validated alternative methods to animal testing.

“The methods involve in vitro tests to measure potential skin and eye irritancy and corrosion, besides other interactions. Laboratories in Brazil have until 2019 to implement the 17 validated methods that replace animals or reduce their use in toxicity testing,” said José Mauro Granjeiro, who chairs CONCEA.

In March, CONCEA convened a public consultation to seek suggestions capable of improving the chapter of the Brazilian guidelines on animal use in teaching and research that addresses the treatment of captive non-human primates. This chapter contains the information required to assure appropriate conditions for animal husbandry, maintenance and use, focusing on the welfare of the animals as well as the quality of the research and teaching methods concerned.

Another speaker at the workshop was Joel Majerowicz, representing ANVISA’s Department of Institutional Relations, who explained how a regulator operates and expressed an interest in policy measures designed to adopt new approaches to toxicity assessment.

The presentations that were delivered by the researchers at the workshop can be downloaded from www.fapesp.br/9310.