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Ethics

Fewer animals in science

New legislation aims to reduce the number of animals used in teaching and research in Brazil

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Universities, businesses, and public and private research institutes have until August to replace animal testing with alternative methods for substances that may irritate or corrode the eyes or damage the human reproductive system, as well as in quality control processes for injectable products—the latter is commonly used to assess the safety of batches of vaccines and injectable drugs. The requirement is part of a resolution published in 2016 by Brazil’s National Council for the Control of Animal Experimentation (CONCEA), a collegiate institution under the Ministry of Science, Technology, and Innovations (MCTI), responsible for creating and enforcing rules on the ethical use of animals. The council issued a similar resolution in 2014 that came into force at the end of 2019, stating that animals must be replaced with one of 17 other testing methods when studying genetic mutations induced by chemical substances, acute toxicity, and skin sensitivity, among other tests.

These resolutions are legally binding, with punishments for violations including warnings, fines, and institutional suspensions. They affect businesses and public and private research institutions equally, especially activities that require approval by the Brazillian Health Regulatory Agency (ANVISA), such as the development of new cosmetics, pesticides, pharmaceutical products, drugs, and vaccines. “Reproductive toxicity tests are widely used by the pharmaceutical industry to check whether a new molecule has an effect on reproduction, while eye irritation tests are often conducted by pesticide manufacturers, among others,” explains biochemist Marize Valadares of the Federal University of Goiás (UFG).

Veterinarian Luciana Honorato, from the Federal University of Santa Catarina (UFSC), adds that each method has a specific application and is aimed at reducing or totally or partially replacing the number of animals used. “Some methods approved by CONCEA do not function as independent substitutes for the use of animals, but as the initial step in a layered testing strategy,” she explains. This is the case with the bovine cornea opacity/permeability test, an approved alternative to the use of rabbits for eye irritation testing. The method uses corneas from freshly slaughtered cattle, which would otherwise be discarded. “The substance under assessment is applied to the surface of the cornea to see whether it causes any damage. If the results are inconclusive, animals may be used.”

The use of animal subjects, however, remains indispensable in many studies (see Pesquisa FAPESP issue no. 144), such as in the field of pharmacokinetics, which evaluates the path that a molecule takes in an organism after its administration. This type of analysis is essential to defining the toxicity parameters related to the safety and efficacy of drug or vaccine candidates, such as those developed against COVID-19. “The tests evaluate how and at what speed a substance is absorbed and metabolized by the organism, as well as whether it accumulates in a certain organ and the potential impacts of this process,” explains Valadares. Because animal subjects offer the chance to study complex interactions in equally complex and interdependent systems, it is difficult to create a robust and internationally standardized method capable of replacing them. One technique that could help overcome this problem is microphysiological systems, formed by human organoids on microfluidic devices: human cells from different organs, interconnected in a closed system on a microchip. “With these systems, we can analyze the systemic impacts of substances on different organs,” explains the UFG researcher.

The CONCEA regulations are a result of efforts initiated in Brazil more than two decades ago by public health expert and former federal deputy Sérgio Arouca (1941–2003). In 1995, he presented a bill proposing mechanisms to ensure the ethical and reasonable use of animals in teaching and research activities in Brazil, as already existed in other countries. The bill remained in Congress for 13 years until it was approved in 2008. Law 11.794, known as the Arouca Law, governs the use of animals in Brazil and establishes the framework for establishing a network of laboratories devoted to finding alternative testing techniques, such as the Brazilian Center for the Validation of Alternative Methods (BRACVAM) in Rio de Janeiro. The law also instituted CONCEA, whose resolutions were all drafted with support from BRACVAM. “Brazil has come a long way in the last decade in terms of legislation on the use of animals in science,” says Valadares.

The Arouca Law was approved after a concerted effort by the academic community, which was unhappy about state and municipal laws prohibiting the use of animals in scientific research without taking into account the damage it could do to Brazilian science. One of the groups leading the push for the law was the Federation of Experimental Biology Societies (FESBE), at the time headed by neuroscientist Luiz Eugênio Mello, current scientific director of FAPESP. “I met frequently with politicians in Brasília to make them aware of the importance of the matter and to build a consensus among party leaders on the text of the bill,” recalls Mello. He believes that the efforts of the scientific community contributed to the law being approved in the form that the researchers had hoped for.

Physician and biophysicist Marcelo Morales, former coordinator of CONCEA, believes one important aspect of the legislation was that it defined the classes of animals to which it applied, namely vertebrates in general, such as fish, amphibians, reptiles, birds, and mammals. “Existing municipal laws were vague and created uncertainty, since they made it illegal to use any animals at all in certain research activities,” says Morales, now secretary of Scientific Education and Research at MCTI. “Studies using insects and worms, often used in genetic research, for example, were compromised. The Arouca Law was important because it supersedes these municipal laws, providing legal and ethical security for the reasonable use of animals.”

Thirteen years after it was approved, experts agree that one of the key advances fostered by the Arouca Law was the formation of Ethics Committees on the Use of Animals (CEUAs) at companies and public and private teaching and research institutions that test on animals. As a result, any researcher wishing to use animal subjects must submit a proposal to their institution’s ethics committee before starting their research. “They assess the most suitable alternative methods that could be used for the proposed experiment,” says biologist Octavio Presgrave, head of BRACVAM.

Just over 190 million animals were used for scientific purposes worldwide in 2015

According to biomedical researcher Monica Andersen of the Federal University of São Paulo (UNIFESP), CEUAs are composed of biologists, veterinarians, and professors and researchers from specific fields, in addition to representatives of Brazilian animal protection societies. “They analyze research proposals that involve the use of animals based on the three Rs,” explains the researcher, referring to the approach conceived in 1959 by zoologist William Russell and microbiologist Rex Burch, both from the UK, which seeks to Replace, Reduce, and Refine the use of animals in research whenever possible.

Biomedic Debora Fior Chadi, advisor to the dean of research at the University of São Paulo (USP), believes CEUAs have helped to improve Brazilian science. “Now, researchers have to describe in detail how they plan to use animals in their study and justify the need to do so, given the alternative methods available,” she says. “The aim is to verify the real need for using animals, the most suitable species to achieve the proposed objectives, and the number required.” The committees also require scientists to create animal welfare protocols, to ensure that they are kept in adequate conditions and to minimize their suffering.

Thanks to CEUAs, the CONCEA resolutions tend to have a small impact on universities, since their researchers are already obliged to use alternative methods when possible and comparably effective. “Research projects can’t go ahead without approval from the ethics committee. All researchers working with animals, even the few that are familiar with the regulations, need to update their methodologies to reduce or replace animal subjects in their experiments and adapt to the latest requirements,” says Liliana Scorzoni, from São Paulo State University (UNESP), São José dos Campos campus.

Some institutions, such as UFSC, also evaluate the harm-benefit relationship of a study. “We ask scientists to tell us how their research will benefit science and society in general, so that we can assess whether the harm that will be caused to animals is justified,” explains Luciana Honorato, president of the Santa Catarina university’s CEUA. “This kind of evaluation isn’t always easy, because many proposals are for basic research, which involve the pursuit of scientific understanding without anticipating any particular benefit.” The measures implemented at UFSC have resulted in a 40% reduction in the number of rats and mice bred by the institution’s labs between 2014 and 2018. “Interestingly, the number of research projects conducted remains unchanged,” she says. The same phenomenon occurred at the Butantan Institute, where the reduction was 30%.

It is estimated that just over 190 million animals were used for scientific purposes worldwide in 2015—there are no consolidated estimates for Brazil specifically, according to Presgrave. The global number represents an increase over 2005, when about 115 million animals were used in research. China, Japan, and the USA use the most animals in science. The trend is reversed in Europe, where the number of animals used for scientific purposes was 9.59 million in 2015, but fell to 9.39 million in 2017. The data are from a 2020 report by the European Commission on the state of animal research in EU member countries since the introduction of Directive 2010/63/EU. In force since 2013, the legislation governs how animals are managed in laboratory environments and promotes tests that minimize the number of animals used and the extent of their suffering. In the USA, the government has been putting pressure on federal funding agencies, such as the National Institutes of Health (NIH), to explore alternatives to animals in research. This extends to the Food and Drug Agency (FDA), which will present a plan within the next five years for reducing the number of monkeys used in research and detailing how they are retired.

In Brazil, the requirements go beyond research practices, also including teaching activities. “Practically all universities and educational institutes have abolished the use of animals in practical undergraduate classes,” says veterinarian Ekaterina Botovchenco Rivera, coordinator of CONCEA. “They can only be used in exceptional cases, such as anesthesiology courses in veterinary medicine. Even so, the lesson plan has to be approved by the CEUA, which will evaluate strategies for reducing the number of animals used.” This is important, she says, because “it has helped to instill a culture change in new researchers.”

The 2008 Brazilian law also stimulated the formation of research groups dedicated to finding alternative methods to the use of animal subjects.  The cosmetics industry has made major progress in this regard, perhaps more than any other, in order to adapt to the requirements imposed by ANVISA—the agency uses CONCEA rules to assess the test results submitted by companies applying for product approval—and in an attempt to improve its image faced with public pressure to end animal testing. “The vast majority of cosmetic companies in Brazil already comply with the CONCEA regulation and have long used other methods to test their products,” says Valadares.

At Butantan, one of the institutions that conducts the most quality control tests for injectable products in the country, a team led by chemist Wagner Quintilio and biochemist Terezinha de Jesus Andreoli Pinto, from USP’s School of Pharmaceutical Science, is working on an assay to detect endotoxins in batches of antiophidic serum, a process which currently uses rabbits. In 2020, UNESP’s School of Pharmaceutical Science, Araraquara campus, inaugurated the Laboratory for the Development and Validation of Alternative Methods for Assessing the Safety and Efficacy of Bioproducts (LAMABIO), led by mycologists Ana Marisa Fusco Almeida and Maria José Giannini. “Since 2004, we have been investing in methodologies for assessing natural products,” says Giannini. “In 2010, we started working on standardizing new methods for assessing the toxicity of pharmaceutical products.”

In the long term, if new techniques and technologies are developed, CONCEA will publish resolutions with more alternative methods. “I see it as a path of no return,” says Valadares.

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