{"id":152760,"date":"2014-06-17T18:32:05","date_gmt":"2014-06-17T21:32:05","guid":{"rendered":"http:\/\/revistapesquisa.fapesp.br\/?p=152760"},"modified":"2014-07-17T18:42:18","modified_gmt":"2014-07-17T21:42:18","slug":"alternative-methods","status":"publish","type":"post","link":"https:\/\/revistapesquisa.fapesp.br\/en\/alternative-methods\/","title":{"rendered":"Alternative methods"},"content":{"rendered":"
\"USP

EDUARDO CESAR<\/span>USP researchers are making artificial skin with the goal of developing a commercial kit for testing cosmeticsEDUARDO CESAR<\/span><\/p><\/div>\n

In Brazil, a link between public laboratories, research groups and government organizations to curtail or eliminate the use of animals for testing product safety and effectiveness is gaining strength.\u00a0 The effort exploded in 2012 when the federal government established the National Network for Alternative Methods (Renama) and when the National Council for Scientific and Technological Development (CNPq) issued a request for proposals for 10 Renama research projects.\u00a0 All of the projects are under way and they focus on different things, such as producing artificial skin kits to test cosmetics for sensitivity, studies of larvae capable of replacing mammals in toxicity tests or reducing the number of rodents used to control vaccine quality.\u00a0 Three laboratories are part of the central core at Renama.\u00a0 One of them is the Brazilian Biosciences National Laboratory (LNBio) in Campinas.\u00a0 The others are in Rio de Janeiro: the National Institute for Quality Control in Health (INCQS), part of the Oswaldo Cruz Foundation; and the\u00a0National Institute of Metrology, Quality, and Technology (Inmetro).<\/p>\n

In March, this organization, still in the consolidation phase, was given the challenge of meeting an ambitious goal: provide support to phase out the use test animals over the next five years, provided a validated alternative exists.\u00a0 For new methods that have yet to be validated, this process will involve the Brazilian Center for the Validation of Alternative Methods (BraCVAM) and all of Renama.\u00a0 The National Council for the Control of Animal Experimentation (Concea), a collegial institution that is part of the Ministry of Science, Technology and Innovation (MCTI), made the decision to replace test animals.\u00a0 Since 2009 it has been in charge of developing animal experimentation standards in Brazil and replacing animals for scientific and educational proposals whenever alternative resources exist.\u00a0 In May, BraCVAM made its first recommendation to Concea on alternative methods that had already been validated and that are internationally accepted.\u00a0 There are 17 techniques that deal with skin sensitivity, the potential for eye irritation and deterioration and toxicity.\u00a0 \u201cWith this resolution, Brazil will be able to actually adopt alternative methods for testing pesticides, cosmetics and drugs,\u201d says the coordinator of Concea, Jos\u00e9 Mauro Granjeiro.<\/p>\n

The greatest potential for replacing animals with alternative methods is not in scientific research in the academic arena, but in tests that the regulatory agencies require to ensure that products are safe and effective.\u00a0 \u201cExperiments with animals that have been conducted to prove scientific hypotheses are designed independently by researchers.\u00a0 Each one has a different question and designs a specific set of experiments to answer the question.\u00a0 Therefore, standardizing the experiments is much more difficult,\u201d explains Eduardo Pagani, researcher and manager of development of pharmaceuticals at the LNBio.\u00a0 \u201cThe tests that agencies everywhere in the world require for cosmetics and other products are always performed in accordance with standardized methods.\u00a0 In these tests there is more room for proposing alternatives that do not use animals,\u201d he observes.\u00a0 The requirement to perform in vivo<\/i> testing to register drugs and cosmetics began in the 1960s after the well-known accident with thalidomide.\u00a0 The drug was sold all over the world as a treatment to control nausea during pregnancy.\u00a0 Thousands of mothers that used the drug gave birth to deformed children.\u00a0 The movement for replacing animal models with alternative methods ramped up in 2003 when Europe proposed banning the use of animals in cosmetic testing, and then it took two decades to implement it.<\/p>\n

\"Production

EDUARDO CESAR<\/span>Production of artificial skin by the group of USP professor Silvya Stuchi-Maria EnglerEDUARDO CESAR<\/span><\/p><\/div>\n

The projects for alternative methods that the MCTI supported in 2012 were divided into two segments.\u00a0 In one segment, the goal was to identify groups that were already working with alternative methods and to support studies they performed.\u00a0 Nine projects of groups in the states of S\u00e3o Paulo, Bahia, Goi\u00e1s, Rio de Janeiro, Santa Catarina and Rio Grande do Sul were selected.\u00a0 The second segment had a specific focus: develop the capability in Brazil to mass produce artificial skin kits used by the cosmetic industry to test the safety of their imported products that became problematic in Brazil.\u00a0 It turns out that the kits with live cells became unusable in just a few days and long waits in customs often made it impractical to purchase them.\u00a0 As a result, companies performed these tests abroad.<\/p>\n

The project that was considered came from a group at the USP School of Pharmaceutical Sciences, led by Silvya Stuchi-Maria Engler, who began producing artificial skin around 2005 with FAPESP support (see Pesquisa FAPESP<\/i> Issue No. 166<\/a>).\u00a0 Produced from cells taken from donors, the skin reproduces the same human biological tissue and can be used to assess the toxicity and effectiveness of new compounds for pharmaceuticals and cosmetics.\u00a0 At first, the purpose of research on artificial skin was to support the other line of investigation in which Stuchi is involved: the study of molecules capable of controlling melanoma, a very aggressive skin tumor.\u00a0 \u201cWe quickly saw that the skin could be useful to businesses,\u201d she says.\u00a0 \u201cThe kits are an alternative for testing cosmetics, but it is good to point out that the use of animals is still vital, for example, in tests for developing drugs,\u201d Stuchi observes.<\/p>\n

The Butantan Institute, whose role is to develop and produce serums and vaccines, is cutting down on the number of animals such as mice and guinea pigs that are used for quality control.\u00a0 This effort has already brought about progress in many areas, including a more than 60% reduction in the use of mice for testing the quality of the Hepatitis B recombinant vaccine by developing an immunosorbent test with equivalent functions.\u00a0 Through its work, the Institute was able to submit a project for the Renama request for proposals that involved many alternative methods to control the quality of vaccines and serums.\u00a0 In one of the areas of research, the goal is to lower the number of animals in the tests in lots of diphtheria and tetanus vaccines by using in vitro<\/i> testing to detect immunogenic activity.\u00a0 In another, the goal is to replace testing using guinea pigs with trials in cells to control the diphtheria anatoxin, a diphtheria toxin that maintains immunogenic activity, although it is no longer toxic.\u00a0 A third focus is to adapt a kit to vaccines that the Institute produces to replace the use of rabbits in tests for pyrogens, \u00a0contaminants that cause fever and that can be derived from microorganisms or protein agglomerations.\u00a0 The fourth area is to attempt to lessen the use of mice in serology for the whooping cough vaccine; the idea is to use for the disease the same animals that are used to dispense antibodies that fight diphtheria and tetanus.\u00a0 The decrease in the number is significant: down from 170 animals per lot of vaccine to just six guinea pigs.<\/p>\n

Finally, Butantan has already seen success with a technique that has the potential to replace the use of mice with immunoenzymatic testing in one stage of production of the rabies vaccine.\u00a0 \u201cThere\u2019s no turning back once you cut down on the number and use of animals,\u201d says chemist Wagner Quintilio, the researcher at Butantan who is in charge of the project.\u00a0 \u201cThere is the pressure from society and research ethics committees that will not accept excessive animal use.\u00a0 There is economic pressure as well.\u00a0 It is expensive to raise animals in adequate conditions and they take up a lot of space,\u201d he says.<\/p>\n

\"Artificial

EDUARDO CESAR<\/span>Artificial skinEDUARDO CESAR<\/span><\/p><\/div>\n

The project of the group led by mycologist Maria Jos\u00e9 Giannini, professor at the Araraquara School of Pharmaceutical Sciences at the S\u00e3o Paulo State University (Unesp), plans to establish the Center for the Development and Validation of Alternative Methods (Cedevam) to develop and test techniques that curb the use of animals.\u00a0 Giannini is the supervisor for post-doctorate student Liliana Scorzoni, who is in charge of research on models that can replace mammals with other organisms in microbe virulence and drug effectiveness tests.\u00a0 The most advanced front is the Galleria mellonella<\/i>, a species of lepidopteron insect whose larva is useful for verifying the activity of certain substances.\u00a0 \u201cIt is easy to handle and can minimize the use of animals,\u201d says Giannini, who is also a member of the FAPESP Board of Trustees.\u00a0 \u201cThe larva has cells similar to the cells in the immune system.\u00a0 When a toxic substance is injected into it, it reacts and darkens,\u201d she says.\u00a0 It is expected that the Galleria<\/i> will replace other animals such as rats and mice for at least some stage of toxicity and virulence testing.<\/p>\n

Another alternative model that the Unsesp group is eying is the C. elegans<\/i>, a nematode that is one millimeter long and is susceptible to infection from pathogenic bacteria and fungi.\u00a0 \u201cIt has an immune system for recognizing and eliminating pathogens that is very similar to the system in vertebrae.\u00a0 In addition, its genome has been completely sequenced, while the genome of the Galleria <\/i>has not,<\/i>\u201d Giannini says.\u00a0 Both models are being tested to evaluate the virulence of Paracoccidioides <\/i>fungi, which are endemic in Latin America.\u00a0 Other models such as zebrafish will be tested.\u00a0 In 2010, the Office of the Dean for Research at Unesp, which Giannini heads, organized an international forum in S\u00e3o Paulo to discuss alternatives to using animals for toxicity testing.\u00a0 The forum brought together authorities such as Thomas Hartung, Director of the Center for Alternatives to Animal Testing at Johns Hopkins University.\u00a0 \u201cThe search for alternative models is also important for developing more efficient methods.\u00a0 Animal models have limitations and sometimes their guarantees of safety are insufficient, as illustrated by drugs that are approved but are eventually withdrawn from the market,\u201d Giannini says.<\/p>\n

The decision by Concea to push for the recognition of validated alternative methods was a response to a petition from\u00a0Humane Society International,<\/i> a nongovernmental organization that demanded banning animal testing for cosmetics.\u00a0 In S\u00e3o Paulo State, the use of animals for testing cosmetics is banned by a state law that was enacted in January 2014.\u00a0 Concea, which did not accept the petition, realized that accelerating the implementation of alternative techniques would be more effective in limiting the use of animals for testing cosmetics than just an exclusive ban on such use, since animals are practically no longer used for this purpose.\u00a0 \u201cA total ban would place public safety at risk,\u201d says physician and biophysicist Marcelo Morales, professor at the Federal University of Rio de Janeiro (UFRJ) and former coordinator of Concea.\u00a0 \u201cA total ban could make it impossible to develop cosmetics with new ingredients or molecules discovered in our biodiversity that contain unknown contaminants,\u201d he says.\u00a0 Luiz Henrique do Canto Pereira, general coordinator of biotechnology and health at the MCTI, states that the ban could interfere with the MCTI strategy of replacing, reducing and improving the use of animals in tests whenever possible.\u00a0 \u201cThe campaign for the ban is destroying the effort we have been making since 2011, when we began to design this program to organize a structured network in Brazil that would be capable of validating and more broadly disseminating alternative methods, and not just for cosmetics, but for pharmaceuticals and pesticides as well,\u201d he says.\u00a0 \u201cEven in Europe there are safeguards that allow tests to be performed if the health of the public is at risk.\u201d<\/p>\n

\"Zebrafish,

EDUARDO CESAR<\/span>Zebrafish<\/em>, whose larvae can replace animals in toxicity tests: alternative modelsEDUARDO CESAR<\/span><\/p><\/div>\n

Some are in favor of shortening the five-year deadline for the replacement that Concea recommends.\u00a0 \u201cRecently we have begun to invest in developing alternative methods here in Brazil and now we run the risk of snatching defeat from the jaws of victory if we fail to achieve results immediately,\u201d says Maria Jos\u00e9 Giannini of Unesp.\u00a0 \u201cBecause they are under deadline pressure, companies may import technicians instead of using home-grown expertise.\u00a0 This is already happening today.\u00a0 Cosmetics companies report that they do not conduct tests with animals in Brazil; instead, they do so in other countries to make sure that the products are safe,\u201d she explains.<\/p>\n

Octavio Presgrave, coordinator of BraCVAM, expects that internationally accepted practices will be approved soon.\u00a0 \u201cFor domestic validation, it will have to be demonstrated that the data already obtained abroad is reproduced in the tests we conduct in our laboratories,\u201d says Presgrave, a researcher at the National Institute for Quality Control in Health (INCQS).\u00a0 According to Presgrave, the five-year deadline is feasible.\u00a0 \u201cIt is enough time for the companies and laboratories to adjust,\u201d he says.\u00a0 In other cases, BraCVAM\u2019s work will take more time. \u00a0This is true, for example, of the Het-Cam protocol, which aims to replace the use of rabbits with a membrane from the eggs of hens to identify compounds that cause deterioration or significant irritation.\u00a0 The method, established in Europe in 1985, is accepted only as a pre-test in France and Germany.\u00a0 The Hat-Cam process will be the first validation study in Brazil that follows international principles, Presgrave says.\u00a0 \u201cWhen we stop using animals in a test, the gain in ethics is major.\u00a0 But a new method also means creating knowledge.\u00a0 We are developing innovations to find methods that are more reliable and sensitive,\u201d he says.<\/p>\n

In another area to curb the use of animals in laboratory testing, the LNBio is receiving funding from the MCTI to create a center for in silico<\/i> testing in order to use fewer animals in research for drugs.\u00a0 In silico<\/i> refers to the silicon used in integrated circuits and means \u201cin computers.\u201d\u00a0 This term was coined as an analogy to the terms in vivo<\/i> and in<\/i> vitro<\/i>, which have been in use for a long time.\u00a0 In silico<\/i> testing involves computer simulations to determine, for example, whether molecules that are being considered for use in new drugs can really be used for this purpose.\u00a0 \u201cComputers are able to compare the structure of the candidate molecule with the structure of others that have already been tested and whose characteristics are stored in databases to determine whether it is worthwhile to continue to develop them,\u201d says Eduardo Pagani from the LNBio.\u00a0 These tests can also assist in determining whether a given molecule, even one with potential, has a real chance of being absorbed by the organism if it is administered orally.\u00a0 According to recognized estimates, of the five to ten thousand molecules that are evaluated initially for potential activity in a target, 250 are synthesized and move on to testing in animals, while five move on to clinical testing in humans, and just one reaches the market as a drug.\u00a0 \u201cThe purpose of in silico<\/i> testing is to further decrease the number of substances that are tested using animals in order to quickly eliminate those that are shown to be not feasible.\u00a0 It is a screening process we use so that we do not waste time or financial resources, and most of all it prevents the unjustifiable use of animals in projects that are predictably doomed to fail.\u201d<\/p>\n

\"Cell

EDUARDO CESAR<\/span>Cell cultures for testing cytotoxicity for a diphtheria anatoxin to replace the use of guinea pigs to control the quality of the diphtheria vaccineEDUARDO CESAR<\/span><\/p><\/div>\n

Last month the LNBio released the results of a request for proposals that gave companies, research institutes and universities the opportunity to conduct in silico<\/i> tests in the laboratory.\u00a0 Seven companies submitted 19 proposals.\u00a0 \u201cAll were accepted and we will begin the testing in the coming months,\u201d says Tiago Sobreira, a bioinformatics researcher at the LNBio in charge of the operational part of the in silico <\/i>tests.\u00a0 The companies expressed interest in submitting proposals and now they will negotiate the terms of their involvement, and this will include protecting industrial confidentiality.\u00a0 Some of the labs being considered are Farmanguinhos, Crist\u00e1lia and Eurofarma, as well as cosmetics companies such as Botic\u00e1rio and Natura.\u00a0 \u201cAnyone who develops pharmaceuticals says that it will take 15 years and cost R$1 billion to bring a product to market.\u00a0 Brazil\u2019s trade deficit in pharmaceuticals is R$6 billion per year.\u00a0 We need to make a public effort so that Brazilians develop drugs here,\u201d Pagani says.<\/p>\n

The implementation of alternative methods depends on the existence of laboratories recognized in the so-called good laboratory practices or GLP, but they are still few and far between in Brazil.\u00a0 \u201cGood practices help with tracking and as a result they make the study more reliable.\u00a0 The reliability of alternative methods will also be ensured by making comparisons among the Renama laboratories,\u201d says the coordinator of Concea, Jos\u00e9 Mauro Granjeiro, who is in charge of this area at Inmetro.\u00a0 Recently, Inmetro coordinated a comparison among five laboratories in the network with support from an experienced international consulting firm \u2013 the European Center for the Validation of Alternative Methods (ECVAM).\u00a0 The results are now being analyzed.<\/p>\n

In order to expand studies of alternative methods, an effort to fund the groups of researchers that are involved will be required, observes Luiz Henrique Canto from the MCTI.\u00a0 \u201cWe have been able to design a structure and are beginning to make progress.\u00a0 The MCTI is making an all-out effort, including going before Congress for support for budget increases to strengthen Renama.\u00a0 We believe that this initiative may be of substantial benefit to the scientific and technological development of Brazil in the area of life sciences,\u201d he says.<\/p>\n","protected":false},"excerpt":{"rendered":"Effort to replace the use of animals in testing is beginning to pay off","protected":false},"author":11,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_exactmetrics_skip_tracking":false,"_exactmetrics_sitenote_active":false,"_exactmetrics_sitenote_note":"","_exactmetrics_sitenote_category":0,"footnotes":""},"categories":[166],"tags":[209,226,230,247],"coauthors":[98],"class_list":["post-152760","post","type-post","status-publish","format-standard","hentry","category-policies-st-en","tag-biology","tag-education","tag-ethics","tag-medicine"],"acf":[],"_links":{"self":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/152760","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/users\/11"}],"replies":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/comments?post=152760"}],"version-history":[{"count":0,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/152760\/revisions"}],"wp:attachment":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/media?parent=152760"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/categories?post=152760"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/tags?post=152760"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/coauthors?post=152760"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}