New Methodological Approaches (NAMs previously referred to as Alternative Methods to Animal Experimentation) are any method, protocol change and set of strategies that reduce the number of animals, reduce the severity of the procedure, or replace the use of laboratory animals, in accordance with the 3 Rs Principle. It is normal for the term to be associated exclusively with substitutive methods; however, its concept is broader.
Do you now Three Rs Principle was created in 1959?
Alternative Methods to Animal Experimentation are any methods or protocol alterations that either reduce the number of animals, reduce the severity of the procedure, or substitute the use of animals, according to the Three Rs Principle. Although this term is commonly associated exclusively with replacement methods, it represents a broader concept.
What is the Three Rs Principle?
In 1959, zoologist William M. Russel and microbiologist Rex L. Burch presented the book "The Principles of Humane Experimental Technique" to the scientific community, in which they proposed the "Three Rs Principle" in scientific research: Reduction, Refinement and Replacement. The researchers defined the three Rs as: Reduction – the minimization of the number of animals used and the reuse of animals by the assessment of multiple endpoints; Refinement – the search for and technological development of methods to minimize the incidence and severity of scientific procedures in animals; Replacement – the use of organisms that are not considered capable of experiencing suffering, such as bacteria, yeasts, earlier stages of animal development, or in vitro cultures of cells and tissues, thus replacing in vivo for in vitro.
Furthermore, alternative methods also aim to improve the prediction of toxic effects in humans. Many assays that employ cells and tissues of human origin present results that are similar to the possible effects in human populations; thus, they offer advantages over the classical animal experiments, and contribute to the assertion of product safety.
Which assays are considered Alternative Methods? Currently, several validated in silico, in chemico, in vitro and ex vivo assays are available for use as alternative methods to classical animal testing. It is important to keep in mind that in vivo methods that focus on reduction and/or refinement are also considered alternative methods. However, they must be outlined according to the project objective and the applicability of each method.
Do you now Brazil have a good regulation?
REGULATION OF ALTERNATIVE METHODS IN BRAZIL
The Arouca Law and the Creation of CONCEA
The enactment of the Arouca Law (11.794/2008) changed the way in which animal experimentation is conducted in Brazil. This law approaches the use of animals in teaching and research activities, regulates their use, management and assay conditions, focusing heavily in animal well-being.
The fundamental point of the Arouca Law was the creation of the National Council for Animal Experimentation Control (CONCEA – Conselho Nacional de Controle de Experimentação Animal), a democratic entity that includes representatives of Scientific Institutions and Legally Constituted Animal Protection Societies and whose main competences are:
to issue and enforce regulations regarding the humane use of animals in teaching and research;
to accredit Brazilian institutions for the breeding or use of animals in teaching and scientific research;
to monitor and evaluate the introduction of alternative techniques to replace the use of animals in teaching and research.
Focusing on the latter, CONCEA has appointed a permanent committee dedicated to alternative methods.
Normative Resolution 17/2014 – CONCEA
This resolution defines the need of approval of alternative tests by CONCEA and turns approved methodologies mandatory after 5 years.
Normative Resolution 54/2022 – CONCEA
In 2022 a new resolution replaced NR 17/2014, with more complete definitions and mentioning the use of internationally recognized methods. According to NR54/2022 all approved methods must be used immediately after approval, bus a 5 year tolerance were maintained.
Normative Resolution 18 – CONCEA
In September 2014, CONCEA issued the Normative Resolution 18 (RN 18), which approved, for the first time, methods considered as alternatives to their original versions. This resolution approved 17 methods, including alternatives for seven toxicological outcomes (toxicological outcome: result of effect monitored by a toxicological study).
The toxicological endpoints covered by RN 18 are:
I – Dermal irritation and corrosion potential
II – Ocular irritation and corrosion potential
III – Phototoxicity Potential
IV – Dermal absorption
V – Potential skin sensitization
VI – Acute toxicity
VII – Genotoxicity
Other normative resolutions had been published since, adding other methods to the list.
Board Resolution N. 35/2015 – Brazilian Health Regulatory Agency (ANVISA)
RN 17 established a period of five years for the approved methods to take priority over their traditional versions. This period became more critical following the issue of the BOARD RESOLUTION (RESOLUÇÃO DA DIRETORIA COLEGIADA, RDC) N. 35/2015 by the Brazilian Health Regulatory Agency (ANVISA), which delineates the acceptance of the alternative methods to animal experimentation recommended by CONCEA. This act conferred regulatory significance to the methods approved by CONCEA; that is, it was not only about their acceptance by the Council, but also by the regulatory agency, thus impacting all the productive sectors regulated by ANVISA.
SEPTEMBER 2019 – FIVE YEARS OF RN 18
In September 2019, five years have passed since the issue of RN 18 by CONCEA. Thereby, whenever experiments are needed to assess any of the toxicological outcomes addressed by RN 18, the alternative method must be the first method of choice, for all activities of basic or applied research in Brazil, from scientific projects to regulatory compliance services.
In August 2016, CONCEA issued the Normative Resolution 31 (RN 31), which approved seven additional methods; beyond the dermal and ocular irritation and corrosion potential (already included in RN 18), the outcomes of Assessment of Reproductive Toxicity and Assessment of Pyrogenic Contamination in Injectable Products were included. These seven methods became mandatory in August 2021.
Is the use of alternative methods mandatory?
Not exactly. They are the priority methods of choice. If there is a scientific justification to support that a given alternative method is not adequate for the tested sample, or that it does not provide all the necessary answers, another version of the assay may be performed. For instance: issues regarding sample solubility might be deterrent for the performance of in vitro tests, or the alternative method might not be applicable for the conclusion of some toxicological classifications. Overall, it is believed that only a small fraction of the cases will suit these justifications.
It is not clear how this will be controlled, but it seems logical that the first filter would be applied by Animal Ethics Committees (AECs). Each institution that performs animal experimentation in Brazil must have an AEC registered at CONCEA. Possibly, the ethics committees would not approve the use of traditional methods without a good justification as to why the alternative method is not appropriate. A second filter may be applied by the regulatory agency, which may inquire or deny dossiers that do not comply with regulations.
Don´t you know the principles of GLP?
GOOD LABORATORY PRACTICE (GLP) AND ALTERNATIVE METHODS
What are the principles of GLP? Good Laboratory Practice (GLP) is a quality system that encompasses the organizational process and the conditions in which non-clinical studies of health and environmental safety are designed, performed, developed, monitored, registered, archived and reported. These principles are applied to all the studies on environmental and health safety required by the regulatory agencies, aiming the register or license for pharmaceutical products, pesticides, cosmetics, veterinary medicines, food additives, feed additives and industrial chemical products, with the exception of products for which these studies are not mandatory.
As any other study of product safety evaluation, alternative methods may, and in some cases must, be performed under GLP conditions. GLP ensures the quality and traceability of data, thus resulting in study robustness and reliability.
Are there Alternative Methods in GLP in Brazil? Most of the 17 methods approved by CONCEA are available in Brazil, with all their toxicological outcomes represented, and they are performed according to Good Laboratory Practice (GLP).
WHAT'S NEXT?
In other countries, several methods are being developed as alternative methods for screening and product registration, employing distinct technologies. Other methods have already been validated, with acknowledged guidelines available, such as those published by the Organisation for Economic Cooperation and Development (OECD). Furthermore, several initiatives are led by governments of the United States and European Communities to promote the development and acceptance of alternative methods.
In Brazil, after the publication of RN 18 by CONCEA, there have been important developments regarding Alternative Methods. We are a reference in South America, our legislation and science are emerging in the continent. In this regard, CONCEA has approved more alternative methods, and several initiatives for the dissemination and implementation of alternatives are underway.
However, it is fundamental that the government, the industry and the academy join efforts to promote the knowledge on alternative methods and the use of assays in Brazil. This encompasses several strategies, from the promotion of courses, training, and legislation updates, to the incentive for implementation and contract of assays.
We have a long way ahead! Let's move forward!
Izabel Villela, PhD - Executive Director
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