APPLIED IN VITRO TOXICOLOGY

Volume 1, Number 00, 2015

Mary Ann Liebert, Inc.
DOI: 10.1089/aivt.2015.0008

ORIGINAL ARTICLE

Alternative Methods to Animal Testing

and Cosmetic Safety:
An Update on Regulations and Ethical
Considerations in Brazil
Leslie Laquieze,1 Marcio Lorencini,2 and Jose Mauro Granjeiro 3

Abstract

Consumers of personal care and cosmetic products have been protected for many years by regulatory organizations, always providing the most advanced science in order to ensure a better safety evaluation of products and
ingredients. Since the middle of the 20th century, ethical considerations have been emerging concerning animal
experimentation. The cosmetic industry was the first to have restrictions on animal experimentation. Bans were
implemented for animal experimentation with regard to ingredients and finished product evaluation in Europe in
2013. As a result, a lot of recent changeovers have been observed in the field of toxicology and in the consideration of alternative methods. This article aims to sum up the evolution of ethical considerations and the establishment of specific regulations worldwide, focusing on alternative methods. At the present day, and as the third
market leader of cosmetics at this time, and considering its promising future, a special attention will be given to
Brazil.
Key words: alternative

methods, cosmetics safety evaluation, ethics and regulations, toxicology

Sir Peter Medawar, a Nobel Prize winner, said in 1969, I think that the use of experimental animals on the present
scale is a temporary episode in biological and medical history, and that its peak will be reached in ten years time, or
perhaps even sooner .

based on Integrated Approaches to Testing and Assessment

(IATA), as observed by the adverse outcome pathway,2
and even in advanced computer-modeling techniques
(in silico).3
In the cosmetic market, animal testing has been officially
banned in the European community since 2013, followed by
other countries, such as Norway, Israel, India, and New Zealand,4 in the global Cosmetic Compliance Summit Report of
2015. In Brazil, significant advances have been observed in
the last few years and the leading cosmetic groups are already working in agreement with this philosophy.
The Brazilian scenario of alternative methods for cosmetic
product evaluation, when considering the regulatory status
and the ethical standards around the globe, and the endpoints
that are required for cosmetic product safety evaluation, in
relation to the existing alternative methods, are reviewed
and discussed here.

Introduction

he use of animals in life sciences comes from medical experiments performed in ancient Greece. However,
today, the consciousness of animal testing from a psychological and a cognitive point of view and the need for more predictive in vitro and in vivo methods1 have to be taken into
account. Current technologies sustain the use of scientifically
valid alternative methods in order to predict local toxicity
and genotoxicity, in agreement with regulatory and ethical
requests. However, single- and repeated-dose systemic toxicity, toxicokinetic, sensitization, reproductive toxicity, and
carcinogenicity tests are not yet replaced by nonanimal
methods.1 Scientists are being challenged to develop and to
use animal replacement methods in order to better understand the biological mechanisms of diseases and to test products. These methods must be relevant to human health and be
1
2
3

Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brazil.

Grupo Boticario, Curitiba, Brazil.
Universidade Federal Fluminense, Niteroi, Brazil.

LAQUIEZE ET AL.

Alternative Methods

The European Union Reference Laboratory for alternatives to animal testing (EURL-ECVAM at the Joint Research
Centre of the Institute for Health and Consumer Protection,
IHCP) was formally established in 2011, in order to develop
and to validate new alternative methods in the European
Union. Alternative methods can be defined as procedures
that completely replace the use of animals, reduce the number of animals required, or lessen the pain or discomfort suffered by them, consolidating in the concept of the 3R.5
However, for regulatory purposes, validated alternative
methods are required to undergo a chemical safety assessment, being a validation defined as being the process by
which the reliability and the relevance of a particular approach, method, process, or assessment is established for a
defined purpose. This definition was agreed at a CAAT/
ERGATT workshop on the principles of the validation of
toxicity test procedures that were held in Amden, Switzerland, in 1990,6 but they owed much to an excellent report
on validation produced by Frazier for the Organization for
Economic Co-operation and Development (OECD),7 and to
discussions that had taken place at a CAAT symposium on
validation held in 1987.8,9
When regulatory authorities formally accept a test method
that is used to provide information to meet a specific regulatory requirement, it reaches its regulatory acceptance, as observed by the Food and Drug Administration (FDA), the
Environmental Protection Agency (EPA), and the OECD
countries, which have recognized the alternative methods
as being valuable tools for modern toxicology. The tests
that agencies everywhere in the world require for cosmetics
and other products are always performed in accordance with
standardized methods.10
Nevertheless, not all valid methods have regulatory acceptance, being valid methods that are applicable, defensible, fitting, proper, and well founded for a purpose, but have not yet
been validated and, for instance, have not been accepted by
the regulatory bodies. In spite of this, they have been proved
to be useful in assessing the local tolerance of cosmetic ingredients. Basically, a sufficient amount of scientific data exists, proving their relevance and reliability, following the
concept of weight of evidence. This can be defined as the
process of considering the strengths and weaknesses of various pieces of information, and when reaching and supporting a conclusion concerning a property of the substance.11
The validation procedure involves a number of welldefined steps. It is important to emphasize that there must
be independence between the groups that perform the distribution, the material analysis, the data analysis, and the samples that have to be coded.12
Thus, the validation process involves the following steps:
 Prevalidation: Interlaboratory study on a small scale,
carried out in order to ensure that the protocol is sufficiently standardized for inclusion in a formal validation
study.
 Phase IRefinement: Where the Lab 1 or leader develops the method, describes the protocol, and defines
the parameters.
 Phase IITransfer: Where Lab 1 disseminates the protocol for Labs 2 and 3. At this stage, it takes advantage
to verify the protocol transferability.

 Phase IIIImplementation: Where the Labs 1, 2, and 3

perform the experimental protocol and where this preliminary data is evaluated.
Validation studies can be considered as prospective or retrospective, depending upon the format that they were carried
out. Basically, a prospective study involves the generation of
new data, while a retrospective study reassesses the existing
data. Subsequent steps must be taken to submit the method
for regulatory consideration and for the possible establishment of regulations and guidelines. Incorporation of the alternative techniques into the OECD guidelines makes them
officially available worldwide for toxicological testing.10
In 2009, the International Cooperation on Alternative
Methods (ICATM) was established. The United States, Canada, Japan, and the EU signed a memorandum of cooperation, with the goal of reducing the number of animals
required for consumer product safety testing worldwide.
The agreement yielded globally coordinated scientific recommendations, on alternative toxicity testing methods, and
that this agreement should speed up their adoption in each
of these countries, thus reducing the number of animals
needed for product safety testing. The ICATM works in
strong collaboration with the following organizations: the
International Conference on Harmonization (ICH) (for the
technical requirements for the registration of pharmaceuticals for human use), the OECD (which has a test guideline
program that deals with chemicals), and the International
Cooperation on Cosmetics Regulation. Briefly, ICATMs
main goals are (1) to establish international cooperation in
the critical areas of validation studies, independent peer reviews, and the development of harmonized recommendations, in order to ensure the worldwide acceptance of the
alternative methods and strategies, and (2) to establish the
necessary international cooperation, in order to ensure that
the new alternative testing methods/strategies are adopted
for regulatory use, and will provide equivalent or improved
protection for people, animals, and the environment, while
replacing, reducing, or refining (causing less pain and distress) animal use, wherever is scientifically feasible.
The EU chemical legislation REACH gives provisions on
testing methods through Regulation (EC) 1907/2006 concerning the registration, authorization, and the restriction
of chemicals. It gives a prominent role to nonanimal methods
and approaches and contains an explicit cross-reference to
principles laid down in Directive 86/609/EEC (now replaced
by 2010/63/EU, on the protection of animals used for scientific purposes)13; REACH implements the 3R and the
last resort principle; it establishes a Regulation on Test
Methods. REACH annexes specify information requirements and applicable test methods. REACH also promotes
the use of alternative methods, in order to reduce animal testing, by obliging registrants to collect and examine existing
data, before performing new tests. It has a far-reaching provision to waive testing by using nontesting approaches: grouping, read-across, QSAR, and a possibility to replace in vivo
data by in vitro test results/weight of evidence assessment.
The Brazilian commitment, with a validation of alternative methods, resulted in the creation of the National Network of Alternative Methods (RENAMA) in July 2012 by
the Ministry of Science, Technology and Innovation
(MCTI) and in September 2012 of the Brazilian Center for

ALTERNATIVE METHODS TO ANIMAL TESTING AND COSMETIC SAFETY

the Validation of Alternative Methods (BraCVAM), a partnership between the National Institute of Health Quality
Control (INCQS/Fiocruz) and the National Health Surveillance Agency (ANVISA). These were the first partnerships
in Latin America to validate and to coordinate a studies substitution, a reduction, or a refinement, of the use of animals in
laboratory tests.
Regulatory Aspects

The European Commission (EC) defined cosmetic products as any substance or mixture intended to be placed in
contact with the external parts of the human body (epidermis,
hair system, nails, lips and external genital organs) or with
the teeth and the mucous membranes of the oral cavity,
with a view to exclusively or mainly cleaning them, perfuming them, changing their appearance, protecting them, keeping them in a good condition, or correcting body odors.13 In
Brazil, the Brazilian Health Surveillance Agency (ANVISA)
use the same definition, but classifies cosmetic products into
two classes, grades 1 and 2, according to the likelihood of unwanted effects due to an inappropriate use of the product, its
formulation, the purpose of use, and for those areas of the
body for which it is intended, together with the precautions
to be observed when using it.14 These products only accidentally enter into the bloods circulation and their chemical
reactivity is not intended to interfere with biological
processes. Around the globe, as a public backlash against
cosmetic testing on animals increases, it is pushing the
cosmetic industry to commit to ultimately eliminating the
need to conduct animal testing.
The seventh amendment of the Cosmetic Directive (CD)
(76/768/EC) that was entered into action in March 2013 banned
all animal tests for those cosmetic products to be sold in the European Union (EU), without reducing the requirements for their
safety assessment. The same provisions were contained in the
Cosmetics Regulation (EU Regulation 1223/2009), which
replaced the Cosmetics Directive, as of July 11, 2013.15
The Brazilian National Council to Control Animal Experimentation (CONCEA) is the legal authority responsible for
regulating the use of animals in teaching and scientific activities (Brazilian Law 11,794/2008).16 The CONCEA aims to
discipline the humanitarian and ethical use of animals, within
the purpose of teaching, research, and technological development; to coin legal security in relation to the use of the animals; to legitimate the implementation of policies related to
the control of animal experimentation (i.e., a policys alternative methods); to define and to make known the penalties
for infractions; and to provide transparency for society regarding the use of animals in teaching and scientific activities. Among the activities of the CONCEA, the main roles
are regulation, registering, and licensing those public and
private institutions that use animals in teaching and scientific
activities. In addition, the CONCEA must provide a legal
basis for the surveillance of those institutions and monitor
the implementation of alternative methods.
The CONCEA established a procedure to introduce validated alternative methods (Normative Resolution RN17/
14),17 regulating the recognition of alternative methods in
the use of animals used in research activities in Brazil,
pausing to consider the infrastructure and the expertise
available, together with acknowledging the regulators

Table 1. Alternative Methods Formally

Accepted in Brazil by the CONCEA
Endpoint

OECD guides

Skin irritation and corrosion

TG 430
TG 431
TG 435
TG 439
TG 437
TG 438
TG 460
TG 432
TG 428
TG 429
TG 442A and 442B
TG 420
TG 423
TG 425
GD 129
TG 487

OECD
OECD
OECD
OECD
Ocular irritation and corrosion OECD
OECD
OECD
Phototoxicity
OECD
Skin absorption
OECD
Skin sensitization
OECD
OECD
Acute toxicity
OECD
OECD
OECD
OECD
Genotoxicity
OECD

CONCEA, Brazilian National Council to Control Animal Experimentation; OECD, Organization for Economic Co-operation and
Development.

perspective. Public or private institutions, such as universities, research centers, and industries, interested in a validation of alternative methods should be associated with the
National Network of Alternative Methods (RENAMA).
The CONCEA recognizes those alternative methods as validated by those centers for validation, or by international
collaborative studies, as published in its official compendia.
The CONCEA plenary is the only instance to recognize validated alternative methods when considering the opinion of
the regulatory bodies.
In 2014, BraCVAM recommended 17 validated alternative methods published by the OECD that were accepted
by the CONCEA after hearing the Brazilian regulatory agencies (RN 18, 2014) (Table 1).18 So, up to the year 2019, the
animals used in those tests must be completely replaced by
the adopted methods.
Despite most of a Brazilian cosmetic companys aims to
follow the testing and marketing procedures required, few
laboratories follow good laboratory practices (GLP) with
qualified facilities, forcing them to perform safety assessment tests abroad. As Brazilian regulatory bodies demand
GLP, those facilities that perform toxicological tests require
well-trained technicians and test providers that are able to
follow the GLP requirements.
In Brazil, all regulators, such as the ANVISA and the Brazilian Institute of Environment and Renewable Natural
Resources (IBAMA), must comply with Law 11.794/2008
and the CONCEA regulation. It is worthwhile to mention
that, even before the CONCEA regulation, the ANVISA
clearly accepts alternative methods for the safety evaluation
of cosmetics, as is observed in its guidelines for the Safety
Evaluation of Cosmetic Products,19 except in case of childrens products, or for the evaluation of new ingredients,
where alternative methods are still unavailable. Also, in
July 2015, the ANVISA Executive Board decided that,
from now, all alternative methods recognized by CONCEA
will be immediately accepted by ANVISA.

4
Evolution of Ethical Standards
and Worldwide Regulation

The development and use of alternative methods in studies

performed with laboratory animals is a longtime goal within
the scientific community. Stephens et al. established a complete time line of the first 40 years of alternative approaches.20 Table 2 summarizes this chronology, including
an update with the events of the last few years.
In 1876, the first law to specifically regulate animal experimentation was enacted in Great Britain. However, the starting point for the reduction, the refinement, and the
replacement (3R) program was in 1954, initiated by The Universities Federation for Animal WelfareUFAWs, which
resulted in the publication of The Principles Humane of
Experimental Techniques in 1959, by Russell and Burch.21
Little attention had been paid to the 3R until the 1980s.
During the 1980s, new laws and protocols had been created
and adopted in several countries, not only in recognizing
the concept of Russell and Burch, but also in identifying
the legal and moral obligations involved in seeking to reduce,
refine, and replace, wherever possible, those procedures involving experimental animals. Several campaigns emerged
in the developed countries, such as the Spira Draize campaign
against cosmetic companies. In return, cosmetic companies
and consumer product companies began seriously investing
in the research and in the development of alternatives.
National governments incorporated the alternative approach
into their animal protection legislation and began funding
the research and development of alternatives. Some companies started developing and marketing alternative testing,
and academic centers devoted to the issue began to be established. The field of in vitro toxicology blossomed.20
During the 1990s, government centers that were devoted
to the validation and regulatory acceptance of alternative
methods were established in Europe and in the United States.
The World Congress on Alternatives and Animal Use in Life
Sciences began, and alternative tests began to be formally
approved and accepted by the regulatory agencies.
In Europe, the Cosmetic Regulation provided the regulatory framework for the phasing out of animal testing for cosmetics purposes. It established a prohibition for testing
finished cosmetic products and cosmetic ingredients on animals (a testing ban); a prohibition to market them in the European Union; and a prohibition for finished cosmetic
products with ingredients that were included in the cosmetic
products and that had been tested on animals for cosmetics
purposes (a marketing ban). This testing ban on finished cosmetic products was applied in September 2004. The testing
ban on ingredients, or on a combination of ingredients, was
applied in March 2009. The marketing ban for all human
health effects was applied in March 2009, with the exception
of repeated-dose toxicity, reproductive toxicity, and toxicokinetic. For these specific health effects, the marketing ban
was applied in March 2013, irrespective of the availability
of alternative nonanimal tests.13
Norway and Israel in 2013, India in 2014, and New Zealand
in 2015 had also adopted a testing and marketing ban.4 The
United States, Australia, and South Korea are also considering
the possibility of prohibiting animal testing. China is projecting, first, not to oblige animal testing for hazard and safety
evaluation and, second, to prohibit the importation of animal-

LAQUIEZE ET AL.

tested products. The Chinese Centre for the Validation of

Alternative Methods (ChCVAM) was created. The Association of South East Asian Nations (ASEAN) is planning to
end animal testing for cosmetics by December 2015.4
Brazil has been the pioneer in Latin America in projecting
to adopt practices related to animal testing. Brazilian discussions on the theme were intensified by the identification of the
need for the establishment of the BraCVAM,22,23 whose
structure was proposed in 2009,24 and was formally established in September 2012, as result of a partnership between
the INCQS and the ANVISA. In parallel, the MCTI established the National Alternative Methods Network, a national
network whose overall goal was to integrate the RENAMA
laboratories to develop and/or use alternative methods, and
was aimed at disseminating validated methods, and to develop and to validate new methods, in order to contribute
to the increased analytical capacity of the country. This action
has the aims to provide industrial and service capacity, overcoming technical barriers, and, in parallel, contributing to the
achievement and expansion of the 3R in Brazil.
The RENAMA consists of 3 central laboratories, as represented by the INCQS, the National Institute of Metrology,
Quality and Technology (INMETRO), and the National Biosciences Laboratory (LNBio), together with 21 associated
laboratories. In the initial phase of the implementation of
RENAMA (20122015), the following stood out: (1) an
implementation of the electronic network site; (2) a mapping
competency of the alternative methods in Brazil; (3) the development of an electronic management system based on an
international computer network, in order to enable the traceability of data on a testing facility recognized by GLP
(GLP++ systems and RENAMA-Flux); (4) an interaction
with Agential National Sanitary Surveillance (ANVISA) to
set internalization strategies of validated methods; (5) an
implementation of the monocyte activation test (MAT), the
opacity and permeability test of bovine corneal (BCOP) cytotoxicity test (OECD Guide 129), and a validation method
for the potency evaluation of vaccines; and (6) the implementation of the Computer In Silico Simulations Center in
the LNBio organization to support the academia and companies development of drugs and cosmetic ingredients.
A relevant and original strategy of RENAMA was the
implementation of interlaboratory comparisons in order to
evaluate and monitor the performances of associated laboratories, and the stimulus toward GLP principles, and providing a confidence to regulators about the expertise of
service providers. As the result of a link between the public
laboratories, the research groups, and the government organizations, the application of alternative methods, to curtail
or eliminate the use of animals for testing a products safety
and effectiveness, is gaining strength, and the research effort
for such a philosophy is beginning to pay off.
The Ministry of Science, Technology and Innovation, together with the CNPq (National Counsel of Technological
and Scientific Development), funded the Central Labs of
RENAMA (of around 1.7 million of reaisroughly 0.85
million dollars), and nine group projects in the states of
Sao Paulo, Bahia, Goias, Rio de Janeiro, Santa Catarina,
and the Rio Grande do Sul, were selected (1.2 million of
BRLroughly 0.6 million US$). These projects had two
main focuses: to support the implementation of alternative
methods in a general way and the development of an artificial

ALTERNATIVE METHODS TO ANIMAL TESTING AND COSMETIC SAFETY

Table 2. Chronological Key Steps for the Ethical Concept

and the Regulation Evolution of Animal Testing
1876
1927
1938
1944
1954
1957
1959
1962
1963
1964
1965
1967
1969

1970
1971

1972
1973
1975
1976
1977
1978

1979

1980

1981

1982
1983

1984

Cruelty to Animals Actthe first law to specifically regulate animal experimentationis enacted in Great Britain.
The LD50 Test is introduced to standardize the potency of digitalis extract.
The Federal Food, Drug, and Cosmetic Act is enacted, marking the first time a US government agency is given the
power to regulate consumer products.
Eye irritancy testing is standardized as the Draize Test.
Universities Federation for Animal Welfare (UFAW) establishes a committee to study humane techniques
used in laboratory animal experiments.
UFAW holds a symposium, Humane Techniques in the Laboratory, at which William Russell presents an
article, marking the first time the 3Rs of replacement, reduction, and refinement are discussed in public.
Russell and Burchs study is published as The Principles of Humane Experimental Technique, which develops
the 3Rs approach at length.
Lawson Tait Trust (UK) is establishedthe first research fund to support the scientific development of
alternatives.
The first edition of The Guide for the Care and Use of Laboratory Animals, written by the National Academy
of Sciences, is published by the National Institutes of Health.
The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement of ethical
principles for medical research involving human subjects, including research on identifiable human material
and data.
The Littlewood Committee Report (UK) concludes that little would be gained by paying special attention to
alternatives.
United Action for Animals is formed in the United States and later campaigns specifically for replacement
alternatives.
The Fund for the Replacement of Animals in Medical Experiments (FRAME) is formed in the United
Kingdom to promote to the scientific community the idea of alternatives.
The Lord Dowding Fund (UK) is established to support alternative research. Sir Peter Medawar correctly
predicts the subsequent worldwide decline in animal use.
FRAME publishes Is the Laboratory Obsolete?which outlines replacement methodologies such as computer
modeling, tissue culture studies, and the use of lower organisms.
Council of Europe Resolution 621 suggests that an alternatives database be established, the first significant
government recommendation on alternatives.
Bruce Ames of the University of California at Berkeley introduces a nonanimal test for detecting mutation-causing
substances, later known as the Ames Test, using a bacterium.
The Felix Wankel Prize for advancing the field of alternatives is offered for the first time.
FRAME begins to publish Alternatives to Laboratory Animals (ATLA).
The U.S. National Academy of Sciences holds the United States first major scientific meeting on alternatives.
Council Directive 76/768/EEC of 1976-07-27 on the approximation of the laws of the member states relating to
cosmetic products (as amended) is the main European Union law on the safety of cosmetics. It was made under
Art. 100 (ex Art. 94) of the Treaty of Rome. By agreement, it is also applicable in the European Economic Area.
The Netherlands Animal Protection Law includes a specific section on alternatives that has grown into a program in
which the government provides the equivalent of hundreds of thousands of dollars to support alternatives research.
FRAME hosts Alternatives in Drug Development and Testing at the Royal SocietyEuropes first big
scientific meeting on alternatives.
David Smyth, president of the United Kingdom Research Defense Society, established to support animal research,
publishes the first book examining alternatives since the publication of Russell and Burchs 1959 work.
At the urging of United Action for Animals, the Research Modernization Act (H.R. 4805), which would redirect
3050% of animal research funding to alternatives, is introduced in Congress. The Swedish government
allocates $90,000 in funding for alternativesthe first government funding for alternatives.
The Dutch Minister of Health states that the government supports the use of alternatives.
American activist Henry Spira launches the Draize campaign against the rabbit-based eye irritancy test. As a result
of the Draize campaign, Revlon gives a $750,000 grant to Rockefeller University to establish an alternatives
research program. The New England Antivivisection Society gives $100,000 for alternatives research on tissue
culture, and a second animal-welfare consortium provides $176,000 for chorio-allantoic membrane (CAM) test
development.
As a result of the Draize campaign, the cosmetics industry gives $1 million to the Johns Hopkins University to
establish the Center for Alternatives to Animal Testing (CAAT) (Avon and Bristol-Myers Squibb were the
leading donors).
Swiss animal legislation specifically requires the consideration of alternatives.
Zbinden and Flury-Roversi publish a critique of the LD50 Test.
Colgate Palmolive provides $300,000 to investigate the CAM system. CAAT holds its first symposium.
Switzerland provides 2 million Swiss francs over 2 years for alternatives research.
The Food and Drug Administration (FDA) formally announces that it no longer requires data from the classical
LD50 Test.
Utrecht University in the Netherlands establishes research and education programs directed toward a further
implementation of the 3R.
FRAME receives 160,000 from the Home Officethe first UK government funding for alternatives research.
(continued)

LAQUIEZE ET AL.

Table 2. (Continued)
1985

1987

1988

1989

1990

1991

1992

1993

The Health Research Extension Act is passed, requiring the NIH to develop a plan for alternatives.
Animal Welfare Act amendments are passed, requiring greater attention to alternatives to research techniques that
cause pain and distress.
Index Medicus, an index of published biomedical studies, adds the subject heading Alternatives to Animal
Testing.
The European Research Group into Alternatives to Toxicity Testing (ERGATT) is formed.
The Soap and Detergent Association (USA) initiates the In Vitro Alternatives Program.
The Organization for Economic Cooperation and Development (OECD) announces changes in its guidelines for
acute oral and dermal toxicity and starts to discuss alternatives.
British Industrial Biological Research Association (BIBRA) increases the funding of alternatives research to
700,000 per annum.
The Industrial In Vitro Toxicology Society (IVTS) is established in the United Kingdom.
The Federal Republic of Germany enacts new laws on animal protection requiring the consideration of alternatives
in animal research.
The Humane Society of the United States publishes an analysis of the historical importance of alternative methods
in biomedical research that were awarded Nobel Prizes.
The Dutch Alternatives to Animal Experiments Platform is established with participation from government,
industry, and animal welfare organizations.
The Swiss Foundation Finanzpool 3 R is established to support alternatives research with 1 million Swiss francs.
A government/industry workshop is held on alternatives in ocular irritancy testing, to review the Soap and
Detergent Associations Alternatives Program.
The Industrial In Vitro Toxicology Group holds its first meeting.
The U.S. Republican presidential platform encourages the implementation of alternatives to animal testing.
The J.F. Morgan Foundation for Alternatives Research is established in Canada.
The Swiss governments Office for Animal Experiments and Alternatives is established.
The Center for the Documentation and Evaluation of Alternative Methods to Animal Experiments, known by its
German acronym ZEBET, is established in Germany.
Procter and Gamble announces that it is contributing $450,000 per year for 3 years to its University Animal
Alternative Research Program.
Avon Products announces that it will no longer use the Draize Test.
The Scandinavian Society for Cell Biology establishes the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC)
to assess alternatives to LD50 testing for acute toxicity.
The Second International Conference on Practical In Vitro Toxicology is held in the United Kingdom.
The Swedish Fund for Scientific Research Without Animal Experiments invests 700,000 Swedish crowns in
alternatives projects.
The Clonetics Corporation begins to market cells and cell testing methods.
The American Anti-Vivisection Society establishes the Demeter Fund (later known as the Alternatives Research
and Development Foundation) in order to support nonanimal research, funding up to $50,000 annually for one or
more projects.
CAAT and ERGATT hold a workshop on the validation of alternative methods.
The University of California Alternatives Center is established at UCDavis.
The Platform for Alternatives to Animal Experiments in the Netherlands allocates the equivalent of $700,000
annually for the promotion and validation of research into the 3Rs and the improvement of housing and care
systems.
The HSUS establishes the Russell and Burch Award for scientists who have made outstanding contributions to
alternative methods.
The Interagency Regulatory Alternatives Group holds a workshop, Eye Irritation Testing Alternatives: Proposals
for Regulatory Consensus, in Washington, DC.
The HSUS presents Alan Goldberg, director of CAAT, with the first Russell and Burch Award.
The OECD accepts the Fixed Dose Procedure as an alternative to the LD50 Test.
Representatives of regulatory agencies in Japan, Europe, and the United States agree to drop the classic LD50 Test
as a required measure of acute toxicity.
The UK Home Office announces a grant program for the funding of alternatives research.
The Second Report of the FRAME Toxicity Committee is published in ATLA.
The Swiss Institute for Alternatives to Animal Testing (SIAT) is established in Zurich.
The European Centre for the Validation of Alternative Methods (ECVAM) is established.
The European Parliament amends the Cosmetic Directive 76/768 to ban the marketing of cosmetics tested on
animals after January 1, 1998 (a decision on the ban is later postponed until June 30, 2000).
CAAT hosts a 10th anniversary conference in Baltimore, MD, giving Founders Awards to Dr. D.A. Henderson,
the CTFA, and Henry Spira.
The NIH Revitalization Act of 1993 directs the NIEHS to establish criteria for the validation and regulatory
acceptance of alternative testing and to outline a process for regulatory review of potential alternative methods;
it also directs the NIH director to establish an alternatives program and to report on its progress annually.
The first World Congress on Alternatives and Animal Use in the Life Sciences: Education, Research, and Testing
takes place in Baltimore.
(continued)

ALTERNATIVE METHODS TO ANIMAL TESTING AND COSMETIC SAFETY

Table 2. (Continued)

1994

1995
1996

1997
1998

1999

2003

2004
2005
2007
2008
2009

2012
2013

2014

Member states of the European Union agree on the goal that everything possible should be done to achieve a reduction
of 50% in the use of vertebrate animals for experimentation and other scientific purposes by the year 2000.
The Interagency Regulatory Alternatives Group holds its second meeting on alternatives, in Washington, DC; Dr.
Michael Balls of FRAME is appointed director of ECVAM.
The U.S. Federal government establishes the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM), co-chaired by William Stokes of NIEHS and Richard Hill of EPA, in response to the 1993
NIH Revitalization Act.
The Netherlands Centre for Alternatives to Animal Use (NCA) is established as a national information center on
alternatives.
The Gillette Company and the HSUS launch a program to fund research and development of alternative methods;
two grants of $50,000 each are awarded annually.
The second World Congress on Alternatives and Animal Use in the Life Sciences is held in Utrecht, the
Netherlands.
The OECD holds a workshop to develop internationally harmonized criteria on validation and regulatory
acceptance.
CAAT, the HSUS, Procter and Gamble, and other organizations establish Altweb, a website devoted to information
on alternative methods.
ICCVAM issues guidelines on criteria for validation and the regulatory acceptance of alternative methods.
The Institute for In Vitro Sciences is established in Gaithersburg, MD.
The HSUS presents the FDAs Neil Wilcox and ICCVAMs William Stokes with the Russell and Burch award for
their contribution to the development of alternatives.
ECVAM accepts the following alternative methods: 3T3 NRU PT test as an alternative for assessing phototoxicity,
Episkin and similar methods for assessing skin corrosivity, and transepithelial electrical resistance (TER) test for
assessing skin corrosivity.
ECVAM endorses in vitro methods as alternatives to the Ascites method for the production of monoclonal
antibodies.
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods
(NICEATM) is established to provide support to ICCVAM.
The third World Congress on Alternatives and Animal Use in the Life Sciences is held in Bologna, Italy.
The HSUS presents Procter and Gamble scientist Dr. Katherine Stitzel with the Russell and Burch award for her
contribution to the development of alternatives.
CAAT holds Test Smart (a humane and efficient approach to regulatory toxicity data) workshops in order to
discuss alternatives to animal testing in the Environmental Protection Agencys High Production Volume (HPV)
chemical testing program.
The EPA announces major changes in its HPV program, including the funding for alternative methods following
the Test Smart workshops and negotiations with animal protection organizations.
ICCVAM endorses Corrositex for the assessment of skin corrosivity and the murine local lymph node assay for the
assessment of allergic contact dermatitis.
7th Amendment of the Cosmetics Directive introduces new provisions related to nonanimal testing of cosmetic
finished products and ingredients.
Brazil adopts the 3R concept.
ANVISA edited a safety guide for cosmetic products.
Prohibition of animal testing for cosmetic finished products in Europe
The Japan Center of Validation of Alternative Methods is established ( JaCVAM).
Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), a European Union Regulation
dated December 18, 2006, entered into force in January 2007. REACH addresses the production and use of
chemical substances and their potential impacts on both human health and the environment.
Lei Arouca 11-794: Creation of the Nacional Center of Animal Testing CONCEA (Conselho Nacional de
Experimentacao Animal) in Brazil
The Korean Center of validation of Alternative Methods (KoCVAM) is established.
CIUCA Decreto 6.899 do CONCEA (National Council of Animal Experimentation Control) created a registration
of the institution using animals for scientific purposes.
Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of November 30, 2009 on cosmetic
products
Brazil enter the OECD MAD Mutual Acceptance of Data for pesticide & chemicals.
The Brazilian Centre for Validation of Alternative Methods (BraCVAM) and The National Network of Alternative
Methods RENAMA are established.
Animal testing is banned in Europe for cosmetics, finished products, and ingredients.
SBMAltthe Brazilian society of alternative methodsaims to simplify the process between the BraCVAM, the
RENAMA, and the CONCEA.
The Cosmetic Regulation replaces the Cosmetic Directive (76/768/EEC).
Animal testing is prohibited in Sao Paulo state for the validation of cosmetic products, perfumes, and corporal
hygiene products (Lei Estadual no. 15.316, 23/01/2014).
European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) was launched
in 2014 for the validation of an ARTA.

skin equivalent for the safety evaluation by the cosmetic and

chemical industry.
From a scientific viewpoint, Brazil was the host country
for the 1st Latin American Congress on Alternative Methods for the Use of Animals in Education, Research and
Industry (Congresso Latino Americano de Metodos Alternativos, COLAMA November 2012), with the presence of
national and international speakers, which presented five
short-time courses, companies, researchers, and undergraduate and graduate students; in total, an audience of
around 100 participants.
Regarding the Brazilian marketing ban, Sao Paulo was
the first state to adopt the testing ban in February 2014,
prohibiting the use of animals in the testing of cosmetic products, in response to activists pressure (State Law no. 15.316
published in January 2014). This law was implemented suddenly, without any public consultation, and being incoherent
to Federal Legislation (Federal Law 11.794/2008). This promoted controversy. The total ban made it impossible to develop cosmetics with any new ingredients or molecules as
discovered by Brazilian biodiversity that contained unknown
molecules and interfered with the MCTI strategy of replacing, reducing, and improving the use of animals in testing
whenever possible. Recently, a study addressed the impact
of the implementation of this law in the state of Sao Paulo
and concluded that the cosmetic industry would transfer its
industrial pole to other federal states. This was in order to
allow the cosmetic industry to continue to follow the federal
legislation that still required animal testing, in order to ensure their quality and to reduce the risk of adverse effects
in consumers.25 In fact, the Brazilian Constitution guarantees
that any federal law could not be modified by a state law,
making the Sao Paulo State Law unconstitutional and, until
it is regulated, it is not in force, prevailing Law 11.794/
2008 and the CONCEA regulations.
From Alternative to Predictive Methods

An alternative method is a single or a combination of methods to reduce, refine, or replace animal testing, for any toxicological assessment.12 For example, according to the Cosmetic
Regulation, the different types of human health effects possibly
induced by topical products are acute toxicity; skin irritation
and corrosion; skin sensitization; skin absorption/penetration;
UV-induced toxic effects; carcinogenicity; subacute and subchronic toxicity; genotoxicity and mutagenicity; toxicokinetic
and metabolism; and reproductive and development toxicity.
Table 3 presents the validated alternative methods for cosmetic
product safety assessment.
Reducing animal testing presents both risks and benefits.
Beyond reducing animal numbers, alternative methods also
provide a better approximation, and permit to take into account the mechanisms of action. But it also presents some
limitations, such as the application areas, the predictability
of the obtained results, the economic impact, and the real incidence on the use of animals. Thus, we should first consider
the origins of animal testing, which answer was expected,
and with which criteria and limits ensued. A decisional
tree might be designed to represent this predictive strategy,
as can be observed in the Globally Harmonized System of
Classification and Labeling of Chemicals,9 to classify physical health and environmental hazards. A predictive strategy

LAQUIEZE ET AL.

is a combination of multiple tools that could be in a systematic literature review, in silico (computerized), in tube (biochemical), in vitro (based on cells and tissue and culture),
ex vivo (based on tissue excision), and in vivo (based on animal testing and a human clinical trial). Different models can
be used depending on the questions asked (i.e., software, cellular lineage, primary cells, or reconstructed tissues).
No stand-alone test method allows for covering the complexity of the physiological processes of a whole body.26,27
Knowledge of the biology and physiology, the metabolism,
and the cellular and tissue engineering culture is the key to
establishing a predictive strategy. Several prerequisites
have to be taken into account. Replacing animal testing by
alternative methods reveals the challenge of combining several methods into an Integrated Testing Strategy (ITS) in
order to obtain equivalent results. An IATA is an approach
that integrates the existing knowledge bases on the classes
of chemicals, with the results of biochemical and cellular assays, computational predictive methods, exposure studies,
and other sources of information, in order to identify the requirements for targeted testing, or to develop assessment
conclusions. The IATA also have the potential to further enhance an understanding of the mechanisms of action, including the consideration of relevant adverse outcome pathways
that provide biological linkages between molecular initiating
events, to adverse outcomes in individual organisms and
populations that are the base for risk assessments. A structured approach that integrates and weighs all of the relevant
existing data and informs about additional data needs to enable any regulatory decision. To this aim, the different steps
that need to be analyzed are in the following order: physicochemical data; in silico data; in chemico data; in vitro data;
in vivo data; information of analog chemicals; information
from other relevant sources; exposure considerations; and
an assessment using weight of evidence, or a predefined approach, or combination of both.
First, the model has to be well defined, depending on the
question and/or the endpoint that we are looking for. Does
a simple model, such as a cell line, is sufficient, or is a
more complex model, like a reconstructed tissue or a zebra
fish, more reliable? Which conditions of culture are more
adapted: static, dynamic, or microfluidic? Which gas, temperature, and humidity parameters? Which characterization:
morphology, functionality, metabolic capacities, or what genetic status? The chosen method mechanistic should be
based on the mode of action of the compound. The protocols
have to be adapted to the physicochemical properties of the
product and its mechanism, the composition of the environment, and the treatment and detection system (flow cytometer, RT-PCR, spectrophotometer, etc.). Additionally, it needs
to be relevant, suitable for standardization, transferrable,
and reproducible intra- and interlaboratory. According to
Kandarova and Letasiova,10 a well-validated test might
consider that its performance characteristics, advantages,
and limitations have been adequately determined, especially when considering some technical parameters, such
as (1) sensitivity (a percentage of positive chemicals correctly identified); (2) specificity (a percentage of negative
chemicals correctly identified); (3) predictivity (a percentage of predictions for a particular correct classification);
(4) accuracy (an overall percentage of correct classifications); (5) reproducibility within laboratories; (6)

ALTERNATIVE METHODS TO ANIMAL TESTING AND COSMETIC SAFETY

Table 3. Validated Alternative Methods for Cosmetic Product Safety Assessment

Guideline references

Year

27,28

29,30

2006
2015

3t3 Nru phototoxicity test

+ application to UV filter chemicals
In vitro human skin model: EPISKIN
SIT, Epiderm EPI-200-SIT, Skinethic
SIT-42bis
In vitro human skin model:
EPISKIN, Epiderm, Skinethic RHE,
EST-1000 RHE now called Epics,
Vitrolife-Skin RHE
Transcutaneous electrical resistance test
(Ter)
Corrositex noncellular membrane
Direct peptide reactivity assay (DPRA)

2015

KeratinosensTM

37,38

2014

h-CLAT (ESAC peer review finalized

in 2014)
In vitro mammalian cell micronucleus
Bacterial reverse mutation (Ames) test
In vitro cell gene mutation test (COMET)
In vitro chromosomal aberration test
In vitro sister chromatid exchange test
In vitro unscheduled dna synthesis test
Saccharomyces cerevisiae gene mutation
assay
S. cerevisiae mitotic recombination assay
Bovine corneal opacity permeability
(BCOP) test

39,40

2012
2012

Cytosensor microphysiometer modified

Fluorescein leakage

49,50

2009

Isolated chicken eye (ICE) 2012 test

51,52

2012

Sequential testing strategy for eye irritation

and corrosion

52,53

OECD GD-129

2010

Skin absorption

2004

UV-induced effect

OECD TG-428
OECD GD 28
OECD Guidance Notes
No. 156
OECD TG-432

2004

Skin irritation

OECD GD-439

2010

Skin corrosion

OECD TG-431

2004

OECD TG-430

2004

OECD TG-435
OECD TG442c
ECVAM Protocol No.154
OECD TG442d
ECVAM Protocol No. 155
OECD draft
ECVAM protocol No.128
OECD TG-487
OECD TG-471
OECD TG 476
OECD TG 473
OECD TG 479
OECD TG 482
OECD TG 480

Genotoxicity and
mutagenicity

Ocular
irritation/corrosion

OECD TG 481
OECD TG-437
OECD Proficiency Standards
OECD GD No. 160
Draft OECD TG
OECD TG-460
OECD Summary Document
No. 180
OECD TG-438
OECD Proficiency Standards
OECD GD No. 160
OECD TG 405

References

Balb/C 3T3 NRU assay

NHK NRU assays
Diffusion cell

Acute toxicity

Skin sensitization

In vitro alternative methods

2010
1997
1997
1997
1986
1986
1986
1986
2009

reproducibility between laboratories; and (7) a probability

for a correct classification.10
Concluding Remarks

The adoption of validated alternative methods (OECD

guides) represents a great advancement in terms of sustainable and ethical processes for the safety evaluation of cosmetics. However, for regulatory purposes, these methods
demand a GLP infrastructure, presenting a well-trained network of skilled technicians, test suppliers, and interested
companies. However, these issues are still few and far between in Brazil. Cosmetic companies should be prepared
for this technical challenge and might consider a change of

28,29

30,31

31,32

32,33
33,34
35,36

40,41
41,42
42,43
43,44
44,45
45,46
46,47
47,48
48,49

50,51

mindset in terms of toxicological evaluation and safety assessment decisions. Furthermore, for an efficient evolution
of this scenario, by the participation of the RENAMA, the
BraCVAM, the CONCEA, the regulatory bodies, and other
playerssuch as the Brazilian Society for Alternative Methods (SBMAlt)supported by the Ministry of Science,
Technology and Innovation, an integrated and strongly cooperative work must be performed for a promising future of
alternative methods in Brazil.
Author Disclosure Statement

The authors do not have any real or potential financial or

commercial conflicts of interest in the context of this review.

10

LAQUIEZE ET AL.

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Address correspondence to:

Dr. Leslie Laquieze
Instituto de Metrologia
Qualidade e Tecnologia
Diretoria de Metrologia Aplicada a`s Ciencias da Vida
Avenida Nossa Senhora das Gracas, 50, Xerem
25250-020 Duque de Caxias
Rio de Janeiro
Brazil
E-mail: leslielaquieze@gmail.com