Alternatives to animal experimentation

REVIEW
Ethics of animal research in human disease remediation, its
institutional teaching; and alternatives to animal
experimentation
Rajkumar Cheluvappa1 , Paul Scowen2 & Rajaraman Eri3
1
Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales, Australia
2
Department of Animal Services, University of Tasmania, Hobart, Tasmania, Australia.
3
Mucosal Biology Laboratory, School of Health Sciences, University of Tasmania, Launceston, Tasmania, Australia
Keywords
alternatives, animal ethics committee, animal
experimentation, animal research, code,
distress, ethics, pain, pathophysiology,
reduction, replacement
Correspondence
Rajaraman Eri, School of Health Sciences,
University of Tasmania, Newnham,
Launceston 7248, Australia.
Tel: +61-3-6324 5467; Fax: +61-3-6324
3995; E-mail: [email protected]
Funding Information
No funding information provided.
Received: 7 May 2017; Accepted: 23 May
2017
Pharma Res Per, 5(4), 2017, e00332,
https://doi.org/10.1002/prp2.332
doi: 10.1002/prp2.332
Abstract
Animals have been used in research and teaching for a long time. However,
clear ethical guidelines and pertinent legislation were instated only in the past
few decades, even in developed countries with Judeo-Christian ethical roots.
We compactly cover the basics of animal research ethics, ethical reviewing and
compliance guidelines for animal experimentation across the developed world,
“our” fundamentals of institutional animal research ethics teaching, and emerging alternatives to animal research. This treatise was meticulously constructed
for scientists interested/involved in animal research. Herein, we discuss key animal ethics principles – Replacement/Reduction/Refinement. Despite similar
undergirding principles across developed countries, ethical reviewing and compliance guidelines for animal experimentation vary. The chronology and evolution of mandatory institutional ethical reviewing of animal experimentation (in
its pioneering nations) are summarised. This is followed by a concise rendition
of the fundamentals of teaching animal research ethics in institutions. With the
advent of newer methodologies in human cell-culturing, novel/emerging methods aim to minimise, if not avoid the usage of animals in experimentation. Relevant to this, we discuss key extant/emerging alternatives to animal use in
research; including organs on chips, human-derived three-dimensional tissue
models, human blood derivates, microdosing, and computer modelling of various hues.
Abbreviations
ECPA, European Crop Protection Association; EFPIA, European Federation of
Pharmaceutical Industries and Associations; HSE, human skin equivalents; ICLAS,
International Council for Laboratory Animal Science; SPCA, Society for Prevention
of Cruelty to Animals.
Introduction
The humanest possible treatment of experimental animals,
far from being an obstacle, is actually a prerequisite for
successful animal experiments.
— Russell & Burch. Principles of Humane Experimental Technique (1959)(Russell and Burch 1959).
The use of animals in pathology, and related research/
teaching is pivotal to the advancement of science, as
animals have been considered to be good model systems
for humans and human disease. Animal models can be
appropriate, or can be approximated to study human
anatomy, physiology, pathology, etc., as animals may have
a biological milieu resembling human homoeostatic conditions.
Research involving animals may include awareness
research (e.g., behavioural, embryological, physiology, and
genetic) which is necessary to contribute eventually (and
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
2017 | Vol. 5 | Iss. 4 | e00332
Page 1
indirectly) to human disease remediation (Cheluvappa
et al. 2007a,b), and applied research (academic or/and
commercial), such as pathology (Cheluvappa et al. 2015),
drug testing, pathogen research (Cheluvappa et al. 2010),
defence research, and toxicology (Cheluvappa et al. 2008).
When animal research ethics are mentioned subsequently
in this work, it will generally refer to the academic areas
which we (the authors) work with, namely; murine models of physiology, pathogenesis, and toxicology.
Animal usage in research and teaching is subject to
strict ethical guidelines all over the developed world. With
the advancement of technology in medical research, we
are now at a stage to consider manifold alternatives to
utilising animals in research and teaching. In this study,
we concisely cover the fundamental principles of animal
research ethics, compliance guidelines for animal experimentation, institutional animal research ethics teaching,
and emerging alternatives to animal research. The
intended targets of this study are scientists and ethicists
interested in animal research, for example, undergraduates, graduate students, medical students, and clinicians.
We emphasise that this work is not intended to be an
elaborate treatise.
The intents of this study are fourfold, with each intent
in contiguity with the next.
(1) The first intent of our study is to provide a concise
summary of previous and extant thought on animal
experimentation ethics.
(2) The second intent of our study is to demonstrate
how previous and contemporary thought on principles/mores pertaining to animal experimentation
ethics, finally translated into concrete legislation to
mandate compulsory review of ethical practices in
animal research.
(3) The third intent of our study is to lay out suggestions, practical recommendations, and teaching
strategies for the lucid inculcation of animal experimentation ethics to interested parties.
(4) The fourth intent of our study is to provide and raise
awareness of available alternatives to animal research.
Ethics in animal experimentation –
the beginnings and the basics
The first intent of our study is to provide a summary of
historical and extant thought on animal experimentation
ethics in human disease elucidation and therapy, inclusive
of extant thought that is more or less accepted around
the developed world. This intent also extends an implicit
encouragement to relevant personnel to conform to these
ethical principles and standards.
Animal ethics are not stringent rules mandating
researchers to conduct animal research in certain ways,
but an arena for promoting the expression of human
moral obligations towards animals used in research. However, Russell and Burch (1959) set of 3Rs (Replacement,
Reduction, and Refinement) is arguably the best known,
and the most utilised set of animal ethics to date.
Despite Greek and Roman references to animal
experimentation by Aristotle (4th century BC) (Cohen
and Loew 1984), Erasistratus (3rd century BC)(Cohen
and Loew 1984), and Galen (2nd century AD)(Greek and
Greek 2000), the earliest reference to animal welfare and
ethics occurs only in the 19th century (Zurlo et al. 1994).
For example, what we know as Society for Prevention of
Cruelty to Animals (SPCA) today, was originally organised in England in 1822 (Zurlo et al. 1994). In 1831, the
first seeds were sown for today’s animal ethics guidelines
by Marshall Hall, a British physiologist (Zurlo et al.
1994). In 1876, the English House of Commons passed
the first bill relating to animal experimentation (the Cruelty to Animals Act 1876 = An Act to Amend the Law
Relating to Cruelty to Animals 1876) following which, a
number of countries including USA followed suit (Zurlo
et al. 1994). In 1959, William Russell, an intelligent young
zoologist (then), psychologist and scholar; and Rex Burch,
a microbiologist, published “The Principles of Humane
Experimental Technique.” Therein, they categorised
humane animal experimentation techniques (Part 2 The
Progress of Humane Technique) under replacement,
reduction, and refinement, now referred to as the 3Rs –
Replacement, Reduction, and Refinement (Russell and
Burch 1959).
Animal experimentation ethics did not emerge de novo.
It evolved over centuries of philosophical traditions. Concepts such as Aristotle’s virtue ethics (Cohen and Loew
1984) (ethical treatment of animals stem from the “character” of individual humans), Hobbe’s 17th century contractarianism (Rowlands 2013) (acceptable if most people
accept the experimental objectives without offence),
Kant’s (1788) deontological approach (beneficence
towards humans vs. non-malfeasance towards animals),
Bentham’s 1789 utilitarianism (Bentham 2007) (acceptable if adequate human benefit is expected), mid-20th
century animal rights (acknowledge animals as having
intrinsic rights, on a varying scale relative to humans),
respect/dignity (Anderson and Perry 1999) as per “the
code” 1978 (acknowledge animals as respectable entities,
on a varying scale relative to humans), the Dutch 1981
legislated inherent value (Brom and Schroten 1993) (acknowledge animals as having intrinsic value, on a varying
scale relative to humans), Francione’s 1996 abolitionism
(Francione 2010) (stopping animal research completely),
Garner’s 2013 justice (“morally fair” treatment of animals), Kantian-derived “fellow creatures” (acknowledge
animals as our counterparts in sharing the world), etc.,
2017 | Vol. 5 | Iss. 4 | e00332
Page 2
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
are elaborately debated, but out of scope of this study
(Brom 2002).
Animal research ethics may also be placed in the wider
context of the human use of animals. It is interesting to
observe the 1986 Council of Europe Convention ETS No.
123 preamble (Council of Europe, 1986) making this
philosophical construct systematically as follows:
“The member States of the Council of Europe, signatory hereto,…..
• Recognising that man has a moral obligation to respect
all animals and to have due consideration for their
capacity for suffering and memory;
• Accepting nevertheless that man in his quest for knowledge, health and safety has a need to use animals where
there is a reasonable expectation that the result will be
to extend knowledge or be to the overall benefit of
man or animal, just as he uses them for food, clothing
and as beasts of burden;
• Resolved to limit the use of animals for experimental
and other scientific purposes, with the aim of replacing
such use wherever practical, in particular by seeking
alternative measures and encouraging the use of these
alternative measures;
• Desirous to adopt common provisions in order to protect animals used in those procedures which may possibly cause pain, suffering, distress or lasting harm and
to ensure that where unavoidable they shall be kept to
a minimum, Have agreed as follows:”
Russell’s and Burch’s replacement principle (first R)
involves “‘non-sentient’ material which may in the history
of experimentation replace methods which use ‘conscious
living vertebrates’” (Russell and Burch 1959). It entails
relative (substitution) or absolute (no animals) replacement. It is to be noted that animal experimentation is
not restricted to “more sentient” vertebrates alone. It
clearly extends to “relatively less sentient” invertebrates as
well. Three important invertebrate species that have contributed substantially to the areas of cell biology and
genetics are the nematode Caenorhabditis elegans, bakers’s
yeast Saccharomyces cerevisiae, and the arthropod Drosophila melanogaster (fruit fly). An excellent workshop
report by Kretlow et al. (2010) summarises the significant
role of these three invertebrates in biomedical research –
the nematode C. elegans (apoptosis, RNA interference,
developmental genetics), baker’s yeast S. cerevisiae (genome sequencing, ageing, mitochondrial diseases), and the
fruit fly D. melanogaster (genetic modelling, developmental biology, transformation, mutation, toxicity screening).
In our own area of research pertaining to Pseudomonas
aeruginosa pathogenesis, the nematode C. elegans has been
used instead of mice models, the standard model for
pathology and mortality studies (Tan et al. 1999).
Reduction (second R) entails the balance of statistically
significant numbers and minimising the number of animals. The “humaneness”(Russell and Burch 1959) of
decreasing the animal numbers required for a study is
quite obvious. Researchers utilising animals in their
research have to substantiate how they conform both to
the minimum statistically significant animal numbers
needed for the study (power analysis), and to the principle of reduction. This information has to be made available both during research grant funding applications, and
during animal ethics approval applications.
The third R, refinement, presents more of a challenge,
owing to its diverse possibilities, in innumerable scenarios. Refinement is so flexible that a unique refinement
may be possible in each study, every time. Refinement
“might be regarded as an art or an ability to improvise”
(Russell and Burch 1959). Morton (1998) defines refinement as, “those methods which avoid, alleviate or minimise the potential pain, distress or other adverse effects
suffered by the animals involved, or which enhance animal wellbeing.” Moreover, refinement not only attempts
to reduce negative states in animals, but promotes “positive mental and physical states.”
Mandatory Institutional Ethical
Reviewing of Animal
Experimentation: Chronological
Sequence of Pioneering Nations
The second intent of our study is to demonstrate how
previous and contemporary thought on principles/mores
pertaining to animal experimentation ethics, finally translated into concrete action, via the implementation of concrete legislation to mandate compulsory review of ethical
practices in animal research.
The goals and strengths of institutional animal ethical
review include setting guidelines for ascertaining whether
animals are necessary for a study in the first place, overseeing humane experimental conduct of animal studies,
ensuring species-specific tailoring of methodology, minimising biological variability, and establishing minimum
discomfort to experimental animals. To start with,
mandatory ethical requirements are needed to ascertain
whether animals are needed for a particular study in the
first place. The projected scientific outcome and impact
must be weighed against the wellbeing of the animals to
be used. This justification must include evaluation of the
maximum number of study parameters that have the
potential to impact negatively on the animal’s wellbeing,
an assessment of the knowledgeability of the investigators
regarding these parameters, and their ability of remediation of the same. Variability in the choice of research
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 3
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
animals reduces the detectability power of a study, and
increases the number of animals required for statistical
reliability. Contrariwise, variability per se may be essential
to the study in question, as in certain toxicology studies
(Biggers et al. 1958). Ethical reviewing must scrutinise
these elements to ensure observance of the 3Rs. The ethics
review is also needed to confirm that putative animalhandlers are familiar with species-specific indicators of
distress, pain, disease, and abnormal behaviour. Additionally, the animal-handlers must be endowed with the ability to assist in obviation of the same.
There are several potential weaknesses and drawbacks
of institutional animal ethical review. Institutional animal
ethical review may or may not have teeth, depending on
the local environment. Legislation conflicts or common
law precedents may dilute penalties in case of infringements of ethics. How “severe” would the punishment
have to be? Suspension of animal experimentation may
just be temporary in some cases. How does a body rehabilitate a “repeat offender”? There may be doubtful efficacy in the monitoring of procedural consistency. It
would be difficult to oversee data and indicators from a
plethora of working individuals under the ambit of an
ethics committee review approval. The interinstitutional
and intrainstitutional variability between the qualities of
animal ethics reviewing will be difficult to ascertain.
Therefore, quality control and consistency will always be
in doubt.
In developed countries, the pioneering nations which
were most concerned about adherence to animal experimentation ethics, systematically and progressively mulled
ideas, discussed them in the legislature, and finally promulgated mandatory institutional ethical reviewing of animal experimentation. The chronology of these events
varied from country to country. In general, it took a few
decades after initially contemplating the ideas, to finally
implementing laws pertaining to mandatory institutional
ethical reviewing of animal experimentation. Developing
countries generally do not have laws mandating institutional ethical reviewing of animal research. However, a
few developing countries like India, regardless of adherence or non-adherence, have introduced legislation similar to their Western counterparts.
The forerunning nations or jurisdictions which pioneered mandatory institutional ethical reviewing of animal experimentation are as follows.
Australia (1978)
Animal experimentation has been governed by the “Australian Code of Practice for the Care and Use of Animals
for Scientific Purposes” (the Code) since 1969 (Anderson
and Perry 1999). However, the requirement of institutions
to establish Animal Experimentation Ethics Review Committees (scientists and non-scientists) was indicated only
in the 2nd edition of this Code (1978) (Ewing 1989). Further details and functional itemisation were done in the
Code’s subsequent editions.
Sweden (1979)
After a 3-year pilot program, animal ethics committees
(six regional committees) were made compulsory (1979)
(Hagelin et al. 2003).
Canada (1980)
In 1980, Canada mandated institutions to establish local
animal care committees to review reviewing ethical features of animal experimentation protocols (CCAC, 1980).
The autonomous Canadian Council on Animal Care
(CCAC) advises and supervises the surveillance of animal
care and experimentation in Canada’s universities, government laboratories and pharmaceuticals (Rowsell 1986).
The USA (1985 edition of 1963-published
“The Guide”)
The “Guide for the Care and Use of Laboratory Animals”
(the Guide) was first published in 1963, under the title
“Guide for Laboratory Animal Facilities and Care”, and
was revised at least six times (Committee for the Update
of the Guide for the Care and Use of Laboratory Animals,
2011). In the 1985 edition, the Guide mandated institutions to appoint committees (with one noninstitution
affiliated member) to evaluate animal care and experimentation (Committee for the Update of the Guide for
the Care and Use of Laboratory Animals, 2011).
India (1998)
The 1998 Indian gazette notification (and its 2001
amended notification) by the Committee for the Control
and Supervision of Experiments on Animals (CPCSEA)
mandates local institutional animal ethics committees
(Committee for the Purpose of Control and Supervision
of Experiments on Animals, 1998).
The UK (2000)
The United Kingdom (UK) Home Office made it compulsory (2000) for institutions to “introduce an animal
experimentation ethical review process” (Home Office,
2014). Prior to this, the Cruelty to Animals Act 1876 (An
Act to Amend the Law Relating to Cruelty to Animals)
was supplanted by the Animals (Scientific Procedures)
2017 | Vol. 5 | Iss. 4 | e00332
Page 4
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
Act 1986 (ASPA) – both of them did not mandate a
requirement for institutional ethical reviewing of animal
use in research (Home Office, 2014). However, it is
indeed noteworthy that the pioneering Cruelty to Animals
Act 1876 first established a licensing system with a relative
degree of prospective evaluation, in addition to the establishment of a monitoring inspectorate. Vestiges of the
Cruelty to Animals Act 1876 (sans local animal ethical
review) are still within the legal framework in a few erstwhile British colonies, but are out of scope of further
elaboration herein.
The EU (1986 & 2010) – Inclusive of the UK
The EU 86/609/EEC directive (1986) was issued by the
European Union (EU) to promote ethical adherence in
animal research (https://conventions.coe.int/Treaty/en/
Treaties/Word/123.doc) (Council of Europe, 1986). The
2010/63/EU directive (2010) on the Protection of Animals
Used for Scientific Purposes (http://eur-lex.europa.eu/legalcontent/EN/TXT/?uri=celex:32010L0063)(European Commission 2010) makes holistic project evaluation (including
harm-benefit analysis) compulsory before conducting animal research. However, both the EU 86/609/EEC directive
(1986) and the 2010/63/EU directive (2010) do not require
institutional review by committees. In 2006, the Federation
of European Laboratory Animal Science Associations
(FELASA) identified 16 EU countries (amongst the 20 EU
countries reviewed by them) as having robust ethical
reviewing as a compulsory prerequisite to conducting animal research (Smith et al. 2007). In their analysis, FELASA
posited that “ethical review should aim to ensure that, at
all stages in scientific work involving animals, there is adequate, clearly explained ethical justification for using animals” (Smith et al. 2007). Therein, FELASA not only
emphasised the necessity of harm-benefit analyses prior to
embarking on research projects involving animals, but also
underscored the importance of “normative” animal
research ethical review processes to reflect diverse ethical
perspectives (Smith et al. 2007).
There is great diversity of the “organisation” of ethical
review processes for animal experimentation in developed countries. For example, please refer to Smith et al.
(2007) for a concise summary (Table 1) of the wide
range of ethical review processes organisation of laboratory animal use in the European Federation of Laboratory Animal Science Associations (FELASA). We have
chosen a few aspects (FELASA countries and elsewhere)
which we found interesting and summarised them below
as points.
(1) There is a high degree of consensus (predominantly
amongst developed countries) on animal research
ethics, harm-benefit analysis (e.g., Bateson’s decision
cube with three research dimensions – quality, suffering, and benefit), and the necessity for systematic animal research ethical review processes. This was
elaborately analysed and described by the International Council for Laboratory Animal Science
(ICLAS) in 2010.(ICLAS – International Council for
Laboratory Animal Science, 2010)
(2) A country may have mandatory ethical evaluation by
a person (chosen from selected and endorsed individuals), but not by a committee (e.g., Norway) (Smith
et al. 2007).
(3) A country may have national ethical review bodies
feature legislated, without mandating local institutional bodies.
(4) A country may have national or professional body
codes/guidelines, but animal experimentation legislation may or may not reference them.
(5) Legislation and regulations are not synonymous,
although regulations generally stem from legislation.
(6) Legislation may be delegated regionally (e.g., Australia
and Canada), but the regional law may or may not
include enforcement of the code of guidelines (White
2007).
Research pertaining to the efficacy of institutional ethical reviewing of animal research is sparse. We speculate
that institutional ethical reviewing may work better in
countries (and circumstances) which are more developed,
have better funding for animal facilities, have lesser
bureaucratic impediments, have simpler/more direct processes, and have flexible common/statutory law providing
allowance for better reviewing and penalty implementation. Institutional ethical reviewing may not work as well
in countries (and circumstances) with the opposite of
what was just mentioned.
Teaching ethics in animal
experimentation pertaining to
human disease remediation
It is indeed important to note that not only generally, but
also specifically in our discipline; animal research per se is
rather distinct from animal research teaching. Our
biomedical/and pathophysiological research aims to judiciously explore and garner hitherto unknown knowledge.
However, animal research (and ethics) teaching aims to
illustrate already known facts. This has fundamental
implications for the ethical reviewing of animal use, and
for the necessity of justifying the indispensability of animal use. Pertaining to teaching animal research ethics, we
have found widespread conceptual similarities in basic
content of animal research ethics courses across multiple
Sydney-based universities. However, the teaching styles
and interactional content differ.
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 5
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
Table 1. An overview of the organisation of ethical review of laboratory animal use in the Federation of Laboratory Animal Science Associations
(FELASA) countries in Europe (Smith et al. 2007).
Country Mandatory processes* Voluntary processes
Austria For academic institutions: National committee of the Ministry of Education,
Science and Culture. Industry: Official veterinarian
Institutional committees
in some facilities
Belgium Institutional committees (which can be shared between institutions) and
Government inspectors (who are members
of the local committees) and a National committee when difficult issues arise
Czech Republic Institutional committees; two National committees: representing (i) all
Ministries involved in animal experiments and (ii)
the Academy of Sciences; final authorisation by a Government committee,
the Central Commission for Animal Welfare and the Environment
Denmark Review by National committee appointed by the Minister of Justice which
directs a Government inspectorate
Four institutional committees
Estonia A National licensing committee was established at the Estonian Ministry of
Agriculture in May 2004. The committee
reviews applications and grants permits for animal experiments; meetings
take place according to the number of applications received
Finland At the time of writing, institutional committees (some are shared between
institutions). Changing to a National Committee
as a result of a change in the law in 2006
France Applications for licences are approved and given by the Ministry of Agriculture.
Government veterinary inspectors from the
local Veterinary Service in each Prefecture check compliance (field of research,
training and competence of researchers).
Painful protocols must be declared to the local Prefecture, and an additional
licence and evaluation is required for use of
non-domestic animals. A National Ethical Committee oversees the good
functioning of the ethical committees (but there
is not as yet a legal requirement for researchers to submit their work for
ethical review by these committees)
Regional committees for public
research (22); Institutional
committee in each
industrial firm#
Germany Review by institutional Animal Welfare Officer (a veterinarian, medical
doctor or zoologist), then by Regional
committee (c. 40) advising the government authorities
Greece Official veterinarian from the Local Veterinary Service in each Prefecture,
who may take advice from scientists in the relevant field of work
Institutional committees in
Medical Faculties and some
research institutions
Ireland Applications for licences must be approved by the Minister for Health and
Children. A local nominated competent
person (preferably a veterinary surgeon) must review each application and
declare that he/she does not envisage any
practical difficulties on welfare grounds and specify any reservations
Institutional committees in
most institutions
Italy A review by a special Commission at the National Institute of Health is
required only for: procedures involving
cats, dogs, non-human primates and/or endangered species; procedures without
anaesthesia; and those for education and training
Institutional committees
in most research centres
Latvia National committee, at the Latvian Council of Science
Lithuania National committee of the State Food and Veterinary Service Institutional committees
in some facilities
Netherlands Local (mostly institutional) committees, plus a National committee which
acts as a ‘court of appeal’ when a local
committee has rejected a proposal (very rare). The law permits the outsourcing
of ethical review, so that ‘institutional’
committees can advise more than one institution, and there can also be
independent committees (there is one at present),
whose services can be hired by institutions do not have their own
(Continued)
2017 | Vol. 5 | Iss. 4 | e00332
Page 6
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
The third intent of our study, therefore, is to lay out
suggestions, practical recommendations, and teaching
strategies for the lucid inculcation of animal experimentation ethics to interested parties, namely scientists, students, and clinicians. This is an extremely important
intent, owing to the necessity for clarity in the transfer of
information, mores, and legalities.
Recommended and/or familiar components
of a typical teaching course on animal
experimentation ethics
Teaching ethics in animal experimentation pertaining to
human disease remediation ought to be concise, but
empathetic. Audiovisuals will certainly make the teaching much more interesting. A typical animal
experimentation ethics course at an institution or university should topically involve the following, or segments of it thereof.
(1) Evolution of thinking in animal ethics
(a) Recognise that animal experimentation ethics is
to be placed in a wider context of the human use
of animals (Council of Europe, 1986), and ethics
as depicted in the chapter by Olssson, et al. in the
2010 edition of the Handbook of Laboratory Animal Science (Olsson 2010).
(b) History of the local institutional animal ethics
committee, and relevant legislation (local and
international)
(2) Russell’s and Burch’s 3R principles (Russell and
Burch 1959)
Table 1. Continued.
Country Mandatory processes* Voluntary processes
Norway Local ‘competent person’ and National committee (National Animal
Research Authority – for review of cases which the
local competent person finds too controversial to make a decision, or
is involved in, field experiments, and painful
experiments where painkillers are withheld (very rare))
Institutional committees in some facilities
A new Animal Welfare Act is currently being drafted
Poland Regional committees (18) set up by the National Ethics Committee on
Animal Experimentation (NEC/AE) which
oversees their work as an appeal authority.
Spain Regional committees in Catalonia, Andalusia and Aragon; institutional
committees in all research centres in Catalonia
and Aragon. From October 2005, a new national law requires institutional
committees in all State (but not other)
research centres, and sets up a State Ethical Commission of Animal Welfare
which must approve and supervise high severity procedures
Institutional committees in
most other research centres
in the remaining regions
Sweden Regional committees (7)
Switzerland Regional committees (10), which advise the Cantonal Authority whether or not
experiments should be authorised; plus a
National committee to advise the cantons in controversial cases and more
general matters. The Federal Veterinary
Office has the right to appeal.
Institutional committees
in some facilities
UK Institutional committees and other local processes review project licence
applications as well as more general matters
pertaining to the care and use of laboratory animals within institutions.
Applications then forwarded to Government
inspectors who, having weighed the likely welfare costs against the potential
benefits, advise the Secretary of State
for the Home Office whether or not they should be granted. There is also a
National committee (the Animal
Procedures Committee) for general advice on the operation of the law and
ethical review of certain classes of licence application
*
Italics indicate countries in which there is not yet a national, mandatory requirement for prior ethical review of all regulated scientific uses of
animals
#
Although not legally required, the organisations involved signed a binding commitment to submit work to these processes for ethical review.
This table summarises the wide range of general organisation of ethical review processeses of laboratory animal use in the Federation of Laboratory Animal Science Associations (FELASA) countries in Europe.(Smith et al. 2007) [License number to reproduce table from SAGE Publications –
4115900032136]
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 7
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
(3) A scientist’s ethical responsibilities
(a) Recognition and relief of distress and pain in
experimental animals
(4) Summary of extant animal models
(a) Well-established alternatives to animal experimentation
(b) Emerging alternatives to animal experimentation
(5) Strategising animal experiments with careful heed to
Russell’s and Burch’s 3R principles (Russell and
Burch 1959)
(6) Tips on writing proposal submissions to the local
animal ethics committee, with emphases on the following:
(a) Clear objective(s)
(b) Targeting non-scientists – Where? What? Why?
How? Who? When?
(c) Clarity in thought process, and sequence of
experiments
(d) Concise and appropriate literature summary
(e) Animal number estimates – minimisation and
justification
(f) Personnel involvement – stratification, description, and justification
(g) Stage of study – pilot experiments? Transitional
stage? Main study corpus?
(h) Record keeping strategies/commitments, and
accountability hierarchy
(i) Interventional techniques, details of surgery,
pain/distress-management
(j) Candid and crystal-clear endpoint(s)
(7) Animal welfare scrutiny and monitoring
(a) Animal experiment record keeping, mishap
reporting/redressal, and feedback addressal
(8) Short lecture on research procedures
(a) Few demonstration sessions and lectures on animal handling, and common techniques (anaesthetisation, blood sampling, euthanasia simulation,
etc.)
(9) Reading material (“homework – whole/links or piecemeal/bibliography”)
(a) Relevant national legislation on animal experimentation ethics
(b) The authorised national code of ethics on animal experimentation
(i) For example, the Australian Code (Ewing 1989;
Anderson and Perry 1999) section on justification
posits thus: “Projects using animals may be
performed only after a decision has been made
that they are justified, weighing the predicted
scientific or educational value of the projects
against the potential effects on the welfare of the
animals”.
(c) Guidelines to common animal experimentation
procedures
(d) Guidelines to promote experimental animal
well-being
Ideal learning endpoints
Utilising a multiple-choice test at the end of a course, the
course participants would be assessed for a “reasonable”
comprehension of (percentile scores or percentage cutoffs):
(1) The spectrum of ethical issues pertaining to animal
experimentation
(2) A scientist’s ethical responsibilities
(3) A practical application of Russell’s and Burch’s 3R
principles (Russell and Burch 1959)
(4) Application submission procedure to the local animal
ethics committee
(5) Recognition and relief of distress and pain in experimental animals
(6) Basic animal handling, anesthetisation, blood collection, drug administration, and euthanasia
We consider these components and endpoints to be
essential as these topics adequately cover the legal, administrative, ethical, statistical (basic), and technical (basic)
aspects of animal experimentation.
Emerging alternatives to animal
experimentation pertaining to
human disease remediation
The fourth and last intent of our study is to provide
available alternatives to animal research to raise awareness
of viable, and at times, even better options outside of animal experimentation. This would directly and indirectly
feedback on our first intent extension of encouraging relevant personnel to better conform to these ethical standards.
Outside of the well-established alternatives to animal
experimentation like tissue culture methods including primary/continuous/immortalised cell lines, explant cultures,
organ cultures, several recent strategies have been recently
mooted to curtail animal experimentation, and simultaneously (and surprisingly) improve efficacy of data-gathering. We used Pubmed searches (using replacement,
in vitro alternatives, human tissue, etc., as search words)
to identify those alternative methods which we thought
2017 | Vol. 5 | Iss. 4 | e00332
Page 8
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
would have “maximum impact” (well-cited research or
high-impact journals on this subject or articles associated
with our reseach).
While alternatives to animal experimentation may
reduce research dependence on animal (through replacement); they currently cannot replace animal testing altogether. This impossibility exists despite several ethical,
political, and financial “incentives” to persevere in this
direction. The extant alternatives serve to complement
animal experimentation in current research.
In vitro models
Organs on chips
The most interesting “animal substitute” to buttress preclinical drug development is the Organs on chips (OOC),
pioneered by scientists from Harvard University and
University of Pennsylvania (Huh et al. 2010). Microfabrication methodology from the computer microchip manufacturers was utilised to devise microengineered systems
capable of supporting living cells. The OOC looks
promising as a pathophysiologically pertinent model of
experimentation. OOCs are “micro-engineered biomimetic systems containing microfluidic channels lined by
living human cells, which replicate key functional units of
living organs to reconstitute integrated human organ-level
pathophysiology in vitro” (Huh et al. 2013). A seminal
protocols paper (Huh et al. 2013) describes the first OOC
innovation. This pioneering model describes not only
how a “‘breathing, elastic’ lung-on-a-chip” (Fig. 1) is
crafted, but also how their protocol can be modified to
develop other human organ chips, like a ‘peristaltic’ guton-a-chip’”.
Human-derived three-dimensional tissue models:
Epidermis–dermis human skin equivalents
In vitro models of skin pathophysiology and drug testing
has been around for some time. Pioneering testing
of human skin equivalents (HSE) included EpiDerm
(Monteiro-Riviere et al. 1997) and full-thickness EpiDerm
(Kubilus et al. 2004). Presently, HSE models include a
huge spectrum, ranging from those used to demonstrate
simple physiology, to those used to analyse model diseases
(from autoimmune disorders to malignancies) (Auxenfans
et al. 2009; Semlin et al. 2011). Contingent on standardisation and quality, these models may be better than animal
models. This is partly because the originating skin samples are human-derived. Additionally, these tissue models
are grown in vitro in a biochemical and physiological
milieu closely simulating human homeostatic conditions.
Human-derived three-dimensional tissue models:
Others
Using Russel and Burch’s principle of replacement (Russell and Burch 1959), several human-derived three-dimensional models have been synthesised, tested, validated (a
few of the several), and used (Sheasgreen et al. 2009).
These 3-D in vitro models have not only an “ethical
edge”, but also a more pathophysiologically relevant edge
owing to the human origin of these tissues. This edge is
obvious as the tissue samples originate from humans, and
are grown in vitro in a homeostatic environment akin to
human biochemical and physiological conditions. Additionally, an animal model (like a commonly used inbred
murine model) has far less biological and genetic differences compared to the complex human genetic/biological
heterogeneity, making human tissue much more physiologically relevant. These three-dimensional human-derived
tissue models include oral epithelia (Klausner et al. 2007),
gastrointestinal epithelia (Sheasgreen et al. 2009), vaginal
epithelia (Ayehunie et al. 2011), ocular tissue (Kaluzhny
et al., 2011), gingival tissue (Hai et al. 2006), respiratory
epithelia (Sexton et al. 2011), and dendritic antigen-presenting cells (Sheasgreen et al. 2009).
Human blood derivatives
The European Partnership for Alternative Approaches to
Animal Testing (EPAA) (Cozigou et al. 2015) is a voluntary collaboration intending to focusing information and
resources to the experimental use of animals in regulatory
testing. The collaborating partners include the European
Commission, individual companies from seven industrial
sectors, and their European trade federations. The European Commission is bound by the EU Treaty to maximally promote 3R principles (Cozigou et al. 2015). The
seven industrial sectors include the European Chemical
Industry Council (Cefic), the European Federation of
Pharmaceutical Industries and Associations (EFPIA), the
European Cosmetic Toiletry and Perfumery Association
(Colipa), the European Association for Bioindustries
(EuropaBio), the International Federation for Animal
Health Europe (IFAH-Europe), the International Association for Soaps, Detergents and Maintenance Products
(A.I.S.E), and the European Crop Protection Association
(ECPA). The EPAA indicated the necessity to include
authorities during the validation of, and the legal acceptance processes pertaining to animal experimentation
alternatives (Montag et al. 2007). The pyrogen complement activation test (human plasma complement activation test) (Sladowski et al. 2001), the alternative pyrogen
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 9
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
test (monocyte activation test) (Schindler et al. 2009),
and the whole blood cytokine-release immunotoxicity test
(Langezaal et al. 2002) are three such processes investigated under EPAA’s auspices. These tests display enormous potential to replace the limulus amebocyte lysate
(LAL) assay which requires a quarter of a million horseshoe crabs to be exsanguinated every year (30% of blood
collected per animal) with a 3–15% post-exsanguination
mortality rate (Anderson et al. 2013).
Pathophysiologically relevant in vitro models serve as
exemplary models of replacement and cost-cutting, but
replacement herein will still be partial, albeit significant.
Animal testing will still be required for the foreseeable
future. For example, in our research, a bacterial toxin
had effects which were different from that on cultured
cells (Cheluvappa et al. 2007c), than its in vivo effects
in a live animal (Cheluvappa et al. 2008). Similarly a
tested drug, owing to a multitude of reasons, may work
fine on an in vitro model, but may not work (or may
work differently) on a live animal. Therefore, in vitro
models will effectuate manifold prescreening processes
prior to animal experimentation, but may only serve
partially in reduction. Furthermore, only in vivo animal
models can account for complex and/or unknown biological systems and pathways that in vitro models cannot encompass.
Computer modelling – in silico and
quantitative structure–activity relationship
analyses
Pathophysiological simulations can now be screened using
high-tech computer modelling programs (in silico modelling) (Martonen et al. 2003; Aguda et al. 2011). Toxicity
screening (Golbamaki et al. 2014) and fundamental pharmacokinetic events such as gut absorption, protein-binding, endothelial barrier passage, etc. can also be done
rapidly in vitro depending on specific in silico modelling
program availability (Raunio et al. 2004). There are addition software-based techniques (quantitative structureactivity relationships or QSARs) (van Leeuwen et al.
2009) that utilise sophisticated estimates of a molecule’s
hazard-inducing capacity, based on its similarity to existing molecules, and extant human physiology. QSAR software (toolboxes) (van Leeuwen et al. 2009) have been
used extensively, either exclusively, or in conjunction with
(A) (C) (D)
(E)
(B)
Figure 1. Organ on Chip – the human “lung-on-a-chip” microsystem (Huh et al. 2010, 2013). (A) The microfabricated lung mimic device uses
compartmentalised polydimethylsiloxane (PDMS) microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane
coated with extracellular matrix (ECM) – fibronectin or collagen. The device recreates physiological breathing movements by applying vacuum to
the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar–capillary barrier. (B) During inhalation in the
living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip), leading to distension of the alveoli and physical
stretching of the alveolar–capillary interface. (C) Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel
microchannels separated by a 10-lm thick PDMS membrane containing an array of through-holes with an effective diameter of 10 lm. Scale bar,
200 lm. (D) After permanent bonding, PDMS etchant is flowed through the side channels. Selective etching of the membrane layers in these
channels produces two large side chambers to which vacuum is applied to cause mechanical stretching. Scale bar, 200 lm. (E) Images of an
actual lung-on-a-chip microfluidic device viewed from above. [License number to reproduce image from The American Association for the
Advancement of Science – 4115900305591].
2017 | Vol. 5 | Iss. 4 | e00332
Page 10
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
reduced animal numbers. Examples include hydrogenated
azoles (Craig et al. 2014), sulphur-containing compounds
(Richarz et al. 2014), pesticide/biocide carcinogenicity
(Devillers et al. 2011), unsaturated aliphatic aldehydes
(Devillers and Mombelli 2010b), aromatic amines (Devillers and Mombelli 2010a), and chemical carcinogens
(Mombelli and Devillers 2010).
Computer modelling is limited in its role in limiting
animal use in research. To start with, animal research is
essential to glean pathophysiological nuances, even before
one starts to play with the keyboards. While available data
may be used for extant in silico models, incorporation of
future data, which may ostensibly be more complex, may
necessitate further animal research. Processor speed and
configuration adaptability are essential not only for
designing intricate simulations, but also for using them
(Zaslavsky et al. 2014). Such simulations generally focus
on major aspects, and tend to overlook smaller, but
equally (if not more) important aspects. Therefore, computer modelling may assist in preliminary vetting surveys
ahead of more concrete experiments involving other
models (including animals). This may partially assist with
the reduction objective, the magnitude of which may fortunately or unfortunately depend on “research and funding priorities/popularity.”
Research involving human volunteers
Positron emission tomography (PET) and functional
magnetic resonance imaging (fMRI) pertaining to brain
activity are the first approaches that come to mind when
the topic of research involving human volunteers is
broached. However, there are several other “human testing” investigative methods which have been used. A classic example is microdosing, the research pertaining to
which, was first published a decade ago. Microdosing is
“an approach to early drug development where exploratory pharmacokinetic data (with or without imaging) are
acquired in humans using inherently safe sub-pharmacologic doses of drug” (Lappin et al. 2013). It is essential to
note that human ethics approval must be obtained prior
to any microdosing experiment. In the US, institutional
review boards (IRBs) come into play with regard to this.
The human ethics review committees (HRECs) are the
Australian equivalent of the US IRBs. It is essential (and
obvious) that microdosing studies ought to be conducted
on human volunteers without coercion. A large number
of drugs (Lappin et al. 2013) have been investigated using
microdosing, and around 80% of microdose pharmacokinetics published is commensurate with those observed at
therapeutic doses, within a twofold difference. It is to be
emphasised that the options for human testing have their
own ethical and legal hurdles. They are not simple or
simplistic substitutions for animal models. However,
owing to the voluntary and cognizant nature of the test
human subjects, a significant proportion of these hurdles
are easily overcome. Microdosing may not have produced
“concrete data” yet, but surely has much more to offer,
being an excellent contributing tool.
While the thought of human PET and MRI scans
“lighting up” when activated under certain sensory/motor
conditions sounds like an appealing example of replacement, we currently still require animals to devise and test
the efficacy and safety of therapeutic approaches as in
mortality or toxicity studies. On the other hand, microdosing inherently cannot predict adverse reactions of
drugs that may occur at therapeutic levels, which animal
studies clearly can. Therefore, microdosing can only assist
in partial reduction of animal use in research.
Conclusions
Animals have been used in research as it is generally purported to simulate human biology. The ethics pertaining
to animal research evolved over centuries of philosophical
traditions, and not rigid rules of operation, but an avenue
to express our moral obligations towards research animals. Russell and Burch set of 3Rs (Replacement, Reduction, and Refinement) are currently the most utilised set
of animal ethics. The countries which were most concerned about adherence to animal experimentation ethics
finally promulgated mandatory institutional ethical
reviewing of animal experimentation. The chronological
sequence (in its pioneering developed nations) of national
legislation in developed countries pertaining to mandatory institutional ethical reviewing of animal experimentation has been charted out in this work. Although
developing countries generally do not have laws requiring
institutional ethical reviewing of animal research, a few
like India have introduced relevant legislation. While
teaching animal research ethics, we must include topics
pertaining to the legal, administrative, ethical, statistical
(basic), and technical (basic) aspects of animal experimentation. In this work, our rendering of the fundamentals of teaching animal research ethics in institutions is
discussed. We have laid out suggestions, practical recommendations, and teaching strategies for the lucid inculcation of animal experimentation ethics to scientists,
students, and clinicians. In addition to “traditional” alternatives to animal experimentation (like tissue cultures),
several innovations have been recently introduced with
the objective to retrench animal experimentation. They
include organs on chips, human-derived three-dimensional tissue models, human blood derivates, microdosing, and computer modelling. However, these alternatives
can only reduce research dependence on animals (through
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 11
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
replacement and reduction) by complementing animal
research. We have a fair way to go!
Author Contribution
Rajkumar Cheluvappa wrote and submitted the manuscript. Paul Scowen assisted in animal research ethics.
Rajaraman Eri proposed the idea and wrote the manuscript.
Disclosure
None.
References
Aguda BD, Marsh CB, Thacker M, Crouser ED (2011). An in
silico modeling approach to understanding the dynamics of
sarcoidosis. PLoS ONE 6: e19544.
Anderson WP, Perry MA (1999). Australian animal ethics
committees: we have come a long way. Camb Q Healthc
Ethics 8: 80–86.
Anderson RL, Watson WH 3rd, Chabot CC (2013). Sublethal
behavioral and physiological effects of the biomedical bleeding
process on the American horseshoe crab, Limulus polyphemus.
Biol Bull 225: 137–151.
Auxenfans C, Fradette J, Lequeux C, Germain L, Kinikoglu B,
Bechetoille N, et al. (2009). Evolution of three dimensional
skin equivalent models reconstructed in vitro by tissue
engineering. Eur J Dermatol 19: 107–113.
Ayehunie S, Cannon C, Larosa K, Pudney J, Anderson DJ,
Klausner M (2011). Development of an in vitro alternative
assay method for vaginal irritation. Toxicology 279: 130–138.
Bentham J. (2007). An introduction to the principles of morals
and legislation. edn. Courier Corporation, 1–157. www.ea
rlymoderntexts.com/assets/pdfs/bentham1780.pdf (Last
accessed on June 2017).
Biggers JD, Mc LA, Michie D (1958). Variance control in the
animal house. Nature 182: 77–80.
Brom FW (2002). Science and society: different bioethical
approaches towards animal experimentation. Altex 19: 78–82.
Brom F, Schroten E (1993). Ethical questions around animal
biotechnology. The Dutch approach. Livest Production Sci 36:
99–107.
Canadian Council on Animal Care (CCAC) (1980). Guide to
the care and use of experimental animals. In Guide to the care
and use of experimental animals, Vol. 1. Canadian Council on
Animal Care, Ottawa, Ontario. 1980.
Cheluvappa R, Hilmer SN, Kwun SY, Cogger VC, DG LEC
(2007a). Effects of old age on hepatocyte oxygenation. Ann N
Y Acad Sci 1114: 88–92.
Cheluvappa R, Hilmer SN, Kwun SY, Jamieson HA, O’Reilly
JN, Muller M, et al. (2007b). The effect of old age on liver
oxygenation and the hepatic expression of VEGF and VEGFR2.
Exp Gerontol 42: 1012–1019.
Cheluvappa R, Jamieson HA, Hilmer SN, Muller M, Le
Couteur DG (2007c). The effect of Pseudomonas aeruginosa
virulence factor, pyocyanin, on the liver sinusoidal endothelial
cell. J Gastroenterol Hepatol 22: 1350–1351.
Cheluvappa R, Cogger VC, Kwun SY, O’Reilly JN, Le
Couteur DG, Hilmer SN (2008). Liver sinusoidal endothelial
cells and acute non-oxidative hepatic injury induced by
Pseudomonas aeruginosa pyocyanin. Int J Exp Pathol 89:
410–418.
Cheluvappa R, Denning GM, Lau GW, Grimm MC, Hilmer
SN, Le Couteur DG (2010). Pathogenesis of the
hyperlipidemia of Gram-negative bacterial sepsis may involve
pathomorphological changes in liver sinusoidal endothelial
cells. Int J Infect Dis 14: e857–e867.
Cheluvappa R, Eri R, Luo AS, Grimm MC (2015). Modulation
of interferon activity-associated soluble molecules by
appendicitis and appendectomy limits colitis-identification of
novel anti-colitic targets. J Interferon Cytokine Res 35: 108–
115.
Cohen BJ, Loew FM (1984). Laboratory Animal Medicine:
Historical Perspectives in Laboratory Animal Medicine.
Academic Press Inc, Orlando, Florida.
Commission European (2010). Directive 2010/63/EU of the
European Parliament and the Council on the protection of
animals for scientific purposes. Off J Eur Union L276: 33–69.
Committee for the Purpose of Control and Supervision of
Experiments on Animals (1998). Breeding of and experiment
on Animals (Control) and (Supervision) Rules, 1998. Gazette
of India, Extraordinary. Part II, Section 3(Sub-section (ii) vide
S.O. No. 809).
Guide for the Care and Use of Laboratory Animals. 8th
edition. (2011). Committee for the Update of the Guide for
the Care and Use of Laboratory Animals. In Guide for the
Care and Use of Laboratory Animals. 8th edition. Washington
(DC): National Academies Press (US); 2011.
Council of Europe (1986). European convention for the
protection of vertebrate animals used for experimental and
other scientific purposes. European Treaty Series – No. 123.
Cozigou G, Crozier J, Hendriksen C, Manou I, RamirezHernandez T, Weissenhorn R (2015). The European
Partnership for Alternative Approaches to Animal Testing
(EPAA): promoting alternative methods in Europe and
beyond. J Am Assoc Lab Anim Sci 54: 209–213.
Craig EA, Wang NC, Zhao QJ (2014). Using quantitative
structure-activity relationship modeling to quantitatively
predict the developmental toxicity of halogenated azole
compounds. J Appl Toxicol 34: 787–794.
2017 | Vol. 5 | Iss. 4 | e00332
Page 12
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.
Devillers J, Mombelli E (2010a). Evaluation of the OECD
QSAR Application Toolbox and Toxtree for estimating the
mutagenicity of chemicals. Part 1. Aromatic amines. SAR
QSAR Environ Res 21: 753–769.
Devillers J, Mombelli E (2010b). Evaluation of the OECD
QSAR Application Toolbox and Toxtree for estimating the
mutagenicity of chemicals. Part 2. alpha-beta unsaturated
aliphatic aldehydes. SAR QSAR Environ Res 21: 771–
783.
Devillers J, Mombelli E, Samsera R (2011). Structural alerts for
estimating the carcinogenicity of pesticides and biocides. SAR
QSAR Environ Res 22: 89–106.
Ewing T (1989). Animal experimentation: Australian code of
practice. Nature 339: 412.
Francione G (2010). Rain without thunder: The ideology of the
animal rights movement. edn. Temple University, Philadelphia,
PA.
Garner R (2013). A theory of justice for animals: Animal rights
in a nonideal world. edn. OUP, USA.
Golbamaki A, Cassano A, Lombardo A, Moggio Y,
Colafranceschi M, Benfenati E (2014). Comparison of in silico
models for prediction of Daphnia magna acute toxicity. SAR
QSAR Environ Res 25: 673–694.
Greek CR, Greek JS (2000). Sacred Cows and Golden Geese.
The Continuum International Publishing Group, Inc., New
York, New York.
Hagelin J, Hau J, Carlsson HE (2003). The refining influence
of ethics committees on animal experimentation in Sweden.
Lab Anim 37: 10–18.
Hai R, Chu A, Li H, Umamoto S, Rider P, Liu F (2006).
Infection of human cytomegalovirus in cultured human
gingival tissue. Virol J 3: 84.
Home Office (2014). Research and testing using animals.
Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin
HY, Ingber DE (2010). Reconstituting organ-level lung
functions on a chip. Science (New York, N.Y.) 328: 1662–1668.
Huh D, Kim HJ, Fraser JP, Shea DE, Khan M, Bahinski A,
et al. (2013). Microfabrication of human organs-on-chips. Nat
Protoc 8: 2135–2157.
ICLAS – International Council for Laboratory Animal Science
(2010). ICLAS Working Group on Harmonization of
Guidelines on the Use of Animals in Science., Ethical. review
of proposals to use animals in science.
Kaluzhny Y, Kandarova H, Hayden P, Kubilus J,
d’Argembeau-Thornton L, Klausner M (2011). Development
of the EpiOcular(TM) eye irritation test for hazard
identification and labelling of eye irritating chemicals
in response to the requirements of the EU cosmetics
directive and REACH legislation. Altern Lab Anim 39:
339–364.
Kant I (1788). The Critique of Practical Reason. edn.
Klausner M, Ayehunie S, Breyfogle BA, Wertz PW, Bacca L,
Kubilus J (2007). Organotypic human oral tissue models for
toxicological studies. Toxicol In Vitro 21: 938–949.
Kretlow A, Butzke D, Goetz ME, Grune B, Halder M, Henkler
F, et al. (2010). Implementation and enforcement of the 3Rs
principle in the field of transgenic animals used for scientific
purposes. Report and recommendations of the BfR expert
workshop, May 18–20 (2009), Berlin, Germany. Altex 27: 117–
134.
Kubilus J, Hayden PJ, Ayehunie S, Lamore SD, Servattalab
C, Bellavance KL, et al. (2004). Full Thickness EpiDerm: a
dermal-epidermal skin model to study epithelialmesenchymal interactions. Altern Lab Anim 32(Suppl 1A):
75–82.
Langezaal I, Hoffmann S, Hartung T, Coecke S (2002).
Evaluation and prevalidation of an immunotoxicity test based
on human whole-blood cytokine release. Altern Lab Anim 30:
581–595.
Lappin G, Noveck R, Burt T (2013). Microdosing and drug
development: past, present and future. Expert Opin Drug
Metab Toxicol 9: 817–834.
van Leeuwen K, Schultz TW, Henry T, Diderich B, Veith GD
(2009). Using chemical categories to fill data gaps in hazard
assessment. SAR QSAR Environ Res 20: 207–220.
Martonen T, Fleming J, Schroeter J, Conway J, Hwang D
(2003). In silico modeling of asthma. Adv Drug Deliv Rev 55:
829–849.
Mombelli E, Devillers J (2010). Evaluation of the OECD (Q)
SAR Application Toolbox and Toxtree for predicting and
profiling the carcinogenic potential of chemicals. SAR QSAR
Environ Res 21: 731–752.
Montag T, Spreitzer I, Loschner B, Unkelbach U, Flory E,
Sanzenbacher R, et al. (2007). Safety testing of cell-based
medicinal products: opportunities for the monocyte activation
test for pyrogens. Altex 24: 81–89.
Monteiro-Riviere NA, Inman AO, Snider TH, Blank JA,
Hobson DW (1997). Comparison of an in vitro skin model to
normal human skin for dermatological research. Microsc Res
Tech 37: 172–179.
Morton DB (1998). The importance of non-statistical design
in refining animal experiments. ANZCCART News 11: 1.
Olsson AS (2010). Ethics of Animal Research. In Handbook of
Laboratory Animal Science Volume 1(3rd Edition: Essential
Principles and Practices), Jann Hau, Steven J. Schapiro, eds.
CRC Press, Taylor & Francis Group.
Raunio H, Taavitsainen P, Honkakoski P, Juvonen R,
Pelkonen O (2004). In vitro methods in the prediction of
kinetics of drugs: focus on drug metabolism. Altern Lab Anim
32: 425–430.
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 4 | e00332
Page 13
R. Cheluvappa et al. Animal Research – Ethics, Teaching, Alternatives
Richarz AN, Schultz TW, Cronin MT, Enoch SJ (2014).
Experimental verification of structural alerts for the protein
binding of sulfur-containing compounds. SAR QSAR Environ
Res 25: 325–341.
Rowlands M (2013). Animal rights. edn. Wiley Online Library,
New Jersey.
Rowsell HC (1986). Regulation of animal experimentation:
Canada’s program of voluntary control. Acta Physiol Scand
Suppl 554: 95–105.
Russell WMS, Burch RL (1959). The Principles of Humane
Experimental Technique. Universities Federation for Animal
Welfare. (Originally: Methuen & Co. Ltd), London.
Schindler S, von Aulock S, Daneshian M, Hartung T (2009).
Development, validation and applications of the monocyte
activation test for pyrogens based on human whole blood.
Altex 26: 265–277.
Semlin L, Schafer-Korting M, Borelli C, Korting HC (2011). In
vitro models for human skin disease. Drug Discovery Today
16: 132–139.
Sexton K, Balharry D, Brennan P, McLaren J, Brewis IA,
BeruBe KA (2011). Proteomic profiling of human respiratory
epithelia by iTRAQ reveals biomarkers of exposure and harm
by tobacco smoke components. Biomarkers 16: 567–576.
Sheasgreen J, Klausner M, Kandarova H, Ingalls D (2009). The
MatTek story – how the three Rs principles led to 3-D tissue
success!. Altern Lab Anim 37: 611–622.
Sladowski D, Kinsner A, Langezaal I, Kay S, Coecke S (2001).
Activation of the complement system as an indicator of
pyrogenic reaction to lipopolysaccharide (LPS). Toxicol In
Vitro 15: 339–342.
Smith JA, van den Broek FA, Martorell JC, Hackbarth H,
Ruksenas O, Zeller W, Experiments FWGoEEoA (2007).
Principles and Practice in Ethical Review of Animal
Experiments across Europe: summary of the report of a
Federation of European Laboratory Animal Science
Associations (FELASA) Working Group on Ethical Evaluation
of Animal Experiments. Lab Anim 41: 143–160.
Tan M-W, Mahajan-Miklos S, Ausubel FM (1999). Killing of
Caenorhabditis elegans by Pseudomonas aeruginosa used to
model mammalian bacterial pathogenesis. Proc Natl Acad Sci
96: 715–720.
White S (2007). Regulation of animal welfare in Australia and
the emergent Commonwealth: entrenching the traditional
approach of the states and territories or laying the ground for
reform. Fed L Rev 35: 347.
Zaslavsky I, Baldock RA, Boline J (2014). Cyberinfrastructure
for the digital brain: spatial standards for integrating rodent
brain atlases. Front Neuroinform 8: 74.
Zurlo J, Rudacille D, Goldberg AM (1994). Animals and
Alternatives in Testing: History, Science, and Ethics. Mary Ann
Liebert Inc, New York.
2017 | Vol. 5 | Iss. 4 | e00332
Page 14
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Animal Research – Ethics, Teaching, Alternatives R. Cheluvappa et al.

---

Get Professional Assignment Help Cheaply

Are you busy and do not have time to handle your assignment? Are you scared that your paper will not make the grade? Do you have responsibilities that may hinder you from turning in your assignment on time? Are you tired and can barely handle your assignment? Are your grades inconsistent?

Whichever your reason is, it is valid! You can get professional academic help from our service at affordable rates. We have a team of professional academic writers who can handle all your assignments.

Why Choose Our Academic Writing Service?

• Plagiarism free papers
• Timely delivery
• Any deadline
• Skilled, Experienced Native English Writers
• Subject-relevant academic writer
• Adherence to paper instructions
• Ability to tackle bulk assignments
• Reasonable prices
• 24/7 Customer Support
• Get superb grades consistently

 

Online Academic Help With Different Subjects

Literature

Students barely have time to read. We got you! Have your literature essay or book review written without having the hassle of reading the book. You can get your literature paper custom-written for you by our literature specialists.

Finance

Do you struggle with finance? No need to torture yourself if finance is not your cup of tea. You can order your finance paper from our academic writing service and get 100% original work from competent finance experts.

Computer science

Computer science is a tough subject. Fortunately, our computer science experts are up to the match. No need to stress and have sleepless nights. Our academic writers will tackle all your computer science assignments and deliver them on time. Let us handle all your python, java, ruby, JavaScript, php , C+ assignments!

Psychology

While psychology may be an interesting subject, you may lack sufficient time to handle your assignments. Don’t despair; by using our academic writing service, you can be assured of perfect grades. Moreover, your grades will be consistent.

Engineering

Engineering is quite a demanding subject. Students face a lot of pressure and barely have enough time to do what they love to do. Our academic writing service got you covered! Our engineering specialists follow the paper instructions and ensure timely delivery of the paper.

Nursing

In the nursing course, you may have difficulties with literature reviews, annotated bibliographies, critical essays, and other assignments. Our nursing assignment writers will offer you professional nursing paper help at low prices.

Sociology

Truth be told, sociology papers can be quite exhausting. Our academic writing service relieves you of fatigue, pressure, and stress. You can relax and have peace of mind as our academic writers handle your sociology assignment.

Business

We take pride in having some of the best business writers in the industry. Our business writers have a lot of experience in the field. They are reliable, and you can be assured of a high-grade paper. They are able to handle business papers of any subject, length, deadline, and difficulty!

Statistics

We boast of having some of the most experienced statistics experts in the industry. Our statistics experts have diverse skills, expertise, and knowledge to handle any kind of assignment. They have access to all kinds of software to get your assignment done.

Law

Writing a law essay may prove to be an insurmountable obstacle, especially when you need to know the peculiarities of the legislative framework. Take advantage of our top-notch law specialists and get superb grades and 100% satisfaction.

What discipline/subjects do you deal in?

We have highlighted some of the most popular subjects we handle above. Those are just a tip of the iceberg. We deal in all academic disciplines since our writers are as diverse. They have been drawn from across all disciplines, and orders are assigned to those writers believed to be the best in the field. In a nutshell, there is no task we cannot handle; all you need to do is place your order with us. As long as your instructions are clear, just trust we shall deliver irrespective of the discipline.

Are your writers competent enough to handle my paper?

Our essay writers are graduates with bachelor's, masters, Ph.D., and doctorate degrees in various subjects. The minimum requirement to be an essay writer with our essay writing service is to have a college degree. All our academic writers have a minimum of two years of academic writing. We have a stringent recruitment process to ensure that we get only the most competent essay writers in the industry. We also ensure that the writers are handsomely compensated for their value. The majority of our writers are native English speakers. As such, the fluency of language and grammar is impeccable.

What if I don’t like the paper?

There is a very low likelihood that you won’t like the paper.

Reasons being:

• When assigning your order, we match the paper’s discipline with the writer’s field/specialization. Since all our writers are graduates, we match the paper’s subject with the field the writer studied. For instance, if it’s a nursing paper, only a nursing graduate and writer will handle it. Furthermore, all our writers have academic writing experience and top-notch research skills.
• We have a quality assurance that reviews the paper before it gets to you. As such, we ensure that you get a paper that meets the required standard and will most definitely make the grade.

In the event that you don’t like your paper:

• The writer will revise the paper up to your pleasing. You have unlimited revisions. You simply need to highlight what specifically you don’t like about the paper, and the writer will make the amendments. The paper will be revised until you are satisfied. Revisions are free of charge
• We will have a different writer write the paper from scratch.
• Last resort, if the above does not work, we will refund your money.

Will the professor find out I didn’t write the paper myself?

Not at all. All papers are written from scratch. There is no way your tutor or instructor will realize that you did not write the paper yourself. In fact, we recommend using our assignment help services for consistent results.

What if the paper is plagiarized?

We check all papers for plagiarism before we submit them. We use powerful plagiarism checking software such as SafeAssign, LopesWrite, and Turnitin. We also upload the plagiarism report so that you can review it. We understand that plagiarism is academic suicide. We would not take the risk of submitting plagiarized work and jeopardize your academic journey. Furthermore, we do not sell or use prewritten papers, and each paper is written from scratch.

When will I get my paper?

You determine when you get the paper by setting the deadline when placing the order. All papers are delivered within the deadline. We are well aware that we operate in a time-sensitive industry. As such, we have laid out strategies to ensure that the client receives the paper on time and they never miss the deadline. We understand that papers that are submitted late have some points deducted. We do not want you to miss any points due to late submission. We work on beating deadlines by huge margins in order to ensure that you have ample time to review the paper before you submit it.

Will anyone find out that I used your services?

We have a privacy and confidentiality policy that guides our work. We NEVER share any customer information with third parties. Noone will ever know that you used our assignment help services. It’s only between you and us. We are bound by our policies to protect the customer’s identity and information. All your information, such as your names, phone number, email, order information, and so on, are protected. We have robust security systems that ensure that your data is protected. Hacking our systems is close to impossible, and it has never happened.

How our Assignment  Help Service Works

1.      Place an order

You fill all the paper instructions in the order form. Make sure you include all the helpful materials so that our academic writers can deliver the perfect paper. It will also help to eliminate unnecessary revisions.

2.      Pay for the order

Proceed to pay for the paper so that it can be assigned to one of our expert academic writers. The paper subject is matched with the writer’s area of specialization.

3.      Track the progress

You communicate with the writer and know about the progress of the paper. The client can ask the writer for drafts of the paper. The client can upload extra material and include additional instructions from the lecturer. Receive a paper.

4.      Download the paper

The paper is sent to your email and uploaded to your personal account. You also get a plagiarism report attached to your paper.

PLACE THIS ORDER OR A SIMILAR ORDER WITH US TODAY AND GET A PERFECT SCORE!!!

---