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Moving forward in the ethical consideration of invertebrates

in experimentation: Beyond the Three R’s Principle

Augusto C. Crespi-Abril

1,3

Tamara Rubilar

2,3

1. Instituto Patagónico del Mar, Universidad Nacional del a Patagonia San Juan Bosco, Boulevard Brown, Puerto

Madryn, Argentina; crespi@cenpat-conicet.gob.ar (*Correspondence).

2. Laboratorio de Química de Organismos Marinos, Instituto Patagónico del Mar. Facultad de Ciencias Naturales y

Ciencias de la Salud. Universidad Nacional de la Patagonia San Juan Bosco. Bv. Brown, Puerto Madryn, Chubut,

Argentina; rubilar@cenpat-conicet.gob.ar

3. Laboratorio de Oceanografía Biológica, Centro para el Estudio de Sistemas Marinos. Boulevard Brown, Puerto

Madryn, Argentina.

Received 11-VII-2020. Corrected 24-X-2020. Accepted 06-XI-2020.

ABSTRACT

Introduction: The Three R´s Principle (Refinement, Reduction, and Replacement), postulated more than 60

years ago, is the main ethical framework currently applied for conducting animal research. This principle has

never been reviewed applying a philosophical reflection during all of these years, even though a variety of ani-

mal ethics studies have presented new insights. The Three R´s Principle was designed to be used as a policy tool

to ameliorate the suffering of animals and to reduce the use of animals in research, but has failed in achieving

these goals. This principle is only applied when using sentience vertebrates, and fails to consider invertebrates

as their capacity to sentience is still disputed. In this way, invertebrates are reified, which has been determined

to be detrimental as their suffering has been consistently denied. As a consequence, new insights are necessary

to improve scientific practices. Epistemology and ethics have always been viewed as opposing approaches.

´Epistemology-based Ethics’ subordinate ethical concern to scientific facts and ‘Ethics-based Epistemology’

purports that ethical practice should guide epistemological practices. Objective: In this paper, we maintain

that unifying both approaches under a broader conceptual framework may result in the view that these are not,

actually, opposite approaches. We propose to progress beyond the Three R’s Principle and extend it to a posi-

tion equal to the level of the ethical and epistemological approaches. We also propose to use the Precautionary

Principle as it is always better to be safe than sorry, and to include two more Rs. Methods: This paper is based

on the analysis of different ethical frameworks used in biology and ecology that can be implemented in inver-

tebrates experimentation. Results: The analysis revealed that different ethical approaches are frequently used

in biological research, but not all of them are implemented in experimental research that involves invertebrates.

We argue that the ethical considerations used in any research field can be implemented in invertebrate research.

Conclusion: We propose a Five R´s Principle: the traditional Refinement, Reduction, and Replacement, used

along with Respect and Responsibility (a respectful relationship with every living being regardless of its com-

plexity and personal commitment to conscientiously apply ethics concepts).

Key words: five R´s Principle; responsibility; respect; precautionary principle; animal ethics.

Crespi-Abril, A.C., & Rubilar, T. (2021). Moving forward

in the ethical consideration of invertebrates in

experimentation: Beyond the Three R’s Principle.

Revista de Biología Tropical, 69(S1), 346-357. DOI

10.15517/rbt.v69iSuppl.1.46366

DOI 10.15517/rbt.v69iSuppl.1.46366

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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 346-357, March 2021 (Published Mar. 30, 2021)

Non-human animals (hereinafter referred

to as animals) have been used in experiments

for more than two thousand years. The majority

of these experiments were done on vertebrate

animals (and some in cases on invertebrates)

as they were considered adequate models of

human anatomy and physiology (von Staden,

1989). During many years, an anthropocentric

view dominated and animals were not con-

sidered subjects of ethical concern. However,

the relationship between humans and animals

has changed through time and ethical concern

regarding animals started to increase due to the

evident suffering animals were experiencing in

order to satisfy scientific enquiry. In this sense,

Bentham (1823) stated: “The question is not,

Can they reason? Nor, Can they talk? But, Can

they suffer?”. Although animal research was

acceptable under his utilitarian ideology (i.e., a

moral action is the one that results in the high-

est overall wellbeing for all stakeholders) as

benefits to mankind justified it, two hundreds

years later Bentham’s statement continues to

exert a great deal of influence in the debate on

animal use in the life sciences in general. The

recognition of animals’ sentience (capable of

experiencing suffering) was an important start-

ing point for the new philosophical thought

that would be crucial for the development of

new ethical perspectives concerning the moral

status of animals. Singer (1975) published his

work claiming for animal rights. This works is

a breakthrough since it provides an exhaustive

philosophical framework about ethics and ani-

mal suffering. In spite of its solid philosophical

ground, Singer’s ideas were controversial, but

they inspired a rising public concern about

animal rights that influenced animal experi-

ments (Horta, 2011). Currently, debate on

animals rights are on the table (Regan, 2003;

Regan, 2004) and significant progress has been

made at the normative level with the inclusion

of some groups of invertebrates (European

Parliament, 2010).

In 1959, Russell & Burch published their

work “The Principles of Human Experimental

Technique” where they postulated the Three

R’s Principle (Refinement, Reduction, and

Replacement) (Russell & Burch, 1959). How-

ever, this principle was largely ignored for more

than 20 years, until Smyth (1978) reinforced

the utilization of the three R’s by postulating

“all procedures which can completely replace

the need for animal experiments, reduce the

numbers of animals required, or diminish the

amount of pain or distress suffered by animals

in meeting the essential needs of man and other

animals”. This definition is not only a restate-

ment of the Three R’s, it also places the respon-

sibility onto researchers to provide robust

evidence that can justify the use of animals in

their research.

At present, the Three R’s Principle rep-

resents the main ethical framework for con-

ducting research with animals. However, this

principle was postulate over 60 years ago and

it is addressed without any major change. Even

though it is currently used to regulate the sci-

entific use of animals (Bayne, Ramachandra,

Rivera, & Wang, 2015; Burden, Chapman,

Sewell, & Robinson, 2015), it has been recent-

ly questioned due to its limitations in consid-

ering all the cases of animal use in research

(Rubilar & Crespi-Abril, 2017; Crespi-Abril &

Rubilar 2018; Hermann & Jayne, 2019). In this

work, we provide new insights that support the

ethical consideration of the majority (if not all)

of cases in which animals are used in research.

Based on our arguments, we propose a broader

principle that can be used as an ethical frame-

work in research with animals.

Invertebrates in experiments

Invertebrates represents a heterogeneous

group of animals that consist in more than 30

phyla ranging from sponges (extremely simple

multicellular organisms) to cephalopods (organ-

isms that display complex behaviors and can

solve simple problems). Invertebrates accounts

for more than 95 % of the total species on Earth

(Kellert, 1993; Wilson, 1999) which represents

more than 1 million different species.

Invertebrates are frequently used in

experiments since they present a series of

advantages over vertebrates such as: relatively

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simple to maintain and culture, simpler and

easier to manipulate organisms, shorter life

cycle (Smith, Scimeca & Mainous, 2011).

Additionally, invertebrates are considered as

adequate biological models for other species

of animals including humans (Horvarth et

al., 2013). The most recognized invertebrates

models are Drosophila melanogaster, Cae-

norhabditis elegans, Aplysia californica, sea

urchins, squids and honeybees. Significant

breakthroughs were conducted in biomedical

research using only this few groups of inverte-

brates (Wilson-Sanders, 2011).

Usually invertebrates (except for cephalo-

pods and decapods) are not included in the leg-

islation and welfare of these animals is mostly

neglected. This leads to a gap of knowledge

regarding the number of individuals used in

experiments since there is no need to record it

(Harvey-Clark, 2011). Although there are no

exact estimations, the numbers of individuals

of particular species (Drosophila melanogas-

ter, Caenorhabditis elegans, Aplysia califor-

nica) may reach millions in a single laboratory

(Harvey-Clark, 2011).

Three R’s principle: current situation

Three R’s Principle introduces the three

basic concepts of Refinement, Reduction and

Replacement which any researcher should

consider when conducting research involving

animals. Basically, Russell and Burch (1959)

defined Replacement as “any scientific method

employing non-sentient material [to] replace

methods which use conscious living verte-

brates”; Reduction as diminishing “the number

of animals used to obtain information of a

given amount and precision”; and Refinement

as a series of practices conducted to “decrease

in the incidence or severity of [...] procedures

applied to those animals which have to be

used”. These three concepts are considered

to incur the same level of significant but

were originally postulated as a sequence to

achieve the goal of total replacement as a

maxim: “[r]efinement is never enough, and we

should always seek further for reduction and

if possible replacement” (Russell & Burch,

1959). Clearly, this principle is only applied

when using vertebrates since they are the only

group of animals that are considered as sentient

animals due to their similarities with humans

in terms of the structure of the nervous system.

In this sense, the principle fails to consider

inclusion of invertebrates, whose capacity to

sentience is still disputed. A strong assumption

underlies the Three R’s: This is that non-human

animals which are lower on the zoological scale

lack sentience (Tomasik, 2014). This represents

a significant constraint, especially when it is

considered that the number (and diversity) of

invertebrates used in science is overwhelming.

Despite the worldwide acceptance of the Three

R’s as a policy tool to ameliorate the suffering

of animals and to reduce the use of animals in

research, the failure in achieving these goals

has been clearly noted (Blattner, 2019). In fact,

the number of animals used for experimental

purposes in the European Union (EU) is cur-

rently similar to the number registered in 1980s

(Taylor & Rego, 2016). The main cause of this

failure is that the language used in legislation

regarding the replacement concept is frequently

laxer that the language used in refinement.

As an example, the EU Directive 2010/63/

EU states “Member States shall ensure that,

wherever possible, a scientifically satisfactory

method or testing strategy, not entailing the

use of live non-human animals, shall be used

instead of a procedure” (European Parliament,

2010, Article 4). This implies that replacement

is only necessary if alternatives exist and are

recognized by the normative. However, this

comprises a limitation as, in practice, replace-

ment alternatives are barely recognized in

legislation and when they are, they do not

refer to replacement in an absolute sense, but

to fewer sentient animals. These conditions

fail to exhort researchers to stop using animals

in research. This, in turn, leads to an implicit

hierarchical understanding of the Three R’s in

which refinement and reduction take priority

over replacement (Gerritsen, 2015).

Relation between Ethics and Epistemol-

ogy: alternative frameworks

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In this section, we introduce the main rela-

tion between ethics and epistemology, which

represents a metaethical analysis. However, it

is not the aim of this paper to address exhaus-

tively the field of metaethics.

Significant advances have been made on

ethical considerations in animal experiments.

Proof of this is seen in the fact that the Three

R’s Principle govern research practices. How-

ever, the principle is not applied to any species,

but only to those considered sentient. In this

sense, it is crucial to gather evidence to ensure

that a particular species is sentient by estab-

lishing objective criteria involving behavioral,

evolutionary and physiological considerations.

Sentience is very complex to demonstrate, and

researchers have been focused on demonstrat-

ing the existence of pain, as it is assumed to

comprise a particularly important form of

suffering (EFSA Panel on Animal Health and

Welfare, 2005). In 2012, it was published the

Cambridge Declaration on Consciousness on

which scientific community concludes that ani-

mals are conscious living beings able of expe-

riencing negative emotional sensations (pain).

The experience of pain is based on the

presence of two components: nociception

(physiological detection of nocive stimuli),

and the experience of pain (negative emotional

sensation produced after a nocive stimuli). If

either of these two components is absent, it is

assumed that there is no possibility of experi-

encing pain. If nociception is absent, stimuli

are not detected, and if emotional sensations

are absent, pain does not occur (nociceptive

reflex responses). Seven criteria are applied to

determine if individuals of a particular species

are capable of experiencing pain (Smith, 1991;

EFSA Panel on Animal Health and Welfare,

2005), and these can be summarized as follows

(Andrews et al., 2013):

1. Possession of receptors sensitive to

noxious stimuli, located in functionally

useful positions on or in the body and con-

nected by nervous pathways to the lower

parts of the nervous system

2. Possession of higher brain centres (in the

sense of integration of brain processing),

especially a structure analogous to the

human cerebral cortex

3. Possession of nervous pathways connec-

ting the nociceptive system to the higher

brain centres

4. Receptors for opioid substances found in

the central nervous system, especially the

brain

5. Analgesics modify the animal’s respon-

se to stimuli that would be painful for a

human

6. An animal’s response to stimuli that would

be painful for a human is functionally

similar to the human response (that is, the

animal responds so as to avoid or minimize

damage to its body)

7. An animal’s behavioral response persists

and it shows an unwillingness to resubmit

to a painful procedure; the animal can

learn to associate apparently non-painful

with apparently painful events

Most vertebrates fulfill these criteria and

there is no doubt as to their capacity of expe-

riencing pain. But in the case of invertebrates

this is not that clear, and it is an area of long-

standing concern and controversy (Fiorito,

1986; Mather, 2016) as the majority of the

established criteria for pain are not met. This is

a fallacious argument, as ‘absence of evidence

is not evidence of absence’. If one considers

the large biodiversity of invertebrates (Kellert,

1993; Wilson, 1999) and the large variability in

‘bauplan’ (which includes the nervous system),

it is likely that the capacity of experiencing

pain can be achieved by other mechanisms

and structures differing from those described

for vertebrates (Riebli & Reichert, 2015). For

instance, cephalopods are considered to be an

exception as it has been already assumed that

they can suffer and they were included in the

ethical normative (Andrews et al., 2013; Smith

et al., 2013; Della Rocca, Di Salvo, Giannet-

toni, & Goldberg, 2015) This applies in spite of

the fact they have a different nervous system as

compared to vertebrates. However, after much

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research effort, controversy still remains due

to the inherent difficulty of demonstrating the

capacity of experiencing pain by this group of

invertebrates (Harvey-Clark, 2011). With this

framework, it is assumed that we first must

evidence that animals are capable of suffering

pain and, then, we should seek ethical consider-

ations. This approach is denominated ‘Episte-

mology-based Ethics’ and subordinates ethical

concern to scientific facts (Chandroo, Yue, &

Moccia, 2004; Griffin & Speck, 2004; Elwood,

2011; Lewbart & Mosley, 2012; Mather, 2016).

A different approach has been proposed

in which the relationship between epistemol-

ogy and ethics is inverted where: ethics should

guide epistemological practices. This implies

that there is an initial need to establish a

respectful relationship with living beings rather

than a facts-based relationship. This, implies a

limit (ethical one) on our actions, resulting in

the need to rethink whether knowledge regard-

ing the biology, physiology, ecology of animals

should take precedence over the welfare of

those living beings. In addition, it is impor-

tant to note in this context that the utilitarian

rationale purporting that human interest takes

precedence over animal suffering is an insuffi-

cient ethical justification for the use of animals

in experimentation as animals and humans

equal rights as living beings. This approach

is denominated ‘Ethics-based Epistemology’

(Cheney & Weston, 1999; Weston, 2009). This

insight has been implemented in research with

invertebrates, particularly regarding echino-

derms (Rubilar & Crespi-Abril, 2017; Crespi-

Abril & Rubilar, 2018). To biologists, this

different approach may sound counterproduc-

tive as the focus of our own research may be,

in fact invertebrates. However, we believe

that science can be exercised within a new

bio-ethical framework that includes these new

concepts and ideas.

Precautionary Principle

The precautionary principle aims to

take actions to avoid harm even though evi-

dence is insufficient to provide certainties on

the magnitude or probability of happening

(Raffensperger, 1999; Kriebel et al., 2001). A

simple way to phrase this is: until the risk of

harm is controlled, be cautious, as it is better

to be ‘safe than sorry’. This rationale guides

the vast majority of human, ordinary activi-

ties. This is not the same in current scientific

practices involving animal experiments. As we

stated above, the current approach is that until

we do have the certainty that animals are capa-

ble of experiencing suffering (harm), we will

not, ethically, give them consideration (action).

Why do we act this way in science? The answer

to this point does not rely on the fact that the

principle leads in the wrong direction, but

in the thought that it leads in no direction at

all and threatens to be paralyzing (Sunstein,

2003). This thought generates resistance in the

researcher community to start considering an

ethical approach in scientific practices. With

vertebrates there is a clear consensus on this

point, but most invertebrates are still not treated

relying on bio-ethical basis.

The precautionary principle is stated by

incorporating different levels of restrictions

that can be grouped into weak or strong ver-

sions. The weak version is less restrictive and

allows preventive actions under the existence

of the risk of harm, but does not require such

preventive actions. Often, cost-benefits analy-

ses are considered in order to postpone preven-

tive actions and these analyses include factors

other than scientific certainty, such as eco-

nomic factors. In this case, the burden of proof

(the requirements to justify) lies with those

advocating precautionary actions. On the other

hand, the strong version requires the taking of

precautionary actions in the presence of the risk

of harm, in spite of the costs incurred in doing

so. Here, the burden of proof is inverted and

lies with those who argue that certain activities

will not cause significant harm. This means

that society is not willing to accept any envi-

ronmental risk, no matter the benefits (social

or economic) that may be achieved (Di Salvo &

Raymond, 2010). Four types of the precaution-

ary principle were distinguished capturing both

weak and strong types by Stewart (2002):

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• Non-preclusion Precautionary Principle:

Scientific uncertainty should not automati-

cally preclude that regulation of activities

posing a potential risk of significant harm.

• Margin of Safety Precautionary Principle:

Regulatory controls should incorporate a

margin of safety; activities should be limi-

ted below the level at which no adverse

effect has been observed or predicted.

• Best Available Technology Precautionary

Principle: Activities that present an uncer-

tain potential for significant harm should

be subject to best technology available

requirements to minimize the risk of harm

unless the proponent of the activity shows

that they present no appreciable risk of

harm.

• Prohibitory Precautionary Principle: Acti-

vities that present an uncertain potential

for significant harm should be prohibited

unless the proponent of the activity shows

that it presents no appreciable risk of harm.

Currently scientific practices involving

animal experiments are guided under a weak

precautionary principle as it is necessary to

demonstrate (to show scientific certainty) the

existence of pain (harm) in order to take

actions to diminish such pain and always only

after a cost-benefit analysis has taken place to

determine the benefit of the scientific practice.

In an opposing position, one finds the ideals

postulated by activists whose claims argue

for a strong version of precautionary principle

requiring the demonstration that pain is not

present (absence of harm). Both of these are

extreme positions and there are several flaws in

the justification of each posture. Here, we pro-

pose to reach a position in the middle ground.

Five R’s Principle:

beyond Russell´s proposal

Problems emerging from an ethical

approach regarding animal experimentation

are more widely appreciated than the episte-

mological ones. Most researchers agree that

subordinating ethics into epistemology (Epis-

temology-based Ethics), provides rigorous sci-

entific ground whilst an empathic approach

with animals (Ethic-based Epistemology) may

bias experiments and conclusions due to the

extreme reduction in the number of animals

used or to the use of inappropriate replace-

ments. In this way, it is assumed that keep-

ing ethics out of the picture will provide the

objectivity that is crucial for science. However,

researcher desensitization may bias the results

of experiments as animals may be kept in inap-

propriate environmental conditions (e.g. small

spaces, intense noise, lack of environmental

stimulation, poor quality of seawater, under

fed, etc.) which produce significant levels of

stress in the animals. This causes epistemologi-

cal problems as stress impacts on the physiol-

ogy of individuals, hence, modifying their

response to treatments and, thereby, affecting

the reliability of scientific data obtained from

the animals (Baldwin, Primeau & Johnson,

2006; Burwell & Baldwin, 2006). None of

these approaches are completely satisfactory

since they present flaws, either at the episte-

mological level or ethical level. Consequently,

there is no difference in our choice of levels to

guide our scientific practices; in either case, we

will not be able to fulfill epistemological and

ethical grounds. One important point that is

supposed to be a barrier in this context is that

both approaches are seen to comprise oppo-

site and irreconcilable frameworks, instead

of complementary. We maintain that unifying

both approaches under a broader conceptual

framework may lead to the possibility of over-

coming previous flaws.

The Three Rs’ Principle was thought to

provide ethical grounds (although limited only

to certain species) for animal experimentation,

but were not to address the epistemological

problems arising from animal manipulation

(Johnson & Degeling, 2012). We maintain that

this principle needs to be extended in order to

unify both approaches into a broader concep-

tual framework that considers the ethical and

epistemological grounds at the same level here

proposed to expand the principle including

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two concepts involving personal commitment:

Respect and Responsibility (Fig. 1).

Respect

Respect points to the establishment a

respectful relationship with any living beings,

regardless of their complexity or the knowl-

edge we have of them. This implies that we

are to be respectful with life, itself, rather

than being in a facts-based relationship. The

use of animals in experimentation implies a

restriction on the ethical consideration to them.

This not only implies leaving out from ethical

consideration several individuals (such as any

invertebrate), but also implies the restriction of

our ethical actions to those animals which are

considered ethically (e.g. individual suffering

is justified if it is in favor of human benefits).

Researchers often carry out procedures that

have been defined and required by others (such

as institutions or colleagues), which means

that their ethical autonomy is not considered

when conducting experiments with animals.

They are trained to desensitize, by reframing

their ethical relationship with animals, in order

to accomplish the job when it involves ani-

mal experiments, as these experiments always

imply animal suffering (Capaldo, 2004). At this

point, researchers are no longer empathetically

related with animals but, instead, take a neutral

observer perspective, intentionally avoiding

any commitment with them. This perspective

distorts the ability to relate to animals by pro-

moting reification of them (Honneth, 2006).

Reification is very risky for those who are

reified, since their interests or suffering are per-

manently denied (Johnson & Smajdor, 2019).

An empathic relationship with animals

does not imply a barrier to achieving knowl-

edge about them or to the use of the animals

according to our needs. Although an empathic

relationship in science is an innovative insight,

it is worth noting that it is not fanciful or

implausible, and is therefore a strategy that is

worth considering. There are solid evidences

among empathic relation and emotions com-

munication between mammals and this could

provide a novel field for studying empathic

relations in other animals groups (Preston & de

Wall, 2002; de Waal & Preston, 2017). There

are two different approaches in which the key

point is empathy. These are the Traditional

Ecological Knowledge (TEK) and Animal-

as-Patients approaches. The first one involves

three aspects of nature: the cosmos (beliefs,

emotions and symbolic representations), the

corpus (general environmental knowledge),

and the praxis (the behaviors carried out in

relation to the use of Nature). Advances com-

ing from ethnoecology revealed that several

cultures relate with environment in a respect-

ful manner (Toledo & Barrera-Bassols, 2008).

They consider that environment is populated

by living beings that have social relation-

ships between themselves and with whom they

establish social relationships. This provides a

conceptual framework allowing a responsible

management of natural resources, transcending

mere utilitarian and economic aspects (Gadgil,

Berkes, & Folke, 1993; Berkes, Colding, &

Folke, 2000). This implies a type of knowledge

that can be fully understood only if symbol-

isms and meanings that are attributed to natural

entities are considered (Reyes-García & Martí,

2007). The second one, is to approach animals

during experiments as patients (Pemberton,

2004; Haraway, 2008). This means to shift our

current actions undertaken during experiments

to treating animals as individual patients rather

than as mass-produced and expendable objects.

This means that animals should be included

in experiments in a similar way as humans

are enrolled into clinical trials (Johnson &

Degeling, 2012). This approach is already

implemented in animal experimentation in vet-

erinary practice. When an animal is put through

an experimental treatment, the care and welfare

of the individual is not only evaluated by the

neutral (professional) position of the veterinar-

ian, but also by the empathy and feelings of

the owner.

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Responsibility

Responsibility when conducting animal

experiments is the fifth concept that completes

our proposal. The essence of scientific work

requires a strong professional ethics since

it is based on the confidence of the scien-

tific community in the honesty of research-

ers. Although there is a rigorous peer review

process, honesty in performing experiments,

conducting data analysis and reporting the

results is crucial. We argue in favor of expand-

ing this responsibility and of including ethical

commitment with animals as a part of our prac-

tices. Responsibility has already invoked by

the Max Planck Society (MPG, 2020), mainly

focusing on promoting animal welfare. In our

proposal, responsibility calls for the personal

commitment of researchers to conscientiously

apply the ethical concepts. This goes beyond

just promoting welfare, but also promotes the

rethinking as to whether knowledge regarding

the biology, physiology, ecology or medicine

should be prioritized over the welfare of the

animals involved in experiments. We need to

act responsibly when using animals in experi-

ments considering the real value of the life of

each living being, and we need to be honest

when asking if the life of any animal worth the

knowledge obtained. The most controversial

point is, of course, the use of animal experi-

ments in medicine (Knight, 2019). There is a

long tradition in using animals as physiological

models in medicine, and currently this is an

unavoidable step in medical trials in spite of

the fact that there is overwhelming evidence

that animals are not appropriate models (Ram,

2019). The unquestioned practice of using

animals in medicine not only has ethical con-

cerns, but also implies a problem in terms of

human health (Greek & Kramer, 2019). This

is a clear example of a scientific practice that

can not stand a critical and responsible review

and would need to be abandoned in most cases

(Archibald, Coleman, & Drake, 2019).

Concluding Remarks

The current framework allows researchers

to relieve their ethical commitment through

the implementation of the Three R’s Prin-

ciple. However, this is not enough for ethically

responsible science. The Three R’s Principle

comprised a starting point in establishing an

ethical approach in science but has remained

almost unmodified for more than 60 years.

As a community, we owe a critical review of

our approaches to animals in experiments. It

is necessary to keep permanently revising our

approaches in order to develop new insights.

We propose to extended the Three R Principle

to a position in which the ethical and episte-

mological approaches are at the same level.

This change may seem difficult to implement,

but this should not be a barrier to developing

new ways of thinking. All significant changes

were seen as impossible when first postulated

and are now commonplace. Scientific practice

is no exception to the rule, it is worth to keep

promoting ethical concern and real commit-

ment in terms of how researchers relate to ani-

mals in the context of their own work and how

this can be improved. We must stop accepting

practices that in the long-term perpetuate the

use of animals in experiments as an accept-

able scientific standard. We are aware that

this may not be seen as a straightforward way

Fig. 1. Five R´s Principle. Equal level of importance of the

ethical and epistemological approaches.

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of implementing changes, but communication

between stakeholders (e.g. researchers, vet-

erinarians, animal welfare activists, competent

authorities) is crucial to promote change and

keep moving forward on ethical commitment in

animal experimentation. It is very important to

include whole society in the debate since public

perception and opinion is a strong influence in

defining scientific practices and it is a chal-

lenge in invertebrates research (Drinkwater,

Robinson, & Hart, 2019).

We propose to use the Precautionary Prin-

ciple along with the Five R´s Principle, includ-

ing Refinement, Reduction, Replacement but

also Respect (to establish a respectful rela-

tionship with any living being regardless its

complexity or the knowledge we have of

that living being) and Responsibility (personal

commitment of researchers to conscientiously

apply ethics concepts). Under this new pro-

posal, invertebrates are at the same level of

ethical concern as vertebrates. This means that

institutional animal care and use committee

(IACUC) should evaluate all research project

that requires the use of any invertebrate, and

not only those projects that involves verte-

brates. The high diversity of invertebrates

requires that IACUC address with particular

attention every case and this challenging for

committee members (Harvey-Clark, 2001).

However, in order for our proposal to succeed,

it must be embraced in all of the different roles

we as researchers assume and execute. First, in

our own practices in the laboratory, then in our

teaching (e.g. universities, schools, courses,

etc) but, most crucially, in our role as review-

ers. We can change the balance within and shift

our own practices, but we can do even more in

influencing others to follow in our tracks and,

in the manner, initiate a real change.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgements

section. A signed document has been filed in

the journal archives.

ACKNOWLEDGMENTS

We would like to thanks to Kathleen

Anderson for the revision of the English and

criticism of the manuscript. In addition, we

thank the anonymous reviewers for their

comments, which improved significantly the

manuscript.

RESUMEN

Avanzando sobre la consideración ética

de los invertebrados en experimentación:

más allá del principio de las tres Rs

Introducción: El Principio de las Tres R (Refina-

miento, Reducción y Reemplazo), postulado hace más

de 60 años, es el principal marco ético que se aplica

actualmente para realizar investigaciones en animales. Este

principio no ha sido revisado desde una reflexión filosófica

a pesar de tantos años y de lavariedad de estudios en ética

animal que han presentado nuevas ideas. Este principio

solo se aplica cuando se utilizan vertebrados sintientes,

y no tiene en cuenta a los invertebrados, ya que su capa-

cidad de sentiencia aún está cuestinada. De esta manera,

los invertebrados son cosificados, lo que ha sido en su

detrimento ya que su sufrimiento ha sido constantemente

negado. El Principio de las Tres R fue diseñado para ser

utilizado como una herramienta de política para mejorar

el sufrimiento de los animales y reducir el uso de anima-

les en la investigación, pero no ha logrado alcanzar estos

objetivos. Como consecuencia, se necesitan nuevas ideas

para mejorar las prácticas científicas. La epistemología y

la ética siempre se han visto como enfoques opuestos. La

concepción ética subordinada de a los hechos científicos se

llama “Ética basada en la epistemología” y, en cambio, la

“Epistemología basada en la ética” pretende que la práctica

ética guie las prácticas epistemológicas. Objetivo: En este

trabajo, proponemos que unificar ambos enfoques bajo un

marco conceptual más amplio y eliminar la percepción

de que son enfoques opuestos. Proponemos avanzar más

allá del Principio de las Tres R , extenderlo y colocar

un mismo nivel a los enfoques éticos y epistemológicos.

También proponemos utilizar el Principio de precaución,

ya que siempre es mejor prevenir que curar e incluir dos

R más. Métodos: El trabajo está basado en un análisis de

diferentes corrientes éticas utilizadas en biología y ecología

que pueden ser implementadas en la experimentación con

invertebrados. Resultados: El análisis de los trabajos reve-

ló que hay diferentes posturas éticas que se usan frecuente-

mente en biología, pero no todas ellas son implementadas

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en la experimentación con invertebrados. Bajos nuestra

argumentación sostenemos que las consideraciones éticas

usadas en cualquier campo de investigación puede ser

implementada en la experimentación con invertebrados.

Conclusión: De esta manera, proponemos un Principio de

las Cinco R: Refinamiento, Reducción, Reemplazo para

ser usados junto con el Respeto y la Responsabilidad (una

relación respetuosa con cada ser vivo independientemente

de su complejidad y un compromiso personal de aplicar

conscientemente los conceptos éticos).

Palabras clave: principio de las Cinco R; responsabilidad;

respeto; principio de precaución; ética; epistemología.

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