3R Principle (ethics)
The 3R Principle, also known as the "3Rs," is a fundamental concept in animal research and experimentation aimed at minimizing harm and improving the welfare of animals used in scientific studies. The 3Rs stand for: Replacement, Reduction and Refinement.
You may also be interested in other methods and concepts that are presented in detail here:
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HUMANE EXPERIMENTAL TECHNIQUE AND THE 3RS
“The principles of humane experimental technique” is an ethical framework that intends to improve laboratory animals' welfare [1, 2]. It was formulated by W. M. S Russell and L. R. Burch in 1959 and will be cited as “Russell and Burch 1959 ” (Figure 1) [1, 2] . The foundation of Russell's and Burch's proposals to reduce experimental animal suffering are the three main principles of Replacement, Reduction, and Reﬁnement of studies with conscious “higher animals” ("The 3Rs") [1-4].
Figure 1: W. M. S Russell & L. R. Burch
The ethical agenda of the 3Rs was first entirely ignored by the scientific community and administrations [3, 5]. This slowly changed in the 1980s, and by now, the principles are globally implemented in all major science funding programs, government initiatives, and enactments . The ethical framework of the 3Rs was adopted almost unaltered since its original publication in 1959 to most regulations . Hence it is appropriate to review and recapitulate the ethical implications of the three Rs from 1959 and to relate them to our current work. In 2009 M. Balls published an revised and updated version of the principles: “The Three Rs and the Humanity Criterion” cited as “Balls, Russell and Burch, 2009” . The author kindly provided me with a copy of his excellent book, which builds the foundation of this summary and helps to evaluate the replacement of small mammals through insects as an alternative inflammation model for high-throughput imaging modalities. Since both publications are out of stock and out of print, direct quotes will be used to allow a genuine understanding of the Three Rs.
On a total of 229 pages, the word insect is only referred to seven times . This shows that the authors have only marginally dealt with invertebrates. In the introduction the authors say: ”[...] we have restricted the discussion entirely to the vertebrates […]. The higher invertebrates perhaps deserve a review to themselves, but they raise many problems which would gravely complicate an account which can otherwise be quite general and confident. Only one group, the insects, are of any numerical consequence in practice; the number of cephalopods used alive for experiment is small, though growing. Many experiments on insects are concerned with the development of substances that can be used to kill them, since the economic status of many insects as pests is urgent. Until a fully humane poison is developed for the actual control of rats, it is plainly premature to devote much thought to the research aspects of insecticides. The privileged status of vertebrates may appear arbitrary when compare, say, lamprey with octopus; but for simplicity and clarity, we shall stick to the traditional division (of animals into vertebrates and invertebrates), which has much to recommend it” (Balls, Russell and Burch, 2009, p 5).
In 1959 it was not clear that insects would emerge as a model system for many human diseases and represent an elementary part of basic biological and medical science [6-10]. But we will see that “The Principles” can still be applied very well to this kind of research.
The Humanity Criterion
Russell and Burch reject the Cartesians point of view, which denied nonhuman consciousness: “We shall not waste any time on those philosophers who would forbid us to speak of consciousness in nonhuman animals. In any case, it is now generally recognized that "consciousness" is a useless concept as an abstraction in its own right. All progress in this field has been achieved not by obsessional worry about what "consciousness" is, but by treating it as a variable and examining its different states.” (Balls, Russell and Burch, 2009, p 10). They defined the human-centric word humane (vs. inhumane) in a “[...] purely objective sense to characterize the kind of treatment actually applied to an animal” (Balls, Russell and Burch, 2009, p 9). By inhumane, they mean inflicting animals with distress . The absence of distress defines humane conditions . “We can therefore define distress of a certain degree (of whatever origin) as a central nervous state of a certain rank on a scale, in the direction of the mass autonomic response which, if protracted, would lead to the physiological stress syndrome. Inhumane procedures are those which drive the animal's mood down in rank towards this point. Removing inhumanity must ultimately mean driving the animal as near the other end of the scale as we can. More humane then means simply less inhumane.” (Balls, Russell and Burch, 2009, p 15). Invertebrates are not mentioned at all in the introduction of “the concept of inhumanity” .
Russell and Burch further distinguish direct and contingent inhumanity. “By the former, we mean the infliction of distress as an unavoidable consequence of the procedure employed, as such, even if it is conducted with perfect efficiency and completely freed of operations irrelevant to the object in view. By contingent inhumanity, on the other hand, we mean the infliction of distress as an incidental and inadvertent by-product of the use of the procedure, which is not necessary for its success. In fact contingent inhumanity is almost always detrimental to the object of the experiment, since it introduces psychosomatic disturbance likely to confuse almost any biological investigation” (Balls, Russell and Burch, 2009, p 29). Russell and Burch mainly address animal husbandry in terms of contingent inhumanity. Back in 1959, standardized animal husbandry was unheard-of, and experimental animals were kept under poor and unhygienic conditions [1, 2]. In addition to the ethical component, Russell and Burch point out that contingent inhumanity also affects the experiments' validity. This is, of course, correct and widely recognized in today's animal legislations and initiatives.
The 3Rs: Replacement, Reduction and Refinement
Russell and Burch described the 3Rs in this order and noted they should also be addressed in this order (Figure 2) [1, 2]. In the end, though, all three Rs should always be archived. Consequently, the replacement of small mammals with insects is a quite relevant subject in terms of the 3R.
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Figure 2: The 3R: Replacement, Reduction and Refinement of Russell and Burch from 1959
“We shall use the term "replacement technique" for any scientific method employing non-sentient material which may in the history of experimentation replace methods which use conscious living vertebrates” (Balls, Russell and Burch, 2009, p 35). Russell and Burch distinguishing three forms of replacement: Comparative Substitution, Absolute, and Relative Replacement.
“Among this non-sentient material, we include higher plants, microorganisms, and the more degenerate metazoan endoparasites, in which nervous and sensory systems are almost atrophied. A more difficult question arises when we consider the free-living metazoan invertebrates. We have arbitrarily excluded them from consideration as objects of humanitarian concern. It remains to consider them in the light of possible substitutes for vertebrate subjects. Such a procedure may be called comparative substitution.
Russell and Burch discuss the use of insects, and the highly-evolved response of polychaete worms and sea anemones, before concluding that they regard comparative substitution as a limited gain, and that, in fact, there does not seem to have been much progress in this direction. The problem is raised only for completeness, and from now on we shall consider only the wholly desirable progress and prospects of replacement proper.
However, they do also point out that, in another context, the octopus has been described as a more suitable subject than the albino rat for studies on the mechanism of visual form discrimination. [It should be noted that Octopus vulgaris is now a ”protected animal” under the Animals (Scientific Procedures) Act 1986.]
They also comment “that to shed obsessional tears over the fate of these organisms would bring the whole concept of humanity into contempt by Samuel Butler's famous reductio ad absurdum (disproof of a proposition by showing that it leads to absurd or untenable conclusions), by referring to the Erewhonian philosopher who inquired whether salt can feel. Erewohn is the title of a book by Butler, published in 1872, which is a satire of Victorian society. Erewohn is a fictional country and its name is based on Nowhere backwards, albeit with the letters “h” and “w” transposed. In the book among many other advantages the narrator visits the College of Unreason and meets, among others, the professor of Worldly Windom, who was also the President of the Society for the Suppression of Useless Knowledge. The Professor of Botany there contended “that vegetables are only animals under another name. The conclusion he drew … was that if it was sinful to kill and eat animals, it was not less sinful to do the like by vegetables or their seed” ” (Balls, Russell and Burch, 2009, p 35-26).
Therefore, the use of insects in pur projects is a comparative substitution (Figure 2). Russell and Burch considered this substitution “as a limited gain”  but mention that insects' implementation would set the concept of humanity ad absurdum. In the Addendum of the Principles, Russell and Burch state ”The comparative substitution of lower for higher animals raises difficult issues. But where great severity is concerned, as in the study and assay of natural venoms, we must be glad to see lower forms substituted for mammals. Comparative substitution is more welcome when it does not entail severe treatment of the substitute. Witt (1952) has studied the effects of a number of neurotropic drugs (including lysergic acid) on web-spinning spiders. The drug-specific effect is objectively and quantitatively recorded in the shape and pattern of the spun web. This technique may be worth further study in the context of experimental psychiatry” (Balls, Russell and Burch, 2009, p 114).
In the original publication from 1959, Russell and Burch are explicitly mentioning experiments with Drosophila melanogaster in venom assays as an adequate substitute for the mice .
Therefore, it can be clearly stated that the usage of insects is clearly in accordance with the 3R principle.
Absolute and Relative Replacement
“In relative replacement, animals are still required, though in actual experiment they are exposed, probably or certainly, to no distress at all. In absolute replacement, animals are not required at all at any stage” (Balls, Russell and Burch, 2009, p 36). For example, Russell and Burch understand the implementation of recovery experiments (e.g. testing a drug with only transient effects) under strong aesthetics as a relative replacement and “totally free from inhumanity” . Absolute replacements “involve work on the isolated cells, tissues, or organs of vertebrates” (Balls, Russell and Burch, 2009, p 38). Russell and Burch see the absolute replacement clearly as “the absolute ideal ” . However, they admit “the progress of replacement is gradual, nor is it ever likely to absorb the whole of experimental biology” (Balls, Russell and Burch, 2009, p 63).
Considering the replacement of laboratory animals, Russell and Burch introduce two important features in which the model can differ from the replaced or substituted original: Fidelity and discrimination . “Fidelity means overall proportionate difference, and discrimination means the extent to which the model reproduces one particular property of the original ” (Balls, Russell and Burch, 2009, p 43). According to Russell and Burch the first can culminate to the so-called “high-fidelity fallacy” . “The high-fidelity fallacy might suggest that, for man, a member of the another placental, mammalian species would be a model of higher fidelity than a bird or a microbe [or an insect]. The major premise states that the highest possible fidelity is always desired in medical research and in testing of biological substances. The stubborn conclusion is that mammals are always the best model ” (Balls, Russell and Burch, 2009, p 45). “The high-fidelity fallacy includes three important assumptions, which brand it as an obsession rather than a principle: the extant of our ignorance may be exaggerated, the fidelity of mammals as models of man may be greatly overestimated and the advantages of correlations may be ignored ” (Balls, Russell and Burch, 2009, p 46). An excellent example of the high-fidelity fallacy is the use of mice in epigenetic research of spermatogenesis. Epigenetics of spermatogenesis varies widely across mammals, and there is no good reason to study this process in mice .
In the light of Russell and Burch's fidelity and discrimination, the model M. sexta is clearly of low fidelity but of high discrimination in terms of development and detection of gastrointestinal inflammation. This is true because M. sexta’s gut structure and the innate inflammatory responses are very similar to those of mammals and hence detectable via CT, MR, and PET. But obviously, there are also many differences between hornworms and mammals (e.g., the metamorphosis, the overall morphology and the regulation of the body temperature). But within these differences lies the strength of this model. To put it in the words of Russell and Burch: “Differences are sometimes more useful than similarities” (Balls, Russell and Burch, 2009, p 44). In this context Russell and Burch mention the words “scouting” and “prescreening”, which is exactly the scope of our work .
“Reduction is desirable in any procedure, however directly humane, which employs large numbers of animals in one laboratory. […] Reduction remains of great importance, and of all the mods of progress, it is the one most obviously, immediately and universally advantageous in terms of efficiency” (Balls, Russell and Burch, 2009, p 63).
First of all, these statements must be seen in the context of time. As mentioned at the beginning, in 1959, laboratories had no standardized animal husbandry and experiments. Because many of the results were not comparable, many trials had to be repeated several times, and so purposeless suffering was caused and resources were wasted. That's why Russell and Burch call for standardization, which fortunately has long been established in most laboratories today.
Russell and Burch extensively discuss the statistical problem of variance to which there was no satisfactory answer at the time. Nevertheless, it should be mentioned that the power analyzes, which are mostly proposed as a solution today, do not solve this problem either. In most cases, the effect size is not known or is set arbitrarily and today it is still not possible to determine the exact number nor the minimum number of experimental animals needed beforehand. But unlike back then, there are now clear statistic conventions for hypothesis significance testing available.
They also criticize the undirected random approach of some research procedures. “Whenever it is possible directly to compare guided and random research, the former is seen to be more efficient. Where such methods are used, it is desirable in terms of humanity, cost, and effort for the trial and error to be applied replacement methods or subject “(Balls, Russell and Burch, 2009, p 65). This statement remains appropriate, and random high throughput screening should be avoided with our established Manduca -based screening system. Our work is clearly hypothesis-driven.
It should be mentioned that the conception of a high throughput screening system is in apparent contradiction to the premise of Reduction. Although I strongly agree with Russell and Burch's ethical framework, I can't entirely agree with the premise of Reduction in an absolute manner.
First, more research will always mean more experimental animals, and it was not the intention of Russell and Burch to constrain the epistemology of science. Second, and this point is also in full agreement with Russell and Burch, scientific experiments which sacrifice experimental animals need specific quality standards in terms of reproducibility. Russell and Burch thought that standardization of experiments and animal husbandry could cope with “the problem of variance” . Sixty years after the publication of “The Principles”, the scientific community is in a methodically “crisis of reproducibility” . The existence of serious and justified concerns about scientific findings' validity is threatening science. Therefore, the conception of an invertebrate-based throughput screening system is contrary to the requirement of the reduction of experimental animals but not in contrast to the intention of the 3Rs. In the end, few experiments with dubious results due to a small number of experimental animals ostensibly reduce the suffering of laboratory animals. However, then these few animals also died in vain and without purpose, which is certainly not in agreement with the 3Rs. Also, it must be noted that in vivo imaging modalities like CT, MR, or PET are significantly reducing the use of experimental animals because mammals or insects tolerate these modalities quite well and can, in principle, be used for other experiments, which is not true for most other experimental techniques (like qPCR).
“Its object is simply to reduce to an absolute minimum, the amount of distress imposed on those animals that are used” (Balls, Russell and Burch, 2009, p 85). Russell and Burch distinguish between neutral and stressful investigations. “In neutral studies, the imposition of any degree of distress, however slight, is likely a priori to disturb the efficiency of the investigation“ (Balls, Russell and Burch, 2009, p 86). In most stressful investigations, the perception of pain or physiological stress itself is the subject of the study. “In stressful investigations, there seems at first sight an irreconcilable conflict between the claims of humanity and efficiency“ (Balls, Russell and Burch, 2009, p 86).
In addition to various aspects that depends on the selected model species, Russell and Burch point out anesthesia's central importance for the refinement of animal experiments. “The most generally important of all is that of anesthesia, the supreme refinement procedure. This has brought about perhaps the greatest single advance in humane technique, and has at the same time been virtually indispensable for the advance of experimental biology ” (Balls, Russell and Burch, 2009, p 87). Fortunately, insects can be treated very well with established anesthetics such as isoflurane or ice.
The work of our lab aims to replace small mammals with the insect larvae of the tobacco hornworm (as a comparative substitution) in high-throughput early preclinical imaging studies. In this in vivo experimental setting, it is not possible to use non-sentient models for hypothesis testing. Therefore, it is desirable to replace the widely used mouse models, as far as possible, with models with presumably lower susceptibility and without a social structure. This Comparative Substitution fully complies with the 3R principles of Russell and Burch (Figure 2) [1, 2].
1. Russell WMS, Burch RL. The principles of humane experimental technique: Methuen; 1959.
2. Balls M, Russell WMS, Burch RL. three Rs and the humanity criterion: FRAME; 2009.
3. Hubrecht RC, Carter E. The 3Rs and humane experimental technique: implementing change. Animals. 2019;9(10):754.
4. Rollin BE. The regulation of animal research and the emergence of animal ethics: a conceptual history. Theor Med Bioeth. 2006;27(4):285-304.
5. Badyal DK, Desai C. Animal use in pharmacology education and research: The changing scenario. Indian J Pharmacol. 2014;46(3):257.
6. Maslova E, Shi Y, Sjöberg F, Azevedo HS, Wareham DW, McCarthy RR. An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models. Front Microbiol. 2020;11:998-. doi: 10.3389/fmicb.2020.00998. PubMed PMID: 32582051.
7. Jennings BH. Drosophila – a versatile model in biology & medicine. Materials Today. 2011;14(5):190-5. doi: https://doi.org/10.1016/S1369-7021(11)70113-4.
8. Mirzoyan Z, Sollazzo M, Allocca M, Valenza AM, Grifoni D, Bellosta P. Drosophila melanogaster: A Model Organism to Study Cancer. Frontiers in Genetics. 2019;10(51). doi: 10.3389/fgene.2019.00051.
9. Yamaguchi M. Drosophila models for human diseases: Springer; 2018.
10. Durieux M-F, Melloul É, Jemel S, Roisin L, Dardé M-L, Guillot J, et al. Galleria mellonella as a screening tool to study virulence factors of Aspergillus fumigatus. Virulence. 2021;12(1):818-34. doi: 10.1080/21505594.2021.1893945.
11. Witt PN. Ein einfaches Prinzip zur Deutung einiger Proportionen im Spinnennetz. Behaviour. 1952:172-89.
12. Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogramming in mammals. Hum Mol Genet. 2005;14(suppl_1):R47-R58. doi: 10.1093/hmg/ddi114.
13. Baker M. Reproducibility crisis. Nature. 2016;533(26):353-66.