Alternatives to Animal Testing in Research — Insights and Future Directions

In April Pro Anima had the opportunity to attend a veterinary congress held at Utrecht (The Netherlands). Salomé Pollet our specialist made a lecture and promoted alternatives to animal testing to a large audience of veterinarians and veterinary students with a poster.

This poster has been selected among the 3 best posters of the congress demonstrating the growing interest in the field of alternatives to animal experimentation.

Introduction

The use of animals in research is getting more and more controversial. Besides the ethical issue related to animal testing, there also exists an argument based on its scientific inaccuracy, when animals are used to predict the efficiency and safety of drugs or treatments used in fine in humans.

The scientific committee Pro Anima, funded in 1989 in Paris, aims at promoting alternative methods to animal testing in research. Bringing together more than 20 biologists, physicians and veterinarians, it supports concrete initiatives, through the financial support of a fund (EthicScience) and lobbying in the European institutions, and publishes a quarterly newspaper in French, ”Sciences Enjeux Sant”, that covers the news in the scientific community around alternatives to animal testing.

Ethical issues related to animal testing

Many authors have highlighted reasons explaining the amorality of using animals in research.

In 1789, Jeremy Bentham writes ”The question is not : Can they reason ? Nor, can they talk ? But, can they suffer ?”. He suggests that, more than the ability to be conscious of their fate, the fact that animals can feel pain through experiments is in itself an argument to avoid using them. The Australian philosopher Peter Singer exposes the theory of equal consideration of interests between species, but through an utilitarian approach : for instance, if the use of a small number of animals leads to the treatment of thousands of human beings, then it could be justifiable according to him. But this is only a matter of the number of lives involved, and not on their characteristics of belonging to a particular species. In opposition to this vision, Tom Reagan argues that each animal has an intrinsic value, that should be considered independently to the use that humans can take advantage of, and states that animal testing is supported by an overestimation of benefits and an underestimation of harms.
Some of these critical views on the ethics of animal testing have led to welfarist approaches aimed at reducing the suffering of animals — such as the development of the 3R (refine, reduce, replace) of Russell and Burch -, while others advocate for abolitionist procedures, with a complete ban of the use of animals in research.

Nowadays, scientists seem to agree that many animals have a form of consciousness and pathways of feeling pain very similar to human beings. This has been publicly acknowledged by a panel of pharmacologists, neurophysiologists, anatomists, and neuroinformaticians in the Cambridge Declaration on consciousness.

Scientific inaccuracy of animal testing

In a globally shared view, even though animals used in experiments suffer, the potential benefit in terms of human health dominate the ethical issues. Science is thus seen as being ethics-free, exempt of stating moral judgments. However, there is no consensus on the scientific community on the efficacy of animal testing for health purposes in humans.

Safety testing

In safety testing, a drug must be proven to be safe for human use : it must be non life-threatening and avoid the occurrence of adverse drug reactions (ADR).

False safety negatives occur when a drug has been tested negative for harmful effects in animals, but found to have some in humans. A study stated that 100,000 hospitalized patients die each year because of ADR, making it the 4th cause of deaths (Lazarou et al., 1998). An estimate of 50 per 100 of drugs are put out of the market because of serious ADR found after their commercialization (Greek and Greek, 2004). An infamous example of this is the Vioxx, an NSAID that has been approved in the USA in 1999 and withdrawn in 2004, after many ADR and deaths. It has been shown that the use of Vioxx increases in humans by 5 times the incidence of heart attacks ; surprisingly, 9 out of 11 studies on mice and rats have found Vioxx to be safe on blood vessels and hearts, and 6 studies on 4 species showed that it was actually protective against heart attack and vascular diseases (PCRM, 2005).

False safety positives occur when a substance has shown to be unsafe on animals but is safe for use in humans. Many classical examples arise in pharmacology, such as penicillin that would not have stand many animal tests (very toxic at low doses in guinea pigs, and various levels of toxicity in mammals).

Efficiency testing

In efficiency testing, a drug must show its therapeutic properties against a specific pathological state at a determined dose (that, combined with safety testing, should heal and not harm the patient).

False efficiency predictors are drugs found effective in animals, but proven to be ineffective in humans.

Many drugs under testing fall into this category : according to A.C. Eschenbach (former commissioner of the FDA from 2006 to 2009), 90 per 100 of drugs that show positive results of efficiency through preclinical nonhuman studies fail during clinical human trials.

False inefficiency predictors are drugs that have been proven effective in treating human diseases, but not in animal models. It is hard to assess how many drugs fall under this bias, because they are often removed from testing when found ineffective in animals. However, the National Cancer Institute (NCI) tested in 2000 12 anticancer drugs that show improvements in humans and found that 8 were ineffective on mice.

The weight of habits

More and more scientists are pointing the fact that it is hard to rely on tests made on another species to gain knowledge for human health, given that the result of these tests can greatly vary among species and in a same species (given the genetic strain, sex, body condition, housing capacity of the animals, etc.). However, the weight of practice still remains heavy in the scientific community. A common argument is that many Nobel Prizes winners have used animal models in their research (18 out of 25 laureates in biology since 1980, according to a report from the Foundation for Biomedical Research). However, these data could also have been obtained without the use of animals, if adequate alternative models had been available to replace them.

Legislation on animals used in experiments

Animals have been used as experiment subjects in the last 200 years, but this practice exploded in the 20th century. In the same time, laws have emerged in order to regulate their use and set some standards to assess the welfare of animals kept in labs. In the USA, the Animal Welfare Act enacted in 1966 provides some guidance in the domain of animal testing. In the UE, the directive 86/609/EEC on the protection of animals used for scientific purposes has been updated in 2010 (directive 2010/63/EU), in a new version that strengthens a welfare legislation and provides a legal frame for the 3R rule of Russell and Burch. In the EU, the Cosmetics directive (regulation 1223/2009), enforced since 2013, fixes a testing ban on finished products and ingredients, which has been delayed for tests that have currently no validated alternative, such as reproductive toxicology or repeated-dose toxicity. In a common effort to reduce the number of animals used in labs, a center for the validation of alternative methods (ECVAM) has been funded in 2011. Each country is also responsible for setting animal ethics committee, that decide if procedures can be conducted under a defined ethical framework.

Perspectives on alternatives to animal testing

A validated alternative for the Draize test

The Draize test assesses the predictability of damages caused by chemicals in contact with the skin. Designed in 1944 and largely used since then, it consists in a cutaneous application of a substance on rabbits, and a record of the skin aspect (erythema, edema, pain, local heat, etc.). However, it has been criticized for being subjective, leading to an overestimation of the toxicity for humans, and harming animals. Alternative models based on human reconstructed epithelium have been found to predict very well the local and systemic toxicity of cutaneous agents, through an objective appreciation of skin reaction (measure of cell viability through enzymatic activity, exposure time that reduces the cell viability by half, concentration of a substance that reduces the cell enzymatic activity by half, etc.).
Several models have been validated by the ECVAM : EpiDerm, SkinEthic, Episkin… They are now routinely used for cosmetic irritation testing, and increasingly for pharmaceuticals.

An alternative under study for the LD50 test

The LD50 test accounts for the determination of the human lethal dose of a substance, through the measure of the lethal dose for 50 per cent of the animals. Nevertheless, given species differences and particular metabolism, it often gives false negative or positive results. Several tests should be combined in order to show an alternative to it : observation of the toxicity on tissues (changes in shape, growth, adhesion, enzymatic activity on cell cultures), changes in RNA levels, quantitative structure-activity relationships, bioinformatic approaches…

Organs-on-chips models

The Wyss Institute, at Harvard University, is developing multidimensional organs at a microscopic scale. Cell populations, organized in a similar way as in tissues, are connected through microfluidic channels, allowing it to mimic organs at a high level of complexity. A first model of lung is currently under test for toxicological assays and seems to offer a very good predictability for respiratory toxicity of chemicals (Esch et al., 2015). Models of kidneys, liver, heart, etc. are also getting started.
In the long run, the Wyss Institute aims to offer a ”human-on-a-chip”, that would allow to take into account interactions between organs and tissues.

Patient-based medicine

The way begins to be paved for a more individualized practice of medicine. Important advancements have been made in understanding molecular mechanisms of differentiation of a stem cell into a cell of a specialized tissue, and it is possible to retrograde a differentiated cell to a stem cell state (iPS, for ”induced pluripotent stem cell”). From a biopsy conducted on a patient, it is possible to test drugs on organods, which are blocks of cells that result from the differentiation of stem cells, and that can recreate precisely the structure of a given organ (Hynds and Giangreco, 2013). Preliminatory results have already been collected in specific cases, such as a test of efficiency for a drug against cystic fibrosis, or chemotherapy for pancreatic cancer (Willyard 2015).

Conclusion

• Animal testing raises scientific as well as ethical concerns.
•  The lack of validated alternative methods and the weight of habits make change slower.
•  A paradigm shift has recently occurred, where advances in technology could offer a future with a research without animal testing.

Salome Pollet,
Pro Anima scientific committee
National Veterinary School of Alfort, Maisons-Alfort, France

References

Bentham J. (1789) An introduction to the principles of morals and legislation. Cambridge University Press, England. Greek C.R., Greek J.S.(2002) Specious science : why experiments on animals harm humans. Continuum International Publishing Inc., U.S.A. Esch E.W., Bahinski A., Huh D. (2015) Organs-on-chips at the frontier of drug discovery. Nature Reviews Drug Discovery, 14(1) : 248 – 260. Hynds R.E., Giangreco A. (2013) The relevance of human stem cell-derived organod models for epithelial translational medicine. Stem Cells, 31(3): 417 – 422. Lazarou J., Pomeranz B.H., Corey P.N. (1998) Incidence of adverse drug reactions in hospitalized patients. JAMA, 279(15) : 1200 – 1205. Low P. (2012) Cambridge Declaration on consciousness. Physicians’ Committee for Responsible Medicine (PCRM) (2005) Vioxx tragedy spotlights failure of animal research. pcrm.org. Reagan T. (1983) The case for animal rights. University of California Press, U.S.A. Russell W.M.S., Burch R.L. (1959) The principles of humane experimental technique. Methuen, London, England. Singer P. (1975) Animal liberation : a new ethics for our treatment of animals. Harper Collins, U.S.A. Willyard C. (2015) The boom in mini stomachs, brains, breasts, kidneys, and more. Nature News Feature, 523(1): 520 – 522.