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Black rat used in an experiment

Experiments with new materials on animals

All products that are expected to or may potentially come into contact with human beings are tested to examine their biocompatibility. Biocompatibility tests measure the way the material interacts with biological systems to ensure that their use will not cause harm to humans. This practice straddles the line between the concerns of industry, biomedical research, and biomaterials research for medical purposes. Examples of new biomaterials that are regularly tested are those materials used in bypass surgeries, pacemakers, plates for trauma operations, and dental procedures. Testing is also done for non-medical materials such as the plastics used to make bottles, bags, and other items used on a daily basis, as well as other materials used in industry whose effects on humans are not known or are only partially known.

Historically, new materials were always evaluated on humans in order to determine their biocompatibility. But now this practice is considered unacceptable, and for a material to be considered biocompatible, it must pass through several stages. The usual test stages are in vitro testing, in vivo testing (which now uses animals rather than humans), and in-use or clinical tests. All levels of testing include various types of procedures and span an array of ethical questions.


Types of testing

In vitro testing

In vitro testing does not use living organisms, but instead uses cells, tissues, and organs. These tests are done in test tubes and/or petri dishes. However, in order to get the body parts used in the test, animals are killed or harmed to remove tissues, organs and other body parts.

In vivo tests

The name “in vivo,” Latin for “within the living,” describes clearly that these tests consist of experimentation on live animals, who go through aggressive contact applications of the materials to be tested. Materials are often implanted into nonhuman animals via subcutaneous (under the skin), intramuscular (in the muscle), intravascular (in a blood vessel) or in bone procedures. In vivo tests are often performed repeatedly even after a material has gone to market in order to check for issues missed by previous studies, or to test new uses or changes to the material.

In-use and clinical tests

In-use testing differs from in vivo testing in that, with in-use testing, the material is always tested under identical conditions to those of their future real-world use. For this reason, in-use testing uses nonhuman animals with physiological similarities to humans, such as dogs and monkeys as opposed to rodents. When in-use testing is done on humans it is usually called clinical testing. In theory, the two kinds of procedure could be basically the same; however, in practice they are not. Humans are treated with respect and risks are minimized. This is not the case with nonhuman animals who are used in whatever way is needed with much less consideration for the pain they suffer. Additionally, they are killed routinely despite the fact that killing the animal post-testing typically has nothing to do with the test itself.


Some procedures used for materials testing

The specific procedures used for materials testing on animals cause them harm and considerable suffering, often including death. Some examples of the standard tests are the following:

Mucous membrane irritation testing

These tests determine if a material can cause inflammation to mucous membranes. At the beginning of the test, the potentially irritating substance is applied to the mucous membrane or implanted into it. Several weeks later, the animals are photographed and killed in order to do histopathological (microscopic) tests on tissue, and to determine the inflammatory response generated by the material.

Cutaneous (skin) sensitivity testing

Cutaneous sensitivity tests are intended to determine if a material may cause inflammation after being injected into the skin. The level of response and percent of animals who show a reaction to the material are the basis for estimating the level of allergenicity of the material.

Dentin barrier tests

These tests are similar to cutaneous testing. However instead of the skin, they test biocompatibility with dental pulp.

Cell membrane permeability

Another way to measure the cytotoxicity (the level of toxicity to cells) of a material is to evaluate the changes in the permeability of the cell membrane. Colorants are used to dye the cell to make measurements and cell identification easier.

Use of barrier materials

These tests consist of a substance tested in vitro (in a test tube), but with an added barrier in between the test substance and the cells on which it is tested. For example, vegetable polymers such as agar-agar (a gelatin derived from red algae) are often used at the barrier. The barrier material blocks contact between the material to be tested and the cells it’s tested on in a certain way. This is done to more closely reproduce what occurs in living organisms, and makes the studies more applicable to clinical realities.

Cellular growth tests

The goal of these procedures is to determine the number of cells that grow during the test as a result of the implantation of a material. The material to be tested is placed in direct contact with the cell culture on a plate. If the material is cytotoxic (toxic to cells), the cells will not grow where it is placed or anywhere near it. Cellular density measurements can be described qualitatively, semi-qualitatively, or quantitatively to determine the level of biocompatibility. These tests are used to measure the antimicrobial capacity of certain materials in relation to pathogenic microorganisms (organisms capable of producing disease). The extent of the cells that are killed by the test substance gives a measure of the cytotoxicity of the material.

Enzymatic biosynthesis

In these tests DNA changes are measured in order to determine the effect of a material on protein synthesis. Cells (precursors) marked with radioisotopes (radioactive chemicals) are added to a cell culture. Observations are then made to measure which precursors become incorporated into the DNA or protein and to count them.

Ames Test

This is a type of in vitro method to evaluate the potential of a material or chemical compound to induce or increase mutation to the cells around it (mutagenic capability). Since cancer is linked to DNA damage, this method also serves as a test to estimate the carcinogenic potential of a compound.

The Ames test measures the ability of a potentially mutagenic material to cause mutations in a strain of modified Salmonella tymphimurium bacteria. Normally, the bacteria have the capacity to synthesize the amino acid histidine. This test uses bacteria with an isolated mutation in the genes that synthesize histidine, which prevents them from synthesizing it.1

Traditionally, rat liver extract is added to the histidine-dependent bacteria at the start of the test, to create something closer to the metabolic conditions found in mammals, though it is now possible to use human liver extract instead.

Since histidine is an essential amino acid for the bacteria and the tested strain lacks the ability to synthesize it, the only way they can survive without it is if they mutate and begin to be able to synthesize it. The test looks at the ability of a new material to cause such a mutation.

During the test, the bacteria are spread over an agar plate with a small quantity of histidine, which allows the bacteria to survive and reproduce only during an initial period until the amino acid runs out. They remain in contact with the potentially mutagenic material that is being tested for 48 hours. Any bacteria that survive and reproduce during this time will be able to do so only because they have mutated so that they have the ability to produce histidine. The number of colonies at the end is compared with the number of colonies in a control that did not have the potentially mutagenic material added to it. The effectiveness of the potentially mutagenic material in causing the mutation is considered proportional to the number of colonies of bacteria at the end of the test. A large number of bacterial colonies indicates high mutagenic potential.

Styles test

This is similar to the Ames test, but it uses mammalian cells instead of bacterial cells.


Testing models

In the past, the standard process for testing materials for biocompatibility followed a precise pyramidal structure. The first level is in vitro testing, which does not necessarily have to be clearly applicable to the final use of the material.2 Next, there is in vivo testing with animals and, finally, in-use or clinical testing. Only the materials that successfully passed the first level could be evaluated at the second level. The model is pyramidal because in the first level all the materials and substances are tested, but some are dismissed, so in the second level there are fewer materials and substances to be tested.

In this model, the initial tests cannot always predict the behavior of the material in actual use. In order to test that, the tests performed in the last level (in-use/clinical) are necessary.

Currently, the pyramidal model is still used, but it’s less strict. This is due to the fact that the tests done at the first and second levels (in vitro tests and in vivo tests with animals) are considered less important than they used to be. Moreover, the research done at each level is more closely related now to what happens at other levels. Rather than having a clear separation into different levels, the whole process is now seen holistically.The different types of testing are part of a continuous process that develops along with clinical experience with the material being tested.

Additionally, efforts made to decrease testing on nonhuman animals have led to an increase in in vitro testing. The strict pyramid model which relied heavily on in vivo testing has been changed due to new developments in in vitro testing which better simulate conditions in living organisms, though these tests still generally use animal products such as tissues and organ extracts.3 There have also been improvements in the use of barrier materials appropriate for specific tests, cell cultures and tissues. Advances in identifying relevant clinical biological markers, such as changes in DNA transcription4 or the presence of certain chemicals, to measure the biological effects of a given material have also improved testing standards.5

Today, the three types of biocompatability tests are often carried out simultaneously. For example, an in vitro test might be used for investigating a specific biological response observed during clinical tests, or after the material has been introduced to the market.

Further readings

Animal Procedures Committee (2003) Review of cost-benefit assessment in the use of animals in research, London: Home Office.

Atchley, F. W. (1991) “Genes trees and the origins of inbred strain of mice”, Science, 254, pp. 554-558.

Cothran, H. (ed.) (2002) Animal experimentation: Opposing viewpoints, San Diego: Greenhaven.

DeGrazia, D. (1999) “The ethics of animal research: What are the prospects for agreement?”, Cambridge Quarterly of Healthcare Ethics, 8, pp. 23-34.

European Commission (2013) Seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the European Union, Brussels: European Commission [accessed on 2 November 2016].

Festing M. (1979) Inbred strains in biochemical research, London: Macmillan.

Frame, J. W. (1980) “A convenient animal model for testing bone substitute materials”, Journal of Oral surgery, 38, pp. 176-180.

Hench, L. L. & Thompson, I. (2010) “Twenty-first century challenges for biomaterials”, Journal of the Royal Society Interface, 7, suppl. 4, pp. S379-S391.

Krug, H. F. & Wick, P. (2011) “Nanotoxicology: an interdisciplinary challenge”, Angewandte Chemie International Edition, 50, pp. 1260-1278.

LaFollette, H. & Shanks, N. (1997 [1996]) Brute science: Dilemmas of animal experimentation, new ed., New York: Routledge.

Langer, R. & Tirrell, D. A. (2004) “Designing materials for biology and medicine”, Nature, 428, pp. 487-492.

MacGregor, J. T. (2003) “The future of regulatory toxicology: Impact of the biotechnology revolution”, Toxicology Science, 75, pp. 236-248.

Muschler, G. F.; Raut, V. P.; Patterson, T. E.; Wenke, J. C. & Hollinger, J. O. (2010) “The design and use of animal models for translational research in bone tissue engineering and regenerative medicine”, Tissue Engineering. Part B, Reviews, 16, pp. 123-145.

Nel, A.; Xia, T.; Mädler, L. & Li, N. (2006) “Toxic potential of materials at the nanolevel”, Science, 311, pp. 622-627.

Shanks, N.; Greek, R. & Greek, J. (2009) “Are animal models predictive for humans?”, Philosophy, Ethics, and Humanities in Medicine, 4 (2), pp. 1-20 [accessed on 17 April 2013].

Tannenbaum, J. & Rowan, A. N. “Rethinking the morality of animal research”, Hastings Center Report, 15 (5), pp. 32-43 [accessed on 17 April 2013].

1 The original articles in which this method was addressed were published four decades ago. See: Ames, B. N.; McCann, J. & Yamasaki, E. (1975) “Methods for detecting carcinogens and mutagens with the salmonella/mammalian-microsome mutagenicity yest”, Mutation Research, 31, pp. 347-364. Maron, D. M. & Ames, B. N. (1983) “Revised methods for the salmonella mutagenicity test”, Mutation Research, 113, 173-215.

2 Many different kinds of invitro tests may be carried out, also known as “test tube” tests they are usually carried out in test tubes, Petri dishes or similar apparatus. To give just one example from the area of the biocompatability of materials used in wound dressings, cells are placed in agarose (a material usually extracted from seaweed) and broken down by other chemicals to realease the DNA within them. They are then examined to test for genotoxicity. See: Keong, L. C. & Halim, A. S. (2009) “In vitro models in biocompatibility assessment for biomedical-grade chitosan derivatives in wound management”, International Journal of Molecular Science, 10, pp. 1300-1313 [accessed on 22 April 2013].

3 Jessen, B. A.; Mullins, J. S.; de Peyster, A. & Stevens, G. J. (2003) “Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression”, Toxilogical Sciences, 75, pp. 208-222 [accessed on 22 April 2013].

4 Johansson, H.; Lindstedt, M.; Albrekt, A.-S. & Borrebaeck, C. A. K. (2011) “A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternativeto animal tests”, BMC Genomics, 12, p. 399 [accessed on 26 November 2012].

5 Orfeas L.; Tighiouart, H.; Perianayagam, M.; Kolyada, A.; Han, W. K.; Wald, R.; Bonventre, J. V. & Jaber, B.L. (2009) “Comparative analysis of urinary biomarkers for early detection of acute kidney injury following cardiopulmonary bypass”, Biomarkers, 14, pp. 423-431 [accessed on 26 September 2012].

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