The search for alternative methods to animal testing is underway in many laboratories across the entire world. While success has been made, the research is far from over. These alternatives have been developed using the concept of the three Rs.
In 1959, William Russell and Rex Burch defined the principle of the three Rs in the book Principles of Humane Experimental Technique. The three Rs are reduction, refinement, and finally replacement (5).
The first concept, reduction alternatives, covers any strategy that will result in fewer animals being used to obtain the same amount of information. Also, reduction refers to maximizing the information obtained per animal so as to limit or avoid the use of more animals. There are several approaches that can help to reduce the use of animals. Some laboratories alert all of the researchers when animals are going to be killed in an experiment. For example, one researcher may be doing a study on livers, and so other researchers may be able to use the kidneys, heart, or brain tissue for other experiments. In some cases it might also be possible to use in vitro methods, which are studies done with cells or tissues cultured in a petri dish, in place of in vivo methods, which are studies done in the living animal (3).
The second principle, refinement, represents the modification of any procedure from the time the laboratory animal is born until its death, to minimize the pain and distress experienced by the animal. Paying attention to issues of animal welfare is not only important in light of ethics, but also in the matter of good science. The experience of pain and other stress is likely to have an effect on the variability of experimental results. In fact, it is in the best interest of the researcher to ensure that conditions in animal facilities are the best possible. It does not require excessive funding to enrich the environment in which the animals live in. For example, toilet paper rolls, egg cartons, and PVC tubing can provide rodents with places to hide. Bales of straw and rubber tires can be used to create an area for rabbits to interact with other members of their species. Dogs can be given numerous toys to play with, and be provided with a raised platform so they are not forced to stand in their own waste.
It is also important for the staff of the facility to be well trained in handling the animals that are being used, and that they have the correct attitude when working with the animals. Anesthesia should be used whenever possible, and at the end of the experiment, the most humane method of euthanasia should be chosen.
The final concept of the three Rs is replacement. Any experimental system that does not use whole, living animals is considered to be a replacement alternative. Some of these techniques still involve the humane killing of an animal for the purpose of obtaining cells, tissues, or organs for in vitro studies. Other techniques involve no use of any biological material from a fully developed vertebrate, non-human animal. In some cases, replacement methods can be used for the total replacement of animals in a study, in others they will complement animal experiments and reduce the total number of animals used in the whole project. Replacement alternatives can be divided into six categories: information; computer-based systems; physico-chemical techniques; the use of lower organisms and embryo stages; human studies; and cell, tissue, and organ cultures (5).
Access to information can prevent the unnecessary duplication of animal work that has already been done. Also, the in vivo data that has already been found to be reliable can be used to validate alternative methods without having to do any new animal studies.
Developments in computer modeling and expert systems that can predict biological activity and toxicity have already revolutionized the process of drug development by eliminating the need to use animals for pre-screening of potential drug candidates. For example, TOPKAT is a mathematical computer model based on physical and chemical structures and properties of a substance (10). This replacement procedure is used to determine the oral toxicity and possible skin and eye irritancy of a substance. The TOPKAT test is currently 75 to 100% accurate, and is regularly used by the Food and Drug Administration, the Environmental Protection Agency, and the United States Army. Computer simulations and multi-media presentations are often used to replace the use of animals for education purposes. Even though these simulations are available, it is very difficult, and in many cases still not possible, to simulate a hands-on experimental situation. In order to improve upon these programs, a huge amount of data from mostly in vivo studies has to be collected and applied to the programs.
Physicochemical data, pH for example, are used in combination with structure-activity relationships to predict the biological effects of chemicals. One product, the Irritection Ocular Assay System, formerly known as Eytex or Skintex, uses a solution that is made up of proteins, glycoproteins, lipids, and low molecular weight components that self-associate to form a complex molecular matrix. The test works by mimicking the reaction of the cornea and human skin when exposed to a foreign substance. If a chemical has a potential to irritate the eye, then it will cause the solution to coagulate. At the present time, the test can determine the toxicity of over 5000 different materials (8). Many cosmetic companies, such as Avon, use this system to screen potential irritants without testing them on animals.
Sometimes it is possible to conduct studies in lower organisms, such as invertebrates, plants and microorganisms, or in vertebrates at early stages of development. Some examples of this type of alternative are the Ames test and the LAL test. The Ames test uses salmonella bacteria to detect any carcinogenicity (the ability to cause or promote cancer) (8). This test has been validated and accepted for screening purposes in toxicology. The LAL test for endotoxins has also been validated for certain purposes. This test detects the presence of fever-inducing endotoxins in intravenous products. This is made possible from blood samples gathered in the wild from horseshoe crabs. The researcher extracts amoebocyte lysate from the samples and mixes it with the endotoxin to see if a reaction occurs (8). Also, hydra can be used to screen for teratogenicity, which is the production of malformations in the embryo. Yeast cells and tobacco plant pollen tubes have also been suggested for toxicity testing. Advances in genetic engineering are opening up even more possibilities to replace the use of higher animals. Genetically engineered roundworms, because they carry human disease genes, have already been used to identify new drugs. Many studies on the development and growth can be carried on animal embryos in vitro rather than in the pregnant mother. Rodents are usually used in these procedures. Fertilized chicken eggs are also used in a test called the HETCAM, which predicts eye irritancy. The researcher can observe the effects a chemical has on the chorioallantoic membrane of the egg (5).
If sufficient consideration is given to ethical and safety issues, studies on humans can replace the use of animals in some cases. Clinical studies in humans have always been required to register drugs, and human volunteers are being used more and more for the skin testing of cosmetics. Non-invasive methods can be used in healthy subjects to investigate disease processes. Some of these methods are nuclear magnetic resonance, electron spin resonance, and positron emission tomography. The great advantage to human studies is that they deal with human beings in their normal environments.
Finally, the use of cell, tissue, and organ cultures are used usually only as relative replacements, because they require freshly obtained animal cells and tissue. However, the animals are used more economically in this way, because one animal can provide tissue for a number of cultures. Human tissue can sometimes be used, but it is difficult to obtain, store, and distribute. Some human tissue is available after surgeries. Human placenta has been suggested as a source of tissue for various types of research. Because it contains mast cells that share certain structures with nerve cells, it can be used in some neurological studies. One drawback is that when human tissue is used, there is a greater risk that it will contain dangerous viruses, and so greater precautions must be taken. One idea is to establish tissue banks, much like blood banks, where the tissue can be screened and then be used to supply researchers.
Because in vitro procedures isolate the system under study from the rest of the organism, they are ideal when trying to avoid the effects of influences such as hormones. This can also be a disadvantage because these external factors may have a crucial effect on the question being studied. There are several different kinds of in vitro systems, and each one has its own advantages and disadvantages.
The first method is to use subcellular fractions of one cell component. An example of this type of in vitro would be to study the role of liver microsomes and their importance to drug metabolism. One drawback to this type is the fact that this system will not provide information about the influence of factors in the cell, let alone in the organ or the entire organism (5).
Primary cell cultures are produced from fresh tissue that has been disrupted to obtain individual cells. These cultures are fairly easy to set up, and the advantage is that they contain normal cells with all the characteristics that determine their specialized functions in the tissue they came from. The disadvantage, however, is that they can only be maintained for a few days, or sometimes weeks, and they tend to lose ability to function with time. This means that fresh tissue is constantly required, and the cultures cannot be used for long-term studies. Another drawback is the fact that these cultures will not permit cell/cell interactions.
Another in vitro system is cell line cultures. These consist of cells that can grow indefinitely. They are often taken from human or animal tumors and some have been kept for decades. They have undergone a process called transformation that makes them able to ignore the control mechanisms that limit the number of times normal cells can divide before dying. Cell line cultures can be kept frozen in liquid nitrogen. They are used a lot because they are so easy to maintain and do not require fresh tissue. The disadvantage of this system is that it has not been possible to produce cell lines of every tissue. A further drawback is that the cells are abnormal in many ways. In some cases, the cells dont even resemble the normal cells from the tissue they came from. One advancement that has been made is the development of embryonic stem cell lines. These cells remain unaffected until the researcher manipulates them. This type of cell line culture could be used to test teratogenicity, which is the ability of a chemical to cause malformations in the fetus of an animal or human. Researchers are also interested in using embryonic stem cell lines for gene knock out studies to identify the roles of specific genes. Cell lines can be genetically engineered in many ways. For example, human genes can be inserted into an animal cell line to give it the same enzyme capabilities as human tissues (5).
Tissue culture is a system that is unique in that cell/cell interactions are still possible through fragments or slices of tissue that maintains the tissues original architecture. For example, very thin slices of liver and kidney can be used to study possible effects of drugs on these organs. This type can also be very economical because human tissue obtained after surgery can be used in tissue culture. However, these cultures have a limited lifespan and a high level of technical skill is needed to set up and maintain them. The latest breakthrough has come with the development of three-dimensional tissue equivalents, which essentially mimic the real tissue. These equivalents are made by culturing tissues on an artificial support matrix. A lot of human skin equivalents have been developed and work is in progress on tissue equivalents for other organs. One alternative, Testskin, is actual human skin grown in a sterile plastic bag. This test is used to measure irritancy, and is being used by Avon, Amway, and Estee Lauder. Epipack, which also uses cloned human tissue, is designed to work much like the Testskin. Finally, the Neutral Red Bioassay test, which is a test performed on cultured human cells, is used to compute the absorption of water-soluble dyes. This can help determine the relative toxicity of the dye (9).
Organ cultures have an advantage in that they allow all of the interactions that take place in the organ. These in vitro procedures are used in a lot of pharmacology studies. The disadvantage, once again, is that they are hard to maintain and are short-lived. Organ cultures also involve killing an animal for an organ, and only one organ can be taken from each of the animals, with the exception of the kidneys.
One in vitro procedure, Corrositex, was developed as an alternative to the rabbit skin test. The test assesses chemical corrosivity using a protein membrane designed to function like the skin. When exposed to a potentially corrosive substance, the biomembrane becomes colored. Results to a Corrositex test are available in as little as three minutes and no longer than four hours. The in vivo rabbit skin method takes two to four weeks to complete (6).
There are many more alternative procedures that are being developed and improved up on every day, and so the hope for replacement of animals in the laboratory continues to grow stronger. Establishments such as the John Hopkins Center for the Alternatives to Animal Testing, the International Foundation for Ethical Research, the Cosmetic, Toiletry, and Fragrance Association, and the Soap and Detergent Association have all started their own programs to validate alternatives.
Although some critics are doubtful that replacement methods will ever be able to take the place of hands-on animal experimentation, the struggle continues for avid animal rights supporters to find alternative testing procedures. The question of whether animals have the right to be protected from such research experiments is a hard one to answer, and has been disputed for many years. The question may be easier to answer when it is asked about testing for such things as cosmetics, but when it comes to cancer studies and the like, the lines arent quite as clear cut. It is up each individual to decide whether the life of an animal is just as important, or more important, than research that benefits mankind. In his autobiography, The Story Of My Experiments, Mohandas K. Gandhi states his opinion on animal experimentation:
To my mind the life of the lamb is no less precious than that of a human being. I should be unwilling to take the life of the lamb for the sake of the human body. I hold that, the more helpless the creature, the more entitled it is to protection by man from the cruelty of man. (9)
Although companies are continuously searching for alternatives to animal research, the number of animals used in experiments actually continues to increase. The old test, the in vivo method, must be done along with the new test, the in vitro method, to ensure that the new test will have results that are consistent with the old test. It is believed that even if the number is increasing at this time, the number of animals that will ultimately be saved after in vitro methods are improved will greatly outweigh the number of those used during the research to perfect such methods.
Until alternative methods can be proven to be just as, if not more, effective than in vivo methods, the question of the rights of animals and experimental research will continue to be debated and questioned. Advancements are being made every day, and, hopefully, one day animals will not have to be used in experiments.
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