Animals and Alternatives in Testing: History, Science, and Ethics
Joanne Zurlo, Deborah Rudacille, and Alan M. Goldberg
TABLE OF CONTENTS
Preface
Acknowledgments
Science and Society
The Eye of Science
Toxicology and Toxicity Testing
Science In Vitro
Animal Experimentation: Ethics and Law
Bibliography
APPENDICES
Appendix A: Methodologies of Vaccine Development
Appendix B: Timeline of In Vitro Toxicology
Appendix C: Timeline of Tissue Culture
Appendix D: United States Animal Welfare Timeline
· Appendix E: Great Britain Animal Welfare Timeline
Chapter 1 Science and Society
The little I have hitherto learned is almost nothing in comparison with that of which I am ignorant.
- Descartes
Although the word "scientist" was not coined until 1840, the desire to understand the natural world and one‘s place in it are basic human aspirations. The scientific knowledge of the ancient world was practical and related to specific ends. Astronomy developed in most ancient societies in order to facilitate calendar making, and the Mesopotamians created a basic algebra and geometry in order to assist them in surveying and developing. The ancient Egyptians, consumed by the afterlife, acquired basic anatomical knowledge through embalming practices and the rudiments of hygiene dysentery, and typhoid (Ronan, 1982). The first attempts to classify and systematize knowledge of the natural world were undertaken by the Greeks.
In medieval Europe, the sciences were considered "magical arts" and were usually based upon ancient authorities and superstition. Roger Bacon, in his Opus Majus, published in 1265, noted the causes of error - authority, custom, popular prejudice, and the concealment of ignorance with the pretense of knowledge. He pointed out that the two methods of acquiring knowledge are argument and experience. Mere argument, he commented, is never enough, for "the strongest argument proves nothing so long as the conclusions are unverified by experience."
Bacon‘s insistence on the gathering of data is one of the hallmarks of science. Systematic attempts to organize data into a coherent system, attempted by early Greek scientists such as Aristotle, Hippocrates, and the astronomer Ptolemy, vanished in the Middle Ages as the quest for knowledge of the natural world was perceived as a challenge to the authority of the church and an inappropriate subject of study. The legacy of the Greeks themselves proved a formidable obstacle to the acquisition of real scientific knowledge when later investigators refused to accept any new information that contradicted the revered yet erroneous doctrines of Aristotle, Ptolemy, and Galen.
The birth of modern science dates to the year 1543 and the publication of Copernicus‘ De Revolutionibus Orbium Coelestium (On the Revolutions of the Heavenly Spheres) and Vesalius‘ De Humani Corporis Fabrica (On the Fabric of the Human Body) which challenged systems of belief dating back to the second century. Copernicus‘ assertion that the earth circled the sun precipitated as spiritual crisis. Ever since Ptolemy, men and women had believed that the earth was the center of the universe and man the pinnacle of earthly creation. Copernicus‘ calculations did nothing to alter the latter perception but began the slow intellectual journey that shattered the first. His ideas were mathematically elaborated, experimentally tested, and finally confirmed by Galileo, Kepler, Brahe, and Newton.
Vesalius‘ impact was not quite as far-reaching but it had a profound effect on the practice of medicine. Until Vesalius‘ detailed dissections of human corpses, medicine was largely based on the teachings of Galen, a revered Greek physician and writer whose knowledge of human anatomy was largely deduced from animal dissections and whose treatment of disease was based upon the doctrine of the four bodily humors. Unlike most medieval instructors in anatomy, Vesalius performed dissections himself. Therefore, he saw firsthand the inaccuracies of Galen‘s descriptions and was able to challenge them. In doing so, he destroyed the foundation of medieval medical practice, which, like astronomy, was based upon ancient tradition and inherited knowledge.
In this he was succeeded by William Harvey whose 1628 book On the Motion of the Heart and Blood demonstrated that the heart was the center of the circulatory process, that the same blood flows through both veins and arteries, and that the blood makes a complete circuit throughout the body. Harvey, whose findings were achieved "by autopsy on the live and dead, by reason and by experiment" (Richardson, 1987), is considered the inventor of modern laboratory science. Paracelsus, a 16th century chemist and physician, also contributed to the decline of Galenic medicine by rejecting the humoral theory and initiating the use of chemicals as treatments for disease.
Harvey‘s discovery of the circulation of the blood led to a more extensive use of vivisection in Europe. An Introduction to the Study of Experimental Medicine by 19th century French physiologist Claude Bernard provided scientific medicine with the philosophic rationale it had previously lacked and greatly accelerated its development and ascendancy over clinical (i.e., empirical) medicine. The increasing use of animals as subjects of scientific research was by no means universally applauded, yet by the early 20th century, the knowledge accruing from such investigations was testimony to the success of the method (Fig. 1, Russell and Burch, 1959).
Historical Perspective on Human Dissection
Dissection of human cadavers, a key element of contemporary medical training, has been viewed as both morally and legally unacceptable throughout much of history. Although practiced in ancient Egypt in the city of Alexandria, where Herophilos, Erasistratos, and others explored the nervous system, circulatory system, genitals, and the eye, human dissection was forbidden throughout Greece and later Rome. Galen‘s work in anatomy and physiology was seriously compromised by his inability to dissect human cadavers, a handicap that led to many errors not redressed until Vesalius‘ corrections 1,400 years later. Dissection seems to have been generally accepted throughout Renaissance Europe, although anatomists (including Vesalius) were sometimes accused of practicing human vivisection and were careful to protect themselves from such charges.
In 1832, the Warburton Anatomy Act legalized the sale of bodies for dissection in England, in an attempt to end the practice of "body snatching" from cemeteries. In The Old Brown Dog (Lansbury, 1985), an analysis of the psychology and sociology of antivivisectionism in Edwardian society, Coral Lansbury notes that opposition to animal experimentation among the poor and working classes was often closely linked to fear of one‘s corpse being sold for dissection. According to Lansbury, this fear was not unfounded as "those who died in the workhouse or the hospital and had neither friends nor family to claim the body were regularly handed over to the surgeons" (Lansbury, 1985).
Many working people were convinced, she notes, that scientists vivisected animals only because they could not vivisect humans and that if the bodies of living human beings were made available, they would be used. Meanwhile, the scientific argument for animal experimentation was based upon the ethical inadmissibility of the use of living human beings for most research, necessitating the use of animals as surrogates. Ruth Richardson‘s scholarly study of the Anatomy Act, Death, Dissection and the Destitute (Richardson, 1987), similarly explores attitudes of the poor toward scientists and surgeons. Like Lansbury, Richardson finds ample evidence that the poor were often victimized by unscrupulous and/or inadequately trained practitioners and that their bodies, in life as in death, were often treated as "teaching material."
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Figure 1. The expansion of science and of animal experimentation
Reprinted from Russell & Burch (1959).
Chapter 2 The Eye of Science
If I have seen further than other men, it is by standing on the shoulders of giants.
-Sir Isaac Newton
The 18th and 19th century physiologists who vivisected animals in order to understand the structure and function of various organs were succeeded by the pioneers of immunology, who used in vitro techniques to create vaccines for anthrax, cholera, and tuberculosis. Many early vaccines were developed using primitive in vitro methodologies and were then tested in animals, so it is difficult to draw a line between what has come to be called in vitro research and that which utilized whole animals, when discussing past achievements (see Appendix A: Methodologies of Vaccine Development).
In fact, in vitro studies have played a key function in increasing biomedical knowledge since Anton von Leeuwenhoek first observed protozoa, sperm, and bacteria under the lens of his self-made microscope in the 17th century. The work of Marcello Malpighi (who in 1660 viewed capillaries through a microscope, thus confirming Harvey‘s prediction of a system of tiny vessels carrying blood to the lungs) is an early example of the way in vitro studies have complemented and completed other forms of experimentation and observation throughout modern biomedical history. The development of cell theory and germ theory in the late 19th century greatly expanded the role of nonwhole-animal methodologies.
In 1665, Robert Hooke called the tiny chambers in a piece of thinly sliced cork he viewed under a microscope "cells" due to their resemblance to monks‘ rooms in monasteries. One hundred and sixty-six years later, Robert Brown observed that all plant cells contain a small body, which he called "little nut" or nucleus, and by 1835, Jan Purkinje had confirmed that animal tissues are also composed of cells. However, German scientists Schleiden and Schwann were the first to realize the significance of the cell as the basic unit of living organisms, with Schleiden proposing in 1838 that all plant tissues are made from cells, and Schwann suggesting one year later that eggs are cells, that animal tissues are also made from cells, and that life begins with a single cell. Rudolph Virchow, who insisted that "all cells arise from cells," augmented the cell theory of Schleiden and Schwann in the 1850s.
Meanwhile, Robert Koch and Louis Pasteur were laying the foundations of germ theory, with Koch discovering in 1876 that the microorganism responsible for anthrax in cattle could be grown in culture, and Pasteur learning in 1879 that weakened cholera bacteria would immunize chickens against its more virulent strain. In 1877, Koch developed a method for obtaining pure cultures of bacteria. By 1880, he was using solid cultures of gelatin or agar to grow microbes. In that same year, Pasteur published On the Extension of the Germ Theory to the Etiology of Certain Common Diseases and presented his findings on vaccination to the French Academy of Medicine.
In reviewing the tremendous progress made in the biomedical sciences over the past 100 years, it is clear that the tripartite approach, incorporating clinical (human) studies, animal experimentation, and in vitro studies, has been an extremely fruitful mode of inquiry. The discovery of a vaccine for polio is a classic example of the success of this approach, in which each of the three methodologies played a key role at different stages of research.
"When the work began, little was known about polio transmission or the mechanism of viral spread through the body. There was no means of prevention; after a bad summer, polio left thousands of children dead or paralyzed" (Fee, 1992). Researchers first attempted to study the route of viral infection using rhesus monkeys, "but the rhesus monkey proved to be a poor model for investigating the human disease" (Fee, 1992). Chimpanzees were used next and they proved a much better model, leading researchers to the key discovery that the initial route of infection was the digestive system, with the virus then spreading through the bloodstream into the nervous system. The work of teams headed by David Bodian at Johns Hopkins and Dorothy Horstman at Yale University led to the development of a killed-polio vaccine, which was tested in 12 children. The inactive vaccine was successful. However, a safe and easy method of growing the virus in large quantities was needed.
"In 1949, John Enders, Thomas Weller, and Frederick Robbins at Harvard were able to culture the polio virus in a variety of tissues and to develop a simple method of identifying the virus in culture" (Fee, 1992). Jonas Salk grew all three types of the polio virus in vitro, and Albert Sabin developed a live vaccine by growing viruses on monkey kidney tissue. The University of Michigan School of Public Health designed large-scale human trials of the vaccine in 1954, and one year later it was clear that the vaccine was a success. Research on the etiology and course of the disease and the development of a safe and effective vaccine required the use of an integrated approach, incorporating clinical, animal, and in vitro studies.
This three-pronged approach to the prevention and treatment of disease remains the standard methodology in biomedical research, as a more recent example illustrates. One of the more promising avenues in current cancer research involves the search for chemoprotectors. Epidemiological studies have indicated that people who eat plenty of green and yellow vegetables are less likely to develop cancer. Paul Talalay, a molecular pharmacologist at Johns Hopkins, has been studying the role of dietary chemicals in cancer protection since the late 1970s. Animal studies illustrated the mechanism by which certain food additives and other chemicals stimulated the production of protective enzymes in cells and demonstrated that increased enzyme activity resulted in higher resistance to cancer-causing chemicals, thus decreasing the incidence of cancer in the animals. This permitted the researchers to develop a quantitative tool to measure enzyme activity.
By 1985, Dr. Talalay and his team had begun to experiment with cell culture systems, using microtiter plates to automate the testing process. Researchers found that a component in broccoli proved most efficient in boosting enzymes that detoxify carcinogens. However, it took several high-tech spectroscopic methods to identify the chemical. Mass spectrometry, nuclear magnetic resonance, and infrared and ultraviolet spectroscopy revealed the structure of the chemoprotector sulforaphane. It will now be necessary to use animal studies to map the metabolic effects of sulforaphane and to correlate sulforaphane‘s effects in vitro with an ability to block the action of carcinogens in vivo. Human studies will inevitably follow.
As these examples indicate, it is not completely correct to say that progress in the biomedical sciences is wholly the result of animal research, nor is it correct to say that animal research has contributed nothing of value and that improvements in human health are solely the result of better diets and hygiene. The truth is that in vivo, in vitro, and clinical studies each provide pieces of the research puzzle and each contributes important information (Stephens, 1987). Progress depends on these three strands of investigation flowing into and feeding each other in an infinity circle of inquiry and information (Fig. 2).
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Figure 2. Infinity circle of research
Chapter 3 Toxicology and Toxicity Testing
Professor Heeresh Chandra, a leading pathologist for the Home Office at Bhopal‘s main Hamidia Hospital, said, "Why hasn‘t Union Carbide come forward and said this is the gas that leaked, this the treatment? Is it not a moral duty to tell us what was used, what is the treatment, what is the prevention? They have not come forward. Somebody has to tell us...A company should put it in the newspapers, a big advertisement on what can be the after-effects."
-Financial Times (December 8, 1984)
Toxicology is the science of poisons. Poisons are chemical substances that are harmful or "toxic" to living things. All substances are poisonous if ingested in sufficient quantities, including such necessities of life as water and oxygen (Table 1). Humans and animals can be exposed to both naturally occurring and man-made chemicals in a variety of ways -- by mouth, skin contact, or inhalation. Toxicological tests measure the effects of a limited exposure of an animal to a substance (acute toxicity) as well as repeated, long-term exposure (chronic toxicity). Substances are also tested for more specific endpoints such as cytotoxicity (ability to damage cells), mutagenicity (ability to cause changes in genetic material), carcinogenicity (ability to cause cancer), and teratogenicity (ability to cause birth defects).
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Table 1: Approximate LD50 for Various Chemical Substances Fed to Humans
Chemical
Equivalent* LD50 for 160 lb. Human
Sugar (sucrose)
3 Quarts
Alcohol
3 Quarts
Salt (sodium chloride)
1 Quart
2,3-D
1/2 Cup
Arsenic (aresenic acid)
1 to 2 Teaspoons
Nicotine
1/2 teaspoon
Dioxin (TCDD)
Speck
Botulinum toxin
Too small to be seen
*From rat LD50
Reprinted from Toxicology: A Primer on Toxicology Principles and Applications. Kamrin, MA, Lewis Publishers, 1988.
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All of these are aspects of risk assessment, the process by which substances are evaluated for their potential impact on human health and safety. Risk assessment can be divided into two segments. Assessment of exposure is an estimate of the number of people who will be exposed to a given chemical, together with concentration, duration, and terms of the exposure. Toxicity testing identifies hazards, determining which adverse effects will ensue from exposure to a chemical, and provides data estimating the quantitative exposure-response relationship for the chemical. The likelihood of a human being or animal developing an adverse response after exposure to a chemical varies depending on the route of exposure (skin contact, ingestion, inhalation) as well as the age, sex, genetic makeup, and health status of the individual. Some, but not all, of the latter group may be assessed by toxicity testing.
Other types of tests establish the toxicokinetic and toxicodynamic properties of chemicals. Toxicokinetic studies trace the absorption, distribution in the body, metabolism, storage, and excretion of chemicals. Toxicodynamic studies chart biological responses that are a consequence of the presence of a chemical in the body. For example, neurological and behavioral tests monitor effects of chemicals on cognitive functions, while tests for phototoxicity determine if sunlight can activate a test chemical and enhance skin irritation. The complete toxicological testing of a single chemical is a complicated, time-consuming, and expensive process (Fig. 3).
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Figure 3. Standard whole animal toxicity tests. Reprinted from Goldberg & Frazier (1989)
Why Test?
On April 15, 1980, a coalition led by animal activist Henry Spira took out a full-page advertisement in The New York Times, which posed the incendiary question, "How many rabbits has Revlon blinded for beauty‘s sake?" Revlon did not offer any figures in answer, but within a year they had donated $750,000 to Rockefeller University to research possible alternatives to the Draize test for ocular irritation, the first corporate funding of the embryonic science of in vitro toxicology.
However, in 1921, the Journal of the American Medical Association did quantify the results of the lack of testing for eye irritation, reporting numerous cases of human blindness and disfigurement (and at least one death) resulting from the use of a synthetic aniline dye called Lash-Lure, which was applied by operators in beauty salons to darken eyebrows and eyelashes. "Just how many women have been injured by the use of this preparation there is no way of knowing; but the Journal of the American Medical Association has reported at least 17 authentic cases, and there have no doubt been many others, for the firm has settled a number of what it termed ‘nuisance‘ cases for small sums. Still other claims for damages have been paid by the beauty shops or their insurance companies..." (Lamb, 1926).
Lash-Lure was not the only cosmetic preparation to cause injury. A hair dye called Inecto Rapid Notox was even more dangerous. "Among the specific injuries recorded in 37 cases where a hair dye containing The Criminal Ingredient was used were swelling of the face, eyelids, and larynx, inflammation of the skin with cracking, blistered scalp, impaired eyesight, swelling of the head and limbs, spreading of infection and eruption over entire body, hospitalization with incapacity for long periods, permanent blindness developing..." (Lamb, 1926) (see Sidebar, U.S. Regulatory Law).
U.S. Regulatory Law
Although regulation of foods in the United States originated during colonial times, and federal controls over drugs were enacted in 1848 (Green/Bradlaw, 1992), federal responsibility for consumer protection did not become a reality until the passage of the Food, Drug, and Cosmetics Act of 1938. The Delaney Amendments to this law, passed in 1958, require manufacturers to furnish data establishing the noncarcinogenicity of a product prior to marketing and sales.
The Toxic Substances Control Act of 1976 gives the Environmental Protection Agency (EPA) (established in 1970) the power to ban or restrict the manufacture or use of any chemical that it deems hazardous. The Act also authorizes the EPA to require testing of potentially harmful chemical substances already on the market. The Federal Insecticide, Fungicide, and Rodenticide Act requires that all pesticides distributed in the United States be registered.
The Occupational Safety and Health Administration (OSHA), created in 1971, is responsible for regulating safety in the workplace. OSHA uses both epidemiological and animal studies to make regulatory decisions regarding toxins. Finally, the Consumer Products Safety Commission (CPSC) is responsible for maintaining a clearinghouse of information about the hazards associated with the use of consumer products.
Each of these agencies is charged with ensuring the health and safety of the American people and each has its counterparts in other nations. One of the current challenges facing government scientists in many nations is finding a way to balance regulatory responsibilities with the desire to reduce animal testing. A recent fact sheet outlining current federal practices and regulations with respect to eye and skin irritation notes that the CPSC, EPA, and Federal Drug Administration do not require Draize tests for products or chemicals known to be corrosive, and that Draize tests are the final, not the initial, analysis done on products or chemicals that might prove irritating to the eyes or skin.
These descriptions illuminate the era in which tests such as the Draize test for eye and skin irritation were developed, when the toxins arsenic, quinine, resorcin, and mercury were commonly used in various cosmetics and personal care products. Drugs, too, often proved dangerous to consumers. Over 100 Americans died in 1937 when sulfanilamide, an early antibiotic, was mistakenly mixed with the toxic solvent diethylene glycol, and marketed as Elixir of Sulfanilamide. Not until enactment of the Federal Food, Drug, and Cosmetic Act of 1938 was a government agency given the power to regulate such products. Legislation existing prior to that time "prohibited interstate commerce in misbranded and adulterated foods, drinks and drugs" (Green and Bradlaw, 1992) but did not grant authority over cosmetics nor require safety testing for new drugs or provide for tolerance levels for poisonous substances.
Public emergencies resulting from the leakage or spillage of toxic chemicals have also occurred with regularity in the modern era. From Nitro, West Virginia in 1947 to Seveso, Italy in 1976 to Bhopal, India in 1984, human beings have been exposed to chemical toxins with tragic results (see Sidebar, Chemical Disasters). Leaving aside for the moment questions of culpability and liability, the fact remains that as long as human beings manufacture and store large quantities of toxic chemicals, the potential for such catastrophe exists and medical staff members will need basic information to treat victims.
Chemical Disasters
On July 10, 1976 an explosion at an Icmesa factory in Seveso, Italy released 1.3 kg of 2,3,7,8-tetrachlorodibenzodioxin, more commonly known as TCDD or dioxin, into the air. Over the next 3 days, vegetation, birds, and animals sickened and died in an area directly downwind from the plant, designated Zone A. Despite this evidence of toxicity, the 735 human residents of Zone A were not evacuated until July 26, 16 days after the accident. Studies later confirmed that the residents of Zone A exhibited the highest levels of dioxin ever found in human serum and that the soil in the zone was heavily contaminated.
Dioxin is a highly toxic synthetic chemical that resists environmental degradation. Since 1961, scientists have known that human beings exposed to dioxin often develop chloracne, a skin disease. The chemical‘s proven effects on animals are considerably more serious and include cancer, birth defects, and severe liver damage. Workers exposed to dioxin after a March 8, 1949 explosion at a Monsanto plant in Nitro, West Virginia developed skin and eye irritations, headaches, dizziness, and breathing problems in the immediate aftermath of the incident. Epidemiological studies following the Nitro and Seveso incidents have not proven excess cancer, birth defects, or any clearly substantiated toxicoses other than chloracne in these human populations. However, anecdotal accounts of neurological damage, decreased sexual potency in men, and lower birthrates in women persist (Whiteside, 1979).
In the early morning hours of December 3, 1984, 200,000 people in Bhopal, India were exposed to the gas methyl isocyanate. The 90-minute exposure resulted in at least 2,500 deaths and countless cases of severe eye and lung damage. Most of the deaths were caused by pulmonary edema (excess accumulation of fluid in the lungs) or its effects. Medical treatment of the victims was handicapped by a lack of information on the effects of methyl isocyanate in human beings; a lack that the accident has, unfortunately, remedied.
Furthermore, normal discharges and emissions may also pose a hazard over the long run. The National Cancer Institute is currently funding a large-scale study of the link between environmental contaminants and breast cancer, based upon the results of a recent study published in the Archives of Environmental Health. This study at Hartford Hospital in Connecticut (Falck, 1992) found that fat samples taken from malignant breast tumors contained more than 50% more PCBs (polychlorinated biphenyls) and DDT (dichlorodiphenyltrichloroethane) than were found in samples taken from women of the same age and weight with noncancerous breast biopsies.
Most of our current understanding about the toxicity of various chemicals comes from animal data, and as Andrew Rowan notes in his critical evaluation of animal research, Of Mice, Models & Men, "there is not doubt that our knowledge of the risks to humans of most chemicals is very inadequate" (Rowan, 1983). He and a number of other commentators have pointed out that an increase in animal testing, such as that which occurred after the thalidomide disaster in 1962, will hardly address the problem. "The pressing need at the moment is for more data on mechanisms and the development of a theoretical framework within which rational decision making can have a chance" (Rowan, 1983) (see Sidebar, The Truth About Thalidomide).
The Truth About Thalidomide
Fetal malformations caused by the use of the drug thalidomide constitute one of the most tragic chapters in modern pharmacology. Over the past decade, many individuals and organizations have used this episode to illustrate the inadequacy of animal testing, pointing out that extensive testing in animals did not reveal the teratogenic potential of the drug in human beings. However, as Rowan revealed in 1984, this claim is erroneous. "The fact is that thalidomide was not adequately tested, and after the tragedy, drug registration authorities around the world immediately increased their animal-testing requirements" (Rowan, 1984).
Although the drug was marketed in 1957, reproductive studies on thalidomide in animals were not started until 1961, after the drug‘s effects on human fetuses had begun to be suspected (MacBride, 1961 and Lenz, 1961, 1962). Initial studies on rats and mice revealed some reproductive abnormalities, notably reduction in litter size due to resorption of fetuses; however, only when the compound was tested in the New Zealand white rabbit did abnormalities similar to those noticed in human babies occur. Studies on monkeys revealed that they were almost as sensitive as humans to the deformative effects of the drug.
Rowan traces the confusion about thalidomide to the publication of Richard Ryder‘s book, Victims of Science (Ryder, 1975), noting that Ryder may have been misled by the claims of the Turkish scientist Aygun that he had evidence of the reproductive toxicity of thalidomide in tissue culture. This allegation has never been substantiated. The Insight Team of The Sunday Times of London revealed (Suffer the Children, 1979) that neither the German makers of the drug nor the British distributors performed any type of premarket teratogenic testing on animals, although such tests were being performed at that time on other sedative drugs such as Miltown and Librium. If anything, the story of thalidomide exhibits the need for tight regulation and extensive testing of new drugs and chemicals. When the German manufacturer of the drug was asked in 1961, four years after the drug went on the market, "when thalidomide is given to women patients, does it cross the placenta" the company‘s answer was "not known." The answer was not known because the necessary tests had not been performed.
Knowledge of the manner in which a chemical‘s structure determines its activity or how it exerts its ultimate toxic effects is not gained through whole-animal tests alone, but also through in vitro studies. In vitro biomedical research attempts to preserve organs, tissues, and cells outside the body. These cell cultures, tissue cultures, or organ cultures can then be used for a number of purposes, including toxicity testing.
The advantages of in vitro systems in toxicity testing are numerous. In vitro tests are usually quicker and less expensive. Experimental conditions can be highly controlled and the results are easily quantified. However, the relative simplicity of nonwhole-animal testing results in limitations as well. Cells or tissues in culture cannot predict the effect of a toxin on a living organism with its complex interaction of nervous, endocrine, immune, and hematopoietic systems. In vitro systems can predict the cellular and molecular effects of a drug or toxin, but only a human or animal can exhibit the complex physiological response of the whole organism, including signs and symptoms of injury.
What Is an Alternative?
As the science of in vitro toxicology has grown over the past few years, some confusion has developed over the exact definition of the word "alternative" as applied to these methodologies. A
replacement alternative is one that entirely eliminates the need for whole-animal testing. The limulus assay for bacterial endotoxins, in which the fever-producing potentialities of intravenous therapies are tested using the (extracted) blood of horseshoe crabs rather than whole rabbits, is one such replacement alternative. The use of in vitro systems for pregnancy testing is another (Fig. 4).
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Figure 4. The three R‘s of alternatives -- replacement, reduction, and refinement.
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A reduction alternative substantially decreases the number of whole animals necessary to perform a particular test or group of tests. A number of in vitro assays are now being used as screening tests for the Draize test for ocular irritation, reducing the number of animals required to fully evaluate the potential irritancy of a chemical. Refinement alternatives are those that improve the design and/or efficiency of the test, therefore lessening the distress or discomfort experienced by laboratory animals. Draize tests are no longer performed on substances with a pH of less than 2.0 or more than 11.5 (very acidic and very basic substances known to be severely irritating to the eye). This is a refinement alternative.
At present, in vitro methods are being developed in laboratories around the world. In a comparatively brief period of time, an international consensus has developed regarding the necessity of developing alternatives to the use of animals in toxicity testing (see Sidebar, European Ban on Animal Testing). This is partly in response to citizen concern about animal welfare, but it is also an aspect of the evolution of science itself.
European Ban on Animal Testing
On February 12, 1992, the European Parliament voted to amend Cosmetics Directive 76/768 to ban the marketing of cosmetics containing ingredients that have been tested on animals after January 1, 1998. The Council of Ministers agreed to consider extending the 1998 deadline for a period of not less than two years, if validated nonanimal tests have not been developed. With 518 members representing 320 million people, the European Parliament represents the world‘s largest trading group, the 12-nation European community. The European legislation will affect all companies in that they will be unable to market in Europe products that have been tested on animals, or containing ingredients that have been tested on animals, even if those products have been manufactured outside of Europe. Industrial chemicals are exempt from the ban.
Directive 76/768 was amended after an extensive campaign by national and international animal protectionist groups. A petition containing 2.5 million signatures was presented to the Chairman of the Parliament‘s Environment committee in June 1992. Rallies and legislative lobbying also contributed to the success of the campaign. At this point, it is not possible to assess the impact of the European legislation on American and international law or industrial practice. However, the amendment of this directive demonstrates that animal protectionism is a potent political force, capable of achieving its goals within large legislative bodies.
In the past 40 years bioscience has endured a change of perspective as profound as that borne by physics when the world was greeted with Einstein‘s famous equation E=mc2. The shift in scientific perspective, which arose in response to Einstein‘s theories of relativity and the contiguously developed quantum theory, has been paralleled by that effected by the discoveries in molecular biology. In both cases the eye of science has been drawn inward to the infinitesimal, the nearly invisible processes that create and sustain life. In physics, the stage in which these processes are enacted is the atom; in biology it is the cell, with its complex organization of molecules and information systems (Fig. 5).
Figure 5. Sketch of a cell with key structures noted, taken from an introductory text in molecular biology
Reprinted from Darnell & Lodish (1986).
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As a result of this shift in perspective and related discoveries, bioscience is in the midst of the same sort of technological revolution that physical science underwent 100 years ago when the discovery of the laws of electromagnetism and thermodynamics led to the production of electric motors, electric lights, telephones, radios, and most of the other modern conveniences that we take for granted today.
Researchers are just beginning to develop the knowledge base to produce the tools necessary to manipulate biological processes and solve the problems associated with disease. However, the biotechnological enterprise is based not on the macrocosm as was the Industrial Revolution, but on the microcosm, the barely perceptible processes that take place in molecules and cells.
As Thomas Kuhn noted in his seminal treatise on scientific paradigms and their replacement, The Structure of Scientific Revolutions, "we do not see electrons, but rather their tracks, or else bubbles or vapor in a cloud chamber" (Kuhn, 1962). Similarly, molecular biologists do not see the process of protein synthesis itself but rather, the evidence of protein synthesis. Bioscience, like physics, has turned inward and penetrated deeper and deeper into a nearly invisible world.
Over the past 20 years it has become clear that the process of writing a sentence involves chemical and molecular interactions as complex and delicate as the physical processes that keep the moon from crashing into earth or the stars from falling from the sky. A message is sent from the brain through the arm to the hand. It sounds simple and familiar enough, but consider the thousands of neural and glial cells, synapses, transmitters, and receptors that carry out the simple process; a single exposure to a potent neurotoxic substance is enough to sabotage the process and turn this sentence to aeua;/dnaa/f;a.
So how does a scientist determine if a new pesticide, or hairspray, or household cleaner is going to disrupt the nervous system, causing the consumer to write and speak gibberish, or walk unsteadily, or fall into a coma? In a typical in vivo study, a scientist trained in toxicology will select a group of animals, use half as a control group, and administer the substance to the experimental group while observing their reactions, prior to sacrificing them and examining their tissues. The toxicologist then renders a report to his or her agency (if a government regulatory toxicologist) or to his or her company (if an industrial toxicologist) and a decision regarding the use of the chemical is made.
This process is an aspect of risk assessment, the primary mechanism by which the health of the public is protected. No one wants to eliminate risk assessment. However, since 1981, a number of individuals and organizations have been working very hard to develop alternatives to the traditional whole-animal models used in risk assessment. In most cases they are attempting to discover the cellular and molecular mechanisms by which a substance exerts its toxic effects as well as recording the effects themselves. This sounds very easy but is in actuality incredibly difficult. Researchers in this new field, in vitro toxicology, are working on the cutting edge of knowledge and technique, which encompasses molecular and cellular biology, complex biochemistry, and biophysics.
In this endeavor they have been supported by the three R concept of replacement, reduction, and refinement first articulated in The Principles of Humane Experimental Technique. Written by W.M.S. Russell and Rex Burch of the Universities Federation for Animal Welfare (UFAW) and published in 1959, The Principles of Humane Experimental Technique marks an important scientific attempt to define and, hence, alleviate the distress suffered by laboratory animals. Russell and Burch stated that good science and animal welfare are not incompatible; rather the two go hand in hand. They found that experimental results taken from stressed animals may be misleading or erroneous. Replacement, reduction, and refinement are therefore essential to the creation of a more precise science, as well as a more humane one.
Serendipitously, Russell and Burch‘s proposal of the three Rs has coincided with the development of molecular biology, as well as with vast improvements in technique for such things as propagating cells in culture and measuring molecular processes. As Rowan notes in Of Mice, Models & Men, "many important discoveries are preceded by the development of one or more techniques of suitable sensitivity and discriminative powers" (Rowan, 1984) (see Sidebar, Biotechnology and Alternatives).
Biotechnology and Alternatives
Biotechnology, the use of biological processes to manufacture products, will have a significant impact on the development of alternatives and has already made contributions to the science of in vitro toxicology. Over the past decade, advances in culture methodology, including the developments of defined media, matrices, and effective growth factors have brought cell and tissue culture to new levels of sophistication. Researchers at The Johns Hopkins School of Medicine recently developed two human cortical neuronal cell lines for use in toxicity testing and basic research. Such a feat was almost inconceivable 20 years ago and considered impossible even a decade ago - the stuff of science fiction. Today it is a reality thanks to basic research and biotechnology.
Biotechnology will drive the development of in vitro methodologies in yet another way. As new biochemical entities, such as human growth hormones, are produced they will be tested, just as prior chemical formulations have been tested. However, in many cases, the only way to evaluate the efficacy and safety of these new compounds will be in cultures of human cells or through epidemiological studies. The nature of the technology will therefore mandate the use of human, not animal, tissues and subjects.
One other factor has contributed to the diminishing use of animals as models in toxicity testing. The animal rights movement and the political pressures brought to bear by animal protectionists have had (and will likely continue to have) noticeable impact on the practice of biomedical research. Ever since the publication of Rachel Carson‘s Silent Spring in 1962 instigated a societal reevaluation of human behavior and its impact on ecosystems, thoughtful people have recognized that the earth and its resources, whether animal, vegetable, or mineral, should be treated with respect and perhaps even reverence. That perspective may not preclude the use of animals in biomedical research or as food or companions, but it certainly mitigates against their being viewed as "test tubes with legs," as the authors of The Animal Rights Crusade: The Growth of a Moral Protest points out (Jasper and Nelkin, 1992).
At this stage in the alternatives debate, most toxicologists are willing to accept the fact that in vitro and other alternatives are a fruitful avenue for research and that at some point in the future they may replace many whole-animal methods in toxicity testing. Most also insist that until that time in vitro and other alternatives should serve as screens for in vivo (within the living organisms) tests. In addition , it is not possible to predict what scientific information will be produced over the next decade and what its effect on the feasibility and timing of replacement methodologies will be.
Those who argue that in vitro tests can never truly replace whole-animal methods illustrate a concept that Thomas Kuhn termed incommensurability. "The proponents of competing paradigms tend to talk at cross purposes - each paradigm is shown to satisfy more or less the criteria that it dictates for itself and to fall short of a few of those dictated by its opponent" (Kuhn, 1962). Critics of whole-animal methods claim that species variability invalidates the animal model, and proponents of traditional in vivo models insist that in vitro tests cannot predict the systemic effects of a toxic substance in a whole animal or human subject.
The problem is exacerbated by the fact that in vitro toxicology is an infant science, hardly mature enough to satisfy the enormous demands being placed upon it by those who would like whole-animal testing to be abolished this year, next year, or the year after that. Kuhn notes that it takes at least a generation for a cycle of change to complete itself. Until the cycle is complete "there will be a large but never complete overlap between the problems that can be solved by the old and by the new paradigm. There will also be a decisive difference in the modes of solution" (Kuhn, 1962) (See Appendix B, Timeline of In Vitro Toxicology.)
Chapter 4 Science In Vitro
When the profession can no longer evade anomalies that subvert the existing tradition of scientific practice - then begin the extraordinary series of investigations that lead the professions at last to a new set of commitments, a new basis for the practice of science. The extraordinary episodes in which the shift of professional commitments occurs are the ones known as scientific revolutions.
-The Structure of Scientific Revolutions
Thomas Kuhn (1962)
For many people, the Draize test for ocular irritation represents the whole of toxicity testing. Although this perception is not factually correct, it is true that the Draize test provides a good illustration of the inherent strengths and weaknesses of the whole-animal approach to toxicity testing. The search for an in vitro replacement to the Draize test also provides an exemplar of the practical difficulties involved in implementing the new methodologies.
The test is named for the late Dr. John Draize, a U.S. government scientist who standardized the scoring system of a preexisting test for ocular irritation in 1944. In the standard Draize test, a liquid or solid substance is placed in one of the rabbit‘s eyes and changes in the cornea, conjunctiva, and iris are observed and scored, with 73% of the score weighted to corneal changes, 18% to conjunctival changes, and 9% to changes in the iris when compared with the untreated eye (Fig. 6). The rabbit‘s eyes are inspected at 24, 48, 72 hours, and at four and seven days. Different regulatory agencies and different nations use modifications of the standard Draize test in which the number of animals, use of topical anesthetics, rinsing of the eye after application of test material, and methods for interpreting the final outcome may vary.
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Figure 6. Cross-section of the Eye
Reprinted from Bruner (1992).
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The rabbit eye, although similar in size to the human eye, differs from it in a number of important respects. These differences in physiology, together with the subjectivity of the scoring apparatus and the variability of results produced by different laboratories (Rowan, 1983), form the basis of the scientific objections; the humane objections are obvious. Nonetheless, the Draize test, when performed by trained personnel, has proven quite accurate in predicting human eye irritants, particularly slightly to moderately irritating substances, which are difficult to identify using other methods.
The lack of scientific elegance in its methodology has not prevented the Draize test from performing its primary function of assessing both the damage and potential for recovery after exposure to irritants. For this reason, many toxicologists and ophthalmologists are reluctant to repudiate the Draize test for ocular irritation, although they recognize its shortcomings. Most experts believe that a battery of in vitro alternatives will be necessary to replace the Draize test, since no one test can measure all the necessary variables (Rougier, Cottin, de Silva, et al, 1992; Balls, 1992; Goldberg and Silber, 1992).
Current tests are not yet developed and validated to the point that they comprise an appropriate battery for all chemical substances. Draize tests are performed in many different industries for many different reasons. The risk assessment needs of the chemical industry, for example, differ greatly from those of the cosmetic industry and both of these differ from the needs of companies who manufacture medicines for the eye. Each of these users is, therefore, likely to create a different role for in vitro tests in their risk assessment procedures.
Many in vitro tests are being used as reduction and refinement alternatives while undergoing validation, the process by which the suitability of a particular test is assessed for a specific purpose, with its reliability and reproducibility verified (Frazier, 1990). At the Center for Alternatives to Animal Testing‘s 10th anniversary symposium, the Cosmetic, Toiletry, and Fragrance Association‘s Director for Toxicology, Stephen Gettings, announced that from 1980 to 1989, the number of rabbits used in irritancy evaluations had been reduced by 87% in the cosmetics industry.
The Interagency Regulatory Alternatives Group, IRAG, composed of representatives from the Consumer Products Safety Commission, Food and Drug Administration, and Environmental Protection Agency, has recommended that the number of animals used per eye irritation test be decreased from six to three. They have also proposed the used of a tiered assessment process (Fig. 7) to determine the irritancy potential of a test substance before it is placed in the rabbit eye. Although these initiatives do not signify the elimination of the Draize test for ocular irritation, they do indicate a departure from sole reliance on the test and a theoretical and practical acceptance of the scientific validity of developing in vitro methodology and implementation of the three Rs.
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Figure 7. Tier-testing approach to assessment of ocular irritation
Reprinted from Frazier, Gad, Goldberg, and McCulley (1987).
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Endpoint Assays
In vitro assays consist of three components -- the biological model, the endpoint measurement, and the test protocol (Table 2). The biological model is the system used for evaluation, for example, hepatocytes (liver cells). The greater the ability of the biological model to represent in vivo structure and function, the more valuable the data. An endpoint measurement is the yardstick used to predict toxicity (e.g., cell death). The test protocol is the schedule of events defining the test -- for example, exposing hepatocytes to a test chemical for a specific period of time and measuring the defined endpoint at various times after rinsing the chemical form from dish of cells.
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Table 2: Existing In Vitro Toxicity Tests for Acute Dermal Irritation Testing
Name
Biological Component
Type of Testinga
Level of Testingb
Referencesd
A. Cell Culture Assays
1. Neutral red uptake
(1) BALB/c 3T3 cells
S
B
(1, 3, 4)
(2) NHEK
S
B
(1, 17)
(3) 3T3 Swiss mouse fibroblast
S
B
(7)
2. Uridine incorporation
BALB/c 3T3 cells
S
B
(16)
3. Total cellular protein
(1) BALB/c 3T3 cells
S
B
(15)
(2) NHEK
S
B
(7)
4. Keratinization
XB-2 cells
S
B
(7)
5. MTT assay
3T3 Swiss mouse fibroblast
S
B
(7, 11)
6. Enzyme leakage
3T3 Swiss mouse fibroblast
S
B
(6)
7. Arachidonic Acid metabolism
NHEK
S
B
(6, 10)
B. Skin culture assays
1. Protein synthesis assay
Human skin
S
B
(12)
Rabbit skin
S
B
(12)
Guinea pig skin
S
B
(12)
2. Nuclear vacuole formation
Human skin
S
B
(12)
Rabbit skin
S
B
(12)
Guinea pig skin
S
B
(12)
3. MTT assay
(1) TESTSKIN (organogenesis)
S
B
(2, 5)
(2) Human skin model (Marrow-Tech)
S
B
(5)
4. Release of inflammatory
TESTSKIN (organogenesis)
S
B
(13)
5. Neutral red uptake
Human skin model (Marrow-Tech)
S
B
(2, 5)
6. Electrical conductivity assay
Epidermal slice
S
B
(14)
C. Other
1. SKINTEX
c
S
B
(9)
2. Computer-based structure-activity relationship
c
S
A
(8)
aS = screening, A = adjunct, R = replacement.
bA = toxic potential, B = potency, C = hazard/risk.
cNo living biological component.
dReferences: 1. Babich et al (1989); 2. Bell et al (1988); 3. Borenfreund and Puerner (1984); 4. Borenfreund and Puerner (1985); 5. Center for Animals and Public Policy (1989); 6. DeLeo et al (1987); 7. Duffy et al (1986); 8. Enslein et al (1987); 9. Gordon et al (1989); 10. Lamont et al (1989); 11. Mol et al (1986); 12. More et al (1986); 13. Naughton et al (1989); 14. Oliver and Pemberton (1985); 15. Shopsis and Eng (1985); 16. Shopsis (1984); 17. Triglia et al (1989).
Reprinted from Dermatoxicology, by F. Marzulli and H. Maibach, Hemisphere Publishing, Washington, DC, 1991.
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The neutral-red assay is an example of an in vitro test designed to provide indication of cell membrane integrity as an endpoint. In this test, cells are cultured in plastic petri dishes and treated with various concentrations of a test chemical. The neutral-red dye, which is added to the cell culture after rinsing out the test chemical, is accumulated and stored by cells. The amount of dye retained by cells indicates the number of living cells in the dish (Fig. 8).
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Figure 8. Microtiter plate used in in vitro studies
Reprinted by permission from Clonetics, Inc.
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The cellular protein assay, which assesses amount of protein present, proceeds in a similar manner except that an analytical reagent called kenacid blue, which reacts with protein in the cells, is added to the cell culture after the test chemical is rinsed away. A dish full of healthy, rapidly growing cells will stain dark blue, while dishes in which cell death has occurred will be lighter in tone, depending upon the extent of the damage. These endpoints may be quantified by sensitive instruments that measure the amount of light absorbed by the dye at specific wavelengths.
Other processes that can be used as endpoint assays are the MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay, a test that assesses energy production by the mitochondria, and the LDH (lactate dehydrogenase) assay that measures the amount of this enzyme leaking out of dead or damaged cells. The introduction of molecules that sense the intracellular environment can be used to detect other toxicological endpoints such as the elevation of intracellular calcium and changes in relative acidity (pH) by fluorescence (the emission of light). All of these very general assays provide measures of cellular responses to chemicals that can, in turn, be interpreted as indications of acute toxicity, particularly if a ranking of known toxic substances deduced from such test agrees with appropriate in vivo data.
A series of chemicals can be tested with a particular in vitro method, such as the MTT assay, and the concentration of each chemical, which affects the endpoint response by 50% (designated the EC50), used to rank that chemical. This ranking reflects the relative toxicity of the series of chemicals in vitro. Whether or not this in vitro rank ordering of chemicals will correspond to the relative potency of the chemicals in vivo will depend on many factors, for example, in vivo kinetics, metabolism, repair, and defense mechanisms. At this time, the techniques needed to extrapolate in vitro data to the in vivo situation are just developing. However, chemicals that demonstrate high toxicity during in vitro cytotoxicity tests relative to well-understood toxins should be flagged for further investigation.
A great deal of in vivo data, both human and animal, has been accumulating over the past 50 years, although much of it is not currently available to the public. The compilation of this vast multitude of data into an easily accessible and comprehensive database would be a tremendous boon to alternatives researchers. Problems associated with the compilation of this database include the inability to access all of the data due to proprietary restrictions imposed by corporations, the lack of identified sources of funding for such a massive undertaking, and the lack of a broad-based unified effort to support such a project (see Sidebar, The LD50 Test).
The LD50 Test
The LD50 test was used worldwide for over 50 years as a means of assessing the acute toxicity of chemicals. The test was introduced in 1927 by a British biologist, Trevan, to assess and standardize the potency of batches of therapeutic substances such as digitalis, insulin, and diptheria toxin. It eventually came to be used as a standard measure of acute toxicity and a key element in the toxicological profile of a chemical or chemical mixture.
The goal of the LD50 test is to determine the amount of a test substance required to kill half of the test animals, hence the acronym (L)ethal (D)ose50. Like the Draize test for ocular irritation, the LD50 has been criticized by scientists as well as animal protectionists, and an International Coalition to Abolish the LD50 was established in 1980. In 1991, representatives of regulatory agencies in Japan, Europe, and the United States agreed to drop the classic LD50 as a required measure of acute toxicity.
New approaches to acute toxicity testing include the determination of Approximate Lethal Dose, the Up and Down Procedure, and acute toxicity testing in the nonlethal dose range. The Approximate Lethal Dose is derived by the administration of graduated doses of the test substance to individual animals, rather than cohorts, and can usually be determined using four to ten animals. The Up and Down Procedure also involves administering a substance to animals one at a time, with doses increased by a factor of 1.3 unless the animal dies, in which case the dose is decreased by 1.3. In such tests, fewer animals are needed compared to the classic LD50.
In 1989, a working group of the British Toxicology Society developed a new approach to acute testing which, rather than using death as the only endpoint, also accepts "sign of toxicity of sufficient severity that treatment at the next higher dose level is likely to lead to mortality." According to the authors of a 1990 paper "the advantage of our approach...is that the whole range of intoxication is shifted to the lower toxicity range and the average pain is much lower, while at the same time the scientific value of the results is much higher" (Taborini, Sigg, and Zbinden, 1990).
Tissue Culture
Although attempted on a primitive scale as far back as 1885, tissue culture was not a practical methodology until the discovery of antibiotics, which inhibit the growth of bacteria (see Appendix C: Timeline of Tissue Culture). It is possible to culture both normal and abnormal tissue and to harvest both cells and tissues from humans and animals.
Cell culture is complicated by the tendency of isolated cells to "dedifferentiate" in culture, taking on the qualities of unspecialized cells instead of keeping the characteristics that define them as cells from a specific organ such as the liver. For that reason, the easiest type of cells to maintain in culture are the less differentiated cells such as fibroblasts. Cell lines can be developed from these proliferating cells and maintained continuously, eliminating the need for further animal or human donors, but due to their relative lack of specific functions, they are not really useful for many types of toxicity testing.
Primary cells, taken from a specific organ in an animal or human donor, are very useful but maintain their differentiated functions only for a few days, or at most a few weeks. The challenge is finding a way to keep primary cells, with all their special structures and functions, from dedifferentiating. One possibility is by co-culturing them with different types of cells from the same organ. Studies have shown that in co-cultures, the cells survive longer and are better able to maintain their differentiated functions. Another potentially useful technique for long-term culture of differentiated cells is immortalization, in which the DNA encoding viral genes are transfected into primary cells. Ideally, the gene can be turned on and off, causing the cell to replicated indefinitely when the gene is turned on and to redifferentiate when the gene is turned off
Tissue slices are another in vitro option (one gaining in popularity) as the cellular architecture of the organ is preserved with all cell types present in the whole organism present in the tissue sample used for testing. However, given the status of current culture techniques, the slice is viable for a relatively brief period of time (hours to days). An additional technique involves the use of isolated organs. At the present time certain organs, such as the liver, can be maintained outside of the animal for several hours via perfusion, the pumping of blood or artificial media through the organ to nourish it. It is then possible to infuse chemicals into the organ and examine their effects.
Each of the above methodologies can be used to measure toxicity in a specific organ. However, to mimic better the effect of a toxic substance in a whole animal researchers, are beginning to co-culture cells from multiple organs. Many toxic substances are metabolized by the liver. Sometimes the metabolism of a nontoxic substance by liver enzymes will result in the formation of a toxic metabolite, which will then affect another organ in the body, for example the kidney. By co-culturing liver and kidney cells and then introducing a toxic chemical, the investigator can observe the process wherein the liver cells metabolize the chemical and the kidney cells respond to the toxic effects of the metabolite.
While cell culture techniques have become increasingly sophisticated over the past decade, a great deal of work still needs to be done to define the optimum culture conditions for different types of cells, so that they may propagate indefinitely and still function and they do in vivo.
Physiologically-Based Toxicokinetic Modeling
Tissue and cell culture alone cannot predict the effects of toxic substances in an animal or human. The cells and tissues must be placed in context within the organism. Physiologically-based toxicokinetic modeling attempts to do just that. Using computer simulation, researchers are attempting to relate the concentration of a chemical that causes toxicity at the cellular level as measured with an in vitro test to the corresponding in vivo exposure level that might have produced that effect. The simulations are performed using a computer to solve the differential equations that describe the model (Fig. 9).
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Figure 9. Toxicokinetics (absorption, distribution, metabolism, storage, and excretion of a chemical) and toxicodynamics (effects of the chemical and its metabolites on an organism
Reprinted from Goldberg & Frazier (1989).
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In this type of modeling, the toxicokinetics of various drugs and chemicals are determined by their tissue solubility characteristics, metabolic rates, and the physiology of the test species. The information required to construct the model can be gained through literature searches, in vitro studies, and in vivo experiments, which are limited to those designed to obtain very specific information relevant to the model. Due to the systemic nature of the model, much of the necessary information retrieved through literature search and databases is the result of past whole-animal studies.
The construction of a model for the first chemical in a class demands a great deal of time and effort, but once the prototype is created and validated, it can be extrapolated and applied to other related chemicals under various exposure conditions. Mathematical modeling offers a tool to predict the concentration of the active form of the toxicant at the site where it generates its effects. However, as researchers working in the field admit, physiologically-based toxicokinetic modeling is, like much of in vitro toxicology, in its infancy with an enormous array of practical and theoretical challenges to be overcome before it is able to serve as a general basis for risk management.
Structure-Activity Relationships and Databases
The hypothesis on which the concept of structure-activity relationships is based states that the structure of a chemical inherently possesses all of the information necessary to predict its toxicity, including the manner in which both the parent chemical and its metabolites will interact with macromolecules in the cell. This principle has been successfully applied to certain classes of carcinogens; however, its broader applications to general toxicity have not yet been established.
In applying structure-activity relationships, biological effects are expressed in qualitative terms. A mathematical equation is prepared to correlate the toxicant‘s chemical properties with the biologic effect. The relationship derived from the equation is used to make predictions about the toxicity of a chemical. Computers are used to establish these relationships. Advances in computer technology over the past 20 years have contributed greatly to the development of both structure-activity relationships and physiologically-based toxicokinetic modeling.
The construction and use of comprehensive databases will also help reduce the number of animals used in testing and refine the testing process. John Frazier of the Johns Hopkins Center for Alternatives to Animal Testing and Mildred Green of Technical Database Services are currently compiling a database that will include both descriptions of in vitro methodology and specific results obtained by testing chemicals with these methodologies. A collection of information about methods, together with a compendium of data produced by these methods, should prove useful for both validation and interpretation of in vitro testing data.
In Germany, ZEBET (The Center for Documentation and Evaluation of Alternative Methods to Animal Experiments) was established at the Institute of Veterinary Medicine of the Federal Health Institute in 1989. The ZEBET Data Bank offers both a compilation of literature on alternative methods in relation to the three Rs (replacement, reduction, refinement), description of the animal experiment that can be replaced, reduced, or refined, names of scientists experienced in the area, and references on the specific alternative methods and on the animal experiment it is designed to replace, reduce, or refine. Most of the information in the ZEBET Data Bank is available only in German; however, both the summary and list of references are available in English. FRAME (Fund for the Replacement of Animals in Medical Experiments) in England and Professor Nicola Loprieno and colleagues at the University of Pisa, Italy are also constructing databases that should prove useful to researchers implementing the three Rs.
In the United States, the Toxicology Data Bank, developed in 1978, lists over 4,000 chemicals and includes data on the production and use of each chemical, a description of its physical properties, and the results of in vivo pharmacological and biochemical experiments. As the Toxicology Data Bank and similar in vitro databases grow, researchers will be able to design and plan better experiments, based upon the knowledge gained from newly accessible data. Conversely, as understanding of toxicological mechanisms increases, the data bank grows larger and more useful.
Validation and the Future of In Vitro Toxicology
The future of alternatives research lies in validation. Validation is the door through which every alternative method must pass before entering the armamentarium of toxicity testing. In validation, a particular test is defined for a specific purpose. The test‘s relevance and reliability are established through intralaboratory and interlaboratory assessment, test database development, and evaluation.
In September 1992, the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing completed a draft framework for validation and implementation of new in vitro toxicity tests. Noting that "continuing advancements in both cellular and molecular biology and bioanalytical and computer techniques" had resulted in a proliferation of in vitro alternatives without a "formal administrative process to organize, coordinate or evaluate validation and implementation of these advancements," the committee proposed a validation plan aimed at increasing scientific and regulatory acceptance of alternative technologies (Journal of American College of Toxicology, In Vitro Toxicology, Xenobiotica, In Vitro: Cellular and Developmental Biology, 1993).
Key elements in the framework for validation include reference laboratories that could evaluate in vitro methods using sets of chemicals provided by chemical banks. Information could then be placed in a continuously updated database, with publication in appropriate peer-reviewed journals. The committee has also proposed the establishment of Scientific Advisory Board review panels. These panels would provide advice and information to researchers interested in developing and validating new procedures, review and recommend the scientific criteria for validation of new testing methods, identify tests ready for validation, and recommend tests as being validated for specific purposes (Fig. 10).
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Figure 10. Framework for validation proposed by the Center for Alternatives to Animal Testing validation committee.
Reprinted from Goldberg, Frazier, Brusick, et al. (1993).
Chapter 5 Animal Experimentation: Ethics and Law
The universities do not teach all things, so a doctor must seek out old wives, gypsies, sorcerers, wandering tribes, and such outlaws and take lessons from them.
- Paracelsus (1493-1541)
In 1831, British physiologist Marshall Hall proposed five principles that he believed should govern animal experimentation. First, an experiment should never be performed if the necessary information could be obtained by observations; second, no experiment should be performed without a clearly defined and obtainable, objective; third, scientists should be well-informed about the work of their predecessors and peers in order to avoid unnecessary repetition of an experiment; fourth, justifiable experiments should be carried out with the least possible infliction of suffering (often through the use of lower, less sentient animals); and finally, every experiment should be performed under circumstances that would provide the clearest possible results, thereby diminishing the need for repetition of experiments.
Hall also proposed the founding of a scientific society to oversee publication of research results and recommended that "the results of experimentation be laid before the public in the simplest, plainest terms" (Rupke, 1987). Hall was castigated by those who disapproved of animal experimentation, both within and without the medical community. He died in 1857, with many of his recommendations formally instituted over a century later in the British Animals (Scientific Procedures) Act and the U.S. Animal Welfare Act.
Laws Regulating Animal Experiments
The first law written specifically to regulate animal experimentation was Great Britain‘s 1876 Cruelty to Animals Act. This act was a much weakened version of the original, extremely bill, which came close to passage in 1875. The 1876 law, which implicitly approved animal experimentation at the same time that it set up a system of licensing and certification, was replaced by the Animals (Scientific Procedures) Act of 1986, which specifically states that "The Secretary of State shall not grant a project license until he is satisfied that the applicant has given adequate consideration to the feasibility of achieving the purpose of the programme to be specified in the license by means not involving the use of protected animals" (Animal Welfare, UFAW, Vol. 1, No. 2, 1992).
In the United States, the 1966 Animal Welfare Act, amended in 1970, 1976, 1986, 1989, and 1991, set standards for the transportation and husbandry of laboratory animals, excluding rats, mice, and birds. On January 8, 1992 the U.S. District Court in Washington, DC ruled that the U.S. Department of Agriculture had been violating the Animal Welfare Act by not enforcing its provisions as they relate to these animals. The case is currently on appeal (see Appendix D: United States Animal Welfare Timeline, and Appendix E: Great Britain Animal Welfare Timeline).
The U.S. Public Health Service Guide for the Care and Use of Laboratory Animals and the Health Research Extension Act of 1985 regulate all research funded by the National Institutes of Health (NIH) and require the submission of regular reports on protocols involving animals. The guide has been revised five times and is being updated once again this year. The NIH also requires accreditation by the American Association of Laboratory Animal Care (AAALAC) or the operation of an institutional animal care and use committee. However, since AAALAC requires an animal care and use committee as well, almost all institutions conducting vertebrate research or testing are now subject to review.
Institutional Animal Care and Use Committees
Under the 1985 amendment to the U.S. Animal Welfare Act, Institutional Animal Care and Use Committees (IACUC) have been established to review all protocols for procedures involving live warm-blooded animals, whether or not pain or distress is likely to occur. If these procedures are acceptable, the IACUC provides institutional approval. In addition, the IACUC must evaluate procedures every year and inspect facilities.
Each research facility must set up an IACUC composed of at least five members, one of whom is a Doctor of Veterinary Medicine, who are responsible for the activities involving animals at the institution. The committee must also include at least one practicing scientist experienced in research involving animals, one member whose primary concerns are nonscientific (i.e., a lawyer, cleric, or ethicist), and one individual who is not affiliated with the institution in any way except through his or her membership on the committee. However, most committees have more than five members, and in academic institutions, it is common to have student representatives as well.
The establishment of animal care and use committees in universities, industrial laboratories, and other research facilities has certainly had an impact on the use of animals in these places. IACUCs are charged with reviewing both the ethical and procedural issues related to animal use, including the choice of animal, numbers of animals, the degree of pain and discomfort animals will experience, and whether or not the investigator has considered the use of replacement, refinement, and reduction alternatives to whole animals in constructing the protocol (Fig. 11).
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Figure 11. University of California (Berkeley) Animal Care and Use of Committee Protocol Review Process
larger image
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Although grumbling has been heard about the "bureaucratization" of science, some researchers are willing to admit that the IACUCs have not only improved conditions for laboratory animals but have also raised the calibre of the research itself. During a recent discussion at an IACUC symposium sponsored by the Office of Research Subjects at the Johns Hopkins University School of Public Health, the chairman of the University of Maryland School of Medicine‘s Animal Care and Use Committee, Michael Lipsky, commented that the animal care and use committee in that institution has facilitated a great deal of interaction and cooperation between researchers in different departments and disciplines. Because the members of the animal care and use committees are familiar with work of every investigator in the institution using animals, they are able to act as liaisons between researchers who might not otherwise connect, resulting in an extremely fruitful exchange of information and expertise.
It would be naive to assume, however, that animal care and use committees are universally popular and well-respected. Although science in the United States is very carefully peer-reviewed, there is increasing public demand for accountability, both ethical and economic. This is sometimes resented and perceived as an infringement of academic freedom. However, as institutions and researchers come to understand the role of the committees, this perception is diminishing.
Surrogate Responsibility
As a number of commentators have pointed out (Scientific Perspectives on Animal Welfare; Dodds and Orlans, 1982) the ultimate responsibility for the humane care and treatment of animal lies with scientists themselves. "It is impossible to police every laboratory in the country for 24 hours every day to ensure that proper treatment is given to these animals...Humane use of the animal is entirely in the hands of the investigators" (Dodds and Orlans, 1982). The vast majority of scientists acknowledge this responsibility and conduct experiments in a humane manner.
Recognizing the need to affirm investigator responsibility early in a student‘s scientific career, the government of The Netherlands has mandated that every doctoral student in the sciences take a course in the history and ethics of animal experimentation prior to graduation (van Zutphen, personal communication). This legislation may ultimately prove far more effective than restrictive covenants in sensitizing individual investigators and disseminating the three Rs approach. Studies in the social sciences have illustrated time and again that a change in both individual attitudes and group norms is essential for effective implementation of reform. If the culture of biomedical research promotes mindfulness and respect for animal life and well-being, the great majority of scientists and scientists-in-training will conform to these norms.
Richard C. Bartlett, President of Mary Kay Cosmetics and an active conservationist, and Frederick S. Carney, Professor of Ethics at Southern Methodist University, have written of the widespread employment of the concept of surrogate responsibility not only by parents on behalf of their children and by spouses and offspring on behalf of mentally incapacitated older persons, but also an increasing number of persons to identify, protect, and maintain threatened species and endangered ecosystems. Bartlett and Carney believe that this concept applies to laboratory animals as well. "The notion of surrogate responsibility...involves a profound respect for animals, a strong commitment to their welfare through proper care and use, and an avoidance or minimization of discomfort, distress and pain... It also calls for rigorous and continuing efforts to find in vitro alternatives to the use of animals" (Bartlett and Carney, 1992).
Bartlett and Carney also note the necessity of "an appropriate and effective institutional framework" in support of surrogate responsibility, including high national standards for the care and use of laboratory animals, monitoring of laboratory conditions, and the development of local review boards, which must approve in advance all use of animals in product safety testing. Professor Carney has stated that "in the United States the institutional animal care and use committee mandated by the 1985 amendment to the Animal Welfare Act would be sufficient, although open to future refinements in the light of ongoing experience and ethical reflection" (personal communication).
Clearly, the animal care and use committee has stimulated a greater degree of sensitivity within the scientific community to the ethical aspects of animal experimentation, just as the campaigns of animal advocates have stimulated a greater awareness among the public of all aspects of human use of animals, from food production to entertainment to the wearing of fur and leather (Fig. 12). Reasonable people may disagree about the morality of human use of animals, just as they disagree about other hotly debated topics in ethics such as capital punishment, abortion, and euthanasia.
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Figure 12. Animals killed annually in the United States. EStimated numbers provided by the U.S. Congress Office of Technology Assessment, the Humane Society of the United States (1988), Animals‘ Agenda (1987), and the OTA (1985).
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In a democratic society, individuals have a right (and a responsibility) to express their convictions on these issues, contributing to the national discourse. However, individuals do not have a right to destroy property or to harass or otherwise threaten fellow citizens who hold different beliefs. The impotence of those who choose violence as a means of protest can and should be contrasted with the pragmatic and highly effective approaches of activists such as Henry Spira, whose Campaign to Abolish the LD50 and Draize Tests has instigated widespread institutional change.
"Many people perceive the movement for animal protection as negative and antiscience," Spira wrote recently. "To counteract this perception we have continually accentuated the fact that members of the science community are part of the nine out of ten people concerning protecting animals. We have attempted to turn walls in to bridges by promoting the shared goals of better science, efficiency, economy, and humanity" (Spira, 1993).
The bridges that Spira and others in the scientific and animal protection communities have built in the past decade will become better traveled as more people in both groups realize that good science and animal welfare are complementary -- not conflicting -- goals and that diplomacy yields far higher dividends than ad hominem attacks. Jerald Silverman, a laboratory animal veterinarian, commented recently that "we spend far too much of our time and effort trying to win the propaganda war of ethical animal use versus no animal use."
Silverman, like Spira, suggests focusing on common goals. "I argue that our goal should be to stop using animals while I readily admit that this is unrealistic in the foreseeable future. Nevertheless, if we do not try to make progress toward that goal, we deny the validity of scientific endeavor... We can therefore share a common goal, differing in our opinions as to the speed with which the goal can be reached...One of the intermediate steps toward reaching this goal must be to make the search for adjuncts and alternatives a well-funded scientific discipline. When funding is available, scientists and scientific acceptance will follow" (Silverman, 1993).
Spira and Silverman, activist and scientist, together point to an important truth -- that idealism tempered by pragmatism achieves measurable progress. The reductions in animal use for toxicity testing, which have been achieved over the past decade, are truly remarkable and have been spurred by scientific, economic, and political factors. Few would argue that the implementation of the three Rs on a broad scale has resulted in a decrease in public safety or poses dangers to human health. In fact, it is widely recognized that alternatives research has moved the science of toxicology forward and will continue to do so.
Whether one chooses to refer to these methodologies as adjuncts or alternatives may not be as important an issue as some would make it. Both science and activism seek the goal -- quantifiable results. If replacement, reduction, and refinement methodologies are able to produce (as they demonstrably have) quantifiable results, then both scientists and activists can, with clear consciences, work together in continued pursuit of that goal. As many have already discovered, the process itself yields rewards.