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Johns Hopkins Bloomberg School of Public HealthCAAT

Animals and Alternatives in Testing: History, Science, and Ethics

Joanne Zurlo, Deborah Rudacille, and Alan M. Goldberg

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).

Table 1: Approximate LD50 for Various Chemical Substances Fed to Humans

ChemicalEquivalent* LD50 for 160 lb. Human
Sugar (sucrose)3 Quarts
Alcohol3 Quarts
Salt (sodium chloride)1 Quart
2,3-D1/2 Cup
Arsenic (aresenic acid)1 to 2 Teaspoons
Nicotine1/2 teaspoon
Dioxin (TCDD)Speck
Botulinum toxinToo small to be seen

*From rat LD50
Reprinted from Toxicology: A Primer on Toxicology Principles and Applications. Kamrin, MA, Lewis Publishers, 1988.

All of these are aspects of , the process by which substances are evaluated for their potential impact on human health and safety. Risk assessment can be divided into two segments. 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. identifies hazards, determining which adverse effects will ensue from exposure to a chemical, and provides data estimating the quantitative 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 and 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 functions, while tests for 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).

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).

Figure 4. The three R's of alternatives -- replacement, reduction, and refinement.

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).

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.)