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Disclaimer • Welcome • Why Alternatives? • Alternative Cancer Therapy Guides www.healingcancernaturally.com On Conventional Medicine • On Modern Medicine • On Cancer Research |
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Cancer Research & Animal Experimentation: an Unholy Union? Better Science: NEAVS' new report is detailed, user-friendly and proactive. It contains substantive and persuasive arguments on the development and use of alternatives; descriptions of tests and their replacements; and details on how regulatory change can immediately reduce the number of animals used in labs. The NEAVS Report effectively refutes specious arguments of the animal research industry, and shows the scientific advantages of alternatives. Bolster your ethical arguments with scientific fact. Permission to use material from this report is granted on condition of acknowledgement. Print full report. I. INTRODUCTIONIn 1959, William Russell and Rex Burch, the founders of the modern alternatives movement, published their landmark book, The Principles of Humane Experimental Technique. Their simple dictum -- “if we are to use a criterion for choosing experiments to perform, the criterion of humanity is the best we could possibly invent” – has survived decades of non-acceptance, suspicion, misunderstanding and derision to become a central principle of the emerging science of alternatives. Alternatives have progressed from being wishful thinking by a few visionary scientists and humane individuals to a mainstream approach to answering questions posed by students, those with commercial interests, and scientists. Such methods have matured from a perceived or fabricated threat to biomedical research to an obvious opportunity for advancement without the pain and distress associated with the use of animals. As Dr. Michael Balls, former Director of the EuropeanCenter for the Validation of Alternative Methods (ECVAM) noted, “this is a time of non-violent revolutions, when alternatives are replacing more traditional and outdated uses of animals to protect public welfare and educate future generations of biological scientists and conduct basic biomedical research.” Throughout the 19th and 20th centuries, there have not been two competing systems – one based on animal models and one derived from humane alternatives – with the animal models found to be superior. Animal experimentation today in large part results from a historical accident rather than an accumulation of successful performances. In Europe the use of animals for safety and product quality purposes has declined significantly for several decades. Unfortunately that trend may be reversed due to politically motivated calls for massive new testing programs in both Europe and the United States (i.e., High Production Volume – chemicals produced in very large quantities; Endocrine Disruptors – chemicals with potential to affect human and/or wildlife reproduction; and the Children’s Health Initiative – consideration of chemical safety related to children’s susceptibility). What is happening in the United States? According to the U.S. Pharmacopoeia, which describes mandatory safety tests for drugs and other pharmaceutical products, animal tests now account for less than two percent of all drug testing to ensure product quality. This is down from 11.2 percent in 1985. During the past decade, with one notable exception, the total number of animals used in the United States laboratories has declined approximately 50 percent, in large part due to the adoption and use of alternatives. This trend, however, has reversed for one group – transgenic animals, mostly rats and mice, which are denied legal protection in the United States and for whom there are no reliable statistics on numbers of animals used. In addition to elucidating the concepts of humane research and the importance of alternatives, Russell and Burch formalized the possible options into three broad categories (3Rs) that are not mutually exclusive. One or all could apply to any research or testing protocol or educational exercise. Replacement methods represent the ultimate goal of the alternatives approach to basic biomedical research, testing and education. Refinement refers to those techniques and attitudes that alleviate or eliminate pain and distress experienced by the animals utilized in laboratory procedures. This may involve environmental and/or behavioral enrichment, humane endpoints (not involving pain, distress and/or death), better veterinary care and expanded use of analgesics and anesthetics. Reduction refers to any changes that contribute to the use of fewer animals, such as better research and statistical design and elimination of duplication. Both Refinement and Reduction are best viewed as interim steps on the way to the ultimate goal of complete Replacement of all animal use (i.e., in vitro tests). Animals are traditionally utilized in four broad categories – each characterized by its own unique set of attitudes, patterns of usage, and degrees of successful application of the alternatives approach. These four areas include biomedical research; production and testing of biologicals; education; and product development and safety testing. Biomedical Research: Basic biomedical research is the largest consumer of animals worldwide and, in the United States, the group most resistant to adopting the alternatives approach to answering their research questions. One factor distinguishing this hesitant response from the more favorable reception of industry may be that the former typically involves the use of someone else’s money and the latter their own in-house funding. Regardless of this history, the variety and sophistication of alternative methods, especially cell and tissue culture techniques (growing cells and tissues in various types of containers in vitro), continues to expand. The current research emphasis on embryonic and adult stem cells -- possibly the ultimate in vitro alternatives -- is the most obvious recent manifestation of this trend. Production and Testing of Biologicals: The production of biologicals, such as vaccines and antibodies, is in large part an alternatives-focused activity, with the safety testing of these products gradually switching to in vitro or physiochemical (basic chemical analysis) methods. Education: By its very nature, educational demonstrations and practice sessions, such as dissections and physiology/anatomy labs, are ideally suited for adoption of the alternatives approach. It is in this category that the development and use of replacement alternatives has been most successful. Product Development and Safety Testing: As noted by Phil Botham, Syngenta Central Toxicology Laboratory, “toxicology offers both a threat and an opportunity for reduction, refinement and replacement alternatives to animal experimentation.” For logistical and economic reasons, such household product and pharmaceutical companies are motivated to develop and use alternative techniques. Rapid progress here depends, however, on regulatory authorities (i.e., the Food and Drug Administration and the Environmental Protection Agency) abstaining from instituting new testing requirements based on outmoded animal-based approaches. Although the majority of toxicological research on biological mechanisms of chemical injury is done using in vitro methods and industry has widely acknowledged the superiority of alternative methods for safety testing, resistance still remains within some national and international regulatory organizations that establish and enforce safety testing requirements. It is clear that animal-based methods currently used in toxicological testing have not provided the assurances of harm or safety needed by the public and have in fact directly contributed to the existing problems of toxic ignorance. To address these historical failings, Replacement alternatives need to integrate rational testing requirements (not the traditional check-box approach that includes all available tests regardless of relevance); maximize use of existing data in both company and government agency files; mathematical predictions; models of physiological, pharmaceutical and toxicological mechanisms; new in vitro and in silico technologies (computer and microchip); and, where appropriate, ethical uses of human volunteers, post-marketing surveillance (reporting of adverse effects of products and drugs on consumers) and epidemiology (correlations between human exposure and health effects). Development of alternative techniques is widely recognized as a legitimate and important area of basic and applied scientific investigation. Regulatory agencies in Europe and to a lesser extent in the United States are finally accepting and promoting new alternative tests that have passed vigorous scientific validation procedures. In contrast, all of the traditional animal-based safety tests were never validated and would be unlikely to pass the level of proof required of new in vitro methods. This perspective has led to an increased emphasis on the importance of new techniques as the source of scientific discovery and advancement. There is a realistic expectation that in the future the use of animals will become the infrequent, reluctant alternative. II. PROBLEMS WITH IN VIVO ANIMAL-BASED METHODSAlternatives have played a critical role in the advancement of biomedical research and modern medical practice. By any objective measure the Nobel Prizes in Physiology and Medicine represent the best and most significant accomplishments in the biomedical sciences. An analysis of the specific projects for which these awards were given since their inception in 1901 documented that more than two-thirds of them were for work that was either partially or entirely based on the use of alternative methods. This percentage is even higher for the past few decades due to the increasing importance of in vitro and mathematical techniques. Nobel Prizes are frequently awarded for the development of major new experimental techniques, not for new animal models. Enders received his Nobel Prize in 1954 for creating an in vitro means, utilizing human cells, for growing the poliovirus. This new method is widely acknowledged as the key event leading to the first successful polio vaccine. Researchers using hundreds of thousands of monkeys to study polio did not receive similar recognition. Historically, the concept of “animal models” of human health problems was formulated in response to legitimate concerns about infectious diseases. The basic assumption was that if animals used in laboratories experimentally contracted an infection and were cured, there was a high probability of stopping the same disease in humans. Although a useful concept at the time, such uses can now be replaced in most instances by available alternatives and clinical studies of naturally-occurring diseases in human and non-human animals. The traditional animal “model” approach to studying human illness rapidly collapses and is most questionable when the focus switches from introduction of a common disease-causing organism to species-specific health problems such as psychopathology, cancer, drug addiction, Alzheimer’s and AIDS. As originally conceived, to be a valid model of human health concerns, the animal disease must have the same biological mechanisms, symptoms and responses to treatment as the theoretically similar human counterpart. Failure to meet one or more of these criteria invalidates the animal “model.” It is not sufficient to artificially produce a condition in an animal in a laboratory that only mimics, resembles, imitates or is similar to the so-called human equivalent. The current epidemic of iatrogenic (disease or injury caused by medical treatments) diseases -- one of the leading causes of death in the United States -- is partly the result of using inappropriate animal “models” to predict human responses to drugs and other treatments. Patients then have unexpected reactions or die from exposure to these supposedly safe drugs and chemicals. In an attempt to overcome the severe limitations of traditional animal “models,” researchers now are genetically engineering animals by either removing or adding genes believed to be related to specific human diseases. The underlying assumption here is that these new genetically constructed animals will be more human-like. The fact that existing animal models need to be genetically “improved” is further evidence of their original lack of biological and/or clinical relevance. The concept of animal models becomes even more tenuous when it is applied to the fields of toxicology and risk assessment. After exposure to potentially toxic or dangerous substances, both the inter- and intra-specific (between and within a species of animal) differences in morphology (anatomy), physiology and biochemistry between humans and the species commonly used in such tests introduce multiple significant biasing factors which cannot be avoided. The data derived from such experiments are not scientifically relevant to the purposes of the tests. Consider that in some carcinogenicity (cancer promotion) studies there is no effective correlation between the results for mice and rats (closely related rodents), let alone relevance to evolutionarily more distantly related humans. Although seldom mentioned, essentially all of the in vivo animal safety and toxicity tests currently in use were never validated and would be unlikely to pass present scientific validation procedures. These in vivo tests continue to be used for reasons of familiarity, tradition and checkbox/six-pack regulatory schemes. They are not used because they are the result of proven relevance and reliability. In vivo tests are subject to a series of basic biasing factors that simply do not exist for their in vitro and in silico (computer) replacements. Differences in lifespan and maturation processes between humans and rodents are significant. There are meaningful contrasts between processes that develop naturally over the course of time versus accelerated laboratory tests of induced, unnatural levels and routes of exposure. Commercial in vivo safety testing usually sacrifices accuracy and relevance for speed and cost. These problems are especially applicable to chronic (long-term) toxicity testing, the results of which may be no more accurate than simply flipping a coin. Because of the multiple, well-documented differences in responses, the use of non-human species in toxicity testing requires the application of often complex mathematical equations to extrapolate the results to potential human exposure. Major differences are associated with simple differences in body size. Extrapolations between species are not and should not be based on such simplistic criteria as length or weight differences. The husbandry conditions under which animals are typically bred, raised and housed seriously biases any data derived from their use. This is true for even the best state-of-the-art laboratory animal facilities. Recent studies suggest that much, if not all, of the research and testing done utilizing captive laboratory species in traditional cage environments may be so biased as to be useless, even if it can be replicated. In vitro replacement alternatives, especially with regard to safety and toxicity testing, have a number of positive characteristics:
For example, several decades ago the National Cancer Institute adopted an in vitro replacement for their standard animal-based procedures to identify potential anti-cancer compounds. This single decision dramatically increased the number of tests conducted; significantly reduced the per unit cost of the program; and saved more than a million rodent lives every year. III. AREAS OF ANIMAL EXPLOITATION AND ADOPTION OF ALTERNATIVESBasic ResearchThe most direct approach to an increased emphasis on alternatives in basic (non-medical) biomedical research is the development of new techniques that are subsequently widely adopted in multiple areas of investigation. For example:
The application or development of new alternatives really is a reflection of the imagination and technical skills of the individual researchers. For example:
Although replacement of flawed animal models with more relevant in vitro, computer and clinical methods is the long-term goal of the alternatives approach in basic biomedical research, the majority of every current protocol could benefit immediately from consideration of Reduction and Refinement alternatives. For example:
IV. TESTING ALTERNATIVESAnimal-based (in vivo) testing is characterized by massive suffering and questionable scientific value. Although safety testing does not involve the largest number of animals utilized in the United States specifically or the world in general, the numbers are still very high. In vivo testing protocols often involve severe levels of pain and distress and account for the majority of animals listed in USDA annual statistics as experiencing pain without anesthesia or analgesics. By its nature, animal-based toxicity testing is deliberately designed to cause injury, pain and/or death to some or all of the animals involved. Dr. Gerhard Zbinden, one of the world’s leading toxicologists, once described a standard in vivo bioassay test as little more than “a ritual mass execution of animals.” Efforts to Refine the severity of the animal’s experience and Reduce the numbers involved are in progress, but have had limited success. Any advances in these activities may be overshadowed by recent calls for massive, new animal-intensive testing programs of dubious necessity. A combination of activities will be needed to replace the use of in vivo toxicity tests with more humane methods. Initially, we need realistic information on human exposure to individual chemicals to determine actual risk, as well as a comprehensive database on past human exposure experience. Little of this type of information is currently available and may not yet exist due in large part to companies and government agencies not sharing in-house data. As new and existing in vitro methods become more widely used and accepted for regulatory purposes, the importance of multi-step, tier-testing strategies will be recognized. Such approaches allow testing for multiple endpoints that more accurately reflect the mechanistic processes involved in toxic exposures. Concomitantly, there will be a shift away from tests (such as the Draize Eye Irritation which involved placing various test substances in one eye of a rabbit and subjectively recording any damage that resulted) that have more to do with symptoms than actual toxic responses. High Throughput Screening (HTS), (now widely used in-house by pharmaceutical companies in research, drug development, candidate screening of potential compounds), followed by Medium Throughput Screening (MTS) methods currently under development, will examine the absorption, disposition, metabolism and excretion of test compounds. Eventually the expanding fields of toxicogenomics and proteonomics may provide quick, accurate “toxicity on a chip” and eliminate all remaining in vivo and much of in vitro techniques. At present the process of hazard assessment can begin with computer (in silico) approaches in association with in vitro cell culture models. This combination either exists, is being developed or is under validation trials for: • eye irritation • skin irritation • phototoxic potential (UV radiation) • nephrotoxicity (kidney) • reproductive toxicity • skin penetration • chronic toxicity • blood-brain barrier • gastrointestinal barrier Tier-testing strategies are either under developed or available for: • physiochemical properties • acute toxicity • skin corrosivity • skin sensitization • carcinogenicity/genotoxicity • xenobiotic metabolism • neurotoxicity • endocrine disruption What follows is a brief review of some key areas of toxicity testing and the current status of associated alternatives development and use in each. Acute ToxicityEssentially all of the basic in vivo toxicity tests measure some aspect of acute exposure with organ- or system-specific endpoints. There are, however, more general acute poisoning studies designed to provide information on substance concentrations necessary to produce death or severe injury. Routes of exposure may vary between oral, skin and inhalation. Such in vivo tests, whether combined with detailed histopathological (microscopic damage to cells and tissues) examination or not, represent little more than graduated, mass poisoning of surrogate species (e.g., mouse, rat, dog) already known to respond differently than humans. The most egregious example of such a useless test was the classical LD50 (Lethal Dose = 50% of animals die). Developed in the early 20th century to standardize the production batches of digitalis (a concern that can now be addressed using non-animal alternatives), it historically acquired a level of toxic significance for which it was never intended and was entirely unsuited. After more than two decades of criticism and documentation of the failures of the LD50, its use as a standard, worldwide test has finally ended. This long-overdue response was, however, delayed for several years due to the refusal of one national regulatory agency (the United States Environmental Protection Agency) to abandon support for its use. Development and validation programs to find in vitro replacements to measure general acute toxic exposure are underway in Europe and the United States. QSAR (computer) models are widely used by industry to either prioritize in vivo or identify the need for further in vitro tests. Since acute systemic toxicity (single exposure) results from cytotoxicity-associated (i.e., cellular damage) responses, measuring cytotoxicity in cell cultures should be predictive of the more general in vivo response. There are large existing databases of cytotoxicity information available in the biomedical literature. The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) program, which NEAVS as well as other national and international animal and scientific organizations funded, identified 69 different methods with potential applications for acute toxicity prediction and a subset of these that has a general predictive ability of 84% for humans. In contrast a standard rodent test might have only 65% accuracy. In some cases these in vitro methods also identify human toxic mechanisms that can only be seen in the alternatives. This may explain why such non-animal methods are so widely used for non-regulatory, in-house purposes. A battery of MEIC-identified tests are currently undergoing validation as in vitro replacements for the now discredited LD50 test. Furthermore, MEIC has initiated a new series of research efforts (EDIT – Evaluation-guided Development of New In Vitro Tests) to expand the earlier work with an emphasis on the use of human cells and improved use of toxicokinetic data. The German government has published a comprehensive Registry of Cytotoxicity (RC) that collects a large volume of relevant data from in vitro cells, comparisons with in vivo LD50 values and the use of linear regression analysis. This database can be used to predict some acute toxicity and reduce the number of animals and compounds utilized for test purposes. Ultimately, acute toxicity testing will be based on a tiered-testing strategy utilizing QSARs, a battery of basal cytotoxicity tests (e.g., MEIC), assessments of biotransformations and cell-specific toxicity protocols. The testing of any compound would end, if a positive result is identified. Using animals as surrogate tasters to identify human poisons will end. Chronic ToxicityOne traditional criticism of in vitro replacement alternatives was their inability to mimic or reproduce the consequences of long-term, chronic human exposure to toxic substances. This is no longer the case. Essentially all of the endpoints measured in animal-based tests can be transferred to appropriate in vitro systems. This is particularly applicable as the mechanistic bases for such endpoints become identified and characterized. In most cases, however, such tests measure the consequences of acute, not chronic exposure. As cell culture technology has evolved, it is now possible to maintain in vitro systems for longer periods of time – weeks or months. It is equally apparent that it is not necessary to maintain such cultures for years, as is done with some typical chronic in vivo tests. Long-term cell and tissue culture techniques now allow the study in vitro of the effects of chronic, repeated exposure to toxic substances, as well as the recovery from such exposure. To be a valid in vitro replacement, such long-term cultures must:
Pilot studies with systems such as Technomouse, Integra and EpiFlow have all demonstrated the feasibility of long-term cell cultures for chronic exposure studies. One test for a low-dose neurotoxin utilizing epithelial cells grown in a hollow-fiber perfusion system gave results that “mirrored” those found in traditional in vivo studies. Other ongoing programs are exploring the use of genetically engineered cells and applications of different co-culture and three-dimensional in vitro systems to address the issue of identifying problems associated with chronic toxicity. Although the MEIC Project was designed to measure the effects of acute exposure, some of the test components appear relevant to chronic situations. MEIC laboratories were able to maintain cultures for up to six weeks. As these long-term culture techniques are refined, validated and come online as in vitro replacements for traditional animal-based methods, they should not be viewed as high-throughput systems. Such cultures will still require longer periods of time. Their use, however, should only be necessary if other in vitro acute exposure tests suggest problems may exist. Computers and ToxicologyAlthough the ability of computers to replace in vivo experiments and test is occasionally overstated, toxicity testing is ideally suited to the application of such in silico approaches. What computers do best is compare and contrast large quantities of quantitative and qualitative data. Safety testing is based on the production and use of such information. There are three basic types of computer applications in safety testing:
QSARs are currently widely used for non-regulatory purposes in the study of skin corrosion, skin irritation, eye irritation, the blood-brain barrier, acute toxicity, metabolic endpoints (to name a few), and as the initial step in tiered testing strategies. Pharmaceutical companies routinely used QSARs to design new chemical agents and drugs, with applications to toxicology only recently adopted (although not yet formally validated for toxicity endpoints). SARs utilize computers to identify portions of molecules that are known to be associated with specific (in this case -- toxic) biological properties. Both QSARs and SARs have the advantage over in vivo of being based on detailed knowledge of chemical structure (determined by physical analysis); easily transferred to computer automation; and extremely quick response times. There are several current limitations on the utility of such computational approaches. These include:
These restraints are being addressed and removed as:
Despite their limitations and continued development, there are already a number of high-powered programs available and in widespread use for non-regulatory purposes. These include: TOPKAT (Toxicity Prediction by Computer-Assisted Technology), CASE (Computer Automated Structure Evaluation), COMPACT (Computerized Optimized Parametric Analysis of Chemical Toxicity), DEREK (Deductive Estimation of Risk from Existing Knowledge), Hazard Expert, ONcologic and Meteor. Several of these systems have, for example, overall accuracies of 60 to 90% for some standard toxicity endpoints (e.g., rodent carcinogens). Ocular ToxicityThe classic Draize Eye Irritancy Test was characterized more than two decades ago as extremely inhumane, of questionable relevance to human or animal exposure to harmful substances, and scientifically flawed with high degrees of inter- and intra-laboratory variability for the same test materials. Computer simulations documented a lower correlation between repeat tests of the same chemicals than would be tolerated for any in vitro test used for regulatory purposes. The Draize is simply not consistently reproducible and thus cannot be reliably used to predict human risks. Although criticized on both ethical (pain and distress to the animals involved) and scientific (multiple, significant anatomical and physiological differences between the eyes of rabbits and humans), there still remains a widespread misconception among some toxicologists and regulatory officials and agencies, that the Draize provides a valid measure of eye irritation potential. The poor quality of Draize data also contributes to difficulties in replacing it. Due to the seriously compromised nature of existing in vivo data, potentially valid alternatives have technically failed validation efforts when compared to bad Draize data. Reanalysis of previously conducted validation studies for in vitro Draize replacements, unbiased by the poor quality animal data, suggest these replacements are adequate to identify potentially hazardous materials, especially if utilized as a tier-testing strategy. A number of in vitro replacements are widely used in-house by industry to eliminate nearly all requirements for the classical Draize test. They are also accepted on a case-by-case basis by several national regulatory agencies other than the United States. In vitro replacements for the Draize Eye Irritancy Test include one or more of the following, done in combination with each other and several QSAR computer analysis programs:
A comprehensive German study of these methods proved that the HET-CAM and NRU tests could identify severe eye irritants and eliminate them from further testing protocols. There remains a need to create new in vitro methods to be added to the existing battery of replacement alternatives to the Draize. These should be based on specific cellular and molecular endpoints associated with positive eye irritation responses. At its simplest level, any substance producing a positive skin irritancy response should be labeled as an eye irritant and not be further tested. Skin ToxicityThere are four basic aspects of skin toxicity that are routinely tested: corrosivity, irritation, sensitization and absorption. Skin corrosivity can be easily measured using in vitro systems such as EPISKIN (a three-dimensional human skin model) and EpiDerm (a reconstructed human skin model) – both of which measure cell viability as an endpoint and have been accepted for regulatory purposes in the European Union and by the OECD. In addition, a non-cellular test, Corrositex, is approved in the EU and the United States, but only for acids, bases and their derivatives. Corrositex utilizes a specialized collagen matrix membrane. There is no longer any justification to do animal testing for this endpoint, especially if the in vitro methods are combined with a tiered approach involving such physical parameters as pH. Skin irritation potential is currently measured using the classic Draize skin test, the lesser-known cousin of its ocular counterpart. Several promising in vitro tests are currently subject to validation programs. These include the EPISKIN and EpiDerm systems, as well as Prediskin (a human skin culture derived from plastic surgery discards) and a variety of sophisticated QSAR models – one of which had a sensitivity of 85% and specificity of 92%. Animal-based tests for this same endpoint should be eliminated in the very near future with an in vitro replacement used immediately for prescreens and priority setting. Skin sensitization assays are designed to identify a substance’s potential to produce contact dermatitis. Increased responses associated with this endpoint have helped to create new or reapply existing replacement alternatives. Several computer techniques (DEREK, TOPKAT and CASE) include this endpoint. L’Oreal is developing a human reconstituted epidermis, multi-cell culture model that includes such unique components as melanocytes, keratinocytes and Langerhans cells. MatTek Corporation is actively working on an in vitro replacement for the Murine Local Lymph Node Assay (LLNA), a reduction alternative currently accepted in the EU, United States and by the OECD. Complete replacement of in vivo skin sensitization tests is a realistic short-term expectation. Percutaneous absorption can be measured using any one of the currently available in vitro reconstituted human skin equivalents since studies show that such methods provide data predictive of human and animal exposure to test substances. In vitro replacements for this endpoint have already been accepted by the OECD. Genotoxicity / Mutagenicity (changes to genes) and Carcinogenicity (cancer-causing)It is now widely accepted by regulatory officials and toxicologists that screening for mutagenic potential can be done via in vitro methods such as the Bacterial Reverse Mutation Test; In Vitro Cell Line Mutation Test, or the In Vitro Chromosomal Aberation Test. There are also several SAR, QSAR and Expert Systems available for this endpoint. The only potential problem with these assays is the existence of mechanisms that produce non-genotoxic carcinogenesis. New in vitro tests based on Syrian Hamster Embryo (SHE) cell lines may address this concern. However, some toxicologists question the significance of such carcinogens to human risk assessment since their activity profiles are often only identified in mice. Toxicokinetics / Biokinetics -- ADMEActual systemic toxicity depends on several variables -- external dose; rate of exposure; absorption, distribution, metabolism and excretion (ADME); and the intrinsic characteristics of the test material. All of these can be identified and modeled using computer and in vitro approaches. Studies focusing on ADME are now human-based, mechanistic protocols that provide both predictive and computerized models. Although classical in vivo toxicity tests are based on dose/response relationships for entire animals, a more realistic approach might focus on concentration/response curves at the actual toxic target within the recipient’s body. In vitro methods are especially useful for such studies on the biological activity and mechanisms of toxic response of chemicals. Programs such as MEIC have provided evidence of the value and utility of this approach. Perhaps most significantly, the creation of toxicokinetic-derived QSAR programs will allow toxic exposure from one test or set of tests to be used to predict the response for other types of tests. This would eliminate the need for the latter and replace the animals used with simple abstinence. As a specific example, the Environmental Protection Agency (EPA) announced on July 14, 2003, that they had conducted a review to determine if chemical companies could use physiologically-based pharmacokinetic computer models to extrapolate data from previous oral toxicity studies to predict potential hazardous consequences of inhalation exposure to the same substance. The EPA endorsed this approach to both reduce the number of costly tests required and to eliminate some current uses of animals. As mechanistic data, in vitro methods, computer simulation and DNA-chip technology continues to improve and intertwine, the justifications offered to defend continued in vivo testing requirements will become more and more tenuous. Pyrogen TestingThis test is designed to identify potential bacterial contamination of injectable products (originally), implants, medical devices, dialysis machines, cellular therapies, recombinant proteins and IV products. Injectable drugs have been around for more than 100 years. Sixty years ago the rabbit pyrogen test (involving injection of test materials to check for reactions to contamination) was developed and subsequently millions of rabbits died. Twenty-five years ago the LAL (Limulus amebocyte lysate) alternative was developed based on the coagulation response of horseshoe crab blood when exposed to bacterial toxins. In theory blood is collected from the crabs, who are then released. In practice, poor technique and carelessness lead to a high percentage of crab fatalities. To avoid killing the crabs and limitations of the LAL test, as well as the need for replacing the 400,000 rabbits still used worldwide, researchers in Europe developed a new pyrogen test based on human isolated blood cells, cell lines and whole blood incubation to detect the presence of fever reaction products – a direct measure of pyrogen contamination that would affect human patients. This approach can also identify both immunostimulants and immunosuppressants. PhototoxicityAlthough it took seven years to complete the validation/approval/adoption process, there now is an in vitro replacement alternative to identify phototoxic (i.e., drugs and chemicals become toxic when human recipients are exposed to sunlight) potential. The 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) utilizes a mouse-derived cell line which measures the degree of cellular damage (cytotoxicity) of the cultures and toxicants when tested in the presence and absence of non-cytotoxic exposure to UVA light. Additional validation tests are currently being conducted on other in vitro methods (e.g., EpiDerm PT) to provide additional phototoxicity alternatives. These in vitro methods are accepted by both the OECD and the European Union testing authorities. There is no further justification for the continued use of in vivo tests for this toxic endpoint. Embryotoxicity / TeratogenicityThere are currently more than a dozen in vitro methods representing various aspects of the reproductive process. The use of immortalized mammalian cell lines, especially embryonic (not derived from therapeutic abortions) stem cells are being used to create in vitro assays for teratogenicity that are directly predictive of human toxic risks. Using rodents for such studies is especially inappropriate due to the major physiological, biochemical and structural differences between human and rodent placentas. The Embryonic Stem Cell Test (EST) has been validated by the EuropeanCenter for the Validation of Alternative Methods (ECVAM) and accepted in the European Union for the identification of embryotoxicants. Of the currently available alternatives, it is the only one suitable for high throughput screening and avoids killing large numbers of pregnant animals. It also identifies three unique endpoints representing the principal reproductive toxicological mechanisms. Endocrine DisruptorsThis represents a newly hypothesized class of potential toxic effects on human and wildlife reproductive systems for which there were no existing animal-based tests. Although there is evidence that humans may be unaffected by endocrine disruptors, there is also evidence of negative impacts on other species (especially wildlife). Current proposals for in vivo-based testing protocols share a set of serious problems including:
For these reasons multiple in vitro screens and QSAR computer models are being developed based on mechanistic endpoints that can only be examined using such alternatives. Unlike most existing in vivo tests, because this area of toxic concern is entirely new, it may be possible to create an alternatives-focused, tier-testing strategy that will “do it right” the first time. Metabolic ToxicitySome chemicals and drugs are essentially nontoxic but become hazardous once ingested and metabolized by the body. For this reason, information from in vitro systems utilizing human cell lines, genetically engineered human cells and subcellular components as well as several computer-based systems (METEOR, Hazard Expert, Metabol Expert, COMPACT) are being utilized to detect metabolism-mediated toxicity. Because of the enormous species differences in metabolic parameters (especially between humans and rodents -- the animals most frequently used for such tests), it is critical that such studies utilize human-based in vitro techniques and human data for computer simulation. This is one area of toxicology for which animal models are widely acknowledged by toxicologists to be inappropriate. Work is currently underway to create a simple microchip that will provide all of the necessary metabolic information simultaneously and in a human-specific context. Nephro (Kidney) ToxicityFor many years primary cultures of kidney cells have been powerful tools to study renal function and toxicity. A number of in vitro toxicity endpoints are currently being investigated with an emphasis on using immortalized renal epithelial cell lines (e.g., MDCK cells originally derived from dogs). In order to reproduce some of the structural and cellular complexity of the kidneys, new perfusion culture techniques (e.g., EpiFlow) were developed that allow longer-term, simultaneous cultures of two or more cell types. Efforts are also underway to replace all of the animal-derived cell lines and cultures with their respective human counterparts and to identify consistently relevant in vitro toxic endpoints. Neuro (Brain/Nerve) ToxicityThe routine use of in vitro methods for research and testing models dates back more than twenty years. Long-term cultures of neural and support cells are currently in use by industry to screen for toxic effects of pharmaceuticals, agricultural chemicals and other compounds. In addition, a large number of in vitro systems are being developed as toxicity screens and indicators of multiple toxicity endpoints. These include neuronal cell lines, genetically engineered cells and reaggregating brain cell cultures (which reproduce some of the in vivo complexity of the brain). Eventually a tiered testing (multiple levels of pass/fail testing) strategy, incorporating several of these in vitro methods should be sufficient to identify neurotoxic hazards. There is also evidence that not all potential endpoints need to be examined to adequately predict substances of concern. ToxicogenomicsIf toxicology is to eventually evolve from its primitive beginning in quantifying the mass poisoning of various species of animals, the final high-tech destination may be in the field of toxicogenomics and its sister disciplines of proteonomics and metabonomics – all of which integrate the interactions between human genes and toxic substances, proteins and metabolic activities respectively. The ultimate goal of toxicogenomics is a single or series of DNA chips that would provide almost immediate toxicity profiles of all test substances. Such chips can provide vast amounts of data on gene expression in response to specific conditions. A single chip can replace the information derived from 20,000 individual experiments. There is evidence that of the mind-boggling number of potential gene expression patterns in the human genome (10 30,000), the number with relevance to toxicologic responses is approximately 317. Once the 256 types of human cells are represented, there only remains another 60 sites of potentially relevant responses to toxic exposures. This is a fairly small number for existing microarray technology. Each microarray includes thousands of tiny pieces of DNA which allow simultaneous examination of overall patterns of gene expression. The goal of toxicogenomics is to determine which of these patterns are associated with each of the classical toxicity endpoints. Once identified, these arrangements could then be used to predict potential toxicity of new substances. It is already known that human genes respond to the presence of a compound and any damage associated with it. Genes also respond in characteristic patterns to changes in levels of metabolically important compounds and the internal environment of the cells in the body. Because of its use of specific gene expression information, microarray-based toxicology would involve a more mechanistic approach to hazard identification and characterization – certainly more relevant to humans than anything currently derived from historically crude animal poison experiments. A recent set of microarray experiments identified a set of twelve diagnostic points that provided 100% predictive accuracy for five different types of toxic substances. It has also been established that gene expression profiles (as on the chips) correlate with results of histopathology, clinical chemistry and known mechanisms of toxicity. Studies are currently underway to apply this technology to the fields of hepatotoxicity (liver), genotoxicity (genes) and nephrotoxicity (kidney). It is also likely that, once fully developed, toxicogenomics will provide the scientific proof that animal-based toxicity testing has little or no relevance to human risk assessment. Use of such chips will become a standard part of any future validation process for in vitro or in vivo safety tests and animal models intended for use in basic biomedical research. Consider the potential consequences of documenting entirely unrelated gene expression profiles for a human disease and its putative animal model surrogate. Microarrays, once validated and widely adopted, should change toxicology into a high-throughput, predictive discipline with unique sets of biomarkers (gene expression patterns) for toxic endpoints and classes of toxicants. This technology is also uniquely suited to interact with existing in vitro methods. For all of these reasons, pharmaceutical and chemical manufacturing companies are investing heavily in the field of toxicogenomics and creation of DNA microarray chips. This is the beginning of a new age of drug and chemical evaluation. Enthusiasm for this new, high-tech approach to toxicology may be premature since the biological relevance of gene expression patterns needs to be established and its predictive abilities validated. Some toxicologists have proposed conducting a limited number of animal toxicity tests in order to create the DNA patterns for the chips. V. ALTERNATIVES AND BIOLOGICALSAlthough often overlooked, the production and testing of biologicals consumes 15% or more of all animals used in United States laboratories specifically and the world in general. For example, a complete batch test for a therapeutic protein can involve 12,000 mice and cost $2.4 million without producing any useful information. Potency tests of such products as vaccines are still routinely based on the principle of protection, i.e., survival or death after exposure, which was first introduced in the 1890s. Many of these tests are exceptionally cruel, involving high levels of pain and distress for a variety of species of rodents, dogs, cats and non-human primates (including chimpanzees). According to 1998 USDA statistics, more than 60% of the animals experiencing unrelieved pain and distress were used for vaccine testing. Essentially all of this work was conducted in industry laboratories. As a category, biologicals include antibodies, blood products, bioactive compounds (e.g., cytokines), hormones, immunosera products, recombinant-DNA proteins and vaccines. Nearly all of these are produced under mandated quality, potency and efficacy controls. Abandonment of the erroneous concept that functional tests in animals corresponds to the same function in a human, production of biological products in a form which allows easy quantification of characteristics and elimination of excessive duplication of national testing requirements will rapidly advance the development and use of alternative replacements for safety testing of biologicals. For example, introduction of a new vaccine presently might require 32 different in vivo testing protocols instead of a few in vitro alternatives. The field of biologicals production and testing includes several glaring anachronisms. Target animal safety tests with sample sizes of two are statistically meaningless and could be eliminated immediately. Similarly, the general or abnormal toxicity tests was one of the first animal-based tests developed; is used for a variety of materials; duplicates data produced by more familiar tests; is widely acknowledged to be useless; and was deleted from the European Pharmacopoeia in 1997 with no negative consequences. It could be eliminated worldwide immediately with a similar harmless outcome. Because some vaccines utilize live pathogens (e.g., oral polio (OPV), MMR, varicella, yellow fever, or are well defined (e.g., influenza), they have always been tested using in vitro methods. Cholera and typhoid vaccines are not tested for potency due to a lack of valid animal models. Neurovirulence tests for OPV, recombinant FSH hormones, tetanus and diptheria vaccines all now have alternative replacements for the previous more traditional animal-based tests. Vaccines and some biologically active compounds are routinely produced using in vitro methods. Perhaps the most successful example of this is the production of monoclonal antibodies (MAbs), which began as a new in vitro technique; was usurped by a very painful and distressful in vivo method; and can now be done entirely via alternative replacement methods. In fact, there are so many in vitro MAb methods currently available, all producing high quality antibodies at lower costs, that the old in vivo approach is prohibited or severely restricted in many countries including the United States. Production of polyclonal antibodies continues to rely on injections of test substances (antigens) into various species of animals and subsequent bleeding to retrieve the antibodies. As an interim step, which will also be replaced, such antibodies can be produced in chicken eggs (eliminating the use and bleeding of mammals). Although somewhat technically difficult and in the early stages of development, within a few years it will be possible to use recombinant DNA techniques to produce all antibodies – mono- and polyclonal. This final evolution of technological methods will completely eliminate an entire area of traditional animal usage (millions of animals per year) and at the same time provide a higher quality, less expensive product. This is the promise and reality of the alternatives approach. VI. ALTERNATIVES AND EDUCATIONBecause educational demonstrations and laboratory sessions are characterized by repetitive exposure to existing knowledge, they represent the ideal situation for the application of replacement alternatives. For this reason the “alternatives approach” to education in the biomedical sciences has had its most widespread success in primary, secondary, undergraduate and graduate/professional curricula. There is no longer any valid educational or scientific justification for the continued use of animals to acquire either basic knowledge or practice manual skills. In the 1990s NEAVS’ educational affiliate, The Ethical Science and Education Coalition, published one of the first resources on available alternatives appropriate to middle and high school level life science classes. NEAVS has also been a major funder of From Guinea Pig to Computer Mouse, second edition (2002) – an exhaustive resource of alternatives appropriate for college, university and professional school levels. Compiled and published by the International Network for Humane Education (InterNICHE), it lists more than 500 alternatives and is available in several languages. For more than a century it has been illegal in the United Kingdom to use animals for such basic educational purposes. When this law was revised in the 1980s (after more than 100 years of successful application to classrooms), only one exception was allowed (microsurgery practice) for which appropriate replacement alternatives are now available. Comparative studies of basic knowledge and professional skills (e.g., veterinary and human surgeons) confirm that individuals trained under the alternatives-focused United Kingdom system are not less qualified than their counterparts exposed to the more animal-based approach still common in the United States. There are no documented disadvantages associated with the exclusive use of humane alternatives. In 1999 NEAVS worked with Tufts University School of Veterinary College to make it the first US vet school to eliminate all terminal labs on all species. The trend in both medical and veterinary schools is rapidly moving toward making all live animal labs either electives or eliminated. It is now possible for a student to go through primary, secondary and undergraduate education (even with an emphasis in biology) and not need to harm, kill living animals or work on already dead animals. For more detailed information on issues associated with educational uses of alternatives and animals, please go to the ESEC website. VII. RESISTANCE TO ALTERNATIVESAlthough the “alternatives approach” represents state-of-the-art scientific methods, superior to traditional animal-based approaches, in the United States there remains considerable resistance among many academic researchers, toxicologists and regulatory officials to making the switch to more humane approaches to experimentation, testing and education. Experts in this field have observed: “Many toxicologists and regulators do not want to question the value of the methods they currently use.” Michael Balls, Ph.D., former Director, ECVAM, 2002 “Regulators appear to be more willing to accept new animal tests which have not been validated than non-animal tests which have.” Michael Balls, Ph.D., former Director, ECVAM, 1998 “Behind all of this there lies a profoundly irrational bias in favor of in vivo tests.” William Russell, Ph.D., founder of modern alternatives movement, 2003 “Extreme views on both sides of the animal research battle have led to a stalemate in which any search for alternatives is too often seen as a concession to animal rightists. As a result, the United States lags far behind Europe in finding and implementing alternatives.” John Rennie, 1997 / Editor, Scientific American There is also a general consensus that resistance to the identification, development, adoption and promotion of humane alternatives reflects several attitudes and biases, which are not mutually exclusive. These include, but are not limited to:
All of these barriers are being overcome at an accelerating, but still frustratingly slow pace. The future of animal-based experimentation, testing and education is questionable. As noted by Nobel Prize winner Sir Peter Medawar: “The use of experimental animals on the present scale is a temporary episode in biological and medical history.” VIII. CHALLENGES TO ALTERNATIVESIn addition to the resistance described above, more direct threats to the adoption of alternatives and promotion of animal-based testing protocols may come from massive new testing programs being proposed by such federal regulatory bodies as the Environmental Protection Agency (EPA). For the past few years, under pressure from politicians as well as consumer and environmental organizations, the EPA has proposed new testing requirements that will kill millions of animals, tailored to:
Nearly identical programs have also been proposed for the European Union (REACH) and internationally (OECD). In all cases the respective regulatory agencies have used the new testing mandates to promote existing or create new animal-based testing procedures – usually without the rigorous levels of validation and proof of principle still required for in vitro replacement alternatives. If needed, the new testing requirements could be used as incentives to finally create an alternatives-based, humane approach | |||||
