Why Choose Alternative Cancer Treatments (22)

"The drugs Prednisone and Vincristine are often hailed as 'curing' childhood leukemia. Both drugs were rejected by the US National Cancer Institute as 'useless' on the basis of animal tests. Prednisone was developed as a result of clinical observation of the effects of adrenal extract. Vincristine is an alkaloid of 'Vincra Rosea', a type of periwinkle plant, and extracts of periwinkle were used in the Roman Empire to 'dry tumors' (Pliny). They were eventually brought to clinical trials. The children cured of leukemia owe their lives to clinical observations and trials - and not to the animal 'model'."
Brandon Reines, DVM, Cancer Research on Animals: Impact and Alternatives

Welcome to page twenty-two of “Why Choose Alternative Cancer Treatment?” detailing more alternatives to experimentation on animals.

Better Science: Alternatives to Animal Research (Part 1)

By John McArdle, PhD., Science Advisor New England Anti-Vivisection Society (NEAVS)

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.

I. INTRODUCTION

In 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 METHODS

Alternatives 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:

  • They were scientifically validated and proven to be relevant to the desired endpoints.
  • They allow multiple, simultaneous tests under a range of concentrations and controlled conditions.
  • They allow larger numbers of tests in shorter periods of time.
  • They are easily adapted to high throughput (high volume and high speed) conditions that cannot be replicated by in vivo methods.
  • They are logistically simpler and economically less costly.

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 ALTERNATIVES

Basic Research

The 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:

  • Nowhere is this more evident today than with the development and use of in vitro methods to produce monoclonal antibodies (MAbs), which are specific to a single structure, chemical or disease organism. Originally developed as an alternative technique, MAbs quickly became the source of large-scale animal pain and distress (ascites) due to the massive swelling of the animals’ abdomens. With the development of new replacement alternatives (several dozen different possibilities), more progressive countries in Europe finally banned the routine use of the ascites method to produce MAbs and required the use of more humane methods. The use of such alternatives is now the mandated first choice approach to MAb production in the United States for anyone receiving funding from the National Institutes of Health. This simple change in perspective, acknowledging that the alternatives were superior to in vivo techniques, will save millions of animal lives.
  • Although still used in some areas of research, polyclonal antibodies (PCA), which attach to multiple research and clinical targets, find their greatest applications in academic and clinical diagnostic kits. PCAs are conventionally produced from the blood of immunized mammals such as rabbits, goats and horses. More than a century ago the possibility of producing such biological compounds via hen’s eggs was suggested. This is becoming a more common practice today. Ultimately all antibodies (MAbs and PCA) will be created and produced using recombinant DNA technology, thus completely eliminating the use of animals for such purposes.
  • Development of a completely virtual human computer simulation is still in progress, but some aspects of the concept are available and in use. Several years ago the National Library of Medicine created a set of serial sections of specially prepared male and female human cadavers. Each section was digitized, allowing a complete, anatomically accurate computerized reconstruction of the human body. This in silico alternative is utilized by both academic and corporate institutions for a variety of research and teaching applications including the development of new and/or refinement of existing surgical techniques.
  • The least publicized but most critical aspect of the development of the first artificial heart was not the use of animal models, but rather the use of five brain-dead, artificially maintained human bodies (neomorts) to establish and practice the final surgical techniques and efficacy of implantation of the artificial heart in a human patient. Use of neomorts has widespread possibilities in both academic and applied research such as toxicity testing, but remains a tightly kept secret within the biomedical research community.
  • Applications of in vitro methods are widespread within the basic biomedical research, with some disciplines entirely dependent on them. Nearly 40% of the research program funded by the National Institutes of Health involves some use of in vitro alternatives. Such techniques are not adjuncts but mainstream state-of-the-art scientific methods. With ongoing developments in perfusion techniques, three-dimensional, multiple cell type culture and immortalized cellular (long-term) methods it will be possible to reproduce and/or simulate in vitro all principal human organ systems and responses.
  • Another rapidly advancing area of technique development and application in basic research is noninvasive imaging. Magnetic Resonance Imaging (MRI), Positron Emission Tomography Imaging (PET), Functional Magnetic Resonance Imaging (fMRI) and combinations of these allow very sophisticated, real-time measurements of associations between structure and function in both humans and animals under a wide variety of experimental conditions. Some imaging units are specifically designed for use with small animals. Despite earlier limitations on these techniques, they are now faster and more accurate with resolutions possible down to single cells. These imaging options have had their most extensive applications in the neurosciences, allowing direct, noninvasive studies of neurophysiology that would be impossible to do with nonhuman animals. On a more fundamental level, for decades many of the animals killed in neuroscience experiments died only to identify the specific site of electrode implantation. Such deaths are not necessary.

The application or development of new alternatives really is a reflection of the imagination and technical skills of the individual researchers. For example:

  • Computer simulations of cancer cells are now used to test drug targets within them.
  • There are several in vitro models for studying the gastrointestinal system, with the most complex being a multi-culture, in vitro simulation of each portion of the digestive system (FIDO) developed in the Netherlands. Each part of the interconnected model contains cell cultures for that particular organ.
  • A researcher in New Jersey developed a multi-dimensional, bioengineered human skin cell culture for the study of burns and ultraviolet exposure. This in vitro model can reproduce any human skin coloration and tans if exposed to the sun.
  • In vitro models of the brain and more recently the blood-brain barrier have existed in one form or another for more than twenty years, being used for studies of neurotransmitter pathways, electrophysiological characteristics, morphological associations of human diseases (i.e., Alzheimer’s, Parkinson’s, Huntington’s, epilepsy), new drug design, receptor targets and modes of action of new pharmaceuticals. Current in vitro models have reached very high degrees of structural and functional sophistication.
  • The Skin Ethics Laboratories in France have developed ten human in vitro tissue models (cornea, oral, eye, esophageal, lung, vaginal and complex dermal simulations) for use in industry (efficacy and safety testing) and academics (basic research). These models are routinely used by such companies as Pfizer, GlaxoSmithKline, Unilever, Kimberly Clark, Avon and 3M.
  • Multilevel, multi-culture in vitro simulations of different parts of the human lung have been developed at the University of South Carolina for distribution studies of aerosols and eventually respiratory toxicology.
  • Cyprotex has developed a software system that accurately predicts the pharmacokinetics of new drug compounds using a virtual human computer simulation.

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:

  • Physical and behavioral enrichment of the animal’s environment is finally being taken seriously despite continued objections from researchers and facility personnel.
  • Efforts are underway to identify, characterize and eliminate pain and distress, which always biases the results of studies in which animals are used.
  • A significant number of experiments continue to use or abuse the wrong statistical tests, sample sizes and appropriate design options.
  • Mouse Specific, Inc. has designed an entirely noninvasive system to monitor cardiovascular health and activity.
  • Bioluminescent imaging allows noninvasive measurement of physiologically relevant processes.

IV. TESTING ALTERNATIVES

Animal-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 Toxicity

Essentially 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 Toxicity

One 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:

  • retain their differentiated functions, as is the case for in vivo models;
  • maintain stable, reproducible conditions;
  • involve perfusion systems with continuous replacement of nutrient media, removal of waste products and allow for periodic exposure to toxic test substances;
  • be based on serum-free cultures of human cell lines, which have unlimited lifespans and are more directly relevant to human exposures;
  • include the possibility of co-cultures of multiple cell types to more closely match organotypic conditions.

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 Toxicology

Although 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:

  • SAR -- Structure Activity Relationships – examine known associations between chemical structures and biological activity with similar data for new substances.
  • QSAR – Quantitative Structure Activity Relationships – creates multivariable mathematical relationships between chemical structure, physiochemical properties and biological activity.
  • Expert Systems are any formal approach (with or without the use of computers) that allows the rational prediction of toxicity of test substances.

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:

  • the absence of good quality toxicological information despite decades of animal tests and human exposure data;
  • overly simplistic models of complex toxicological mechanisms; and
  • extrapolation beyond the area covered by the available data.

These restraints are being addressed and removed as:

  • there is more reliance on mechanisms of toxicity than simple chemical structure;
  • increased use of human cells and tissues;
  • better characterization of cellular receptor structure and properties;
  • improvements in computer databases; and
  • incorporation of information from the human genome project and toxicogenomics.

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 Toxicity

The 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:

  • use of chorioallantoic membrane of a fertilized hen’s egg (does not damage the embryo) (HET-CAM test) is the only one to include responses of the circulatory system.
  • fluorescein leakage (utilizing MDCK dog kidney monolayer cell cultures and measuring dye leakage due to damaged inter-cellular connections.
  • neutral red uptake (NRU) and neutral red release (NRR), which are indicators of cytotoxicity (cell injury/death) and are excellent predictors of responses to severe irritants.
  • agarose diffusion, which measures cell death in cultures exposed to test materials.
  • EpiOcular, which is a sophisticated reconstituted human corneal epithelial cell culture that provides data on cell viability, release of indicators for inflammation and changes in membrane permeability.

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.

continued on next page

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