Alternatives to Animal Research
By John McArdle, PhD
Science Advisor
New England Anti-Vivisection Society
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 European Center 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when alternatives are replacing more traditional and outdated uses of animals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.
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.
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.
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.
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.
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.
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).
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.
There 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.
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.
Actual 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.
This 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.
Although it took seven years to complete the validation/approval/adoption process, there now is an in vitro replacement alternative to ident