Transcript The Four R`s In Experimental Toxicology

Experimental Toxicology
Prof. Dr. Şahan SAYGI
NEU Faculty of Pharmacy
Department of Toxicology
CONTENTS
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Introduction
Models for toxicity testing and research
The Four R’s In experimental toxicology
Animal models in toxicology
Current animal studies
Origins of predictive animal testing
Selecting an animal model
Husbandry and care
Choosing species and strains
Dosing
Animal physiology
Introduction
• Toxicology is the science concerned with identifying and
understanding the mechanisms of agents adversely
affecting the health of humans, other animals, and living
portions of the environment.
• Toxicology is concerned with those man-made chemical
agents adversely affecting the health of humans.
• The current test methods designed and used to evaluate
the potential of manmade materials to cause harm to the
people.
• On the one hand, our society is not only critically
dependent on technologic advances to improve or maintain
standards of living, but it is also intolerant of risks, real or
potential, to life and health that are seemingly avoidable.
• On the other hand, the traditional tests (with both their
misuse and misunderstanding of their use) have served as
the rallying point for those individuals concerned about the
humane, ethical, and proper use of animals.
• This concern has caused all testing using animals to come
under question on both ethical and scientific grounds, and
it has provided a continuous stimulus for the development
of alternatives and innovations.
• Since 1980, tremendous progress has been made in our
understanding of biology down to the molecular level.
• This progress has translated into many modifications and
improvements in in vivo testing procedures that now give
us tests that:
– Are more reliable, reproducible, and predictive of potential
hazards in humans,
– Use fewer animals
– Considerably more humane than are earlier test forms.
• Various terms are used to describe the different kinds of
testing and research performed by the model systems used.
• In vivo is used to denote the use of intact higher organisms
(vertebrates).
• in vitro is used to describe those tests using other than intact
vertebrates as model systems.
• These tests include everything from lower organisms (planaria
and bacteria) to cultured cells and computer models.
• In between clearly in vivo and in vitro models are the
“alternatives.”
• This term has a different meaning to different people.
• In its broadest sense, it incorporates everything that
reduces higher animal usage and suffering in the existing
traditional test designs.
• This definition includes use of the following range of
situations:
– A reduced volume of test material in a rabbit eye irritation
test
– A limited test design to characterize lethality in the rat
– Earthworms instead of rats or mice for lethality testing
– Fish instead of rats or mice for carcinogenicity bioassays
– Computerized structure activity models for predicting
toxicity
– True in vitro models
Models for toxicity testing and research
 In vivo (intact higher organism)
 Lover organisms (earthworms, fish)
 Isolated Organs
 Cultured cells
 Chemical/biochemical systems
 Computer simulations
In vivo (intact higher organism)
• Advantage; Full range of organismic response.
• Disadvantage; Costs, ethical/ animal welfare concerns,
species-to-species variability.
Lover organisms (earthworms, fish)
• Advantage: Range of integrated organismic responses.
• Disadvantage: Frequently lack responses of higher organism,
animal welfare concerns.
Isolated organs
• Advantages: Intact isolated tissue and vascular system,
controlled environmental and exposure conditions.
• Diadavantages: Donor organisms still required, time
consuming and expensive, no intact organisms responses,
limited lenght of viability.
Cultured cells
• Advantages: No intact animals directly involved, ability to
carefully manipulate system, Low costs, wide range of
variables can be studied.
• Disadvantages: Instability of system, limited enzymatic
capabilities and viability of system, no or limited integrated
multicell or organismic responses.
Chemical/biochemical systems
• Advantages: No donor organism problems, Low costs, longterm stability of preparation, wide range of variables can be
studied, specificity of response.
• Disadvantages: No de facto correlation to in vivo system,
limited to investigation of single defined mechanism.
Computer simulations
• Advantages: No animal welfare concerns, speed and low perevaluation cost.
• Disadvantages: Problematic predictive value beyond narrow range
of structures, Expensive to establish.
The Four R’s In Experimental Toxicology
1.
2.
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4.
Replacement
Reduction
Refinement
Responsibility
• The first and most significant factors behind the interest in
so-called in vitro systems have clearly been political
campaign by a wide spectrum of individuals concerned
with the welfare and humane treatment of laboratory
animals.
• The historical beginnings of this campaign were in 1959.
Replacement
• Using methods that do not use intact
animals in place of those that do.
• For example, veterinary students may use a canine
cardiopulmonary-resuscitation simulator, Resusci-Dog, instead of
living dogs.
• Cell cultures may replace mice and rats that are fed new products
to discover substances poisonous to humans.
• In addition, using the preceding definition of animal, an
invertebrate (e.g., a horseshoe crab) could replace a vertebrate
(e.g., a rabbit) in a testing protocol.
Reduction
• The use of fewer animals.
• For instance, changing practices allow toxicologists to
estimate the lethal dose of a chemical with as few as
onetenth the number of animals used in traditional tests.
• Reduction can also refer to the minimization of any
unintentionally duplicative experiments, perhaps through
improvements in information resources.
Refinement
• The modification of existing procedures so that animals are
subjected to less pain and distress.
• Refinements may include ;
– administration of anesthetics to animals undergoing otherwise
painful procedures,
– administration of tranquilizers for distress,
– humane destruction before recovery from surgical anesthesia,
– careful scrutiny of behavioral indices of pain or distress,
followed by cessation of the procedure or the use of
appropriate analgesics.
Responsibility
• To toxicologists, this is the cardinal R.
• They may be personally committed to minimizing animal use and
suffering and to doing the best possible science of which they are
capable, but at the end of it all, toxicologists must stand by their
responsibility to be conservative in ensuring the safety of the
people using or exposed to the drugs and chemicals produced and
used in our society.
ANIMAL MODELS IN TOXICOLOGY
• The use of animals in experimental medicine,
pharmacology, pharmaceutical development, safety
assessment, and toxicological evaluation has become a
well-established and essential practice.
• Animal experiments also have served rather successfully as
identifiers of potential hazards to and toxicity in humans for
synthetic chemicals with many intended uses.
CURRENT ANIMAL STUDIES
• The current regulatory required use of animal models in
acute testing began by using them as a form of instrument
to detect undesired contaminants.
• For example, miners used canaries to detect the presence
of carbon monoxide, a case in which an animal model is
more sensitive than humans.
• In 1907, FDA started to protect the public by the use of a
voluntary testing program for new coal tar colors in foods.
This was replaced by amandatory program of testing in
1938, and such regulatory required animal testing
programs have continued to expand until recently.
• The Society of Toxicology (SOT) and the American College of
Toxicology (ACT) have both established Animals in Research
Committees, and these have published guidelines for the use of
animals in research and testing.
• In general, the purpose of these committees is to foster thinking on
the four Rs of animal-based research: reduction, refinement,
(research into) replacements, and responsible use.
• The media frequently carry reports that state that most animal
testing and research is not predictive of what will happen in people,
and therefore such testing is unwarranted.
• Many animal rights groups also present this argument at every
opportunity, and reinforce it with examples that entail seemingly
great suffering in animals but add nothing to the health, safety, and
welfare of society.
• Our primary responsibility (the fourth R) is to provide the
information to protect people and the environment, and without
animal models we cannot discharge this responsibility.
• The problem is that toxicology is a negative science.
• The things we find and discover are usually adverse.
• If the applied end of our science works correctly, the results are
things that do not happen, and therefore are not seen.
• For example, if we correctly identify toxic agents (using animals
and other predictive model systems) in advance of a product or
agent being introduced into the marketplace or environment,
generally it will not be introduced (or it will be removed) and
society will not see death, rashes, renal and hepatic diseases,
cancer, or birth defects,.
ORIGINS OF PREDICTIVE ANIMAL TESTING
• The “Lash Lure” Case:
• Early in the 1930s, an untested eyelash dye containing pphenylenediamine (Lash Lure) was brought onto the market in the
United States.
• This product rapidly demonstrated that it could sensitize the
external ocular structures, leading to corneal ulceration with loss of
vision and at least one fatality.
• A woman known as "Mrs. Brown", in 1933, trying to
beautify her appearance before going to a social party in
Dayton , Ohio, was encouraged in a beauty shop to try an
eyelash dye to enhance her eyes.
• Lash Lure was the name of the product.
• At the next morning, she couldn't open her eyes, they
were completely infected, with ulcers and scars, and in
three months she became permanent blind.
• Advertisements of Lash Lure Eye Lash and Brow Dye were
saying, in 1933, that their "new and improved mascara will
give you a radiating personality, with a before and an
after"...
• This last part was true: the "before" was the regular
appearance, and the "after" was a horror film, a cosmetic
disaster, with melted ocular globes, the flesh around them
with multiple scars, blinded people with infected ulcers, and a
woman died in the hospital with septicemia, blood poisoning.
• The Elixir of Sulfanilamide Case:
• In 1937, an elixir of sulfanilamide dissolved in ethylene
glycol was introduced into the marketplace.
• One hundred and seven people died as a result of ethylene
glycol toxicity.
• The public response to these two tragedies helped prompt
Congress to pass the Federal Food, Drug, and Cosmetic Act
of 1938.
• It was this law that mandated the premarket testing of
drugs for safety in experimental animals.
• Thalidomide:
• The use of thalidomide, a sedative-hypnotic agent, led to
some 10,000 deformed children being born in Europe.
• This in turn led directly to the 1962 revision of the Food,
Drug and Cosmetic Act, requiring more stringent testing.
• Current testing procedures would have identified the
hazard and prevented this tragedy.
• In fact, it has not occurred in Europe or the United States
except when the results of animal tests have been ignored.
• For example, birth defects have occurred with isotretinoin
(Accutane) where developmental toxicity had been clearly
established in animals and presented on labeling, but the
drug has continued to be used by potentially pregnant
women.
• Isotretinoin is used for severe acne treatment.
• This drug should not be used by pregnant patients.
• Patients who have already used Isotrotinoin, discontinue
the drug before 1 month of earlier and the whole
pregnancy period.
SELECTING AN ANIMAL MODEL
• Choosing the appropriate animal model for a given problem is
sometimes guesswork and too often a matter of convenience.
• For example, the rat is probably a poor model for studying the
chronic toxicity of any new nonsteroidal anti-inflammatory
drug (NSAID) because the acute gastrointestinal (GI) toxicity
will probably mask any other toxic effects.
• The guinea pig is less sensitive to most NSAIDs than the rat,
and would therefore be a more appropriate species for
investigating the chronic (nongastrointestinal) toxicity of an
NSAID.
HUSBANDRY AND CARE
• Inappropriate handling could result in unhealthy animals
and an experiment yielding variable and irreproducible
results.
• All animals have optimal temperature, humidity, light cycle,
light intensity, cage size and bedding, and dietary
requirements.
• Rabbits, for example, have a different optimal temperature
range than rats.
• Rats and ferrets have completely different dietary
requirements.
• Albino rodents have very sensitive eyes, and lights of too high
power can cause ocular damage, especially in those animals
on the top row of a cage rack.
FERRET
Caging
• Caging deserves special mention for two reasons. First, not
all animals can be group housed.
• Hamsters, for example, are notoriously antisocial. Even
breeding pairs cannot be left in the same small cage
together for prolonged periods.
• Guinea pigs, on the other hand, flourish when group
housed. Obviously these factors need to be considered
when designing an experiment.
• Second, cage size is important because the animal rights
movement has made it important.
• Many caging systems currently in use would no longer be
permitted and their replacement would be very expensive.
• This is just an example of how the animal rights
movement, and the resultant animal care laws, could
affect the conduct of pharmacologists and toxicologists.
CHOOSING SPECIES AND STRAINS
• Not only is it important to pick the correct species for an
experiment, but sometimes the correct strain as well.
• In some cases, an inbred strain might provide qualitative
and specific characteristics that make it a good disease
model, such as the spontaneously hypertensive rat.
• There are other more quantitative strain-related
differences such as size, color, temperament, and
background disease.
• For example, the Fischer 344 rat is smaller than the
Sprague-Dawley rat.
• These differences might make a particular strain more
appropriate for one experiment than others.
• The Fischer 344 rat has a high rate of spontaneous Leydig cell
tumors as compared to the Sprague-Dawley rat, which would
make the latter less appropriate for determining if a chemical
is a testicular carcinogen.
• Rats and mice provide the greatest array of strains from which
to choose, including outbred and some inbred.
DOSING
• Dosing is the act of introducing a drug or chemical into a living
organism.
• It requires active interaction between man and animal.
• There are, however, passive dosing techniques that are also used
frequently in which the chemical is placed in the animal’s air, water, or
feed, and the animal doses itself by breathing, drinking, or eating.
• Administering an antibiotic intravenously is active dosing;
giving it in the feed is passive dosing.
• The main routes used for active dosing are oral,
intravenous, intraperitoneal, dermal, and subcutaneous.
• The dose is the total amount of test article given, such as
1,000 mg.
• The dosage is a rate term and is the dose divided by the
weight of the test animal; for example, 1,000 mg/10 kg (for
a dog) = 100 mg/kg.
ANIMAL PHYSIOLOGY
• All animal species and strains have their own distinctive
physiology.
• As a result, values belonging to blood pressure, breathing
rates, ECGs, rectal temperatures, and normal clinical
laboratory parameters often vary between species.
• Clearly, appropriate interpretation of an in vivo experiment
requires a firm understanding of these baseline data.
• For example, there are well-established differences between
species with regard to red blood cell size: What is normal for a
dog would be high for a rat. The converse is true for breathing
rates.