Experimental Toxicology

Prof. Dr. Şahan SAYGI

NEU Faculty of PharmacyDepartment of Toxicology

CONTENTS

• 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, long-term 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 per-evaluation cost.

• Disadvantages: Problematic predictive value beyond narrow range of structures, Expensive to establish.

The Four R’s In Experimental Toxicology

1. Replacement2. Reduction3. Refinement4. 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 p-phenylenediamine (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.