Download Introduction to Toxicology

Introduction to Toxicology
JOHN H. DUFFUS
(Fundamental Toxicology, 2nd Ed. 2006; pp. 1 – 3)
1.1 INTRODUCTION
Toxicology is the fundamental science of poisons. A poison is generally considered to be any
substance that can cause severe injury or death as a result of a physicochemical interaction
with living tissue. However, all substances are potential poisons since all of them can cause
injury or death following excessive exposure. On the other hand, all chemicals can be used
safely if exposure of people or susceptible organisms to chemicals is kept below defined
tolerable limits, i.e. if handled with appropriate precautions. If no tolerable limit can be defined,
zero exposure methods must be used. Exposure is a function of the amount (or concentration)
of the chemical involved, and the time and frequency of its interaction with people or other
organisms at risk. For very highly toxic substances, the tolerable exposure may be close to
zero. In deciding what constitutes a tolerable exposure, it is essential to have data relating
exposure to the production of injury or adverse effect. A problem often arises in deciding what
constitutes an injury or adverse effect. An adverse effect is defined as an abnormal, undesirable
or harmful change following exposure to the potentially toxic substance. The ultimate adverse
effect is death but less severe adverse effects may include altered food consumption, altered
body and organ weights, visible pathological changes or simply altered enzyme levels. A
statistically significant change from the normal state of the person at risk is not necessarily an
adverse effect. The extent of the difference from normal, the consistency of the altered property
and the relation of the altered property to the total well-being of the person affected have to be
considered. An effect may be considered harmful if it causes functional or anatomical damage,
irreversible change in homeostasis or increased susceptibility to other chemical or biological
stress, including infectious disease. The degree of harm of the effect can be influenced by the
state of health of the organism. Reversible changes may also be harmful, but often they are
essentially harmless. An effect which is not harmful is usually reversed when exposure to the
potentially toxic chemical ceases. Adaptation of the exposed organism may occur so that it can
live normally in spite of an irreversible effect.
In immune reactions leading to hypersensitivity or allergic effects, the first exposure to the
causative agent may produce no adverse effect, although it sensitizes the organism to respond
adversely to future exposures, often at a very low level. The amount of exposure to a chemical
required to produce injury varies over a very wide range depending on the chemical and the
form in which it occurs. The extent of possible variation in harmful exposure levels is indicated
in Table 1.1, which compares median lethal dose (LD50) values for a number of potentially toxic
chemicals. The LD50 value is more descriptively called the median lethal dose and is defined
below. The LD50 is the statistically derived single dose of a chemical that can be expected
to cause death in 50% of a given population of organisms under a defined set of experimental
conditions. Where LD50 values are quoted for human beings, they are derived by extrapolation
from studies with mammals or from observations following accidental or suicidal exposures.
The LD50 has often been used to classify and compare toxicity among chemicals but its value for
this purpose is limited. A commonly used classification of this kind is shown in Table 1.2. Such a
classification is entirely arbitrary and has some intrinsic weaknesses. For example, it is difficult
to see why a substance with an LD50 of 200 mg kg_1 body weight should be regarded only as
harmful while one with an LD50 of 199 mg kg_1 body weight is said to be toxic, when the
difference in values is minimal. Further, there is no simple relationship between lethality and
sublethal toxic effects. In particular, there is no simple relationship between lethality and
effects of great concern, such as cancer or abnormal development of the human embryo. Even
in relation to lethality, it is not helpful because it gives no measure of the minimum dose that can
be lethal and thus no guide to what might be a ‘safe’ exposure level.
In decisions relating to chemical safety, the toxicity (hazard) of a substance is less important
than the risk associated with its use. Risk is the predicted or actual frequency (probability) of a
chemical causing unacceptable harm or effects as a result of exposure of susceptible
organisms or ecosystems. Assessment of risk is often assessment of the probability and likely
degree of exposure.
By comparison with risk, safety is the practical certainty that injury will not result from exposure
to a hazard under defined conditions; in other words, the high probability that injury will not
result. Practical certainty is defined as a numerically specified low risk or socially acceptable risk
applied in decision making for risk management.
In assessing permissible exposure conditions for chemicals, uncertainty factors are applied. A
threshold of exposure above which an adverse effect can occur (and below which no such
effect is observed) is defined from the available data, and this is divided by an uncertainty factor
to lower it to a value that regulatory toxicologists can regard as safe beyond doubt. An
uncertainty factor may be defined as a mathematical expression of uncertainty that is used to
protect populations from hazards that cannot be assessed with high precision. For example, the
1977 report of the US National Academy of Sciences Safe Drinking Water Committee proposed
the following guidelines for selecting uncertainty (safety) factors to be used in conjunction with
no observed effect level (NOEL) data. The NOEL should be divided by the following uncertainty
factors:
1. An uncertainty factor of 10 should be used when valid human data based on chronic
exposure are available.
2. An uncertainty factor of 100 should be used when human data are inconclusive, e.g.
limited to acute exposure histories, or absent, but when reliable animal data are
available for one or more species.
3. An uncertainty factor of 1000 should be used when no long-term, or acute human data
are available and experimental animal data are scanty.
This approach is subjective and is being continually updated. Safety control often involves the
assessment of ‘acceptable’ risk since total elimination of risk is often impossible. ‘Acceptable’
risk is the probability of suffering disease or injury that will be tolerated by an individual, group,
or society. Assessment of risk depends on scientific data but its ‘acceptability’ is influenced by
social, economic and political factors, and by the perceived benefits arising from a chemical or
process.
INTRODUCTION TO TOXICOLOGY
(Cassaret and Doull’s)
Toxicology is the study of the adverse effects of chemical or physical agents on living
organisms. A toxicologist is trained to examine and communicate the nature of those effects on
human, animal, and environmental health. Toxicological research examines the cellular,
biochemical, and molecular mechanisms of action as well as functional effects such as
neurobehavioral and immunological, and assesses the probability of their occurrence.
Fundamental to this process is characterizing the relation of exposure (or dose) to the
response. Risk assessment is the quantitative estimate of the potential effects on human health
and environmental significance of various types of chemical exposures (e.g., pesticide
residues on food, contaminants in drinking water). The variety of potential adverse effects and
the diversity of chemicals in the environment make toxicology a broad science, which often
demands specialization in one area of toxicology. Our society’s dependence on chemicals and
the need to assess potential hazards have made toxicologists an increasingly important part of
the decision-making processes.
Different Areas of Toxicology
The professional activities of toxicologists fall into three main categories: descriptive,
mechanistic, and regulatory (Fig. 2-1). Although each has distinctive characteristics, each
contributes to the other, and
all are vitally important to chemical risk assessment (see Chap. 4). A mechanistic toxicologist is
concerned with identifying and understanding the cellular, biochemical, and molecular
mechanisms by which chemicals exert toxic effects on living organisms (see Chap. 3 for a
detailed discussion of mechanisms of toxicity). The results of mechanistic studies are very
important in many areas of applied toxicology. In risk assessment, mechanistic data may be
very useful in demonstrating that an adverse outcome (e.g., cancer,
birth defects) observed in laboratory animals is directly relevant to humans. For example, the
relative toxic potential of organophosphate insecticides in humans, rodents, and insects can be
accurately predicted on the basis of an understanding of common mechanisms (inhibition of
acetylcholinesterase) and differences in biotransformation for these insecticides among the
different species. Similarly, mechanistic data may be very useful in identifying adverse
responses in experimental animals that may not be relevant to humans.
For example, the propensity of the widely used artificial sweetener saccharin to cause bladder
cancer in rats may not be relevant to humans at normal dietary intake rates. This is because
mechanistic studies have demonstrated that bladder cancer is induced only under conditions
where saccharin is at such a high concentration in the urine that it forms a crystalline precipitate
(Cohen, 1998). Dose–response studies suggest that such high concentrations would not be
achieved in the human bladder even after extensive
dietary consumption.
Mechanistic data are also useful in the design and production of safer alternative chemicals and
in rational therapy for chemical poisoning and treatment of disease. For example, the drug
thalidomide was originally marketed in Europe and Australia as a sedative agent for
pregnantwomen. However, itwas banned for clinical use in 1962 because of devastating birth
defects that occurred if the drug was ingested during a critical period in pregnancy. But
mechanistic studies over the past several decades have demonstrated that this drug may have
a unique molecular mechanism of action that interferes with the expression of certain genes
responsible for blood vessel formation (angiogenesis). With an understanding of this
mechanism, thalidomide has been “rediscovered” as a valuable therapeutic agent that may be
highly effective in the treatment of certain infectious diseases (e.g., leprosy and AIDS), a variety
of inflammatory diseases, and some types of cancer. This provides an interesting example of
how a highly toxic drug with selectivity toward a specific population (pregnant women) can be
used safely with proper precautions.
Following its approval for therapeutic use in 1998, a program was established that required all
clinicians, pharmacists, and patients that receive thalidomide to enroll in a specific program
(System for Thalidomide Education and Prescribing Safety, STEPS). The population at risk for
the potential teratogenic effects of thalidomide (all women of childbearing age) were required to
use two forms of birth control, and also have a negative pregnancy test within 24 hours of
beginning therapy, and periodically the patients registered with the STEPS program, 6000 were
females of childbearing age. Remarkably, after 6 years of use, only one patient actually
received thalidomide during her pregnancy. She initially tested negative at the beginning of
therapy; on a subsequent test she was identified as positive, and the drug was stopped. The
pregnancy ended up as a miscarriage (Uhl et al., 2006). Thus, a clear understanding of
mechanism of action led to the development of strict prescribing guidelines and patient
monitoring, thereby allowing a potentially dangerous drug to be used safely and effectively to
treat disease in tens of thousands of patients who would otherwise not have benefited from the
therapeutic actions of the drug (Lary et al., 1999).
In addition to aiding directly in the identification, treatment, and prevention of chemical toxicity,
an understanding of the mechanisms of toxic action contributes to the knowledge of basic
physiology, pharmacology, cell biology, and biochemistry. The advent of new technologies in
molecular biology and genomics now provide mechanistic toxicologists with the tools to explore
exactly how humans may differ from laboratory animals in their response to toxic substances.
These same tools are also being utilized to identify individuals who are genetically susceptible
to factors in the environment or respond differently to a chemical exposure. For example, it is
nowrecognized that a small percentage of the population genetically lacks the ability to detoxify
the chemotherapeutic drug, 6-mercaptopurine, used in the treatment of some forms of
leukemia. Young children with leukemia who are homozygous for this genetic trait (about one in
300) may experience serious toxic effects from a standard therapeutic dose of this drug.
Numerous genetic tests for polymorphisms in drug metabolizing enzymes and transporters are
now available that can identify genetically susceptible individuals in advance of pharmacological
treatment (Eichelbaum et al., 2006). These new areas of “pharmacogenomics” and
“toxicogenomics” provides an exciting opportunity in the future for mechanistic toxicologists to
identify and protect genetically susceptible individuals from harmful environmental exposures,
and to customize drug therapies that enhance efficacy and minimize toxicity, based on an
individual’s genetic makeup.
A descriptive toxicologist is concerned directly with toxicity testing, which provides information
for safety evaluation and regulatory requirements. The appropriate toxicity tests (as described
later in this chapter and other chapters) in cell culture systems or experimental animals are
designed to yield information to evaluate risks posed to humans and the environment from
exposure to specific chemicals. The concern may be limited to effects on humans, as in the
case of drugs and food additives. Toxicologists in the chemical industry, however, must be
concerned not only with the risk posed by a company’s chemicals (insecticides, herbicides,
solvents, etc.) to humans but also with potential effects on fish, birds, and plants, as well as
other factors that might disturb the balance of the ecosystem. Descriptive toxicology is of course
not divorced from mechanistic studies, as such studies provide important clues to a chemical’s
mechanism of action, and thus contribute to the development of mechanistic toxicology through
hypothesis generation. Such studies are also a key component of risk assessments that are
used by regulatory toxicologists. The recent advent of so-called “omics” technologies
(genomics, transcriptomics, proteomics, metabonomics, etc.) form the basis of the emerging
subdiscipline of toxicogenomics. The application of these new technologies to toxicity testing is
in many ways “descriptive” in nature, yet affords great mechanistic insights into how chemicals
produce their toxic effects. This exciting new area of toxicology is discussed
in more detail later in the chapter.
A regulatory toxicologist has the responsibility for deciding, on the basis of data provided by
descriptive and mechanistic toxicologists, whether a drug or other chemical poses a sufficiently
low risk to be marketed for a stated purpose or subsequent human or environmental exposure
resulting from its use. The Food and Drug Administration (FDA) is responsible for allowing
drugs, cosmetics, and food additives to be sold in the market according to the Federal Food,
Drug and Cosmetic Act (FFDCA). The U.S. Environmental Protection Agency (EPA) is
responsible for regulating most other chemicals according to the Federal Insecticide, Fungicide
and Rodenticide Act (FIFRA), the Toxic Substances Control Act (TSCA),
the Resource Conservation and Recovery Act (RCRA), the Safe Drinking Water Act, and the
Clean Air Act. In 1996, the U.S. Congress passed the Food Quality Protection Act (FQPA)
which fundamentally changed the pesticide and food safety laws under FIFRA and FFDCA
requiring stricter safety standards particularly
for infants and children, who were recognized as more susceptible to health effects of
pesticides. The EPA is also responsible for enforcing the Comprehensive Environmental
Response, Compensation and Liability Act [CERCLA, later revised as the Superfund
Amendments Reauthorization Act (SARA)], more commonly called the Superfund Act. This
regulation provides direction and financial support for the cleanup of waste sites that contain
toxic chemicals that may present a risk to human health or the environment. The Occupational
Safety and Health Administration (OSHA) of the Department of Labor was established to ensure
that safe and healthful conditions exist in the workplace. The National Institute for Occupational
Safety and Health (NIOSH) as part of the Centers for Disease Control and Prevention (CDC) in
the Department of Health and Human Services is responsible for conducting research and
making recommendations for the prevention of work-related injury and illness. The Consumer
Product Safety Commission is responsible for protecting consumers from hazardous household
substances, whereas the Department of Transportation (DOT) ensures that materials shipped in
interstate commerce are labeled and packaged in a manner consistent with the degree of
hazard they present. Regulatory toxicologists are also involved in the establishment of
standards for the amount of chemicals permitted in ambient air, industrial atmospheres, and
drinking water, often integrating scientific information from basic descriptive and mechanistic
toxicology studies with the principles and approaches used for risk assessment (see Chap. 4).
In addition to the above categories, there are other specialized areas of toxicology such as
forensic, clinical, and environmental toxicology. Forensic toxicology is a hybrid of analytic
chemistry and fundamental toxicological principles. It is concerned primarily with the
medicolegal aspects of the harmful effects of chemicals on humans and animals. The expertise
of forensic toxicologists is invoked primarily to aid in establishing the cause of death and
determining its circumstances in a postmortem investigation (see Chap. 31). Clinical toxicology
designates an area of professional emphasis in the realm of medical science that is concerned
with disease caused by or uniquely associated with toxic substances (see Chap. 32). Generally,
clinical toxicologists are physicians who receive specialized training in emergency medicine and
poison management. Efforts are directed at treating patients poisoned with drugs or other
chemicals and at the development of new techniques to treat those intoxications. Public contact
about treatment and prevention is often through the national network of poison control centers.
Environmental toxicology focuses on the impacts of chemical pollutants in the environment on
biological organisms. Although toxicologists concerned with the effects of environmental
pollutants on human health fit into this definition, it is most commonly associated with studies on
the impacts of chemicals on nonhuman organisms such as fish, birds, terrestrial animals, and
plants. Ecotoxicology is a specialized area within environmental toxicology that focuses more
specifically on the impacts of toxic substances on population dynamics in an
ecosystem. The transport, fate, and interactions of chemicals in the environment constitute a
critical component of both environmental toxicology and ecotoxicology.
CLASSIFICATION OF TOXIC AGENTS
(Cassaret and Doull’s)
Toxic agents are classified in a variety of ways, depending on the interests and needs of the
classifier. In this textbook, for example, toxic agents are discussed in terms of their target
organs (liver, kidney, hematopoietic system, etc.), use (pesticide, solvent, food additive, etc.),
source (animal and plant toxins), and effects (cancer, mutation, liver injury, etc.). The term toxin
generally refers to toxic substances that are produced by biological systems such as plants,
animals, fungi, or bacteria. The term toxicant is used in speaking of toxic substances that are
produced by or are a by-product of anthropogenic (human-made) activities. Thus, zeralanone,
produced by a mold, is a toxin, whereas “dioxin” [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)],
produced during the production and/or combustion of certain chlorinated organic chemicals, is a
toxicant. Some toxicants can be produced by both natural and anthropogenic activities.
For example, polyaromatic hydrocarbons are produced by the combustion of organic matter,
which may occur both through natural processes (e.g., forest fires) and through anthropogenic
activities (e.g., combustion of coal for energy production; cigarette smoking). Arsenic, a toxic
metalloid, may occur as a natural contaminant of groundwater or may contaminate groundwater
secondary to industrial activities. Generally, such toxic substances are referred to as toxicants,
rather than toxins, because, although they are naturally produced, they are not produced by
biological systems.
Toxic agents may also be classified in terms of their physical state (gas, dust, liquid), their
chemical stability or reactivity (explosive, flammable, oxidizer), general chemical structure
(aromatic amine, halogenated hydrocarbon, etc.), or poisoning potential (extremely toxic, very
toxic, slightly toxic, etc.). Classification of toxic agents on the basis of their biochemical
mechanisms of action (e.g., alkylating agent, cholinesterase inhibitor, methemoglobin producer)
is usually more informative than classification by general terms such as irritants and corrosives.
But more general classifications such as air pollutants, occupation-related agents, and acute
and chronic poisons can provide a useful focus on a specific problem. It is evident from this
discussion that no single classification is applicable to the entire spectrum of toxic agents and
that combinations of classification systems or a classification based on other factors may be
needed to provide the best rating system for a special purpose. Nevertheless, classification
systems that take into consideration both the chemical and the biological properties of an agent
and the exposure characteristics are most likely to be useful for regulatory or control purposes
and for toxicology in general.
CHARACTERISTICS OF EXPOSURE
(Cassaret and Doull’s)
Toxic effects in a biological system are not produced by a chemical agent unless that agent or
its metabolic breakdown (biotransformation) products reach appropriate sites in the body at a
concentration
and for a length of time sufficient to produce a toxic manifestation. Many chemicals are of
relatively low toxicity in the “native” form but, when acted on by enzymes in the body, are
converted to intermediate forms that interfere with normal cellular biochemistry and physiology.
Thus, whether a toxic response
occurs is dependent on the chemical and physical properties of the agent, the exposure
situation, how the agent is metabolized by the system, the concentration of the active form at
the particular target site(s), and the overall susceptibility of the biological system or subject.
Thus, to characterize fully the potential hazard
of a specific chemical agent, we need to know not only what type of effect it produces and the
dose required to produce that effect but also information about the agent, the exposure, and its
disposition by the subject. Two major factors that influence toxicity as it relates to the exposure
situation for a specific chemical are the route of exposure, and the duration, and frequency of
exposure.
Route and Site of Exposure
The major routes (pathways) by which toxic agents gain access to the body are the
gastrointestinal tract (ingestion), lungs (inhalation), skin (topical, percutaneous, or dermal), and
other parenteral (other than intestinal canal) routes. Toxic agents generally produce the greatest
effect and the most rapid response when given directly into the bloodstream (the intravenous
route). An approximate descending order of effectiveness for the other routes would be
inhalation, intraperitoneal, subcutaneous, intramuscular, intradermal, oral, and dermal. The
“vehicle” (the material in which the chemical is dissolved) and other formulation factors can
markedly alter absorption after ingestion, inhalation, or topical exposure. In addition, the route of
administration can influence the toxicity of agents. For example, an agent that acts on the CNS,
but is efficiently detoxified in the liver, would be expected to be less toxic when given orally than
when given via inhalation, because the oral route requires that nearly all of the dose pass
through the liver before reaching the systemic circulation and then the CNS. Occupational
exposure to toxic agents most frequently results from breathing contaminated air (inhalation)
and/or direct and prolonged contact of the skin with the substance (dermal exposure), whereas
accidental and suicidal poisoning occurs most frequently by oral ingestion. Comparison of the
lethal dose of a toxic substance by different routes of exposure often provides useful information
about its extent of absorption. In instances when the toxic dose after oral or dermal
administration is similar to the toxic dose after intravenous administration, the assumption is that
the toxic agent is absorbed readily and rapidly. Conversely, in cases where the toxic dose by
the dermal route is several orders of magnitude higher than the oral toxic dose, it is likely that
the skin provides an effective barrier to absorption of the agent. Toxic effects by any route of
exposure can also be influenced by the concentration of the agent in its vehicle, the total
volume of the vehicle and the properties of the vehicle to which the biological system is
exposed, and the rate at which exposure occurs. Studies in which the concentration of a
chemical in the blood is determined at various times after exposure are often needed to clarify
the role of these and other factors in the toxicity of a compound. For more details on the
absorption of toxicants.
Duration and Frequency of Exposure
Toxicologists usually divide the exposure of experimental animals to chemicals into four
categories: acute, subacute, subchronic, and chronic. Acute exposure is defined as exposure to
a chemical for less than 24 hours, and examples of exposure routes are intraperitoneal,
intravenous, and subcutaneous injection; oral intubation; and dermal application. Whereas
acute exposure usually refers to a single administration, repeated exposures may be given
within a 24-hours period for some slightly toxic or practically nontoxic chemicals. Acute
exposure by inhalation refers to continuous exposure for less than 24 hours, most frequently for
4 hours. Repeated exposure is divided into three categories: subacute, subchronic, and chronic.
Subacute exposure refers to repeated exposure to a chemical for 1 month or less, subchronic
for 1 to 3 months, and chronic for more than 3 months, although usually this refers to studies
with at least 1 year of repeated dosing. These three categories of repeated exposure can be by
any route, but most often they occur by the oral route, with the chemical added directly to the
diet. In human exposure situations, the frequency and duration of exposure are usually not as
clearly defined as in controlled animal studies, but many of the same terms are used to describe
general exposure situations. Thus,workplace or environmental exposures may be described as
acute (occurring from a single incident or episode), subchronic (occurring repeatedly over
several weeks or months), or chronic (occurring repeatedly for many months or years). For
many chemicals, the toxic effects that follow a single exposure are quite different from those
produced by repeated exposure. For example, the primary acute toxic manifestation of benzene
is central nervous system (CNS) depression, but repeated exposures can result in bone marrow
toxicity and an increased risk for leukemia. Acute exposure to chemicals that are rapidly
absorbed is likely to produce immediate toxic effects but also can produce delayed toxicity that
may or may not be similar to the toxic effects of chronic exposure. Conversely, chronic exposure
to a toxic chemical may produce some immediate (acute) effects after each administration in
addition to the long-term, low-level, or chronic effects of the toxic substance. In characterizing
the toxicity of a specific chemical, it is evident that information is needed not only for the singledose (acute) and long-term (chronic) effects but also for exposures of intermediate duration.
The other time-related factor that is important in the temporal characterization of repeated
exposures is the frequency of exposure. The relationship between elimination rate and
frequency of exposure is shown in Fig. 2-2. A chemical that produces severe effects with a
single dose may have no effect if the same total dose is given in several intervals. For the
chemical depicted by line B in Fig. 2-2, in which the half-life for elimination (time necessary for
50% of the chemical to be removed from the bloodstream) is approximately equal to the dosing
frequency, a theoretical toxic concentration (shown conceptually as two Concentration Units in
Fig. 2-2) is not reached until the fourth dose, whereas that concentration is reached with only
two doses for chemical A, which has an elimination rate much slower than the dosing interval
(time between each repeated dose). Conversely, for chemical C, where the elimination rate is
much shorter than the dosing interval, a toxic concentration at the site of toxic effect will never
be reached regardless of how many doses are administered. Of course, it is possible that
residual cell or tissue damage occurs with each dose even though the chemical itself is not
accumulating. The important consideration, then, is whether the interval between doses is
sufficient to allow for complete repair of tissue damage. It is evident that with any type of
repeated exposure, the production of a toxic effect is influenced not only by the frequency of
exposure but may, in fact, be totally dependent on the frequency rather than the duration of
exposure. Chronic toxic effects may occur, therefore, if the chemical accumulates in the
biological system (rate of absorption exceeds the rate of biotransformation and/or excretion), if it
produces irreversible toxic effects, or if there is insufficient time for the system to recover from
the toxic damage within the exposure frequency interval.