Principle and Kinetics of

Toxicology
 Toxicology
Toxicology is the science of poisons and has as its focus the study of the adverse effects of chemicals on living
organisms.
What is Poison? — All substances are poisons; there is none that is not a poison. The right dose differentiates a poison and
a remedy. Paracelsus (1493-1541)
Adverse effect — any change from an organism’s normal state
 dependent upon the concentration of active compound at the target site for a sufficient time.
 Xenobiotic — any compound not occurring within the normal metabolic pathways of a biological system
 Toxicant — any substance that causes a harmful (or adverse) effect when in contact with a living organism at a sufficiently
high concentration.
 Toxin — any toxicant produced by an organism (floral or faunal, including bacteria); that is, naturally produced toxicants.
Phytoxins, - Zootoxin, - bacteriotoxin
An example would be the pyrethrins, which are natural pesticides produced by pyrethrum flowers (i.e., certain
chrysanthemums) that serve as the model for the man made insecticide class pyrethroids.
 Types of Toxic effects

Reversible toxicity — an adverse or undesirable effect that can be reversed once exposure is stopped. Reversibility
of toxicity depends on a number of factors, including the extent of exposure (time and amount of toxicant) and the
ability of the affected tissue to repair or regenerate. An example includes hepatic toxicity from acute acetaminophen
exposure and liver regeneration.
• Dermatoxic – affects skin
• Hemotoxic – affects blood
• Hepatotoxic- affects liver
• Nephrotoxic – affects kidneys
• Neurotoxic – affects nervous system
• Pulmonotoxic- affects lungs
Irreversible toxicity — an adverse or undesirable effect that cannot
be reversed.

• Carcinogen - cause cancer

• Mutagen - cause chromosome (gene) damage
• Teratogen - cause birth defects
Acute exposure—exposure over a brief period of time (generally less than 24 h). Often it is considered to be a single
exposure (or dose) but may consist of repeated exposures within a short time period.
Subacute exposure—resembles acute exposure except that the exposure duration is greater, from
several days to one month.
Subchronic exposure—exposures repeated or spread over an intermediate time range. For animal testing, this time range is
generally considered to be 1–3 months.
Chronic exposure—exposures (either repeated or continuous) over a long (greater than 3 months) period of time. With
animal testing this exposure often continues for the majority of the experimental animal’s life, and within occupational
settings it is generally considered to be for a number of years.
Acute toxicity—an adverse or undesirable effect that is manifested within a relatively short time interval ranging from
almost immediately to within several days following exposure (or dosing). An example would be chemical asphyxiation from
exposure to a high concentration of carbon monoxide (CO).
Chronic toxicity—a permanent or lasting adverse effect that is manifested after exposure to a toxicant. An example would
be the development of silicosis following a long-term exposure to silica in workplaces such as foundries
• Local toxicity — an adverse or undesirable effect that is manifested at the toxicant’s site of contact with the
organism. Examples include an acid’s ability to cause burning of the eyes, upper respiratory tract irritation, and skin
burns.

• Systemic toxicity — an adverse or undesirable effect that can be seen throughout the organism or in an organ with
selective vulnerability distant from the point of entry of the toxicant (i.e., toxicant requires absorption and
distribution within the organism to produce the toxic effect). Examples would be adverse effects on the kidney or
central nervous system resulting from the chronic ingestion of mercury.

Safety — the measure or mathematical probability that a specific exposure situation or dose will not produce a toxic
effect.

Risk — the measure or probability that a specific exposure situation or dose will produce a toxic effect.

Risk assessment — the process by which the potential (or probability of) adverse health effects of exposure are
characterized.
 Chemistry of Toxicants
Chemistry of a poison it is a major determinant in the Solubility and Reactivity of substance

Solubility is defined as the maximum amount of a substance that will dissolve in a given amount of solvent at a specified
temperature.
Reactivity: the quality or condition of being reactive. - the relative capacity of an atom, molecule, or radical to undergo a
chemical reaction with another atom, molecule, or compound.
  - Chemistry can dictate the vehicle used to administer the substance in testing
 - and can predict to some extent the duration of the test,
 - as well as any potential products of biotransformation (metabolism).

 For small molecules, knowledge of chemistry is relatively simple to gain by examining the chemical
formula and performing tests such as an octanol/water partitioning experiment to find the relative lipid
solubility.
 However, the coming generation of drugs will be composed of many large polymeric compounds such as
proteins and nucleic acids, which are more challenging to study chemically.
 There is also a trend to return to botanical products, which are often highly complex mixtures with
multiple active ingredients. This can complicate the administration and interpretation of testing for toxicity
 Toxicity Testing Methods

• A wealth of information can be gathered from toxicity testing by carefully observing animals during and
following exposure. These data may provide evidence for the mode of action of the substance and provide
clues as to which physiological system, organs, and tissues could be affected.

Although specific protocols for toxicity testing have been developed by various regulatory agencies (such
as The Food and Drug Administration (FDA) and Environmental Protection Agency (EPA), they share
many characteristics in common.
 Handling and treatment of animals - Humane and same
 Tagged for identification - using either simple numbered, metal ear tags, or more sophisticated devices
such as electronic transponding implants.
 Animals must be housed in clean, comfortable conditions with access to adequate food and water.
 These cages are equipped with a separator for collection and measurement of urine and feces, so that
consumption of food and water can be measured more accurately.
• Body weight of the test animals is measured either daily or periodically
• Animals are observed for behavior (comparing behavior of treated with control animals) and symptomology
(such as tremors or convulsions, for example).
 During the exposure period, animals are monitored closely for symptoms of poisoning, as well as timing of
appearance of those symptoms, which might suggest the mechanism of poisoning of the substance. A slow
onset of poisoning, for example, might suggest a bioactivation of the substance to a more toxic metabolite,
or product, which accumulates as the parent substance is converted.

 Following an exposure period, the animals are sacrificed and necropsy is performed. This is a procedure
in which the treated and control animals are dissected and organs are weighed and examined for toxic
effects in gross morphology and physiology. Sections of tissue samples may be sliced on a microtome and
examined under the microscope for evidence of histopathology (which is any abnormality in cell or
tissue). Tissue samples may also be analyzed for the presence of biochemical indicators of pathology.
 Factors to Be Considered in Planning Toxicity Testing

1. Through what physiological route does exposure occur (in other words, how does the

substance get into the body)?

2. How much of the substance is necessary to produce toxicity?

3. Over what period of time does exposure occur?

 Routes of Exposure
Various means of administration or routes of exposure are used in toxicity testing.
• Oral toxicity is of primary concern when considering a substance that might be ingested in food, such
as the residue, or a pesticide or food additive, or taken orally as a drug.
Dosing through the mouth is technically described as the peroral or per os (po) method.
o In some cases, the substance to be tested may be added directly to the animal’s food or water.
o Alternatively, it may be dissolved in water, vegetable oil, or another vehicle (depending on the
solubility of the test substance) and introduced directly into the esophagus or stomach through use of
a curved needle-like tube (a process called gavage).
• Dermal administration may be considered for a substance that might be handled by workers, such as paints, inks,
and dyes, or for cosmetics applied to the skin. The test substance is painted onto the skin, covered with a patch of
gauze held with tape, and plastic is wrapped around the body to prevent ingestion of the substance.
• Finally, respiratory administration should be considered in testing industrial solvents or cosmetics applied in an
aerosol spray.
Toxicity may also be assessed by direct injection of the substance, using a syringe and needle.
Intraperitoneal (ip) injections are made into the body cavity
Intramuscular (im) injections are placed into a large muscle of the hind leg
Subcutaneous (sc) injections are placed just beneath the skin
Intravenous (iv) injections are made directly into a large vein.
Data derived from these injections are especially useful in estimating doses for investigations of drugs that
eventually may be injected by an analogous method in human patients.

IP
 Determining the Responses to Varying Doses of a Substance

Several terms are used to describe levels of exposure to toxicants.

The terms dose and dosage have been used nearly interchangeably,
 although dose commonly refers to the amount of a chemical administered,
 and dosage refers to the amount of chemical administered per unit body weight of the recipient.
Thus, a dose of a drug might be expressed in milligrams, while the dosage would be expressed as milligram per
kilogram of body weight.
o In toxicity testing most chemical amounts are calculated and administered as dosages, which allows better
standardization of the amount of chemical received, and allows a better basis for comparison of effects between
individuals and species of widely varying body size.
o In respiratory exposures, exposure levels are usually measured by the concentration of the substance in the
environment (in parts per million).
 Quantitative toxicology involves challenging test animals with the substance to be evaluated, which is applied in an
ordered series of doses. The dose is controlled by the toxicologist; therefore, it is considered to be the independent
variable. Response of the animals may be measured in many different ways, and is generally dependent on the dose
applied (i.e., it is the dependent variable).
Responses can be scored and related to dose in order to determine the dose vs. response relationship.
1. One response considered in toxicology is the death of the animal. This is scored as a quantal value, alive (no
response: 0) or dead (response: 1), and recorded as mortality. A dose producing mortality is a lethal dose of
the substance.
2. In other experiments, the observed response may be a continuous variable that can be measured in each
subject. Examples of continuous variables include consumption of oxygen, time to onset of convulsions,
degree of inhibition of an enzyme, and loss of weight.
 A basic principle of toxicology is that response varies proportionally to a geometric, not arithmetic, increase
in dose. This means that to test a substance that produces responses in a small proportion of animals at 1 to 2
mg/kg, a geometric dosing range (1, 2, 4, 8, and 16 mg/kg) would be used rather than an arithmetic range (1,
2, 3, 4, and 5 mg/kg). Because of this, graphs relating dose and response are generally plotted with the
response value on the y-axis and the logarithm of the dose on the x-axis.
 Timing of Exposure
Often, the first of many considerations in toxicity testing is to assess the acute toxicity of the chemical. Acute toxicity is the
toxicity that results from a single exposure to the substance. Typically, animals are dosed with a single dose and then
observed for up to 14 days. One example of an acute toxicity test is the LD50, which will be discussed next in this chapter.
Subacute toxicity testing measures the response to substances that are delivered through repeated or continuous exposure
over a period that generally does not exceed 14 days; subchronic toxicity testing involves repeated or continuous exposure
over a period of 90 days. The final category of exposure is chronic toxicity testing, which refers to repeated or continuous
exposures that last for more than 90 days.
• To ensure sufficient challenge, animals are often exposed to the maximum tolerated dose, the greatest dose that neither
kills nor causes incapacitating symptoms. While very high doses are used so that any chronic toxicity of the test
compound will be observable, some experts consider that effects seen at large doses may be due to massive physical
damage or mitogenesis (regeneration due to cell death), and thus may not accurately predict a substance’s toxicity at
lower doses.
 The LD50 Experiment
Testing
What is LD50?
Traditionally, the median lethal dose has been used as a general measure of acute toxicity of any substance.
This is the predicted dose at which one half of the individuals in a treated population would be killed. The
median lethal dose is determined by exposing groups of uniform test animals to a geometric series of doses
of the substance of interest under controlled environmental conditions. The abbreviation LD50, for lethal
dose 50, is often used for the median lethal dose. The standard laboratory animal commonly used in this test
is the white Norway rat, Rattus norwegicus.
Dose Choice
The dose is expressed in dosage units of milligrams of active ingredient of the test substance administered
per kilograms of body weight of the test organisms (mg/kg). The highest dose administered in a typical
LD50 experiment is chosen so that 90% or more of the animals in the highest dose group will be killed. The
choice of the highest dose can be estimated from previous results with chemically similar substances, or by a
pilot, range-finding experiment in which a smaller number of animals are exposed to a wide range of
dilutions. Then serial dilutions of that dose are used to produce a gradient of intermediate responses over
four or five doses. Typically, at least 10 animals (ideally, 5 males and 5 females) are exposed at each of six
doses, plus there is always a negative control group exposed only to the vehicle.
Toxicant Grade
In LD50 determinations, the test substance is usually applied as technical grade, the practical grade as
manufactured for sale and use (usually of approximately 95% purity). In some cases, an agency might
require additional tests with the analytical grade (which is greater than 99% pure). If necessary, a sample
can be tested by gas chromatographic or high-performance liquid chromatographic analysis to verify the
purity. A vehicle and route of administration must be chosen, based in part on the physical properties of the
test substance, including solubility in water, organic solvents, and corn oil; melting point, boiling point,
and vapor pressure; and color and odor.
Test Conditions
 To perform the dosing, animals of similar weight are chosen, fasted overnight, numbered, and then assigned to
treatment groups using a table of random numbers. Doses are applied beginning with the negative control,
which is vehicle only, and then increasing doses of the test substance. (In this way, the same syringe can be
used with no risk of contaminating a lower dose with the residue of a higher dose.) For each dose, a solution is
prepared so that a small volume of vehicle (e.g., no more than around 2.0 ml) will contain the intended dose
when administered to the animal of average size.
For example, if a dose of 10 mg/kg is intended, and the average rat weighs 200 g (0.2 kg), then the concentration
of the test substance in oil solution should be 1.0 mg/ml. If the subject rat weighs 200 g, then 2.0 ml administered
will result in the intended dose of 10 mg/kg. If the next rat weighs only 190g, then the amount administered can
be reduced to 1.9 ml to maintain the desired dose of 10 mg/kg.
Analysis
Heterogeneity
In any population, a very small proportion of individuals are very susceptible, while another very small
proportion of individuals are very tolerant to the same dose of the same poison. Some variability is actually
experimental error due to such factors as the precision in administering the dose, environment of the animal,
and condition of the animal, such as fasting, handling of the animal, etc. True heterogeneity is due to the
genetic variability in physiological characteristics of the animals (although it is expected that inbred strains of
rodents used in laboratory experiments would be much less heterogeneous in response than wild populations
of the same species).
Normal distribution
o This variation in response is observed as the long tails of the dose vs. response histogram (Figure 1.1).
If tolerances of individuals are normally distributed (values are symmetrical around the mean, with
68% of the values falling within one standard deviation of the mean and 98% of the values falling
within two standard deviations of the mean) in the population of rats tested (as commonly assumed).
Sigmoid Curve
o then a sigmoid curve would describe the accumulated percentage mortality plotted vs. the logarithm of
the dose (Figure 1.2). The increasing mortality observed at each higher dose is the result of
accumulating responses of less tolerant to more tolerant individuals in each group.
Probit mortality
The median lethal dose is often calculated by transforming the accumulated percentage mortality at each
dose to a probit mortality score. This is a type of probability transformation in which “one probit unit is
defined as being equal in magnitude to one standard deviation unit of the response”. Probit mortality scores
plotted vs. the logarithm of the dose will produce a straight line from which the median lethal dose can be
predicted (Figure 1.3 and Figure 1.4).
The slope of the probit plot line is related to the uniformity of response within the animal population. If the
slope of the response were 2.0, a rather typical value, then approximately 68% of the population would be
expected to respond to a tenfold range in doses centered at the median lethal dose. On the other hand, if the
slope of the response were 6.0, then more than 99.6% of the population would be expected to respond to a
tenfold increase in dose centered at the median lethal dose. As an example, an extremely homogeneous
response was observed in CF1 mice exposed to an organophosphorus agent; there was a change of 18.8
probit units of response per 1.0 log10 increase in dose (Figure 1.4).