Transcript Richards_CH20x

Chapter 20:
Toxicity Testing
Toxicity Testing
• There are two purposes of toxicity testing.
– There is a quantitative effort to elucidate a
dose–effect relationship
– There is a qualitative determination of the
toxicity of the agent relative to other known
chemicals.
Toxicology testing, cont.
• Both purposes are accomplished using
laboratory animals and in vitro methods.
– There are ethical concerns associated with
whole animal studies as the intent of toxicity
testing is to produce harm to the animal and
then extrapolate the results to humans.
– Extrapolation magnifies error, so standards
have been developed using uncertainty
factors and modifying factors.
– Application of the results from animal testing
can improve safety and help prevent injury.
Toxicity Testing
• The toxic effects of chemicals are
determined by:
– The nature of the chemical hazard
– The dose or quantity to which the individual is
exposed
– The pathway(s) of exposure
– The pattern of the exposure
– The duration of the exposure
Toxicology testing, cont.
• In toxicity testing the importance of the
dose or concentration and the hazardous
nature of the chemical may vary
considerably depending on the route of
exposure.
Toxicology testing, cont.
• A chemical may be poorly absorbed through the
skin but well absorbed orally.
– Because of such route-specific differences in
absorption, toxicants are often ranked for hazard in
accordance with the route of exposure.
– For example, a chemical may be relatively nontoxic
by one route of exposure and highly toxic via another
route of exposure.
– Accordingly, toxicity testing should be conducted
using the most likely routes for human exposure.
Toxicity Information
• Toxicity information is obtained primarily
by
– Use of laboratory animals (in vivo studies)
– Surrogate animal models such as cell culture
systems [SARs] (in vitro studies)
– Human data obtained from intentional or
accidental exposures to chemical agents
– Nonbiological models (computers, structure–
activity relationships [SARs])
Toxicity Information, cont.
• A great deal of information is available on
the toxicity of chemicals from whole animal
studies, in vitro studies, and
epidemiological studies. There are
advantages and disadvantages for each of
these types of studies.
Exposure Durations
One of the most important considerations
in toxicology is the duration and frequency
of exposure to a chemical. This is also an
important consideration in developing
toxicity tests. There are basically four
types of exposure durations:
1. Acute
2. Subacute
3. Subchronic
4. Chronic exposure
Exposure Duration: Acute
1. Acute: Generally refers to an exposure
lasting less than 24 hours, and in most cases
it is a single or “continuous” exposure over a
period of time within a 24-hour period. For
example, a single oral exposure to 10 ml of an
organophosphate pesticide or the inhalation
of toluene in the air that we are breathing at
150 ppm over a period of 3 hours would
constitute examples of acute exposures.
Exposure Duration: Subacute
2. Subacute: Generally refers to
repeated exposure to a chemical
for a period of 1 month or less.
Exposure Duration: Subchronic
3. Subchronic: Generally refers to
repeated exposure for 1–3 months.
Exposure Duration: Chronic
4. Chronic exposure: Generally
refers to repeated exposure for
more than 3 months.
Toxicity Studies
• Toxicity studies are conducted for chemicals that
have the potential for public exposure; however,
the extent of the toxicity testing and therefore the
complexity of the study depend on several
considerations:
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The specific type of chemical hazard
How it is to be used
The projected levels of human exposure
The extent of its release into the environment
Toxicity Studies, cont.
• As you might anticipate, any study
involving chemicals such as food
additives, agricultural chemicals,
pharmaceuticals, and veterinary drugs
would undergo more extensive toxicity
testing than chemicals that have limited
use, perhaps in a specific industrial or
research application.
Toxicity Studies, cont.
• Toxicity testing using laboratory animals is
often the only initial means by which
human toxicity can be predicted and is
often the only acceptable means for safety
testing that satisfies certain regulatory
requirements.
Toxicity Testing
To be meaningful, any test for toxicity must
contain the following three stipulations:
– An appropriate biological model
– An end-point that can be qualitatively
and quantitatively assessed
– A well-developed test protocol
Meaningful Toxicity Testing
An appropriate biological model
• The model represents the system that is used for
evaluation.
• This may involve the use of whole animals (in vivo
testing) or an appropriate in vitro test system.
• When in vitro models are used, one should be
selected that best represents what is believed to
be occurring in the whole animal.
Meaningful Toxicity Testing
An end-point that can be qualitatively and
quantitatively assessed
• The measurement end-point is an appropriate
parameter that can be used to predict toxicity.
• Toxicological end-points are the biological responses
to chemical insult.
• They represent a measure of interaction between
toxicant and living system.
• The term toxicodynamics is sometimes used to refer
to this dynamic interaction.
• This end-point can be as crude a measure as lethality
or as subtle as a nonclinically detectable change in
cellular DNA.
Meaningful Toxicity Testing
A well-developed test protocol
• The test protocol is the schedule that
defines the conditions related to dosing and
time, and provides all experimental details,
including statistical methodology.
Toxicity Studies
• Toxicity studies using laboratory animals
thus provide a basis for:
– Understanding how a chemical may potentially
produce an adverse response in humans
– Demonstrating a range of exposure levels and
gradation of toxicity from no observable effects
to severe toxicity
– Justification for public health risk assessments
Toxicity Studies
• It must be recognized, however, that
although the intent of such studies is to
provide information that would be
predictive of effects in humans, responses
vary between animal species because of
anatomical, physiological, and biochemical
differences, and this limits to some extent
our confidence as to human applicability.
Extrapolation of Animal Results
to Humans
Equivalent Dose Levels
in Several Species
Acute Local Toxicity
• Irritation and corrosion tests are examples
of local tissue responses.
– The chemical being tested is applied to the skin
of the test animal and over a period of time,
generally hours to a few days, the skin is
examined for signs of inflammation.
– When these types of tests are performed on the
eyes, it is referred to as the Draize test.
Irritation and corrosion tests, cont.
– The ocular toxicity of irritants is determined by the
brief application of the substance to the eyes of
several test animals, which are usually rabbits.
– Examination of the eyes is conducted over a period of
3 days to assess for any injuries that may have been
produced to the conjunctiva, cornea, or iris.
– Substances have been demonstrated to produce a
range of effects from no observable reaction or simple
reversible irritation to severe irritation and corrosion.
Acute Local Toxicity, cont.
• Some chemicals have the potential to produce a
direct irritating/inflammatory skin response while
others may need to first be processed by
immunological sensitization.
– In the latter process, skin injury is not the direct effect
of the chemical on the skin, but rather an indirect
response from the release of mediators of
inflammation upon reexposure in the sensitized
individual.
– Rabbits are generally used for these types of studies.
Acute Local Toxicity, cont.
• To determine whether the chemical produces a
primary irritant response (contact dermatitis), the
substance is applied to the skin and any
changes are observed over the course of
several days.
• To assess for an immunological response,
guinea pigs are first treated with the chemical by
its topical application to the skin for several
hours (sensitization phase).
– There should be no inflammatory changes to the skin
over the course of a week or two.
– The substance is then reapplied to the skin (skin
challenge) and observations are made over a period
of one to several days.
Acute Systemic Toxicity
• Toxicological prechronic tests typically use
rodents of both sexes, over a period of either 24
hours (acute), 14 days (the subacute or 2-week
study), or 90 days (the subchronic or 13-week
study).
– A simple end-point measure used for many years is
the LD50.
– This is a dose (generally orally administered) that is
statistically derived from laboratory animals and
represents the dose at which 50% of the test animals
would be expected to die.
– In the late 1920s the LD50 test was developed as a
measure of the toxicological potency of chemicals
intended for human use such as insulin and digitalis.
Acute Systemic Toxicity, cont.
• The use of the test was expanded to one
that was generally recognized as an
acceptable in vivo animal surrogate to
rank chemical toxicity and became
accepted for regulatory purposes as an
important source of safety information for
new chemicals, including drugs,
household products, pesticides, industrial
chemicals, cosmetics, and food additives.
LD50 Curve
Efficacy, Toxicity, and Lethality
• For many chemicals that we intentionally
use, some benefit is derived from their use.
– For example, a prescribed medication is
anticipated to produce a beneficial effect if
properly taken.
– The level of benefit (efficacy) can also be
quantitatively measured; thus an ED50 would
represent the lowest dose that is beneficial
(efficacious) in 50% of the test population.
Efficacy, Toxicity, and Lethality, cont.
• It should be apparent that for a chemical
intended to produce some benefit to the
body at a certain dose, the likelihood of
some toxicity may also result from the
same chemical at some dose beyond
therapeutic.
– Any dose that results in a toxic end-point
(nonlethal) can be abbreviated TD.
– Thus, a TD50 would represent the dose of a
chemical toxic to 50% of the population.
Efficacy, Toxicity, and Lethality, cont.
• For chemicals that produce a beneficial
effect, e.g., a drug, a comparison of the
doses that produce efficacy and those that
produce toxicity can yield important
information regarding its safety.
Common Abbreviations of
Beneficial, Toxic, and Lethal Doses
Margin of Safety
• Perhaps a better designation to describe
the safety of a drug is that of the margin of
safety (MOS), which overcomes the
problem of any significant differences in
the response slopes between toxicity and
efficacy curves.
– The MOS represents the ratio of lethality at a
very low level (e.g., 1%) compared with
efficacy at the 99% level (MOS= LD1/ED99).
– The higher the value, the safer the drug.
Human Studies
• Toxicity information from human studies may come
from a number of sources:
– Case reports from individuals that have been accidentally
or intentionally poisoned
– Reported adverse reactions to drugs
– Clinical studies from various sized groups of individuals
that have been intentionally exposed to an investigational
chemical, such as a new pharmaceutical
– Epidemiological studies that attempt to determine
whether a causal relationship exists in a study population
that has been exposed to a substance that may produce
adverse health effects when compared with an
unexposed population that has been matched for such
factors as age, gender, race, and economic status.
Human Studies, cont.
• An example of such a study might be to
determine whether a greater incidence of
a specific disease (e.g., asthma) in a
community is associated with the
discharge of pollutants from a specific
geographical area.
Human Studies
Although epidemiological studies offer obvious
advantages over laboratory studies, there are
nonetheless a number of disadvantages:
– The tests are often expensive to conduct.
– Good quantification of exposures is frequently difficult.
– Large numbers of individuals are acquired for
meaningful statistical evaluation.
– Exposure quantification in humans is frequently
difficult because of simultaneous exposures to
multiple chemical, physical, and biological agents.
– Epidemiological studies generally require long periods
of time before information is made available through
the appropriate published resources.
Alternatives to Animal Testing
• Toxicologists as well as other scientists
who use animals for research and testing
purposes have been encouraged to
explore the “3R’s” of animal alternatives:
– Replace the animal with another appropriate
test.
– Reduce the total number of animals used.
– Refine the study to reduce the distress of
laboratory animals.
Alternatives to Animal Testing:
in vitro limitations
• The replacement of laboratory animals
with an appropriate in vitro test is often not
a viable option.
– Accepting an in vitro methodology as a
suitable surrogate for an in vivo test requires
its validation.
– The in vitro methodology must be
implementable by multiple laboratories, and
consistent results must be produced, that is,
the new methodology must be validated.
Alternatives to Animal Testing:
in vitro limitations
– Validation may be defined as a process by
which the credibility of a new test is
established for a specific purpose and its
reliability and reproducibility have been
verified by independent sources.
– Although a large number of in vitro tests are
available, most of them have not been
validated and are unacceptable for regulatory
purposes.
In Vitro Methodologies
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Mutagenicity and Chromosome Damage
Tumor Promotion
Cytotoxicity
Eye Irritation
Cardiac Muscle Toxicity
Nephrotoxicity
Hepatotoxicity
Endocrine Toxicity
Respiratory Toxicity
Reproductive Toxicity
Ecological Toxicity Tests
Websites
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Chemical Toxicity Database:
http://wwwdb.mhlw.go.jp/ginc/html/db1.html
National Toxicology Program:
http://ntp-server.niehs.nih.gov/
The Centers for Disease Control:
http://www.cdc.gov/
The Department of Health and Human Services:
http://www.hhs.gov/
The Environmental Protection Agency:
http://epa.gov/
The Food and Drug Administration:
http://www.fda.gov/ :
The National Toxicology Program
http://ntp.niehs.nih.gov/
U.S. Department of Labor Occupational Safety & Health Administration:
http://osha.gov/
U.S. FDA Center for Food Safety and Applied Nutrition:
http://www.cfsan.fda.gov/