Toxicity assessment methods include:

 Exposure assessment: Determines the type, magnitude, duration, and source of

exposure to a chemical, physical, or biological agent

  Quantification of toxicological effects: Measures the effects of a chemical exposure,

such as developmental delay
  Hazard identification: Discusses the toxic properties of a chemical
  Intrinsic toxicity evaluation: Determines the toxicity of a substance, such as a
medicinal plant, based on the presence of toxic compounds
  QSAR methodologies: Uses computational models to assess the toxicity of organic
chemicals
  Chemical analyses: Evaluates the toxicological profile of a substance to determine a
safe dose
  In-vitro toxicological assessment: Uses in vitro tests to assess the safety of a substance,
such as a food additive
  Risk assessment: Assesses the effects of a substance on population health

Toxicity assessment methods are important for determining the safety of substances, such as
food additives and medicinal plants.

Toxicity testing is a series of experiments that evaluate how chemicals affect living systems.
There are many types of toxicity tests, including:

 Carcinogenicity: Determines if a substance can cause cancer. These tests are similar
to chronic toxicity tests, but they last longer and involve more animals.

  Subchronic toxicity: Studies the toxicity of a substance on a large group of species over
a period of time that's usually shorter than chronic toxicity.
  Developmental toxicity: Determines if a substance can harm a developing fetus. These
tests look for structural and functional changes in the fetus from the zygote stage to birth.
  Genotoxicity: Determines if a substance can cause DNA damage, genetic mutations, or
chromosomal abnormalities. These tests help identify substances that could cause genetic
disorders or cancer.
  Reproductive toxicity: Determines if a substance can harm an organism's reproductive
system.
  Genetic toxicity: Determines if a substance can cause mutations in an organism's DNA.
  Immunotoxicity: Determines if a substance can cause an allergic reaction.

When designing a toxicity test, it's important to consider things like:

 Scientific basis: The test should be based on sound scientific principles

  Repeatability: The test should produce similar results when repeated in different
laboratories
 Sensitivity: The test should be able to measure the effects of a substance as precisely
as possible

Chapter 3
Toxicity Assessment Basics

Before there can be a risk assessment, there must be toxicity assessments.

Scientists use different procedures to assess toxicity, depending on
whether they are looking for carcinogenic or non-carcinogenic effects.
Awareness of these procedures will help reporters ask questions to clarify
the significance and relevance of a toxicity assessment.

Toxicity Assessment Overview

Toxicity assessment is a major component of risk
assessment. *
A toxicity assessment
provides an estimate of how
A toxicity assessment is a tool to investigate the much of a substance causes
potential for a substance to cause harm--and how much what kind of harm.
causes what kind of harm.

All substances are toxic in quantity. Many therapeutic

medications are acutely toxic, but beneficial when used at the appropriate level. Vitamin D,
table salt, oxygen, and water are toxic in quantity. Thus, the mere presence of a substance
does not automatically imply harm. This is why toxicity assessment is concerned with the
type and degree of harm caused by differing amounts of a substance.

There is no one measure of toxicity. Effects may occur in the short term (acute effects) or
after repeated exposures over a long time (chronic effects). They may affect only one part of
the body or many, and they may vary greatly in severity. **
 * * Other components are exposure assessment ( Chapter 2 ) and
hazard identification and risk characterization ( Chapter 1 ).

** **The term toxicity refers to the inherent potential of a

substance to cause systemic damage to living organisms. The term
hazardous is very different. It refers to the potential of a substance
to (1) cause any of several kinds of harm, through toxicity,
flammability, explosiveness, corrosiveness etc., and (2) the ease
with which people can come in contact with it. Hazardous is nota
synonym for toxic.

Toxic Effects
Toxic effects are classified as either acute or chronic.

 Acute effects happen very rapidly after a single There are two types of toxic
exposure has occurred (food poisoning, effects--acute and chronic.
breathing fumes from a chlorine spill).
Sweating, nausea, paralysis, and death are
examples of acute effects.
 Chronic effects happen only after repeated long-term exposure (cigarette smoking,
eating foods with low levels of contaminants, breathing polluted air). Cancer, organ
damage, reproductive difficulties, and nervous system impairment are examples of
chronic effects.

These chronic effects fall into two categories: carcinogenic effects andnon-carcinogenic
effects.

Examples of non-carcinogenic chronic effects:

 Organ damage: cirrhosis of the liver from long-term alcohol consumption;

emphysema from long-term tobacco smoking.
 Reproductive difficulty: decreased fertility from the pesticideDBCP
(dibromochloropropane).
 Nervous system impairment: mental retardation in people exposed to high levels of
lead during early childhood.
Assessing Toxicity
All quantitative toxicity assessments are
based on the dose-response concept: as
you increase the dose (exposure), the The dose-response concept is
response (toxicity) also increases. the basis for all toxicity
assessments. It is used
Scientists perform studies to determine differently to evaluate
acute effects and chronic
exactly how high a dose causes what effects.
kind of a response, or effect. The
smaller the dose needed to cause an
effect, the more potent (toxic) the
substance is.

For all compounds other than cancer-causing agents (carcinogens), it is assumed that
there is a dose below which no effect occurs (a threshold). This is similar to a drug
where too small of a dose has no beneficial effect.

For carcinogens, it is often assumed that even the smallest dose can cause an effect (
no threshold ).

Although the dose-response concept is used in all types of toxicity assessments, it is

used somewhat differently for each of them.

Acute toxic effects are estimated by LD50 studies or observation of accidental

exposures.

Chronic toxic effects are estimated by dose-response studies on animals.

Carcinogenic effects are estimated by a type of dose-response study called a
carcinogenesis bioassay .

Assessing Acute Toxicity

Most information about acute toxicity of

chemicals to humans comes from
accidental poisonings or exposures, such Acute toxicity is assessed
as drug overdoses or chemical spills. using observations of
Physicians/researchers know or estimate accidental human exposures
the level of exposure and observe and or by conducting LD50 tests
on experimental animals,
document the effects. usually rodents.

Scientists also use animal tests called

LD50 (L-D-fifty) studies to assess acute
toxicity. These studies determine the amount of a substance that will kill half the test
animals in 14 days. This amount is called the LD50--Lethal Dose for 50% of the
animals.

LD50 is stated in milligrams per kilogram (mg/kg): milligram of chemical per

kilogram of body weight. * The lower the LD50-the lower the lethal dose-the more
toxic the substance.

* The term LC50-Lethal Concentration-is used to measure the

toxicity of gases. The LC50 is stated in milligram of chemical per
liter (or cubic meter) of air.

Example: A reported "rat oral LD50 of 50 mg/kg" means that half of the rats that
ingested a dose of 50 milligrams of the substance per kilogram of body weight died
within 14 days.

LD50 values are unknown for humans,

since LD50 experiments are not
conducted on humans. If a LD50 LD50 values are unknown for
statement is applied to humans, it must humans, but animal LD50
in actuality be either: values can be used to
estimate lethal amounts for
humans.
o an animal LD50.
o the "average lethal dose" (ALD),
calculated from the effects of
accidental poisonings and exposures. Also called mean lethal dose (MLD).
o the "lethal amount" calculated from an animal LD50. This calculation is done
by multiplying the LD50 by a number representing average human weight.

Example of lethal amount: If the LD50 is 50 mg/kg: The lethal amount for a child
would be 50 mg/kg times 10 kg, which equals 500 mg (about 1/8 tsp.)
The lethal amount for an adult would be 50 mg/kg times 70 kg, which is 3,500 mg
(about 3/4 tsp.) *

* The 10 kg and 70 kg in the example above come from the weight

of the "average" person-10 kg (22 lbs) for a child; 70 kg (154 lbs)
for an adult.
Assessing Chronic Toxicity

Chronic toxicity can be divided

into two categories:
Chronic toxicity is measured
o cancer (carcinogenic toxicity). in two ways--depending on
whether the concern is
o all other effects (non- cancer or other chronic
carcinogenic toxicity). effects.

Cancer is in a separate category because

public concern about it is so great.
People want to know if even one person in a million persons who are exposed to a
substance will get cancer. To discover this, researchers conduct a specific type of
dose-response study--a carcinogenesis bioassay .

Non-carcinogenic effects are usuallyassessed with a different type of dose-response

study.

Non-Carcinogenic Assessment

Introduction

Scientists assess non-carcinogenic

chronic toxicity by administering
varying amounts of a substance (dose) Non-carcinogenic chronic
to laboratory animals and noting the toxicity is assessed by
effects (responses), if any, at each dose. studies to determine the
smallest dose that causes
any detectable effect.
Essentially, the scientists look for the
smallest dose that causes any detectable
effect. This smallest dose is called the
Lowest Observable Effect Level (LOEL). *

To conduct these dose-response studies, scientists:

o Administer different small doses of a substance to several groups of test

animals every day over a lifetime.
o Periodically examine and finally autopsy the animals to determine if any
effects have occurred. The effects may be:
 damage to an organ,
 behavioral modifications,
 change in the level of an essentialbody chemical.
o Determine the smallest dose at which an effect occurs--the Lowest Observable
Effect Level (LOEL). **
o LOEL is measured in milligrams (mg) of substance per kilogram (kg) of body
weight, or in parts per million (ppm) of substance in food.
 * In addition to LOEL, the term LOAEL (Lowest Observable
Adverse Effect Level) is sometimes used. The term LOAEL implies
a judgement that the effect is adverse. A LOEL refers to any effect
and may or may not be judged to be adverse.

** This dose may still be a high dose compared to environmental

exposures.

Determining Safe Levels

When performing the experiments just

described, scientists also determine the
highest dose at which no effects occur-- To protect the public,
the No Observable Effect Level (NOEL). scientists also determine
the highest dose at which no
* effects occur.

The NOEL is considered the "safe level"

for that chemical in the species studied.

The NOEL is not necessarily the "safe level" for humans, because:

o humans may be more/less sensitive to the substance than the animals studied.
o humans have more genetic, health, age, and other variabilities, which may
affect individual human reactions. **
To account for these differences, public health officials divide the NOEL by a safety
factor, usually 100, to arrive at a presumed "safe level" for humans. If the NOEL for a
substance were 100 mg/kg, the "safe level" for humans would be considered 1 mg/kg.

This "safe level" is most likely lower than scientists' best estimate of the NOEL in
humans. However, it is the number risk managers use to establish regulations, such as
the maximum amount of a chemical allowed in drinking water, and to create
guidelines such as fish consumption advisories.

* The term No Observable Adverse Effect Level (NOAEL) is

sometimes used. The term NOAEL implies a judgment that the
effect is adverse. A NOEL refers to any effect and may or may not
be judged to be adverse.
 ** Lab animals are bred to be similar to one another, and an
experiment will be conducted on animals of the same age and
health.

Human Sensitivity and Variability

The "safe level" calculation for humans

assumes that humans are more sensitive
than animals, but humans are not more The "safe level" calculation
sensitive in all cases. Dividing the for humans assumes that
NOEL by a safety factor assumes that humans are more sensitive
humans are more sensitive than animals. than animals, but humans are
not more sensitive in all
But humans are not always more cases.
sensitive. For some substances, they
may be less sensitive, or less sensitive
than some species.

This variation is usually due to the different degrees and rates of absorption,
metabolism, and/or excretion of the substance by the different species.

Although it might be reasoned that the most humanlike animals-monkeys-would be

the best test animals, they re-act to some substances more differently from humans
than other animals. For example, dogs react to nitrobenzene sim-ilarly to humans,
while monkeys do not.

Knowledge is still incomplete regarding the best test animals for different types of
substances and whether humans can be expected to be more or less sensitive to any
particular compound.

Examples of human sensitivity:

o More sensitivity: The drug Thalidomide caused no adverse effects in the

animals studied, but caused severe birth defects in humans.
o Less sensitivity: Insecticides are often developed to be more toxic to insects
than to humans. Since people are less sensitive to these chemicals, they can
use them without injuring themselves.
o Genetic variability: People vary widely in their reactions to bee venom. Some
show almost no reaction to a bee sting; others may die without immediate
medical treatment.

Carcinogenesis Bioassay

Introduction
Scientists assess carcinogenic toxicity
very differently than they assess non-
carcinogenic toxicity. This is in Officially accepted methods
response to public fear about cancer. of assessing carcinogens
assume there is no safe
People want to know if even one in a level.
million individuals will get cancer from
exposure to a suspected carcinogen. To
find this out with any degree of
confidence by traditional dose-response studies, scientists would have to use several
million test animals. The impracticality of such experiments has led to the
development of the carcino-genesis bioassay. *
With a carcinogenesis bioassay, scientists are not looking for the safe level of
exposure (NOEL). Rather, harm is assumed, and they are looking for the incidence, or
risk, of harm.

* The carcinogenesis bioassay is a method of testing substances for

carcinogenic effects that utilizes high-dose studies on laboratory
animals to look for even the rare case of cancer. It is not necessarily
the best scientific approach to assess the carcinogenic effects of
chemicals. Instead it is a way to respond to public concerns by
generating carcinogenic risk values with large margins of safety.

Methodology

Scientists assess carcinogenic toxicity

by feeding large doses of the substance
in question to animals in an effort to To measure carcinogenic
find even the rare case of cancer toxicity, scientists try to
resulting from exposure to it. find even the rare case of
cancer.
o Test animals are administered
different large doses of a
substance daily over a lifetime
(24-30 months in rats).
o At the end of the study, the animals are examined to see if cancer can be
found.
o If cancer is found, scientists use available data and mathematical models to:
 estimate the cancer incidence at the lower doses more likely to occur in
the environment.
 estimate the effect of the size and sensitivity differences between the
test animals and human beings.
Example of a carcinogenesis bioassay: A carcinogenesis bioassay was performed
for benzene on both rats and mice. Both sexes of each species got leukemia at the
high doses administered. Extrapolating the cancer incidence at high dose to low dose
and from rodents to humans resulted in the risk estimate that a benzene dose of 1
mg/kg/day will result in 3 cancers per 100 people exposed daily for a lifetime to that
dose. This dose is much higher than anyone would be exposed to in the environment
under normal conditions.

Mathematical Models Vary

A mathematical model is a set of

equations that mimic a real situation and
predict what will happen under different The choice of model has a
circumstances. strong influence on the
outcome of the study.
In toxicity assessments, scientists do not
know what will happen to humans
exposed to the low doses found in the
environment. So models are developed to apply information gained in animal studies
to the human condition. Many educated assumptions must be made in developing
these models. *
The choice of model will have a strong influence on the outcome of the toxicity
assessment, because when scientists apply different models to identical data, they will
get different results.

Non-Threshold vs. Threshold Models

Two fundamentally different types of

mathematical models are applied to
carcinogenic risk assessment. One is Public health officials
called a non-threshold model and is generally rely on non--
based on the assumption that even one threshold models--models
molecule of a cancer-causing agent can that make worst-case
assumptions.
lead to the disease. This type of model is
also referred to as a "one-hit" model.

The second type of mathematical model is called a threshold model and is based on
the premise that repeated exposures to a chemical are needed before a threshold of
exposure is reached and cancer follows.

Scientists in regulatory agencies generally use non-threshold models for

carcinogenesis bioassays. These assign to a substance a higher estimate of cancer
potency than would threshold models. (However, the threshold model is currently
used to assess risk of all non-carcinogenic chemicals.)
 * These assumptions regard: similarities and differences between
animal and human reactions; the effects of genetic, age, health, and
other variations in humans; and whether only one molecule or many
molecules of a carcinogen are sufficient to start a cancer process
under appropriate conditions (see next page). Since not all
carcinogens work the same way, a particular type of model may
give a fairly realistic risk value for some, but a very unrealistic risk
value for others.

Role of Toxicity Assessment

In the end, a toxicity assessment

provides information on how much of a
chemical causes what kind of harm. The risk assessor estimates
real world risk by combining
If the toxicity assessment is based on an information on toxicity and
animal study, the degree of harm to exposure.
humans must be extrapolated using
mathematical models based on a variety
of assumptions. Thus the toxicity
assessment provides only an estimate of the harm to humans.

As more toxicity studies on a particular chemical are conducted-dose-response studies

on different species of animals for example, or epidemiological and in vitro (test tube)
studies-scientists become more confident in their characterization of the toxicity of
the substance.

The risk assessor's job is to determine the real world risk to humans of a substance by
combining information on toxicity and exposure. This job is made more complicated
if data are collected from many different studies, but the results will be more likely to
reflect the best estimates scientists can make.

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