Doses.pdf

Chapter 4
The Dose-Response
Relationship

Introduction
• The need to establish some criteria to base the relative safety of
chemicals is essential.
• Several methods are used to obtain data in order to establish safe
levels and provide information about the relative toxicity of
chemicals.

Acute Toxicological Studies (1)
• Information concerning the toxicity of a substance is obtained
primarily from either acute or chronic toxicity studies.
• Acute toxicity studies are the most commonly performed studies for
obtaining information on the effects of chemical exposure. They are
short-term, relatively inexpensive tests with death of the test animal
being the useful observed effect.
• Information obtained from acute toxicity tests can be used to
– determine the relative toxicity of different chemicals comparing the respective LD50
values, which are the doses that prove to be lethal for 50 percent of the test animals;
– identify the target organs (heart, liver, kidneys, etc.) that are affected as a result of
exposure; and
– determine the appropriate doses for long term, chronic studies.

• To perform acute toxicity studies the test animal population is divided
into several groups, with an equal number of individuals (usually 10 to
50) in each group. One group of test animals –referred to as the
control group – is exposed to all the same experimental conditions
except exposure to the toxic substance.
• The groups are observed for 14 days, and the number of deaths in each
group is recorded.

• LC50 is a measure of chemical toxicity resulting from inhalation. LC
means “lethal concentration” and the subscript has the same meaning as
described previous for LD. The unit of measure for the LC50 is usually
expressed as part per million (ppm).

Chronic Toxicological Studies (1)
• Chronic toxicity tests may be performed over a period of months, years,
or the lifetime of the test animal. Doses of the toxic substance are
selected to assure that most of the animals will survive the entire time
the study is performed.
• Different species of test animals may be more or less sensitive to the
same toxic chemical.
• Males and females of the same species may respond differently to the
same substance.
• Although acute and chronic studies provide useful information in
evaluating chemical toxicity, it is important to understand that they are
not truly representative of the environment to which people are exposed
in everyday life.

Chronic Toxicological Studies (2)
• Characteristics of animal studies
– A single dose
– A single route of exposure
– The number of animals exposed is small.
– The genetic make-up of the population is not very diverse.
– Only individuals in good health and/or of the same sex are selected.
• In reality
– Several different routes
– Exposure to more than one chemical at a time
– Greater genetic variability in a larger population – the range of
responses to a given chemicals more diverse.
– The collective interactive effect of all these factors on toxicity
makes it difficult to establish a cause-effect relationship.

Dose-Response Curves (1)
• The major purpose for performing acute and chronic toxicity studies is
to establish a cause-effect relationship between exposure to a toxic
substance and an observed effect in order to determine a safe exposure
level.
• A curve can be drawn that illustrates the relationship between the dose
administered and the observed response. This curve is referred to as
the dose-response curve.
• A dose-response curve can be developed form most chemicals. From
these curves the threshold level and the relative toxicity of chemicals can
be obtained to help establish safe levels of chemical exposure.

• The threshold is the dose below which no effect is detected
or above which an effect is first observed.
• The threshold information is useful information in
extrapolating animal data to humans and calculating what
may be considered a safe human dose for a given toxic
substance.
• The threshold dose (ThD0.0) is measured as mg/kg/day. It
is assumed that humans are as sensitive as the test animal
used. To determine the equivalent dose in man the ThD0.0
is multiplied by the average weight of a man, which is
considered to be 70 kg.

• The calculation used to determine the safe human dose
(SHD) is as follows:
substance
toxic
of
mg/day
Amount
SF
(kg)
70
ThD
SHD 0.0



Where
SHD = Safe Human Dose
ThD0.0 = Threshold dose at which no effect is observed.
70 Kg = Average weight of a man
SF = Safety factor (ranges from 10 to 1000, which varies
according to the type of test and data used to
obtain the ThD0.0.

Terminology Associated with
• No observed effect level (NOEL)
– The highest tested dose of a substance that has been
reported to have no harmful (adverse) health effects on
people or animals.
• No observed adverse effect level (NOAEL)
– It denotes the level of exposure of an organism, found by
experiment or observation, at which there is no
biologically or statistically significant (e.g. alteration of
morphology, functional capacity, growth, development
or life span) increase in the frequency or severity of any
adverse effects in the exposed population when
compared to its appropriate control.

• lowest-observed-effect-level (LOEL)
– Lowest concentration or amount of a substance, found
by experiment or observation, that causes any alteration
in morphology, functional capacity, growth, development,
or life span of target organisms distinguishable from
normal (control) organisms of the same species and
strain under the same defined conditions of exposure
• lowest-observed-adverse-effect-level (LOAEL)
– Lowest concentration or amount of a substance, found
by experiment or observation, which causes an adverse
alteration of morphology, functional capacity, growth,
development, or life span of a target organism
distinguishable from normal (control) organisms of the
same species and strain under defined conditions of
exposure.

• Frank-effect level (FEL)
– Level of exposure that produces unmistakable and
irreversible effects (such as impairment or mortality)
at statistically significant increased severity or
frequency.
– The level where overt effects are observed is referred to
as the Frank-effect level.

Threshold Dose
• Dose below which no adverse effects are observable in a
population of exposed individuals.
• Threshold dose approximated by a NOAEL (No Observed
Adverse Effect Level)

Non-threshold Effects
• Thresholds are assumed not to exist for genotoxic
carcinogens

Safe Dose for Threshold Effect
• NOAEL approach
– Identify the adverse effect that occurs at the lowest dose
– Determine the NOAEL or LOAEL for that endpoint
– Divide NOAEL/LOAEL by uncertainty factors

Acceptable Daily Intake
• ADI estimated (maximum) amount of an agent, expressed
on a body mass basis, to which a subject may be exposed
over his lifetime without appreciable health risk (also TDI,
tolerable daily intake)
• ADI derived from NOAEL by the use of uncertainty
factors UFs (or safety factors SFs)

Reference Dose (RfD)
• RfD is an estimate of the daily exposure that is likely
to be without appreciable health effect even if
continued exposure occurs over a lifetime.

Uncertainty Factors
• Default values:
UFinterspecies = 10 , UFhuman variability = 10

Additional Uncertainty Factors
• Sometimes applied:
– UFLOAEL-NOAEL = 3 or 10
– UFsubchronic-chronic = 3 or 10
– UF database insufficiences = up to 10
• MF, modifying factor for professional judgement: up
to 10
• RfD = NOAEL (or LOAEL) / UF1 UF2 UF3 MF

Benchmark Dose Approach (1)
• NOAEL approach uses only single points, shape of
dose-response is ignored.
• Bench mark dose (BMD) is calculated from the curve
fitted to the dose-reponse data, so all information is
used.
• BMD can only be used when available data are
suitable for modelling.
• Not replacement for NOAEL, but additional tool

Benchmark Dose Approach (2)
1. A mathematical model is applied to the
experimental data to produce a dose-response curve
of best fit.
2. By statistical calculation an upper 95% confidence
limit of the curve is determined
3. The Benchmark Response is defined as 10% (or 5%,
or 1%).
4. The Benchmark Dose corresponds to the bench
mark response on the upper confidence limit curve.

Non-threshold?
• Absence of a detectable effect at low doses is either
because the dose is below a threshold or because the
response is below the level that can be detected by the
test sytem.

Threshold or Non-threshold
• Presence of a threshold cannot be proven from
experimental data.
• Conclusions about existence of a threshold are based
on biological plausibility and expert judgement.
• Genotoxic effects (DNA dammage leading to cancer)
are thought to be possible at any level of exposure, so
no threshold.

Low-dose Extrapolation
• Dose-response curve is use to extrapolate to doses lower
than then the experimental region.

Calculated Risk or Safe Dose
• Extrapolation may be used to calculate:
– the risk associated with a known intake.
– intake associated with a de minimis risk, e.g. an
increased life time risk of developing cancer of 1
in 106 (‘virtually safe dose’, VSD).

Linear Low-dose Extrapolation
• Assumption: linear relationship between dose and
biological response

Linear Extrapolation from LED10
• LED10
– low confidence limit of dose with response of 10%

From LED10 to VSD
• If LED10 = 57 mg/kg/day (response 10% or 0,1)
• then VSD (virtual safe dose, response 1 in 106 or 10-6)
= 57 x 10-6 / 0,1 = 5,7 x 10-4 mg/kg/day

ALARA Approach
• For genotoxic carcinogens (non-threshold effects)
also the ALARA-approach may be followed:
exposure to be reduced to as low as reasonably
achievable (ALARA) or as low as reasonably
practical (ALARP)

Relative Toxicity (1)
• A comparison of the dose-response curves for two or more different
chemicals – administered to the same type of test animal – can be used
to determine the relative toxicity of the chemicals.
• The LD50 for chemical A is less than the LD50 for chemical B.
Therefore, chemical A is considered more toxic than chemical B.

• Chemical A is more toxic than chemical B at the higher
doses, but at the lower doses chemical B is more toxic than
chemical A.

• Relative toxicity also be evaluated by comparing the slope
of each dose-response curve. A steep dose-response curve
is indicative of a chemical that is rapidly and extensively
absorbed and slowly detoxified: therefore, the chemical is
more toxic.

Variables Influencing Toxicity
• These factors include:
– the dose-time relationship,
– the route of exposure,
– the physical and chemical properties of the toxic substance,
– the chemical interactions,
– the interspecies and intraspecies variability,
– genetics,
– age,
– sex, and
– health of the individual collectively.
• The toxicity of a given substance is the net result of the
complex interaction between all of these various factors.

Dose-Time Relationship
• Not only is the dose of a toxic substance important in
determining toxicity, but so is the frequency and length of
exposure.
• The rapid elimination of the substance from the blood
stream decreases the probability of an adverse effect.
• Gradual accumulation increases the probability that an
adverse effect will occur.
– Symptoms of acute exposure to sublethal quantities of chlorinated
solvents, such as chloroform or carbon tetrachloride, are primarily
associated with the nervous system, such as excitability, dizziness
and narcosis.
– Chronic exposure to these solvents is primarily associated with
liver damage.

Routes of Exposure
• LD50 values for various organochlorine and
organophosphate pesticides resulting from ingestion and
skin exposure.
• The different absorption rates for various regions of the
skin in the human male

Physical and Chemical Factors
• Chemical toxicity can be affected by the arrangement of
the atoms in a molecule.
• The structural differences can result in different toxic
effects being observed as a result of exposure.
– The LD50 for 1,1-dichloroethylene is 5,750 mg/kg and the LD50 for
the 1,2-dichloroethylene isomers is 770 mg/kg.
– Based on animal studies, 1,1-dichloroethylene is a suspected human
carcinogen. Its structure is similar to a known human carcinogen,
vinyl chloride.
– The 1,2-dichloroethylene isomers are not carcinogenic, but they do
have different effects. The cis isomer is an irritant and acts as a
narcotic (causes drowsiness). The trans isomer affects the central
nervous system (brain and spinal cord) and the gastrointestinal
system causing weakness, tremors, cramps, nausea; it may also
cause liver and kidney damage.

Interspecies and Intraspecies Variability
• Interspecies variability
– The variability in response between species is primarily the result
of the evolution of different biological mechanisms involved in the
metabolism of toxic substances.
– The differences in toxicity may be due to the rate at which
metabolism occurs, or whether metabolism produces chemical
intermediates more toxic than the parent compound.
• For example, the pesticide malathion is metabolized at a different rate in
mammals than in insects. In mammals, the chemical intermediates are rapidly
metabolized to deliver end products that are excreted from the body. In insects
metabolize malathion more slowly and produce a chemical intermediate ---
malaoxon --- that is toxic to the insect.
• Methanol intoxication results in ocular damage and blindness. However, in
other species methanol is less toxic and the symptoms associated with human
intoxication are not observed.
• Rats do not have a vomiting reflex. Rodenticides, such as squill, are more
effective if they are ingested because the rat can not discard the substance by
vomiting.

Intraspecies (individual) variability
• Individuals within a population ---exposed to a single dose
--- will demonstrate a wide array of responses.
• This type of variability can be caused by differences
associated with individual genetics and sex of the
individuals within the population.

Genetics
• The differences in genetics makeup both within species and
between species markedly influence the disposition and the
metabolism of toxic substances.
– For example, some individuals have an enzyme (glucose-6-
phosphate dehydrogenase) deficiency resulting in their red blood
cells being more fragile than normal. Consequently, red blood cells
of these individuals are more susceptible to chemicals such as
phenylhydrazine and primaquine, which cause hemolysis.
– The enzyme aryl hydrocarbon hydroxylase (AHH) transforms the
chemical benzo[a]pyrene to its carcinogenic metabolite. Some
individuals produce lower amounts of AHH; benzo[a]pyrene is
therefore less toxic to these individuals. Similarly, low AHH
individuals may be less likely to develop cancer from cigarette
smoke.

Age
• In general, younger and developing individuals are more
sensitive to toxic substances than older individuals. The
difference can be primarily attributed to differences in
metabolisms; specially to differences in the functioning of
the liver.
• In younger individuals the mechanisms for detoxifying
substances are less developed than in older individuals.
However, some substances are less toxic to the young than
to adults.
• The lead absorption rate is four to five times greater in
young individuals than in adults; the cadmium absorption
rate is 20 times greater than in adults.

Sex
• The difference in toxicity between genders seems to be
primarily influence by the sex hormones (estrogen and
testosterone), which can affect metabolism.
• Some organophosphate pesticides are more toxic to
females than males.
– Parathion is metabolized more rapidly in females resulting in a
higher concentration of the more toxic intermediate, paraoxon.
– Male rats are 10 times more susceptible to liver damage than
female rats as a result of chronic oral exposure to DDT.

State of Health
• The liver and the kidney are important organs for
detoxifying and removing toxic substances. Therefore,
conditions that lead to liver or kidney disease enhance the
toxic effects of substances normally detoxified by these
organs.

Chemical Interactions (1)
• The interaction between two or more chemicals and their
subsequent effects have not been widely studied; not
because these interactions are not important, but rather
because the results are difficult to interpret and
extrapolate, and also because the studies are longer and
costlier.
• The presence of other chemicals at the time of exposure or
as a result of previous exposure can affect the response to
the specific toxicant being studied. These effects can be
additive, synergistic, antagonistic, or they may
demonstrate the characteristics associated with
sensitization or potentiation

• Additive effects
– An additive effect occurs when the combined effect of
the two chemicals is equal to the sum of the effects of
each individual chemical.
• Synergistic effect
– A synergistic effect occurs when the observed effects are
greater than what would be predicted by simply
summing the effects of each individual chemical.

• Antagonistic effects
– Antagonistic effects are the basis on which antidotes are
developed to counteract the effects of toxic substances
that enter the body.
– Antagonistic effects occur when the sum of the effects of
each individual chemical is less than the sum of the
effects for both chemicals.

• Four major types of antagonism
– Functional antagonism
• It refers to a type of reaction two chemicals have on a specific
physiological function in the test organism, usually producing
counterbalancing or opposite effects.
– Chemical antagonism
• It may be referred to as chemical inactivation. This type of
interaction is the result of a reaction between two chemicals to
produces a less toxic product.
– Dispositional antagonism
• The toxic substance is either transformed into a less toxic
substance or it is eliminated from the body more rapidly, then
the observed effects will be less than the expected ones.

– Receptor antagonism
• It occurs when two chemicals require binding to the same
receptor in order to exert their effect. The result is that when
the two chemicals are administered together the observed effect
is less than the sum of the effects for each chemical.
– Sensitization
• Initial exposure to toxic chemicals does not always result in an
observable or measurable effect. However, subsequent
exposure to the same chemical may result in an observable or
measurable effect.
• Sensitization occurs as a result of the interaction of the body’s
immune system with the toxic chemical during the initial
exposure.

– Potentiation
• At first glance it would appear that synergism and potentiation
are the same. In A potentiation, only one of the chemicals has
an observable effect when administered alone.
• Chemical A has no effect on mortality (0%). Chemical B
causes 25% mortality. When the two chemicals are
administered together the effect is greater than the sum of their
individual effects (0% + 25% = 100 %).

LD50 of
Chemicals
Substance Animal, Route LD50 Reference
Sucrose (table sugar) rat, oral 29,700,000,000 ng/kg [7]
Vitamin C (ascorbic acid) rat, oral 11,900,000,000 ng/kg [8]
Grain alcohol (ethanol) rat, oral 7,060,000,000 ng/kg [10]
Melamine rat, oral 6,000,000,000 ng/kg
Table Salt rat, oral 3,000,000,000 ng/kg [12]
Paracetamol (acetaminophen) rat, oral 1,944,000,000 ng/kg [13]
Metallic Arsenic rat, oral 763,000,000 ng/kg [15]
Aspirin (acetylsalicylic acid) rat, oral 200,000,000 ng/kg [17]
Caffeine rat, oral 192,000,000 ng/kg [18]
Cadmium oxide rat, oral 72,000,000 ng/kg [22]
Nicotine rat, oral 50,000,000 ng/kg [23]
Strychnine rat, oral 16,000,000 ng/kg [24]
Arsenic trioxide rat, oral 14,000,000 ng/kg [25]
Metallic Arsenic rat, intraperitoneal 13,000,000 ng/kg [26]
Sodium cyanide rat, oral 6,400,000 ng/kg [27]
Beryllium oxide rat, oral 500,000 ng/kg [30]
Aflatoxin B1 (from Aspergillus flavus) rat, oral 480,000 ng/kg [31]
Dioxin (TCDD) rat, oral 20,000 ng/kg [33]
Polonium-210 human, inhalation 10 ng/kg (estimated) [37]
Botulinum toxin (Botox)
human, oral,
injection, inhalation
1 ng/kg (estimated) [38]
Ionizing radiation human, irradiation 6 Gy

Typical Costs of Various Types of Toxicity Tests

Typical Testing protocols for Acute and Chronic
Toxicity Studies

LD50 Values of Organochlorine Pesticides

LD50 Values of Organophosphate Pesticides

Regional Differences of Dermal Absorption in Human Male

Toxicity of various Compounds to
Different Test Species

No-effect Levels on
a Dose-Response Curve

Acute Toxicity of N-hydroxy
acetylaminofluorene in Various Strains of Rats

Five Threshold Doses
• 美國環境保護署（EPA）對動物毒性實驗的反應程度確立了五種界限值（threshold），
用以評估毒性強弱而發展出各類毒性物質的每日允許攝取量。這五個界限值為：
• 一 ﹑沒有可觀察到的影響值（no-observed-effect level，NOEL）。
• 二﹑沒有可觀察到的不良影響值（no-observed-adverse-effect level，NOAEL）。
• 三﹑可觀察到有影響的最低值（lowest-observed-adverse-drrect level，LOEL）。
• 四﹑可觀察到有不良影響的最低值（lowest-observed-adverse-effect level，LOAEL）。
• 五﹑有直接顯著影響值（frank effect level，FEL）。
• 美國是以NOAEL值經過體重權衡後，除以安全因素而得到每日允許攝取量。而這一限
值就是表示，對設定毒性物質可允許它每天進入體內之總和量。
•
•
圖片連結-各種劑量反應關係各種劑量反應關係圖片連結-各種劑量反應關係

LOEL
• 最低作用值（LOEL，Lowest-observed effect level）
• 是指在研究人體或動物接觸某種物質時產生任何反應的頻率或嚴重性在生物學上顯著
增加的最低劑量。相關名詞
• 最低作用值:不良效應級（有害作用劑量）：最低濃度或數量的物質，通過實驗或觀察
發現，這會導致不利的改變形態，機能，生長，發育，壽命或生物體的目標分辨正常
（對照）的生物物種和同一菌株在規定條件下的暴露。逆轉錄不利影響，最低的觀察
效果級別，不遵守，不利的效應級別，無觀測效應水平
• 在劑量反應曲線中，我們常常會應用到幾個相關名詞，包括：
• 無反應劑量（no-effect level, NEL）或無觀察反應劑量（no-observed effect level, NOEL
）：無顯著反應的最低劑量。
• 最低觀察反應劑量 (最低作用值)lowest observed effect level,LOEL）：可以觀察到反應
之最低劑量，不過此反應為次要之反應而非引起主要想要測量之反應。
• 無觀察危害反應劑量（no observed adverse effectlevel, NOAEL）：一些非欲觀察之毒性
反應（危害性低）發生之劑量，但在高劑量下觀察得到的反應，在此劑量並不會出現
。（無顯著危害的最高劑量，並非無反應而是與對照組無統計差異）
• 最低觀察危害反應劑量（lowest observed adverseeffect level, LOAEL）：第一個可以觀
察得到危害反應的最低劑量濃度。
• 法蘭克反應劑量（frank effect level, FEL）：引起嚴重危害反應發生之劑量稱之。訂定

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