Download 2_01 Food chemicals Toxicity Text

Chapter 2: Food chemicals – their toxicity
Content: Understanding the steps from intake of food chemicals, their metabolism, the observed
toxic effects, assessing the severity of effects in animals and humans.
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Absorption, distribution, metabolism, and excretion (ADME)
Toxicokinetics
Toxicodynamics (Effects)
Tests
Dose-response observations
Threshold vs non-threshold mode of actions
Examples of chemicals and toxicity endpoints
1. Absorption, distribution, metabolism, and excretion (ADME)
In order to assess the risk of a food chemical on humans it is of central importance to
understand some basic processes within the human organism: the absorption, distribution,
metabolism and the excretion of food chemicals.
Absorption
Bioavailability
First-pass effect
Nutritional state
There are different ways how a chemical compound can enter the human body: through
ingestion of food or water, inhalation via the lungs, epidermal through the skin, or through
injection (Greim, Snyder 2008). For the chemical risk assessment of food chemicals only the
adsorption of orally administered substances is of interest.Upon oral intake of food or drinks etc
a chemical compound can be absorbed from the oral cavity, the stomach, the small intestine
and the large intestine (Brimer 2011). The absorption can either be passive by diffusion etc. or
active as facilitated by a carrier under the use of energy in the form of ATP. In the following we
will look at two important factors for the absorption of food chemicals: the chemical speciation
of a substance and the nutritional state of the individual (EU_Hansen_ ADME). The former is
decisive for the bioavailability of a compound, thus the fraction of the administered dose, which
is systematically available (Greim, Snyder 2008). An example concerning bioavailability:
humans take up almost 100% of methylmercury whereas the uptake of metallic mercury is
limited. In order to understand the concept of bioavailability one must understand some basic
processes of human physiology concerning the ingestion of food. After ingestion the food will
first reach the stomach, where the food enters an acid environment and can thus be rapidly
hydrolysed if the food chemical is not stable in acid (Greim, Snyder 2008). This will result in a
reduced uptake of the genuine compound. After the stomach the chemical compounds reach
the intestine where they will be taken up via the intestinal walls and are transported to the liver,
i.e. before entering the systemic blood circulation the blood, from the gastrointestinal organs,
first flows via the portal vein to the liver. Any metabolism happing in the liver as a result of this
is called the “first-pass effect”(first-pass metabolism or presystemic metabolism) and usually
results in a partial removal of the administered/absorbed dosage. Because the chemical
compound thus is transformed before reaching the systemic blood circulation, the bioavailability
is reduced (Greim, Snyder 2008). Some substances also undergo a first biotransformation in
the intestinal walls of the gastrointestinal tract (Greim, Snyder 2008). The nutritional state of an
individual is especially of interest with regard to divalent metal ions like calcium, zinc and iron.
Iron deficiency thus increases absorption of cadmium, lead, and aluminum. Figure 1 shows an
overview of the different pathways for adsorption, distribution, metabolism and excretion of food
chemicals.
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Figure 1: Different pathways for adsorption, distribution, metabolism and elimination of food chemicals in the human
body. (Greim, Snyder 2008), page 20.
Distribution
Protein binding
Crossing of cell
membrane
Perfusion
Accumulation
After the absorption of the food chemicals into the blood the distribution within the body starts.
Generally it can be said: In order to harm or damage an organ the food chemical needs to reach
the respective organ. The main transport medium in the human body is the blood cells and the
plasma. The plasma contains a lot of proteins, to which chemical compounds can bind . The
degree of protein binding has been investigated thoroughly for many drugs and can vary
considerably. Alternatively a chemical compound can also bind to the erythrocytes (red blood
cells). An example: When the toxic heavy metal lead is introduced to the body, 99% of the lead
is bound to the erythrocytes, while only 1% is found in the plasma, where most of it is bound to
albumin (Brimer 2011). For the further distribution in the body the extent of protein binding is
crucial because only the unbound fraction can diffuse through the capillary wall into cells and
produce systemic effects (Brimer 2011). For the passive diffusion of the unbound fraction of
food chemicals through membranes it is furthermore of paramount importance whether a
chemical compound is hydrophilic or lipophilic. Hydrophilic compounds are highly water-soluble
and will in general distribute relatively slowly into the cells because they are transported rather
slowly through the phospholipid bilayer, which forms the biological membrane of tissues and
organs. Lipophilic substances, which are highly lipid-soluble chemical compounds, may cross
cell membranes more rapidly (Brimer 2011). Alternatively compounds can be filtered through a
membrane through pores in the double lipid layer due to hydrostatic or osmotic pressure
(Brimer 2011).
Beside the ability of the compound to cross a cell membrane, the blood flow to the organ in
question (the perfusion) is decisive for the rate at which unbound compounds will enter the
target organ (Brimer 2011). Thus, when trying to assess the distributin rate of a substance the
perfusion of organs or tissues needs to be accounted for. However the perfusion of tissues and
organs can vary considerably. There are well-perfused tissues like the liver, muscle and the
lungs, while the adipose tissue exhibits a more restrictive blood flow (Greim, Snyder 2008). Yet
the adipose tissue can store compounds effectively by dissolving them in the fatty matrix of the
tissue leading to accumulation of chemical compounds. The accumulation of chemical
substances can also take place in the bone marrow, or the peripheral or central nervous
system (PNS and CNS). The accumulation in the fat tissue or the bone marrow is, per se, no
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problem as long as the stored substances are not mobilized. However if fat is mobilized due to
starvation, the concentration of the substance in the plasma can rise drastically, which can lead
to acute intoxication (Brimer 2011). Examples of substances that accumulate in the fatty tissue
are chlorinated pesticides (e.g. DDT), pollutants (e.g. PCBs) or metal-organic compounds like
methylmercury (Brimer 2011).The accumulation of substances in the peripheral or the CNS on
the other hand is always an issue (Brimer 2011). In order to quantify the distribution of a
chemical compound radioactive tracers or microscopy can be used, thus lead accumulated in
the kidneys may be seen as protein-lead bodies in the cells. As an example of the tissue
dependent distribution of a compound Figure 2 shows the concentration of thiomebumal in the
plasma, the muscles, the brain and in lipids after intravenous injection as a function of time
(Brimer 2011).
Figure 2: Example for the concentration of thiomebumal in the plasma, the muscle, the brain and in lipids after
intravenous injection as a function of time. (Brimer 2011) page 37.
Physiological
barriers
However there are some important physiological barriers of toxicological importance that need
to be mentioned: the blood-brain barrier, the blood-testis barrier and the placental barrier
(Brimer 2011).
Metabolism
After the absorption and distribution of a substance in the human body, it is of great importance
to understand the metabolism (biotransformation) of a substance. After a substance has
entered the human body and is distributed it is usually metabolised by means of enzymecatalysed chemical reactions, which may alter the structure and reactivity of the compound
(Brimer 2011). Generally metabolism may occur in all cells of the body. However the main
detoxification pathway in humans (and most animals) is via the liver, which is the major
metabolising organ. Other important sites for metabolism are the kidneys, the lungs and the
skin. Metabolism reactions can be divided in two types: phase I and phase II reactions (Brimer
2011). Phase I reactions are generally degradation reactions, where a substance is oxidised,
reduced or hydrolysed. Phase II reactions are termed conjugation reactions. This kind of
reaction comprises the formation of a conjugate that is biosynthesized from the toxicant or from
one of its metabolites. Additionally an endogenous metabolite is involved to form the conjugate.
It is possible but not necessary that a phase I reactions precedes a phase II reactions. However
it is also possible that a substance undergoes only a phase I or phase II reaction before being
excreted. The polarity of the genuine compound determines among others which pathway
Metabolism
reactions
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prevails. The rate and the type of metabolism also depend on the species, age, gender,
environment, and the ingested food (Brimer 2011).
Excretion
Urine
Faeces
Lungs
Mammary glands
Toxicokinetics
There are different ways how the metabolised compounds can be excreted: via faeces, urine,
air, sweat or as constituents in hair, nails or dead skin cells (Brimer 2011). In order to be
released via urine the substance has to be (or be metabolised to) a water soluble structure and
pass through the kidneys. The functional units of the kidneys, the so called nephrons, in total
filter about a quarter of the cardiac blood output (about 180 litre) per day) for an average adult
person) and substances that are leaving the blood are excreted with the urine if not
reabsorbed in the tubuli (happens e.g. for small proteins, phosphorous etc. that are essential for
the body). Examples of compounds that are secreted by the kidneys are Penicillin G, atropine,
and quinine (Brimer 2011). The kidneys primarily excrete small and water-soluble molecules,
while the biliary excretion favours compounds with very high polarity, compounds bound to
plasma proteins, and compounds with high molecular weight. The bile is produced by the liver
and is then released to the small intestine. Some compounds may be reabsorbed from the small
intestine to the liver and again secreted into the bile (enterohepatic circulation). If a compound
does not take part in such an enterohepatic circulation it is readily excreted via the faeces.
Another excretion pathway is by air via the lungs. Pulmonary excretion is mainly used for
volatile metabolites of certain selenium compounds, for example of dimethylselenium (Brimer
2011); still another elimination pathway is via the mammary glands. Particularly slowly
metabolized, highly lipophilic compounds are eliminated by means of the mammary glands, as
are to a certain extend weak bases such as alkaloids (e.g. caffeine). The latter is due to the
slightly lower pH of mother’s milk (7.0-7.4) compared to that of the organism (7.4) (Greim,
Snyder 2008). The pH of cow’s milk is around 6.6. Many medicines (e.g. antibiotics) and
everyday drugs (e.g. nicotine, ethanol) as well as heavy metals are to a certain extent
eliminated with the milk. Thus special caution should be taken in order to spare breast-fed
babies form toxic influences (Greim, Snyder 2008). For all pathways a compound may remain
unchanged and leave the body by 100% while others are excreted as a mixture of the pristine
compound and metabolites (Brimer 2011).
2. Toxicokinetics
What is now still lacking is more detailed knowledge concerning i) the concentration-time
relationship of the chemical compound and its metabolites in different target and non-target
organs, ii) the mechanisms of toxicity on cellular, subcellular and molecular levels, and iii) a
description of the concentration-effect correlation for a given toxicant and its effects (Brimer
2011). The first point is covered in toxicokinetics where the uptake, the distribution, metabolism
and elimination of a food chemical is of interest. The second and the third point belong to the
toxicodynamics and will be discussed in the following chapter. In Figure 3 below the
differentiation between toxicokinetics and toxicodynamics is illustrated.
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Figure 3: Conceptual pathway of toxicokinetics and toxicodynamics (Wikipedia1).
Rout of
administration
Because we only discuss food chemicals we will focus on the orally administered compounds.
Yet it is important to mention that the concentration-time relationship of a chemical compound
within the body strongly depends on the rout of administration (Brimer 2011). Figure 4 shows an
example of how the time-blood concentration curve may look for a given compound for different
kinds of administration-
Figure 4: “Plasma concentration as a function of time after intravenous injection (IV), intramuscular injection (IM),
subcutaneous injection (SC), or dermal (D, percutaneous) or oral (PO, per os) administration of a hypothetical
compound with an adsorption fraction of 100%.” (Brimer 2011) page 54.
Enterohepatic
circulation
For the orally administered compounds it is important to account for the “first-pass effect”. As
discussed before the primary compound may undergo biotransformation in the intestinal wall or
the liver before they reach the general blood circulation. This is due to the fact that the blood
from the gastrointestinal organs first reaches the liver via the portal vein before entering the
systemic blood circulation (see Figure 5). After reaching the general circulation fractions of the
chemical compound still will a re-enter the liver. (Fig. 5).
1http://en.wikipedia.org/wiki/File:Diagram_showing_the_conceptual_pathway_of_toxicokinetics_and_toxicodynamics.pn
g#mediaviewer/File:Diagram_showing_the_conceptual_pathway_of_toxicokinetics_and_toxicodynamics.png
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Figure 5: “Routes of exposure and systematic distribution of a compound within the organism.” (Greim, Snyder 2008)
page 9.
Concentrationtime relationship
First-order
reaction
In toxicokinetics we try to assess the concentration-time relationship of a compound in different
organs/tissues including the blood.. For convenience The concentration is usually measured in
the blood/bloood plasma and is then plotted over time in an x-y-graph. The shape of the curve
for this concentration-time relationship strongly depends on both the chemical structure and the
physico-chemical properties of the compound (Gupta 2007; Brimer 2011). Normally for the
adsorption and the elimination of a compound first-order reactions (kinetics) are assumed. A
first-order chemical reaction depends on the concentration of one reactant and the rate is
proportional to the amount of this reactant. The concentration of the reactant over time usually
varies between different tissues and organs (Brimer 2011).
3. Effects (Toxicodynamics)
Adverse and
non-adverse
effects
After looking at the the fate of a given chemical compoun in the human body we will now look at
the effects of this chemical in the body, the toxicodynamics. Effects of chemicals on the human
body include changes in the morphology, physiology, growth, development, reproduction or life
span of an organism (EU_Sharma_Intro_risk_analysis), and are differentiated in non-adverse
and adverse effects. Many fluctuations in enzyme levels or other biochemical parameters, a
decrease in body weight or gain due to palatability of feed, some discolorations of organs or
tissue, or other effects with no statistical significance will be regarded as non-adverse..
Examples for adverse effects are cancer, damage on organs, damage on DNA (genotoxicity),
damage on the central nervous system (CNS) (neurotoxicity) or damage on the reproductive
system. However not all damage on organs, DNA or the CNS is irreversible (Greim, Snyder
2008). There is still the possibility that the damage is repaired or that the affected cells die
without giving rise to any further disturbance (apoptosis).
In the case of primary DNA damage the cell may still repair this. If the cell is not repaired and
undergoes division mutation occurs. At this point the mutated cell can still be repaired or die
(apoptosis). If neither occurs there are different consequences of a DNA damage, which may
either concern somatic cells or germ cells. Any cell that forms the body of an organism is a
somatic cell with the expectation of germ cells, which give rise to gametes and thus the
offspring of an organism that reproduces sexually. Damage in somatic cells can lead to cancer,
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Genotoxicity
Somatic cells
Germ cells
Genotoxic and
non-genotoxic
carcinogens
Example
genotoxic: PAH
premature ageing, cardio-vascular diseases or damage of the immune system. Cancer is
initiated if tumour suppressor genes are affected. Damaged DNA of germ cells on the other
hand can lead to heritable diseases, malformation or decreased fertility. Genotoxic effects can
occur through direct and indirect pathways. For direct genotoxicity the administered chemical or
its metabolites are DNA reactive and causes immediate damage. Chemicals with this mode of
action are also called genotoxic carcinogens. For indirect genotoxicity first a chronic
inflammation occurs which releases reactive species, which then damage the DNA. Compounds
with an indirect pathway are called non genotoxic carcinogens and first need to overcome a
threshold before adverse effects occur. In order to discriminate between genotoxic and nongenotoxic carcinogens genotoxicity tests can be used. An example of a substance that is
genotoxic is the polyaromatic hydrocarbon (PAH) benzopyrene, which can be produced when
cooking meat at high temperatures like barbequing. After entering the body the benzopyrene is
transformed by enzymes (Cytochrom P450) to metabolites that react with the guanine base of
the DNA and are thus carcinogenic.
Neurotoxicity concerns the adverse change in the structure or a functional alteration of the
nervous system originating from exposure to chemicals, biological and physical agents.
Neurotoxic damage can be reversible or irreversible and strongly depends on the dose and the
duration of the exposure to a chemical, on the genetic pre disposition, the liver function and the
developmental stage of the targeted organism and other factors. (Quelle: Berlin Vortrag,
Nikolopoulou). The signal transporting cells of the nervous system are called neurons and are
the functional units of the nervous system. In order to understand the possible toxic effects on
the nervous system the fundamental structure needs be understood. Neurons are highly
branched cells that consist of cell bodies and appendices; i.e. several dendrites and one axon..
Dendrites are responsible for receiving the communication from other neurons and are highly
branched (Greim, Snyder 2008). Thus, each neuron receives signals from other neurons and
transmits the information further through the long axon. For this communication the action
potential needs to be transferred along the axon.Therefore neurons exhibit a high density of ion
channels along the axon and at the synapses, which are specialized junctions through which
cells of the nervous system signal to one another and to non-neuronal cells such as muscle
cells. In the synapses the signal is forwarded by means of a chemical transmitter, called a
neurotransmitter, which are released in the synaptic cleft and diffuse to the postsynaptic site
where they bind to postsynaptic receptors. Upon binding to the postsynaptic receptors a new
action potential is induced by means of a biochemical response and the information can be
forwarded (Greim, Snyder 2008). Parts of the axon are surrounded by myelin layers that have
high electric impedance. The myelin shields are disconnected at the Ranvier nodes, which
occur every 1.5 mm along the axon (Greim, Snyder 2008). The myelin layers around the axons
allow for a fast signal transmission because the electrical signals jump from one Ranvier node
to another (Greim, Snyder 2008).
An impairment of the CNS, consisting of the brain and the spinal cord, is troubling to the fetal
brain still under development but also to because neurons of the adult brain which are terminally
differentiated cells that cannot undergo proliferative responses to repair damage. Beside the
CNS there is the peripheral nervous system (PNS), which consists of sensory neurons that
transmit information from peripheral receptors to the CNS and motor neurons that transfer
information from the CNS to the muscles and glands. In the PNS damage can be reduced if
there are surviving neurons, which expand their territory by axonal branching and can thus
overtake the territory of dead neuronal cells.
Although new cells cannot be developed in the CNS the brain is protected by other adaptive
mechanisms that provide the brain with considerable capacity for structural and functional
modification. Dysfunctions in selected areas of the brain can be compensated for by other
areas. Yet there are specialized areas of the brain where no compensation is possible.Overall it
can be said that neurons are very active cells that exhibit a very high metabolic demand. Some
neurons overcome very long distances with their axons and present a very effective system for
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Neurotoxicity
Neurons
Dendrites and
distributing metabolites between the cell body, the dendrites, and the axons. Neurons are thus
especially susceptible to chemicals that destroy the myelin layers or the cytoskeleton of the
cells, or interfere with the energy supply (Greim, Snyder 2008).
A common toxicant for nervous tissue is lead, which is known to be a toxic agent for the
cognitive development of children and the mental abilities of adults by environmental,
occupational or food exposure (Greim, Snyder 2008). Well known neurotoxic effects of lead are
abnormal myelin formation, altered neurotransmitter release and receptor density, the disruption
of the blood-brain barrier and lowered IQ.
axons
Synapses
Neurotransmitter
Axons
Myelin layers
Ranvier node
Impairment of
CNS
Impairment of
PNS
Damage of
neurons
Example: lead
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Figure 6: Schematic diagram of neurons. From (Greim, Snyder 2008), page 251.
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4. Experimental data acquisition
Different
methods
There are different approaches in order to measure the adverse effects of a specific chemical
on a target organism. It is possible to conduct studies as experimental animal assays (in vivo) or
as cell culture assays (in vitro) or to analyse epidemiological data from groups of humans;
exposed e.g. through their work. The results of the experiments/studies should help to identify
and risk assess the adverse effects that a chemical compound can initiate in a target organism.
When talking about food constituents, the overall goal of experiments is to simulate lifelong
exposure of humans to a specific chemical.
OECD guidelines
The first international organization that developed internationally agreed guidelines for the
testing of chemicals with regard to their toxicity was the Organisation for Economic Co-operation
and Development (OECD) in 19812 (Brimer 2011). Meanwhile guidelines were published by
other organisations amongst others the US FDA. Yet most international and national
organizations stipulate that their tests are performed according to the OECD guidelines for the
testing of chemicals (Brimer 2011).
In vivo
In the following we will focus on in vivo experimental studies, because they are frequently used
when assessing the risk of food chemicals for humans. Results from suchexperimental animal
studies need to be extrapolated to other species (e.g. man) or to a longer time span than the
exposure time in the experiment (Gupta 2007). This is done by division with uncertainty
factors.The use of animal testing for the risk assessment of food chemicals, drugs or cosmetics
is increasingly criticized, however. In line with tis, the EU, to take an example, supports the
development of alternative (typically in vitro methods) that can reduce, replace or refine (RRR)
the use of animal experiments (Brimer 2011).
RRR
Experimental
design
Toxicokintetics
experiment
The experimental design must be adapted to the specific hypothesis that should be tested. At
this point two experimental designs for in vivo experiments will be presented. For all
experiments it is important to consider the rout of administration because the distribution and
the effect of a toxicant may vary depending on the type of administration (see Figure 4).
Figure 7:” Schematic rendering of an experimental design for evaluating the kinetics of an administered toxicant” (Gupta
2007), page 15.
The first experimental setting here described should render results that help to understand the
link between the exposure and the internal dose and can be used to better understand the
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OECD (revised 1993) Guidelines for the Testing of Chemicals, Sections 1–5.
http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm
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toxicokinetics of a substance (see Figure 7). Data rfom this kind of experiments is used to
create a mass balance between the administered quantity and the quantity recovered. For this
all the animals are exposed to a specific dose of a possible toxic substance. At different time
points after the administration a certain number of experimental animals will be euthanized and
tissue is collected. Like this a dynamic profile can be created of how the body handles the
administered chemical (Gupta 2007). The time points when the animals are sacrificed are
guided by the anticipated kinetic profile of the compound and its metabolites (Gupta 2007). A
possible output of such an experiment can be seen in Figure 8, which shows an example for the
blood concentration plotted against time. The graph is purely hypothetical.
Figure 8: An example for an output of an experiment to understand the toxicokinetics. Blood concentration-time curve
(―) as the sum of input (- - -) and output (…) in linear coordinates. (Brimer 2011), page 55.
Dose-response
observations
In order to make dose-response observations another experimental setting is needed (Gupta
2007). Also here the administration route should be chosen according to the most likely
exposure conditions to be encountered; i.e. in our case exposure through food or beverages. In
this example we will look at a 2-year assay that is typically used to evaluate carcinogenicity in
rats or in mice. However, the same study design can be used to evaluate other endpoints and to
conduct shorter-term studies (Gupta 2007).Common study durations include the evaluation of
acute effects, and further study durations such as 28 days, 90 days, and maybe finally chronic
studies, which last at least one year or two years for carcinogenicity in rats or mice. It is critical
that multiple exposure levels are used (see Figure 9). The choice of the exposure levels is very
important, because they are used for the identification of threshold values for regulatory
documents and laws. In this experimental design multiple sacrifice times are used for all
exposure levels (Gupta 2007). This setting can provide valuable insight into the progression of
disease processes. As the name of this kind of experiments already states the output are doseresponse relationships that are plotted in an x-y-graph, which are discussed in the following
chapter.
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Figure 9: "Schematic rendering of an experimental design for evaluating exposure (dose)- response relationships for a
toxicant. (Gupta 2007), page 15.
In vitro
Skin irritation test
Ames test
Beside in vivo experiments it is common to conduct in vitro experiments, which for example can
be used to test for acute general cytotoxicity and for genotoxicity (Brimer 2011). In vitro
experiments are biological experiments that are conducted outside the intact live organism
using cultures of isolated organs (also termed ex vivo), primary cells,cells in continuous culture
or subcellular fractions (Brimer 2011). The development of in vitro methods was enhanced by
the implementation of the Animal Welfare Guidelines in 1986. At this time the EU institutions
started to support the development of alternatives to in vivo methods by supporting the RRR
principle (see beginning of this chapter). Common effects (end-points) that are tested with in
vitro methods concern cell morphology, cell viability, cell metabolism, cell membrane integrity,
cell proliferation, cell adhesion but also genotoxicity (Brimer 2011). It is also possible to conduct
some skin irritation tests with in vitro methods and thus replacing in vivo methods where acute
dermal irritation/corrosion is tested. An example for an in vitro test for skin irritation is the nonbiological test called SKINTEX TM, which is commercially available. In this test the chemical
compound is administered to a membrane of collagen, keratin and colorant, which is in contact
with a proteinous reagent. If the compound is irritating it may alter the membrane and provoke a
colorant liberation, and/or pass through the membrane r reacting with the reagent inducing
different levels of precipitation, according to the sample irritating capacity (Bason et al. 1992).
Anr example of an in vitro experiment for the test of genotoxicity is the Ames test, which is used
to test whether a chemical substance induces mutation. For this bacteria, usually salmonella
typhimurium, are deprived of their ability to produce a specific amino acid, namely the amino
acid histidine. The salmonelle typhimurium strain is then administered onto an agar plate where
only very little histidine is present in the growth medium, just sufficient to allow the bacteria to
grow for an initial time and have the opportunity to mutate. Some of the plates do not contain
any of the chemical compound to be investigated (control plates), while two or three groups are
made containing different concentrations of the compound. In case the compound is mutagenic,
it will among others back-mutate the bacteria to the histidine producing “wild-type”, which within
the next 48-72 hours will form visible colonies. The mutagenicity of a substance is proportional
to the number of colonies observed. It is also possible to use Escherichia coli for such a test yet
Salmonelle typhimurium is more suitable because it has a defect in the DNA repairmen system,
thus mutations cannot be eliminated. Additionally the permeability of the membrane of
Salmonelle typhimurium is higher so that mutagenic substances are not detained by it.
The third possibility to assess the toxicity of chemicals is through observations in
humans/human studies(Integrated Risk Information System (IRIS) 1993). For this different
methods can be used: case reports, studies in volunteers (normally only low dose studies of the
kinetics), occupational experiences, and more general epidemiological studies (Nielsen et al.
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Human studies
Case reports
Studies in
volunteer
Occupational
experiences
Epidemiological
studies
2008). Human studies (data) obviate the extrapolation from animals to humans and are thus
given first priority whenever available. Case reports describe particular observed effects in
individuals or groups that have been exposed, usually accidentally or in suicidal attempts, to a
substance (Nielsen et al. 2008). Therefore case reports are mainly used to assess acute toxic
effects and clinical symptomsOccupational experiences refer to the monitoring of workers in
their working environment with regard to the compliance of occupational exposure to the limits
required by national laws (Nielsen et al. 2008). In epidemiological studies the distribution and
determinants of health-related states and events in human population is scrutinized (Greim,
Snyder 2008). It is a non-experimental approach that presumes that a certain group of
individuals happened to have been exposed to a specific compound. Commonly measured
endpoints are mortality, morbidity, medical visits or hospital admissions, and clinical signs and
symptoms (Nielsen et al. 2008). Epidemiological studies can be used to assess long-term
effects from repeated exposure for a long time or can help to reveal effects from short-term
exposure.
5. Dose-response observations
Dose-response
relationship
In order to assess the effects of a toxic substance, dose-response relationships are plotted. The
dose response curve describes the change in effect on an organism caused by different levels
of exposure (dosage) to a chemical compound. The dose is plotted on the x-axis and the
response or the effect on the y-axis. As the toxic effect of a substance is the function of the
dose and time, the dose-response curve is referring to a certain exposure duration, and thus
independent of time. The response(s) (effect(s)) is defined in each study and can be
physiological or biochemical responses, or number of fatalities (mortality). The effect can
furthermore be quantified at different levels (molecular, cellular, tissue or organ etc.) and may
thus be expressed in different entities (Brimer 2011). A commonly used measurement for the
effect is percentage of responses in the target organism. A formerly frequently used
measurement is the percentage of fatalities, whereas the dose where 50% of the experimental
animals died after specified test duration is termed as the LD50 (Brimer 2011). Today a number
of alternative assays have been developed to reduce the number of animals and their
sufferings. Figure 10 shows a hypothetical dose-response curve for two different toxicants with
the dosage on the x-axis and the response in percent on the y-axis.
Figure 10: Hypothetical dose-response curves for two different toxicants, A and B. Extracted from Brimer (2011), page
66.
6. Threshold vs non-threshold mode of actions
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Non-threshold
mode of action
Normally we differentiate between two modes of action that can lead to adverse effects: the
direct and the indirect pathways. The direct mode of action has no threshold and can be
displayed as a linear relationship between the dose of a food chemical and the adverse effects
(see figure 11, left graph). This relationship describes the mode of action of direct genotoxicity,
where a small number of molecular events can evoke changes in a single cell that can lead to
uncontrolled cellular proliferation – cancer (Brimer 2011).
effect
effect
threshold
Figure 11: Dose-effect dose
relationship without (left) and with a threshold (right).
Threshold mode
of action
dose
In the indirect mode of action the organic homeostatic, compensating and adaptive mechanisms
need to be overcome before a toxic effect occurs. Thus there is a threshold that needs to be
reached before adverse effects take place (Brimer 2011). These kinds of effects are also
referred to as “systemic toxicity”. They describe effects other than gene mutation related cancer
o, which on the other hand are often treated as non-threshold processes as described above
(Integrated Risk Information System (IRIS) 1993).
7. Examples of chemicals and toxicity endpoints for risk assessment
NOAEL
Until now we have looked at several basic toxicological principles and assay methods. In this
last chapter an important output of the risk assessment is discussed. For the utilisation of the
bulk of results obtained from different assays and observations (e.g. case reports etc.) for
regulatory purpose,s it is important to generate key figures. There are different measures for
each toxicity endpoint that can be used in this regard.
The most prominent through time one is probably the NOAEL (No Observed Adverse Effect
Level.), The NOAEL depends on the adverse effect endpoint of the experiment. For example: If
the endpoint of an experiment is skin irritation and the subject under study exhibits skin irritation
at the dosage of 50 ppm but none at the next lower level of 30 ppm, then the NOAEL is 30 ppm.
Normally the NOAEL is determined for different endpoints (e.g. skin irritation, genotoxicity, etc.)
for the same chemical compound. In the end the lowest NOAEL of all the experiments is chosen
(Brimer 2011). The NOAEL can only be determined for chemical compounds that follow a
threshold mode of action (United States Environmental Protection Agency (EPA) 2012).
As an example to illustrate the above discussed issues we can take a look at the results of a
study conducted by Littlefield et al. (1980) where over 24’000 mice and 81 different treatment
groups were used to determine the shape of the dose-response relationship for two different
adverse effects of the exposure to 2-acetylaminofluorine (2-AAF) (Casarett et al. 2001). The
mice were exposed to 2-AAF to one of seven doses between 30 to 150 ppm (in the food) plus
one control group with 0 ppm. Figure 12 shows the dose-relationship between 2-AAF exposure
and liver and bladder cancer at 24 months of exposure. Although for both types of tumours the
incidents increase with increasing dose the shape of the curves are dramatically different. For
liver tumours no evident threshold can be determined, whereas for bladder tumours an apparent
threshold is evident. For the bladder tumours the NOAEL was set at 75 ppm.
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Figure 12: Dose-response relationship for carcinogens adapted from (Littlefield et al. 1980), extracted from (Casarett et
al. 2001),page 22.
LOAEL
If the NOAEL is not available the Lowest Observed Adverse Effect Level (LOAEL) can be used.
Both methods have the advantage that they do not depend on a mathematical model, are easy
to understand and that it can be applied to all data. On the other hand they only provide
knowledge at the dose level of the experimental study and are thus strongly dependent on the
study design.
Figure 13: Illustration of the NOAEL, the LOAEL and the benchmark dose (BMD) approach. Extracted from Brimer
(2010) page 261.
Benchmark dose
Alternatively the benchmark dose (BMD) can be assessed, based on the statistically bestfitting dose-response curve derived from the experimental results. . The BMD is the dose that
produces a predetermined change (usually 5-10%) in the rate of an adverse response
compared with the background level. The mathematical model applied is fitting the experimental
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data within the observable range and refers to the central estimate of the dose that is expected
to yield the benchmark response (BMR). The statistical method applied in the BMD approach
thus uses the information in the complete dataset instead of making pair wise comparison using
subsets of the data (Brimer 2011). The BMD is thus less dependent on the experimental setting
than the NOAEL and is thus conceptually superior.
ADI
The determined NOAEL or BMD(L) can be used in the deviation (an estimate) of the
acceptable daily intake (ADI) for humans of a substance. For this, the selected NOAEL
(BMDL) is divided by an uncertainty factor (range 10 -100). The lowest NOAEL (BMDL) of the
different experiments within a study should be chosen. For example: In a study the NOAEL was
determined for the effects on the liver and the germ cells,for the testis weight reduction, and for
reduced F2 generation. The NOAEL for the liver experiment was the lowest, thus the calculation
of the ADI should be based upon this NOAEL (Brimer 2011). The ADI represents the maximum
dose that is believed a person can ingest throughout his/her life without any adverse effects.
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Brimer, Leon (2011): Chemical food safety. Nosworthy Way Wallingford Oxfordshire UK ;,
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Casarett, Louis J.; Doull, John; Klaassen, Curtis D. (2001): Casarett and Doull's toxicology. The
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introduction / edited by Helmut Greim and Robert L. Snyder. Chichester: John Wiley.
Gupta, Ramesh C. (2007): Veterinary toxicology. Basic and clinical principles / Ramesh C.
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checked on 13/01/2015.
Littlefield, N. A.; Farmer, J. H.; Gaylor, D. W.; Sheldon, W. G. (1980): Effects of dose and time in
a long-term, low-dose carcinogenic study. In J Environ Pathol Toxicol 3 (3 Spec No), pp. 17–34.
Nielsen, Elsa; Østergaard, Grete; Larsen, John Christian (2008): Toxicological risk assessment
of chemicals. A practical guide. New York: Informa Healthcare.
United States Environmental Protection Agency (EPA) (2012): EPA Risk Assessment - Human
Health Risk. Step 2 - Dose-Response ASsessment. United States Environmental Protection
Agency (EPA. Available online at http://www.epa.gov/risk_assessment/dose-response.htm,
updated on 31/07/2012, checked on 23/01/2015.
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