Toxicity Tests
Alternative Methods in
Toxicology
Prof. Dimitrios Kouretas
Toxicity Tests
Toxicity tests are performed to assess
the safety or hazards of several
substances such as industrial chemicals,
pharmaceuticals and consumer care
products.
Toxicity tests characterize the toxicity
and the level of toxicity. They help to
find out the dose-response and the
target organ.
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Toxicity Tests
 Toxicity tests differ from each other;
 *Acute,
 *Subchronic
 *Chronic ( life-time, long-term ) toxicity
tests evaluate all potential toxicity.
 Certain
toxicity
tests
such
as
teratogenicity,
mutagenicity,
carcinogenecity, reproductive toxicity,
immunotoxicity,
neurotoxicity…..evaluate
certain type of toxicity.
 Many of the current toxicity testing
methods use laboratory animals (e.g. mice,
rats, rabbits).
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Toxicity Tests
 Animals are useful models to predict the
toxicity of chemicals in humans because
they have similar cell organelles, cells and
organs.
 If the model is not close to human,
uncertainity of results increase.
 Toxicity tests can be performed in several
laboratories and the same results must be
obtained in different labs (tests must be
objective ).
 Toxicity tests must be fast and cheap
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Alternative Test Methods
Alternative test methods are test methods
that reduce, refine and replace animal use.
Reduction, refinement, and replacement
are commonly referred to as
 “the 3Rs (4Rs) of alternatives”.
 The concept of 3Rs was first proposed by
William Russell and Rex Burch in the
“Principles of Humane Experimental
Technique”.
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Alternative Test Methods
1R= Replacement of test animals
2R=Refinement ( better tests)
3R=Reduction ( decrease of the
number of animals)
4R=Responsibility ( tests
scientifically acceptable)
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Alternative Test Methods
 A
reduction
alternative
decreases the number of
animals required for a test
method,
while
remaining
consistent
with
scientific
practices necessary to obtain
valid results.
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Alternative Test Methods
 A refinement alternative uses
procedures that lessen or
eliminate pain or distress in
animals or enhances animal wellbeing.
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Alternative Test Methods
 A replacement alternative uses non-animal
systems instead of animals, or uses a
phylogenetically lower species of live animal than
the current test.
 Among all approaches the use of alternative
techniques replacing animals has potential for the
future research.
Acute Toxicity Tests
LD50 TEST
 The lethal dose 50 (LD50) test was first
introduced in 1927 by Trevan (1927), for
testing substances intended for human use
such as digitalis and insulin.
 It covers the application of one high dose
or several low doses during 24hours.
Effects are observed for 14 days
 The endpoint is the death of animals.
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REDUCTION
LD50 TEST
 By 1970s, the test, which aims to find the
single lethal dose of a substance which kills
half the animals in a test group, had
become generally accepted as a basis of
comparing and classifying the toxicities of
chemicals, and gradually became a required
test for various regulatory bodies
concerned with new drugs, food additives,
cosmetic ingredients, household products,
industrial chemicals and pesticides.
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REDUCTION
LD50 TEST
The test required up to 100 animals,
sometimes for each of two species
(normally the rat but also the mouse
when a second species was needed)
for each substance tested.
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REDUCTION
 In 1981, the Organisation for Economic
Cooperation and Development (OECD)
incorporated the LD50 test into its new
Test Guidelines.
 By this time, it was generally agreed that
the statistical precision of the LD50 value,
together with its confidence intervals and
the slope of the dose–mortality curve, which
this classical LD50 test could provide, were
not needed for normal hazard and risk
assessment purposes.
REDUCTION
Hence, the 1981 guideline for
acute oral toxicity (OECD 401,
1987) required the use of only
five animals per sex per dose
group, with three dose groups per
test which were chosen, from
sighting
studies
or
from
historical data, to span the LD50
value.
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REDUCTION
 An upper dose level limit of 5000 mg/kg
was also introduced and, for essentially
non-toxic substances, the concept of a
‘Limit Test’ was included which required,
for those substances with LD50 values
greater than 5000 mg/kg, only this upper
dose level to be tested.
 Similar guidelines were also published for
acute dermal (OECD 402, 1987) and
inhalation toxicity.
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REDUCTION
 After many years of controversy and
debate, the LD50 test was finally
suspended by the end of 2002.
 Three alternative animal tests, the “Fixed
Dose Procedure (FDP)”, the “Acute Toxic
Class Method (ATC)” and the “Up and Down
Procedure (UDP)” have been developed
which give rise to significant improvements
in animal welfare.
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REDUCTION
FDP,ATC and UDP have recently
undergone revision to improve their
scientific performance but more
importantly
to
increase
their
regulatory acceptance. They can now
be used within a strategy for acute
toxicity testing for all types of test
substances and for all regulatory and
in-house purposes.
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REDUCTION
 On the other hand, in vitro cytotoxicity tests
could be used as adjuncts to these alternative
animal tests within the next years or so to
improve dose level selection and thus give
further modest improvements in the numbers
of animals used.
 However, the total replacement of animal
tests requires a considerable amount of
further test development, followed by
validation, and is at least 10 years away.
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Fixed Dose Procedure
(OECD 420)
 These studies showed that the FDP
was able to provide results that
enable substances to be ranked
according to the EU system of
classification.
 The FDP causes less compoundrelated mortality and subjects those
animals which are used to less pain
and distress.
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Fixed Dose Procedure
(OECD 420)
The FDP also provided the necessary
information on the nature, time to
onset, duration and outcome of signs
of toxicity that is required for risk
assessment purposes.
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Fixed Dose Procedure
(OECD 420)
In FDP, the test substance is given at one of
the four fixed-dose levels (5, 50, 500, and
2000 mg/kg) to five male and five female
rats. The objective is to identify a dose
that produces clear signs of toxicity but no
mortality.
Fixed Dose Procedure
(OECD 420)
 Depending on the results of the first test, either
no further testing is needed or a higher or lower
dose is tested: If mortality occurs, retesting at a
lower dose level is necessary (except if the
original dose chosen is 5 mg/kg). If no signs of
toxicity occur at the initial dose, it is necessary to
retest at a higher dose level. The results are thus
interpreted in relation to animal survival and
evident toxicity and it becomes possible to assign
the chemical to one of the OECD classification
categories.
Acute Toxic Class Method (ATC)
(OECD 423)
 In 1996, a second alternative method, the
ATC was adopted (OECD 423, 2001).
 The ATC also uses the concept of fixed
dose levels but retains mortality as a
principal endpoint.
 The oral ATC method is a sequential
testing procedure with the use of three
animals of one sex per step.
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Acute Toxic Class Method (ATC)
(OECD 423)
 During the development of the new study
protocol the starting doses (25, 200 or
2000 mg/kg b.w.) were chosen mainly from
the class limits for classification of the EU
at that time and modified at a later stage to
5, 50, 300 or 2000 mg/kg b.w. based on the
class limits of the Globally Harmonized
Classification System (GHS).
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Acute Toxic Class Method (ATC)
(OECD 423)
 The ATC method is a sequential testing
procedure using only three animals of one
sex per step at any of the defined dose
levels. Depending on the mortality rate
three but never more than six animals are
used per dose level. This approach results
in the reduction of numbers of animals
used in comparison to the LD50 test by 40–
70%.
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Acute Toxic Class Method (ATC)
(OECD 423)
The
ATC
method
has
been
successfully used in Germany and in
2003 >85% of all tests on acute oral
toxicity testing was conducted as oral
ATC tests.
In member states of the EU, the ATC
method is used in the range of 50%
of all tests conducted.
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Up and Down Procedure (UDP)
(OECD 425)
The AITC was was followed in 1998 by
the UDP.
The UDP, as its name suggests, aims to
estimate the LD50 value by testing
individual animals sequentially, with the
dose for each animal being adjusted up
or down depending upon the outcome for
the previous animal.
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COMPARISON OF OECD 420, 423 AND
425
FDP (OECD 420)
ATC (OECD 423)
METHODOLODY
Single bolus dose. Young adult
rats (one sex). Oral gavage
with constant volume or
concentration, clinical
observations, body weight and
mortality over 14 days.
Necropsy at termination.
Single bolus dose. Young adult
rats (one sex). Oral gavage
with constant volume or
concentration, clinical
observations, body weight and
mortality over 14 days.
Necropsy at termination.
Single bolus dose. Young adult
rats (one sex). Oral gavage with
constant volume or
concentration, clinical
observations, body weight and
mortality over 14 days.
Necropsy at termination.
SIGHTING
STUDY
Yes
No
No
DOSE LEVELS
Fixed doses of 5, 50, 300,
2000 and (5000) mg/kg 5 rats
per dose level
Fixed doses of 5, 50, 300,
2000 and (5000) mg/kg 3 rats
per dose level
Starting at best estimate of
LD50 (or 175 mg/kg) and using
dose progression factor 3.2
single animals dosed until one
of the three stopping criteria
met
AIM
Identify lowest fixed dose
causing evident toxicity
Identify lowest fixed dose
causing mortality
Estimate LD50
OUTPUT



Range estimate of
LD50
Signs of acute toxicity
Target organ(s).



Range estimate of
LD50
Signs of acute toxicity
Target organ(s).
UDP (OECD 425)



Point estimate of LD50
with confidence
intervals.
Signs of acute toxicity
Target organ(s).
 Acute toxicity tests are useful in getting
information for
 *subchronic and chronic toxicity tests
 *risk assessment of acute effects,
 *treatments of poisoning cases,
 *regulatory toxicology and classification,
labelling and transportation,
 *about toxicity mechanisms and structure
activity
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Replacement
New Technologies and New Models:
A number of new emerging fields and
techniques are contributing major new
insights for replacing sentient animal
use within biomedical research and
toxicity testing. These can be classified
as follows:
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1.Computerized Modeling: Computerized
modeling based on (Q)SAR, in silico
models in the future may replace some
of the animal tests at least.
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2. Physiology and Pharmacokinetic
Modeling: This kind of modeling
predicts the disposition of
xenobiotics and includes.





ADME parameter predictors
metabolic fate predictors
metabolic stability predictors
cytochrome p450 substrate predictors
physiology-based pharmacokinetic (PBPK)or biokinetic
(PBBK) modeling software
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3. Microarray Technology: Microarray
consists of DNA or protein fragments
placed onto a slide, which are then
used as “miniaturized reaction areas”.
Its aim is to detect any changes in
gene or protein expression patterns
in cells or tissues.
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4. Omics Technology: The aim of each
“omics” technology is to extract
information that has mechanistic and
predictive value. These include:
 Genomics: The study of genes and their function.
 Proteomics: The study of proteins.
 Metabonomics: The study of molecules involved in
cellular metabolism.
 Transcriptomics: The study of the mRNAs.
 Glycomics: The study of cellular carbohydrates.
 Lipomics: The study of cellular lipids.
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5.Non Mammalian Models: The use of
invertebrates such as drosophila,
freshwater snails and Caenorhabditis
elegans are widely used for the
assessment of toxicity of several
xenobiotics.
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In Vitro ModelsCell Cultures
In Vitro Models
Cell Cultures
There has been an increasing scientific
interest in developing more innovative
and non-animal experiments as an
alternative approach to toxicity testing.
Several international centers have
dedicated their work to the
development and validation of these
non-animal alternatives.
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In Vitro Models
Cell Cultures
Between 1998 and 2007, 24 distinct
tests or categories of test methods
that could replace, reduce or refine
laboratory animal use were scientifically
validated and registered to the
“European Centre for the Validation of
Alternative Methods (ECVAM)” and EC
organization and thirteen of them
achieved regulatory acceptance.
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In Vitro METHODS
The are several advantages as well as
disadvantages of culture studies. It is
possible to examine cells under the
microscope and to investigate the
changes
quantitatively
and
simultaneously.
ADVANTAGES OF In Vitro
METHODS
1.It is possible to report each change
that takes place by changing the
environmental conditions. For example,
it
is
possible
to
change
pH,
temperature, amino acid and vitamin
concentration of the medium and to
clarify the effects of such conditions.(
Controlled test circumstances )
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ADVANTAGES OF In Vitro
METHODS
2.It is possible to obtain higher growth
and development especially by cell lines
and this enables more work with less
time consumption.
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ADVANTAGES OF In Vitro
METHODS
3. It is possible to obtain similar results
with 100 cell culture flasks to 100 rat
or human.
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ADVANTAGES OF In Vitro
METHODS
4.It is possible to choose the appropriate cell line for
the endpoint that the researcher want to measure.
For example, for drug metabolism studies hepatic
cell cultures, for excretion studies renal cell cultures
or for drug accumulation studies muscle cell cultures
can be used.
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ADVANTAGES OF In Vitro
METHODS
In vitro tests;
*reduces animals use in the tests
*tests cheap and fast
*test compound needed in trace amounts
*human tissues and cells can be used
*suitable for screening
*possibility to use same doses in other tests
*time response can be tested
*toxicity mechanisms can be studied
*decrease biological variability
*human genes can be moved to cells
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DISADVANTAGES OF In Vitro
METHODS
1. Some cell cultures have
low proliferation capacity
and high phenotipically
changing capability. It is
not possible that in vitro
tests may represent in
vivo conditions under such
circumstances.
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DISADVANTAGES OF In Vitro
METHODS
2. Some cell cultures
especially primary cell
cultures cannot show
clonal growth and show
loss of viability in short
periods of time. Such cell
lines cannot be used for
chronic toxicity studies.
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DISADVANTAGES OF In Vitro
METHODS
3. Animal cell cultures
do not always
represent similar
results with human cell
cultures because of
the interspecies
differences. It is
difficult and costly to
use human cell cultures.
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DISADVANTAGES OF In Vitro
METHODS
4. There are not
definitive and precise
test procedures for in
vitro toxicity tests
given by regulatory
authorities.
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DISADVANTAGES OF In Vitro
METHODS
*common harmful effects
like weight loss can not be
measured
*systemic effects can not
measured in in vitro tests
*specific organ effects can
not be studied
*how tissues and organs work
together can not be
tested in in vitro tests.
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The advantages and disadvantages of different
in vitro models
Models
Subcellular preparations
Advantages

Disadvantages
Enables molecular level
studies
 Toxic metabolite
formation and covalent
binding in macromolecules
can be assessed

Only quantitative
information available
Isolated cells

Retain original capabilities
and properties
 Mimic in vivo response

Loss of influences such as
hormones and immunity
Multicellular tissue

Retention of 3D structure
 Cell-cell interaction

Poor retention of viability
 Oxygen and chemicals
cannot penetrate
(primary cultures, cocultures)
(slices, cubes, aggregates, explants)
Isolated organs

Stem cells
High reproducibility

Short period of viability

Pluripotent
Cell lines

Can be maintained for a
prolonged period
Immortalized cell lines

Capable of extended and
indefinite growth in vitro

Can form tumors
Prolongation results in
decreased metabolic
capacity and altered cellular
function


Immortalization alters their
characteristics and
functions
Target Organ/Tissue assays
Cells from the desired species and
target tissues can be used to predict
toxicity of xenobiotics. Especially,
these assays are used in ocular toxicity
and skintesting.
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The Ocular Toxicity Assays
The ocular toxicity assays are bovine
corneal capacity and permeability
(BCOP) test, isolated rabbit eye test
(IRE) and chicken enucleated eye test
(CEET).
Bovine Corneal Capacity and
Permeability (BCOP) Test
In BCOP test, test materials are applied
to epithelial surface of living bovine
corneas. The end points to be tested
are the changes in opacity and
permeability which are measured by
optical devices.
Damaged Bovine Cornea. Loss of
superficial layers of stratified
corneal epithelium.
Rinsing of cornea mounted in
BCOP Chamber and then the
opacity is read using an
opacitometer.
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Isolated Rabbit Eye Test (IRE)
(Draize Test)
 In IRE test, test substances are applied
topically to the eyes of the rabbit and the
effects are observed with slit lamp
biomicroscope. With IRE, extremely potent
irritants are evaluated.
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Chicken Enucleated Eye Test
(CEET)
In CEET test, material is
directly applied to the isolated
eye of the chicken and eye
irritation potential of the test
compound is evaluated as the
primary end point.
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Skin Irritation Tests
Skin irritation tests are
Episkin, Epiderm, pig ear, and
PREDISKIN.
Episkin
Episkin is a 3D model of human skin
with reconstructed epidermis and a
functional stratum corneum. Test
material is applied to this layer.
Toxicity is assessed by using MTT
assay. The primary end point is cell
viability.
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Epiderm
Epiderm
incorporates
normal
human keratinocytes cultured on
permeable Milipore membranes.
Toxicity is assessed by using
MTT assay. The primary end
point is IC50.
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Pig Ear Test
In pig ear test, non-perfused pig
ear test material for 4 hr to
distinguish between irritants and
non-irritants. The end point is
increase in trans-epidermal loss.
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PREDISKIN
In PREDISKIN test, human skin
cultures are exposed to test
material and toxicity is assessed by
MTT assay and the primary end
point is percentage of cell viability.
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The Skin Corrosion Tests
The skin corrosion tests are
Corrositex, Transcutaneous
electrical resistance (TER)
and SKINETHIC.
Corrositex
Corrositex
is
a
protein
membrane and it can measure
the penetrating rate of a
chemical in the simulated skin
barrier and the primary end
point is the color-change in
the membrane.
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Transcutaneous electrical resistance (TER)
 In TER assay, corrosive materials are
identified by their ability to produce a loss of
integrity and the end point is the reduction in
TER of layers of skin to an applied current.
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SKINETHIC
 SKINETHIC is the assay that involves the
estimating of the loss of viability of human
epidermal keratinocyte cultures overtime
when substances are applied topically. The
primary end point is the loss in cell viability
and the release of cytokines.
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The Skin Sensitization Test
There is only one skin sensitization test
widely used and it is the murine local
lymph node (LLNA) assay.
It measures sensitization of a chemical
in mice as function of proliferative
activity induced in lymph nodes draining
the site of exposure to the test
chemical. The end point is the increase
in thymidine (3H-TdR) incorporation.
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The Use of Non-Invasive Matrices in
Toxicology
 The use of non-invasive matrices in toxicology
studies can reduce the usage of animals.
 Use of hair, nails, breast milk, saliva,
meconium, urine, semen, placenta and bones
can be a very useful tool for human
biomonitoring though blood is still the ideal
matrix for most chemicals due to its contact
with the whole organism and its equilibrium
with organs and tissues where chemicals are
stored.
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Human Hair
 Human hair is a stable matrix that
presents numerous advantages for human
biomonitoring, such as easy collection, low
cost, easy transport and storage,
information about short- and long-term
exposure and the temporal exposure
pattern by segmental analysis.
 Methyl Hg, Cd and Pb can be detected in
hair.
 Organic pollutants could also be measured
in human hair.
 Correlations were also determined between
some PCDDs, PCDFs and coplanar PCBs
levels in blood and hair.
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Nail
 Nails have been used historically in forensic
science.
 As poisoning and to a lesser extend in
monitoring other inorganic chemicals such as
heavy metals.
 Although both fingernails and toenails can be
employed, some authors consider that toenails
are better than fingernails because they are
less exposed to external contamination.
 Cd and Pb can be detected in nails.
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Breast Milk
 Breast milk is a very commonly used matrix in
human biomonitoring as breast milk measurements
give information concerning the exposure levels of
both the mother and her child.
 Breast milk is usually employed for monitoring
lipophilic chemicals due to its high fat content.
 When breast milk is employed for human
biomonitoring, it is important to take into account
the process of depuration, that is, the reduction
of chemicals in milk during lactation.
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Breast Milk
 Although most studies determine organic
pollutants, some studies have also determined the
level of heavy metals in breast milk.
 Unlike POPs, heavy metals tend to accumulate in
blood more than in breast milk.
 It was reported that Cd has a lesser tendency to
associate with blood and breast milk than Pb and
Hg.
 A significant association has been found to exist
between Cd in breast milk and smoking. More than
90% of Pb body burden is accumulated in skeleton
so the mobilization of Pb from bones during
pregnancy and lactation is an important process to
mobilise lead in the body.
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Breast Milk
 The presence of Hg in breast milk has also
been studied in relation to amalgam fillings,
diet and mercury exposure in polluted areas.
 POPs have been determined in breast milk in
numerous studies.
 Breast milk has also been employed monitoring
different pesticides and others compounds.
 Emerging chemicals such as phthalates and
PBDEs can be found in breast milk.
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Saliva
 Saliva is an easy-to-collect low-cost matrix that is
very useful for screening large populations.
 The presence of a chemical in saliva depends on its
chemical characteristics, both lipophilic and nonionized molecules pass from blood to saliva better
than hydrophilic and ionized molecules.
 Saliva has a very high water content and low protein
content, which means that strongly protein-bound
chemicals are unlikely to be present in this matrix.
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Saliva
Many factors, such as circadian
rhythms, exercise, medication or age,
can influence the flow and physiological
characteristics of saliva.
Heavy metals (Pb, Cd) and PCBs can be
detected in saliva.
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Meconium
 The main advantage of meconium is its easy
collection, the large amount of sample that
can be collected and the information it can
give regarding long-term exposure.
 A foetus can be exposed to different
chemicals, most of which are deposited and
accumulated in the meconium.
 Pb, Cd and Hg have been detected in
meconium by different authors.
 Besides, organochlorine pesticides, PCBs and
phthalates can also be detected in meconium.
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Urine
 Urine is probably the second most common matrix for
human biomonitoring after blood, particularly for
water-soluble chemicals.
 Two different types of urine samples can be
collected, namely spot samples or 24-h samples. The
collection of spot samples is easier, therefore they
are employed more often.
 However, spot samples have the disadvantage of
varying volume and chemical concentration, both of
which mean that spot samples must be adjusted. This
adjustment can be performed by different methods,
but the most commonly used is the creatinine
concentration adjustment
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Urine
 Urine is the preferred non-invasive matrix in heavy
metals (Pb, Cd) biomonitoring.
 Urine is not a useful matrix for monitoring POPs (i.e.
DDT, PCBs).
 Many metabolites of PAHs can be measured in urine.
Recent exposure to PAHs, for example, is often
determined by the presence of 1-hydroxypyrene (1OHP).
 Phthalate metabolites are also usually measured in
urine.
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Semen
 Semen is used to assess effect biomarkers in
most studies.
 Exposure biomarkers are measured in other
matrices and then related with semen quality
parameters.
 A minority of studies, however, determine
heavy metals in semen or seminal plasma.
 Moreover,
organochlorinated
pesticides,
dioxins, and phthalates can also be
determined in semen.
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Others
Placenta (PCBs, PAHs, phthalates)
Bones (Pb)
Faeces (Pb, Cd, PCBs)
Teeth
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Thanks for listening…..