toxicity tests alternative methods in toxicology prof. dimitrios kouretas

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Toxicity Tests Toxicity Tests Alternative Alternative Methods Methods in in Toxic Toxic ology ology Prof. Prof. Dimitrios Kouretas Dimitrios Kouretas

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Page 1: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

Toxicity TestsToxicity TestsAlternative Alternative Methods Methods

in in ToxicToxicologyology

Prof.Prof. Dimitrios Kouretas Dimitrios Kouretas

Page 2: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

2

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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Page 3: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Toxicity TestsToxicity 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).

Page 4: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Toxicity TestsAnimals 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

Page 5: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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”.

Page 6: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Alternative Test Methods

1R= Replacement of test animals2R=Refinement ( better tests) 3R=Reduction ( decrease of the

number of animals)4R=Responsibility ( tests

scientifically acceptable)

Page 7: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 8: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Alternative Test Methods

A refinement alternative uses procedures that lessen or eliminate pain or distress in animals or enhances animal well-being.

Page 9: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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.

Page 10: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Acute Toxicity TestsLD50 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.

Page 11: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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REDUCTIONLD50 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.

Page 12: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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REDUCTIONLD50 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.

Page 13: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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.

Page 14: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 15: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 16: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 17: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 18: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 19: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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 compound-related mortality and subjects those animals which are used to less pain and distress.

Page 20: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 21: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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.

Page 22: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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.

Page 23: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 24: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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).

Page 25: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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%.

Page 26: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 27: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 28: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

COMPARISON OF OECD 420, 423 AND 425

FDP (OECD 420) ATC (OECD 423) UDP (OECD 425)

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).

Point estimate of LD50 with confidence intervals.

Signs of acute toxicity Target organ(s).

Page 29: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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

Page 30: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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:

Page 31: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 32: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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2. Physiology and Pharmacokinetic Modeling: This kind of modeling predicts the disposition of xenobiotics and includes.

ADME parameter predictorsmetabolic fate predictorsmetabolic stability predictorscytochrome p450 substrate predictorsphysiology-based pharmacokinetic (PBPK)or

biokinetic (PBBK) modeling software

Page 33: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 34: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 35: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 36: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

In Vitro Models-Cell Cultures

Page 37: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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In Vitro ModelsCell 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.

Page 38: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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In Vitro ModelsCell 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.

Page 39: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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.

Page 40: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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 )

Page 41: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 42: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

42

ADVANTAGES OF In Vitro METHODS

3. It is possible to obtain similar results with 100 cell culture flasks to 100 rat or human.

Page 43: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 44: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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

Page 45: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

DISADVANTAGES OF In Vitro METHODS

Page 46: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

DISADVANTAGES OF In Vitro METHODS

Page 47: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

DISADVANTAGES OF In Vitro METHODS

Page 48: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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4. There are not definitive and precise test procedures for in vitro toxicity tests given by regulatory authorities.

DISADVANTAGES OF In Vitro METHODS

Page 49: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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*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.

DISADVANTAGES OF In Vitro METHODS

Page 50: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

The advantages and disadvantages of different in vitro models

Models Advantages Disadvantages Subcellular preparations Enables molecular

level studies Toxic metabolite formation and covalent

binding in macromolecules can

be assessed

Only quantitative information available

Isolated cells(primary cultures, cocultures)

Retain original capabilities and

properties Mimic in vivo response

Loss of influences such as hormones and

immunity

Multicellular tissue(slices, cubes, aggregates, explants)

Retention of 3D structure

Cell-cell interaction

Poor retention of viability

Oxygen and chemicals cannot penetrate

Isolated organs High reproducibility Short period of viability

Stem cells Pluripotent Can form tumors

Cell lines Can be maintained for a prolonged period

Prolongation results in decreased metabolic capacity and altered

cellular function

Immortalized cell lines Capable of extended and indefinite growth

in vitro

Immortalization alters their characteristics and

functions

Page 51: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 52: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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).

Page 53: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 54: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 55: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 56: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

Skin Irritation Tests

Skin irritation tests are Episkin, Epiderm, pig ear,

and PREDISKIN.

Page 57: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 58: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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EpidermEpiderm incorporates normal

human keratinocytes cultured on permeable Milipore membranes. Toxicity is assessed by using MTT assay. The primary end point is IC50.

Page 59: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 60: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 61: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

The Skin Corrosion Tests

The skin corrosion tests are Corrositex,

Transcutaneous electrical resistance (TER) and

SKINETHIC.

Page 62: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 63: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 64: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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SKINETHICSKINETHIC 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.

Page 65: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

65

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.

Page 66: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 67: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

67

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.

Page 68: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 69: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 70: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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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.

Page 71: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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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.

Page 72: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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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 non-ionized 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.

Page 73: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 74: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 75: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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

Page 76: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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 (1-OHP).

Phthalate metabolites are also usually measured in urine.

Page 77: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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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.

Page 78: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

EUROTOX Advanced Toxicology Course-2013-Volos-Greece

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Others

Placenta (PCBs, PAHs, phthalates)Bones (Pb)Faeces (Pb, Cd, PCBs) Teeth

Page 79: Toxicity Tests Alternative Methods in Toxicology Prof. Dimitrios Kouretas

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