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Toxicity Tests Alternative Methods in Toxic ology

Toxicity Tests Alternative Methods in Toxic ology. Toxicity T est s. Toxicity test s are performed to assess the safety or hazards of several substances such as industrial chemicals, pharmaceuticals and consumer care products.

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Toxicity Tests Alternative Methods in Toxic ology

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  1. Toxicity TestsAlternative Methods in Toxicology

  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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  3. 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). EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  4. 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 EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  5. 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”. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  6. 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) EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  7. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  8. Alternative Test Methods • A refinement alternative uses procedures that lessen or eliminate pain or distress in animals or enhances animal well-being. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  9. 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.

  10. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  11. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  12. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  13. 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.

  14. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  15. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  16. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  17. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  18. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  19. 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. • TheFDP causes less compound-related mortality and subjects those animals which are used to less pain and distress. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  20. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  21. 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.

  22. 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.

  23. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  24. 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). EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  25. 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%. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  26. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  27. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  28. COMPARISON OF OECD 420, 423 AND 425

  29. 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 EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  30. 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: EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  31. 1.ComputerizedModeling: Computerized modeling based on (Q)SAR, in silico models in the future may replace some of the animal tests at least. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  32. 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 EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  33. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  34. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  35. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  36. In Vitro Models-Cell Cultures

  37. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  38. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  39. 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.

  40. 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 ) EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  41. 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. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  43. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  44. 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 EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  45. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  46. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  47. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  48. DISADVANTAGES OF In Vitro METHODS 4. There are not definitive and precise test procedures for in vitro toxicity tests given by regulatory authorities. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  49. 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. EUROTOX Advanced Toxicology Course-2013-Volos-Greece

  50. The advantages and disadvantages of different in vitro models

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