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

2. 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.

3. 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).

4. 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

5. 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”.

6. 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)

7. 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.

8. 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.

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

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

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

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. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 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.OECD 401, 1987

15. 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.OECD 402, 1987

16. 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.

17. 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.

18. 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.

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

20. 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.

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. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 23 Acute Toxic Class Method (ATC) (OECD 423) In 1996, a second alternative method, the ATC was adopted (OECD 423, 2001).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.

24. 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).

25. 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%.

26. 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.

27. 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.

28. 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. SIGHTING STUDY YesNo 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).

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

30. 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:

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

32. 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

33. 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.

34. 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.

35. 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.

36. In Vitro Models- Cell Cultures

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

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

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

41. 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.

42. 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.

43. 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.

44. 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

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

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

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

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

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

50. The advantages and disadvantages of different in vitro models ModelsAdvantagesDisadvantages 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

51. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 51 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.

52. 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).

53. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 53 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.

54. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 54 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.

55. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 55 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.

56. Skin Irritation Tests Skin irritation tests are Episkin, Epiderm, pig ear, and PREDISKIN.

57. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 57 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.

58. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 58 Epiderm Epiderm incorporates normal human keratinocytes cultured on permeable Milipore membranes. Toxicity is assessed by using MTT assay. The primary end point is IC50.

59. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 59 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.

60. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 60 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.

61. The Skin Corrosion Tests The skin corrosion tests are Corrositex, Transcutaneous electrical resistance (TER) and SKINETHIC.

62. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 62 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.

63. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 63 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.

64. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 64 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.

65. 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.

66. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 66 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.

67. 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.

68. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 68 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.

69. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 69 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.

70. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 70 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.

71. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 71 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.

72. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 72 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.

73. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 73 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.

74. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 74 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.

75. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 75 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

76. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 76 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.

77. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 77 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.

78. EUROTOX Advanced Toxicology Course-2013- Volos-Greece 78 Others Placenta (PCBs, PAHs, phthalates) Bones (Pb) Faeces (Pb, Cd, PCBs) Teeth

79. Thanks for listening…..