WELCOME

1. WELCOME • Course in applied toxicology November 2007 • The course programme • Course material • Presentation of participants • General overview of Toxicology

2. Me – Grete Østergaard • DVM in 1983, ph.d. in 1997, MLAS 2006 • 1983-85 – vet practice, slaughterhouse • 1985-88 – clinical research associate • 1988-2003 – toxicologist National Food Agency • (research area behavioural studies) • 2003- ….. – lab animal vet at Copenhagen University

3. Course material • Toxicological assessment • List of abbreviations commonly used in risk assessment • List of adopted OECD guidelines (chemicals) • List of adopted OECD guidance and review documents • List of EU guidelines (chemicals) • List of ICH guidelines (human medicine) • List of VICH guidelines (veterinary medicine) • TGD content • TGD effects assessment • EU ”labelling guide” • OECD principles on good laboratory practice • What to include in a toxicological summary • Necropsy guide • List of literature

5. Course participants: Please present yourself • Anna Cecilie Skovgaard • Mia Birkhøj Kjærstad • Camilla Taxvig • Morten Kobæk Larsen • Janne D. Olling • Maja Myren • Line Mathiesen • Lone Kielberg • Marie Louise Hagen • Ann Dyreborg Larse • Rasmus Lundquist • Marie Frederiksen • Morten Hellmers Fjordholt • Janne Kjærsgaard Folkmann

6. Aim of the course • To provide you with a solid background for work with toxicological risk assessment • Realistic goals in 2 weeks: • Necessary basic knowledge • Examples • Reference to further reading

7. The lectures • Toxicological endpoints and guidelines: Irritation, sensitisation, acute toxicity…. • Elements of guideline studies: Necropsy, haematology, clinical chemistry… • Practical advice: Study planning, how to find data…. • General items: GLP, overview of lab animals…. • Areas of regulation: Pharmaceuticals, Working environment… • Risk assessment… • Visit to contract test lab Scantox

8. Toxicology The Greek word for bow is "toxon" and something bow-like or pertaining to the bow is "toxikos." It was discovered that it was far more effective against the enemy to smear a little poison on the end of the arrow, thus making toxicon pharmakon a poison for (smearing) arrows. • The study of adverse effects of chemical substances on living systems • The prediction of effects in man based on data from animals or other test systems

9. Toxicologist • in Denmark not a person with a particular education • Background in natural sciences + special knowledge which can be acquired in various ways – primarily by working with toxicological topics • Veterinarian, biologist, pharmacist, medical doctor, human biologist, engineer • Possibility to become MSc. Applied Toxicology e.g. U. of Surrey

10. What can a toxicologist do? • Make a scientifically based opinion about what can be expected to happen if a human being is exposed to a chemical substance • Can calculate a ”safe” / ”dangerous” dose for human beings • From data from animal experiments and human studies

11. Categories of Toxicology • Regulatory toxicology (pharmaceutical, cosmetic, food, agrochemical, chemical) • Industrial toxicology (pharmaceutical, cosmetic, agrochemical, chemical) • Academic toxicology • Occupational toxicology • Medical/clinical toxicology • Forensic toxicology • Ecotoxicology • Environmental toxicology

12. Eurotox http://www.eurotox.com/ Education

13. Dansk Selskab for Farmakologi og Toksikologi http://www.dsft.dk/

14. Regulatory Toxicology • Important decisions have to be made • a. stop development of a promising new drug thereby losing what has already been spent along with future profit • b. encourage further progress thereby promoting clinical trials with bad results and resulting loss of enormous amount of money • a. prohibit the sale to the public of a household chemical thereby preventing people of a useful remedy and companies of business • b. allow sale thereby risking consumer’s safety & putting damage costs onto the public • a. clean up a building site at enormous cost • b. allow building resulting later on in unsellable houses • Big money is involved – what you say has consequences

15. What can you do? • Use recognized internationally established principles, or be able to explain your chosen strategy convincingly • Use scientific arguments • Be able to explain why you hold a particular view • Argue against views you believe are wrong using scientific arguments • Be consistent – facts are better than beliefs – don’t be pushed • Know when to keep quiet !

16. You only find what you are looking for • Study results refer to a particular situation: E.g. a few young male animals exposed to a low concentration for a few weeks where 5 endpoints were evaluated. • A negative study is only negative for what has been looked for. • In this case: • Conclusions about females, about older individuals, about higher concentrations, about longer exposure durations, about endpoints not among the 5 evaluated • CANNOT be made with certainty.

17. Interspecies comparison

18. Can effects in animals predict effects in humans? • Cellular structure and biochemistry similar among species • Large amount of data shows similarities in toxic effects e.g. cancer, foetal toxicity • Pharmaceuticals for man are developed in animals • Differences are often related to kinetics (absorption, distribution, metabolism, excretion) • Allometry • No animal species is ”most like man” • Certain species may be ”most similar to man” with respect to e.g. sensitivity to certain toxicants

19. Theophrastus Phillippus Aureolus Bombastus von Hohenheim • ”All substances are poisons; there is none that is not a poison. The right dose differentiates a poison and a remedy” ”Paracelsus” 1493-1541 (”the equal of Celsus", an early Roman physician)

20. ”Danger” and ”Risk” • Danger: Inherent or intrinsic dangerous property of a chemical - basis for classification in the EU • Risk: Danger + Exposure • A dangerous chemical can be used in a way where risk is low: • e.g. a carcinogenic substance can be used in a closed industrial processes • Thalidomide can be used by males and non-pregnant females

21. Central dogma Threshold is ”dose or exposure concentration of a substance below that a stated effect is not observed or expected to occur” (OECD 2003)

22. NOAEL and LOAEL Ex. In a 28-day rat study increased frequency of smal foci of liver cell necrosis is the adverse effect.

23. ADI acceptable daily intakeTDI tolerable daily intake • Traditionally calculated on the basis of the NOAEL as • NOAEL / safety factor, often set to 100 • The figure 100 is not scientifically based • In the example ADI is = 100 mg/kg/day : 100 • = 1 mg/kg/day

24. Benchmark dose • Like NOAEL/LOAEL defines a ”point of departure” for establishemt of tolerable exposure • The dose that corresponds to a defined change in an adverse response (benchmark response) e.g. 10% increase above control response • Curve fitting to experimental data - software

25. Benchmark dose modelling illustrating the “Benchmark dose” (BMD), the “Benchmark Response” (BMR), and the “Benchmark dose lower limit” (BMDL).

26. Hormesis The “inverted U” curve, characteristic of the hormetic response of low-dose stimulation and high-dose inhibition.

27. Characterisation of alterations • “adaptive,” exaggerated normal physiologic response; • “pharmacologic,” expected alteration in response to the desired action of the test article • “adverse,”alterations that are generally undesired, progressive and deleterious to the normal function

28. Adversity • Irreversible effects: e.g. lung fibrosis, disturbed development • Progressive effects: e.g. cancer • Heritable effects: mutation • Loss of (important) function: e.g. widespread necrosis, paralysis • Effects that are part of a larger complex of disease or known to be associated with serious health effects

29. How large a deviation can be before it becomes adverse….. • Statistical significance vs. biological significance • When you evaluate studies: Always calculate the percentage of the changes that are found • Case-by-case (which parameter), use 5-10% as a starting point for discussion • Example: Smoking-related retardation in foetal growth: • On average a reduction in birth weight of 200 g (at 3500 g equal to 5.7%)

30. Mechanism of action: Exact molecular mechanism is known, usually includes mention of the specific molecular targets to which the toxicant binds, such as an enzyme or receptor. • some organophosphorus and carbamate pesticides • PCDDs, PCDFs, PCBs • Mode of action: Key events known

31. Animal experiments

32. The traditional ”full picture” of a substance Routes oral inhalation dermal • Endpoints • Acute toxicity • Irritation • Sensitisation • Repeated dose toxicity • Mutagenicity • Carcinogenicity • Reproduction

33. Types of toxicity studies • Single dose vs. Repeated dose • Multiple effects vs. Single effects • Target organ toxicity: integument, digestive, urinary, cardiovascular, haematopoietic, immune, musculoskeletal, nervous, endocrine, respiratory, male reproductive, female reproductive, mammary gland, eye, ear, nose

34. OECD guideline terms • Dosage is a general term comprising the dose, its frequency and the duration of dosing. • Dose-response is the relationship between the dose and the proportion of a population sample showing a defined effect. • Dose-effect is the relationship between the dose and the magnitude of a defined biological effect either in an individual or in a population sample.

35. Elements of toxicity studies • Clinical examination • Behavioural assessment • Clinical chemistry • Haematology • Ophthalmoscopy • Pathology

36. Future: Toxicogenomics? • ”Omics”: Genomics – Proteomics - Metabolomics • determine whether gene, protein or metabolite expression profiles or ”signatures” can serve as markers to predict toxicity • US NIEHS National Center for Toxicogenomics established in 2000 • http://www.niehs.nih.gov/nct/home.htm • Textbook: Hamadeh HK, Afshari CA: Toxicogenomics – Principles and applications. John Wiley & Sons, inc. Hoboken, New Jersy, 2004. • OECD SERIES ON TESTING AND ASSESSMENT Number 50 • REPORT OF THE OECD/IPCS WORKSHOP ON TOXICOGENOMICS • Kyoto, 13-15 October 2004

37. Precautionary principle • “The precautionary principle is a moral and political principle which states that if an action or policy might cause severe or irreversible harm to the public, in the absence of a scientific consensus that harm would not ensue, the burden of proof falls on those who would advocate taking the action.” • “Guilty” unless proven innocent • rather than • “Innocent” unless proven guilty

39. Classification • Evaluation of the hazard of a substance Communication (label) Downstream regulation

40. Endpoints in EU health classification • † acute toxicity • † subacute, subchronic or chronic toxicity • † corrosive and irritant effects • † sensitising effects • † specific effects on health (carcinogenicity, mutagenicity and reproductive toxicity, ”CMR”)

41. Example: • Carcinogenic substances are placed into category 1,2 or 3 based on strength of evidence • Category 1 or 2 => assigned symbol T • Category 3 => assigned symbol Xn

42. Labelling Pictograms • Old EU system: Directive 67/548/EEC, 1999/45/EC New system: GHS Danger Toxic Harmful Warning Corrosive

43. Downstream regulation • § 26. Meget giftige og giftige stoffer og produkter må kun sælges til købere, der afgiver rekvisition efter reglerne i §§ 31-35. Dette gælder dog ikke salg til de i § 27 nævnte virksomheder, institutioner og personer. •     Stk. 2. Meget giftige og giftige stoffer og produkter må ikke sælges til personer under 18 år eller i øvrigt overlades til personer, som må antages at ville forvolde skade på sig selv eller deres omgivelser. •     Stk. 3. Kemiske stoffer, der er optaget på listen over farlige stoffer og klassificeret som kræftfremkaldende, mutagene og reproduktionstoksiske i kategori 1 og 2 og tildelt farebetegnelsen »Giftig«, må ikke sælges en detail til offentligheden. Bekendtgørelse om klassificering, emballering, mærkning,salg og opbevaring af kemiske stoffer og produkter

44. End