Emerging Contaminants – do we need to change the paradigm?

1. Emerging Contaminants – do we need to change the paradigm? Maria Fürhacker University of Natural Resources and Applied Life Sciences, Vienna Muthgasse 18, A-1190 Wien maria.fuerhacker@boku.ac.at

2. Content • Introduction • Current approach of risk assessment • New approaches • TTC concept • German, • IWA Conference • Toxicity testing in the 21st century • Conclusions

3. Chemicals in the market • 100 000 substances registered in 1981 in the EU as „existing substances“ • 8700 different food additives are in use • 3300 pharmaceuticals in human medicine • In addition: unintentionally produced substances, by-products, reaction intermediates, degradation products etc. …resulting in complex mixtures of environmental contaminants

4. Problem I: Delay in action PCB banned US 1977 EU 1989 Russia 1999 Problem II global distribution WFD: PBT criteria UNEP: PB, long range transport

5. Personal care products Drugs, hormons, antibiotics fragrances sun screens repellents Industrial chemicals PFC, PFOS, PFOA – perflourinated compounds QAC – quarternery ammon compounds BPA – bisphenol A (Nitro)PAH – nitro polycyclic aromatic compounds Oxygenates – MTBE, ETBE Pesticides Nanomaterials NP,NPEO-nonylphenolethoxylates PBC – polybrominated compounds TBT – tributyltin Perchlorate And many more……. What are „Emerging Contaminants“ „ new chemicals or new because of the development of new analytical methods….“ „ contaminants that are currently not included in routine monitoring programs and which may be candidates for future regulation, depending on their (eco)toxicity, public perception and monitoring data revealing their occurrence in different environmental compartments“ (TT21C) Food ingredients, additives • Artificial sweeteners • Capsaicine, solanine, etc. Explosives e.g. TNT and degradation products

6. The dilemma: It is hard to keep pace with the newly detected and newly released chemicals in the environment - we have only minimum understanding of toxicity for most chemicals!Precautionary principle :: risk assessment – are we on the right track at all?

7. Basic approaches for standard setting Risk assessment Predicted environmental Concentration (PEC) Predicted no effect conc. (PNEC) Precautionary principle DW-values 0.1 µg/l for pesticides and metabolites (DWD, 1998) improving static European Medicines Agency EMEA, 2006 0.01 µg/l GW-values 0.01 µg/l PEC/PNEC > 1 LAWA, 2004 0.01 µg/l In case the knowledge for RA is not available at a proper time, no action is taken. PP acts in advance // RA is lacking behind Take action

8. Can we drink the water? bacterial toxins animal toxins viral toxins plant toxins bacterial toxins pesticides animal toxins viral toxins PPCP plant toxins drugs pesticides DBP PPCP drugs DBP risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk risk

9. Do we ask the right questions?

10. Are food/water contaminated – what is in? aflatoxine B1 Symptoms include nausea, diarrhea, vomiting, stomach cramps, burning of the throat, cardiac dysrhythmia, headache and dizziness. In more severe cases, hallucinations, loss of sensation, paralysis, fever, jaundice, dilated pupils, hypothermia and death have been reported. highly carcinogenic solanine High-level aflatoxin exposure produces an acute hepatic necrosis, resulting later in cirrhosis, and/or carcinoma of the liver. Toxic for humans: 3 to 6 mg per kilogram of body weight Normal concentration: <0.2 mg/g Green potatos: 1 mg/g or more

11. From the uptake to the effect Mode of toxic action External concentration Concentration on site of action Adverse toxic response Toxikodynamics Receptor binding Protein binding Cellular changes Energy allocation Physiological compensation Toxikokinetics Uptake Adsorption Distribution Metabolism Excretion Toxicant interaction with target Cellular response Organ response Organism response Population response

12. First step: mode of action and target identificationTranscription factors: sensor and transducer Oxidative stress response Heat shock response DNA damage response Hypoxia - deprived of adequate oxygen supply Endoplasmatic reticulum pathways Specific endogenic receptor pathways Estrogen, androgen and thyroid hormone signaling Second step: link concentrations to excessive perturbations (likely to lead to adverse effects) Toxicity Pathways (Bhattacharya et al., 2011) There is a finite number of core stress response pathways to either maintain homeostasis or apoptosis Goal: moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Toxicity testing in the 21st century (TT21C) (Bhattacharya et al., 2011)

13. Suggestions for biomarkers: Aryl hydrocarbon receptor (AhR), Cytochromes and chaperons A biomarker, is a substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. • AhR is a ligand-activated-transcription factor – key regulator of the cellular response to xenobiotic exposure. AhR is strongly activated by organic compounds: e.g. PPCD/F, PCB, PAH also endogenous and natural compounds • Cytochrome P450 superfamily (CYP) is a large and diverse group of enzymes. The function of most CYP enzymes is to catalyze the oxidation of organic substances such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. • Many Chaperones many of them are heat shock proteins - expressed in response to elevated temperatures or other cellular stresses • In mammals 48 nuclear receptors (NR) are involved in all vital functions – fetal development, metabolism, homeostasis, reproduction, response to xenobiotic substances

14. Effects of priority concern For single substances or mixtures Problem: margin of safety

15. What are the emerging trends on micropollutants issues?

16. The threshold of toxicological concern (TTC) is based on the concept that there are levels of exposure to chemicals that will result in no appreciable risk to health. TTC identifies de minimis values for chemicals which share similar structural characteristics Metabolitescould potentially be excluded from the residue definition as not being toxicologically significant – if they meet a threshold relevant to their chemical structure Threshold of toxicological concern (TTC) This decision would not require additional toxicological testing! (?)

17. TTC values for different endpoints The data show that the distribution of 10-6 risk doses for carcinogens is significantly different from the distribution for the non-cancer NOELs/100. None of the non-cancer endpoints studied was more sensitive than cancer endpoints.

18. TTC - Concept Threshold of regulation based on carcinogenic risk (below 10-6 risk): 1.5 μg/person/day (1.5 kg food and 1.5 l beverage/d, safety factor 100) is highly protective • For migration of chemicals from food packaging. • Munro et al. 1996 extented to low molecular flavouring additives • Application of the TTC concept to wider food safety evaluation • (Non relevant) Metabolites of pesticides found in groundwater • Genotoxic impurities in pharmaceuticals • Cosmetics • Consumer products and household care products • Industrial chemicals - REACH high potency substances are (e.g. N-nitroso compounds, strained heteronuclear rings, heavy metals, hydrazines and azoxy compounds)

19. German approach for non rel. pesticides and biocides in DW (UBA 2003) … the German UBA propose permanently tolerable health-related indication values (HRIV) based on the TTC-concept …. for all substances which are not primarily gentoxic, but cannot yet be toxicologically assessed on the basis of chronic or subchronic animal experiments, and which show no signs, however, of neurotoxic, immunotoxic or germ-cell toxic potential. It has the regulatory function of a precautionary value

20. Escher, (2011) used different bioassays for the monitoring of WWTP bioluminescence inhibition assay (baseline toxicity), acetylcholinesterase (AChE) inhibition (neurotoxicity), combined algae test (phytotoxicity), E-SCREEN (estrogenic effects), AhR CAFLUX (binding to Ah receptor), umuC (SOS response, genotoxicity) and glutathione assay with E.coli (reactive toxicity) Effect monitoring (IWA conference 2011)

21. Effect monitoring (IWA conference 2011) Leusch (2011) tested water from reclamation plants in the full battery of bioassays (human relevance), (monitoring of 39 priority compounds) Assays for basal cellular toxicity (cell viability of gastro-intestinal, liver and white blood cells), mutagenicity (Ames test), genotoxicity (micronucleus test), interference with liver function (CYP induction in liver cells), endocrine effects (estrogenic, androgenic, glucocorticoid, progestagenic and thyroid activity in reporter gene assays), neurotoxicity (acetylcholinesterase assay) and immunotoxicity (monocyte cytokine production assay). The results indicate that bioassay data were in agreement with chemical trends. For example, endocrine activity was correlated with natural and synthetic hormones, neurotoxicitywith insecticides, and genotoxicity, mutageninicity and immunotoxicity with disinfection by-products.

22. Alternatives to animal models: cosmetic industry and the animal testing ban • Biomarkers • Application of “Omics” in risk assessment (genomics, proteomics, metabolomics) • Epigenetics in risk assessment • Risk assessment in complex samples Combination of scientifically reliable alternatives, as well as the evolution of regulatory initiatives to evaluate, integrate, and accept these alternatives.

23. Toxicity testing in the 21st century (TT21C) (Bhattacharya et al., 2011) Comparison of current (A) and proposed (B) toxicity testing paradigms.

24. Conclusions I Yes, we need to change the paradigm – • (develop) and apply test systems based on relevant mode of actions • change the RA approach from animal based toxicity tests to “in vitro” tests • focus on the investigation of complex mixtures

25. Conclusions I Yes, we need to change the paradigm – • and seek the cooperation • in the scientific community including toxicology, medicine, molecular biology, biology, biochemistry …. • and between science, administration and politics

26. Thank you for your attention maria.fuerhacker@boku.ac.at