1. Which information identifies a chemical as endocrine disrupting? Poul Bjerregaard Institute of Biology University of Southern Denmark Odense and Danish Centre on Endocrine Disrupters UNEP EDC Advisory Group meeting, Geneva, September 25-26, 2015 1

2. WHO EDC definition 1.Endocrine mechanism 2.Adversity 3.Plausible link between the two 2

3. Adversity according to WHO/IPCS 2004 A change in morphology, physiology, growth, reproduction, development or lifespan of an organism which results in impairment of functional capacity –or impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other environmental influences 3

4. Adversity? Risk assessment of chemicals –What do we want to protect? Human risk assessment: –Every individual Environmental risk assessment: –The structure and function of the ecosystem 4

5. Environmental risk assessment Protection of ecosystem structure and function The aim is to protect 95% of the species ‘Adversity ’defined from population effects –Not effects on the individual 5

6. Environmental risk assessment Protection of ecosystem structure and function The aim is to protect 95% of the species ‘Adversity’ defined from population effects –Not effects on the individual 6

7. Relevant OECD test guidelines Human risk assessment –TG 416 or 433 (mammals) E.g. malformed male genitals, altered AGD Environmental risk assessment –TG 234 Fish Sexual Development Test 7

8. Altered content of yolk proteins –Controlled by oestrogen Indicates endocrine mechanism - not adversity Altered sex ratio –Indicates endocrine mechanism and adversity –OECD Guidance Document 150 8

9. Danish suggestion for EDC-criteria Category 1 – Endocrine Disrupter –Adverse in vivo effects –ED mode of action in vivo clearly linked to adverse in vivo effects Category 2a – Suspected Endocrine Disrupter –Adverse effects in vivo where an ED MoA is suspected –ED MoA in vitro combined with toxicokinetic in vivo data –ED MoA in vivo suspected to be linked to adverse effects in vivo Category 2b – Indicated Endocrine Disrupter –in vitro/in silico evidence indicating potential for ED in vivo 9

10. Exemplified by chemical UV filters Danish 2012-assessment Category 2a Category 1 BP-3: Indications of oestrogenic and anti-androgenic effects. Not consistent TG234 needed 10

11. Sexual development in zebrafish Hatch ♀ ♂ 20 days40 days 60 days 11

12. Sex ratio altered – more ♀ Kinnberg et al. 2015. Environ.Toxicol.Chem. In press. 12

13. No consistent effect on yolk proteins 12 d exposure 13

14. Conclusion Exposure to benzophenone 3 skews sex ratio –so Benzophenone 3 is an EDC 14

15. Is BP-3 oestrogenic or anti-androgenic? Does it matter ? Depends on the requirement for detailed knowledge about the mechanism of action Detailed knowledge about ‘Adverse Outcome Pathways’ is desirable But not always obtainable 15

16. A chemical may have multiple AOPs Prochloraz Fungicide 16

17. Prochloraz: More male zebrafish % 0 20 40 60 80 100 Female Male Intersex Undifferentiated Control 16  g/l Prochloraz 64  g/l Prochloraz 202  g/l Prochloraz 73 72 69 * Kinnberg et al. 2007. Comp. Biochem. Physiol.145C, 165-170 17

18. Prochloraz is anti-androgenic in rats Laier et al. 2006. Toxicol.Appl.Pharmacol. 213, 160-171 18

19. Effect of prochloraz Cholesterol Testosterone Oestrogen Prochloraz Inhibits aromatase in zebrafish (?) Prochloraz Anti-androgenic in rats 19

20. Conclusion Detailed knowledge on ‘Adverse Outcome Pathways’ is not necessarily needed to reach conclusions on endocrine disrupting activity 20