1. ENDOCRINE DISRUPTION DOES THIS POSE SPECIAL DIFFICULTIES WHEN ASSESSING RISK? Sue Barlow Independent Consultant in Toxicology

2. Scope Distinguish between endocrine disrupter and potential endocrine disrupter Distinguish between endocrine disrupter and potential endocrine disrupter Discuss the special difficulties with ED evidence Discuss the special difficulties with ED evidence Discuss the WHO/IPCS model for evaluating evidence on EDs and illustrate with an example Discuss the WHO/IPCS model for evaluating evidence on EDs and illustrate with an example [Summary evidence on pesticides with ED activity] [Summary evidence on pesticides with ED activity]

3. DEFINITIONS Endocrine disruptor Endocrine disruptor A substance or mixture that alters function(s) of the endocrine system causing adverse effects in an intact organism, or its progeny, or (sub)populations Potential endocrine disruptor Potential endocrine disruptor A substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub)populations

4. WHAT ARE THE SPECIAL DIFFICULTIES? Is endocrine disruption a new phenomenon? Is endocrine disruption a new phenomenon? Are there more endocrine-active chemicals than we thought? Are there more endocrine-active chemicals than we thought? Do standard toxicity tests address endocrine-related endpoints? Do standard toxicity tests address endocrine-related endpoints?

5. WHAT ARE THE SPECIAL DIFFICULTIES? Can they have unusual dose-response curves? Can they have unusual dose-response curves?

6. Dose-response curves

7. Non-monotonic dose- response curve Effect of hexachlorobenzene (an androgen agonist) on androgen response in prostate cancer cells The red line is the level of response obtained by DHT without any HCB present. At levels of HCB exposure around 1 nM (parts per billion) there was up to a doubling of the androgenic response in the presence of DHT. But at very high levels, the androgenic response was repressed.

8. WHAT ARE THE SPECIAL DIFFICULTIES? Can they be active at low doses? Can they be active at low doses? Should all effects be seen as adverse? Should all effects be seen as adverse?

9. How can the evidence be assessed?

10. INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY The IPCS GLOBAL ASSESSMENT of the State of the Science OF ENDOCRINE DISRUPTORS (GAED) 2002 www.ehponline.org/who/

11. CAUSAL CRITERIA FOR ASSESSING ENDOCRINE DISRUPTORS GAED used causal criteria for assessing EDs adapted from GAED used causal criteria for assessing EDs adapted from the Bradford-Hill criteria (1965), widely used for assessing human epidemiological evidence

12. PURPOSE OF THE CAUSAL CRITERIA Provide a framework to assemble and review the body of knowledge on an adverse health event with a potential endocrine-related basis Provide a framework to assemble and review the body of knowledge on an adverse health event with a potential endocrine-related basis Use multiple lines of evidence to bring considerable amounts of information to bear Use multiple lines of evidence to bring considerable amounts of information to bear Focus on the underlying biological alterations in the direct line between an exposure and an adverse outcome Focus on the underlying biological alterations in the direct line between an exposure and an adverse outcome Assess the overall coherence and strength of the evidence that a particular situation is, or is likely to be, due to an alteration in an endocrine system Assess the overall coherence and strength of the evidence that a particular situation is, or is likely to be, due to an alteration in an endocrine system Identify research gaps Identify research gaps

13. CAUSAL CRITERIA FRAMEWORK Statement of Hypothesis Outcome of concern Stressor of Concern Assessment Factors Temporality Strength of association Consistency Biological Plausibility Recovery Overall Strength of Evidence For the outcome For the hypothesis For an EDC mechanism

14. CAUSAL CRITERIA ASSESSMENT FACTORS Temporality Does the presumed cause of the outcome of concern precede its appearance ? Strength of Association What is the incidence rate ? What is the risk attributable to exposure ? Shape of dose-response curve ? Consistency of Observations Are there similar or dissimilar findings in the literature ? Is it seen in multiple geographic areas and/or species ? Does it occur at similar doses ? Biological Plausibility Is there a possible/known endocrine mechanism of action ? Evidence of Recovery Is the adverse outcome reversible when exposure diminishes?

15. CASE STUDY Human Sperm Quality Hypothesis Hypothesis Global reductions in human semen quality over time are related to exposure to oestrogenic and/or anti-androgenic chemicals during critical phases of testis development Global reductions in human semen quality over time are related to exposure to oestrogenic and/or anti-androgenic chemicals during critical phases of testis development

16. The initial evidence Sperm Counts

17. Trends in human sperm quality Temporality No data Strength of association No data for causal association Moderate for effect Consistency of with other observations No data for causal association Weak for effect Several studies show significant decline in sperm quality over time but no data on preceding chemical exposures, especially during early development No data relating possible cause (chemicals) to ↓ sperm Carlsen et al. meta-analysis 1938-1990 shows 50% decline over 50 years 1.5% per year in USA; 3.5% per year in Europe No data on consistency of effect and exposure Longitudinal studies in single centres: 10 show decline 10 show decline 6 improvement 6 improvement 8 no change 8 no change Two ‘time to pregnancy’ studies not consistent with decline

18. Trends in human sperm quality Biological plausibility StrongRecovery No data Endogenous oestrogens control testis development (but prenatal exposure to DES, OCs no effect on human fertility) Support from related trends in human testis cancer and male reproductive tract abnormalities Support from animal studies (e.g. prenatal exposure to oestradiol, nonylphenol, methoxychlor, vinclozolin, phthalates, dioxins) (e.g. prenatal exposure to oestradiol, nonylphenol, methoxychlor, vinclozolin, phthalates, dioxins) No relevant data

19. Overall strength of evidence on sperm quality Human health outcome Putative stressors of concern Strength of evidence for hypothesis Strength of evidence for hypothesis Strength of evidence for EDC mechanism Decline in human sperm quality Oestrogens & anti- androgens No data Weak

20. CONCLUSIONS Interpreting data on endocrine disruption requires good knowledge of endocrinology, mechanisms of action and toxicology Interpreting data on endocrine disruption requires good knowledge of endocrinology, mechanisms of action and toxicology In vitro evidence can indicate possible hazard but is insufficient by itself to demonstrate risk to humans In vitro evidence can indicate possible hazard but is insufficient by itself to demonstrate risk to humans In vivo evidence from animal studies shows several pesticides are EDs In vivo evidence from animal studies shows several pesticides are EDs Conventional risk assessment approaches can be applied to in vivo data and may allow setting of acceptable exposure limits Conventional risk assessment approaches can be applied to in vivo data and may allow setting of acceptable exposure limits Lack of studies on human health outcomes with adequate pesticide exposure history precludes conclusions on causality Lack of studies on human health outcomes with adequate pesticide exposure history precludes conclusions on causality

21. Annex on pesticides with endocrine activity

22. PESTICIDES WITH ENDOCRINE ACTIVITY ENDOCRINE ACTIVITY EFFECTSEXAMPLES Oestrogenic in vitro &/or in vivo (ERα,β agonists) Bind to oestrogen receptors Proliferation in breast cancer cell lines ↑ Uterine weight in uterotrophic assay Chlordane o,p’-DDT DieldrinEndosulfanFenarimolFenvalerateToxaphene Methoxychlor metab

23. PESTICIDES WITH ENDOCRINE ACTIVITY ENDOCRINE ACTIVITY EFFECTSEXAMPLES Inhibition of aromatase (Converts testosterone to oestradiol) Inhibit male mating behaviour FenarimolImazalilProchlorazConazoles Induction of aromatase Feminisation Delays puberty in males Atrazine Inhibition of 5α- reductase (Converts testosterone to dihydrotestosterone) ↓ Prostate growth Atrazine

24. PESTICIDES WITH ENDOCRINE ACTIVITY ENDOCRINE ACTIVITY EFFECTSEXAMPLES Suppression of luteinising hormone and prolactin surges (Hypothalamus) Delay puberty Disturb oestrous cycles Reduce implantation Earlier onset of mammary tumours Atrazine Androgenic (AR agonists) Masculinisation of females No known pesticide examples

25. PESTICIDES WITH ENDOCRINE ACTIVITY ENDOCRINE ACTIVITY EFFECTSEXAMPLES Anti-androgenic (AR antagonists) In male offspring: ↓ Anogenital distance Nipple retention Hypospadias ↓ Testis and other sex organ weights ↓ Sperm count, fertility Delay puberty Hershberger assay in immature males: ↓ 2ry sex organ growth p,p’-DDE FenarimolFenitrothionFenvalerateLinuron Methoxychlor metab ProchlorazProcymidoneVinclozolin

26. REGULATORY ACTIONS Atrazine: banned in EU in 2003 because of unavoidable water contamination Atrazine: banned in EU in 2003 because of unavoidable water contamination TBT: Most antifoulant uses phased out by 2003, remaining uses by 2008 TBT: Most antifoulant uses phased out by 2003, remaining uses by 2008 Alkyl phenols and their ethoxylates: EU Directive prevents use as co-formulants in new products from 2005; voluntary UK agreement to replace AP(E)s in existing pesticide formulations Alkyl phenols and their ethoxylates: EU Directive prevents use as co-formulants in new products from 2005; voluntary UK agreement to replace AP(E)s in existing pesticide formulations Vinclozolin, Procymidone, Fenarimol: Vinclozolin, Procymidone, Fenarimol: EU discussing phasing out all uses