1. Endpoints for prenatal exposures in toxicological studies Alberto Mantovani Dept. Food Safety and Veterinary Public Health Istituto Superiore di Sanità, Rome, Italy ++39 06 4990 2658; alberto@iss.italberto@iss.it Website: http://progetti.iss.it/inte/ http://progetti.iss.it/inte/

2. WHY DEVELOPMENTAL TOXICOLOGY ? Unique sensitivity of the developing organism Dynamic sequence of events: one event delayed/disrupted leads to persistent sequelae Highly sensitive to factors affecting Proliferation/differentiation

3. STANDARD APPROACHES: Prenatal toxicity test: Specific attention to organogenesis/pregnancy (OECD 414) One and Two-generation test: Overall assessment of effects from fertilization through to postnatal

4. PRENATAL TOXICITY (OECD 414) birth defects AND minor anomalies (may signal severe effects at higher doses) at external/skeletal/visceral level, AND fetal growth AND viability AND degree of ossification (marker of maturation) ANATOMICAL LEVEL

5. PRENATAL TOXICITY (OECD 414) Importance for risk assessment: short-term, even single, exposures in susceptible developmental phases e.g., pharmaceuticals (thalidomide) Workplace Food outbreaks (PCBs in Far East)

6. PRENATAL TOXICITY More attention to compounds with target-organ toxicity (e.g., organs needing plasticity to respond to environmental stimuli: brain, kidney) Mantovani & Calamandrei 2001Current Pharm Design,7: 859- 82 Taylor-made assessments based on functional maturation markers ?

7. IN VITRO TESTS MAY HAVE A ROLE ? YES. Several intersting ones ( rodent embryonic stem cells or whole embryos) NONE may mimic the complexity of in vivo development BUT they may be combined in a battery (EU project REPROTECT At http://ecvam.jrc.it/index.htm)

8. IN VITRO TESTS Problems to be faced: - Metabolic activation systems (multiple mechanisms/pathways) - Incorporation of molecular biology tools (useful for subtle effect ?) - Mechanisms are important for Susceptibility Prevention/treatment Interactions with other factors (antiepileptics potentiated by CYP inhibitors: Tiboni et al., Toxicol Lett. 2003 145:219- 29.

9. ATTENTION SHIFTS more and more towards subtle, late-appearing effects of low exposure levels relevant to the general population, such as intakes of food contaminants.

10. THE 2-GENERATION STUDY (OECD 416) Continuous exposure of Parental Generation, producing Generation 1, producing Generation 2 Generation 1 is exposed during the whole development, from gamete stage through to sexual development.

11. ENDOCRINE DISRUPTERS A lot of knowledge accumulating, Still a good challenge due to critical developmental role of hormone homeostasis

12. POTENTIAL to induce Long-term of effects upon prenatal exposure at dose levels well below those inducing “teratogenicity” or “fetotoxicity” e.g., Impaired spermatogenesis by DES and Lindane (Traina et al., Reprod Toxicol 2003, 17; 25-35) Impaired histogenesis of thyroid and adrenals by methyl Thiophanate (Maranghi et al., Reprod Toxicol 2003, 17: 617-23)

13. Multiple developmental effects: -Reproductive (antiandrogens such as dicarboximide fungicides: Gray et al., Adv Exp Med Biol. 2004;545:217-41) - Immune (AhR agonists such as TCDD; Nohara et al., Toxicology 2000 154:123-33; ER agonists such as Bisphenol A, Yoshino et al., Immunology 2004 112:489-95) - Neurobehavioral (thyroid inhibitors such as persistent organochlorines, Bowers et al., Toxicol Sci. 2004 77:51-62)

14. Multiple developmental effects (cont): -Impaired differentiation of target tissues that might linked to increased susceptibility to cancer: e.g., breast (Bisphenol A: Markey et al., Biol Reprod. 2001 65:1215-23; TCDD: Fenton et al., Toxicol Sci. 2002 67:63-74) Testis (di-n-butyl phthalate: Mahood et al., Endocrinology, 2005 146: 613-23

15. MANY PARAMETERS Histology-histomorphometry Functional: - neurobehavioral, - sperm analysis, - oestrus cycling Molecular biology AND prolonged observation time !

16. The 2-generation assay may become an organizational nightmare How many studies of reasonable quality in a reasonable time ? TIERED AAPPROACH as endorsed by OECD (Gelbke et al., Toxicology. 2004 205:17-25)

17. In vitro/in vivo screening battery (ER ? AR ? Thyroid ? Aromatase ?) Identify most relevant actions Target endpoints of 2-generation study, that will follow-up late outcomes identify most relevant NOELs

18. HOT TOPICS in the field of Developmental Toxicology Examples of rodent/human differences - fetal period “compressed”: newborn rats as 8-moth human fetuses (incomplete brain-blood barrier, etc.) but are outside from mom’s body ! -reduced intrauterine growth associated with pubertal precocity in humans (Neville & Walker 2005 Arch Dis Child. 90:258 61), pubertal delay in rats (van Weissenbruch 2005 Endocr Dev. 8: 15-33)

19. LATE OUTCOMES -Refinement of neurobehavioral testing (not only learning, also social interactions; extrapolation ?) - Immune development battery (what kind of challenges ? Autoimmunity ?)

20. LATE OUTCOMES - Late outcomes of effects on growth (body composition ?): highly relevant to fetal origins of adult disease http://www.som.soton.ac.uk/research/fo ad/centre - Prediction of cancer risk: Assess modulation of relevant genes ? Transgenic animals might help ?

21. PERSPECTIVES -Tiered approaches to pinpoint issues (Compounds/effects) that do matter - Innovative tools, the “omics”: fascinating and fashionable BUT validation/standardization/quality issues More developmental biology inputs to understand the meaning of toxicological endpoints