Use of Developmental Neurotoxicity data in Risk Assessment at EPA: Current Status and Future Efforts

1. Use of Developmental Neurotoxicity data in Risk Assessment at EPA: Current Status and Future Efforts Kathleen Raffaele, EPA/OPP William Mundy, EPA/ORD

2. Overview • Developmental Neurotoxicity (DNT) Guideline Development • Overview of DNT Study design • Current status of DNT review in OPP/HED • Ongoing Efforts • OECD Draft TG426 • Part 158 Revisions • ILSI Projects • OPP/ORD Collaborative Efforts

3. DNT Guideline Development • 1986 - US EPA OPPT published first draft DNT protocol for peer review and public comment • 1991 - US EPA OPPTS published final DNT guideline (§83-6) • 1995 - OECD Working Group on Reproduction and Developmental Toxicity (Copenhagen) recommended development of OECD Developmental Neurotoxicity Test Guideline • 1996 - OECD Expert Consultation Meeting (Copenhagen) provided recommendations for development of Draft OECD 426 • 1998 - US EPA OPPTS issued minor revisions and harmonization of DNT guideline (OPPTS 870.6300) • 1998 - Draft TG 426 submitted to National Coordinators for expert review and comment • 2000 - OECD Expert Consultation meeting (Washington) held to review technical issues • 2003 - Draft TG 426 submitted to National Coordinators for expert review and comment • 2005 - OECD Expert Consultation Meeting (Tokyo) convened to respond to remaining comments on Draft TG 426

4. Gestation Day 0 22 6 Postnatal Day 0 21 11 ~60 Sexual Maturation Growth & Survival Functional Observations Motor Activity Auditory Startle Learning & Memory Brain Wt & Neuropathology No Treatment Offspring Evaluations Treatment Optional (Instead of PND 11) Preferred Extension of Treatment EPA Developmental Neurotoxicity Study - OPPTS 870.6300 -

5. Use of DNT Studies in OPP • Infrequent submission prior to 1996 • Passage of Food Quality Protection Act • Increased emphasis on evaluating risk to children from pesticide exposure • Focus on neurotoxicity • Data call-in for DNT studies on organophosphate pesticides (1999)

6. DNT Studies submitted to OPP

7. Use of DNT Studies in Single Chemical Assessments(Preliminary analysis) • Study Reviews Completed – 58 chemicals • Chemicals with risk assessments based on DNT-related endpoints – 8 • Potential impact in future risk assessments - 18 • [Abstract submitted for presentation at SOT 2007]

8. Ongoing DNT-related Efforts • OECD GD426 • Part 158 Revisions • ILSI • OPP/ORD Collaborative Efforts • Positive control data evaluations • Historical control data comparisons • Retrospective Review • ORD Efforts • Prioritization/Screening protocols

9. OECD Draft TG426 • 1996 – DNT Guideline development initiated, with EPA as lead country • 1998 – Draft TG426 first submitted for comment • 2005 – Expert meeting in Tokyo to resolve outstanding issues • Fall 2006 – Recirculate for comments • 2007 (projected) – Finalization

10. Differences between current EPA and draft OECD DNT Guidelines

11. Revision of Part 158 Pesticide Toxicity Testing Requirements • DNT Guideline (870.6300) added to table as ‘Conditionally Required’ • A DNT study would be required using a weight-of-evidence approach when considering: • i. The pesticide causes treatment-related neurological effects in adult animal studies (i.e., clinical signs of neurotoxicity, neuropathology, functional or behavioral effects). • ii. The pesticide causes treatment-related neurological effects in developing animals, following pre- and postnatal exposure (i.e. nervous system malformations or neuropathy, brain weight changes in offspring, functional or behavioral changes in the offspring). • iii. The pesticide elicits a causative association between exposures and adverse neurological effects in human epidemiological studies. • iv. The pesticide evokes a mechanism that is associated with adverse effects on the development of the nervous system (i.e. SAR relationship to known neurotoxicants, altered neuroreceptor or neurotransmitter responses) • Projected publication in April, 2007

12. ILSI Project: Evaluation and Interpretation of Neurodevelopmental Endpoints for Human Health Risk Assessment • Initiated in 2004 • Workgroups evaluating 5 areas: • Application of developmental neurotoxicity testing to public health protection • Undertaking a Positive Control Study as part of a Developmental Neurotoxicity testing procedure • Identification and interpretation of treatment-related effects in developmental neurotoxicity testing • Framework for Determining Normal Variability for Endpoints measured in a Developmental Neurotoxicity Test • Statistical techniques appropriate for Developmental Neurotoxicity Testing • Seminar at NBTS Meeting in June, 2005 • Publication in peer-reviewed literature (expected 2007)

13. OPP/ORD Collaborative Efforts – Positive Control Data Evaluation • DNT Guideline requires submission of positive control data to support laboratory ability to detect treatment-related effects • Positive control (PC) studies are submitted to EPA along with DNT studies • OPP (HED)/ORD completed an initial evaluation of the completeness and quality of these data, with the following findings : • Submissions were incomplete for many laboratories • Many reporting deficiencies were identified for PC studies • Some submitted studies did not adequately demonstrate sensitivity of methods (Crofton et al., 2004) • HED is currently reviewing status of supporting positive control data for all submitted DNT studies

14. OPP/ORD Collaboration – Historical Control Data Evaluation • Analysis of historical control data from submitted studies to evaluate: • Data variability • Baseline stability • Data reporting • Completeness of individual results • Completeness of methods

15. Historical Control Data Evaluation:Methods • Identify available data for a given endpoint • Multiple studies from the same laboratory • Studies from multiple laboratories • Tabulate data • Methodology • Control means • Control variability • Summarize results and compare within and across laboratories

16. Historical Control Data Evaluation:Results • Results presented as a series of SOT posters: • Positive control data (Crofton et al., 2004) • Motor activity (Raffaele et al., 2003) • Auditory startle (Sette et al., 2004) • Learning and memory testing (Raffaele et al., 2004, 2006) • Morphometry (Crofton et al., 2001; Raffaele et al., 2005) • Direct dosing (Makris et al., 2005, 2006)

17. Historical Control Data Evaluation (Results, continued) • Methodology • Varied considerably from lab to lab • Different devices used • Multiple types of activity chambers for motor activity • 7 different learning and memory tasks • Procedural differences within tasks • Different stimulus intensity for auditory startle • Different duration for motor activity testing • Reporting was incomplete for some labs • Procedural information was sometimes incomplete • Not all data were reported for some endpoints (e.g., in some cases, only selected trials were reported for learning tasks) • Improved reporting would enhance data interpretation

18. Historical Control Data Evaluation (Results, continued) • Variability • Very high for some labs for some parameters • Not consistent from study to study within labs • For brain morphometry and brain weight • Coefficients of variation (CVs) were lower than CVs for body weight • Corpus callosum measurements were more variable than other brain measures. • For motor activity testing • Decreased with age • No apparent association with device type or session length • For auditory startle habituation • Increased with age • No apparent association with device type or rate • Baseline stability • Varied among labs

19. Historical Control Data Evaluation (Results, continued) • Treatment-related effects on brain morphometric parameters are not predicted by changes in qualitative neuropathological evaluations or by changes in brain weight. • Direct dosing of pre-weaning rat pups (PND 7-21 or 11-21) did not result in increased mortality, increased clinical signs, decreased body weight gain, or differences in brain morphometry.

20. Historical Control Data Evaluation (continued) • Future Efforts • Update motor activity and auditory startle analyses, to include more recent submissions • Continue analysis of learning and memory to include other task types

21. OPP/ORD Collaboration Retrospective Review of DNT Data • To be initiated Winter 2006-7 • Evaluation will include: • Status of positive control data submissions • Data from both control and treated animals • Separate analyses by endpoint • Neuropathology (qualitative and quantitative) • Motor activity • Auditory startle • Learning and memory • Other endpoints as appropriate

22. OPP/ORD Collaboraton – Retrospective Review of Submitted DNT Data • Results will be used to: • Provide a historical control database for reviewer use • Develop a Standard Evaluation Procedure • Develop recommendations for Guideline revision, if appropriate • Assess impact of DNT data on pesticide risk assessment

23. OPP Elizabeth Mendez John Doherty Jess Rowland Louis Scarano Kelly Schumacher ORD/OSCP William Sette ORD/NCEA Susan Makris ORD/NHEERL Kevin Crofton Mary Gilbert Ginger Moser Contributors

24. Developmental Neurotoxicity Testing: Alternatives for Screening and Prioritization William Mundy, Kevin Crofton, and the DNT Team Neurotoxicology Division, USEPA

25. Current Status of Toxicity Testing • Large numbers of chemicals identified for testing (e.g., pesticide inerts, HPVs, CCLs) with no risk-based criteria for setting testing priorities • Different regulatory authorities/different testing requirements with no scientific basis for flexible testing approach • Current guideline testing is expensive, time consuming and requires large numbers of animals

26. Research Challenge • Develop alternative testing approaches that are fast and efficient • Use in vitro cell culture or in silico models • Use alternative species (non-mammalian) • Provide data for prioritization of chemicals for further testing (targeted?) • Such an approach will: • Reduce costs and animal use • Facilitate screening of large numbers of chemicals (high-throughput)

27. Addressing the problem - DNT Objective: Develop and validate relatively rapid, cost-effective, and predictive methods for screening and prioritizing chemicals for their potential to produce developmental neurotoxicity Science Questions: • Can in vitro systems be used to model critical events in normal brain development? • Can we predict developmental neurotoxicity in mammals using a non-mammalian test species? • Can we apply technological advances in high-throughput and genomic technologies to DNT testing?

28. Research Approach - In Vitro • In vitro tests based on key events of brain development • proliferation, differentiation, growth, synaptogenesis, myelination, apoptosis • Endpoints amenable to high throughput testing • cell-based endpoints, biomarkers, molecular signaling • Show predictive ability based on “training set” of developmental neurotoxicants

29. High Throughput Cell-Based Assays using the Cellomics ArrayScan • High Content Analysis • HighThroughput Imaging(automated microscope, image-analysis software) • Data obtained at cell level • Not currently used in developmental neurotoxicology Output: Automated analyses of 96 wells in 20-30 min (200 cells x 96 wells x 10 endpoints per cell = 192,000 data points)

30. Cell-Based Endpoint: Neurite Outgrowth • NS-1 Cells (Clonal PC12 cells) • NGF stimulates neurite outgrowth • Fix and stain 4 days later • Automated assessment in 30 min NGF Concentration Response

31. Research Approach - Alternative Species • Use non-mammalian species (e.g., fish, worms) for development of DNT methods • Key - Intact nervous system (development analogous to mammals) • Assessment of behavior, brain morphology/pathology, and molecular changes (integration of studies at different levels of biological organization)

32. ♂ ♀ High Throughput Testing using Alternative Species: Medaka fish • APPROACH • use Medaka and/or Zebrafish • develop medium to high throughput methods for exposure and assessment • advantages include high fecundity, external fertilization, rapid development, small size, intact nervous system, embryos are transparent from http://nh.kanagawa-museum.jp/tobira/5-1/5-1.html • METHODOLOGY • adapt fish embryo larval assay • use specialized 96 well plates • assess toxicity and development • Oxendine et al, Neurotoxicology, 2006

33. Markers of Developmental Neurotoxicity in Fish • Image-based (automated analysis!?) • Brain morphology (size, shape) • Brain pathology (stains for specific cells or events: α-tubulin in neurons, cell division, apoptosis) • Molecular (candidate gene approach – pick conserved genes critical to neural development) • neuronal fate (Shh in neural plate) • glial fate (Nkxx in brain) • Behavior (swimming, feeding) Shh at stage 25 acetylated α-tubulin at stage 40 (hatched fry)

34. GRADN List Validation Requires Positive Controls • What are the “gold standards’ for developmental neurotoxicity? Goal: • To develop a list of chemicals that can be used to determine the predictive validity of alternative assays • GRADN – list of chemicals that are “generally regarded” as developmentally neurotoxic

35. GRADN List Process: • Step 1: Criterion for inclusion and decisions • Step 2:Review available literature • Step 3: Output • Determine level of evidence • One page summaries • One liners Progress to Date • Started with list of ~250 chemicals • Weekly group reviews • Finished 40% Goals • Peer review • Publish the list • Working with NTP to collect chemical stocks • List of chemicals that everyone working in the area can use • Integrate with CompTox ToxREF database

36. Moving Forward • Developing a framework for collecting and evaluating data • Working with partners (scientists, industry, regulators) • Collaborators: Reference Chemicals NHEERL (MED) NCCT OPPTS (K. Raffaele/OPP) NIEHS (NTP, J. Freedman) Alternative Species NHEERL (MED) Duke (D. Hinton) Phylonix In Vitro Models NHEERL (RTD, ETD) NCCT NIEHS (NTP) Johns Hopkins (CAAT) DOW (S. Marty, J. Maurissen) ECVAM