1

Use of Developmental Neurotoxicity data in Risk Assessment at EPA:

Current Status and Future Efforts

Kathleen Raffaele, EPA/OPPWilliam Mundy, EPA/ORD

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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

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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

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EPA Developmental Neurotoxicity Study - OPPTS 870.6300 -

Optional (Instead of PND 11)

Gestation Day

Postnatal Day

No Treatment

Treatment

0 6

0 11 21 ~60

Offspring Evaluations

Growth & Survival

Functional Observations

Motor Activity

Auditory Startle

Learning & Memory

Brain Wt & Neuropathology

Preferred Extension of Treatment

Sexual Maturation

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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)

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DNT Studies submitted to OPP

Years Non-OP DNTs OP DNTs

1993-1996 8 0

1997-2000 7 1

2001-2004 21 21

2005-2006 13 1

Total: 49 23

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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]

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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

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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

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Differences between current EPA and draft OECD DNT Guidelines

Element EPA OECD Dosing period Gestation day 6 through postnatal day

11 (PND21 recommended) Gestation day 6 through lactation (PND21)

Functional observations

Specific days recommended Recommended to be weekly pre-weaning and bi-weekly post-weaning

Minimum group size for pup behavioral assessments

10/sex/dose for most tests 20/sex/dose for most tests

Early neuropathology assessment

Post-natal day 11, with immersion fixation (postnatal day 21 usually accepted, with perfusion fixation);

Between PND11 and 22, either perfusion or immersion fixation

Behavioral ontogeny1

Not discussed At least two measures required

Motor activity Specific days recommended (PND 13, 17, 21, and around 60)

1-3 times pre-weaning, once during adolescence (around PND 35) and once for young adults (PND60-70)

Motor and sensory function

Auditory startle habituation specified Quantitative sampling of sensory modalities and motor functions specified, auditory startle habituation listed as example

Neuropathology – number of animals

6/sex/dose specified (10/sex/dose recommended)

10/sex/dose

Direct dosing to pups

Not discussed (recommended in some situations)

Should be considered for some situations

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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

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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)

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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

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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

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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

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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)

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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

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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

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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.

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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

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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

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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

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Contributors• 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

Developmental Neurotoxicity Testing: Alternatives for Screening and Prioritization

William Mundy, Kevin Crofton, and the DNT TeamNeurotoxicology Division, USEPA

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

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)

Addressing the problem - DNTObjective: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?

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

High Throughput Cell-Based Assays using the Cellomics ArrayScan

High Content AnalysisHigh Content Analysis

• HighThroughput ImagingHighThroughput Imaging(automated microscope, (automated microscope, image-analysis software)image-analysis software)

• Data obtained at cell levelData obtained at cell level

• Not currently used in Not currently used in developmental developmental neurotoxicologyneurotoxicology

Output: Automated analyses of 96 wells in 20-30 minOutput: Automated analyses of 96 wells in 20-30 min(200 cells x 96 wells x 10 endpoints per cell = 192,000 data points)(200 cells x 96 wells x 10 endpoints per cell = 192,000 data points)

0.01 0.1 1 10 100 10000

20

40

60

80

NGF (ng/ml)

Tota

l Neu

rite

Len

gth

(mic

rons

)

NS-1 Cells (Clonal PC12 cells)NS-1 Cells (Clonal PC12 cells)• NGF stimulates neurite outgrowthNGF stimulates neurite outgrowth• Fix and stain 4 days laterFix and stain 4 days later• Automated assessment in 30 minAutomated assessment in 30 min

Cell-Based Endpoint: Neurite Outgrowth

NGF Concentration Response

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)

High Throughput Testing using Alternative Species: Medaka fish

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♂ ♀APPROACHAPPROACH • use Medaka and/or Zebrafish use Medaka and/or Zebrafish • develop medium to high throughput methods develop medium to high throughput methods

for exposure and assessment for exposure and assessment • advantages include high fecundity, external advantages include high fecundity, external

fertilization, rapid development, small size, fertilization, rapid development, small size, intact nervous system, embryos are intact nervous system, embryos are transparenttransparent

METHODOLOGYMETHODOLOGY • adapt fish embryo larval assay adapt fish embryo larval assay • use specialized 96 well plates use specialized 96 well plates • assess toxicity and developmentassess toxicity and development Oxendine et al, Neurotoxicology, Oxendine et al, Neurotoxicology,

20062006

Markers of Developmental Neurotoxicity in Fish

acetylated α-tubulin at stage 40 (hatched fry)

Shh at stage 25

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)

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

GRADN ListProcess:Process:• Step 1: Criterion for inclusion and decisions Step 1: Criterion for inclusion and decisions • Step 2:Review available literatureStep 2:Review available literature• Step 3: OutputStep 3: Output

Determine level of evidenceDetermine level of evidence One page summariesOne page summaries One liners One liners

Progress to DateProgress to Date• Started with list of ~250 chemicalsStarted with list of ~250 chemicals• Weekly group reviewsWeekly group reviews• Finished 40%Finished 40%

GoalsGoals• Peer review Peer review • Publish the listPublish the list• Working with NTP to collect chemical stocksWorking with NTP to collect chemical stocks

List of chemicals that everyone working in the List of chemicals that everyone working in the area can usearea can use

Integrate with CompTox ToxREF databaseIntegrate with CompTox ToxREF database

Human and AnimalData

OnlyAnimal Data

amphetaminechlordiazepoxide

cocainediphenylhydantionhexachlorophene

ethanollead

methylmercuryPCBs

terbutalinethalidomide

trans-retinoic acid

acrylamidebenzenecadmium

chlorpyrifos (oxon)dieldrin

ketaminemethadone

methamidazolemethanol

methylazoxymethanolpropylthiouraciltrichloroethylene

triethyltintrimethyltinvalproic acid

Moving Forward

Reference Chemicals

NHEERL(MED)

NCCTOPPTS

(K. Raffaele/OPP)

NIEHS(NTP, J. Freedman)

In Vitro Models

NHEERL(RTD, ETD)

NCCT

NIEHS(NTP)

Johns Hopkins(CAAT)

DOW(S. Marty, J. Maurissen)

ECVAM

Alternative Species

NHEERL(MED)

Duke(D. Hinton)

Phylonix

• Developing a framework for collecting and evaluating data• Working with partners (scientists, industry, regulators)• Collaborators: