Use of Developmental Neurotoxicity
data in Risk Assessment at EPA:
Current Status and Future Efforts
Kathleen Raffaele, EPA/OPP
William Mundy, EPA/ORD
1
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
2
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
3
EPA Developmental Neurotoxicity Study
- OPPTS 870.6300 Gestation Day 0
6
Postnatal Day
22
0
11
Growth & Survival
21
~60
Sexual
Maturation
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
4
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)
5
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
6
Use of DNT Studies in Single
Chemical Assessments
(Preliminary analysis)
• Study Reviews Completed – 58 chemicals
• Chemicals with risk assessments based on DNTrelated endpoints – 8
• Potential impact in future risk assessments - 18
• [Abstract submitted for presentation at SOT 2007]
7
Ongoing DNT-related Efforts
•
•
•
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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
8
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
9
Differences between current EPA and
draft OECD DNT Guidelines
Element
Dosing period
Functional
observations
Minimum group
size for pup
behavioral
assessments
Early
neuropathology
assessment
Behavioral
ontogeny1
Motor activity
Motor and sensory
function
Neuropathology –
number of animals
Direct dosing to
pups
EPA
Gestation day 6 through postnatal day
11 (PND21 recommended)
Specific days recommended
10/sex/dose for most tests
OECD
Gestation day 6 through lactation (PND21)
Recommended to be weekly pre-weaning and bi-weekly postweaning
20/sex/dose for most tests
Post-natal day 11, with immersion
fixation (postnatal day 21 usually
accepted, with perfusion fixation);
Not discussed
Between PND11 and 22, either perfusion or immersion fixation
Specific days recommended (PND 13,
17, 21, and around 60)
Auditory startle habituation specified
1-3 times pre-weaning, once during adolescence (around PND 35)
and once for young adults (PND60-70)
Quantitative sampling of sensory modalities and motor functions
specified, auditory startle habituation listed as example
10/sex/dose
6/sex/dose specified (10/sex/dose
recommended)
Not discussed (recommended in some
situations)
At least two measures required
Should be considered for some situations
10
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
11
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)
12
OPP/ORD Collaborative Efforts –
Positive Control Data Evaluation
• DNT Guideline requires submission of positive control
data to support laboratory ability to detect treatmentrelated 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
13
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
14
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
15
Historical Control Data Evaluation:
Results
• Results presented as a series of SOT posters:
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–
–
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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)
16
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
17
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
18
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 721 or 11-21) did not result in increased mortality,
increased clinical signs, decreased body weight
gain, or differences in brain morphometry.
19
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
20
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
21
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
22
Contributors
• OPP
–
–
–
–
–
Elizabeth Mendez
John Doherty
Jess Rowland
Louis Scarano
Kelly Schumacher
• ORD/NHEERL
– Kevin Crofton
– Mary Gilbert
– Ginger Moser
• ORD/OSCP
– William Sette
• ORD/NCEA
– Susan Makris
23
Developmental Neurotoxicity Testing:
Alternatives for Screening and Prioritization
William Mundy, Kevin Crofton, and the DNT Team
Neurotoxicology Division, USEPA
Current Status of Toxicity
Testing
• Large numbers of chemicals identified for testing
(e.g., pesticide inerts, HPVs, CCLs) with no riskbased 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 (highthroughput)
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?
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 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)
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
Total Neurite Length
(microns)
80
60
40
20
0
0.01
0.1
1
10
NGF (ng/ml)
100
1000
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
♂
♀
from http://nh.kanagawamuseum.jp/tobira/5-1/5-1.html
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
METHODOLOGY
• adapt fish embryo larval assay
• use specialized 96 well plates
• assess toxicity and development
Oxendine et al, Neurotoxicology,
2006
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)
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
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
GRADN List
Human and Animal
Data
Only
Animal Data
amphetamine
chlordiazepoxide
cocaine
diphenylhydantion
hexachlorophene
ethanol
lead
methylmercury
PCBs
terbutaline
thalidomide
trans-retinoic acid
acrylamide
benzene
cadmium
chlorpyrifos (oxon)
dieldrin
ketamine
methadone
methamidazole
methanol
methylazoxymethanol
propylthiouracil
trichloroethylene
triethyltin
trimethyltin
valproic acid
Moving Forward
• Developing a framework for collecting and evaluating data
• Working with partners (scientists, industry, regulators)
• Collaborators:
In Vitro Models
Reference Chemicals
Alternative Species
NHEERL
(RTD, ETD)
NHEERL
(MED)
NHEERL
(MED)
NCCT
NCCT
NIEHS
(NTP)
OPPTS
(K. Raffaele/OPP)
Duke
(D. Hinton)
Johns Hopkins
(CAAT)
NIEHS
(NTP, J. Freedman)
DOW
(S. Marty, J. Maurissen)
ECVAM
Phylonix