04Zeise Ramazzini tox test - Collegium RamazziniMicrosoft PowerPoint - 04Zeise Ramazzini tox...
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11/19/2015 1 New Data Streams in Toxicity Testing: Limitations and Uses in Decision-Making Lauren Zeise, Gina Solomon, Shoba Iyer, Nathalie Pham, Ed Hackett, Martha Sandy, Melanie Marty Office of Environmental Health Hazard Assessment California Environmental Protection Agency October, 2015 Carpi, Italy The views in this presentation do not necessarily reflect those of the CalEPA Office of Environmental Health Hazard Assessment 2 Type of information needed depends on risk decisions: Differing burdens of proof Site mitigation Advisories Permitting Standards Questions: Is it safe? Pesticide Registration Chemical Substitution or Design 3 Population responses Population responses Individual responses Individual responses Organ responses Organ responses Tissue responses Tissue responses Cell responses Cell responses Biological molecule interactions Biological molecule interactions Internal exposure Internal exposure Metabolism and Pharmacokinetics Metabolism and Pharmacokinetics External exposure External exposure TYPES OF VARIABILITY Genetic heredity Epigenetic heredity Lifestage Existing health conditions Co-exposures Food and nutrition Psychosocial stressors In vitro methods : In vivo methods animals in vivo methods human New tools to interrogate exposure to outcome continuum 4 Hi gh Throughput Testing in the US Federal Government Cell components and cells Mostly immortalized cells derived from cancer cell lines 96-well plates or higher Quantitative response read-out at each concentration Simultaneous cytotoxic measures (when using cell-based assays) Can run test hundreds or thousands of chemicals at a time 4 Tox21 ToxCast I and II E1K ~10,000 chemical in 25 assays US EPA, NIEHS/NTP, NIH, NCATS, FDA 1,068 chemicals 700+ assays 880 chemicals ~50 assays prioritization for screening 81 5 Kevin Crofton, US EPA, 2013 Endosulfan neurotoxicity Established in vivo/in vitro ToxCast High Throughput Inactive: bovine-derived GABARα1α, rat-derived GABARα1α, GABA A R agonist, and GABA A R nonselective, cell-free bovine GABA A Rα5 Active: guinea pig and human DAT assays Inactive: human AChE Noncompetitively binds to GABA A R, blocking chloride conductance Hyper-excitation of central nervous system, humans, several other species Decreased dopamine in weanling rat hippocampus; decreased dopamine active transporter in offspring Neurobehavioral outcomes (e.g., decreased ability to learn, retain a required task) Acetylcholinesterase inhibition Zebrafish: abnormal behavior (prolonged/spastic swimming behavior, disorientation,slower response, and shorter distance swam in response to touch)
Transcript of 04Zeise Ramazzini tox test - Collegium RamazziniMicrosoft PowerPoint - 04Zeise Ramazzini tox...
11/19/2015
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New Data Streams in Toxicity Testing:
Limitations and Uses in Decision-Making
Lauren Zeise, Gina Solomon, Shoba Iyer, Nathalie Pham, Ed Hackett, Martha Sandy, Melanie Marty
Office of Environmental Health Hazard Assessment California Environmental Protection Agency
October, 2015
Carpi, Italy
The views in this presentation do not
necessarily reflect those of the CalEPAOffice of Environmental Health Hazard Assessment
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Type of information needed depends on risk decisions:
Differing burdens of proof
Site mitigation AdvisoriesPermitting
Standards Questions:
Is it safe?
Pesticide Registration Chemical Substitution or Design
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Population responsesPopulation responses
Individual responsesIndividual responses
Organ responsesOrgan responses
Tissue responsesTissue responses
Cell responsesCell responses
Biological molecule interactionsBiological molecule interactions
Internal exposureInternal exposure
Metabolism and PharmacokineticsMetabolism and Pharmacokinetics
External exposureExternal exposureTYPES OF
VARIABILITY
Genetic heredity
Epigenetic
heredity
Lifestage
Existing health conditions
Co-exposures
Food and
nutrition
Psychosocial stressors
In vitro methods
:
In vivomethodsanimals
in vivo methods
human
New tools to interrogate exposure to outcome continuum
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High Throughput Testing in the US Federal Government
� Cell components and
cells
� Mostly immortalized cells
derived from cancer cell
lines
� 96-well plates or higher
� Quantitative response
read-out at each
concentration
� Simultaneous cytotoxic
measures (when using
cell-based assays)
� Can run test hundreds or
thousands of chemicals
at a time4
Tox21
ToxCast I and II
E1K
~10,000 chemical in 25 assays
US EPA, NIEHS/NTP, NIH, NCATS, FDA
1,068 chemicals700+ assays
880 chemicals ~50 assays
prioritization
for screening
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5Kevin Crofton, US EPA, 2013
Endosulfan neurotoxicity
Established in vivo/in vitro ToxCast High Throughput
� Inactive: bovine-derivedGABARα1α, rat-derived GABARα1α, GABAAR agonist, and GABAAR nonselective, cell-free bovine GABAARα5
� Active: guinea pig and human DAT assays
� Inactive: human AChE
� Noncompetitively binds to GABAAR, blocking chloride conductance� Hyper-excitation of central nervous
system, humans, several other species
� Decreased dopamine in weanling rat hippocampus; decreased dopamine active transporter in offspring � Neurobehavioral outcomes (e.g.,
decreased ability to learn, retain a required task)
� Acetylcholinesterase inhibition
Zebrafish: abnormal behavior (prolonged/spastic swimming behavior,
disorientation,slower response, and shorter distance swam in response to touch)
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Methidathion
neurotoxicity
Established in vivo/in vitro
�Acetylcholinesteraseinhibitor
�Requires metabolic activation for toxicity
Toxcast High throughput
� Inactive: human AChE
Endosulfan Endocrine Disruption
In vivo and standard in vitro
♀Decreased ovarian weight and functionality in adult females
♀Decreased ovarian and uterine weights in pups
♀Agonist activity in various ER-related assays
♂Decreased sperm count andvarious indicators of anti-androgenic activity
♂Delayed sexual maturation in boys aged 10 to 19
♂Anti-androgenic in AR-related assays
ToxCast High Throughput
♀Estrogen pathways activity determination dependent on scoring system�Five assay threshold: Inactive
�Gene score: Active
� AUC for ER < 0.1 � Inactive
♂Androgen pathways determination dependent on scoring system�Five assay threshold: Inactive
�Gene score: Active
� AUC for AR pathways: Inactive
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Decreased AR activity at target tissue
Interference with androgen
mediated development
Reproductive tract malformations�AGD
Nipple
Retention
Hypospadias
� Sperm
quality
Leydig cell
tumors Cryptorchism Other reproductive
tract malformations
Other Decreased Decreased Blockade of Androgen Mutated
Stressors Testosterone Dihydrotestosterone Receptor (AR) Receptor
“Phthalate Syndrome”
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Tox Cast Process target activities of phthalates: Chain length
Short Chain
Medium Chain(4-6 carbons)
Long Chain
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Phthalates: ToxCast activity in parent vs. monoester
metabolite
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Phthalate ToxCast assay activities:
Straight vs cyclic side chain dihexyl phthalates
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A few observations for ToxCast assays of phthalates
� Phthalates show a broad spectrum of activities in ToxCast.
� There were no clear patterns for carcinogenicity and endocrine
toxicity for phthalates that exhibit these activities in vivo
� In vivo anti-androgenicity not captured for phthalates
exhibiting phthalate syndrome in vivo
� Little indication that di(isononyl) phthalate would be
carcinogenic or antiandrogenic, as seen in vivo
� Overall biologic activities exhibited, e.g.
� Differed between straight and cyclic side chain dihexyl
phthalates.
� Medium-chain length phthalates were the most active group
�generally consistent with in vivo observations
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Half full? Half empty?
However…
Case studies reinforce concerns about over-reliance on high throughput assays
• Well established toxicities not reflected in bioassays
o Major neurotoxicity endpoints
missed for neurotoxic pesticideso Estrogen-related pathway activities
equivocal in vitro
o Phthalate syndrome missed� Concern led to bans of
phthalates in children’s products
• Opportunities for false negatives
o Lack of metabolic capacity
o Limited biological coverageo Endpoints requiring more integrated
systems
� Some may be captured in vivo in short term tests
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Bisphenol A Analogues
BPA TBBPA BPAF BPE
BPCBPZBPB
TCBPA
TGSA PHBB BPSBPF
TMBPAAssay
Key
ER agonist
PPARγ
AhR
stressAR agonist
ER antagonist
GR
from Mike DeVito NIEHS
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Hazard Traits
GenotoxicCarcinogenic
Cardiovascular
Developmental
Neurologic
Hepatic
Renal
Gastrointestinal
Endocrine
Metabolic Disease
Respiratory
Reproductive
Hematopoietic
Immunologic
Musculoskeletal
Dermal
Structure
Mechanistic test
activity profiles
Metabolite
prediction
persistence
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Hazard Traits
Developmental
Neurologic
Hazard Warning Flags
https://upload.wikimedia.org/wikipedia/commons/f/fa/Balanced...
Evidence Actions
Structure
Mechanistic test
activity profiles
Metabolite
prediction
persistence
