-------
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United States
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List Acronym * List Name
EPAPFASNONDW
PFAS|EPA: New EPA Method Non-Drinking
Water
EPAPFASRESEARCH PFAS|EPA EPA PFAS Research List
EPAPFASRL PFAS|EPA Cross-Agency Research List
* Last Updated
2019-04-17
2019-05-03
2017-11-16
Number of Chemicals " List Description
24 EPA is developing and validating a new method for detecting these PFAS in non-drinking water st
The list of PFAS EPA is currently researching using v
is scientific approaches.
EPAPFASRL is a manually curated listing of mainly straight-chain and branched PFAS (Per- & Poly-fluorinated alkyl substances)
compiled from various internal, literature and public sources by EPA researchers and program office representatives.
7866 PFAS (as of 11 Nov 2019) captured on the PFASMASTER list
PFASMASTER
PFAS Master List of PFAS Substances
2019-11-11
7866
PFASMASTER is a consolidated list of PFAS substances spanning and bounded by the below lists of current interest to researchers
and regulators worldwide.
PFASNTREV19
PFAS: PFAS in Non-Target HRMS Studies (Liu
et al 2019)
2019-04-17
127
List of PFAS substances detected in non-target HRMS reviewed by Liu et al 2019
PFASOECD
PFAS: Listed in OECD Global Database
2018-05-16
4729
OECD released a New Comprehensive Global Database of Per- and Polyfluoroalkyl Substances, (PFASs) listing more than 4700
new PFAS
PFASOECDNA
PFAS|NORMAN: List of PFAS from the OECD
Curated by Nikiforos Alygizakis
2019-05-19
3213
List of PFAS released by the OECD, provided by Zhanyun Wang, curated and mapped to structures by Nikiforos Alygizakis
Unique_Acronym
Showing 11 to 20 of 22 records
PFESA Byproduct 2
kemikalieinspektionen.se
DTXSID 8071354
DTXSID408 81350
Of
Potassium perfluorobutanesulfonat
Perfluorobutanesulfonic acid
Perfluorobutanesulfonate
Sodium perfluorodec
!25??9-i
'p Afft o rt^STftS n s uffo nlte
2,2,3-Trif I u oro-3-(l,l,2,2B,3-lpMfl u or
Potassium perfluoro-l-butanesulfonate
Perfluorobutanesulfonic acid
Perfluorobutanesulfonate
Sodium perfluoro-l-dec
cms
~f APFF
ADONA parent acid
Antony Williams, Ann Richard, Chris Grulke, and Chemical Curation Team
-------
«liyii«
4j#r
r#0"
=§»
—a
#4™
Attempted to procure ~3,000 based on
chemical diversity, Agency priorities, and
other considerations
Obtained 480 total unique chemicals
• 430/480 soluble in DMSO (90%)
• 54/75 soluble in water (72%)
(incl. only 3 DMSO insolubles)
Issues with sample stability and volatility
Categories initially assigned based on three
approaches
• Buck et al., 2011 categories
• Markush categories
• OECD categories
Kathy Coutros, Chris Grulke, Grace Patlewicz and Ann Richard
-------
oErov PFAS List Overlap
United States
Environmental Protection
Agency
OECD
PFAS
STRUCT
PFAS
430INV
PFAS150
OECD
4729
PFASSTRUCT
3682
6648
PFAS430INV
310
360
428
PFAS150
119
134
146
146
-------
3 members
• Lots of singletons
Data collection:
Pre-defined
structural
categories
Step 0:
Characterizing the
PFAS library
Maximizing Read-across
A
On Wkgrp-31 list;
Availability of in vivo data
On EPA-PFAS list;
Availability of in vivo data
Availability of
in vivo data
r
Capturing Structural Diversity
A
EPA interest
in vivo data lacking
Characterizing the
PFAS Landscape
Step 1: Select
substances from
categories of greatest
interest to the Agency
Step 2: Select
substances from
categories of interest
the Agency
5 structural categories
Agency interest
tto^
Step 3: Select
substances from
remaining categories
with in vivo data
Step 4: Select
substances from
categories of interest
to the Agency
+2 structural categories
T
' Step 5: Select \
m > substances from \
rest remaining 7
categories /
+6 structural categories
¦2i
che
+5 structural categories
'2 categories contained only 1
chemical, so were not included
+10 structural categories
Availability of in vivo data
53 structural
categories
53 substances: 12 categories
9 substances:
13 substances
6 categories
10 categories
-------
oE
United States
Environmental Protection
Agency
In Vitro Toxicity and Toxicokinetic Testing
Toxicoloaical Response
Assay
Assay Endpoints
Purpose
Hepatotoxicity
2D HepaRG assay
Cell death and transcriptomics
Measure cell death and changes
in important bioloqical pathways
Developmental Toxicity
Zebraf ish embryo assay
Fertilisation, lethality, and
structural defects
Assess potential teratogenicity
Immunotoxicity
Bioseek Diversity Plus
Protein biomarkers across
multiple primary cell types
Measure potential disease and
immune responses
Mitochondrial Toxicity
Mitochondrial membrane
potential (HepaRG)
Mitochondrial membrane
potential
Measure mitochondrial health
and function
Developmental
Neurotoxicity
Microelectrode array assay (rat
primary neurons)
Neuronal electrical activity
Impacts on neuron function
Endocrine Disruption
ACEA real-time cell proliferation
assay (T47D)
Cell proliferation
Measure ER activity
General Toxicity
Attagene cis- and trans-
Factorial assay (HepG2)
Nuclear receptor and
transcription factor activation
Activation of key receptors and
transcription factors involved
in hepatotoxicity
High-throughput transcriptomic
assay (multiple cell types)
Cellular mRNA
Measures changes in important
bioloqical pathways
High-throughput phenotypic
profiling (multiple cell types)
Nuclear, endoplasmic reticulum,
nucleoli, golgi, plasma
membrane, cytoskeleton, and
mitochondria morpholoqy
Changes in cellular organelles
and general morphology
Toxicokinetic Parameter
Assay
Assay Endpoints
Purpose
Intrinsic hepatic
clearance
Hepatocyte stability assay
(primary human hepatocytes)
Time course metabolism of
parent chemical
Measure metabolic breakdown
by the liver
Plasma protein binding
Ultracentrifugation assay
Fraction of chemical not bound
to plasma protein
Measure amount of free
chemical in the blood
* Assays being performed by NTP and EPA
-------
"SEPA .
ss=i~°" Objectives
•To inform
-Chemical Category and Read-across approaches
-Bioactive Dose Level (BDL) Approach (in to extrapolation
to define administered dose equivalent (ADE) values)
In order to:
Translate learnings to make inferences for a broader landscape of
PF AS
Initially use structural categories to evaluate the degree of
concordance in NAM results (per technology) within categories and
across categories as a means to qualitatively and quantitatively infer
in vivo toxicity
-------
Characterising PF A using structural
Agency
categories
• Structural categories were assigned by visual inspection and whilst
nominally consistent since only one individual was making the
assignments, the approach was prone to error and not easily
reproducible.
• The assignments provided by OECD were similar in their genesis -
they were manually assigned by the same person.
• Indeed, authors of many of the published literature studies on
PF AS have often end up deriving bespoke naming conventions for
categories which leads to the generation of a lot of parallel
nomenclature that differ, creating unintended barriers to
effective communication among scientists
• Urgent need exists to develop a reproducible objective means of
developing structure-based categories
-------
PF AS Structure - based Oote^orisation
• Reconcile the different structural categories schemes
initially used by creating a harmonised set of structure-
based categories
• Category assignments should be computationally generated
from structure only reproducible, transferable,
standardised, extendable
• Permits nested & overlapping categories such that categories
can be tailored to different datasets (i.e. the various NAM
data streams being generated) and decision contexts
-------
PF AS Structure-based Categorisation: ToxPrints
• Publicly available tools exist to generate download ToxPrints e.g.
ChemoTyper, CompTox Chemicals Dashboard
• Provides excellent coverage of PFAS chemical space
• Nested, hierarchical nature lends itself to creating flexible categories
tailored to problem at hand, i.e., "fit for purpose"
• Can augment with computed structure properties (s.a., MW, size, etc.)
• Intuitive, easy to work with
ring:aromatic.biphenyl 587
c c c
c
ring:fused_steroid_gene 602
•Kj5.6j5.fl. c ^ ,c
• • c
..c. ..c.. _.c-.r
c c c c
c.. .C-. ,.c
c c
ring:fused [5 7) azulene 591
C I f
ring:polycycle_t>icydoJ 714
33.2]decane C
V'H
bond:PO_phosphite 258
bond:quatN_alkyLacydi 264
bond:CX_halide_alky1-X 170
trihalo_(1_1 2-)
?yN
?
bond:QQ(Q-0_S)_sulfi 262
de_di-
S
s
bond:CX_halide_alkyl-F 149
perfluoro tiexyl
I Fs/ \f
chain:alkaneLinear_octyl 442
C8
>Cv
ctiaimalkanelinear.hexa 446
decyl_C 16
f' V'N -r
bond;CX_haiide_alkyi-X 153
.aromatic.al kane
ToxPrints:
s 729 chemical features
s Chemically interpretable
s Coverage of diverse chemistry
s Includes scaffolds, functional
groups, chains, rings, bonding
patterns, atom-types
Clear, reproducible means for defining regions of local chemistry, i.e.
categories!!
-------
PF AS Structure - based Oote^orisation
• Reconcile the different structural categories schemes
initially used - by creating a harmonised set of structure-
based categories
• Category assignments should be computationally generated
from structure only reproducible, transferable,
standardised, extendable
• Permits nested & overlapping categories such that categories
can be tailored to different datasets and decision contexts
• ToxPrints have been used to develop 34 structural categories
(TxP Categories) which cover >90% of the different PF
inventories
-------
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Txp_cats
No_cat
TxP_PFAS_
TxP_PFA5_
TxPPFAS
TxP_PFAS_
TxP_PFAS
TxPPFAS
TxP_PFAS_
TxPPFAS
TxP_PFAS_
TxPPFAS
TxP_PFAS
TxPPFAS
TxPPFAS
TxPPFAS
TxPPFAS
TxP_PFAS_
TxPPFAS
TxP_PFAS_
TxPPFAS
TxP_PFAS
TxPPFAS
TxP_PFAS
TxPPFAS
TxPPFAS
TxP_PFAS_
TxP_PFAS
TxP_PFAS
TxPPFAS
TxP_PFAS_
TxPPFAS
TxPPFAS
TxPPFAS
TxP_PFAS
TxP PFAS
alcohol
sulfonyl
sulfonylhalide
perFoctyl
amine
alcoholprimary
alcohol_sulfonylamide
sulfonamide
sulfonamide_alcohol
ether
alcohol_primary_FTn2
sulfonate
oxidehydroxy
COOH
perFhexyl
alkylXtertiaryxCO
acrylate
sulfonate_FTn2
acylhalide
ethylene_xCO
COOR
carboxamide
alkylXprimary
alcohol_primary_FTnl
aldehydeanhydride
amine_ether
alcohol_polyF
ketone
silane
amineprimary
COOHether
alcoholprimaryFTdiol
COOHJFT
COOHethylene
0.05 0.10
OECD Count
0.000 0.025 0.050 0.075
PFASSTRUCT Count
0.00 0.05 0.10
INV Count
-------
-
-50 -
-100 -
PF AS Coverage based on structure
100 - • . • • pfasstruct
Tag
PFAS
• PFAS150
• OC_lssues
.. • •i.ij* '* ^ • PFASINV
. * V ,tji\ K
• A 2D representation constructed using t-
t *» % • ^ . -. ¦ _ . > t . > > mm a a •
n % % ... ••
. .* /' •* V *
Distributed Stochastic Neighbour Embedding
/j_ /»K I r~"\ L I . .fV • ¦ _ I • 1
(t-SNE) based on 729 ToxPrints as chemical
1 •<•»'#/&'
w ¦¦ "7* ^
.Atw'.'iV V *
.* Jy'>•."•••• " • •» « '« -
0 • n" ^ • MV il.T* — i. ' •>
fingerprints
i. i V . • ¦> • „ '• % V* W
».' V * .* *•"¦¦¦'¦VsjTSc*. ¦" 4
• PFAS430 inventory well distributed across
«• . #
i>i
the PFASSTRUCT inventory
-• V i.» • , * •* •*
jw •* . v W \«Vi. * • '•
.! J,« * •• ** #" *
• #t .[%» * « # • • • * «|
• •••. *• • .
V l. .Sf*
f & '* f>' • *
-100 -50 0 50 100
X
-------
Current PFAS Structural Grouping Approaches
Use Different Levels of Aggregation
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Chemical Categories/Group
Available source in vivo tox study
-------
oE
United States
Environmental Protection
Agency
In Vitro Toxicity and Toxicokinetic Testing
Toxicoloaical Response
Assay
Assay Endpoints
Purpose
Hepatotoxicity
2D HepaRG assay
Cell death and transcriptomics
Measure cell death and changes
in important bioloqical pathways
Developmental Toxicity
Zebraf ish embryo assay
Fertilisation, lethality, and
structural defects
Assess potential teratogenicity
Immunotoxicity
Bioseek Diversity Plus
Protein biomarkers across
multiple primary cell types
Measure potential disease and
immune responses
Mitochondrial Toxicity
Mitochondrial membrane
potential (HepaRG)
Mitochondrial membrane
potential
Measure mitochondrial health
and function
Developmental
Neurotoxicity
Microelectrode array assay (rat
primary neurons)
Neuronal electrical activity
Impacts on neuron function
Endocrine Disruption
ACEA real-time cell proliferation
Cell proliferation
Measure ER activity
General Toxicity
Attagene cis- and trans-
Factorial assay (HepG2)
Nuclear receptor and
transcription factor activation
Activation of key receptors and
transcription factors involved
in hepatotoxicity
High-throughput transcriptomic
assay (multiple cell types)
Cellular mER^™
Measures changes in important
bioloqical pathways
High-throughput phenotypic
profiling (multiple cell types)
Nuclear, endoplasmic reticulum,
nucleoli, golgi, plasma
membrane, cytoskeleton, and
mitochondria morpholoqy
Changes in cellular organelles
and general morphology
Toxicokinetic Parameter
Assay
Assay Endpoints
Purpose
Intrinsic hepatic
clearance
Hepatocyte stability assay
(primary human hepatocytes)
Time course metabolism of
parent chemical
Measure metabolic breakdown
by the liver
Plasma protein binding
Ultracentrifugation assay
Fraction of chemical not bound
to plasma protein
Measure amount of free
chemical in the blood
* Assays being performed by NTP and EPA
17
-------
AEPA
United States
Environmental Protection
Agency
Incorporating Mechanistic and Toxicokinetic
Data to Inform PF AS Category Aggregation
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Biochemical, Molecular
Bioactivity Categories/Group
Toxicoloqical Response
Hepatotoxicity
Developmental Toxi< ity
ISDJHe^aR^assa^
Mitochondrial Toxic ty
Endocrine Disruption
Zebr^isj^mbr^issa^
Bioseek Diversity Plus
<4ssav EndDoints
Cell death and transcriptomics
J Measure cell death and changes
in im sortant bioloaical pathways
Fertilization, lethality, and
structural defects
Mitochondrial membrane
(HepaRS)
Protein biomarkers across
multiple primary cell types
Ass« ss potential teratogenicity
Microelectrode array assay (rat
primar^ieurons^^
Mitochondrial rremcirane
potential and o> /gen
consumption
Mea, :ure potential disease and
immi ne responses
Mea :ure mitochondrial health
and ¦ unction
Impi cts on neuron function
ACEA real-time cell proliferation
assay nt/u)
[jCell^proliferatiJi
Me a :ure ER activity
Toxicokinetic Parameter
Intrinsic hepatic
clearance
Plasma protein binding
Attagene cis- and trans-
High-throughput transcriptomic
assay (multiple cell types')
High-tl- roughput phenotypic
prof ilin i (multiple cell tvpes)
Hepatocyte stability assay
(primary human hepatocytes)
Nuclear receptcjr and
transcription fa
tor activation
nRNA
Ultracentrifugation assay
j Nuclear^ endoplasmic reticulum,
nucleoli, golgi, p asma
membrane, cytc skeleton, and
[mitochondria morohcloov
Assay End points
Time course metabolism of
parent chemical
Acti 'ation of key receptors and
tran icription factors involved
in he patotoxicitv
Mea, :ures changes in important
biolc iical pathways
Fraction of chemical not bound
to plasma protein
Char ges in cellular organelles
and general morphology
Measure metabolic breakdown
by the liver
Measure amount of free
chemical in the blood
"Assays being performed by NTP and EPA
• Map HTTK <& TK to Adverse Outcome Pathways (AOP) to facilitate context
18
-------
PFAS Category Aggregation that incorporates
Structural, Mechanistic and Toxicokinetic Data
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Needed in vivo tox study Available source in vivo tox study
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Structural categories provide a useful
lens to start exploring the assay data
Are there differences across the
structural categories for specific
assays?
Which assays are enriched for which
categories?
18 categories out of the 34 had >3 members
13 of 82 AT& assays had a STD > 0.7 wrt AC50 values
Substances with problematic QC excluded
Primary categories (12) are shown
20
-------
Pairwise similarity on the basis of
values using Euclidean distance (higher
distances reflect increasing
dissimilarity)
Within a structural category - how
similar in terms of bioactivity are the
category members?
-------
oE
United States
Environmental Protection
Agency
Similarity within the TxP_COOH category wrt
to ERa
F F F
Substance_Pair
Pairwi se_Eucl ideari_Di stance
('DTXSID00379268',
'DTXSID00379268')
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' DTXSID1037303')
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' DTXSID20375106')
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DTXSID00892442
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DTXSID20375106
DTXSID20874028
22
-------
oE
United States
Environmental Protection
Agency
Preliminary Category-Based Analysis of available in
vivo toxicity data
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collected within ToxValDB
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23
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Preliminary Category-Based Analysis of available in
Pairwise similarity on the basis of
median logNOAEL values for each
substance using Euclidean distance
(higher distances reflect increasing
dissimilarity)
Within a structural category - how
similar in terms of toxicity are the
category members?
What combination of features
(bioactivity, metabolic, structural,
physchem) can account for the
differences to facilitate a read-across
approach?
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Similarity within the TxP COOH category wrt
Environmental Protection W w
to in vivo toxicity (based on median logNOAEL
values) y
Substance Pair
Pairwise Euclidean Distance
(' DTXSID3031862',
' DTXSID3031862')
0
(' DTXSID3031862',
' DTXSID3052856')
0.397940009
(' DTXSID3031862',
'DTXSID40108559')
1.301029996
(' DTXSID3031862',
' DTXSID8031863')
5.346787486
(' DTXSID3031862',
'DTXSID8031865')
5.346787486
Na
F F
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DTXSID40108559
DTXSID3031862
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DTXSID40108559
F OH
F F
F F
F F
F F
F F
HO ^-0
DTXSID8031863
DTXSID8031865
26
-------
oE
United States
Environmental Protection
Agency
Take Home Messages
•o I1
V
F F
'H' ' '
o^\
• Chemical curation efforts are important to harmonise
structure, naming, and identifiers across the PFAS
space
• A chemical library of 430 PFAS has been assembled
for chemical screening, analytical method
development, and other research needs
• A subset of 150 PFAS selected for in vitro toxicity
and toxicokinetic testing to refine/support read
across categories
• In vitro toxicity and toxicokinetic testing and analysis
are underway and demonstrate the diverse biological
activities and toxicokinetic properties of PF AS
• More information at https '• //www. epa. qov/chem ical -
research/pf as - chem ical -1 i sts - and - tiered - testi nq -
methods - descriptions
27
-------
oE
United States
Environmental Protection
Agency
Acknowledgements and Questions
EPA ORD
Grace Patlewicz
Josh Harrill
Keith Houck*
Katie Paul-Fried man*
Barbara Wetmore
Monica Linnenbrink
Reeder Sams
Johanna Nyffeler
Antony Williams*
Ann Richard*
Ryan Lougee*
Chris Grulke*
Kathy Coutros*
Stephanie Pad ilia
Tim Shafer
Jason Lambert
Mark Strynar
Ron Hines
Annette Guiseppi-Elie
Marci Smeltz
Richard Judson
Imran Shah*
Indira Thillainadarajalr
Brian Meyer*
Jason Brown*
Andy Gillespie
Scott Clifton
Matt Henderson
Anna Kreutz
Evgenia Korol-Bexell
Larry McMillan
Chris Mazur
Rusty Thomas
Michael Devi to
National Toxicology Program
• David Crizer
• Suramya Waidayanatha
• Steve Ferguson
• Nisha Sipes
-------