oEPA
United States
Environmental Protection
Agency
The ENTACT Story: Using US EPA
Resources to Evaluate and Enhance
Non-target Workflows
Jon Sobus1, El in Ulrich1, Jarod Grossman2, Alex Chao2,
Seth Newton1, Antony Williams1, Ann Richard1, Chris Grulke1,
Andrew McEachran2, Randolph Singh2, Hussein Al-Ghoul2
1 Center for Computational Toxicology and Exposure
2 ORAU/ORISE Participant
Office of Research and Development
-------
1) Research drivers for NTA/NTS
2) ENTACT genesis & study design
3) Progress to date (at EPA)
4) Progress to date (outside EPA)
5) Ongoing & future work
-------
xvEPA
United States
Environmental Protection
Agency
High-Throughput Risk Characterization
Many industrial & commercial chemicals are covered by the
Toxic Substances Control Act (TSCA), which is
administered by EPA.
TSCA updated in June 2016 to allow
evaluation of existing and new chemicals.
Characterization of risk requires exposure and hazard data.
EPA's Office of Research and Development (ORD) is
developing new approach methodologies (NAMs) for rapid
risk characterization.
NTA is a promising NAM, but requires careful evaluation
and implementation
'70,000 Chemicals on the TSCA
Inventory
Risk-Based
Prioritization
2
Office of Research and Development
-------
oEPA
United States
Environmental Protection
Agency
NTA State-of-the-Sc ence
Science of the Total Environment 670 (2019) 814-825
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Prioritizing potential endocrine active high resolution mass spectrometry
Check for
(HRMS) features in Minnesota lakewater
Meaghan E. Guyader3, Les D. Warren b, Emily Greena, Craig Buttc, Gordana Ivosev d, Richard L. Kieslinge
Heiko L. Schoenfuss b, Christopher P. Higginsl*
a Colorado School of Mines. Golden, CO, USA
b St. Cloud State University. St. Cfoud. MN, USA
c Sciex, Boston, MA, USA
d Sciex, Toronto, Canada
e US. Geological Survey. Mounds View. MN, USA
ences
e
L
cnnoiogg
Cite This: Environ. Sci. Techno/. 2018, 52, 11975-11976
Viewpoint
pubs.acs.org/est
Is Nontargeted Screening Reproducible?
Ronald A. Hites*
School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana 47405, United States
Karl J. Jobst*
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada
"The novelty of nontarget analysis, particularly its
current lack of implementation by regulatory agencies,
has prevented the establishment of streamlined quality
assurance and quality control (QA/QC) procedures."
"A/o single analytical technique is suitable for the
analysis of all compounds, and successful
nontargeted screening will require the development
of multiplatform approaches, facilitated and validated
through interlaboratory collaborations."
-------
xvEPA
United States
Environmental Protection
Agency
Key Research Needs
Anal Biaanal Chan (2015) 407:6237-6255
DOI 10.1007/s00216-0! 5-86SI -7
REVIEW
Non-target screening with high-resolution mass spectrometry:
critical review using a collaborative trial on water analysis
Emma L. Schymanski1 ¦ Heinz P. Singer1 ¦ Jaruslav Stobodnik:
Martin Krauss"1 * Tobias: Sehulze1• Peter Haglund4 • Thomas Leteel5
lldiko M. Ipnlyi2 * Peter Oswald2 <
Nikolaos S. Thomaidis*' • Anna Bletsou'
Sylvia Crosse"
Christian Zwiener ' • Maria Ibafiez* ¦
Tania Portoles* ¦ Ronald de Boer* • Malcolm J. Reid 10 ¦ Matthias Onghena11
Uwe Kunkel12 • Wolfgang Schu Iz
Philippe Bados
13
Amelie Guillon14 ~ i\a'ike Novon14 * Gaela Leroy15
16
Sara Bogialli17 • Draienka Stipanicev18 ¦
Pawel Rostkowski19 • Julia nt1 Hollender
- 18 Institutes
- 12 Countries
- 1 river water extract
- Workflows & Methods:
- Analytical -> well harmonized
- Data processing not harmonized
Clearly expressed needs for:
1) More tightly defined interlaboratory comparisons
2) The use of spiked samples
3) The shared use of comprehensive suspect lists
Office of Research and Development
-------
oEPA
United States
Environmental Protection
Agency
EPA/ORD Takes a Leadership Role
Non-Targeted Analysis Workshop
Home Agenda Registration Abstract Submission Logistics
The U.S. Environmental Protection Agency (EPA) will host the Non-Targeted Analysis Workshop
August 1S-19, 2015 at EPA's Research Triangle Park Campus.
Environmental Protection Agency
(EPA) 2018
The U.S. Environmental Protection Agency (EPA) hosted a workshop
focused on EPA's Non-Targeted Analysis Collaborative Trial (EMTACT).
ENTACT was designed to assess the characteristics and performance
of cutting-edge non-targeted analysis (NTA) methods using a set of
highly controlled synthetic mixtures and reference samples. This
workshop brought together ENTACT participants, NTA experts, and
key stakeholders to discuss findings from ENTACT as well as next
steps for the NTA research community.
n
August 13-15,2018
Durham, NC, USA
oEPA
United States
Environmental Protection
Agency
Environmental Topics Laws & Regulations About EPA
Related Topics: Science Matters
CONTACT US SHARE (J) (*) (p)
EPA's ENTACT Study Breaks New Ground with
Non-Targeted Research
Published July30,2018
EPA scientists are leading a multi-phase project to evaluate the ability of
non-targeted analysis laboratory methods to consistently and correctly
identify unknown chemicals in samples. EPA's Non-Targeted Analysis
Collaborative Trial (ENTACT) was formed in late 2015 and includes nearly
30 academic, government, and industry groups. Non-targeted analysis
involves analyzing water, soil and other types of samples to identify
unknown chemicals that may be present, without having a preconceived
idea of what chemicals may be in the samples.
"One of our main goals is to figure out what scientists are doing with non-
targeted analysis as a group at large, particularly which chemicals we
correctly identify and why," says Elin Ulrich, an EPA scientist who co-leads
ENTACT with EPA's Jon Sobus.
-------
xvEPA
United States
Environmental Protection
Agency
Science Questions for Research Community
How variable are tools and results from lab to lab?
Are some methods/tools better than others?
How does sample complexity affect performance?
What chemical space does a given method cover?
How sensitive are specific instruments/methods?
OQQ
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OQQ
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o<
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i O IICMH 'O Q
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oo ¦' di! omw o
q bcq"' O O
QQOOO
It^QSi
EPA's Non-Targeted Analysis
6
Office of Research and Development
-------
oEPA
United States
Environmental Protection
Agency
Original ENTACT Concept
ToxCast k
Chemicals v
100-400 100-400 100-400 100-400 100-400 100-400 100-400 100-400 100-400 100-400
chemicals chemicals chemicals chemicals chemicals chemicals chemicals chemicals chemicals chemicals
Why are certain
chemicals only found
with certain methods?
Lab B measurement space
Lab A measurement space
What impurities/
interaction products
found?
Can we model these
behaviors?
Can we expand
coverage?
Lab C measurement space
? "other" space (missing chemicals)
-------
x>ERft ENTACT Part 1 ENTACT Part 2
United States
Environmental Protection
Agency
Chemicals from ToxCast Library
~1200 ToxCast Chemicals
(highest quality)
10 Mixtures L,
(100-400 chemicals each)
Multi-Well Plates*
# *x • • •
a
a
-25 Collaborators & 5 Contractors*:
1st: Blinded analysis
2nd: Unveiling of chemicals
3rd: Unblinded evaluation
Reference & Fortified House Dust
1
|contami]W|!j
I'louse Dust
Reference & Fortified Human Serum
w
& es
w.'
—
Reference & Fortified Silicone
Wristbands
8
-------
oEPA
United States
Environmental Protection
Agency
Design of ENTACT Mixtures
400
350
V)
g 300
0 250
O 200
O
CD
JD
E
150
100
50
0
5 NTA method replicates
Grade A - replicate 90 set
Grade A - unique to mix
i Grade A - all isobaric set (replicated)
Grades B,C - lower purity mix
499
500 501
502
503 504
505 506
507 508
Mixture Number
Office of Research and Development
-------
vvEPA
United States
Environmental Protection
Agency
Resources Provided to Participants
4 SOPs for sample handling, analysis, and data submission
4 Procedures used for sample preparation
4- Up to 16 samples with eventual (unblinded) chemical mappings
4- MS-Ready DSSTox list (671,852 unique) with .mol files
4 MS-Ready ToxCast list (4,248 unique) with .mol files
4 Method and Data reporting templates
4 FTP site, accounts, and instructions
~~~
Office of Research and Development
-------
_ EPA Methods for ENTACT Mixtures
Environmental Protection
Agency
Agilent 6530B Q-TOF
Agilent ZORBAX Eclipse Plus C8 column (2.1 x 50 mm, 1.8 |jm)
A: 5% methanol, 95% water (0.4 mM ammonium formate)
B: 95% methanol, 5% water (0.4 mM ammonium formate)
Waters Acquity UPLC® BEH C18 column (2.1 x 50 mm, 1.7 pm)
A: water (0.1% formic acid)
B: acetonitrile (0.1% formic acid)
11
10 ENTACT Mixtures
3 ENTACT Mixtures
-------
oEPA
United States
Environmental Protection
Agency
EPA Analysis Workflow
Sample Preparation
3 Dilutions, 3 Replicate Injections, 6 Blanks
U
Anal Biraual Chan (2017) 409:1729-1735
DOl 10.1007/s00216-016-0139-z
RAPID COMMUNICATION
Sample Analysis
LC-QTOF/HRMS: ESI+and ESI-, MS1
Identifying known unknowns using the US EPA's CompTox
Chemistry Dashboard
Peak Picking & Alignment
Agilent MassHunter Profinder Software
Andrew D. McEachran • Jon R Sobus" * Antony J, Williams"
Formula Assignment
Agilent Mass Profiler Professional Software,
DSSTox Unique MS-Ready Formula List
McEachran et of. J Cheminform (2018) 10:45
hitfpsi/Vdoi.org/10.1186/s 13321-01 fi-0299-2
METHODOLOGY
Journal of Cheminformatics
Open Access
"MS-Ready" structures for non-targeted
high-resolution mass spectrometry screening
studies
Andrew D. McEachran u*, Kamel Mansouri ¦2-3, Chris Grulke2, Emma L Schyrrianski'1, Christoph Ruttkies5
and Antony J. Williams-
Feature Filtering & Flagging
Custom Script:
Blank Subtraction, Fold-Change Thresholds,
Formula Match Score Cut-Off
Candidate Structure Selection
EPA CompTox Chemicals Dashboard:
Batch Search, Data Source Ranking
ti
Chemical Structure Corroboration
LC-QTOF/HRMS:
DDA MS2 Using Preferred Ions List,
Agilent Reference MS2 libraries
-------
xvEPA
United States
Environmental Protection
Agency
EPA Initial Results
By Feature (total = 26K)
By Substance (total = 1,269)
44%
52%
26%
49%
14%
Artifacts ¦ Unmatched Features ¦ Matched Features
Not Observed P Limited Evidence ¦ Observed (MSI) ¦ Observed (MS2)
< 5% of Observed
Features Matched to a
Spiked Substance
~ 75% of Spiked
Substances were
Observed
13
Only 48% of ENTACT substances were in reference MS2 library
-------
oEPA
United States
Environmental Protection
Agency
Generation of in Spectra
CFM-ID v2.0
Competitive fragmentation modeling of ESI-MS/MS
spectra for putative metabolite identification
Authors
Authors and affiliations
Felicity Allen S, Russ Greirver, David Wishart
Linking in silico MS/MS spectra with
chemistry data to improve identification
of unknowns
Andrew D. McEachran llya Balabin, Tommy Cathey, Thomas R. Transue, Hussein Al-Ghoul, Chris
Grulke, Jon R. Sobus & Antony J. Williams ™
Machine Learning
Fragmentation
Prediction
Model
Training Set:
Metlin MS2 spectra
and structures
DSSTox MS-Ready
Structures
(-765,000)
DSSTox MS2
spectra
(10, 20, 40v)
McEachran, Andrew D., etal. Scientific data 6.1 (2019): 1-9
Allen, Felicity, et al. Metabolomics 11.1 (2015): 98-110.
-------
xvEPA
United States
Environmental Protection
Aaencv
Reference vs. in silicoLibrary Coverage
PCDL
88
CFM-ID
111
77
101
MS2 Library
% of "Pass"
Compounds
Identified
Agilent PCDL
53%
CFM-ID Top Hit
50%
PCDL and/or
CFM-ID Top Hit
73%
ji
Pass" Compounds
PCDL -> Agilent reference MS2 library
15
"Pass" compounds (n=377) -> ENTACT
chemicals observed with MS2 data
-------
oEPA
United States
Environmental Protection
Agency
Who Else is Working on ENTACT?
Contractors:
Vendors:
fcol UNIVERSITY OF
W ALBERTA 1
Duke
UNIVERSITY
,V I EMORY
\y UNIVERSITY
Oregon State
UNIVERSITY 1
P® San Diego State
JL University
i wi 3 b
19 Blind
submissions
15 Unblinded
submissions
y The
(^1 S C R I P P S
^ Research
W Institute
Agilent Technologies
Thermo
SCIENTIFIC
IBC® AB SCI EX
General Participants:
lllll.lll UFI FLORIDA
UNIVERSITE DU
LUXEMBOURG
Wisconsin State
Laboratory of Hygiene
UNIVERSITY OF WISCONSIN-MADISON
(©)
RECETOX
UW TACOMA
A
Nisr
CalEPA
California Environmental
Protection Agency s'^>
NC STATE
UNIVERSITY
Mount UNIVERSITY^
Sinai BIRMINGHAM aquatic research Wooo
FIU
-------
oEPA
United States
Environmental Protection
Agency
Comparing Reported Features (n=16 labs)
ToxCast Mixtures
Fortified Matrices
1
2
3
4
5
6
7
8
9
10
Dust
Serum
Band
Act.
95
95
95
95
185
185
365
365
95
365
365
95
185
Lab 1
128
148
166
187
292
269
318
470
177
410
NR
NR
NR
2
142
154
102
129
250
242
401
399
105
452
NR
NR
NR
3
48
40
48
59
110
101
97
130
37
109
NR
NR
NR
4
301
130
375
341
408
404
719
687
198
327
NR
NR
NR
5
65
66
74
72
105
118
193
215
54
162
NR
NR
NR
6
587
552
596
554
798
846
1327
1274
509
1176
NR
NR
NR
7
93
114
116
106
182
201
360
374
73
330
236
92
124
8
337
372
303
365
321
363
466
505
510
463
259
222
313
9
135
130
125
154
188
195
284
295
100
153
270
54
101
10
70
57
64
66
105
115
176
125
35
159
NR
NR
NR
11a
595
486
571
630
746
669
899
910
588
792
1009
614
NR
lib
66
170
51
41
272
116
214
101
163
404
861
145
557
12
51
37
35
39
74
59
124
109
42
105
124
52
76
13
137
65
45
74
68
234
413
408
120
317
389
178
88
14
215
249
212
249
207
275
245
254
140
253
NR
NR
NR
15
1298
1258
1304
1209
1651
1641
2520
2538
1202
2193
NR
NR
NR
16
153
217
221
199
254
321
523
651
496
396
NR
NR
NR
= under reported
= near actual
= over reported
NR = not reported
Ulrich etal. 2019. https://doi.org/10.1007/s00216-018-1435-6
-------
xvEPA
United States
Environmental Protection
Agency
Comparing Identified Compounds fn=3 labs)
1.269 Spiked Substances
GC = gas chromatography
ESI- = neg. electrospray ionization
(liquid chromatography)
ESI+ = pos. electrospray ionization
(liquid chromatography)
18
Ulrich et al. 2019. https://doi.org/10.1007/s00216-018-1435-6
-------
vvEPA
United States
Environmental Protection
Agency
1)
2)
3)
4)
Extraction
Extraction
Extraction
Experiments with SRM Dust
Solvent spike
(best case)
: Post-extraction
high spike
: Pre-extraction
high spike
: Pre-extraction
low spike
(ENTACT sample)
400
\
observed
24% lost (matrix)
(extraction)
\33% lost (conc.)
:s*
s*
s*
s*
s*
Jo'
OQ
Jo'
¦?>
Jo'
Jo'
¦?>
19
Newton et al. [in preparation]
-------
oEPA
United States
Environmental Protection
Agency
Experiments with SRM Dust
Results for Unfortified SRM Dust
Chemical
All Reported
Reported
Observed
Class
Compounds
Using LC-ESI
Using NTA
PAHs
69
0
0
PCBs
44
0
0
PFAS
31
31
12
BFRs
30
3
0
OCPs
15
0
0
OPEs
12
9
4
Phthalates
7
0
2
Total
208
43
18
12585
Organic
nKnin*1*
House JDu-^
flnr ^
Newton et al. [in preparation]
o
o
1 o9-
O)
1 o8-
CD
C
<4-1
1 o7-
3
Q
1 o6-
£
C
ID
o
O
<-»
ra
1 o4-
9>_
-t-i
c
0)
1 o3-
o
c
o
1 o2-
o
1 o1-
1 o0-'
O Reported using LC-ES!
® Not observed using NTA
O Observed using NTA
O Spiked in ENTACT samples
O Spiked at higher conc.
O Est. to cause bioactivity in children*
S
q-o-Lo
o
c
°Oo
Ooo5a^
O o
P°°
Non-fortified SRM Dust
Fortified SRM D ust
* "..the dose that would be needed in the most-sensitive 5% of the population to
produce a steady-state plasma concentration equal to [the 10th] percentile of the
ToxCast AC50 distribution across assays for the given chemical."
Ring et al.: https://doi.Org/10.1016/j.envint.2017.06.004
-------
oEPA
United States
Environmental Protection
Agency
Analytical arid Bioanalytical Chemistry (2019) 411:853-866
https^/doi jorg/10.1007/S00216-018-1435-6
Publications to date
RESEARCH PAPER
#
Cross Mark
EPA's non-targeted analysis collaborative trial (ENTACT): genesis,
design, and initial findings
Elin M. Ulrich1 ¦ Jon R. Sobus1 • Christopher M. Grulke2 ¦ Ann M. Richard2 • Seth R. Newton1 ¦ Mark J. Strynar1
Kamel Mansoun3'4 • Antony J. Williams2
Received: 30 July 2018/Revised: 14 September 2018 / Accepted: 17 October 2018/Published online: 6 December 2018
<: This is a U.S. Government work and not ur>der copyright protection in the US; foreign copyright protection may apply 2018
£°J CHROMATOGRAPHY . , L
¦mi—— Tnn*v February / March 2018
Comprehensive, Non-Target
Characterisation of Blinded
Environmental Exposome Standards
Using GCxGC ana High Resolution
Time-of-Flight Mass Spectrometry
by Lome Fell\ Todd Rjcbards andJoe Btnkley
LECO, &i tut Joseph, AUcbt&tn, USA
* Corresponding Author: lorne JeB@ Jei o.t om
Analytical and Bioanalytical Chemistry (2019) 4113835-851
hltps J'/dOLOrg/lO.10O7/SOO216-01 &-1526-4
RESEARCH PAPER
Crcss.Vlajk
Using prepared mixtures of ToxCast chemicals to evaluate non-targeted
analysis (NTA) method performance
Jon R. Sobus '© • Jarod N. Grossman2,3 • Alex Chao2 • Randolph Singh" • Antony J. Williams5 ¦ Christopher M. Grulke5 ¦
Ann M. Richard5 * Seth R. Newton1 • Andrew D, McEachran4 • Elin M. Ulrich1
Received: 19 September 2018 /Revised: 14 hkivember 2018 / Accepted: 27 November 2018 /Published online: 5 January 2019
f This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019
JCIM
JOURNAL OF
CHEMICAL INFORMATION
AND MODELING & Cite This: J. Chem. inf. Model. 2D19, 59, 4052-4060
pubs.acs.ocg/jcim
Evaluation of In Silico Multifeature Libraries for Providing Evidence
for the Presence of Small Molecules in Synthetic Blinded Samples
Jamie R. Nunez, Sean M. Colby, Dennis G. Thomas, Malak M. Tfaily, ~ Nikola Tolic,
Elin M. Ulrich, Jon R. Sobus, Thomas O. Metz,* " Justin G. Teeguarden,* ' ^
and Ryan S. Renslow
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
~U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, Research
Triangle Park, North Carolina 27711, United States
^Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331, United States
"""Department of Environmental Science, University of Arizona, Tucson 85712, United States
-------
oEPA
Summary of ENTACT Findings
United States
Environmental Protection
Agency
• NTA methods are suitable for detecting many ToxCast chemicals
• False positives can greatly outweigh true positives
• False Pos /True Pos ~ 10x
• Work needed on feature credentialing
• True Positives: <75%
• Will miss some chemicals that are present in samples
• Why? Which ones? Always?
• Multiple methods required for broad characterization
• No "one size fits all" method
• Subtle method changes affect measurable chemical space
• Concentration, media, and extraction techniques will affect performance
• Goal reached when we can make these statements:
• "When a compound is observed, we're confident it's really there!"
• "When a compound isn't observed, we're confident it's not there!"
22
-------
®!S»_ Ongoing and Future Work
Agency
¦ Full cross-lab performance evaluation
• Primary focus true positives, false negatives, confidence levels
• Secondary focus -> unexpected true positives
¦ Database development
• Enable user queries, additional analyses, model development
¦ Global summary report
• Provide guidance and acceptance criteria for NTA studies
¦ The benefits of ENTACT will be proportional to the level of effort!
23
Office of Research and Development
-------
&EPA
United States
Environmental Protection
Agency
Contributing Researchers
This work was
supported, in
part, by ORD's
Pathfinder
Innovation
Program (PIP)
and an ORD
EMVL award
EPA ORD
Hussein Al-Ghoul*
Alex Chao*
J a rod Grossman*
Kristin Isaacs
Sarah Laughlin*
Charles Lowe
James McCord
Jeff Minucci
Seth Newton
Katherine Phillips
Tom Purucker
Randolph Singh*
Mark Strynar
Elin Ulrich
* = ORISE/ORAU
EPA ORD (cont.)
Chris Grulke
Kamel Mansouri*
Andrew McEachran*
Ann Richard
John Wambaugh
Antony Williams
Agilent
Jarod Grossman
Andrew McEachran
GDI!
Ilya Balabin
Tom Transue
Tommy Cathey
24
Office of Research and Development
-------
oEPA
United States
Environmental Protection
Agency
Questions?
# 52 vo
I® I
% r
------- |