oEPA
United States
Environmental Protection
Agency
EPA's Non-Targeted Analysis Research
Program: Building Tools to Enable Rapid
Exposure Surveillance
Jon R. Sobus, Ph.D.
Center for Computational Toxicology and Exposure
Office of Research and Development
-------
Key Drivers for 21st Century Exposure
Environmental Protection ^ ^ ®
Agency
Science
1) Understanding causes of disease
.. 70-90% of disease risks are
probably due to differences in
environments"
EPIDEMIOLOGY
Environment and Disease Risks
Stephen M. Rappaport and Martyn T. Smith
Although the risks of developing
chronic diseases are attributed to
both genetic and environmental fac-
tors, 70 to 90% of disease risks are probably
due to differences in environments {J—3). Yet
epidemiologists increasingly use genome-
wide association studies (GWAS) to investi-
gate diseases, while relying on questionnaires
to characterize "environmental exposures."
This is because GWAS represent the only
approach for exploring the totality of any risk
factor (genes, in this case) associated with dis-
ease prevalence. Moreov er, the value of costly
genetic information is diminished when inac-
curate and imprecise environmental data lead
to biased inferences regarding gene-environ-
ment interactions {4), A more comprehensive
and quantitative view of environmental expo-
School of Pubtk Heallh, University of California, Berkeley,
CA 94720-7356, USA. E-mait srappaport@berkeley.edu
sure is needed if epidemiologists are to dis-
cover the major causes of chronic diseases.
An obstacle to identifying the most
important environmental exposures is the
fragmentation of epidemiological research
along lines defined by different factors.
When epidemiologists investigate environ-
mental risks, they tend to concentrate on a
particular category of exposures involving
air and water pollution, occupation, diet
and obesity, stress and behavior, or types
of infection. This slicing of the disease pie
along parochial lines leads to scientific
separation and confuses the definition of
"environmental exposures." In fact, all of
these exposure categories can contribute to
chronic diseases and should be investigated
collectively rather than separately.
To develop a more cohesive view of envi-
ronmental exposure, it is important to recog-
nize that toxic effects are mediated through
A new paradigm is needed to assess how a
lifetime of exposure to environmental factors
affects the risk of developing chronic diseases.
chemicals that alter critical molecules, cells,
and physiological processes inside the body.
Thus, it would be reasonable to consider
the "environment" as the body's internal
chemical environment and "exposures" as
the amounts of biologically active chemi-
cals in this internal environment. Under this
view, exposures are not restricted to chemi-
cals (toxicants) entering the body from air,
water, or food for example, but also include
chemicals produced by inflammation, oxida-
tive stress, lipid peroxidation, infections, gut
flora, and other natural processes (5, 6) (see
the figure). This internal chemical environ-
ment continually fluctuates during life due
to changes in external and internal sources,
aging, infections, life-style, stress, psychoso-
cial factors, and preexisting diseases.
The term "exposome" refers to the total-
ity of environmental exposures from concep-
tion onwards, and has been proposed to be a
460
22 OCTOBER 2010 VOL 330 SCIENCE www.sciencemag.org
Published by MAS
2) Ensuring chemical safety
GIVE A DOG A PHONE
Technology for our furry friends
NewScientist
We've made
150,000 new chemicals
9
We touch them,
we wear them, we eat them
But which ones should
we worry about?
SPEC IA I. REPORT, pa$e 34
THE GOOD FIGHT CHAMBER OF SECRETS IS IT AUVE>
Mott violence The greatest ever find Artificial worm could
is also virtuous of eirty human bones be first Ogital arwmaJ
-------
oEPA
United States
Environmental Protection
Agency
High- j hroughput 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
-------
oEPA
United States
Environmental Protection
Agency
For Which Chemicals Must We Assess Exposure?
Well-known chemicals
• 100s - 1,000s (e.g., NHANES)
• Quality exposure data
Known but data-poor chemicals
• 1,000s - 1,000,000s (e.g., TSCA)
• Limited exposure data
Chemicals not vet known to exist
• Unknown #
• No exposure data
Targeted Analysis
-------
Targeted Analysis for Quantitation of Knowns
Environmental Protection ^
Agency
Standards/Samples
Lab Analysis
Calibration
Quantitation
C o n ce ntratio n
C o n ce ntration
0=S OH
II
o
C o n ce ntration
C o n c e n tratio n
-------
NTA for Chemical Discovery
Environmental Protection +
Agency
1) Prioritize "molecular features"
2) Correctly assign formulas
3) Correctly assign structures
4) Predict chemical concentrations
5) Determine chemical sources
(4)
12 |jg/mL
1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 33 40 41 42 43 44
Counts (%) vs. Acquisition Time (min)
Samples
A Chromatogram Results
* » * 9. 1 & Tcfl 3 Q. 4 ^[yjn A lit
-ESI ED880.2529) Scan Frag=80.0VWorklistData05.d
1 Sample
1 ionization Mode
300 Extracted "Molecular Features"
High-
-liLr
Resolution MS
-------
vvEPA
United States
Environmental Protection
Agency
How does High Resolution MS Work?
Atom
Natural
Abundance
Exact Mass
1H
99.9885%
1.007825
2H
0.0115%
2.014102
12C
98.93%
12.000000
13Q
1.07%
13.003355
14N
99.632%
14.003074
15N
0.368%
15.000109
16Q
99.757%
15.994915
17Q
0.038%
16.999131
18Q
0.205%
17.999159
19p
100%
18.998403
32 S
94.93%
31.972072
33S
0.76%
32.971459
34 S
4.29%
33.967868
36 S
0.02%
35.967079
35CI
75.78%
34.968853
37CI
24.22%
36.965903
Example: Fipronil
Molecular Formula: C^H^CIjFg^OS
Monoisotopic Mass: 435.938706
= (12.0000*12 Carbon) + (1.007825*4 Hydrogen) +
(34.968853*2 Chlorine) + (18.998403*6 Fluorine) +
(14.003074*4 Nitrogen) + (15.994915*1 Oxygen) +
(31.972072*1 Sulfur)
100
CD
o
c
<0
"O
c
3
_Q
<
m/z
-------
oER
-------
oEPA
United States
Environmental Protection
Agency
Exposure Surve llance for Consumer Products
fj Cite This: invkon. Scl. Technol. 201S, 52, 3125-3135
pubsj3cs.org/est
Suspect Screening Analysis of Chemicals in Consumer Products
Katherine A Phillips. Alice Yau, Kristin A. Favela, Kristin K Isaacs, Andrew McEachran,
Christopher Grulkc.' Ann M. Richard,' Antony J. Williams,' Jon R. Sobus, Russell S. Thomas,"
and John F. Wambaugh* "
National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental
Alexander Drive, Research Triangle Park, North Carolina 27711, United States
Southwest Research Institute, San. Antonio, Texas 78238, United States
Oak Ridge Institute lor Science and Education (ORISE), Oak Ridge, Tennessee 37830, United Stal
National Center for Computational Toxicology, Office of Research and Development, U.S. Environr
T. W. Alexander Drive, Research Triangle Park, North Carolina 27711, United States
19% of chemicals
identified by NTA are on
consumer product
chemical lists
Articles
Confirmed or tentative
chemical identification
Previously known chemical
related to consumer products
Formulations
Foods
Carpet
Cotton Clothing
Carpet Padding
Fabric Upholstery
Shower Curtain
Vinyl Upholstery
Plastic Children's Toy
Lipstick
Toothpaste
Sunscreen
Indoor House Paint
Shaving Cream
Hand Soap
Skin Lotion
Baby Soap
Deodorant
Shampoo
Glass Cleaner
Air Freshener
Cereal
¦KTh
¦n?
CD-
IQ-
^03-
|—C±Z
-OZF-
H-
-D>
—m
i—t-
HXh
-m-
-M
250
200 150 100 50
Unique Chemicals
-4
-2
Iogio0jg/g)
-------
oEPA
United States
Environmental Protection
Agency
Chem cal Prioritization for Drinking Water
Enviionmenul Pollution 234 (2018) 297 306
ELSEVIER
Contents lists available at ScienceDirect
Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
EXvmoNMDrrAJ.
POLLUTION
Suspect screening and non-targeted analysis of drinking water using
point-of-use filters'*
Seth R. Newton a' \ Rebecca L McMahen Jon R. Sobus J, Kamel Mansouri J'c- \
Antony J. Williams c, Andrew L). McEachran b c, Mark J, StrynarJ
a Unt£t?d ifcires fiiWrortmenuii Protection Agency. National Exposure Research Laboratory, Research Triangle Park, JVC 27709, United Stales
b Oak Ridge Institute for Science and Education Research Participant, 109 TW. Alexander Drive, Research 'Triangle Park, NC 27709. United States
c United States Environmental Protection Agency, National Center for Computational Toxicology, Research Triangle fork. NL 27709, United States
H)
HRT1A
3
III
Top 20 Priority
Compounds
L-. I 11 ¦ 11 i
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
Compound
ToxPi
Score
1
l,2-8enzisothiaiolh-3-one*
2.99
2
Diethyleneglycol
2.38
3
N-[3-(Dimethylamino (propyl)
2.32
methacrylamide
4
Nonylparaben
2.22
5
Dipentylphthalate
1.89
5
2-[2-(2-Butoxyethoxy)
1.85
ethoxyJethanoT
7
N,N-Dimethyldodecan-
1-amine*
1.81
8
Sucralose
1.80
9
PFOS*
1.79
10
2-(2-Ethoxyethoxy)
1.76
ethyl acetate*
11
TDCPP*
1.71
12
Zearalano)
1.67
13
PFOA*
1.66
14
Butyl paraben
1.66
15
Noristerat
1.65
16
p-Synephfine
1.55
17
Alprostadil
1.55
18
Sclareol
1.55
19
PFDA*
1.51
20
Simvastatin
1.50
•Confirmed with standard
Top 100 Priority Compounds
-------
oEPA
United States
Environmental Protection
Agency
Exposure Forensics for Recycled Products
Ubiquitous chemicals in articles (e.g., phthalates)
i « i.'./" i t
Jf -to?,
||i«
Fragrances in recycled paper products
Product Category
Construction
Fabric and Home Goods
Food Contact
Paper
¦¦ Plastic Home & Auto
Recycled Tire Products
Toys & Ray Mats
Jd
mi r
Chemicals (variety of functions) that only occur in recycled tire products
Fragrances in toys
IE*
!
%
;! i
Tentatively Identified and Confirmed Chemicals
Figure by C. Lowe and K. Isaacs
Toys/Play Mats
Recycled Tire
Products
Household
Products
Paper
Products
Food Contact
Materials
Fabric Home
Goods
Construction
Materials
0 5 10 15
Mean Number of Chemicals per Sample
Classification Recycled Virgin
-------
Altered Cell Signaling
The Placental Exposome
(via LC-HRMS)
Different Environmental
Exposures
B omarker D scoverv for Placenta Samples
United States
Environmental Protection
Agency
29 in
Preeclampsia
~
Impaired Angiogenesis
*
-5 0 5
Log2 Fold Change
controls
508 n cases
11
Collaboration with J. Rager (UNC Chapel Hill) and J. Grossman (Agilent)
-------
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."
-------
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
omm
OQQ
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i O IICMH 'O Q
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q bcq"' O O
QQOOO
It^QSi
EPA's Non-Targeted Analysis
14
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
16
-------
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
-------
oEPA
United States
Environmental Protection
Agency
Who is Working on ENTACT?
Contractors:
Vendors:
fcol UNIVERSITY OF
W ALBERTA 1
Duke
UNIVERSITY
,V I EMORY
\y UNIVERSITY
Oregon State
UNIVERSITY 1
San Diego State
JL University
1 Wi 3 m
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
hPA Results for 10 Synthetic Mixtures
N
1200
1000
800
600
400
200
0
Spiked Substances -> ~1,200
Observed Features -> ~26,000
0
Sobus et al. 2019.
https://doi.org/10.1007/s00216-018-1526-4
10 15
RT (min)
20
-------
oEPA
United States
Environmental Protection
Agency
hPA Results for 10 Synthetic Mixtures
1200
1000
Real Features -> -12,000
Noise/Artifacts^ ~14,000
Kept
Removed
800
N
600-
400-
200
• ••ti •• •••¦
0
0
Sobus etal. 2019.
https://doi.org/10.1007/s00216-018-1526-4
10 15
RT (min)
20
-------
oEPA
United States
Environmental Protection
Agency
hPA Results for 10 Synthetic Mixtures
N
E
1200
1000
800-
600-
400-
200-
True Positives -> ~1,000
False Positives? -11,000
©.
Pass
Other
•o
0
0
Sobus etal. 2019.
https://doi.org/10.1007/s00216-018-1526-4
10 15
RT (min)
20
-------
xvEPA
United States
Environmental Protection
Agency
Method Comparison (n=3 methods)
1.269 Spiked Substances
GC = gas chromatography
ESI- = neg. electrospray ionization
(liquid chromatography)
ESI+ = pos. electrospray ionization
(liquid chromatography)
22
Ulrich et al. 2019. https://doi.org/10.1007/s00216-018-1435-6
-------
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 and Conclusions
United States
Environmental Protection
Agency
• 21st century exposure science demands higher-throughput monitoring techniques
• HRMS enables rapid chemical characterization in all tested media
• NTA methods represent a viable "first-pass" monitoring solution
• Methods must be selected and implemented with care
• Not a panacea, but a means of collecting provisional exposure data
• NTA well-suited for current "research" endeavors
• Much more evaluation needed to establish "reference" methods
• Successful implementation requires close coordination between
• Analytical chemists
• Environmental/exposure modelers
• Cheminformaticians
• Programmers/Developers
• Subject matter experts
• and others...
-------
&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
25
Office of Research and Development
-------
Questions?
sobus.jon@epa.gov
r»
*
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n|.j
"ll I»#
ir ¦
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5"*rnm ?i «i is it
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& r*^-
*• .
Jr
The views expressed in this presentation are those of the
authors and do not necessarily represent the views or policies
of the U.S. Environmental Protection Agency.
------- |