United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas, NV 89193-3478
Research and Development
EPA/600/S8-91/045 Feb. 1992
&EPA Project Summary
Selection of Adduct-Forming
Chemicals for Human Monitoring
Studies
S. Evans, C. Nauman, M. Waters, and S. Nesnow
The U. S. EPA, through its Environ-
mental Monitoring Systems Laboratory-
Las Vegas (EMSL-LV) and its Health
Effects Research Laboratory-Research
Triangle Park (HERL-RTP) has been ex-
ploring the feasibility of using biologi-
cal markers to monitor exposure to en-
vironmental chemicals. Among the can-
didate biomarkers of exposure and ef-
fect are the adducts formed by reac-
tion of carcinogenic electrophiles with
DMA and/or protein.
In 1987, the staffs of HERL-RTP,
EMSL-LV and Oak Ridge National Labo-
ratory (ORNL) conducted a study de-
signed to identify those chemical ex-
posures of interest to the Agency which
may, potentially, be effectively moni-
tored using adduct-based techniques.
The participants began by compiling a
list of chemicals of known or suspected
health hazards and for which the mas-
ter list was then systematically evalu-
ated for (1) the potential for adduct-
formation in vivo, (2) the availability of
supportive adduct research data, (3)
the identifiability of population(s), and
(4) the level of genetic activity. After
considering all the relevant data, the
participants selected and prioritized for
further study a small group of chemi-
cals considered to have the greatest
potential for use in pilot, adduct-based,
biological monitoring studies in human
populations.
Introduction
The U. S. Environmental Protection
Agency (EPA) has been exploring the fea-
sibility of using biological markers to
monitor exposure to environmental chemi-
cals. Among the candidate biomarkers of
exposure and effect are the adducts
formed by reaction or carcinogenic
electrophiles with DMA and/or protein. Cer-
tain carcinogens form electrophilic spe-
cies that bind covalently to macromol-
ecules such as DMA, RNA, and protein.
New analytical methodologies including
monoclonal antibody techniques, 32P
postlabeling, and gas chromatography-
mass spectrometry (GC/MS) have been
developed to identify these chemical ad-
ducts. Application of these techniques may
permit detection and quantitation of hu-
man exposure.
It was the objective of this project to (a)
identify and rank adduct-forming chemi-
cals of interest to the U.S. EPA, and (b) to
select the most promising of those chemi-
cals for further evaluation as candidates
for use in future molecular epidemiologi-
cal and exposure studies.
In 1987, the staffs of the Health Effects
Research Laboratory-Research Triangle
Park (HERL-RTP), the Environmental
Monitoring Systems Laboratory-Las Ve-
gas (EMSL-LV), and Oak Ridge National
Laboratory (ORNL) met to discuss and
conduct a study designed to identify those
chemical exposures of interest to the
Agency which may, potentially, be effec-
tively monitored using adduct-based tech-
niques.
Chemical Selection-The Initial
Process
Section 2 describes the process em-
ployed to identify the chemicals that would
be of interest to the Agency. The process
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of chemical selection was divided into four
steps based on the following four selec-
tion criteria: (1) interest to the Agency, (2)
availability of supportive adduct research
data, (3) availability of identified exposed
population(s), and (4) genetic activity (i.e.,
genotoxicity). The first step was to com-
pile a list of prioritized chemicals of inter-
est to the EPA, i.e., chemicals which rep-
resent known or suspected health haz-
ards and for which the potential for hu-
man exposure exists. The procedure used
was to request a list of chemicals consid-
ered to be of high priority in terms of their
potential health hazard from each of the
EPA Program Offices.
The lists from each Program Office were
prioritized, based on the number of times
each chemical appeared. All of the lists
were then combined into a common priori-
tized list. The final prioritized list (Appen-
dix B) consisted of approximately 1,585
chemicals. The prioritized list of chemicals
was then cross referenced with a list of
131 chemicals (Appendix C) that were
known to form adducts based upon a re-
view of the literature. Sixty-three of the
chemicals listed in Appendix B had at
least some data indicating that they form
adducts. The potential candidates for fur-
ther research and evaluation were prima-
rily selected from this list of 63 compounds
(Appendix D).
The second step was to identify supple-
mental sources of additional chemicals not
listed in Appendix D. Appendix F lists 167
chemicals which, though not known to form
adducts, are classified as known or sus-
pected human carcinogens. Some of these
chemicals may either (1) not yet have
been tested for adduct-formation, or (2)
the adducts they form were not yet de-
tectable with the methods applied. Appen-
dix G lists adduct-forming, suspect car-
cinogens that do not appear on the priori-
tized list. Most of these are drugs or re-
search chemicals which are not subject to
regulation by the EPA.
Initially, the lists of chemicals in Appen-
dix D were screened for chemicals that
could be eliminated a priori for one or
more of the following reasons: (1) adduct
formation by the chemical was considered
unlikely for structural reasons, (2) the lit-
erature contained insufficient adduct infor-
mation to support the chemicals's candi-
dacy, or (3) the adduct formed by the
chemical was a small alkylation product
that lacked sufficient specificity for expo-
sure monitoring. It should be kept in mind,
that these alkylating agents may still be
useful in screening studies to identify ex-
posure to certain classes of chemicals
(rather than to individual chemicals). In
addition, some of these chemicals might
display a specific adduct-binding pattern
that would increase their specificity and
sensitivity, thus making them useful for
monitoring exposure.
Chemicals in Appendix D were marked
with a plus sign (+) if (1) they were fairly
well represented in the literature on ad-
duct research, or (2) sufficient preliminary
data (i.e., ongoing research) existed to
support their candidate potential. In gen-
eral, it was decided that the chemical(s)
selected for the initial study should be
one(s) that form chemical-specific adducts
that can be identified with relative ease.
Section 2.2.3 describes the process
used to identify and characterize poten-
tially exposed human populations, the third
step in the chemical selection process.
The chemical list used in this step was a
subset of a combined list of chemicals
from two databases: (1) the GENETOX
database and (2) the Genetic Activity Pro-
file database. The selected chemicals
tended to be those that were either known
or considered likely to form adducts, based
on chemical structure and available
genotoxicity data. Appendix I lists the 253
chemicals that were subsequently exam-
ined and characterized for the availability
of exposed populations for study.
The following criteria were used in scor-
ing the chemicals on their human popula-
tion exposure potential:
(1) population availability
(2) population size (best estimate)
(3) level of exposure
(4) quality of exposure history
(5) degree of confounding multiple
exposures
Each criterion was assigned a score from
0 to 3 and the resulting overall score for a
chemical was the product of the criterion
scores. Only 33 chemicals had a score
greater than zero and the remaining 195
chemicals received no score due to the
absence of sufficient data.
The selected adduct-forming chemicals
of interest to the Agency (Appendix D)
were then re-examined in light of the rel-
evant human population exposure data.
Section 2.2.4 describes the process of
using genetic activity profiles in ranking
the chemicals selected, the fourth step in
the chemical selection process. The ge-
netic toxicological activity of a compound,
as measured by various bioassays, is rel-
evant to both (1) the potential hazard it
poses to human health and, hence, its
interest to the EPA, and (2) the likelihood
that the chemical will form detectable mac-
romolecular adducts. Genetic activity pro-
files were particularly useful in rating those
chemicals for which little adduct informa-
tion was available.
Priorltization of Chemicals
Selected for Further Study
Section 2.3 describes those chemicals
that were not eliminated during the four
steps of the chemical selection process
described previously. Twenty- three chemi-
cals listed in Table 1 were assigned high,
medium, or low priority based on adduct
data, current research status, population
availability, and genetic activity. This sec-
tion contains a discussion of the assigned
priority of each chemical. An unclassified
category for reconsideration was estab-
lished for those chemicals for which the
potential for study existed, but for which
the information available at the time was
insufficient to support a judgement.
Chemicals that were classified as mix-
tures were considered separately. While
there is considerable interest in the poten-
tial for monitoring exposure to some of
these chemicals, it was also recognized
that (1) it would be difficult, due to the
presence of confounding exposures, to
attribute health effects to any specific com-
ponent of a mixture (e.g., N-nrtrosonomica-
tine in tobacco smoke), and (2) that po-
tentially high background levels of adducts
to ubiquitous chemicals such as benzene,
benzo(a)pyrene, and 4-aminobiphenyl
might preclude detection of all but the
highest exposures.
Conclusions and
Recommendations
The selection of Adduct-Forming Chemi-
cals for Human Monitoring Studies docu-
ment presents a strategy for selecting
chemicals for further study. The selected
chemicals in Table 1 should be further
reviewed with particular emphasis on the
following 13 chemicals: styrene, ethylene
oxide, 4-4'methylene bis(2-chloroaniline)
(MOCA), benzidine, vinyl formaldehyde,
chlordane,nitropyrene,1,3-dichloropropene,
and ethylene dichloride. It was considered
that certain low priority chemicals (e.g.,
chloroform, 2,4,-toluene diisocyanate, ben-
zyl chloride, o-toluidine, and malathion)
might be of potential use in exposure moni-
toring studies based on measurement of
protein adducts, but more research data
were needed. It was recommended that
adduct research on pesticides be reviewed.
Finally, it was suggested that the chemi-
cal-adduct research data for each chemi-
cal be entered into a computer informa-
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tional system. The efforts of the 1987
workgroup represented an initial step in
the development of the Biomarkers Pro-
gram of the U.S. EPA. Additional program
development studies have, in the inter-
vening time, followed up on the
workgroup's initial conclusions and rec-
ommendations. The reports on these
projects, which represent subsequent up-
dates and refinements of the chemical
selection process, are briefly described in
Section 3.
The information in this document has
been funded wholly or in part by the United
States Environmental Protection Agency
under Contract No. 68-CO-0049 to
Lockheed Engineering and Sciences Com-
pany. It has been subjected to the
Agency's peer and administrative review,
and it has been approved for publication
as an EPA document.
Mention of trade names or commercial
products does not constitute endorsement
or recommendation for use.
Table 1. Prioritized Chemicals Selected for Further Study
Number •
1.(11)
2. (4)
3. (29)
4. (18)
5. (2)
Chemical
Styrene
Ethylene oxide
4,4-Methylene
Ibis (2-chloro-
aniline) (MOCA)
Benzidine
Vinyl chloride
Rating b
H
H
M/H
M/H
M/H
Data Summary
Genetic activity = high. Good population data. Supportive adduct research studies.
Genetic activity = high. Available populations: hospital sterilization workers and
patients receiving treatment through the sterilized equipment used for cases such
as renal dialysis, which provides more consistent dosing than bypass surgery.
Numerous human studies using Hb adducts have been conducted.
Genetic activity = low. Available population. Active research support.
Genetic activity = high. Available population. Supportive adduct research data.
Genetic activity = high. Population available, but numerous confounding exposures include
6. (19)
7. (25)
8. (3)
9. (15)
10. (N/A)
12. (6)
13. (N/A)
14. (N/A)
15. (N/A)
16. (55)
17. (5)
18. (41)
19. (40)
20. (N/A)
Epichlorohydrin M/H
Propylene oxide M/H
Formaldehyde L/M
Acrylonitrile L/M
Pentachlorophenol L
2,4-toluene
diisocyanate
Chloroform
U/Ex
U/Ex
Chlordane U/Ex
Nitropyrene U
1,3-dichloropropene U
Toluidine, O- U/L
Ethylene dichloride U
Benzyl chloride L/M, U/Ex
Dimethyl carbamoyl U/L
chloride
Malathion
U/L
vinyl bromide, acrylonitrile, 1,2-dichloroethane, 1,2 dibromoethane, ethylene halohydrins,
and urethane. Also, exposure might be low and it must be determined if adducts would
be formed at these levels. Additional research data is necessary for support.
Genetic activity = medium. Reasonable population in production workers. Needs further
validation from animal studies.
Genetic activity = similar to ethylene oxide profile, although not as much information has been
collected. Ubiquity could be a problem. Needs more supportive research data.
Genetic activity = high. Ubiquitous. Changed from H to L/M because it was believed that the
separation of endogenous/exogenous adduct formation would be difficult and some reactiv-
ity may be reversible. (CUT)
Genetic activity = positive in vitro, questionable in humans. Available population.
Genetic activity = minimal. Availability of unconfounded exposed population is questionable.
Indirect adduct formation. Additional data needed.
Genetic activity = inadequate information. Population available. Need more information,
particularly on protein adducts as exposure biomarkers.
Genetic activity = low. Exposed populations require further study. Investigate potential of
protein adducts.
Review all criteria.
Review all criteria.
Review all criteria.
Genetic activity = low. Population questionable. Minimal adduct informtion. Investigate protein
adduct formation.
More information is needed on all criteria. Could demonstrate a unique adduct.
Genetic activity needs to be determined. Additional information is needed.
Genetic activity needs to be determined. Population data were questionable. Minimal
suportive research data.
Investigate protein adduct formation.
6U.S. GOVERNMENT PRINTING OFFICE: 1992 - 64K-O80/40163
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Table 1. (concluded)
Number *
21. (33)
22. (57)
23. (38)
Chemical Rating
Mephalan
Mitomycin C
Thioacetamide
Data Summary
Put aside for possible use in other studies.
"
..
The chemicals in Table 1 were rated on their genetic activity, adduct formation, exposed population availability, and research status. Definitions of the notation
used are as follows:
* (#) The number in parentheses is the sequential number as it appeared in Appendix D.
(N/A) Indicates that the chemical is not listed in Appendix D.
b L Indicates a low priority ranking.
M Indicates a medium priority ranking.
H Indicates a high priority ranking.
U Indicates an unclassified rating. Assigned to chemicals for which the potential for study existed, but for which the information available
at the time was inadequate to support any judgement..
Ex Indicates that further information is desired on exposure monitoring of protein adducts.
The EPA authors, S.J. Evans and C.H. Nauman (also the EPA Project Officer, see
below) are with the Environmental Monitoring Systems Laboratory, Las Vegas, NV
89193, and M. Waters and S. Nesnow are with the Health Effects Research Labora-
tory, Research Triangle Park, NC 27711.
The complete report, entitled "Selection of Adduct-Forming Chemicals for Human
Monitoring Studies," (Order No. PB92-132877/AS; Cost: $19.00, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, NV 89193-3478
United States
Environmental Protection
Agency
Center for Environmental
Research Information
Cincinnati, OH 45268
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POSTAGE & FEES PAID
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Penalty for Private Use $300
EPA/600/S8-91/045
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