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
      BULK RATE
POSTAGE & FEES PAID
          EPA
   PERMIT No. G-35
Official Business
Penalty for Private Use $300
 EPA/600/S8-91/045

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