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 Printed on Recycled Paper ------- 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- ------- 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 ------- 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 ------- |