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United States Environmental Protection Agency Office of Health and Environmental Assessment Washington DC 20460 EPA/600/8-84/006F September 1 985 Final Report Research and Development ___________________________________________________ Health Assessment Document for 1,2-Dichloroethane (Ethylene Dichloride) EPA/600/8-84/006F September 1985 Final Report Health Assessment Document for 1,2-Dichloroethane (Ethylene Dichloride) U S. ENVIRONMENTAL PROTECTION AGENCY Office of Research and Development Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Research Triangle Park, North Carolina 27711 CMA. 051555 DISCLAIMER This document has been reviewed in accordance with the U.S. Environmental Protection Agency's peer and administrative review policies and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ii CMA 051556 PREFACE The Office of Health and Environmental Assessment has prepared this health assessment to serve as a "source document" for EPA use. The health assessment document was originally developed for use by the Office of Air Quality Planning and Standards to support decision-making regarding possible regulation of ethylene dichloride as a hazardous air pollutant. However, the scope of this document has since been expanded to address multimedia aspects. In the development of the assessment document, the scientific literature has been inventoried, key studies have been evaluated and summary/conclusions have been prepared so that the chemical's toxicity and related characteristics are qualitatively identified. Observed effect levels and other measures of doseresponse relationships are discussed, where appropriate, so that the nature of the adverse health responses is placed in perspective with observed environmental levels. Any information regarding sources, emissions, ambient air concentrations, and public exposure has been included only to give the reader a preliminary indication of the potential presence of this substance in the ambient air. While the available information is presented as accurately as possible, it is acknowledged to be limited and dependent in many instances on assumption rather than specific data. This information is not intended, nor should it be used, to support any conclusions regarding risks to public health. If a review of the health information indicates that the Agency should consider regulatory action for this substance, a considerable effort will be undertaken to obtain appropriate information regarding sources, emissions, and ambient air concentrations. Such data will provide additional information for drawing regulatory conclusions regarding the extent and significance of public exposure to this substance. iii CMA 051557 AUTHORS AND REVIEWERS AUTHORS The EPA's Office of Health and Environmental Assessment (OHEA) is responsible for the preparation of this health assessment document. The OHEA Environmental Criteria and Assessment Office (ECAO-RTP) had overall responsibility for coordination and direction of the document (Dr. Robert M. Bruce, Project Manager). The chapters addressing physical and chemical properties, sampling and analysis and toxic effects were either rewritten or revised by Syracuse Research Corporation. The pharmacokinetics chapter was written by Dr. I.W.F. Davidson of the Bowman Gray School of Medicine, Wake Forest University and Dr. Jean C. Parker, Carcinogen Assessment Group, U.S. Environmental Protection Agency. The air quality chapters addressing sources, emissions and ambient concentrations were originally written by Syracuse Research Corporation and revised by Radian Corporation under a contract with the Office of Air Quality Planning and Standards. The principal authors of the chapters prepared by Syracuse Research Corporation are: Stephen Bosch Life and Environmental Sciences Division Syracuse Research Corporation Syracuse, New York 13210-4080 D. Anthony Gray, Ph.D. Life and Environmental Sciences Division Syracuse Research Corporation Syracuse, New York 13210-4080 Joseph Santodonato, Ph.D., C.I.H. Life and Environmental Sciences Division Syracuse Research Corporation Syracuse, New York 13210-4080 IV CMA. 051558 The OHEA Carcinogen Assessment Group (CAG) was responsible for preparation of the sections on carcinogenicity. Participating members of the CAG are listed below (principal authors of present carcinogenicity materials are designated by an asterisk [*]). Roy Albert, M.D. (Chairman) Elizabeth L. Anderson, Ph.D. Steven Bayard, Ph.D. David L. Bayliss, M.S. Robert P. Beliles, Ph.D. Chao W. Chen, Ph.D.* Margaret M.L. Chu, Ph.D. I.W.F. Davidson (consultant) Herman J. Gibb, M.S., M.P.H. Bernard H. Haberman, D.V.M., M.S. Charalingayya B. Hiremath, Ph.D.* Robert E. McGaughy, Ph.D. Jean C. Parker, Ph.D.* Charles H. Ris, M.S., P.E. Dharm V. Singh, D.V.M., Ph.D. Todd W. Thorslund, Sc.D. The OHEA Reproductive Effects Assessment Group (REAG) was responsible for the preparation of sections on mutagenicity, teratogenicity and reproductive effects. Participating members of REAG are listed below (principal authors of present sections are indicated by an asterisk [*]). The Environmental Mutagen Information Center (EMIC), in Oak Ridge, TN, identified literature bearing on the mutagenicity of EDC. Eric D. Clegg, Ph.D. John R. Fowle III, Ph.D.* David Jacobson-Kram, Ph.D. K.S. Lavappa, Ph.D. Sheila L. Rosenthal, Ph.D. Carol N. Sakai, Ph.D.* Lawrence R. Valcovic, Ph.D. Vicki L. Vaughan-Dellarco, Ph.D. Peter E. Voytek, Ph.D. (Director) v CMA 051559 REVIEWERS The following individuals provided peer review of this draft and/or earlier drafts of this document: U.S. Environmental Protection Agency Karen Blanchard Office of Air, Noise and Radiation Office of Air Quality Planning and Standards Research Triangle Park, NC Robert M. Bruce, Ph.D. Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Research Triangle Park, NC Larry T. Cuppitt, Ph.D. Office of Research and Development Environmental Sciences Research Laboratory Research Triangle Park, NC James W. Palco, Ph.D. Office of Health and Environmental Assessment Exposure Assessment Group Washington, DC Lester D. Grant, Ph.D. Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Research Triangle Park, NC Thomas G. McLaughlin, Ph.D. Office of Health and Environmental Assessment Exposure Assessment Group Washington, DC David Patrick Office of Air, Noise and Radiation Office of Air Quality Planning and Standards Research Triangle Park, NC David J. Reisman Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Cincinnati, OH Jerry F. Stara, D.V.M. Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Cincinnati, OH vi CMA 051560 CONSULTANTS, REVIEWERS AND CONTRIBUTING AUTHORS: Joseph F. Borzelleca, Ph.D. Virginia Commonwealth University Richmond, VA Mildred Christian, Ph.D. Argus Research Laboratories, Inc. Perkasia, PA Herbert H. Cornish, Ph.D. The University of Michigan Ypsilanti, MI I.W.F. Davidson, Ph.D. Bowman Gray School of Medicine Wake Forest University Winston Salem, NC Larry Fishbein, Ph.D. National Center for Toxicological Research Jefferson, AR Mark M. Greenberg Office of Health and Environmental Assessment Environmental Criteria and Assessment Office Research Triangle Park, NC Derek Hodgson, Ph.D. University of North Carolina Chapel Hill, NC George R. Hoffman, Ph.D. Holy Cross College Worcester, MA Rudolph J. Jaeger, Ph.D. Consultant Toxicologist 7 Bolgert Place Westwood, NJ Marshall Johnson, Ph.D. Thomas Jefferson Medical College Philadelphia, PA Bruce Kleinstein, Ph.D. 1500 Locust Street, Suite 3504 Philadelphia, PA John L. Laseter, Ph.D. Environmental Affairs, Inc. New Orleans, LA vii CMA 051561 Edmond J. LaVoie, Ph.D. American Health Foundation Valhalla, NY Marvin S. Legator, Ph.D. University of Texas Medical Branch Galveston, TX P.D. Lotilaker, Ph.D. Fels Research Institute Temple University Medical Center Philadelphia, PA Bernard Schwetz, Ph.D. National Institute of Environmental Health Sciences Research Triangle Park, NC Sam Shibko, Ph.D. Health and Human Services Division of Toxicology Washington, DC Charles M. Sparacino, Ph.D. Research Triangle Institute Research Triangle Park, NC Daniel S. Straus, Ph.D. University of California Riverside, CA Darrell D. Sumner, Ph.D. CIBA GEIGY Corporation High Point, NC Robert Tardiff, Ph.D. 1423 Trapline Court Vienna, VA Norman M. Trieff, Ph.D. University of Texas Medical Branch Department of Pathology, UTMB Galveston, TX Benjamin Van Duuren, Ph.D. New York University Medical Center 550 First Avenue New York, NY James Withey, Ph.D. Room 315C Environmental Health Center Division of Environmental and Occupational Toxicology DeLa Colonbine Tunney's Pasture Ottawa, Ontario CANADA K1A 0L2 viii CMA 051562 SCIENCE ADVISORY BOARD ENVIRONMENTAL HEALTH COMMITTEE CHLORINATED ORGANICS SUBCOMMITTEE The content of this health assessment document on ethylene dichloride was independently peer-reviewed in public session by the Environmental Health Committee of the Environmental Protection Agency's Science Advisory Board. ACTING CHAIRMAN. ENVIRONMENTAL HEALTH COMMITTEE Dr. Seymour Abrahamson, Professor of Zoology and Genetics, -Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706. EXECUTIVE SECRETARY. SCIENCE ADVISORY BOARD Dr. Daniel Byrd III, Executive Secretary, Science Advisory Board, A--101 F, U.S. Environmental Protection Agency, Washington, DC 20460. MEMBERS Dr. Ahmed E. Ahmed, Associate Professor of Pathology, Pharmacology and Toxicology, the University of Texas Medical Branch at Galveston. Dr. George T. Bryan, Professor of Human Oncology, K4/528 C.S.C. Clinical Sciences, University of Wisconsin, 500 Highland Avenue, Madison, Wisconsin 53792. Dr. Ronald D. Hood, Professor and Coordinator, Cell and Developmental Biology Section, Department of Biology, The University of Alabama, and Principal Associate, R.D. Hood and Associates, Consulting Toxicologists, P.0. Box 1927, University, Alabama 35486. Dr. K. Roger Hornbrook, Department of Pharmacology, P.0. Box 26901, University of Oklahoma, Oklahoma City, Oklahoma 73190. IX CMA 051563 TABLE OF CONTENTS DISCLAIMER.................................................................................................................... Page ii PREFACE.......................................................................................................................... iii AUTHORS AND REVIEWERS................................................................................................ iv LIST OF TABLES............................................................................................................ xii LIST OF FIGURES.............................................................................................. xvii 1. SUMMARY AND CONCLUSIONS.................................................................................. 1-1 2. INTRODUCTION....................................................................................................... 2-1 3. PHYSICAL AND CHEMICAL PROPERTIES................................................................. 3-1 3.1. NAME............................................................................................................. 3.2. CAS REGISTRY, RTECS, AND STORET NUMBERS.......................................... 3.3* DESCRIPTION................................................................................................ 3.4. STRUCTURE................................................................................................... 3.5. PHYSICALPROPERTIES OF PURE ETHYLENE DICHLORIDE............................. 3-1 3-1 3-1 3-1 3-1 4. SAMPLING AND ANALYSIS OF ETHYLENE DI CHLORIDE.......................................... 4-1 4.1. SAMPLING..................................................................................................... 4.2. ANALYSIS..................................................................................................... 4-1 4-1 4.2.1. 4.2.2. 4.2.3* 4.2.4. 4.2.5. 4.2.6. Ethylene Dichloride in Air.................................................. Ethylene Dichloride in Water................................................ Ethylene Dichloride in SolidSamples................................... Ethylene Dichloride in Blood................................................ Ethylene Dichloride in Urine................................................. Ethylene Dichloride in Tissue............................................... 4-1 4-2 4-3 4-3 4-3 4-4 5. SOURCES IN THE ENVIRONMENT............................................................................. 5-1 5.1. PRODUCTION PROCESSES............................................................................... 5.2. ETHYLENE DI CHLORIDE PRODUCERS............................................................. 5.3. ETHYLENE DICHLORIDE PRODUCTIONAND TRENDS......................................... 5.4. ETHYLENE DI CHLORIDE USES....................................................................... 5.5. SOURCES OF EMISSIONS.............................................................................. 5-1 5-2 5-2 5-5 5-9 5.5.1. Production and Related Facilities....................................... 5.5.2. Dispersive Uses......................................................................... 5.5.3. Conclusions................................................................................ 5-12 5-13 5-14 x CMA 051564 TABLE OF CONTENTS (cont.) 6. TRANSPORT AND FATE IN THE ENVIRONMENT........................................................ 6-1 6.1. ATMOSPHERE.................................................................................................. 6.2. AQUATIC MEDIA............................................................................................ 6.3. SOIL............................................................................................................. 6.4. SUMMARY....................................................................................................... 6-1 6-7 6-9 6-10 7. ENVIRONMENTAL LEVELS AND EXPOSURE............................................................... 7-1 7.1. ENVIRONMENTAL LEVELS............................................................................... 7-1 7.1.1. Atmospheric Levels................................................................... 7.1.2. Ground and Surface Water Levels.......................................... 7.1.3* Soil and Sediment Levels......................................................... 7-1 7-9 7-14 7.2. ENVIRONMENTAL EXPOSURE........................................................................... 7-14 7.2.1. Exposure from Air..................................................................... 7.2.2. Exposure from Water................................................................. 7.2.3. Exposure from Food................................................................... 7--18 7-20 7-20 7.3. CONCLUSIONS................................................................................................ 7-20 8. ECOLOGICAL EFFECTS............................................................................................ 8-1 9. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTALANIMALS................................. 9-1 9.1. PHARMACOKINETICS...................................................................................... 9-1 9.1.1. 9.1.2. 9.1.3. 9.1.4. Absorption and Distribution.................................................. Excretion.................................................................................... Metabolism.................................................................................. Summary and Conclusions.......................................................... 9-1 9-18 9-32 9-55 9.2. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY............................................ 9-57 9.2.1. Effects in Humans..................................................................... 9-57 9.2.2. Effects in Animals................................................................... 9-86 9.2.3. Summary of Acute, Subchronic and Chronic Toxicity........ 9-115 9.3. REPRODUCTIVE AND TERATOGENIC EFFECTS................................................ 9-122 9.3*1. Summary and Conclusions........................................................... 9-129 9.4. MUTAGENICITY.............................................................................................. 9-131 9.4.1. 9.4.2. 9.4.3. 9.4.4. Gene Mutation Studies............................................................. Chromosome Aberration Studies.............................................. Other Evidence of DNA Damage................................................ Summary and Conclusions.......................................................... 9-131 9-152 9-157 9-162 xi CMA 051565 TABLE OF CONTENTS (cont.) Page 9.5. CARCINOGENICITY........................................................................................ 9-164 9.5.1. 9.5.2. 9.5.3* 9.5.4. 9.5.5. Animal Studies........................................................................... Epidemiologic Studies............................................................. Risk Estimation fromAnimal Data.......................................... Summary........................................................................................ Conclusions................................................................................ 9-164 9-204 9-205 9-257 9-260 Appendix A. Comparison Among Different Extrapolation Models.................... 9-263 Appendix B. Risk Calculation Based on Time-to-Event Data............'............. 9-265 10. REFERENCES.......................................................................................................... 10-1 xii cMft_ 051566 LIST OF TABLES Table 5-1 Major Manufacturers of Ethylene Dichloride....................................... 5-2 U.S. Production and Sales of Ethylene Dichloride............................ 5-3 Ethylene Dichloride Uses.......................................................................... 5-4 Consumption of Ethylene Dichloride in 1979 and 1974...................... 5-5 Uses of Ethylene Dichloride for Intermediate Purposes................... 5- 6 Estimated Environmental Releases of Ethylene Dichloride in 1979.................................................................................... 6- 1 Rate Constants for a Few Chlorinated Ethanes at Room Temperature..................... 7- 1 Ambient Atmospheric Levels of Ethylene Dichloride.......................... 7-2 Ambient Concentrations of Ethylene Dichloride in Urban Areas... 7-3 Reported Occurrence of Ethylene Dichloride in Groundwater Systems -- Combined Federal Data (NORS, NOMS, NSP, CWSS, GWSS). 7-4 Reported Occurrence of Ethylene Dichloride in Surface Water Systems -- Combined Federal Data (NORS, NOMS, NSP, CWSS)............ 7-5 State Data on Ethylene Dichloride in Groundwater............................ 7-6 Estimated Respiratory Intake of Ethylene Dichlorlde by Adults and Infants.................................................................................... 7-7 Maximum Concentration Level in the Vicinity of Various Emission Sources........................................................................................ 7-8 Total Estimated Population (in Thousands) Exposed to Ethylene Dichloride in Drinking Water at the Indicated Concentration Ranges................................................................................. 7-9 Estimated Drinking Water Intake of Ethylene Dichloride by Adults and Infants............................................................................... 9-1 Physical Properties of Ethylene Dichloride and Other Chloroethanes............................................ 9-2 Partition Coefficients for Ethylene Dichloride................................ Page 5-3 5-4 5-6 5-7 5-8 5-10 6-3 7-2 7-8 7-15 7-16 7-17 7-19 7-19 7-21 7-21 9-3 9-4 9-3 Fate of 14 C-EDC in Rats 48 Hours After Oral (150 mg/kg) or Inhalation (150 ppm, 6-hr) Exposure................................................ 9-6 xiii CMA. 051567 LIST OF TABLES (eont.) Table 9-4 Peak Blood and Tissue Levels After Single Oral Dosage of EDO in Male Rats.................................................................................... Page 9-8 9-5 The Absorption Rate Constants (k ) and Area Under Curve (AUC) for Rats After Single OralaDoses of EDC in Oil and in Water as Vehicle and After Intravenous Administration.............................................................................................. 9-9 9-6 EDC Tissue Levels After 50 ppm Inhalation Exposure......................... 9-14 9-7 EDC Tissue Levels After 250 ppm Inhalation Exposure........................ 9-15 9-8 Blood Tissue Levels of EDC in Male Rats 2 Hours After Single Oral Doses in Corn Oil.................................................................... 9-17 9-9 Percent Distribution of Radioactivity Excreted (48-hr) by Mice Receiving 1,2-Dichloroethane- 14 C................................................... 9-20 9-10 9-11 Pharmacokinetic Parameters of EDC Administered as Single Bolus Intravenous Injections in Saline to Sprague-Dawley Male Rats. Parameters Calculated from a 2-Compartment Open Model........................ 9-24 Pharmacokinetic Parameters of EDC Administered as Single Oral Doses in Corn Oil and Water to Sprague-Dawley Male Rats. Parameters Calculated from a 2-Compartment Open Model..................... 9-26 9-12 Pharmacokinetic Parameters of EDC Following Termination of Steady State Inhalation Conditions (5-hr Exposure) of 50 and 250 ppm to Male Sprague-Dawley Rats................................................ 9-29 9-13 Pharmacokinetic Parameters of EDC Following Termination of Steady State Inhalation Conditions; 6-Hr Exposure, 150 ppm to Male Osborne-Mendel Rats. Parameters Calculated from a Two-Compartment Model................................................................................... 9-30 9-14 9-15 Identified Metabolites of Ethylene Dichloride and Ethylene Bromide.......................................................................................... Percent Distribution of Radioactivity Excreted (48-hr) as^ Urinary Metabolites by Mice Receiving 1,2-Dichloroethane- C or 2-ChloroaceticAcid- 1C4 ......................................................................... 9-34 9-37 9-16 Total Macromolecular Binding and DNA Binding in Selected Tissues of Rats After Exposure to 14 C-EDC by Oral or Inhalation Routes...................................................................................... 9-52 xiv CM* 051568 LIST OF TABLES (eont.) Table 9-17 Effect of Dietary Disulfiram Upon the 14 C Content of Liver Nuclei Isolated 24 or 48 Hours After Administration of a Single Oral Dose of 15 mg/kg [U-^C]EDB................................... Page 9-54 9-18 Effects Associated with Acute Lethal Oral Doses of Ethylene Dichloride in Humans............................................................... 9-58 9-19 9-20 Effects of Acute Oral Ingestion of 1,2-Dichloroethane . (Survey Results)........................................................................................ Effect of Ethylene Dichloride Exposure on Eye Sensitivity to Light................................................................................. 9-65 9-72 9-21 Morbidity and Lost Workdays of Aircraft Industry Gluers Exposed to Ethylene Dichloride.............................................................. 9-78 9-22 Concentrations of Ethylene Dichloride in Oil Refinery Mineral Oil Purification Process Air.................................................. 9-80 9-23 Effects Observed in Polish Oil Refinery Workers............................. 9-81 9-24 Effects of Acute Exposure to Ethylene Dichloride............................ 9-87 9-25 9-26 Effects of Ethylene Dichloride on the Cornea................................... Effects of Subchronic Exposure to Ethylene Dichloride.................. 9-95 9-102 9-27 Summary of Mutagenicity Testing of EDC: Gene Mutations in Bacteria..................................................................................................... 9-132 9-28 Summary of Mutagenicity Testing of EDC: Higher Plants................ 9-143 9-29 Summary of Mutagenicity Testing of EDC: Gene Mutation Tests in Insects............................................................................................ 9-145 9-30 Summary of Mutagenicity Testing of EDC: Mammalian Systems........ 9-151 9-31 Summary of Mutagenicity Testing of EDC: Chromosomal Aberrations Tests.......................................................................................... 9-153 9-32 Summary of Mutagenicity Testing of EDC: PolA Assay..................... 9-158 9-33 Summary of Mutagenicity Testing of EDC: DNA Binding Studies............................................................................................................. 9-161 9-34 Design Summary for 1,2-Dichloroethane (EDC) Gavage Experiment in Osborne-Mendel Rats........................................................ 9-166 9-35 Terminal Survival of Osborne-Mendel Rats Treated With 1,2-Dichloroethane (EDC)............................................................................ 9-170 xv CMA. 051569 LIST OF TABLES (cont.) Table 9-36 Squamous Cell Carcinomas of the Forestomach in OsborneMendel Rats Treated With 1,2-Dichloroethane (EDC)........................... Page 9--171 9-37 Hemangiosarcomas in Osborne-Mendel Rats Treated With 1,2-Dichloroethane (EDC)............................................................................ 9-172 9-38 Adenocarcinomas of the Mammary Gland in Female OsborneMendel Rats Treated With 1,2-Dichloroethane(EDC).......................... 9-173 9-39 Design Summary for 1,2-Dichloroethane (EDC) Gavage Experiment in B6C3F1 Mice.......................................................................... 9-175 9-40 Terminal Survival of B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)............................................................................ 9-176 9-41 9-42 Hepatocellular Carcinomas in B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)............................................................................ Alveolar/Bronchiolar Adenomas in B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)................................................................... 9-179 9-180 9-43 Squamous Cell Carcinomas of the Forestomach in B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)................................................... 9-181 9-44 Adenocarcinomas of the Mammary Gland in Female B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)................................................... 9-182 9-45 9-46 Endometrial Polyp or Endometrial Stromal Sarcomas in Female B6C3F1 Mice Treated With 1,2-Dichloroethane (EDC)............................ Characterization of 1,2-Dichloroethane (EDC) Inhalation Experiment in Sprague-Dawley Rats........................................................... 9-183 9-184 9-47 Design Summary for 1,2-Dichloroethane (EDC) Experiment in Sprague-Dawley Rats................................................................................ 9-185 9-48 Survival of Sprague-Dawley Rats Exposed to EDC at 52 and 104 Weeks......................................................................................................... 9-186 9-49 Tumor Incidence in Sprague-Dawley Rats Exposed to 1,2-Dichloroethane (EDC)............................................................................ 9-188 9-50 Mammary Tumors in Sprague-Dawley Rats Exposed to 1,2-Dichloroethane (EDC)............................................................................ 9-192 9-51 Design Summary for 1,2-Dichloroethane (EDC) Experiment in Swiss Mice................................................................................................ 9-195 xvi CMA 051570 LIST OF TABLES (cont.) Table 9-52 9-53 9-54 9-55 9-56 9-57 9-58 9-59 9-60 9-61 .9-62 9-63 9-64 9-65 9-66 Survival of Swiss Mice Exposed to 1,2-Dichloroethane (EDO at 52 and 78 Weeks.................................................................................. Tumor Incidence in Swiss Mice Exposed to 1,2-Dichloroethane (EDC)........................................................................................................... Pulmonary Tumor Response in Strain A/st Mice Injected with 1,2-Dichloroethane(EDC)................................................................ Mouse Skin Bioassay of 1,2-Dichloroethane (EDC) and Chloroacetaldehyde................................................................................... Incidence Rates of Hemangiosarcomas in the Circulatory Systems of Male Osborne-Mendel Rats and of Hepatocellular Carcinomas in Male B6C3P1 Mice............................................................. Disposition of 14 C-EDC in Rats After Single Administrations of 150 mg/kg Orally and 150 ppm for 6 Hours by Inhalation........ Comparison of the Metabolism and Pharmacokinetics of the "High" Doses in Rats of the Maltoni et al. Inhalation and NCI Gavage Bioassays............................................................................... Evidence of Nonlinear Kinetics for EDC in Rats After Gavage, Intravenous, and Inhalation Dosage..................................... DNA Covalent Binding in Rats Exposed to 14 C-EDC........................... Lifetime Average Human Equivalent Doses Based on the NCI Mouse Bioassay.......................................................................................... Lifetime Average Human Equivalent Doses Based on the NCI Rat Bioassay.............................................................................................. Incidence Rates of Hemangiosarcomas in the Circulatory System of Male Osborne-Mendel Rats.................................................. Incidence Rates of Hepatocellular Carcinomas in Male B6C3F1 Mice................................................................................................ The 95$ Upper-Bound Estimate of Risk at 1 mg/kg/day Using Three Different Models........................................................................... Relative Carcinogenic Potencies Among 54 Chemicals Evaluated by the Carcinogen Assessment Group as Suspect Human Carcinogens.................................................................................... Page 9-196 9-197 9-201 9-203 9-207 9-210 9-211 9-219 9-227 9-233 9-235 9-245 9-245 9-246 9-253 xvii CMA 051571 LIST OF FIGURES Figure Page 9-1 EDO levels after single oral administration in rats.......... 9-7 9-2 Top: Blood levels of EDO observed during and following a 6-hour inhalation exposure to 150 ppm EDC. Bottom: Semilogarithmic plot of EDC blood levels vs. time after exposure termination................................................................................. 9-12 9-3 Blood levels of EDC in rats after i.v. administration...... 9-23 9-4 Levels of EDC in rats after inhalation exposure to 50 ppm (top) and 250 ppm (bottom). Levels were measured at termination of 5-hour exposure period.................................................. 9-27 9-5 Postulated microsomal oxidative metabolism of 1,2-dihaloethanes. 9-39 9-6 Further metabolism of 2-chloroacetaldehyde and 1-chloroso2-chloroethane from microsomal oxidation................. ......................... 9-41 9-7 Postulated cytosolic metabolism of ethylene dichloride.... 9-44 9-8 Growth curves for male and female Osborne-Mendel rats administered EDC by gavage...................................................................... 9-168 9-9 Survival comparisons for male and female Osborne-Mendel rats administered EDC by gavage...................................................................... 9-169 9-10 Growth curves for male and female B6C3F1 mice administered EDC by gavage................................................................................................ 9-177 9-11 Survival comparisons for male and female B6C3F1 mice administered EDC by gavage...................................................................... 9-178 9-12 Relationship in mice between dose of EDC (mg/kg) and the amount of EDC exhaled unchanged and the amount of the dose metabolized to urinary metabolites plus CO^.......................................................... 9-214 9-13 Relationship in Sprague-Dawley rats between the area under the blood EDC concentration-time curve (AUC) and the gavage dose of EDC in corn oil (mg/kg) or inhaled doses of EDC (ppm for 6 hours)......................................................................................................... 9-218 9-14 Linear relationship for EDC between the logarithm of the area under the blood C,t curve (AUC) and the logarithm of gavage dose in corn oil given to Sprague-Dawley rats...................................... 9-221 9-15 Relationship in Sprague-Dawley rats between gavage doses of EDC in corn oil and the ratio from the area under the blood EDC concentration-time curve (AUC) divided by the administered dose. 9-223 xviii CMA 051572 LIST OF FIGURES Figure 9-16 Blood levels of EDC following exposure to EDO by inhalation (150 ppm, 6 hours) or gavage (150 mg/kg)................................................. 9-225 9-17 Histogram representing the frequency distribution of the potency indices of the 5^ suspect carcinogens evaluated by the Carcinogen Assessment Group..................................................... ............... 9-252 xix CMA 051573 1. SUMMARY AND CONCLUSIONS Ethylene dichoride (EDC) is a clear, colorless, volatile liquid with a pleasant odor. EDC has a molecular weight of 98.96, a boiling point of 83.7C and a vapor pressure of 64 torr at 20C. Unlike more highly chlorinated hydrocarbons, EDC has a flashpoint (17C closed cup), an autoignition tempera ture (413C) and explosive limits (6.2-15.6$ by volume in air). It has a water solubility of 8820 mg/2, and a log octanol/water partition coefficient of 1.48. EDC is analyzed best by gas chromatography using either an electron capture or halogen specific (microcoulometric or electrolytic conductivity) detector. Alternatively, a gas chromatograph/mass spectrometer may be used. EPA methods 502,1, 601, 624 and 8010 detail the analysis of EDC from water, wastewater and solid samples. Variations in these methods provide for analysis of EDC in biological media (e.g., blood, urine and tissue). EDC in air is analyzed by condensing the volatiles in a previously collected air sample in a GC column, followed by temperature programming the column. EDC is produced commercially by the direct chlorination or oxychlorination of ethylene. Most of the production capacity, which exceeds nine million metric tons annually, is located in Texas and Louisiana. Roughly 90$ of the production is captively consumed by the producers. Production during 1980, 1981 and 1982 totalled 5.037, 4.523 and 3.455 million metric tons, respectively. A major portion (84$) of the United States EDC production is consumed to make vinyl chloride monomer. It is also used to make chlorinated solvents, vinylidene chloride and ethyleneamines. The major source of EDC emissions to the environment is from vent gas streams during the manufacture of EDC and its derivatives. Significant amounts are also emitted to the environment from dispersive uses in gasoline, paints and 1-1 CMA 051574 cleaning agents. In 1979, an estimated 6696 metric tons of EDC were released to the atmosphere from EDC and derivative production, while 4944 metric tons were released from dispersive uses. The most likely removal mechanism for EDC from the atmosphere is reaction with hydroxyl free radicals. Based on available kinetic data and average tropospheric hydroxyl free radical concentrations, the half-life of EDC has been estimated to range from 36-127 days. Due to different factors that may cause variations in 0H concentrations in the troposphere, the persistence of EDC could vary somewhat from the estimated value. Chloroacetyl chloride is probably the principal product resulting from the reaction of EDC with 0H radicals. EDC is not expected to play a significant role in stratospheric ozone destruction reactions due to its relatively short tropospheric half-life. However, chloroacetyl chloride may have sufficient stability to diffuse to the stratosphere and may participate in UV reactions, producing chlorine atoms. In the aquatic environment, volatilization appears to be the most significant removal mechanism. The half-life for this process has been estimated to be =4 hours. From the little information that is available regarding the fate of EDC in soil, it can be surmised that both volatilization from and leaching through soil may be two significant removal mechanisms for EDC. The highest atmospheric concentrations that have been monitored for EDC have been detected near production and use facilities. Mean levels as high as 27.5 ppb have been monitored near production-use facilities in Lake Charles, LA. However, atmospheric exposure appears to vary greatly from one location to another. The available monitoring data, which includes levels detected in general ambient urban areas, indicate that most locations have concentrations of <0.5 ppb. The data are not sufficient to determine regional variations in 1-2 CMA 051675 exposure levels. Using Federal Reporting Data System information, it has been projected that EDC levels in all groundwater and surface water systems in the United States fall below 10 and that most are <1.0 pg/S.. No data are available indicating the presence of EDC in finished foods. Pharmacokinetic studies in animals indicate that EDC is rapidly and extensively absorbed following oral and inhalation exposure. Dermal absorption is negligible in most vapor exposure situations, although absorption by this route may be significant with direct liquid contact. Complete tissue distribution of EDC is consistent with its lipophilic nature; the chemical crosses the blood/brain and placental barriers and concentrates in breast milk. Up to 90$ of low oral or inhalation doses are metabolized by rats and mice, with biotransformation occurring by multiple pathways; EDC is metabolized to 2-chloroacetaldehyde, S-(2-chloroethyl)-glutathione, and other putative reactive metabolites capable of covalent binding to cellular macromolecules. Metabolism is dose-related; as the dose increases, the percent metabolized decreases, although the absolute amount of EDC metabolized increases until saturation of metabolic pathways occur. Elimination of unmetabolized EDC occurs almost exclusively via the lung, with a greater percentage being exhaled at higher doses. Total body elimination of EDC is relatively rapid and is consistent with a two-compartment system and Michalis-Menten kinetics. Significant bioaccumulation of EDC in blood and tissues is not expected to occur. The effects of acute inhalation exposure to EDC are similar in humans and animals. Immediate symptoms of toxicity are CNS depression and irritation of the respiratory tract and eyes. Death was usually ascribed to respiratory and circulatory failure, and pathologic examinations typically revealed congestion, degeneration, necrosis and hemorrhagic lesions of most internal organs (e.g., liver, kidneys, spleen, lungs, respiratory tract, gastrointestinal tract). 1-3 CMA 051576 Several papers describing human health surveys appear in the literature; adverse effects are largely associated with the gastrointestinal and nervous systems. The exposure information in these studies is not well documented, but taken together indicate that adverse effects have likely occurred in humans at EDO levels below 100 ppm, although probably not as low as 10 ppm. Subtle neurological effects (e.g., fatigue, irritability, sleeplessness) may be more prevalent than overt symptoms of CNS toxicity at lower concentrations. Studies with multiple species of animals have shown that subchronic or ohronic exposure to EDO at vapor concentrations of <100 ppm did not produce treatment-related adverse effects on survival, growth, hematology, clinical chemistry, organ weight or histology. Toxic effects were apparent at higher concentrations and were exposure-related; exposure to 400-500 ppm produced high mortality and histopathological alterations in rodents within a few exposures. Limited data indicate that the toxic response to acute oral exposure is similar to that of inhalation exposure in humans and animals. Subchronic/chronic oral administration of EDC at daily dosages of =200 mg/kg and higher caused decreased growth rate and mortality in mice, which may have been tumor-related. Chronic oral administration of lower dosages of EDC (=34 mg/kg/day) to rats produced mortality that appeared to be due to non-neoplastic lesions including bronchiopneumonia and endocardial thrombosis. The available evidence suggests that EDC does not adversely affect the reproductive or development process in laboratory animals except at maternally toxic levels. However, additional laboratory testing is needed, as well as epidemiological studies, to conclusively establish that EDC is not a human teratogen and does not cause adverse reproductive effects. Positive responses in different test systems representing a wide range of organisms indicate that EPC is capable of causing gene mutations in prokaryotes 1-4 CMA 051577 and eukaryotes. Several of its putative metabolites, thought to be formed in rats and mice, are judged to be more potent mutagens than EDC. EDC has not been adequately tested for its ability to cause chromosomal aberrations or heritable effects. Further testing is needed to assess its ability to cause these effects. EDC was shown to be carcinogenic in a lifetime gavage bioassay that was conducted by the National Cancer Institute, producing tumors in both rats (forestomach carcinomas, cirulatory system hemangiosarcomas, subcutaneous fibromas, mammary gland adenocarcinomas) and mice (hepatocellular carcinomas, alveolar/bronchiolar adenomas, mammary carcinomas, endometrial tumors). EDC produced an elevated but not a statistically significant increase in the incidence of lung adenomas in strain A mice when administered intraperitoneally. EDC induced statistically significant benign lung tumors in mice when applied to the skin, but statistically significant skin tumors were not observed. No statistically significant increases in tumors occurred in rats or mice following lifetime inhalation exposure. No case reports or epidemiologic studies concerning EDC were available in the published literature for analysis. From a weight-of-evidence approach, the direct and supporting evidence for carcinogenicity includes: 1) multiple tumor types in an oral rat bioassay and an oral mouse bioassay, 2) suggestive evidence in two other animal bioassays, 3) demonstrated evidence of reactive metabolites of EDC and formation of a DNA adduct, and 4) evidence that EDC is also a mutagen. The U.S. Environmental Protection Agency is using, on an interim basis, a proposed classification scheme for evaluating the weight of evidence for carcinogenicity. Using this classification, the positive findings in the oral rat and mouse studies would be considered a sufficient level of evidence in experimental animals. The sufficient level of animal evidence, together with an absence of epidemiologic data, provides a basis for an overall weight-of- % 1-5 CMA 051578 evidence ranking of Group B2, meaning that EDC is a "probable" carcinogen in humans. Applying the International Agency for Research on Cancer (IARC) classification scheme, which EPA has used in the past, the positive finding in the experimental animals and the absence of epidemiologic data provide an overall weight-of-evidence ranking of Group 2B, meaning that EDC is a "probable" carcinogen in humans. While the "probable" carcinogenicity conclusion obviously applies to ingestion exposure since the positive evidence comes from gavage experiments, it is likely that EDC is a "probable" carcinogen for humans via inhalation exposure. In view of the evidence, it is prudent to consider EDC to be carcinogenic via inhalation exposure although the potency may vary from that estimated from gavage studies. Assuming that EDC is carcinogenic for humans, data from gavage studies using both rats and mice can be used to estimate the carcinogenic potency and unit risks. The development of the potency and unit risk values Is for the purpose of providing an estimator to assess the possible magnitude of the public health impact if EDC is carcinogenic for humans. The carcinogenic potencies estimated on the basis of hemangiosarcomas in rats and hepatocellular carcinomas in mice are comparable when the linearized multistage model is used. The upper-bound estimate of EDC potency is 9.1 x 10_2 /mg/kg/day based on the hemangiosarcoma response in rats using a time-to-death adjustment and an adjusted dose derived from the metabolism/kinetic evaluation. The upper-bound estimate of the incremental cancer risk due to 1 \ig/Z of EDC in drinking water is 2.6 x 10 Two upper-bound estimates of the incremental cancer risk for the inhalation of 3 _5 1 pg/m of EDC are calculated: 2.6 x 10 on the basis of the gavage potency value, and 1.0 x 10~ on the basis of a negative inhalation study. The 1-6 CMA 051579 inhalation unit risks are presented as two values in recognition of the uncertainties associated with the different data sets used, there being no certain reasoning that one value has more scientific merit than the other value. The upper-bound nature of the unit-risk estimates is such that the true risk is not likely to exceed these values and may be lower. Expressed in terms of relative potency, EDC ranks in the fourth quartile among 54 carcinogens evaluated by EPA's Carcinogen Assessment Group. 1-7 CMA 051580 2. INTRODUCTION EPA's Office of Research and Development has prepared this health assess ment to serve as a "source document" for Agency use. This health assessment was originally developed for use by the Office of Air Quality Planning and Standards to support decision-making regarding possible regulations of ethylene dichloride under Section 112 of the Clean Air Act. However, based on the expressed interest of other agency offices, the scope of this document was expanded to address ethylene dichloride in relation to sectors of the environment outside of air. It is fully expected that this document will serve the information needs of many government agencies and private groups that may be involved in decision-making activities related to ethylene dichloride. In the development of the assessment document, existing scientific litera ture has been surveyed in detail. Key studies have been evaluated and summary and conclusions have been prepared so that the chemical's toxicity and related characteristics are qualitatively identified. The document considers all sources of ethylene dichloride in the envi ronment, the likelihood for its exposure to humans, and the possible effect on man and lower organisms from absorption. The information found in the document is integrated into a format designed as the basis for performing unit risk assessments. When appropriate, the authors of the document have attempted to identify gaps in current knowledge that limit risk evaluation capabilities. The basic literature search for this document is complete through 1983; however, selected publications have been included in the sections on mutagenicity and carcinogenicity through 1984. 2-1 CMA 051581 3. PHYSICAL AND CHEMICAL PROPERTIES 3.1. NAME Ethylene dichloride (EDC) is the common name for 1,2-dichloroethane. It is also commonly referred to as ethylene chloride and S-dichloroethane (Archer, 1979). 3.2. CAS REGISTRY, RTECS, AND STORET NUMBERS CAS Registry: 107-06-2 RTECS: KI05250000 STORET: 34531 3.3. DESCRIPTION Ethylene dichloride is a clear, colorless, volatile liquid with a pleasant odor, and is stable at ordinary temperatures (Archer, 1979). 3.4. STRUCTURE cich2ch2ci 3.5. PHYSICAL PROPERTIES OF PURE ETHYLENE DICHLORIDE The physical properties of pure ethylene dichloride were taken from Archer (1979) and Weast (1980), unless otherwise stated. Molecular weight: Melting point: Boiling point: Density at 20C: Viscosity: Flashpoint: Closed cup Open cup Explosive limits in air at 25C: 98.96 -35.3C 83.7C 1.2529 g/mfl, 1.4451 m Pa S (cP) 17C 21 C 6.2-15.6$ by volume 3-1 CMA 051582 Autoignition tempera ture in air: Vapor pressure: 413C C 10 20 30 kPa 5.3 8.5 13-3 Torr 40 64 100 Solubility in water: 0.0891 M (Valvani et al., 1981) 8,820 mg/i, Log Octanol/Water Partition Coefficient: 1.48 (Valvani et al., 1981) Henry's Law Constant atm nr mol~ : 9.14 x 10"4 (Mabey et al., 1981) Blood/Air Partition Coefficient C37C): 19.5 + 0.5 (Sato and Nakajima, 1979) 3-2 CMA 051583 4. SAMPLING AND ANALYSIS OF ETHYLENE DICHLORIDE 4.1. SAMPLING Taking environmental samples representative of the area in question is a complex task beyond the scope of this document. A number of documents either devoted exclusively to sampling or containing considerable information relating to the subject are listed below and should be consulted prior to initiating environmental sampling. For air, Singh et al. (1979, 1983b), Pellizzari (1978), and Pellizzari et al. (1979) have detailed sampling methodologies that describe and/or show equipment and strategies. Water and wastewater sampling is described by Berg (1982) and solids sampling by U.S. EPA (1982b). 4.2. ANALYSIS 4.2.1. Ethylene Dichloride in Air. Ethylene dichloride in air can be analyzed by a number of methods; however, the method of Singh et al. (1980) appears to be substantially free of artifact problems and completely quantitative. In this method, an air sample in a stainless steel canister at 32 psig is connected to a preconcentration trap consisting of a 4" x 1/16" ID stainless steel tube containing glass beads, glass wool, or 3$ SE-30 on acid washed 100/120 mesh Chromosorb W. The sampling line and trap, maintained at 90C, are flushed with air from the canister; then the trap is immersed in liquid 0^ and air is passed through the trap, the initial and final pressure being noted (usually between 30 and 20 psig) on a high-precision pressure gauge. The ideal gas law can be used to estimate the volume of air passed through the trap. The contents of the trap are desorbed onto a chromatography column by backflushing it with an inert gas while holding the trap at boiling water temperature. Suitable columns include 10$ SP1000 on Supelcoport (100/120 mesh, 15' x 1/8" stainless steel) and 0.2$ CW- 4-1 CMA 051584 1500 on Carbopack C (80/100 mesh, 10' x 1/8" Ni). Both columns can be operated at 45C with a carrier gas flow of 25 mll/minute on the former column and 40 mSVminute on the latter. An electron capture detector operating at 330C was found to be optimum. It should be noted that the above authors found Tenax to be unsuit able for air analyses because of the presence of artifacts in the spectrum from oxidation of the Tenax monomer. In addition, when Tenax is used as a sorbent, safe sampling volumes (i.e., that volume of air which, if sampled-over a variety of circumstances, will not cause significant breakthrough) should be used. Brown and Purnell (1979) determined the safe volume for ethylene diehloride per gram Tenax to be 27 8. (flow rate 5-600 m8/minute; ethylene dichloride cone. <250 mg/nr; temp, up to 20C) with a safe desorption temperature of 90C. The detection limits of this method were not specified and are dependent on the volume of air sampled. Analyses as low as 33 ppt have been reported using this method (Singh et al., 1980). 4.2.2. Ethylene Dichloride in Water. Ethylene dichloride in water can be analyzed by the purge-and-trap method (Method 502.1) as recommended by the Environmental Monitoring and Support Laboratory of the U.S. EPA (1981). In this method, an inert gas is bubbled through 5 ml of water at a rate of 40 miVminute for 11 minutes, allowing the purgable organic compounds to partition into the gas. The gas is passed through a column containing 3J OV-1 on Chromosorb W, Tenax GC silica gel, and coconut charcoal at 22C, which traps most of the organics removed from the water. The adsorption column is then heated rapidly to 180C and backflushed with helium (20-60 miVminute, 4 minutes) to desorb the trapped organics. The effluent of the column is passed into an analytical gas chromatography column packed with 1$ SP-1000 on Carbopack-B (60/80 mesh, 8' x 0.1" ID) maintained at 40C. The column is then temperature programmed starting at 45C for 3 minutes and increasing at 8C/minute until 220C is 4-2 CMA 051585 reached; it is then held there for 15 minutes or until all compounds have eluted. A halogen-specific detector (or GC-MS) having a sensitivity of 0.10 pg/2, with a relative standard deviation of <10K must be used. This method is similar to EPA method 601,624 for use with wastewater (Longbottom, 1982). 4.2.3. Ethylene Dichloride in Solid Samples. Ethylene dichloride in solid samples may be analyzed by EPA method 8010 "Halogenated Volatile Organics" (U.S. EPA, 1982b). With this method, a portion of the sample is dispersed in poly ethylene glycol or methanol. The dispersion is then mixed with water and purged in a manner identical to the analysis of EDC in water. The trap contains 3$ 0V-1 on Chromosorb W (60/80 mesh), Tenax GC (60/80 mesh), Grade 15 silica gel (35/60 mesh), and activated coconut charcoal (6/10 mesh); desorption is performed at 180C for 4 minutes with a gas flow of 20 to 60 m2,/minute. Gas chromatographic conditions are identical to the analysis of ethylene dichloride in water. 4.2.4. Ethylene Dichloride in Blood. Ethylene dichloride in blood can be analyzed by using a modified purge-and-trap method (Pellizzari et al., 1979). This method involves diluting an aliquot of whole blood (with anticoagulant) to =50 m2, with prepurged, distilled water. The mixture is placed in a 100 m2. 3-neck round bottom flask along with a Teflon-lined magnetic stirring bar. The necks of the flask are equipped with a helium inlet, a Tenax trap, and a thermometer. The Tenax trap is a 10 cm x 1.5 cm ID glass tube containing pre-extracted (soxhlet, methanol, 24 hrs) and conditioned (270C, 30 m2,/minute helium flow, 20 minutes) 35/60 mesh Tenax ( = 1.6 g, 6 cm). The sample is then heated to 50C and purged with a helium flow rate of 25 m2,/min for 90 minutes. Analysis can be performed as indicated in Section 4.2.2. 4.2.5. Ethylene Dichloride in Urine. Ethylene dichloride in urine can be analyzed by using an apparatus identical to the one described in Section 4.2.4, using 25 m2, of urine, diluted to 50 m2,, instead of blood. 4-3 CMA 051586 4.2.6. Ethylene Dichloride in Tissue. Ethylene dichloride in tissue can be analyzed by using an apparatus identical to the one described in Section 4.2.4, using 5 g of tissue, diluted to 50 m2., instead of blood and macerated in an ice bath. The purge time is reduced to 30 minutes. 4-4 CMA 051587 5. SOURCES IN THE ENVIRONMENT 5.1. PRODUCTION PROCESSES Two commercial processes account for virtually all of the EDC produced currently. One is the direct chlorination of ethylene with chlorine. The other is an oxychlorination process in which ethylene, hydrogen chloride and oxygen are reacted to form EDC. Currently, most production centers around large manufac turing plants employing a balanced combination of both of these two processes. Over 80$ of the EDC produced is used to make vinyl chloride monomer via dehydrohalogenation of EDC. Most EDC producers have nearby vinyl chloride production facilities. The balanced plants use the hydrogen chloride recovered when the EDC is dehydrohalogenated to vinyl chloride as feed to the oxychlorination reactor. This requires roughly a 50-50 split between direct chlorination and oxychlorination for a completely balanced operation assuming all EDC is cycled to the vinyl chloride facilities. In this manner, there will be no net production of hydrogen chloride. As of 1974, *58$ of the total EDC production capacity was based upon direct chlorination and *42$ was based upon oxychlorination (Pervier et al., 1974). Current capacities are judged to be roughly the same. In the direct chlorination process, EDC is produced by the catalytic vaporor liquid-phase chlorination of ethylene as follows (Archer, 1979): FeCl CHp = CH,, + Cl- (5$ air)------- --------- C1CH-CH-C1 40-50C dd Most liquid-phase processes use ferric chloride as the catalyst. The chlori nation is carried out at 40-50C with 5$ air added to prevent substitution chlorination of the product. 5-1 CMA 051588 The oxychlorination process isusually incorporated into anintegrated vinyl chloride plant in which hydrogen chloride (which is recovered from cracking the EDC to vinyl chloride) is recycled to an oxychlorination unit (Archer, 1979). The hydrogen chloride by-product is used as the chlorine source in the chlorina tion of ethylene in the presence of oxygen and copper chloride catalyst as follows: CuCl 2CH = CH,, + 4HC1 + CL--------2C1CH-CH-C1 + H50 d. 270C dd d 5.2. ETHYLENE DICHLORIDE PRODUCERS The producers of EDC in the United States are listed in Table 5-1 along with their respective production capacities. As can be noted from Table 5-1, most of the production capacity is located in Texas and Louisiana. These production facilities also eaptively consume the major portion of the EDC produced, as in the manufacture of vinyl chloride and, to a much lesser extent, in the manufacture of vinylidene chloride and various chlorinated solvents. From Table 5-2, it can be noted that only =10$ of the EDC production has been sold on the open market in most recent years. 5.3. ETHYLENE DICHLORIDE PRODUCTION AND TRENDS U.S. production volumes and sales of EDC are listed in Table 5-2. Blackford (1974) and the U.S. International Trade Commission (1974) have stated that the production volumes as listed may be somewhat smaller than the actual production because some EDC may be produced, but not separated or accurately measured (and therefore not reported) by some producers. Blackford (1974) has estimated actual productions on the order of 10$ higher than the reported productions. The demand for EDC for the years 1982 and 1983 has been estimated to be 4.127 and 4,716 million metric tons, respectively, while the demand for the year 1987 is expected to be 5.487 million metric tons (CMR, 1983). Production of EDC 5-2 CMA 051589 TABLE 5~ Major Manufacturers of Ethylene Dichloride Manufacture Plant Sites Annual Capacity (Millions of Metric Tons) Atlantic Richfield (ARCO) Borden, Inc. Dow Chemical USA E.I. duPont (Conoco) Ethyl Corp. Formosa Plastics Corp. Georgia-Pacific Corp. B.F. Goodrich Co. PPG Industries Shell Chem. Co. Union Carbide Corp. Vulcan Materials Co. Port Arthur, TX Geismar, LA Freeport, TX Oyster Creek, TX Plaquemine, LA Lake Charles, LA Baton Rouge, LA Pasadena, TX Baton Rouge, LA Point Comfort, TX Plaquemine, LA Deer Park, TXb La Porte, TX Calvert City, KY Convent, LA Lake Charles, LA Deer Park. TX Norco, LA Taft, LAd . Texas City, TX Geismar, LA Total 0.200 0.230 0.725 0.500 0.840 0.525 0.320 0.110 0.240 0.385 0.735 0.110 0.720 0.450 0.360 1.230 0.620 0.540 0.070 0.070 0.160 9.140 Note: Capacities are flexible depending on finishing capacities for vinyl chloride and chlorinated solvents. aSource: SRI, 1983; CMR, 1983 bOperated under a toll agreement with Diamond Shamrock Corp. Q Closed for an indefinite time period because of the temporary closing of Shell's vinyl chloride monomer plant located there. Captive use only. 5-3 CMA 051590 TABLE 5-2 u.s. Production and Sales of Ethylene Dichloride Year 1982 1981 1980 1979 1978 1977 1976 1975 1974 1973 1972 1971 1970 1965 I960 1950 Millions of Metric Tons Production6 Sales 3.455 4.523 5.037 5.349 4.989 4.987 3.647 3*617 4.156 4.214 3-541 3.428 3.383 1.113 0.575 0.138 0.640 0.401 0.510 0.633 0.469 0.692 0.617 0.345 0.596 0.613 0.656 0.595 0.596 0.140 0.198 0.021 aSource: USITC, USTC, 1952-1982 ^Production totals may be understated in some years because some ethylene dichloride is produced but not separated or accurately measured (and therefore not reported) by some producers. 5-4 CMA 051591 in 1982 was sharply lower than in preceding years (see Table 5-2). This was largely due to a recession in the vinyl chloride market and a decision of producers to use their EDC inventories during the winter of 1982 (Chemical and Engineering News, 1982, 1983). Production of EDC is expected to rebound during 1983. Historically, the demand for EDC grew at a rate of =1$ per year during the period from 1973-1982. Demand is anticipated to grow at a rate of 4$ per year through 1987 (CMR, 1983). 5.4. ETHYLENE DICHLORIDE USES A major portion (84$) of U.S. EDC production is converted to vinyl chloride monomer. The major uses of EDC for the years 1983, 1980, 1977 and 1974 are given in Table 5-3. Consumption of EDC for specific end uses is given in Table 5-4. In addition to vinyl chloride, EDC is used as a starting material in the production of chlorinated solvents such as 1,1,1-trichloroethane, trichloro ethylene and perchloroethylene and as intermediate in the production of ethyleneamines and vinylidene chloride (Archer, 1979; Blackford, 1974). Table 5-5 lists the users of EDC for these purposes. In general, these users are also producers of EDC. EDC is used as an additive (lead scavenger) in tetraethyl lead antiknock mixtures for gasolines. Scavenging agents are used in gasolines to transform the combustion products of lead alkyls to forms that are more likely to be vaporized from the engine surfaces. The most important lead scavengers used for this purpose are EDC and ethylene dibromide (McCormack et al., 1981; Blackford, 1974). The sale of tetraalkyl lead compounds is always in admixture with EDC and/or ethylene dibromide. Conventional motor-mix formulations contain 1 mol of EDC and 1/2 mol of ethylene dibromide per mol of tetraethyl lead (McCormack et al., 1981), This use of EDC is expected to decline in future years due to EPA regulations concerning the use of leaded gasolines. CMR (1982) projects that the 5-5 CMA 051592 TABLE 5-3 Ethylene Dichloride Uses Use Vinyl chloride monomer Chlorinated solvents Vinylidene chloride Exports Lead scavenger Amines Miscellaneous 1983 84 4 2 9 -- -- 1h Percent of Total 1980 1977 1974 84 80 78 7 10 8 2---- 5---- --33 ---- 2,,b _7c 2 9-C aSource: CMR, 1974, 1977, 1980, 1983 ^Includes lead scavenger cIncludes exports 5-6 CHA 051593 TABLE 5-4 Consumption of Ethylene Dichloride in 1979 and 1974a Consumption (Thousands of Metric Tons) 1979 % 1974 * Vinyl chloride monomer 1,1,1-Trichloroethane Trichloroethylene Perchloroethylene Vinylidene chloride Ethyleneamines Lead scavenger Exports Preparation of polysulfides Paints, coatings and adhesives Extraction solvents Cleaning of fabric and polyvinyl chloride equipment Grain fumigation Other uses Total 4420 -- 89.4 -- 118.7 168.1 81.7 179.0 0.5 1.36 1.05 0.91 0.46 0.46 5199.44 85.0 -- 1.7 -- 2.3 3-2 1.6 3.4 -- -- -- -- -- 99.8 3894.2 153.3 132.8 124.7 97.0 131.5 97.0 167.3 -- -- -- __ -- 6.8 4804.4 81.0 3.2 2.8 2.6 2.0 2.7 2.0 3.5 -- -- -- -- 0.1 99.9 aSource: Seufert et al., 1980; Blackford, 1974 5-7 051594 CMA TABLE 5-5 a Users of Ethylene Dichloride for Intermediate Purposes Vinylidene 1,1,1-Trichloroethane Trichloroethylene Perchloroethylene Ethyleneamines Chloride Dow Chem. USA Freeport, TX Plaqueraine, LA PPG Industries Lake Charles, LA Union Carbide Co. Taft, LA Texas City, TX Diamond Shamrock Corp. Deer Park, TX E.I. duPont Corpus Christi, TX X X -- -- X X XX -- X -- X XX X -- -- X -- X X -- X -- -- aSource: SRI, 1983; Blackford, 1974 X = produces this chemical from ethylene dichloride 051595 demand for lead in gasoline will decrease at a rate of 1151 per year (average) through 1986. Exports of EDC have been increasing in recent years (see Table 5-6) and may grow by as much as 8% per year through the mid 1980s (CMR, 1983)* The Japanese are expected to become major importers of EDC as they close down significant amounts of chlorine capacity (mercury cell method) because of environmental regulations (some of the chlorine made by this method is used to make EDC). However, part of that demand will be filled by Shell's one billion pounds/year EDC plant in Jubail, Saudi Arabia, which is due on stream in 1985. EDC has a variety of relatively small miscellaneous uses. It is used as a solvent in applications that include textile and PVC cleaning, metal degreasing, extractions, and use in paints, coatings and adhesives. As an intermediate, EDC is used to produce polysulfide elastomers and ethylenimine (aziridine). Dow chemical captively consumes several million pounds per year of EDC at their Freeport, TX, facility to produce ethylenimine. EDC is used as a grain fumigant; Auerbach Associates (1978) estimated grain fumigant uses; Metcalf (1981) lists EDC as a fumigant for use in household and soil applications. Other uses mentioned for EDC include varnish and finish removers, soaps and scouring compounds, wetting and penetrating agents and ore flotation (Hawley, 1981). 5.5. SOURCES OF EMISSIONS EDC can enter the environment through atmospheric emissions, waste effluents to waterways, and land disposals of liquid and solid wastes. EDC in liquid and solid waters evaporates rapidly because of its high volatility; consequently, releases to land and water can be expected to enter predominantly into the atmosphere. The sources of EDC emissions are during EDC manufacture, intermediate use of EDC in production of other chemicals, and dispersive uses of EDC in end-point product applications. 5-9 CMA 051596 TABLE 5-6 Estimated Environmental Releases of Ethylene Dichloride in 1979a Application Releases (metric tons) Air Water Solid waste Production of 1,2-dichloroethane Indirect production Feedstock uses Trichloroethylene Tetrachloroethylene 1,1-Dichloroethylene Ethyleneamines Preparation of polysulfides Vinyl chloride Exports Dispersive uses Lead scavenging Fabric and PVC equipment cleaning Paints, coatings, adhesives Extraction solvents Grain fumigation Miscellaneous uses Total 6,154 65 73 138 86 104 5 136 180 956 864 1,364 1,000 460 300 11,885 61 191 -- -- -- -- -- -- -- -- -- -- -- -- -- 252 -- -- -- -- -- -- 5 -- -- -- 46 -- 50 -- -- 101 aSource: Seufert et al., 1980 5-10 CJ4A 051591 A number of estimates have been generated that predict the amount of EDC that is released to the environment. These estimates are based, in large part, on a sampling of monitoring data and a variety of engineering estimates. In many instances, the impact of current control technology may not have been adequately assessed. Therefore, the estimates that have been generated for EDC releases should not be interpreted as exact measurements, but are best regarded as order of magnitude estimates. Estimates of releases are in Table 5-6. Table 5-6 lists sources of EDC emissions in 1979 and estimates of the amounts released in air, water, and solid waste. Total 1979 emissions to the atmosphere, water, and solid waste amounted to 12,238 metric tons. The data in Table 5-6 indicate that gaseous emissions from EDC production amounted to 52.3$ of total atmospheric releases, while feedstock uses, dispersive applications, and exports accounted for 12,6, 336r and 1.5$, respectively (Seufert et al., 1980). Utilizing data from EDC producers, state and local emissions control agencies and the open literature, Hobbs and Key (1978) estimated 1978 emissions of EDC at ==11,000 metric tons. The degree of current emissions control on domestic processes involved in the primary production of EDC was assessed by Hobbs and Key (1978). Estimates by SRI International (U.S. EPA, 1979a) indicated that total domestic emissions of EDC from primary production, fugitive sources, and tank storage are 44,000 metric tons or =0.8$ of the amount produced. The impact of current control technology was not addressed in the report. Eimutis and Quill (1977) estimated process emissions of EDC for 1977 at 50,000 metric tons. Presumably, this estimate did not take into account current control technology. 5-11 CMA 051598 For 1974, Patterson et al. (1975) estimated total domestic emissions at 74,000 metric tons. Total domestic emissions for 1973 were reported at 54,000 metric tons by Shamel et al. (1975). The sources of EDC releases are discussed below. 5.5.1. Production and Related Facilities. In general, EDC is produced commer cially at an integrated manufacturing facility that uses some or all of the produced EDC to make vinyl chloride and, in some cases, other derivatives. In a typical vinyl chloride manufacturing facility, waste streams are generated by the three distinct processes: direct chlorination of ethylene, oxychlorination of ethylene, and dehydrochlorination to vinyl chloride; these are typically combined at a given facility for recovery, treatment and disposal. The specific number of point sources of releases at a manufacturing site is a function of plant design. Point sources of EDC loss from an integrated vinyl chloride plant include direct and oxychlorination reactor vent streams, light-ends distillation column vent, heavy-ends from the EDC recovery tower, wastewater from drying columns and scrubbers, and fugitive emissions from storage, pumps, seals, etc. (Catalytic, 1979; Drury and Hammons, 1979)- Releases from these manufacturing sites are mostly to the atmosphere and arise largely from vent gas streams (Drury and Hammons, 1979). Control devices used to limit EDC escape to the atmosphere include thermal oxidizers, catalytic oxidizers, vent condensers, scrubbers, and vent gas post-reactors (Hobbs and Key, 1978). Combined wastewaters that are generated from EDC manufacture (vent gas scrubbers, water produced during oxychlorination, and washwater) are treated in several ways depending upon the plant (Catalytic, 1979). Treatments include pre treatment and steam stripping prior to biological treatment, incineration of a portion of the waste stream, neutralization and chemical treatment, secondary treatments and final discharges to surface waters, to public owned treatment 5-12 CMA 051599 works, or to deep-well injection. Estimates of the amounts of EDC released to the surface waters are dependent upon engineering estimates of the overall success of treatments and the volume of wastewater flow. Solid wastes generated at an integrated vinyl chloride plant are usually treated to recover organic compounds present. Wastes are subsequently disposed of in a landfill or incinerated, recovering chlorine as hydrogen chloride (McPherson et al., 1979). Some solid wastes, possibly tars and heavy-ends, may be a suitable feedstock for tetrachloroethylene/carbon tetrachloride via a chlorination process. 5.5*2. Dispersive Uses. 5*5.2.1. LEAD SCAVENGING -- Seufert et al. (1980) estimated that 956 metric tons of EDC were released to the environment in 1979 from lead scavenging applications. Releases occur during blending of the gasolines, refueling of automoblies, filling and evaporation from gasoline storage tanks, and combustion of the gasoline. Approximately 1$ of the EDC used for lead scavenging is estimated to be released into the environment; the remainder is converted to hydrogen chloride and then to lead chloride during combustion. 5.5.2.2. PAINTS, COATINGS, ADHESIVES -- In this application, EDC is used as a solvent that is allowed to evaporate. Therefore, all of the EDC is emitted to the environment. 5.5.2.3. GRAIN FUMIGATION -- Fumigants are defined as gaseous pesticides. They must remain in the gas or vapor state and in sufficient concentration to be lethal to the target pest species. Therefore, all of the EDC used for this purpose will be vented to the atmosphere, as there are no control methods used. 5.5.2.4. FABRIC AND PVC CLEANING -- It is presumed that most EDC used for cleaning purposes will eventually be emitted to the atmosphere. Some cleaning wastes from PVC reactors may be drummed for landfill disposal. 5-13 05160 CMA 5.5.2.5. OTHER DISPERSIVE USES -- In other dispersive uses, it has been assumed that most EDO will eventually be emitted to the atmosphere. Some may be landfill, as with extraction solvents, and some may be incinerated. 5.5.3. Conclusions. The major sources of EDC emissions are from vent gas streams released during the manufacture of EDC and subsequent integrated produc tion of vinyl chloride monomer, and other EDC derivatives. Significant amounts of EDC are also released to the environment from dispersive uses.- In 1979, an estimated 6,696 metric tons of EDC were released to the atmosphere from EDC production and feedstock uses, while an estimated 4,944 metric tons were released to the atmosphere from dispersive uses (see Table 5-6), 5-14 CMA 051601 6. TRANSPORT AND FATE IN THE ENVIRONMENT The transport and fate of EDC in the environment depend on the medium in which it is present. The fate and transport of EDC in three environmental media (atmosphere, water and soil) are discussed below. Since a large percent of EDC emitted into the environment is in the atmospheric medium (see Chapter 5), the fate and transport of the chemical in this medium deserve special attention. 6.1. ATMOSPHERE The fate of EDC in the atmosphere is dictated by its ability to undergo chemical and physical removal processes in this medium. Reaction with *0H radicals is the principal chemical process by which many organic compounds, including EDC, are removed from the atmosphere (Crutzen and Fishman, 1977; Singh, 1977; Altshuller, 1979; Cupitt, 1980). Photolysis of 0^ in the troposphere produces singlet atomic oxygen [01p] that then reacts with water vapor to produce OH radicals. The tropospheric half-life (t1/2) of a compound is related to the OH radical concentration according to the expression t 1/2 = [0.693] [K(0H)]_1 where K is the rate constant of the reaction and [*0H] is the concentration of OH radicals. It is obvious from the above equation that an estimation of the tropospheric half-life for EDC due to *0H radical reaction requires that the values of both K and [ OH] be known. There are only two measurements of EDC reaction rate constant with *0H radicals. The absolute rate constant for EDC reaction with 0H radicals was determined by Howard and Evenson (1976) in a conventional discharge flow system. The value of the rate constant obtained in the system at pressures ranging from 0.7 to 7 mm Hg and at a temperature of 23C was 22 + 5 x 10 -- i U 0 11 (standard deviation of average) cnr molecule sec 6-1 CMA 051602 The rate constant for the reaction of 0H radicals with EDC in the presence of 0^ and was determined by Butler et al. (1978). At 29.5C and a total pressure of 1>00 mm Hg, the reaction rate constant was determined to have a probable value of 6.5 x 10-14 cm^ molecule-1 sec-1, with an upper limit value of 29 x 10-14 cm^ molecule-1 sec-1. Although Butler et al. (1978) argued that the presence of oxygen and nitrogen at a pressure of 400 mm Hg was more represen tative of actual tropospheric conditions, the presence of 02 and N2 actually complicated the reaction scheme through side reactions and made the extraction of kinetic data more difficult. It is for this reason that Butler et al. (1978) failed to determine the precision of the determined rate constant value. Therefore, the measured rate constant value of 22.0 + 5 x 10-14 cm3 molecule-1 sec~ 1 for EDC reaction with *0H radicals as reported by Howard and Evenson (1976) appears to be more accurate than the value of 6.5 x 10-14 cm3 molecule-1 sec-1 given by Butler et al. (1978). In Table 6-1, the rate constants for a number of chlorinated ethanes, including EDC, at room temperature have been shown. It seems clear from this table that a value of 22.0 x 10-14 cm3 molecule-1 sec-1 is more consistent with the measured K values for other chlorinated ethanes than a value of 6.5 x 10 -14 cm3 mo-lecule-- 1 sec-1 On the basis of the above discussion, it is reasonable to accept a value of 22 x 10-14 cm3 molecule-1 sec-1 as the rate constant value for EDC reaction with OH radicals at 296K (room temperature). It should be recognized that the value of K is dependent on temperature. The temperature in the troposphere is dependent on latitude, altitude and seasonal variations. The average annual tropospheric temperature weighted over 6-2 051603 Compound ch3ch2ci ch3chci2 ch2cich2ci ch2cichci2 TABLE 6-1 Rate Constants for a Few Chlorinated Ethanes at Room Temperature ill Rate Constant x 10 cm3-1 molecule-1 sec-1 39, 44 26 22, 6.5 33 Reference Howard and Evenson, 1976; Butler et al., 1978 Howard and Evenson, 1976 Howard and Evenson, 1976; Butler et al., 1978 Singh et al., 1981 6-3 CMA 051604 those variables is close to 265K, or -8C (Altshuller, 1979). Therefore, the evaluation of reaction rate at this temperature representing an average tropospheric reaction rate must incorporate the rate constant value at 265K. When the rate constant is not known, Altshuller (1979) estimated it by dividing the K value at 298K by 1.75. This is an empirical relationship and is applicable only to saturated organic compounds. Therefore, the rate constant for EDC reaction with *0H at 265K can be estimated as -2-2----x----1-0- -1c4m3 molecule-1 sec-1 , -14 3 -1 -1 or 12.6 x 10 cm molecule sec 1*75 The second factor required for the evaluation of tropospheric half-life for EDC reaction with *0H radicals is the average *0H concentration. Besides hemispheric difference, the concentration of *OH radicals in the troposphere is dependent on latitude, altitude and seasonal variations. The concentration of tropospheric 0H radicals in the Northern Hemisphere across the latitudes of the continental United States has been estimated by different investigators to range from 0.2 to 0.9 x 10 molecules cm--3 annually (Logan et al., 1981; Crutzen and Fishman, 1977; Neely and Plonka, 1978). Singh et al. (1983a) have recently estimated a mean hydroxyl radical concentration of 0.4-0.6 x 106 molecules cm 5 over the troposphere of the continental United States. Therefore, a value of 0.5 x 10 molecules cm-*5 is a good estimate for the average tropospheric hydroxyl radical concentration over the continental United States. It should be emphasized that the *0H concentration can vary substantially from the average 6 _o value of 0.5 x 10 molecules cm . Calvert (1976) estimated the average ambient g level of *0H radicals in the morning hours in Los Angeles to be -2.6 + 2 x 10 molecules cm-3 . Assuming equal periods of daylight and darkness, the average 6 *3 concentration for a 24-hour day should be =1 x 10 molecules cm-3. The calculations of tropospheric half-life for EDC reaction with *0H radical can be made under two scenarios. In the first case, a rate constant value at ambient temperature (22 x 10-11* cm^ molecule-1 sec-1) and an *0H 6-4 CMA 051605 concentration of 1 x 10 molecule cm"^ can be used to derive the half-life value of 36 days. In the second case, a rate constant value of 265K corresponding to annual average tropospheric temperature over the continental United States (12.6 x 10-14 cm^ molecule"^ sec-^) and a value of *0H concentration of 0.5 x 10 molecules cm J corresponding to the annual average tropospheric concentration of OH radical over the continental United States can be used to derive a half-life value of 127 days. Therefore, the tropospheric half-life for EDC reaction with OH may vary from 36 to 127 days. The reactivity of EDC with chlorine atoms was studied by Spence and Hanst (1978). These investigators found that the chlorine-sensitized photooxidation of 10 ppm EDC with 4 ppm Cl^ at 22.5C and irradiated by UV lamp (365 nm wavelength max.) and sun lamp (310 nm wavelength max.) produced a number of reaction products. Although hydrochloric acid (6.5 ppm), carbon monoxide (1 ppm), formyl chloride (3.5 ppm), C02 (2 ppm) and chloroacetyl chloride (0.5 ppm) were formed, no phosgene could be detected as a reaction product. EDC was found to have a relatively low reactivity toward Cl atoms compared to other chlorinated ethanes. That a chlorine-sensitized photooxidation is representative of actual tropospheric conditions has been contested by Singh (1978). This investigator estimated that chlorine atom concentrations in simulation chambers can be 10J to 10 times larger than in the troposphere. Thus, the role of chlorine atoms would be minimal under actual tropospheric conditions where *0H radicals are predominant. The role of halocarbons in the catalytic destruction of ozone (0^) is well documented (Altshuller, 1979; Altshuller and Hanst, 1975). Any halogenated compound with a sufficiently long lifetime may be transported to the stratosphere where photolytic mechanisms may release halogen atoms to participate in the 6-5 CMA 051606 destruction of ozone. The rate of reaction of *0H radical with halocarbons is one mechanism that competes with stratospheric photolysis as a halocarbon sink. Since transport to the stratosphere is a relatively slow process, the reported half-life for EDC suggests that only a small fraction released in the troposphere will reach the stratosphere. Thus, emissions of EDC would not be expected to play a significant role in the catalytic destruction of ozone. However, in the view of Altshuller and Hanst (1975), EDC should -be considered a threat in this regard since the production volume of EDC is so large. These investigators cite chloroacetyl chloride as the sole product of EDC reaction with OH. The possibility that this reaction product may have sufficient stability to travel to the stratosphere was raised. If formed in the troposphere, most of the chloroacetyl chloride probably will be washed out by rain. If it travels to the stratosphere, however, it may undergo photodissociation to form atomic chlorine. The three most likely physical processes that may participate in the removal of EDC from the atmosphere were studied by Cupitt (1980). The half-life for EDC removal by rain droplets was estimated to be 390 years. EDC in the vapor phase may be adsorbed on aerosol particles and removed from the atmosphere with the aerosol. However, the half-life of this removal process was estimated to be ^13 years (Cupitt, 1980). It has been estimated by Cupitt (1980) that compounds with _7 saturation vapor pressures of <10 mm Hg are likely to be adsorbed on atmospheric aerosols. Since the vapor pressure of EDC is much higher, it is not expected to be adsorbed by aerosol, and the probability of its removal by this process seems to be remote. The third physical removal mechanism, dry deposition of EDC, was also studied by Cupitt (1980). He estimated a half-life value of 25 years for this process. Therefore, it is not expected to be a significant EDC removal mechanism from the atmosphere. 6-6 CMA 051607 It is apparent from the above discussion that physical processes are not significant for the removal of EDC from the atmosphere. Of the chemical processes, the most significant reaction for removal of EDC from the atmosphere is its reaction with *0H radicals. The half-life for this reaction has been estimated to be =36 to 127 days. Therefore, it is anticipated that EDC will persist for a sufficiently long time in the atmosphere that it may participate in intramedia transport from its source of emissions, but not sufficiently long to allow for transport to the stratosphere. 6.2. AQUATIC MEDIA The fate of EDC in aquatic media is expected to be determined by its chemical, physical and microbiological removal mechanisms. The fate of EDC with respect to its possible chemical reactions has been reported by Callahan et al. (1979). Both photolysis and hydrolysis of EDC in aquatic media are expected to be insignificant fate processes (Callahan et al., 1979). Based on the oxidation rate constant data given by Mabey et al. (1981) and the concentration of free radicals in aquatic media given by Mill et al. (1982), the oxidation of EDC by singlet oxygen (K360 M"1hr--1 ) and peroxy radicals (1 M--1hr--" 1 ) can be predicted to be environmentally insignificant processes. The degradation possibilities of EDC by microorganisms in aquatic media have been studied by several authors. From the results of the laboratory tests conducted by several investigators with activated sludge or sewage seed as microbial inoculum in aquatic media, it can be concluded that EDC may biodegrade slowly under these conditions (Price et al., 1974; Tabak et al., 1981; Ludzack and Etlinger, I960; Henckelikian and Rand, 1955; Stover and Kincannon, 1983). Under methanogenic conditions, Bouwer and McCarty (1983) observed up to 50$ degradation of EDC to CO2 over a 25 week period. In a river die-away test, Mudder et al. (1982) reported no degradation of EDC at concentrations of =1 ppm and 6-7 CMA 051608 above. Stucki et al. (1981, 1983) isolated an organism from soil capable of utilizing EDC as a sole source of carbon. The bacterium could not grow on solid media but grew in liquid culture. No degradation studies were performed in soil so that no assessment can be made of the ability of this organism to degrade EDC in the environment. It can be concluded from these discussions that biodegradation will be an insignificant fate process for EDC in ambient aquatic media. The two likely physical processes that may remove EDC from aquatic media are sorption and volatilization. The removal of EDC by sorption on sediments and the subsequent sedimentation is probably not a significant process (Callahan et al., 19T9; Mabey et al., 1981). A calculation based on EXAMS modeling by the method of Burns et al. (1982) with the input parameters given in Section 3 also shows that EDC is not likely to be significantly sorbed onto particulate matter in water. The evaporation half-life for EDC from water at a depth of 1.6 cm, a concentration of 2 ppm, and a temperature of 24C was estimated to be 8 minutes under stirring (Chiou et al., 1980). Under somewhat more realistic conditions of a wind speed of 3 m/sec, water current of 1 m/sec, and a water depth of 1 ra, Lyman et al. (1982) calculated the evaporation half-life of EDC as 4 hours. Both of these methods, however, neglect a number of important environmental variables such as sorption, advection, and diffusion. These are particularly important in bodies of water deeper than one mile. The bioconcentration factor (BCF) for EDC in aquatic organisms was estimated as 6.03 by Kenaga (1980) from the water solubility and a linear regression equation. In a 14-day exposure experiment with bluegill sunfish (Lepomis macrochirus). Barrows et al. (1980) determined a BCF of 2.0 for this compound. Therefore, EDC is not likely to bioconcentrate in aquatic organisms. 6-8 CMA 051609 It can be concluded from the discussion above that the primary removal mechanism of EDC from aquatic media is probably volatilization. The half-life for volatilization is such that the compound may persist in aquatic media for a reasonable period and may participate in transport in aquatic bodies. 6.3. SOIL Data pertaining to the fate of EDC in soil are very limited. If one considers the chemical reactivity of EDC in aquatic media (see Section 6.2), it is possible to speculate that chemical reactions of EDC may be faster in soil than in water, particularly with soil acclimatized with EDC. However, Wilson et al. (1981) found virtually no biodegradation of EDC with soils collected near Ada, OK, that contained an average sand of 92J6 and organic carbon of 0.09%. The sorption constant (KUv ) of EDC on soils was predicted by Kenaga (1980) to be 43 (standardized with respect to soil organic content) from regression equation and solubility of this chemical in water. Chiou et al. (1979) estimated a value of 19 for KQQ with Willamette silt loam containing 1.6% organic matter, 26$ clay, 3.3? sand and 69$ silt. Therefore, the compound is not expected to be sorbed strongly onto soils, particularly onto soils containing low organic matter, and may percolate through the soil column. This has been confirmed by Wilson et al. (1980), who found =50$ percolation of EDC through a column of 140 cm depth containing the previously described Oklahoma soil. The fact that Page (1981) reported the detection of EDC in 10$ groundwater samples in New Jersey also indicates that the compound may transport downwards through some soils. The volatilization of EDC from soil may be an important removal mechanism. However, very few investigative data are available on this subject. Again, if one considers the investigation of Wilson et al. (1981) that reported the loss of 50$ EDC from the soil column through volatilization, one can conclude that volatilization may be one of the most significant physical removal processes for EDC from soil. 6-9 CMA 051610 6.4. SUMMARY The most likely removal mechanism for EDC from the atmosphere is reaction with hydroxyl free radicals. Based on available kinetic data and average tropospheric hydroxyl free radical concentrations, the half-life of EDC has been estimated to range from 36-127 days. EDC is not expected to play a significant role in stratospheric ozone destruction reactions due to its relatively short tropospheric half-life. However, chloroacetyl chloride may have suffficient stability to diffuse to the stratosphere and may participate in UV reactions producing chlorine atoms. In the aquatic environment, the most significant removal mechanism appears to be its volatilization. The half-life for this process has been estimated to be =4 hours. Therefore, EDC may persist in the aquatic media for a reasonable period and may participate in its transport in water bodies. From the little information that is available regarding the fate of EDC in soil, it has been concluded that both volatilization from and leaching through soil are the two significant removal mechanisms for EDC. 7. ENVIRONMENTAL LEVELS AND EXPOSURE 7.1. ENVIRONMENTAL LEVELS EDO contamination of the environment can result from the manufacture, U3e and disposal of the chemical. Sources of environmental release and quantitative estimates of releases are discussed in Section 5 of this report. This section is concerned with the levels of EDO that have been detected in .various areas and monitoring sites. Sizable quantities of EDO were judged to be released to the air during manufacture and conversion to vinyl chloride at an integrated manufac turing site. This judgment is borne out by the high ambient concentrations of EDC found near several of these sites. 7.1.1. Atmospheric Levels. In addition to atmospheric releases of EDC from manufacture and feedstock uses, EDC can be released to the air from dispersive uses such as grain fumigation, solvent uses in paints, coating and adhesives, cleaning applications, and gasoline uses related to lead scavenging. Recent air monitoring data for EDC are listed in Table 7-1. These data indicate that EDC is often present in ambient air in urban and industrial areas, especially near plants manufacturing or using the chemical. Measurements made at sites in three geographical areas central to EDC production and user facilities indicated EDC concentrations as high as 184 ppb (Elfers, 1979). These levels were associated with industrial sources and occurred during atmospheric conditions of calm or low wind speeds. Measurements were made during 10- to 13day periods at Lake Charles and New Orleans, LA, and at Calvert City, KY. The highest concentrations were reported for the Lake Charles area, with the highest single concentration being 184 ppb and the average detectable concentration being 27.5 ppb. In Calvert City, ambient concentrations ranged from <0.12 to 7-1 CMA 051612 TABLE 7-1 Ambient Atmospheric Levels of Ethylene Bichloride Location Type of Site Date ALABAMA Birmingham urban April, 1977 Birmingham urban April, 1977 ARIZONA Phoenix Grand Canyon urban rural April-May, 1979 Nov-Dec, 1977 CALIFORNIA Dominguez urban May, 1976 Los Angeles Oakland Riverside Upland urban urban urban urban April, 1979 June-July, 1979 July, 1980 Upland urban Aug-Sept, 1977 COLORADO Denver urban June, 1980 ILLINOIS Chicago urban April, 1981 KENTUCKY Calvert City 12 cities near Aug-Sept, 1978 chem. plant LOUISIANA Baton Rouge Baton Rouge Geismar Iberville Parish Lake Charles urban urban Industrial urban urban March, 1977 March, 1977 Feb, Mar, 1977 Jan, Feb, 1977 June-Oct, 1978 Detection Method GC/MS GC/MS GC/ECD GC/MS GC/ECD GC/ECD GC/ECD GC/MS GC/MS GC/ECD GC/ECD GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS Number of Samples 2 7 (4 ND) NR 7 (7 ND) 1 102 8 16 (14 ND) 95 NR 88 25 (9 ND) 18 (2 ND) 10 {1 ND) 11 98 Concentration (cot, v/v)a Max. Min. Avg. Reference 98.6 99 1951.1 50.7 0 38.8 ----- 74.7 35 216.3 -- 1359.8 893-0 580 212.9 210 173-0 38.3 89 61.8 0 3662.3 519.2 82.5 360 110.3 27 980 100 240 2820 17800 22 <120 195 5000 Pellizzari and Bunch, 1979 Pellizzari, 1979a Singh et al., 1980 Pellizzari, 1979a Pellizzari and Bunch, 1979 Singh et al., 1980 Singh et al., 1980 Singh et al., 1982 Pellizzari and Bunch, 1979 Pellizzari, 1979a Singh et al., I960 Singh et al., 1982 Elfers, 1979 630 2556.5 2559.5 1169.2 61000 0 19-3 24.7 2.2 150 270 429.4 760.4 320.9 27000 Pellizzari et al., Pellizzari, 1978a Pellizzari, 1978a Pellizzari, 1978a Pellizzari, 1979b 1979 051613 051614 TABLE 7-1 Ccont.) Location Type of Site Date Detection Method Number of Samples LOUISIANA Ccont.) Lake Charles Lake Charles Nek Orleans Plaquemine urban June, 1978 12 sites near chem* plant near chem. plant urban Sept-Oct, 1978 October, 1978 Jan-Feb, 1977 GC/KS GC/MS GC/MS GC/MS 6 110 91 11 MISSOURI St. Louis NEW JERSEY Batsto Bound Brook Bridgeport Bridgewater Burlington Camden Carlstadt Clifton Deepwater East Brunswick Edison Edison urban May-June, 1980 rural Feb-Dec, 1979 off highway March, 1976 September, 1977 July-Aug, 1978 September, 1977 urban Apr-Oct, 1979 urban urban September, 1979 March, 1976 near chem. plant marina June, 1977 July, 1976 urban Mar-July, 1976 near waste Mar-July, 1976 disposal area GC/ECD 90 GC/FID-ECD-MS GC/MS GC/MS NR GC/MS GC/FID-EDC-MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS 42 (40 ND) 1 2 22 (18 ND) 1 23 (21 HD) 16 (7 ND) 1 6 (3 traces) 2 33 (20 ND) 16 (1 trace) Concentration '(ppt, v/v)a Max. Min. Avg. Reference 307.3 184000 4)700 921.4 5.2 <120 <120 2.2 86.5 27500 2900 337.2 Pellizzari and Bunch, 1979 Elfers, 1979 Elfers, 1979 Pellizzari and Bunch, 1979 260 65 120 Singh et al., 1980 -- -- -- 380 -- -- 998.0 ----- -- 13.1 85.8 3400 14091.5 -- **---- 0 -- 33.4 5.9 37.1 8.6 53-6 Trace (2 samples) Trace Trace 170 Trace Trace (2 samples) 537.2 15950 7.4 61.6 1600 2712 Bozzelli et al., 1980 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Bozzelli et al., Pellizzari and Bunch, 1979 Bozzelli et al., 1979 1980 Pellizzari, 1978b Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 TABLE 7-1 (cont.) Location Type of Site Date Detection Hethod Humber of Samples NEW JERSEY (cont.) Elizabeth Elizabeth Fords Hoboken Linden Linden Middlesex Newark Newark Newark urban near indus trial plant urban urban industrial urban suburban urban urban urban Sept 1970-Dec, 1979 GC/FID-ECD-KS Jan-Dec, 1979 GC/FID-ECD-MS 71 (53 ND) 54 (7 trace, 46 ND) March, 1976 GC/MS 1 March, 1976 GC/MS 2 (2 ND) June, Nov, 1977 GC/MS 16 (1 trace) June, 1977 July, 1978 Jan-Dec, 1979 March, 1976 GC/MS NR GC/FID-ECD-MS GC/MS 11 (1 ND) 18 (13 ND) 37 (9 trace, 28 ND) 1 Mar, 1976-Dec, 1979 GC/FID-ECD-MS 160 (112 ND) Concentration (ppt, v/v)a Hax. Min. Avg. 2200 -- -- 0 48.2 4.9 290 -- -- 5000 0 --- -- -- 0 2.0 4.9 20 -- -- 0 220 2202.7 Trace 0 14.3 4.9 110 Trace Trace 450 Pa33aic Paterson Rahway Rutherford Rutherford Sayreville Somerset South Amboy NEW YORK Niagara Falls Staten Island Staten Island suburban suburban industrial March, 1976 GC/MS March, 1976 GC/MS September, 1978 GC/MS May, 1978-Dec, 1979 GC/FID-ECD-MS May, 1979 July, 1976 GC/FID-ECD-MS GC/MS July, 1978 Jan-Dec, 1979 NR GC/FID-ECD-MS 1 1 16 196 (141 ND) 46 <3 trace, 42 ND) 1 29 (17 ND) 48 (4 trace, 44 ND) residential (Love Canal) urban urban Februaryt 1978 November, 1976 Mar-Apr, 1901 GC/MS GC/MS GC/ECD 9 (2 trace, 7 ND) 3 NR -- -- 775.5 3200 -- -- 1000 -- --~ 50.7 4312 -- -- 165.4 0 -- -- 0 -- Trace Trace 525.8 370 593-3 9372.8 490 Trace """ 46.0 55 Trace 48.2 256 ReTerence Bozzelli et al., 1900 Bozzelli et al., 1900 Pellizzari and Bunch, 1979 Pellizzari, 1977a; Pellizzari et al., 1979 Pellizzari and Bunch, 1979 Pellizzari, 1970c Bozzelli et al., 1979 Bozzelli et al., 1900 Pellizzari and Bunch, 1979 Bozzelli and Kebbekus, 1979; Bozzelli et al., 1900; Pellizzari, 1977 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari, 1978b,d Bozzelli et al., 1979, I960 Bozzelli et al., 1980 Pellizzari and Bunch, 1979 Bozzelli et al., 1979 Bozzelli et al., 1900 Pellizzari, 19780^ Pellizzari and Bunch, 1979 Singh et al., 1902 CMA 051615 CMA 051616 TABLE 7-1 (cont.) Location Type of Site Date Detection Method NORTH CAROLINA Chapel Hill OKLAHOMA Liberty Mound Tulsa Vera PENNSYLVANIA Bristol Marcus Hook N. Philadelphia Pittsburgh TEXAS Aldine Beaumont Deer Park El Paso Freeport Houston Hous ton Houston La Porte Pasadena UTAH Magna June, 1980 NR rural urban July-Sept, 1977 July-Sept, 1977 July, 1977 GC/MS GC/MS GC/KS urban urban urban August, 1977 August, 1977 August, 1977 April, 1981 GC/MS GC/MS GC/MS GC/ECD urban industrial urban Industrial urban urban streets, parks, rural highway urban June-Oct, 1977 March, 1980 August, 1977 Apr-May, 1978 August, 1977 July 1976-May, 1980 NR July, 1976; JuneJuly, 1978 August, 1976 July, 1976 GC/MS NR GC/MS GC/MS GC/KS GC/ECD GC/KS GC/KS GC/MS GC/MS rural Oct-Nov, 1977 GC/MS Number of Samples 6 2 (2 ND) 2 (2 ND) 1 (1 ND) 2 2 4 NR 3 (3 ND) 11 6 (3 trace) 22 (19 ND) 2 99 (5 ND) 30 10 (1 trace) 1 1 9 (9 ND) Concentration (ppt,, v/v)a Max. Min. Avg. Reference 110 110 110 000 000 000 -- -- 238.6 237 -- -- 41.3 66 63-8 48.2 139-2 121 0 670 16390.6 29 1112.5 3000 16390.6 109.8 -- 39 0 670 1002.5 0 815.8 50 73.4 30.4 -- 39 0 670 6353-5 4.3 964.2 1300 893.0 63.0 192.3 39 0 00 Wallace, 1981 Pellizzari, 1978e Pellizzari, 1978e Pellizzari, 1978e Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Singh et al., 1982 Pellizzari et al., 1979 Wallace, 1981 Pellizzari and Bunch, 1979 Pellizzari, 1979a Pellizzari and Bunch, 1979 Pellizzari et al., 1979 Singh et al., 1980 Pellizzari, 1978b Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari et al., 1979 Pellizzari, 1979a 051617 TABLE 7-1 (cont.) Location VIRGINIA Front Royal WEST VIRGINIA Charleston Charleston Institute Institute Nitro Nitro St. Albans St. Albans S. Charleston S. Charleston W. Belle W. Belle WASHINGTON Pullman Pullman Type of Site Date Detection Method industrial Oct-Nov, 1977 GC/MS industrial industrial Industrial Industrial industrial industrial industrial industrial industrial industrial industrial Industrial Sept, Nov , 1977 Sept-Nov, 1977 November, 1977 November, 1977 Oct, Nov, 1977 Sept-Oct, 1977 October, 1977 Sept-Nov, 1977 Mar, Sept-Nov, 1977 Mar-Nov, 1977 Sept-Nov, 1977 Sept-Nov, 1977 GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS rural rural Dec, 1974 -Feb, 1975 November, 1975 GC/MS GC/ECD Number of Samples 16 3 4 (4 ND) 2 (2 ND) 3 4 6 (6 ND) 1 4 (4 ND} 6 16 (15 ND) 6 (6 HD) 4 NR 1 aA3sume ambient temperature of 25C and atmospheric pressure of 760 mmHg. ND = Not detected n NR = Not reported ECD = Electron capture detection FID = Flame ionization detection GC = Ga3 chromatography MS = Mass spectrometry Concentration (ppt. v/v)a Max. Min. Avg. Reference 86.0 37.3 48.2 -- 0 0 -- 63.8 0 -- 0 63.8 37 0 61.8 -- 0 0 -- 37-3 0 -- 0 37.3 0 0 37.3 57.1 0 0 37.3 46.7 0 48.2 0 52.4 2.3 0 47.0 -- -- <5 10 -- -- Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari, 1978e Pellizzari, 1978e Pellizzari and Bunch, 1979 Pellizzari and Bunch, 1979 Pellizzari, 197&e Pellizzari and Bunch, 1979 Pellizzari, 1978e Pellizzari and Bunch, 1979 Pellizzari, 1979a; Pellizzari, 1978e Pellizzari, 197Be Pellizzari and Bunch, 1979 Grlasrud and Rassmussen, 1975 Harsch et al., 1979 17.8 ppb. Levels recorded in the New Orleans study area ranged from <0.12 to 41 .7 ppb. Analyses done by Elfers (1979) were performed by collecting ambient air on charcoal tubes followed by desorption by carbon disulfide. Detection and quanti tation was made by gas-chromatograph-mass spectrometry. Quality control check sample analysis indicated that recovery of standard concentrations was highly variable. The detection limit of the gas chromatographic method was reported as 1 pg. The relative standard deviation of replicate standard solutions was found to be 3?. The precision of the analytical and sampling methods together was 6%. The accuracy of the analytical method varied between 72 and 97t. The highest reported reading for a single sample listed in Table 7-1 is 191 .5 ppb, which was recorded in Lake Charles, LA, the site for several EDC-vinyl chloride manufacturing facilities. Concentrations in excess of 15 ppb for a single sample were also reported near manufacturing facilities in Calvert City, KY; New Orleans, LA; Deer Park and Houston, TX. Recent studies have measured general urban ambient concentrations of EDC in ten cities (Singh et al., 1980, 1981, 1982). The results of these studies are summarized in Table 7-2. The averaging time for the mean concentrations in these studies was 2 weeks. Electron capture detector-gas chromatography was the primary means of analysis. Low levels of EDC were recorded in a year-long monitoring program of five cities in industrial northern New Jersey (Bozzelli et al., 1980). Samples were collected at the five sites regularly throughout 19 79. Only two of the 208 samples analyzed had EDC concentrations higher than trace (41 ng/m^ or 10 ppt). In a survey of air contaminants in the rural northwest, Grimsrud and Rasmussen (1975) found EDC concentration of <5 ppt. 7-7 CMA 051618 TABLE 7-2 a Ambient Concentrations of Ethylene Dichloride in Urban Areas City Los Angeles, CA Phoenix, AZ Oakland, CA Houston, TX St. Louis, MO Denver, CO Riverside, CA Staten Island, NY Pittsburgh, PA Chicago, IL Ethylene Dlehloride Concentrations (ppt) Mean Maximum Minimum 519 216 83 1512 124 241 357 256 121 195 1353 1450 842 7300 607 2089 2505 4312 237 2820 173 39 38 50 45 56 63 55 66 22 aSource: Singh et al., 1980, 1981, 1982 7-8 CMA 051619 7.1.2. Ground and Surface Water Levels. EDC can be released to the water environment via wastewaters generated during production of ethylene dichloride and its derivatives. It is also possible that EDC may be inadvertently produced in the water environment due to chlorination processes of public water supplies or chlorination of sewage or wastewaters (Versar, 1975; Seufert et al., 1980). One chemical reaction that could lead to EDC production during water treatment is the reaction of alkenes with hypochlorite. However, it is expected that industrial discharges to surface water and leaching from solid waste are the primary causes of EDC contamination in drinking water (Letkiewicz et al., 1982). Most discharges of EDC are judged to ultimately reach the atmosphere because of its high volatility (Letkiewicz et al., 1982). The identification and levels of EDC that have been found in the ground and surface water of the United States are discussed below. EDC was reported to be a principal contaminant in finished water in 1975 by Dowty et al. (1975). Because the gas chromatographic peak could not be resolved, it was not possible to quantitate EDC. It was indicated that EDC levels could be increased as a result of the water treatment processes and could pass the treat ment plant without removal. The water analyzed in this study was obtained from the Mississippi River. Deinzer et al. (1978), citing the results of a 1975 national drinking water survey, reported EDC concentrations ranging from 0 to 6 ppb. While sampling for pollutants in 14 heavily-industrialized river basins in the United States, Ewing et al. (1977) found that EDC was present in 53 of the 204 samples purged for volatile organic analysis. The majority of the 81 purgeable organic compounds detected were C^ to Cg halogenated hydrocarbons. Only chloro form, trichloroethylene and tetrachloroethylene were found with greater 7-9 CMA 051620 frequencies. Identification was made by a GC-MS procedure. The limit of sensi tivity was <1 ppb. In a compilation of pollutants found in water, Shackleford and Keith (1976) identified EDC as having been found in industrial effluents, in finished drinking water, and in river waters, Letkiewicz et al. (1982), in a recent study for the U.S. EPA, discuss the results of six federal surveys in which a number of public water supplies were selected for analysis of chemical contaminants, including EDC. The six Federal drinking water surveys providing data on EDC include the National Organics Reconnaissance Survey (NORS), the National Organics Monitoring Survey (NOMS), the National Screening Program for Organics in Drinking Water (NSP), the 1978 Community Water Supply Survey (CWSS), the Groundwater Supply Survey (GWSS), and the Rural Water Survey (RWS). All surveys sampled both ground and surface waters except for the GWSS. The scope and methodology of each survey is outlined below, along with the identification of EDC presence. The National Organics Reconnaissance Survey (NORS) was conducted in 1975 to determine the extent of the presence of EDC, carbon tetrachloride, and four trihalomethanes in drinking water supplies from 80 cities across the country (Symons et al., 1975). Another stated objective of the study was to determine the effect of raw water source and treatment practices on the formation of these compounds. The water samples were collected in 50 m2, sealed vials, with vola tilization prevented by the absence of head space. Samples were subsequently shipped on ice to the EPA Water Supply Research Laboratory in Cincinnati for analysis. Analyses were performed by purge-and-trap gas chromatography with an electrolytic conductivity detector. A population base of 36 million from 80 cities across the country was covered during the study. 7-10 051621 In the NORS study, 16 groundwater systems were analyzed for EDC contamina tion. None of the 16 systems contain detectable levels. Of the 64 surface water samples analyzed for EDC, six were found to contain quantifiable levels of EDC at 0.2-6.0 The National Organics Monitoring Survey (NOMS) was instituted to identify contaminant sources, to determine the frequency of occurrence of specific drinking water contaminants, and to provide data for the establishment of maximum contaminant levels (MCL's) for various organic compounds in drinking water (U.S. EPA, 1977). The NOMS was conducted in three phases, March-April 1976, May-July 1976, and November 1976-January 1977. Drinking water samples from 113 communi ties were analyzed for 21 different compounds by purge-and-trap gas chromato graphy with electrolytic conductivity detectors. During Phase I the samples were collected in 25- or 40-mi, open-top screw cap septum vials with no head space, a feature that prevented volatilization of the compounds of interest. These samples were then stored at 2-8C for 1-2 weeks prior to analysis. Samples were collected in a similar fashion for Phases II and III, but were held at 20C for periods up to 6 weeks. Sodium thiosulfate was added to a number of samples from Phases II and III to reduce any residual chlorine present. Of 18 groundwater systems analyzed for EDC during Phase I of the NOMS study (March to April 1976), none contained quantifiable levels. When these systems were sampled again during Phase II (May to July 1976), one system was found to contain EDC, at 0.02 No detectable levels of EDC were found during Phase III of the study (November 1976 to January 1977). In Phase I of the NOMS (March to April 1976), water samples from 87 surface water systems were analyzed for EDC. Of these 87 systems, only one was found to contain EDC, at 2.0 ^g/#,. When these systems were sampled again during the second phase of the survey, one was found to be contaminated, at 1.8 \ig/Z. 7-11 CMA 051622 During the third phase of the NOMS (November 1976 to January 1977), analyses revealed EDC contamination in one system, at 1.25 pg/S.. In the National Screening Program for Organics in Drinking Water (NSP), conducted from June 1977 to March 1981, both raw and finished drinking water samples from 169 water systems in 33 states were analyzed for 51 organic chemical contaminants (SRI, 1981). Analyses were carried out by gas chromatography with various detection methods, including the mass spectrometer, halogen-specific detector, and flame ionization detector. Thirteen groundwater supplies were tested for EDC contamination during the NSP study. Of these systems, one was found to be contaminated with EDC at 0.2 pg/Ji. Surface water samples for 107 drinking water systems were analyzed for EDC. Of these, only one system was found to be contaminated with EDC, at 3-8 pg/JU In the Community Water Supply Survey (CWSS), carried out in 1978, 110 surface water and 330 groundwater supplies were examined for contamination by volatile organic chemicals (U.S. EPA, 1981a). Fourteen purgeable organic compounds were analyzed, and total organic carbon levels were determined. At the time of analysis, the samples were 1 to 2 years old. Long storage periods may have resulted in the loss of some aromatics and unsaturated halooarbons due to biological action. The analytical method of choice for the CWSS was purge-andtrap gas chromatography with an electrolytic conductivity detector for halocarbons and a flame ionization detector for aromatic analysis. The CWSS survey provided information on EDC levels in finished groundwater supplies from 312 systems. Of the samples taken from these systems, three contained detectable quantities of EDC with levels of 0.57, 1.06 and 1.1 pg/&. Of the 110 surface water systems tested, only one contained quantifiable EDC, with a concentration of 0.62 pg/H. 7-12 CMA 051623 The Groundwater Supply Survey of 1980 (GWSS) was initiated to provide a clearer picture of the extent of contamination of groundwater supplies with volatile organic compounds (U.S. EPA, 1982a). A total of 945 systems was sampled, of which 466 were chosen at random and 479 were picked from locations near potential sources of contamination. Samples were collected at points close to the actual distribution source, with mercuric chloride added as a preservative to prevent biodegradation of aromatics. Sodium thiosulfate was'also added to the vials to reduce any residual chlorine, thus preventing its reaction with organic matter. Analyses were made for 37 volatile organic compounds by purge-and-trap gas chromatography with electrolytic conductivity detectors. Of the 466 randomly chosen water systems, seven were contaminated with EDC, at concentra tions ranging from 0.29-0.57 pg/Jl. Of the seven positive samples, six were from systems serving populations in excess of 10,000 people. The average for all randomly chosen systems was 0.5 pg/8,. Of the 479 nonrandom locations sampled, nine were contaminated with EDC, at concentrations between 0.33-9.8 |ig/A. Of the nine positive samples, four were from systems serving populations in excess of 10,000 people. The average EDC level for the positive nonrandom systems was 2.7 ug/t. The Rural Water Survey (RWS), conducted in 1978 (Brass, 1981), was carried out in response to Section 3 of the Safe Drinking Water Act, which mandated that EPA "conduct a survey of the quantity, quality and availability of rural drinking water supplies." A total of 800 of the 2655 samples collected in the RWS was analyzed for volatile organic chemicals. Three hundred of the samples were selected by a random generation procedure and 500 were picked randomly by state (10 per state). There were 633 samples from groundwater, 47 samples from surface water, and 81 samples from other water source categories. Of 633 groundwater samples, five were found to have EDC present (minimum quantification limit generally 0.5-1.0 pg/2,). The range of concentrations was 7-13 CMA 051624 0.5-18 pg/&; the mean and median concentrations of the positive values were 5.6 and 1.7 pg/2,, respectively. Both the mean and median values of all samples were <0.5 pg/Ji. Of the 47 surface water samples, only one (2.1$) was found to have EDC present (minimum quantification limit generally 0.5-1.0 ug/2,). The concentra tion of the one positive sample was 19 pg/S,. The mean and median values of all samples were both <0.5 pg/&. The combined EDC groundwater data and surface water data-from the above Federal studies are summarized in Tables 7-3 and 7-4, respectively. The RWS study was deleted from these summaries because the RWS data were recorded by a number of service connections rather than population served. From the data presented in Tables 7-3 and 7-4, Letkiewicz et al. (1982) have projected that EDC levels in all groundwater and surface water systems in the United States fall below 10 pg/L, and that most are below 1.0 pg/H. In addition to the six Federal surveys discussed by Letkiewicz et al. (1982), six states (California, Connecticut, Deleware, Indiana, Massachussetts and New Jersey) provided the U.S. EPA with information concerning EDC contamina tion in groundwater supplies. These data are listed in Table 7-5. The only state-supplied surface water Information on EDC was from New York; one sample from Poughkeepsie assayed positive at 5.9 pg/2. (Letkiewicz et al., 1982). 7.1.3- Soil and Sediment Levels. No data were available on monitoring of EDC concentrations in soils, sediments or solid wastes in the sources consulted for this report. 7.2. ENVIRONMENTAL EXPOSURE The general population can be exposed to EDC from three sources, air emissions, drinking water and consumed foods. Some individuals may be exposed to EDC from sources other than the three considered here, such as in occupational settings and in the use of consumer products containing EDC. However, this 7-14 CMA 051625 TABLE 7-3 Reported Occurrence of Ethylene Dichloride in Groundwater Systems -- Combined Federal Data (NORS, NOMS, NSP, CWSS, GWSS)a System size (population served) Number of systems in U.S. Number of positivg systems Number of systems sampled Positive systems (*) Number undetected <1.0 pg/Jl Number of systems with measured concentration (u/Jl) of: <1.0 1.0-5 >5-10 >10 <100 19,632 1/175 0.6 174 10 00 101-500 15,634 0/220 0.0 220 00 00 501-1,000 4,909 0/114 0.0 114 00 00 1,001-2,500 4,331 3/151 2.0 148 12 00 2,501-3,300 881 0/40 0.0 40 00 00 3,301-5,000 1,065 2/79 2.5 77 11 00 5,001-10,000 1,159 1/114 0.9 113 01 00 10,001-50,000 1,101 11/296 3.7 285 64 10 50,001-75,000 68 1/37 2.7 36 10 00 75,001-100,000 16 0/12 0.0 12 00 00 >100,000 58 1/43 2.3 42 10 00 Source: Letkiewicz et al., 1982 ^Positive systems are those with quantified levels of ethylene dichloride. 051626 System size (population served) <1 00 101-500 501 -1,000 1 ,001 -2,500 2,501-3,300 3,301-5,000 5,001 -13,000 1 0,001-50,000 50,001 -75,000 75,001-100,000 >100,000 TABLE 7-4 Reported Occurrence of Ethylene Dichloride in Surface Water Systems -- Combined Federal Data (NORS, NOMS, NSP, CWSS)a Number of systems in U.S. Number of positivg systems0 Number of systems sampled Positive systems (1) Number undetected <1.0 [ig/H Number of systems with measured concentration (usc/5.) of: <1.0 1.0-5 >5-10 >10 1 ,412 2,383 1 ,341 1 ,911 51 4 720 912 1,306 156 85 218 0/4 0/19 0/13 1/22 0/6 1 /I 5 0/1 3 1/38 2/22 0/14 5/1 02 0.0 0.0 0.0 4.5 0.0 6.7 0.0 2.6 9.0 0.0 4.9 4 00 00 19 00 00 13 00 00 21 10 00 6 00 00 14 01 00 10 00 00 37 10 00 20 11 00 14 00 00 97 31 10 CMA 0 5 1 6 2 7 aFour systems reported as undetected with the unusually high detection limit of 2.0 yg/?. were deleted. bPositive systems are those with quantified levels of ethylene dichloride. Source: Letkiewicz et al., 1992 TABLE 7-5 St State Data on Ethylene Dichloride in Groundwater Location CALIFORNIA Baldwin Park Morada Unspecified Unspecified CONNECTICUT Colchester Danbury DELAWARE Collins Park Midvale Newark (North and South) INDIANA Elkhart Granger MASSACHUSETTS Acton Belchertown Dartmouth Rowley NEW JERSEY Bergen County Essex County Fair Lawn Morris County Passaic County 9 counties 12 counties Water type Mean (Hg/SL) Range (pg/i) Number of samples N/S 21 N/S 12 N/S 1.2 N/S 1.2, 5.3 D ND D 7.8 F ND F ND F ND 1 1 2 (1 ND) 2 1 1 1 1 2 F, N/S 777 N/S 30-2,100 19, 160 11 (4 ND) 15 (13 ND) F ND 4 F 14.5 10.1-19.1 10 (7 ND) F 11.6, 18.2 2 F ND 1 N/S N/S F, N/S N/S N/S N/S N/S N/S N/S N/S ND ND 1.3 ND ND ND 1.1-1.9 2.0-2.1 0.1-0.9 1-10 10-100 4 7 15 13 (11 ND) 1 154 228 4 4 1 Source: Letkiewicz et al., 1982 ND = Not detected; N/S = Not specified; F = Finished; D = Distribution 7-1 7 CMA 051628 section is limited to air, drinking water and food since these are considered to be general sources common to most individuals. It must be noted that individual exposure will vary widely depending upon factors such as where the individual lives, works or travels, or what the individual may eat or drink. Individuals living in the same neighborhood can experience vastly different exposure patterns. Unfortunately, methods for estimating the exposure of identifiable popula tion subgroups from all sources simultaneously have not yet been developed (Letkiewicz et al., 1982). Exposure from the three sources considered here is discussed below. 7.2.1. Exposure from Air. Atmospheric exposure to EDC appears to vary greatly from one location to another. Mean levels as high as 27.5 ppb have been monitored near production and use facilities in Lake Charles, LA (Elfers, 1979). However, the available monitoring data (see Section 7.1) indicate that most locations have EDC concentrations of 0.5 ppb or less. The monitoring data presented are not sufficient to determine regional variations in exposure levels for EDC. The majority of high values reported were for samples taken near production and use facilities. Letkiewicz et al. (1982) have estimated respiratory intake of ethylene dichloride by adults and infants; these estimates are summarized in Table 7-6. From available monitoring data, Letkiewicz et al. (1982) estimated that low, a intermediate and high exposure levels for EDC in air were 0.25, 2.5 and 25 pg/m , respectively. Daily intake of EDC for adults in intermediate exposure levels was estimated at 0.82 pg/kg. Dispersion models have also been used to estimate ambient concentrations. Based on modeled results for individual plants, maximum concentration levels in the vicinity of various emission sources have been calculated (SRI 7-18 CMA 051629 TABLE 7-6 Estimated Respiratory Intake of Ethylene Dichloride by Adults and Infants3 Exposure Level pg/mJ (ppb) Intake (ug/kg/daz) Adult Infant Low: Intermediate: High: 0.25 2.5 25 (0.062) (0.62) (6.20) 0.08 0.82 8.21 0.06 0.57 5.71 o Assumptions: 70 kg*man, 3.5 kg-infant, 20 nr of air inhaled/day (man), 0.8 nr of air inhaled/day (infant). aSource: Letkiewicz et al., 1982 TABLE 7-7 Maximum Concentration Level in the Vicinity of Various Emission Sources3 Source EDC Production Facilities End Use Production Facilities Gasoline Service Stations Automobile Emissions Automobile Refueling Maximum Annual Average EDC Concentration Level (ppb) >10b 0.60 - 0.99 0.01 - 0.029 0.01 - 0.029 <0.01 Source: SRI International, 1979 b. An accurate estimate cannot be determined from this reference, but it is thought to be in the range of 10-15 ppb. 7-19 CMA 051630 International, 1979). Table 7-7 shows a summary of estimated maximum concentrations that people may be exposed to in the vicinity of specific emission sources. 7.2.2. Exposure from Water. Letkiewicz et al. (1982) have projected that EDC levels in all groundwater and surface water systems in the United States fall below 10 pg/Jl, and that most are below 1.0 (ig/2- (see Section 7.1*2). The total estimated population exposed to EDC from both ground and surface water sources is shown in Table 7-8. The values in the table were obtained by use of the Federal Reporting Data System data on populations served by primary water supply systems and data on the estimated number of these water systems that contains a given level of EDC (see Section 7.1.2). Letkiewicz et al. (1982) have also estimated daily intakes of EDC from drinking water. These estimates are given in Table 7-9. 7.2.3* Exposure from Food. EDC is used as a fumigant for grain, so contamina tion of flour and bread is possible. Several studies on this question have indicated that EDC dissipates when the bagged flour is exposed to air, and that no detectable EDC remains after baking (U.S. EPA, 1981b). No further information on the presence of EDC in food was uncovered in the literature search. 7.3. CONCLUSIONS Most EDC exposures result from the production and use of the chemical. The highest atmospheric concentrations monitored for EDC have been detected near production and use facilities. Therefore, it appears that the risk for popula tion exposure to EDC is greatest in the vicinity of these sources. 7-20 CMA 051631 TABLE 7-8 Total Estimated Population (in Thousands) Exposed to Ethylene Dichloride in Drinking Water at the Indicated Concentration Ranges3 System type Total served in U.S. (thousands) Groundwater Surface water TOTAL ($ of total) 69,239 126.356 195,595 (100$) aSource: Letkiewicz et al., 1982 Population (thousands) exposed to concentrations (uk/2.) of: <1.0 to 10 >10 69,239 126.356 195,595 (100$) 0.0 0.0 0.0 (0.0$) TABLE 7-9 Estimated Drinking Water Intake of Ett^rlene Dichloride by Adults and Infants3 Exposure Level Intake(ug/kg/day) Adult Infant 1.0 5 10 100 0.029 0.14 0.29 2.9 0.24 1.2 2.4 24.0 Assumptions: 70 kg-man, 3*5 kg-infant, 2 l of water/day (man), 0.85 S. of water/day (infant). aSource; Letkiewicz et al., 1982 7-21 CMA 051632 8. ECOLOGICAL EFFECTS A variety of studies have demonstrated that EDC, in the role of a fumigant, is toxic to insects infesting stored grains (Wadhi and Soares, 1964; Ellis and Morrison, 1967; Vincent and Lindgren, 1965; Snapp, 1958; Krohne and Lingren, 1958; Finnegan and Stewart, 1962; Lindgren et al., 1954; and Bang and Telford, 1966). Little is known, however, of the effects of EDC to soil micoflora. EDC has been reported to be non-toxic to many economically important plant species when directly applied to growing plants (Gast and Early, 1956). LeBlanc (1980) reported the 24-hour LC^0 for Daphnia magna to be 250 mg/S, (nominal concentration) and the 48-hour LCcn to be 220 mg/S, (nominal concentration). A no bo discernible effect concentration was estimated to be <68 mg/S,. Toxicity of EDC to barnacles (Barnacle nauplii) and unicellular algae has been reported (Pearson and McConnell, 1975). The LC,.q for Barnacle nauplii was reported to be 186 mg/S,. The effective concentration needed to reduce the photosynthetic ability of unicellular algae by 50$ was 340 ppm (340 mg/S,). A 24-hour median tolerance limit of 320 ppm (320 mg/S,) was reported for brine shrimp under static test conditions (Price and Conway, 1974). Static 24-hour and 96-hour LCc_ concentrations of >600 and 430 mg/S,, respectively, have been determined for the freshwater bluegill (Lepomis macrochirus) (Buccafusco et al., 1981). Garrett (1957a,b) reported that the concentration of EDC needed to produce >50$ mortality in the marine pinperch (Lagodon rhomeboides) under static conditions was 175 mg/S,. An acute LCD_U- of 115 mg/S, was reported for the marine flatfish (Limanda limanda) (Pearson and McConnell, 1975). In acute tests with another marine species, static 24-, 48-, 72- and 96-hour LC^^s in the range of 130-230 mg/S, 8-1 CMA 051633 were determined for sheepshead minnows C Cyprinodon variegatus) (Heitmuller et al., 1981); the observed no-effect concentration was reported to be 130 mg/Z. The estimated acceptable concentration of EDC in a 32-day early life stage toxicity test with freshwater fathead minnows (Pimephales promelas) lies in the range of 29-59 mg/Z (Benoit et al., 1982). 8-2 CMA 051634 9. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS 9.1. PHARMACOKINETICS 9.1.1. Absorption and Distribution. Ethylene diehloride (EDC; 1,2-dichloro~ ethane) is a colorless, oily liquid with a sweet taste and with a chloroform-like odor detectable over a range of 6-40 ppm. It is appreciably soluble in water (0.869 g/100 mil) (Von Oettingen, 1964), with a vapor pressure of 64 torr at room temperature (20C). Consequently, EDC is rapidly and extensively absorbed through the lungs in its vapor form and from the gastrointestinal tract in solution. Inhalation is considered the primary route of entrance into man from occupational exposure and air pollution. Absorption after oral ingestion is of particular interest for EDC as a contaminating component of drinking water and foodstuffs. Skin absorption is negligible in most industrial vapor exposure situations, although absorption may be significant by this route with direct liquid contact, as evidenced by toxic symptomatology in man (Section 9.2), and also quantitative animal measurements. 9.1.1.1. DERMAL ABSORPTION -- Tsuruta (1975, 1977) studied the percuta neous absorption of a series of chlorinated organic solvents (including EDC) applied to a standard area of shaved abdominal mouse skin for 15 minute periods. Absorption was quantified by the presence of the compound in total mouse body plus expired air, as determined by gas chromatography (GC). For all solvents, percutaneous absorption linearly increased with time and the rate was directly 2 related to water solubility. For EDC, the absorption rate was 479 nmoles/min/cm skin, second highest of 8 solvents measured. Tsuruta concluded that skin absorp tion from liquid contact could be a significant route for EDC entry into the body. 9-1 CMA 051635 Jakobson et al. (1983) also carried out dermal absorption studies with guinea pigs for 10 chlorinated organic solvents including EDC. Liquid contact (skin area, 3.1 cm ) was maintained for up to 12 hours and solvent concentration monitored in blood during and following dermal application. Dermal absorption, as reflected by blood concentration profile, was again observed to be related to the water solubility of the solvent. For solvents like EDC, which are relatively hydrophilic [300-900 mg/dl], the blood concentration increased steadily during the entire exposure. EDC reached 20 pg/mfl, blood in 12 hours. With extended dermal exposure, the blood concentration eventually became fatal. This pattern of steady blood accumulation indicates that dermal absorption occurs faster than body elimination by metabolism or pulmonary excretion. Following dermal expo sure, the EDC concentration in blood declined in a manner consistent with a twocompartment kinetic model, i.e., the sum of two exponential terms. 9.1.1.2. ORAL ABSORPTION -- In man, gastrointestinal absorption of EDC has not been specifically studied, although the absorption rate after oral ingestion has been determined in animal studies. As expected from the neutral and lipo philic properties of EDC (Tables 9-1 and 9-2), transmucosal diffusive passage occurs readily. There are numerous reports of poisoning in humans as a result of accidental or suicidal ingestion of EDC and the peroral LDp-u,, approximates 0.2-1 g/kg (NIOSH, 1976). In animals, extensive gastrointestinal absorption of EDC has also been clearly demonstrated by the biological effects produced by peroral administra tion of a wide range of dosages and dosing schedules in toxicity studies in rats, mice, guinea pigs and dogs (Section 9.2) and in metabolism studies in rats (Reitz et al., 1980, 1982; Spreafico et al., 1978, 1979, 1980). Reitz et al. (1980, 1982) found that C-EDC in corn oil given perorally to rats (150 rag/kg) was completely absorbed by virtue of a complete recovery of radioactivity in exhaled 9-2 CMA 051636 TABLE 9-1 Physical Properties of Ethylene Dichloride and Other Chloroethanesa Vapor Pressure at 25C, torr. 1,2-Dichloroethane 1,1-Dichloroethane 1,1,1-Trichloroethane 80 250 125 Ostwald Solubility Coeff.. 37C Water/ Air Blood/ Air Olive Oil/ Air 11.3 2.7 0.93 19.5 4.7 3.3 447 187 356 aSource: Sato and Nakajima, 1979 9-3 CMA 051637 TABLE 9-2 Partition Coefficients for Ethylene Dichloride MAN Blood/air, 37C Adipose tissue/blood, 25C 19.5 56.7 RAT, 250 ppm inhalation exposure3 Blood/air, 37C, 250 ppm 37C, 150 ppm Adipose tissue/blood Lung tissue/blood Liver tissue/blood 30.3 14.9 8.7b 0.45b 0.72b Sato and Nakajima, 1979 Bonitenko et al., 1977 Spreafico et al., Reitz et al., 1980 Reitz et al., 1980 Reitz et al., 1980 Reitz et al., 1980 aConversion factors: At 25C/760 torr, 1 ppm in air = 4.05 mg/m 3 1 mg/liter air = 247 ppm b Dose-dependent 9-4 CMA 051638 air, urine and carcass (Table 9-3)* Spre&fico et al. (19 78, 19 79 > 1980) found that 25, 50 and 1 50 rag/kg administered orally to rats in corn oil was rapidly absorbed with peak blood levels occurring within 20 minutes. Their data, given in Figure 9-1 , show the time-course of blood, liver, lung and adipose tissue following oral administration of the three dose levels. The peak blood levels, which, according to linear kinetics, should be proportional to the amount of EDO absorbed into the rat body, are summarized in Table 9-4. Although the peak blood levels in this study do not deviate drastically from linearity, the peak tissue levels are not linearly proportional to the three oral doses. These results strongly suggest a passive transport across the gastrointestinal tract of EDC and the occurrence of nonlinear elimination kinetics for EDC, i.e., a nonlinear dosedependency related to saturation of liver metabolism of EDC. An influence of the dose on kinetic parameters has also been found for halogenated ethylene compounds (e.g., vinyl chloride, vinylidene chloride) by other investigators (Gehring et al., 1976, 1977, 1978; Bolt, 19 78 ; McKenna et al., 1978; Reichert and Henschler, 1978; Filser and Bolt, 1979). Table 9-5 gives the absorption rate constants (k ) and areas under the blood concentration curves (AUC) found by Spreafico et al. (1978, 1979, 1980) for rats given a single oral dose of EDC. The rate constant for absorption was dosedependent and was probably influenced by liver metabolism. A markedly lower value was observed for the highest dose (1 50 mg/kg) at which saturation of liver metabolism probably occurs. Nonetheless, these data show that EDC is absorbed from the gastrointestinal tract rapidly, with one-half the dose absorbed within 3.3 minutes for the lowest dose given in corn oil (25 mg/kg) and 6.4 minutes for the highest dose in corn oil (150 mg/kg). As expected, absorption of EDC occurred significantly faster with an oral dose in water than in oil [k3 , 0.299 9-5 CMA 051639 TABLE 9-3 Fate of ^C-EDC in Rats 48 Hours After Oral (150 mg/kg) or Inhalation (150 ppm, 6-hr) Exposurea pmole/kg Oral % metabolites Inhalation pmole/kg % metabolites Body burden (total radioactivity) 1539 + 391 Charcoal trap (B) 447 + 60 Total metabolites (A-B) Urine C0_ trap Total carcass (48 hr after exposure) Feces Cage wash (1092) 926 + 348 83.1 + 11.9 46.9 + 14.7 26.3 + 14.7 12.5 + 6.37 - -- (100) 85.7 7.7 4.3 2.1 1.1 512 + 135 9.4 + 0.4 9.4 + 0.4 (503) 432 + 121 36.1 + 6.89 22.7 + 3-38 8.90 + 2.84 3.34 + 1.34 - (100) 84.4 7.0 4.4 1.7 0.7 Values are mean + S.D., with n = 4 for each route^of exposure and are pinole equiva lents of EDC, based on the specific activity of 1HC-EDC (3*2 mCi/mM). aSource: Reitz et al., 1980 9-6 CMA 051640 EDC. tiglmi or w>9 100 EDC. pgfml or pgfQ 10 1 0.1 100 EDC. pg/ml or jjgfg 10 1 0.1 ih 2h 3h 6h h Figure 9-1. EDC levels after single oral administration in rats: Top panel, 25 mg/ kg; middle, 50 mg/kg; bottom, 150 mg/kg dose. Adipose tissue ( ), blood ( o ), liver ( ), lung ( ). Source: Spreafico et al. (1978, 1979, 1980). 9- 1 CMA 051641 TABLE 9-4 Peak Blood and Tissue Levels After Single Oral Dosage of EDC in Male Rats3 lose lg/kg 25 50 150 Blood 13.29 31.94 66.78 Adipose tissue pg/ml or pg/g 110.67 148.92 259.88 Lung 2.92 7.20 8.31 ^Source: Spreafico et al., 1980 Liver 30.02 55.00 92.10 CMA 051642 9-8 TABLE 9-5 The Absorption Rate Constants (k ) and Area Under Curve (AUC) for Rats After Single OrL Doses of EDC in Oil and in Water as Vehicle and After Intravenous Administration3 Dose (mg/kg) ka min-^ AUC (blood) pg x min x ml~^ Oral 25 0.299 (water) 466 0.209 (oil) 466 50 0.185 (oil, male) 1700 0.181 (oil, female) 1685 150 0.109 (oil) 7297 Intravenous (water) 1 5 25 aSource: Spreafico, 1978, 1979, 1980 " 9 54 595 9-9 CMA 051643 Comparison of the area under the blood concentration curve (AUC) after intravenous administration with AUC after the same oral dose provides a measure of the extent or completeness of oral absorption. Table 9-5 shows that the AUC was 595 pg x min x mS,-1 after a 25 mg/kg intravenous dose and was 466 pg x min x _1 m!L after 25 mg/kg oral dose. These results indicate that absorption of the oral dose was very extensive, averaging J8% of complete absorption, or 100$ absorption if it is assumed that the first-pass effects in liver (-metabolism) and lung (elimination) after oral absorption decreased EDC appearance in systemic blood (Reitz, 1980, 1982). It is notable that the AUCs for differing intravenous doses, where intestinal absorption is not a factor (Table 9-5), are not linearly proportional, providing further evidence of the occurence of Michaelis-Menten elimination kinetics for EDC. Withey et al. (1982) investigated the effect of the dosing vehicle on intestinal absorption of EDC in fasting rats (400 g) following intragastric intubation of equivalent doses (100 mg/kg) in =4 mi of water or of corn oil. The post-absorptive peak blood concentration averaged 5 times higher for water vehicle than corn oil (84.6 vs 15.9 pg/m); moreover, the peak blood concentration was reached 3 times faster for water solution than for oil solution (3.2 vs. 10.6 minutes). The ratio of the areas under the blood concentration curves for 5 hours after dosing (AUC, 5 hr) was 3, water:corn oil. These results are in general agreement with those of Spreafico et al. (1978, 1979, 1980) (Tables 9-4 and 9-5), who found that intestinal absorption was faster with an oral dose in water than in oil. The observations of these investigators stress again the dependence of oral absorptive rate in rat3 not only on the dose, but also on the vehicle. While these factors are unlikely to affect the pharmacokinetics of EDC in man in any practical way, they are of importance to interpretation of data from long-term carcinogenicity tests of EDC in rodents 9-10 CMA 051644 where the modes of intragastric dosing employed may differ by both dose and vehicle. 9.1.1.3. PULMONARY ABSORPTION -- EDO has a moderately high vapor pressure (80 torr at 25C; Table 9-1) and, in man, a high blood/air partition coefficient compared to chloroethanes (19.5 at 27C; Table 9-2). Hence, its vapor in ambient air is a primary mode of exposure, and the lungs are a principal route of entry into the body. The total amount absorbed via the lungs (as for all vapors) can be expected theoretically to be directly proportional to: (1) the concentration of the inspired air; (2) the duration of exposure; (3) the blood/air Ostwald solu bility coefficient; (4) the solubility in the various body tissues; and (5) physical activity which increases pulmonary ventilation rate and cardiac output. Hence, the basic kinetic parameters of pulmonary absorption of EDC and its equilibrium in the body are as valid for the very low concentrations expected in urban ambient air as for the higher vapor concentrations found in the industrial environment and the workplace. However, these parameters of pulmonary absorp tion have not been studied in any detail in man, although some information is available from pharmacokinetic studies in rats. Urosova (1953) reported that women exposed during a normal day in the work place to =15.5 ppm EDC in air accumulated the chemical in breast milk and that initial concentrations in exhaled air following daily exposure were 14.5 ppm. These observations indicate that the women absorbed EDC through their lungs and reached blood and total body equilibrium with inspired air concentration within the daily work period. The deficiencies in the reporting of this work makes the results only suggestive. Spreafico et al. (1978, 1979, 1980) and Reitz et al. (1980, 1982) have studied the kinetics of pulmonary absorption of EDC in rats. Figure 9-2 shows the time-course of blood concentration of EDC, observed by Reitz et al. (1980, 9-11 CMA 051645 ooo 3 es a 01 TIME Imlnutiil TIME (minutes) Figure 9-2. Top: Blood levels of EDC observed during and following a 6-hour inhalation exposure to 150 ppm EDC. Data from four male Osborne-Mendel rats were fitted to a two-compartment open model as described by Gehring et al. The computer plot is shown. The arrow indicates exposure termination. Bottom: Semi-logarithmic plot of EDC blood levels vs. time after exposure termination. Source: Reitz et al. (1980, 1982). 9-1 d CMA 051646 1982) during a 6-hour inhalation exposure to 150 ppm EDC. Blood and body equilibrium with the blood concentration maintained a plateau level of 9 pg/mJl (blood/air partition coefficient, 14.9). Spreafico et al. (1980) exposed their rats to 50 and 250 ppm EDC for 6 hours. Their data are given in Tables 9-6 and 9-7. Blood and body equilibrium (liver, lung and adipose tissue) was established at *2 hours for 50 ppm exposure, and at 3 hours for 250 ppm exposure. During inhalation of EDC, when equilibrium is achieved, the arterial blood concentra tion of EDC should theoretically always be directly proportional to inspired air concentration. This fixed relationship is defined by the blood/air Ostwald solubility coefficient for EDC. As calculated from the data for 50 and 250 ppm exposures of Spreafico and co-workers, the blood/air partition coefficients for EDC are 6.3 and 30.3, respectively. Including the data of Reitz et al. (1980) for 150 ppm exposure in rats (Figure 9-2), these results do not demonstrate a direct proportional relationship between inspired air concentration and blood concentration of EDC, i.e., a constant blood/air partition coefficient value. Furthermore, linear kinetics for EDC after inhalation exposure cannot be presumed, and indeed these data suggest otherwise, and are in accord with the evidence of nonlinear kinetics after oral and Intravenous dosing as noted above (Table 9-5). 9.1.1.4. TISSUE DISTRIBUTION -- After pulmonary or peroral absorption, EDC is distributed into all body tissues. As expected from its general anesthetic properties in man and in animals, EDC readily passes the blood-brain barrier. The compound crosses the placental barrier and has been found in the fetus (Vozovaya, 1975, 1976, 1977). EDC also distributes into human colostrum and mature breast milk. Urusova (1953) found EDC concentrated in breast milk (5.4-6.4 mg/5,) in the workplace. The compound remained in breast milk for 18 hours following workday exposure, even though its concentration in exhaled air 9-13 CMA 051647 TABLE 9-6 EDC Tissue Levels After 50 ppm Inhalation Exposure Time of Exposure 30 minutes 1 hour 2 hours 4 hours 6 hours EDC pg/g or pg/ml + S.E. Blood Liver Lung 0.48+0.05 0.32+0.02 0.14+0.02 0.92 + 0.09 0.67 + 0.04 0.27 + 0.03 1.34+0.09 0.84+0.09 0.34+0.03 1.34+0.11 1.14+0.17 0.42+0.05 1.37+0.11 1.02+0.10 0.38+0.02 Adipose Tissue 2.91+0.22 7.49 + 0.60 10.31+0.94 11.08+0.77 10.19+1.00 Source: Spreafico et al., 1980 9-14 CMA 051648 TABLE 9-7 EDC Tissue Levels After 250 ppm Inhalation Exposurea Time of Exposure 30 minutes 1 hour 2 hours 3 hours 6 hours EDC pg/g or pg/ml + S.E. Blood Liver Lung Adipose Tissue 6.33+1.04 3.82+0.78 2.19+0.21 26.75+3.12 11.65+1.12 7.34+0.74 6.40+0.20 82.64+2.19 23.64+0.91 16.39+1.18 14.07+0.47 151.53+12.17 29.36+1.01 20.83+2.21 14.47+1.12 252.18+14.62 31.29+1.19 22.49+1.12 14.14+0.90 273.32+12,46 Source: Spreafico et al., 1980 9-' 5 CMA 051649 fell to 4 ppm. The lack of detailed reporting of this study precludes adequate evaluation. Sykes and Klein (1957) have demonstrated the presence of EDC in cows' milk after oral administration. There is little information on EDC distribution and concentration in the various body tissues of man after exposure, and few controlled exposure studies in animals investigating the distribution of EDC in body tissues and defining dose-dependent tissue concentrations. Spreafico et al. (1978, 1979, 1980) determined EDC levels in rat blood, liver, lung and epididymal adipose tissue after single oral administration of 25, 50 and 150 mg/kg. The time-course of EDC concentrations are shown in Figure 9-1- This figure shows that tissue accumula tion occurs most rapidly in the liver where peak concentrations were reached within 10 minutes of administration. Following complete absorption and body equilibration after 2 hours, the decay in tissue concentrations of liver, lung and adipose tissue parallels the first-order decline in blood concentration. Table 9-8 summarizes blood and tissue concentrations determined at 2 hours following administration. Blood levels and tissue levels in liver and adipose tissues were not found to be linearly proportional to dose, but increased exponentially, providing evidence of liver metabolism saturation and nonlinear kinetics after oral administration. At oral dose levels of 25, 50 and 150 mg/kg, adipose tissue displayed the greatest concentration of EDC with adipose tissue/blood partition coefficients of 17, 9 and 7, respectively. No other tissue, including liver, demonstrated a partition coefficient >1.0. The very low concentration levels of EDC in lung tissue may occur as a result of EDC elimina tion by this route. Table 9-8 also gives blood concentrations after chronic oral administration to rats (11 daily doses) of 50 mg EDC/kg. Comparison of these tissue levels with those following a single oral administration of 50 mg/kg EDC provides no evidence of blood or tissue accumulation with chronic administration. 9-16 CMA 051650 TABLE 9-8 Blood and Tissue Levels of EDC in Male Rats 2 Hours After Single Oral Doses in Corn Oila Tissue (pg/ml, or jig/g + SE) ___________________________Dose, mg/kg___________________ 25 50 150 Blood Liver Kidney Brain Spleen Lung Adipose 0.46 + 0.04 0.32 + 0.01 <0.05 7.80 + 0.72 5.89 + 0.41 3.81 + 0.45 3.43 + 0.26 2.96 + 0.36 1.79 + 0.35 1.44 + 0.10 55.78 + 6.88 26.60 + 1.23 21.62 + 3-99 1.62 + 0.25 178.50 + 24.66 Blood and Tissue Levels of EDC in Male Rats 2 Hours After the Last of 11 Daily Oral Doses in Oil (50 mg/kg) Blood Liver Lung Adipose 8.11 + 0.23 3.95 + 0.48 2.24 + 0.47 53.71 + 10.07 aSource: Spreafico et al., 1978, 1979, 1980 9-1 7 CMA 051651 Spreafico and his colleagues (1978, 1979, 1980) also investigated blood and tissue concentrations in rats after inhalation exposure to 50 and 250 ppm EDC. The results of these experiments are given in Tables 9-6 and 9-7. Blood and tissue equilibrium (liver, lung and adipose tissue) occurred at 2 hours and 3 hours, respectively, for the two inspired air concentrations. In general, the blood and tissue distribution of EDC at body equilibrium is similar to the results obtained with oral administration given in Table 9-8. A .very clear dose dependence in tissue levels of EDC was again found with the two inhalation concentrations, with differences in EDC concentrations on the order of 20-30 times when blood, liver, lung and adipose tissue are considered. The highest absolute levels of EDC were measured again in adipose tissue with concentrations that were between 8 and 9 times greater than those measured in the blood. As previously noted for oral administration (Table 9-8), liver and lung concentra tions were less than blood concentrations. A comparison of the absolute blood and tissue concentrations at body equili brium for oral and inhalation modes of administration (Tables 9-6, 9-7 and 9-8) indicates that 50 ppm inhalation roughly equates to 25 mg/kg oral, while 250 ppm inhalation provides blood, liver and adipose tissue levels slightly greater than a 150 mg/kg oral dose. 9.1.2. Excretion. Elimination of EDC from the body is perforce the sum of metabolism and excretion of unchanged EDC via pulmonary and other routes. Unmetabolized EDC is excreted almost exclusively through the lungs; however, metabolism of EDC is extensive, with the proportion excreted unchanged dependent on body doses. While no controlled experimental studies have been made on the kinetics of excretion of EDC in man, recent studies have been performed on experimental animals. 9-18 CMA 051652 9.1.2.1. PULMONARY EXCRETION -- Urusova (1953) reported that women exposed to =15.5 ppm EDC in ambient air of industrial environs demonstrated initial EDC concentrations in exhaled air of 14.5 ppm. The breath concentration declined to =3 ppm after 18 hours. These values lead to an approximation of 9 hours for the half-time of pulmonary elimination of EDC in man. Although this human data was difficult to evaluate, similar observations have been made for animals (monkey, dog, cat, rabbit, rat and guinea pig) in early investigations-of the anesthetic properties and toxicities of EDC (Heppel et al., 1945; Kistler and Luckhardt, 1929; Lehman and Schmidt-Kehl, 1936). In controlled studies in mice, Yllner (1971a) found that up to 45$ of an intraperitoneal injected dose of EDC (170 mg/kg) was recoverable unchanged in exhaled air (Table 9-9). The percentage of EDC recovered unchanged in exhaled air increased exponentially with the dose, indicating a limited capacity of biotransformation, i.e., non-linear kinetics. Thus, in mice, pulmonary excretion of EDC is a major route of elimination, increasing in importance with higher body doses. Similar observations have been made for the rat by Reitz et al. (1980, 14 1982). In a balance study utilizing C-EDC given orally (150 mg/kg) and by inhalation (150 ppm, 6 hours), these investigators found that for the oral dose, 29$ was recovered unchanged in exhaled air, and that only 1.8$ of the lower inhalation dose was excreted by the pulmonary route (see Table 9-3). Sopikov and Gorshunova (1979) observed that after an intraperitoneal 250 mg/kg dose to rats, 30$ of the EDC dose was eliminated in the exhaled air within 5 hours. 9.1.2.2. OTHER ROUTES OF EXCRETION -- EDC is not ordinarily eliminated in significant amounts from the body by any route other than pulmonary. However, Shchepotin and Bondarenko (1978) identified EDC by gas chromatography (GC) methods in the urine of persons with severe symptoms of EDC poisoning. Studies of chlorinated compounds in the urine after EDC inhalation exposure or peroral 9-19 CMA 051653 TABLE 9-9 Percent Distribution of Radioactivity Excreted (48-hr) by Mice Receiving 1,2-Dichloroethane-1HCa (exhaled air) Dichloroethane (exhaled air) Urinary metabolites 0.05 13 11 73 aSource: Yllner, 1971a Dose g/kg 0.10 0.14 84 21 46 70 48 0.17 5 45 50 9-20 CMA 051654 dosage to experimental animals have failed to detect unchanged EDC (Spreafico et al., 1980; Yllner, 1971a). Mutagenic metabolites of EDC have been reported in rat and mouse bile (Rannug and Beije, 1979; Rannug, 1980a), but EDC itself has not been found. Diffusion of volatile haloalkane anesthetic agents into bowel space and through skin is known to occur (Stoelting and Eger, 1969), and these routes of excretion may also be significant for EDC because of similarity of structure and comparable high lipid/blood partition coefficients. 9.1.2.3. KINETICS OF EXCRETION -- While the kinetic parameters of elimina tion of EDC for each of the various routes of excretion, i.e., pulmonary, metabo lism, etc., are not well defined for either man or experimental animals, several investigative groups have determined the kinetic parameters for whole-body and tissue compartment excretion of EDC in the rat (Retiz et al., 1980; Spreafico et al., 1978, 1979, 1980; Withey and Collins, 1980). Withey and Collins (1980) determined the kinetics of distribution and elimination of EDC from blood of Wistar rats after intravenous administration of 3, 6, 9, 12 or 15 mg/kg of EDC given in 1 raS, water intrajugularly. For the two lower doses (3 and 6 mg/kg), the blood decay curves exhibited two components of exponential disappearance and best fitted a first-order two compartment model with kinetic parameters of: ke> 0.24 min ; Vd, 43 m.l; k12 and k21, 0.02 and 0.04 min \ respectively. For higher doses (9, 10 and 15 mg/kg), these investigators found their data best fitted a first-order, three-compartment model; they suggest that the shift from two to three-compartment kinetics may have occurred either because of a dose-related alteration in the kinetic mechanisms of uptake, distribution, metabolism and elimination, or that three-compartment kinetics were followed at all dose levels but with lower doses; the third exponential component, representing the third compartment, was obscured by the limit of analytical sensitivity. Average kinetic values for the three-compartment model 9-21 CMA 051655 were: kg, 0.094 min-1; Vd, 67.9 mJl; k12, k21, k13, k31; 0.06, 0.08, 0.01 and 0.01 min-1, respectively. Withey and Collins state that they found no evidence in their kinetic analysis of the disposition of intravenous bolus injections of EDC into the rat of nonlinear or dose-dependent Michaelis-Menten kinetics. These workers suggested that a dose of 15 mg/kg EDC in the rat is below hepatic metabolism saturation. Spreafico et al. (1978, 1979, 1980) intravenously administered EDC in water to Sprague-Dawley rats as bolus injections of 1, 5 and 25 mg/kg and determined the whole blood concentration of EDC as it decayed with time. Figure 9-3 shows the semilog plot of the results of these experiments. A biphasic decline of EDC blood concentration was evident for all doses, indicative of a two-compartment system, with a distributive phase (a) and an excretion phase (B)* The data for intravenous treatment were analyzed by computer fitting to a two-compartment open model with the results shown in Table 9-10. Of immediate note is the fact that the kinetic parameters are not independent of the dose as required by linear kinetics. Thus the half-time (T.^) bodY elimination from central or blood compartment increases with the dose, while whole-body clearance (Cl) and the volume of distribution (Vd.) decrease with an increase of the dose. These results indicate that the rate of whole-body excretion of EDC is largely determined by the metabolism of EDC, a saturable process and a major route of elimination. Spreafico and his co-workers (1978, 1979, 1980) have also investigated the whole-body kinetics of oral administration of EDC in rats. Figure 9-1 shows semilog plots of blood and tissue concentrations during and after absorption (excretory phase) following single oral doses of 25, 50 and 150 mg/kg given in corn oil. In general, the slopes of the disappearance curves (excretion compo nent) for adipose, liver and lung tissues roughly parallel and reflect the blood decay curve. The data for blood were computer-fitted to the kinetic equations 9-22 CMA 051656 Figure 9-3. Blood levels of EDC in rats after i.v. administration. ( ) 25 mg/kg i.v.; (O ) 5mg/kg i.v.; (A) 1 mg/kg i.v. Source: Spreafico et al. (1978, 1979, 1980). 9-23 051657 TABLE 9-10 Pharmacokinetic Parameters of EDC Administered as Single Bolus Intravenous Injections in Saline to Sprague-Dawley Male Ratg. Parameters Calculated from a Two-Compartment Open Model. Parameter 1 CQ, |ig x ml"1 Kel, min-1 K12, min-1 Kg1, min 1 AUC, pg x min x ml*1 T 1/2 8 min Clearance ml x m" Vol. distr., ml 1.50 0.277 0.121 0.158 9 7.30 21.98 231 Dose, mg/kg 5 25 8.00 0.142 0.063 0.140 54 38.12 0.078 0.040 0.118 595 9.49 17.46 14.07 7.98 239 162 ^el (inhalation) or Vm/ (Km + C) (oral) aSource: Spreafico et al., 1979, 1980 9-24 CMA 051658 for a two-compartment open model for oral absorption; the results are given in Table 9-11. Like the kinetics observed for intravenous dosing, the values for the parameters, T1/2, Cl and Vd, are dose dependent. Furthermore, the oral dosing vehicle also affects the parameters. Comparison of the absorption rate constants, Ka, half-times of elimination, T.^ and volumes of distribution, Vd, for 15 mg/kg EDC in oil and 25 mg/kg EDC in water indicates a faster absorption of EDC in water, a shorter half-time of body elimination and a small volume of distribution. Nonetheless, the AUCs indicative of the total absorbed dose were similar for oil and water vehicles, as were the body clearance rates of EDC. On the other hand, no significant differences in the kinetic parameters were seen for male and female rats given the same dose, nor were significant differences observed between a single dose and 11 daily administrations of the same dose (50 mg/kg) of EDC. Spreafico et al. (1980) included in their comprehensive studies on the pharmacokinetics of EDC in the rat, experiments to determine the kinetics of EDC after inhalation dosing. Rats were exposed to 50 and 250 ppm EDC for 5 hours, a period sufficiently long to establish and maintain whole body steady-state conditions with these inspired air concentrations (Tables 9-6 and 9-7). The exposure was terminated, and blood and other tissues were sampled to determine the decay of EDC concentrations in these tissues with time. The results of these experiments are presented as semilog plots of tissue concentrations versus time in Figure 9-4. As for oral dosing, the slopes of the tissue decay curves parallel and reflect generally the blood concentration curves. In contrast to intravenous and oral dosing, which have several components to their curves representing absorption and/or distribution, the curves after steady-state inhalation are monophasic, representing only excretion. Thus, the pharmacokinetic parameters calculated from the blood decay curves following the 9-25 CMA 051659 TABLE 9-11 Pharmacokinetic Parameters of EDC Administered as Single Oral Doses in Corn Oil and Water to Sprague-Dawley Male Rats. Parameters Calculated from a 2-Compartment Open Model. Parameter 25 Oil Vehicle Kfl, min-1 K ., min-1 el AUC, pg x min x ml ^ T 1/2 6 min Clearance, ml x min"^ Vol. Distr., ml. 0.209 0.029 446 24.62 10.64 367 Dose, mg/kg 50 0.185 0.017 1700 44.07 5.58 328 150 0.109 0.010 7297 56.70 3-90 390 Aqueous Vehicle K , min-^ Kel' min_1 AUC, pg x min x ml T 1/2 B min -1 Clearance, ml x min Vol. Distr., ml. 0.299 0.046 446 14.12 10.20 221 aSource: Spreafico et al., 1979, 1980 9-26 CMA 051660 mm EDC. pg/ml or pg/g EDC. tig/ml or i*q/g Figure 9-4. Levels of EDC in rats after inhalation exposure to 50 ppm (top) and 250 ppm (bottom). Levels were mea sured at termination of 5-hour exposure period. Adipose tissue ( ), blood ( o ), liver ( ), lung ( a ). Source: Spreafico et al. (1978, 1979, 1980). 9-27 CMA 051661 i ,'c dy-state inhalation conditions are essentially those applicable to a single c^arartment open model. The kinetic parameters are given in Table 9-12. The hcdy burden or "dose" of EDC in the animals at termination of inhalation exposure is not known; however, a comparison of the areas under the blood decay curves, a reflection of body burden, suggests that the body burden after 250 ppm exposure was 40 times, not the 5 times expected of the 50 ppm exposure. Similarly, the half-life of EDC and the zero-time blood concentrations are dose-dependent. These results from inhalation exposure are in accord with the dose dependent pharmacokinetics exhibited after intravenous and oral dosing of EDC. They strongly indicate that the kinetics of absorption, distribution and elimination of EDC are appropriately described by nonlinear kinetics that take into account saturable processes, presumably in this case the metabolism of EDC by the organism. The pharmacokinetics of EDC after inhalation have also been explored by Reitz et al. (1980, 1982) in male Osborne-Mendel rats. These investigators determined the whole-blood levels of EDC during and following a 6-hour Inhalation exposure to 150 ppm EDC. Figure 9-2 shows the data obtained from 4 rats, computer-fitted to the equations of a two-compartment model and the idealized computer-fit plotted. Within 2 hours of exposure, the animals approached a steady-state blood concentration (9 pg/m$,) with the inspired air concentration; but, following termination of exposure at 6 hours, the blood concentration of EDC rapidly decayed to near zero within 10 hours. Figure 9-2 shows also the semilog plot of computer-fitted data of EDC blood concentration levels vs. time after exposure termination. In contrast to the observation of Spreafico et al. (Figure 9-4) that only a monophasic plot was obtained following inhalation exposure, Reitz et al. found a biphasic elimination with an initial rapid alpha phase and a slower beta phase. Table 9-13 gives the pharmacokinetic parameters calculated from a two-compartment open model. These results may be compared with data obtained by Spreafico et al. for inhalation exposure (Table 9-12). 9-28 CMA 051662 TABLE 9-12 Pharmacokinetic Parameters of EDC Following Termination of Steady State Inhalation Conditions (5-hr Exposure) of 50 and 250 ppm to Male Sprague-Dawley Rats. Parameter -1 C , ug x ml Ft^, min ** T 1/2, min AUC, pg x min x ml-"1 Exposure Concentration, ppm 50 250 1.42 0.0561 12.69 26 30.92 0.0313 22.13 1023 aSource: Spreafico et al., 1979, 1980 9-29 CMA 051663 TABLE 9-13 Pharmacokinetic Parameters of EDC Following Termination of Steady State Inhalation Conditions; 6-Hr Exposure, 150 ppm to Male Osborne-Mendel Rats. Parameters Calculated from a Two-Compartment Model.3 Parameter Cq, jig x ml"1 Ke,l, min-1 K12, min"1 K21, min-1 AUC, fig x min x ml-1 T1/28 min Vol. Distr, ml. aSource: Reitz et al., 1980 9.0 0.092 0.019 0.025 3018 35 513 9-30 CMA 051664 Reitz et al. (1982) also determined in the rat the pharmacokinetics after single oral dosing (150 mg/kg in corn oil). In this case, a semilog plot of blood EDC levels after absorption produced an elimination curve typical of nonlinear kinetics. These workers therefore computer-fitted the data to a two-compartment nonlinear model and calculated the rate of elimination from the central compart ment (blood) according to Michaelis-Menten kinetics. Table 9-13 gives the pharmacokinetic parameters calculated from the model. Several differences exist between the observed data and calculated parameters of Reitz et al. for inhalation vs. oral exposure (Table 9-13) as well as those of Spreafico et al. (Table 9-12). The EDC inhalation concentration used by Reitz et al. (150 ppm) was intermediate to those of Spreafico et al. (50 and 250 ppm). Plateau blood concentrations in the three inhalation studies (1.4 at 50; 8.3 at 150; and 30.9 pg/mi, at 250 ppm), clearly demonstrate saturation of an elimination process, presumably metabolism. Reitz et al. found peak blood levels following oral dosing (30-44 pg/m& at 150 mg/kg) to be similar to those found by Spreafico et al. after a 250 ppm inhalation exposure. Reitz et al. also found a biphasic elimination after both oral and inhalation exposure with the second or 8 phase essentially equal (T^^ = 28 min), and similar to the monophasic elimination calculated by Spreafico et al. for the 250 ppm exposure (T^2 = ^2 min). The relatively slow alpha elimination phase (T^g, 90 min) following oral dosing of 150 mg/kg suggested to Reitz et al. a saturable elimination process. The data of Spreafico and co-workers are generally in agreement with this treatment; however, several kinetic parameters are not in good agreement between the studies. Of particular note are the biphasic and monophasic elimination curves and the large discrepancy of AUC. The explanation for these differences may lie in the different kinetic models, species differences and inability of linear kinetics to accurately describe the kinetics of EDC body elimination. 9-31 CMA 051665 9,1.2.4. BIOACCUMULATION -- There is no definitive experimental evidence in the literature concerning bioaccumulation in man after chronic or repeated mily exposure to EDC. While EDC has a moderately high fat tissue/blood parti,,.on coefficient (Table 9-2), its half-time of elimination in man appears to be =6-8 hours (Urusova, 1953)- In the rat, Spreafico et al. (1978, 1979, 1980) and Reitz et al. (1980, 1982) have provided estimates of half-time of whole-body elimination of 25-57 minutes for acute oral dosing, and 13-35 minutes after 5-6 hour inhalation exposures. The half-time of elimination of EDC from the epididymal adipose tissue was essentially the same as whole-body elimination. These relatively short half-times of elimination indicate that the risk of significant bioaccumulation of EDC is small. Based on the pharmacokinetic data from rats, these investigators predict essentially complete elimination of EDC from the body in 24 hours following acute oral exposure or five 6-hour exposure periods via inhalation. Furthermore, Spreafico et al. (1979, 1980) found no significant differences in the kinetic parameters of EDC elimination between a single dose and multiple daily oral administration of EDC. 9.1.3. Metabolism 9.1.3.1. KNOWN METABOLITES -- Metabolism of EDC in man during or after exposure has not been studied. However, one of the earliest suggestive reports of an active mammalian biotransformation of EDC is related to human exposure. In 1945, Bryzkin reported that EDC underwent rapid transformation to an "organic chloride" in patients who died after ingesting 150-200 m2.; EDC itself was not detectable in tissues at autopsy. Although Heppel and Porterfield demonstrated as early as 1948 that both ethylene dichloride and dibromide were dehalogenated by rat liver enzyme preparations, current knowledge of mammalian metabolism of EDC derives primarily from studies performed within the last 10 years. Metabo lites that have been identified either in vivo in mice and rats or in liver and 9-32 051666 kidney tissue crude enzyme systems are listed in Table 9-14. In addition to the very active metabolite, 2-haloacetaldehyde, other possible reactive interme diates formed during the metabolism of 1,2-dihaloethylenes by glutathione-depen dent reactions, such as an episulfonium ion, may be involved in the covalent binding to tissue macromolecules leading to tissue damage (Rannug and Beije, 1979; Hill et al., 1978; McCann et al., 1975; Guengerich et al., 1980; Anders and Livesey, 1980). 9.1.3.2. MAGNITUDE OF EDC METABOLISM -- The capacity of mammalian organisms to metabolize EDC has been studied only in the mouse and rat. In these two species, the extent of biotransformation of single doses of EDC has been estimated by balance studies utilizing 14 C-EDC. Yllner (1971a) administered intraperitoneally doses of 50-170 mg/kg of 14 C-EDC to mice in metabolism cages and collected the expired CO2 and volatile organic metabolites using the appropriate solvent traps. Yllner recovered 97-100$ of radioactivity in exhaled air and urine within 24 hours. The results of his balance studies are summarized in Table 9-9. For the low dose of 50 mg/kg, Yllner found that EDC was metabolized to CO^ (13$) and water and to other metabolites appearing in the urine (73$)- Unchanged EDC did not appear in the urine, but 11$ of the dose was eliminated unchanged in exhaled air. An increase of the intraperitoneal dose resulted in a smaller percentage of the d .se being metabolized. For the largest dose, 170 mg/kg, 55$ was metabolized to CO2 (5$) and urine metabolites (50$). These results indicate that extensive biotransfor mation (55-86$) occurs in the mouse. Furthermore, the extent of metabolism is dose dependent, suggesting a limited capacity for biotransformation with satura tion of the metabolizing system(s). Saturation of metabolism to CO^ and urinary metabolites occurred at a dose level of between 100-140 mg/kg and 50-100 mg/kg, respectively. 9-33 CMA 051667 TABLE 9-14 Identified Metabolites of Ethylene Dichloride and Ethylene Bromide. Metabolites System Inorganic halide co2 2-Chloroethanol rat liver cytosol mouse, rat exhaled air mouse, rat urine, blood 2-Chloroacetic acid mouse urine Bromoacetaldehyde rat liver microsomes Thiodiglycolic acid mouse, rat urine Ethylene rat liver cytosol S-(2-hydroxyethyl)-cysteine rat liver cytosol, rat urine N-acety1-S-(2-hydroxyethyl)cysteine N-acetyl-S-(2-hydroxyethyl)oxide rat urine blood S-carboxymethyl cysteine mouse urine S-(2-hydroxyethyl)-glutathione S-(2-hydroxyethyl)-S-oxide rat liver cytosol, tissue S, S'-ethylene-bis-glutathione rat liver Reference Heppel and Porterfield, 1948 Nachtomi, 1970 Yllner, 1971b Reitz et al., 1980 Yllner, 1971b Kokarovtseva and Kiseleva, 1978 Yllner, 1971b Hill et al., 1978 Yllner, 1971b Spreafico et al., 1979 Livesey and Anders, 1979 Nachtomi et al., 1966 Edwards et al., 1970 Edwards et al., 1970 Yllner, 1971b Nachtomi, 1970 Nachtomi, 1970 9-34 CMA 051668 Reitz et al. (1980, 1982) have found that EDC is also extensively metabo14 lized in the rat. These investigators conducted balance studies with C-EDC in Osborne-Mendel rats after oral (150 mg/kg) and inhalation (150 ppm for 6 hours) exposure. Their results are summarized in Table 9-3. In the oral balance study, 150 mg/kg of ^C-EDC (or, 1520 pmoles/kg) were administered. The sum of radio activity recovered in exhaled air, urine and feces, and that remaining in the carcass was 101$, or complete recovery. Some 29$ of the dose was excreted unchanged in exhaled air; 5$ was metabolized completely to 14CO^ and 60$ of the dose was metabolized to 14 C-metabolites appearing in the urine. Less than 3$ radioactivity remained in the body 48 hours after dosing (probably as covalently bound metabolites). These results indicate that the extent of total metabolism of the oral dose was =70$ in these rats. For the inhalation exposure study, the absolute dose administered by a 150 ppm, 6-hr exposure was unknown. From the recovery after termination of exposure of ^C-radioactivity in exhaled air, urine and feces, and radioactivity remaining in the carcass 48 hours after exposure, it was estimated that the inhalation dose was 50.5 mg/kg (512 pmoles/kg) or one-third of the oral dose. Of this dose, 2$ was excreted unchanged in exhaled air, 7$ was metabolized completely to 14 COg, and 84$ was metabolized to 14 C-metabolites appearing in the urine. Less than 5$ of the radioactivity was left in the body 48 hours after inhalation exposure. Ignoring the pharmacokinetic differences between acute oral and inhalation dosing, the results of Reitz et al. (1980) demonstrate extensive biotransformation in the rat (70-91$) and also suggest a dose dependency with greater metabolism for lower doses, as Yllner (1971a) observed in the mouse. The balance studies of both Yllner (oral dosing) and Reitz et al. (oral and inhalation dosing) demonstrate that the primary route of elimination of EDC is by metabolism. Renal excretion of nonvolatile metabolites predominates; however, 9-35 CMA 051669 5-10$ is metabolized completely to C02 and water. It is of interest to note that a small amount of EDC or its metabolites appear in the feces (<2$), even from inhalation exposure. Yllner (1971a) identified the metabolites appearing in the urine of mice after dosing with 11(C-EDC (170 mg/kg) and found six ^C-metabo- lites; the three principal metabolites were S-carboxymethylcysteine (45$), thiodiacetic acid (33$) and chloroacetic acid (15$) (Table 9-15). However, Spreafico et al. (1979) dosed rats with 50 and 150 mg/kg 14 C-EDC and found the principal metabolite to be thiodiacetic acid; chloroacetic acid was not detected and S-carboxymethylcysteine was found only in trace amounts. 9.1.3.3* PATHWAYS OF METABOLISM -- A number of studies have been carried out describing the metabolism of 1,2-dihaloalkanes. Two principal pathways, involving microsomal and cystosolic enzymes, respectively, have been proposed. These pathways account for the formation of the metabolites identified in both in vivo and in vitro studies listed in Table 9-14 and also suggest the nature of the reactive intermediates involved in the covalent binding to cellular macromolecules (protein and DNA) which has been demonstrated in both in vivo and in vitro studies (Section 9.1.3.5). 9.1.3.3.1. Microsomal Reactions -- Yllner (1971a) originally proposed that the degradation of 1,2-dichloroethane to 2-chloroacetic acid involved a primary reaction in which chlorine was removed from one of the carbon atoms (hydrolytic dehalogenation) to yield 2-chloroethanol. As evidence for this reaction, he found chloroethanol to be a minor metabolite in the urine of mice injected with EDC (Table 9-15). Following 2-chloroethanol formation, Yllner proposed that alcohol was enzymatically converted to 2-chloroacetic acid (a major urinary metabolite) via 2-chloroacetaldehyde. Kokarovtseva and Kiseleva (1978) have also identified chloroethanol in the blood of rats (T.^ ca. 9 hours), and in rat liver tissue within 1 hour and for 24-48 hours after oral 9-36 CMA 051670 TABLE 9-15 Percent Distribution of Radioactivity Excreted (48-hr) as Urinary Metabolites by Mice Receiving 1 ^-Dichloroethane-^C or 2-Chloroacetic Acid- 4C.a Metabolite Chloroacetic acid 2-chloroethanol S-carboxymethylcysteine Conjugated S-carboxymethyl cysteine Thiodiacetic acid S,S-ethylene-bis-cysteine Glycolic acid Oxalic acid aSource: Yllner, 1971a,b After dichloroethane (0.17 g/kg) 16 0.3 45 3 33 0.9 -- -- After chloroacetate (0.10 g/kg) 13 -- 39 3 37 -- 4 0.2 9-37 CMA 051671 administration of EDC (750 mg/kg). Van Dyke and Wineman (1971), and subsequently Salmon et al. (1978, 1981), found that rat microsomal fractions, in the presence of Og and NADPH, dechlorinated a series of haloalkanes including EDC. The reaction for EDC had a Vmax of 0.24 nmol/min/mg protein with a Km of 0.14 mM (Salmon et al., 1981>. Thus, EDC and other haloalkanes interact with cytochrome P-450 to give a Type I difference spectra associated with the metabolism of these substrates by direct C-hydroxylation (Ivanetick et al., 1978). Besides EDC, Ivanetick et al. (1978) observed that chloroacetaldehyde and chloroethanol also interacted with cytochrome P-450 with Ks values of 10.3, 30 and =15, respectively. Hill et al. (1978) reported that 1,2-dibromoethane was activated to an irreversibly bound species by microsomal mixed-function oxidases, and also provided evidence that 2-bromo-acetaldehyde was formed in such reactions. More recently, Guengerich et al. (1980) showed that 2-chloroethanol was a product of rat microsomal mixed-function oxidation of EDC, requiring 0^ and NADPH. The reaction was blocked by classic P450 inhibitors and increased by pre-treatment with phenobarbital. Guengerich et al. (1980) proposed that part of the microsomal mixed-function oxidative metabolism of EDC proceeded from oxygen insertion into a C-H bond to form an unstable chlorohydrin which spontaneously dehydrohalogenates to form 2-chloroacetaldehyde. Figure 9-5 illustrates the proposed reaction and pathway. Chloroacetaldehyde is then reduced to chloroethanol by alcohol dehydrogenase in the presence of NADH, or oxidized to 2- chloroacetic acid by aldehyde dehydrogenase. Johnson (1967) previously had observed that chloroethanol was readily dehydrogenated to chloroacetaldehyde by alcohol dehydrogenases from yea3t or horse liver. The reaction, therefore, proceeds in either direction depending on enzyme, substrate and cofactor concentrations. Williams (1959) also had suggested that chloraeetic acid appeared in vivo via chloroacetaldehyde. 9-38 Obi672 S' P .t-c-d-x---4-5--0-0-- rx-1c*-c-H-x1 NADPH L HJ -HX BINDING IH PROTEIN. DNA-2 ^|o,c,t- aldahyda * Aid. D H, H. 2-haloathanol 2-haloaeatic acid 1* *} P450.0, T *?* *?* + "1 x-c-c-ci nadph"*[x-C-c-ci-oJ H. Cl-C--C-OH 2 haloacttlc acid Figure 9-5 Postulated microsomal oxidative metabolism of 1,2-dihaloethanes. Source: Adapted from Guengerich et al. (1980) and Anders and Livesey (1980). 9-39 CMA 051673 Further evidence for this reaction sequence has been provided by Guengerich et al. (1980). These workers observed that covalent binding to microsomal protein and DNA was inhibited 30-40$ by inclusion of alcohol or aldehyde dehydro genases in the reaction mixture, strongly indicating that chloroacetaldehyde was the reactive intermediate responsible for this portion of the total irreversible binding of EDC. Chloroethanol itself, when added to the microsomal system, gave only a low level of irreversible binding. In order to account for the remainder of the microsomal mixed-function oxidative irreversible binding of EDC, Guengerich et al. (1980) proposed the oxidative formation of the reactive meta bolite 1-chloroso-2-chloroethane, which spontaneously rearranges to 1-chloro acetaldehyde via a hypochlorite. The reaction is illustrated in Figure 9-5. These workers noted that a reactive chloroso compound could react directly with macromolecules, or hydrolyze (with release of hypochlorite ion) to form 2-chloroethanol. As a consequence of microsomal P-450-mediated oxidation of EDC as illustrated in Figure 9-5, further metabolism of the oxidative metabolites, 2-chloroacetaldehyde and 1-chloroso-2-chloroethane, leads either to a "detoxification" or to a further formation of reactive intermediates, respectively, as illustrated in Figure 9-6. Regarding detoxification reactions of chloroacetaldehyde with glutathione, Johnson (1955, 1966a,b, 1967) found that chloroethanol, administered orally to the rat, caused rapid depletion of liver glutathione (GSH) with a concomitant formation of S-carboxymethylglutathione. In vitro, the reaction with a rat liver cytosol fraction required stoichiometric amounts of GSH (1 mole) and NAD (2 moles). Since pyruvate was also required for reaction, Johnson (1967) postulated that chloroethanol was converted by alcohol dehydrogenase to chloroacetaldehyde, which then conjugated with GSH to give 9-40 CMA 051674 ci-4'-c* *!4i. x-c-ef tiH OH 2<hioroecetaldehyde 2-chloroaeetate 1 -chloroao-2-chloroethane GSS-formylmethylglutethione OH Ij0 GS-C-C' 'OH S-carboxymethyl glutathione glutathionaaa (liver, kidneyl IjiHj * HOOC--CH--CH*--S--CH,--COOH S-carboxymethyl cyateine i COOH S CH*--COOH Thiodiglycollc acid S-12 chloroethyl) glutathione HOH -Cl~ 05`<1 B" I-ND IN--G PROTEIN. ONA epiaulfonium Ion GSH GS--CH,--CH*--SG S.S'-ethylene-bit- glutathione HOH GS-CH.-CH.-OH S42-hydroxyethyl) glutathione GSH, glutathione; Aid. 0. aldehyde dehydrogenate; GT, GSH t/anferaae; OH. dehydrogenate Figure 9-6. Further metabolism of 2-chloroacetaldehyde and 1-chloroso-2-chloro* ethane from microsomal oxidation. Source: Adapted from Guengerich et al. (1980) and Anders and Uvesey (1980). 9-41 CMA 051675 S-formylmethylglutathione, and thence by an NAD requiring dehydrogenation to S- carboxymethylglutathione. Johnson (1966b) reported that ohloroacetaldehyde also rapidly conjugates with GSH in vitro by a non-enzymatic reaction at pH 7.0. He also has shown that S-carboxymethylglutathione is rapidly degraded by rat kidney homogenate to yield glycine, glutamic acid and S-carboxymethylcysteine, part of which is further metabolized to thiodiglycolic acid. Yllner (1971a) found that these latter two metabolites were the two major urinary compounds after administering EDC to mice (Table 9-15). Since these two compounds were also the major urinary metabolites after administering 2-chloroacetate to mice (Table 9-15), Yllner (1971b) proposed that 2-chloroacetic acid could also conjugate with GSH with chloride excision forming S-carboxymethylcysteine and thereby enter the pathway (Figure 9-6). Spreafico et al. (1979) found that after rats were given oral doses of 50 and 150 mg/kg EDC, 2-chloroacetic acid did not appear in the urine, but thiodiglycolic acid did appear as the major urinary product. The difference between the urinary metabolites found in mice by Yllner and those observed in rats by Spreafico et al. may be explained by the availability of GSH in the livers of the animals in the two studies. Reactions of putative 1-chloroso-2-chloroethane: Guengerich et al. (1980) noted that the chloroso compound that they proposed was formed by microsomal P450 oxidative metabolism of EDC, would be extremely reactive and could be expected to either rearrange to 2-chloroacetaldehyde, hydrolyze to 2-chloroethanol, or react directly with microsomal protein (Figure 9-5). In addition, these workers postulated that the chloroso compound may react with GSH to form S-(2- chloroethyl)glutathione, a half sulfur mustard, which could react via an episulfonium ion intermediate with macromolecules (Figure 9-6). However, in an Ames test with S. typhimurium TA1535, EDC activated with rat liver microsomes,. NADPH, and added GSH, did not enhance mutagenic activity. In contrast, the 9-42 CMA 051676 use of 100,000 g liver supernatant containing GSH and GSH transferases markedly increased the number of revertants per 1,2-dichloroethane plate. These results suggested that either the putative chloroso compound is not formed in the micro somal system, or that differences in the subcellular systems contribute to differences in covalent binding and mutagenicity. 9.1.3.3.2. Cytosolic reactions -- Heppel and Porterfield (1948) obtained an enzyme preparation from rat liver capable of hydrolyzing the carbon- halogen bonds of chloro derivatives of methane and ethane, including EDC. Bray et al. (1952) also studied the dehalogenation of dichloroethane and other halogenated hydrocarbons by rabbit liver extracts and nonenzymatic dechlorination by direct interaction with sulfhydryl groups of GSH and cysteine. Nachtomi et al. (1966, 1970), found that an enzyme system from the soluble supernatant fraction of rat liver catalyzed a reaction between EDC and GSH. The formation of inorganic halide in these studies could occur as a consequence of attack by GSH resulting in the excision of halide, and the formation of S-(2- haloethyl)-GSH (Figure 9-7)* This metabolite is a reactive intermediate, a half sulfur mustard, which, via its episulfonium ion and further reaction with a second GSH or with water, may be expected to form S,S-ethylene-bis-GSH or S-(2hydroxyethyl)-GSH. The S,S'-ethylene-bis-GSH may be presumed to be subject to degradation to S,S-ethylene-bis-cysteine by glutathione in liver and kidney. Yllner (1971a,b) found small amounts of S,S*-ethylene-bis-cysteine in the urine of mice injected with EDC (Table 9-15). Nachtomi et al. (1970) found that the products of the soluble fraction of rat liver reaction with EDC were S,S'- ethylene-bis-glutathione and S-(1-hydroxyethyl)glutathione. Nachtomi et al. (1970) identified the same two compounds plus the sulfoxides of the former in urine of EDC- and dibromoethane-treated rats. Nachtomi et al. (1966) have also identified N-acetyl-S-(2-hydroxyethyl)cysteine in the urine of EDC- and 9-43 CMA 051677 V* V* Cl-C-C-Cl CT GSH GS-CH,--CH,--Cl ^CH,-CH,*GSSG --Cl S-(2-chloroothyl>-GSH Ethylene HOH -cr <GS; Epitulfonlum ion I HOH GS --CH --CH,OH S-12 hyd'OxyethyDGSH GSH glutathionase GS-CH.-CH.OH Oxide GS-CH,-CH,-SG S.S'*ethylene bis GSH NH, HOOC--CH-CHt-S--CH,-CH,OH S*<2 hydroxyethyl) cysteine Acaryl CoA Transferase N-acatyl S*(2 hydroxyethyl) cyttaina and oxlda Figure 9*7. Postulated cytosolic metabolism of ethylene dichloride. Source: Adapted from Anders and Livesey (1980). 9-44 CMA 051678 1.2- dibromoethane-treated rats. Jones and Edwards (1968) and Edwards et al. (1970) confirmed the work of Nachtomi with dibromoethane, and moreover, isolated the sulfoxides of both S-(2-hydroxyethyl)cysteine and its mercapturic acid from the urine of dibromoethane-treated rats. The half sulfur mustard, S-(2-chloroethyl)-GSH, and its episulfonium ion formed by cytosolic GSH-dependent transferase metabolism (Figure 9-7) has been suggested by Rannug et al. (1978, 1979, 1980a) to be responsible for the mutagenic action of EDC in S. typhimurium TA1535. These workers found that activation of EDC occurred only with the soluble fraction of rat liver homogenates or with purified GSH transferase enzyme in the presence of GSH. Their findings have been confirmed by Guengerich et al. (1980). In addition, Rannug and co-workers demonstrated that S-(1-chloroethyl)cysteine and N-acetyl- S-(2-chloroethyl)-cysteine produced direct mutagenic effects when tested on Salmonella, whereas S-(2-hydroxethyl)cysteine showed no mutagenic effect. These results showed that the enzymatic degradation of the GSH moiety does not abolish the mutagenic properties of the conjugate, whereas a substitution of the chlorine with a hydroxyl group does. 9.1.3.3.3. Formation of ethylene -- Livesey and Anders (1979) have identified ethylene in rat liver and kidney cytosol incubated with EDC. Ethylene was produced in only small amounts which were, however, linearly independent on the cytosolic protein concentration. The enzymatic conversion was glutathione dependent and specific for this thiol. Studies of substrate specificity with 1.2- dihaloethanes showed that reactivity following the halide order with C-Br bond cleavage and elimination occurring at a faster rate than C-Cl bond breakage. Whereas EDC conversion was primarily an enzymatic reaction, ethylene formation occurred equally well from 1,2-dibromoethane by a nonenzymatic reaction with GSH. The microsomal P-450 inhibitor, SKF 525-A, had no effect on EDC metabolism, 9-45 CMA 051679 but the reaction was inhibited by p-chloromercuribenzoic acid, methyl iodide and diethylmaleate. Livesey and Anders proposed that the intermediate in the conver sion of EDC to ethylene was S-(2-chloroethyl)glutathione (Figure 9-7). An analog of this intermediate, S-(2-chloroethyl) cysteine, was nonenzymatically converted to ethylene in the presence of glutathione and other thiols. 9.1.3.3- 4. Oxidation of EDC to CO^ -- Following oral administration of ^C-EDC to mice, Yllner (1971a), and rat, Reitz et al. (1982), found 5-10? of the dose administered was metabolized to COg. Yllner (1971b) also observed that after 2-chloroacetate administration, small amounts of glycolic and oxalic acids appeared in urine (Table 9-15). Since these acids are known to be metabolized to CC^, Yllner proposed that 2-chloroacetate, arising from EDC metabolism (Figure 9-5) is enzymatically hydrolyzed to glycolate by dehydrohalogenation, a portion of which is further oxidized to oxalic acid. 9.1.3- 3-5. Comparison of the Metabolism of EDC to Vinyl Chloride -- Rannug and Beije (1979) have drawn attention to the similarities in the biotransformation pathways of EDC and vinyl chloride. For both metabolisms, S- (2-chloroethyl)cysteine, thiodiglycolic acid, S-(2-hydroxyethyl)cysteine and its mercapturic are involved as end products. Guengerich et al. (1980) considered the possibility that reductive dechlorination of EDC would yield chloride ion plus a chloroethyl radical, which could either react with macromolecular targets, or lose a hydrogen atom to form vinyl chloride. Alternatively, some other dehydrohalogenation mechanism (-HC1) could give rise to vinyl chloride. Vinyl chloride could then be microsomally oxidized to 1- chloroethylene oxide, which can either react with macromolecules, or be rearranged to 2-chloroacetaldehyde and proceed through the pathway of Figure 9-6. However, Guengerich et al. found that reductive dechlorination of EDC to a chloroethyl radical was an unlikely reaction because metabolism and binding of EDC was dependent upon the presence of 0^ in microsomal incubations containing 9-46 CMA 051680 NADPH. Guengerich et al. calculated that the rate of EDC conversion to either a total nonvolatile or to an irreversibly bound metabolite was -25-50 times too high to support an obligatory role for vinyl chloride as an intermediate. Moreover, they found that irreversible binding of label from vinyl chloride to microsomal protein was inhibited 95$ by either alcohol or aldehyde dehydrogenase in microsomal incubations while these conditions produced only 30-40$ inhibition of binding for EDC, These investigators concluded that the mfctabolism of EDC probably does not involve vinyl chloride as an intermediate. 9.1.3.4. METABOLISM, TOXICITY AND MODIFIERS -- Dichloroethane metabolites, chloroacetaldehyde, chloroethanol (oral LD,-^ for rats, 96 mg/kg), and chloroacetic acid (oral LDb_u- for rats, 76 mg/kg) are several times more toxic than EDC itself (oral LDb,u,, for rats, 770 mg/kg) (Heppel et al., 1945, 1946; Woodward et al., 1941; Ambrose, 1950; Hayes et al., 1973). Since the symptoms of poisoning from both accidental and occupational exposures of humans and experimental exposures of animals to these metabolites are very similar to those resulting from EDC, it can be assumed that the toxicity of EDC for both man and animals is in large part the result of biotransformation to these metabolites. Johnson (1967) was the first to suggest that chloroacetaldehyde may be the premier toxic metabolite, since this very reactive compound is capable of both enzymatic and non-enzymatic interaction with cellular sulfhydryl groups (Figure 9-6). However, Yllner (1971a,b) found that chloroacetic acid also reacted extensively with sulfhydryl compounds in vivo. Heppel et al. (1945, 1946, 1947) found a high mortality (35$) in rats given 1.3 g/kg EDC orally. Mortality was reduced by pre- or post-administration of methionine, cysteine and other sulfhydryl compounds. Sulfur-containing amino acids, cysteine and methionine, also protected young rats from inhalation 9-47 CMA 051681 exposure. This protective effect of sulfhydryl compounds is clearly related to the marked depletion of glutathione levels that occurs in the livers of rats given EDC, chloroethanol or chloroacetaldehyde (Johnson, 1965, 1966, 1967) and to the enzyme pathways of metabolism of EDC (Figures 9-5 to 9-7). Johnson (1965, 1967) has noted that the morbidity and mortality of young rats given chloroethanol orally was reduced by concomitant administration of ethanol. He postulated that the protective effect of ethanol was due to simple substrate competition for alcohol dehydrogenase which catalyzes the conversion of chloroethanol to chloroacetaldehyde (Figure 9-5). Ethanol also inhibited early effects of chloroethanol on liver glutathione depletion in these animals. This author also suggested that the minimal toxicity observed with chronic low inhalation doses of EDC in different animal species may be simply explained by the rapid replenishment of tissue glutathione. Similar observations on the relation of the level of glutathione in liver and other tissues, and toxicity of the chlorocarbons, have been made by Jaeger and his co-workers (1974, 1979). Like EDC, vinylidene chloride is detoxified by glutathione-dependent pathways. Jaeger et al. (197*0 found in rats that an 18hour overnight fast decreased the LC^^ from a 4-hour inhalation concentration of vinylidene chloride from 15,000 ppm (fed) to 600 ppm (fasted) and decreased the concentration of vinylidene chloride that produced a significant plasma eleva tion of a-ketoglutarate transaminase, evidence of liver cytoxicity (2000 ppm, fed; 150 ppm, fasted). Increased susceptibility to hepatotoxicity was shown to be related to a decreased hepatic glutathione concentration associated with the fasting. Jaeger (1979) also demonstrated in rats a circadian rhythm for tissue glutathione concentration of liver, blood, lung and kidney. For rats maintained on a 12-hour light-dark cycle (6 pm to 6 am, dark), hepatic glutathione content was lowest at 6-10 pm and highest at 4-12 am; these periods correspond to the 9-48 CMA 051682 greater and lesser hepatotoxieity respectively associated with vinylidene chloride exposure during these two periods, Nakajima and Sato (1979) studied the metabolism of the chlorocarbons In vitro with microsomes from livers of fed and fasted male rats and found that the metabolism of EDC increased 1.5-fold as the result of a 24-hour fast, although fasting produced no significant increase in the microsomal protein and cytochrome P-450 liver contents. Microsomes from livers of fasted female rats displayed even more active metabolism of EDC (3.6-fold over fed rats). These observations suggest that the increased toxicity of EDC that occurs with food deprivation may be due not only to decreased liver glutathione content, but also to a greater production of toxic metabolites: chloroaeetaldehyde, chloroethanol and chloroacetic acid. Sato et al. (1980) have studied the effect of chronic ethanol consumption on hepatic metabolism of chlorinated hydrocarbons in rats to provide information on the effect of ethanol consumption on the toxic effects of chlorinated hydro carbons in the work environment. Male rats were maintained on a daily intake of ethanol amounting to 30% of their total energy intake for 3 weeks. The hepatic microsomal metabolism of EDC was increased 5.5-fold over microsomes from livers of ethanol-free rats, although ethanol feeding produced only a slight increase in the microsomal P-450 content. The increase in enzyme metabolism of EDC occurred only with microsomal fractions and not with cytosolic fractions, and one-day withdrawal of ethanol feeding almost completely abolished the effect of chronic alcohol consumption. Sato et al. (1981) also studied the effects of an acute single dose of ethanol (given as an aqueous solution by gastric intubation) on the metabolism of chlorinated hydrocarbons in rats. Hepatic metabolism of EDC (by a 10,000 g liver fraction) added in vitro was accelerated (up to 2.5-fold) by doses of ethanol up 9-49 CMA 051683 to 4 mg/kg without causing any increase in P-450 content. The stimulation was not seen and even suppression occurred with higher doses of ethanol. Increased metabolism was most marked 16-18 hours after ethanol administration. Ethanol added directly to the incubation mixture, however, depressed EDC metabolism. These investigators suggest that ethanol is capable of exerting a dual effect on EDC-metabolizing enzymes, i.e., inhibition and stimulation, depending on ethanol concentration. 9.1.3.5. METABOLISM AND COVALENT BINDING -- The metabolism of EDC results in the production of reactive metabolites of an electrophilic nature such as those proposed to be formed in the metabolic schemes of Figures 9-5 to 9-7. The occurrence of these metabolites, chloroacetaldehyde, the putative 1-chloroso-2chloroethane, the half-mustard S-(2-chloroethyl)glutathione and its episulfonium ion, provides a theoretical basis for the covalent binding observed with EDC to cellular macromolecules such as protein and DNA with consequent possibility of damage to cellular integrity and genetic apparatus. Such covalent binding reac tions are known to be related to teratogenesis, mutagenesis and carcinogenesis, although chemical reactivity alone is not evidence of genetic damage (Lutz, 1979). The amount of covalent binding, and hence damage potential, is related to the pharmacokinetics of EDC exposure. At lea3t in the rat, there is ample evidence from pharmacokinetic studies of EDC that the metabolic capacity for EDC metabolism is saturable (Sections 9.1.2 and 9.1.3). It may therefore be specu lated that the organism when confronted with low quantities of EDC utilizes glutathione conjugation detoxification reactions (Figure 9-6) and nonelectrophilic metabolites are primarily produced, but when confronted with higher EDC doses, increasing amounts of electrophilic metabolites are produced to a saturating and limiting rate for their enzymatic pathways (Figures 9-6 and 9-7). Reitz et al. (1980, 1982) determined total covalent binding and binding 9-50 CMA 051684 to DNA in rats exposed to l4C-EDC by the oral (150 mg/kg) and inhalation (150 ppm, 6 hour) routes. The results of their study with these presumably saturating doses are given in Table 9-16. These results show no striking difference between the two routes of exposure in the distribution of covalent binding and DNA binding among the various organ tissues, although the tissue levels of covalent binding are generally higher after inhalation exposure for total binding and after gavage exposure for DNA binding. The exposure doses used in these studies of Reitz et al. are comparable to the oral doses of EDC use in the lifetime NCI carcinogenicity study (NCI, 1978) and to the inhalation doses of the carcinogenicity studies in rats of Maltonl et al. (1980). No excess tumors were reported by Maltoni et al., while liver and forestomach were sites of malignant tumors in the NCI study. Recently, Storer et al. (1982) determined DNA damage produced in vivo in the livers of male B6C3F1 mice following single oral doses of EDC (100 mg/kg). DNA damage was evaluated by sedimentation of liver nuclei in alkaline sucrose density centrifugation, and by DNA recovery, which measure single strand breaks in DNA or alkaline label sites. DNA from EDC-treated mice isolated 4 hours after dosing sedimented more slowly and the total DNA recovery was decreased 16$ from untreated mice. However, dimethyl nitrosamine, used as a positive control, decreased recovery of DNA 61$. In contrast to the low covalent binding potential of EDC in vivo, inhalation exposure of rats to only 20 ppm, 1,2-dibromoethane (EDB; 7 hours/day, 5 days/week for 18 months) produced tumors in the liver and kidney, sites of covalent binding (Hill et al., 1978). Furthermore, Plotnick et al. (1980) found that the carcino genicity of EDB was enhanced by the dietary addition of disulfiram (0.05$ by weight; an inhibitor of aldehyde dehydrogenase) which blocks the further oxida tion of bromoacetaldehyde formed in the metabolism of EDB (Figure 9-5) and which presumably results in increased tissue levels of bromoacetaldehyde with an 9-51 CMA 051685 TABLE 9-16 Total Macromolecular Binding and DNA Binding in Selected Tissues 1 || ^ of Rats After Exposure to C-EDC by Oral or Inhalation Routes Nanomole equivalents EDC/g tissue oral inhalation (150 rag/kg) (150 ppm 6 hr) Total Binding (n=4) Liver Kidney^ Spleenu Lung k Forestomach Stomach DNA Binding (n=3) Experiment 1 Liver^ Spleen Kidney Stomach Experiment 2 Liver b Spleen Kidney Stomach 175 + 24 183 + 25 65 + 21 106 + 34 160 + 19 90+2 21.3 + 7*4 5.8 + 0.7 17.4 + 2.3 14.9 13-9 + 2.1 2.5 + 0.3 14.5 + 6.2 6.7 268 + 45 263 + 48 130+22 147 + 16 71 + 19 156 + 29 8.2 + 3-3 1.8 + 0.3 5.2 + 3-7 2.8 3.3 + 1*2 1.8 + 0.5 2.0 + 0.3 1.9 Results are reported as nanomole equivalents of EDC/g tissue + S.D. Animals were sacrificed 4 hr after oral dosing or immediately following a 6-hr inhalation exposure. aSource: Reitz et al., 1982 ^Site where malignant tumors were observed in the NCI study (1978) after EDC was given by gavage, also forestomach. No excess tumors were reported in the inhala tion carcinogenicity bioassay (Maltoni et al., 1980). 9-52 CMA 051686 increase of covalent binding. Plotnick et al. (1980) showed that dietary disulfiram markedly increased covalent binding of 14 C-EDB in the nuclei of liver cells in rats dosed with EDB (Table 9-17). Covalent binding of metabolites from both oxidative microsomal and cyto solic metabolism of EDC has been demonstrated in vitro by several investigative groups. Van Duuren and his colleagues (Banerjee and Van Duuren, 1979; Banerjee and Van Duuren, 1978, Banerjee et al., 1979, 1980) observed microsomal-activated covalent binding of both EDC and EDB to native DNA from salmon sperm, and to liver and lung microsomal protein from rats and mice. Binding to DNA did not occur in the absence of microsomes or in the presence of denatured microsomes. Cytosolic metabolism produced insignificant metabolic activation and binding. The microsomal-activated binding was enhanced by phenobarbital and 3methylcholanthrene pretreatment, whereas the addition of glutathione to the microsomal reaction reduced the binding. Similar results were obtained by Sipes and Gandolfi (1980). Van Duuren and his co-workers suggested that EDC and EDB were biotransformed by microsomal oxidative reactions to the reactive transient metabolites, 2-haloacetaldehyde, 2-haloethanol or haloethylene oxide, all of which are electrophilic in nature. Banerjee et al. (1979) demonstrated that both 2-bromoacetaldehyde and 2-bromoethanol bind covalently to protein and DNA without metabolic activation. Guengerich et al. (1980) have found that ^C-EDC is metabolized by both microsomes and cytosolic systems to metabolites that covalently bind to protein and calf thymus DNA. Cytosolic metabolism depended upon the presence of glutathione and involved glutathione transferases (Figure 9-7), although glutathione inhibited microsomal-activated binding to protein but stimulated binding to DNA. These investigators also produced evidence that 2chloroacetaldehyde, S-(2-chloroethyl)glutathione, and their putative 1chloroso-2-chloroethane are the metabolites involved in the covalent binding. 9-53 CMA. 051887 TABLE 9-17 lU Effect of Dietary Disulfiram Upon the C Content of Liver Nuclei Isolated 24 or 48 Hours After Administration of a Single Oral Dose of 15 mg/kg [U-^CJEDB3, Time Interval 24 hours 48 hours Control 687 + 82b 460 + 42 Disulfiram 1773 314 1534 197 aSource: Plotnick et al., 1980 ^Results are expressed as dpra/pellet (mean + S.E.M.) of duplicate determinations on 6 animals per group at 24-hr and 5 animals per group at 48 hr. 9-54 CMA 051688 9.1.4. Summary and Conclusions. At ambient temperatures, EDC is a volatile liquid with appreciable solubility in water, and hence the principal routes of entry to the body are by pulmonary and oral absorption. The pharmacokinetics of absorption and excretion of EDC have been studied extensively in the mouse and rat. Absorption from the gastrointestinal tract is rapid and complete, occurring by first order passive processes with a half-time of <6 min. A dose-dependent first-pass effect with pulmonary elimination of unchanged EDC occurs with oral ingestion, thus decreasing the amount reaching the systemic circulation. At the low concentration of EDC existent in food and water, nearly complete extraction by the liver is expected. With inhalation exposure, a blood/air partition coefficient of *20 at 37C has been observed. Tissue distribution of EDC is consistent with its lipophilic nature. The chemical crosses the blood brain and placental barriers and distributes into breast milk. The adipose tissue/blood partition coefficient varies from 7-57 depending on dose. Excretion of unmetabolized EDC is almost exclusively via the lungs; however, metabolism and excretion of the metabolites by other routes is extensive and dose related. In the mouse and rat, after both oral and inhalation exposure, the half-life of EDC in blood increases with dose, although it is <60 min at high doses. The parameters of total body elimination are compatible with a 2-compartment system and Michaelis-Menten kinetics. The short half-life of EDC suggests that the risk of bioaccumulation from intermittent multiple oral and inhalation exposures is small. Daily oral dosing of rats does not result in significant bioaccumulation in blood or other tissues. Pharmacokinetic parameters on multiple doses of EDC are very limited. Treatment of animals with EDC prior to 14 C-EDC may alter the detoxification mechanisms, thus leading to enhanced toxicity and carcinogenicity. Biotransformation of EDC has been shown to occur by multiple pathways, in both 9-55 CMA 051689 liver microsomal and cytosol fractions. In the mouse and rat, up to 90$ of low oral or inhalation doses (<50 mg/kg) are metabolized, with a decreasing percentage of the dose metabolized as the dose approaches and exceeds saturation of metabolic capacity. Metabolism produced 2-chloroacetaldehyde, S-(2-chloroethyl)glutathione (a half sulfur mustard) and other putative reactive metabo lites capable of covalent binding to cellular macromolecules, as well as nonreac tive glutathione conjugates from "detoxification reactions." The intensity of covalent binding of reaction metabolites to proteins, lipids and DNA, however, is considerably less than that observed with the carcinogenic bromine analog and other known carcinogens. Exposure to EDC decreases liver levels of reduced glutathione in a dosedependent manner. Glutathione plays an important role through glutathione conjugation detoxification mechanisms in protecting against binding and cellular toxicity. Covalent binding, hepatotoxicity and mortality of experimental animals are all reduced by administration of sulfhydryl amino acids or gluta thione. Conversely, toxicity is enhanced by low liver and tissue concentrations of glutathione. Phenobarbital and other inducers of P-450 metabolism increase the metabolism of EDC, the toxicity and covalent binding. Fasting reduces gluta thione tissue content and increases both the metabolism and toxicity of EDC. Ethanol, acutely or chronically administered, may either enhance or inhibit EDC metabolism and toxicity depending on the tissue alcohol concentration. 9-56 CMA 051690 9.2. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY 9.2.1. Effects in Humans. The preponderance of reports on the toxicity of EDC to humans was published in the foreign literature (primarily German, Russian, and Polish). Many of these reports involved exposure to high levels of compound, involved single cases or small numbers of individuals, were anecdotal in nature and discussed observations that related to the overall toxicity of this chemical in man, and/or provided little detailed correlation between toxic effects and the amount or duration of exposure. Since detailed reviews of the foreign studies were available from NIOSH (1976) and the U.S. EPA (1979)* these sources were used in part as a basis for the following discussion of human health effects. Trans lations were obtained and evaluated independently for all foreign studies that provided dose-response data of relevance to human risk assessment. Reports in which exposures were not primarily to EDC (i.e., most reports of mixed solvent exposures) were not discussed. 9.2.1.1. ACUTE EXPOSURES 9.2.1.1.1. Case Reports and Surveys 9.2.1.1.1.1. Oral Exposure. Information on the effects of ingested EDC is available solely from clinical case reports and surveys involving accidental and intentional exposures. Many of the cases involved fatalities and described symptoms and signs of poisoning that preceded death. The progression of signs/symptoms and the outcome/findings of cases in which the quantity of EDC ingested leading to death was reported are summarized in Table 9-18. Ingestion of quantities of EDC estimated to range from 8 to 200 mil (-143-3571 mg/kg, assuming 70 kg body weight) were reported as lethal to adult males. Following ingestion there was often a latent period, generally 30 minutes to 3 hours, prior to the onset of symptoms. Symptoms were indicative of CNS effects and gastro- 9-57 CMA 051691 051692 Patient8 Sex/Age 4 males/20 to 29 years 3 males/19 to 27 years 00 Male/KS8 Hale/63 years TABLE 9-18 Effects Associated with Acute Lethal Oral Doses of Ethylene Dichloride in Humans Amount Ingested Symptoms and Signs Outcome and Findings Reference 150 to 200 mi {=188 to 250.6 g) 70, 80 and 100 m (=87.7, 100.2 and 125.3 g) =82 mi (*102.7 g) mixed with coffee and beer 2 oz. (=75.2 g) mixed with orange Juice and ginger ale Symptoms were not specified, but appeared after 3 to 4 hours Onset of symptoms within a few minutes: vomiting; weakness; dizziness; lost consciousness Intoxication; vomiting; diarrhea; unconsciousness; dypsnea Deaths after 10, 15, 33 and Bryzhin, 1945 35 hours. Punctuate hemorrhaging in the epl- cardium, pleura, and mucous membranes of the stomach and duodenum; varying degrees of liver damage with focal hemorrhaging in one case; yellow- white fibrinous bundles of blood in the heart cavities and lesser circulatory ressels; hemolytic Jaundice of the endocardium, aortal intima, and dura mater; evidence of decomposition of circulating erythrocytes Deaths after 5 to 8 hours Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs Kaira, 1966b Death after 6 hours Hyperemia and hemorrhagic lesions (see text) Noetzel, 1944b Onset of symptoms after 2 hours: nausea; faintness; vomiting. Subsequently, cyanosis; dilated pupils; coarse rales; weak and rapid pulse; diarrhea; increased cyanosis; absence of pulse and heart sounds; dypsnea Death after 22 hours Hueper and Smith, attributed to circulatory 1935 failure. Extensive hemorrhagic colitis; nephrosis with calcifications of the tubular epithelium and tubular and vascular elastic membranes; fatty degeneration of the liver; spleen and lungs; multiple perivascular hemorrhages of the brain CMA 051693 Patient3 Sex/Age Male/16 years Hale/80 years Male/18 years Male/57 years Amount Ingested 50 ml (=62.7 g) 50 mA (=62.7 g) 50 mA (=62.7 g) 40 mA (= 50.1 g) TABLE 9-18 (cont.) Symptoms and Signs Outcome and Findings Reference Vomiting; epigastric pain; muscle spasms, hiccups, rapid pulse and lack of eyelid response to light on the 4th day Death after 91 hours Elevated serum enzymes; LDH; Death within a few hours SGOT; SGPT; alkaline phosphatase glutamic dehydrogenase; RHAase. Roubal, 1947b Secchl et al., 1968b Onset of symptoms after 1 hour: somnolence and cyanosis. Diarrhea after 4 hours. Impaired blood coagulation after 5.5 hours: increased prothrombin time; decrease in clotting factors II and V; thrombocytopenia; no increase in fibrinolysis Somnolence; vomiting; sinus tachycardia; ventricular extrasystoles; regained consciousness after 14 hours; dypsnea; loss of blood pressure; cardiac arrest. Impaired blood coagulation observed after 24 hours; prolonged bleeding from venipunctures; reduction in activity of clotting factors II, V, VII and VIII; complete defibrination; thrombo cytopenia ; increased thrombin time Death after 17 hours attributed to circulatory shock. Intravascular thrombosis were not found. Schonborn et al., 1970 Death after 24 hours. Thrombi in the pulmonary arterioles and capillaries; hemorrhages into the mucosa of the esophagus, stump of the stomach, rectum and in the subepicardial, subendocardial and myocardial tissues. The coagulation disorder with thrombocytopenia attributed to disseminated intravascular coagulopathy and hyperfibrinolysis Martin et al., 1969** 051694 Patient Sex/Age Hale/30 years Hale/50 years Male/55 years (asthmatic) Male/NS Hale/NS Amount Ingested 40 at ( = 50. g) =30 at (=37.6 g) 20 ml (=25.1 g) =20 at (=25.1 g) =20 at (=25.1 g) TABLE 9-18 (cont.) Symptoms and Signs Outcome and Findings Reference Slight cough; reddened conJunetlvae; shock; weak, rapid pulse. Regained consciousness after 3 hours; hyperactivity alternated with seaicoaatose condition Onset of symptoms after 30 minutes: unconsciousness; vomiting; cyanosis; dilated and fixed pupils; pulmonary edema; extreme dypsnea Epigastric pain; extreme dizziness; sleeplessness; vomiting; slow pulse Onset of symptoas after 1 hour: repeated vomiting. Cyanosis and dypsnea after 12 hours Symptoms not reported Death after 26 hours Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs Death after 10 hours Diffuse hemorrhagic gastritis; bilateral pulmonary congestion; acute necrotizing bronchiolitis and bronchitis; acute toxic nephrosis; diffuse hepatic necrosis; hypereala of the brain with scattered peri vascular hemorrhages in the pons Death after 24 hours Death after 13 hours Death within 12 hours Garrison and Leadinghaa, 1954 Lochhead and Close, 1951 Roubal, I947b Flowtow, 1952b Flowtow, 1952b 051695 Patient3 Sex/Age Hale/14 years Male/32 years Male/32 years Amount Ingested 15 ml (3 18.8 g) 8 ml (*10 g) glass TABLE 9-18 (cont.) Symptoms and Signs Outcome and Findings Onset of symptoms after 2 hours: Death after 6 days Progressive appearance of Extensive mid-zonal liver severe headache; staggering; necrosis; renal tubular lethargy; periodic vomiting; necrosis; focal adrenal decreased blood pressure; degeneration and necrosis oliguria; dypanea; somnolence; Increased dypsnea; and oliguria, hemorrhagic nasogastric aspirate; ecchymoses; sinus tachycardia; cardiac arrest, pulmonary edema; refractory hypotension. Hypoglycemia on day 2 and hypercalcemia on day 4. Progressive decrease in blood coagulation ability: increased prothrombin time; all clotting factors except Till were markedly decreased on day 4. Burning sensation in mouth, Death after 56 hours throat and stomach; drank milk and vomited; weakness; speeoh retardation; lethargy; asthenia; cold sweat; muffled heart sound; weak and rapid pulse. 22 hours after ingestion: excitation; restlessness; delirium; flushed faoe; systolic murmur; respiratory depression; circulatory weakness; anuria Vomiting, weakness, stomach Death on the 3rd day pains. On 3rd day: rest Hyperemia and hemorrhagic lessness, coated and dry lesions (see text) tongue; bloody diarrhea; abdomen painful on palpitation; enlarged liver; dry, moist rales; rapid, weak pulse; anuria; increasing cyanosis; un consciousness Reference Yodaiken and Babcock, 1973 Agranovich, 1948 969tS Patient3 Sex/Age Male/27 years CeTji a Male/43 years (alcoholic) Male/43 years (alcoholic) Male/NS TABLE 9-18 (cont.) Amount Ingested half glass 4 drinks diluted with orange Juice0 4 drinks diluted 0 with orange Juice several mouthfuls Symptoms and Signs Outcome and Findings Reference Onset of symptoms after 2.5 hours: Unconsciousness that was regained after 12 hours; vomiting (dark vomitus); burning sensation in the digestive tract; dyp3nea; nausea; cyanosis; depressed respiratory rate; moist rales; muffled heart sounds; rapid pulse; extrasystoles; anuria Death after 19 hours Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs Unconsciousness Death after 8 hours Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs and lungs. Death after 24 hours. Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs and lungs Confusion; deep sleepiness; unconsciousness; vomiting with blood Death after 24 hours Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organa and lungs Onset of symptoms after Death on the 5th day 30 min: vertigo; nausea; vomiting; slightly muffled heart sound; tachycardia. After 17.5 hours: headache; 3ubsternal pain; cyanosis; rapid, weak pulse; dry rales; . decreased blood pressure; vomiting with bile; diarrhea; subconjunctival hemorrhage; oliguria; paranephric hemorrhage. 5th day pulmonary edema and unconsciousness Bogoyavlenskl et al., 1968 Hulst et al., 1946b Hulst et al., 1946b Morozov, 19 58b 051697 Patient3 Sex/Age Male/63 years Amount Ingested 1 or 2 3ips Male/1 1/2 years Male/79 years Male/2 years Male/23 years 1 alp 1 sip 1 sip 1 sip aNS = Not stated **Data cemptied from NIOSH, 1976 cPresumably EDO was mixed instead of alcohol TABLE 9-18 (cont.) Symptoms and Signs Outcome and Findings Reference Unconsciousness shortly after ingestion, but soon regained; strong vomiting; period of Improvement; unconsciousness again after 10.5 hours; falling blood pressure Extreme weakness; comatose; vomiting Vomiting; weakness; pale; cyanosis; scarcely conscious; vagueness; rapid, regular pulse; blood pressure not measurable Vomiting; diarrhea; tonic spasms; Increasing loss of consciousness; dypsnea; Impaired circulation Onset of symptoms after 1 hour: dizziness; nausea; unconscious ness; vomiting; cyanosis; no pupil reaction; no corneal reflex; dypsnea; strong motor unrest Death after 1*1 hours attributed to circulatory failure. Hyperemia and hemorrhagic lesions (see text) Freundt et al., Death the next day Hyperemia and hemorrhagic lesions (see text) Death after 40 hours attributed to heart and circulatory failure. Hyperemic and hemorrhagic lesions (see text) Death after 20 hours Hyperemia and hemorrhagic lesions (see text) Keyzer, 1944** Weiss, 1957b Reinfried, 19581 Death after 8 hours attributed to respiratory and circulatory failure Hyperemia and hemorrhagic lesions (see text); evidence of overt bleeding into the visceral organs Reinfrled, 19581 intestinal disturbances, and frequently included dizziness, nausea, headache, periodic vomiting, diarrhea, epigastric pain or tenderness, dilated pupils and lack of corneal reflex, rapid and weak pulse rates, progressive cyanosis, dyspnea and unconsciousness. These symptoms of toxicity are characteristic of those produced by many of the chlorinated aliphatic hydrocarbons. Somnolence, oliguria and anuria, muffled heart sounds and motor unrest have also been described in a number of cases. The results of several health surveys of large numbers of orally exposed individuals have recently been reported by Russian investigators, and summarized by PEDCo Environmental, Inc. 0979) (Akimov et al., 1976, 1978; Shchepotin and Bondarenko, 1978; Bonitenko et al., 197^ 1977; Luzhnikov et al., 197-4; Luzhnikov and Savina, 1976) and Chemical Abstracts (Andriukln, 1979). Information regarding the design of these surveys is not available, and dosage information is either not available or not adequately reported (i.e., not correlated with effects) (Table 9-19). The clinical symptoms and signs of exposure recorded in the surveys are consistent with those described in single case reports (see Table 9-18) (i.e., neurological effects and evidence of liver and kidney dysfunction), but there appears to be a more frequent indication of cardiovascular insufficiency. The range of doses that elicited effects in the surveys ( = 12.5 to 250.6 g) also appear to be consistent with the single case reports (see Table 9-18), but incidences of fatalities were not presented in the available summaries. Although none of the effects reported in the surveys were correlated with specific doses, several of the studies indicated that the severity of poisoning was related to blood levels of EDO (Bonitenko, 1974, 1977; Luzhnikov et al., 1974, 1976). The clinical syndrome of oral EDC poisoning in children is similar to that seen in adults, but the lethal dose range is somewhat lower (0.3 to 0.9 g/kg) (Hinkel, 1965). This range of doses did not always cause death, and is derived 9-64 CMA 051698 TABLE 9-19 Effects of Aoute Oral Ingestion of 1,2-Dichloroethane (Survey Results) Route No. of Subjects Dose Principal Findings Reference Oral 121 20 to 200 at {=*25 to 250.6 g) Oral Inhalation 211 37 VOIOl kJl NR NR x 20 to 30 Minutes NRa 110 NR Mild to severe poisoning characterized by pronounced DCE odor on breath (91%), dry skin (75%), hypotension (71l%), extreme pupil dilation (72%), mucosal cyanosis (67%), tachycardia (62%), respiratory difficulty (59%), muscular hypotonia (46%), decrease in tendon reflexes (46%), loss of consciousness (42%). Neurological syn dromes noted in 118 subjects: comatose (42%), atactic (42%), asthenic with autonomic vascular Insufficiency (27%), extrapyramidal (23%), oonvulslve (7.4%), psyahotio (5%). Lethality not reported. Aggregate findings for 248 total patients reported. Neurological disorders (lncl. unconsciousness, respiratory depression) (100%), cardiovascular insufficiency (lncl., arrhythmias, hypotension, reduced cardiac output, decreased peripheral resistance) (60%), liver dysfunotlon (incl., enlargement, hyperbilirubinemia, increased serum albumin and asparagine transaminase (35%). Naphropathology (oliguria, proteinuria, azotemia, aoute renal failure with disturbed aoid/baae balance) particularly associated with inhalation exposures. Gastroenteritis milder in subjects exposed via inhalation. No correlation between severity of poisoning and the blood or urine concentration or the amount Inhaled. Aoute gastritis with vomiting (77%), neurological disorders including coma (81%), acuta oardiovascular insufficiency (57%), hepatitis (56%) with liver enlargement and dysfunotlon (abnormal bromosulfalein, plasma bilirubin, plana glutamine - aparagine transaminase). Clinical symptoms of poisoning ware observed at blood concentrations of 0.5 mg %, and coma developed at 5 to 7 mg %. Akimov et al., 1976, 19TB Sobepotin and Bondarenko, 1976 Luzhnlkov et al., 1976, 1978 CMA 051699 TABLE 9-19 (cont.) Route No. of Subjects Dose Principal Findings Reference NSa I60b NA Oo'\. Hemodynamic shock due to myocardial dysfunction. Characterized by compensatory (Increased peripheral resistance, normal or slightly Increased arterial blood pressure, decreased cardiac output and blood volume, decreased cardiac Isometric contraction, increased ventricular expulsion time, asynchronous contractions) and decompensatory (pronounced and progressive hypotension, decreased cardiac output, normal or slightly decreased peripheral resistance, decreased myocardial contractile force during ventricular systole, prolonged periods of asynchronous contractions) phases. BKG changes including arrhythmias observed in both compensated and decompensated shock. Pathologic changes In the myocardium charac terized by capillary endothelial cell edema, capillary lumen stenosis, edema of the myocardial Interstices with leukocyte accumulation and miorofocal hemorrhages, decreased glycogen, degenerative changes, and decreased mitochondrial enzyme activities. Luzhnlkov et al., 1976, 1978 Oral Inhalation 24 3 10 to 100 ait (=12.5 to 125 g) NR 17 patients exhibited manifestations of light/mild poisoning: headache, vertigo, abdominal pain, nausea, vomiting, and signs of cardiac insufficiency (increased contraction rate, decreased minute volume, decreased circulating blood volume, decreased cardiac Index); 5 of 17 had hepatomegaly, ioterio skin, solera, and increased bilirubin levels. The other 10 patients were comatose and had more pronounced symptoms of oardiao insufficiency. Andrlukin, 1979 Oral 32 O NR Gastroenteritis and proteinuria. Elevated leukocyte count and elevated alanine/aspartate aminotransferase activities correlated with severity of poisoning. Coma was associated with blood concentrations of 15 to 30 mg %, and consciousness returned at levels below 8 to 10 mg J. Adipose tissue and blood levels of 68 and 1.2 mg S, respectively, at autopsy. Bonltenko et al., 1974, 1977 051700 TABLE 9-19 (oont.) Route Oral0 No. of Subjects 7 children (1 to 6 years) NR Dose Principal Findings Clinical syndrome similar to that in adults: severe and persistent vomiting (within 1 hour), and subsequent (immediate to 10 to 12 hours) narcotic effects (e.g., somnolence, motor unrest, reflex Increases, convulsions or coma). Less frequent indications of oiroulatory failure, kidney and liver functional disturbances, and tachycardia. No remarkable hematologic changes. Typical pathological anatomical findings. LD^o reportedly ranged from 0.3 to 0.9 g/kg. Reference Hinkel, 1965 aRoute is probably oral, but not specifically stated in the EPA (1979) summary of this study. bThe EPA summary of this study stated that "at least" 160 patients were studied. cExposure to a "nerve balsam" medicine that was 75$ 1,2-diohlorethane (other components not stated in EPA summary). CIO n 051701 from a review of seven cases of accidental poisoning in children that ranged in age from 1 to 6 years. Foreign reports of individual non-fatal cases of EDC ingestion (Ienistea and Mezincesco, 1943; Bloch, 1946; Stuhlert, 1949; Flowtow, 1952; Kaira, 1966; Rohmann et al., 1969; Gikalov et al., 1969; Pavlova et al., 1965; Agranovich, 1948) were in most instances not detailed in the NIOSH (1976) or U.S. EPA (1979) reviews, presumably because the effects were similar to those that preceded death with lethal exposures, and because the quantities ingested were not known. Bloch (1946) did note bloody diarrhea and evidence of adverse liver (enlargement, slightly increased serum bilirubin, urobilinogen, abnormal galactose and alcohol load test results) and kidney (oliguria, albumin and casts in the urine, temporary retention of nitrogenous substances) effects in one man who claimed to have swallowed "only a small amount" of EDC. Death was usually ascribed to circulatory and respiratory failure, and autopsies revealed tissue congestion, cellular degeneration, necrosis and hemorrhagic lesions of most organs, including the stomach, intestines, liver, kidneys, spleen, heart, lungs, respiratory tract and brain (see Table 9-18). There was occasionally evidence of gross bleeding into the visceral organs or lungs. Several of the more recent reports (Martin et al., 1969; Schonborn et al., 1970; Yodaiken and Babcock, 1973) indicated that the hemorrhagic effects may have been exacerbated by a condition known as disseminated intravascular coagulation (DIC). DIC is characterized by a depletion (consumption) of clotting factors, hypofibrinogenemia and thrombocytopenia, and the resultant effect is a severe bleeding tendency. The results of the studies by Luzhnikov and coworkers (1976, 1978) suggest that EDC can exert a direct toxic effect on the myocardium (see Table 9-19). Pathologic examinations of EDC-exposed individuals (amount ingested and number 9-68 CMA 051702 examined not available) revealed significant edema in the capillary endothelial cells and stenosis of the capillary lumens, pronounced edema of the myocardial interstices with accumulation of polymorphonuclear leukocytes, microfocal hemor rhages, diminished glycogen content, and degenerative changes. Mitochondrial damage was indicated by a decrease in enzyme activities. Myocardial functional changes (e.g., decreased contractile force, asynchronous contractions) were also observed prior to death. The cardiotoxic effect of EDC apparently precipitated a clinical state of shock; hemodynamic disorders commonly observed in at least 160 patients included increased peripheral resistance, decreased cardiac output, hypotension and EKG changes including arrhythmias. 9.2,1.1.1.2. Inhalation Exposure. Reports of numerous cases of acute occupational exposure to EDC vapor have been published (NIOSH, 1976). These acute exposures have involved both fatal (27 cases) (Wendel, 1948; Brass, 19-49; Hadengue and Martin, 1953; Ollivier et al., 1954; Domenici, 1955; Salvini and Mazzuchelli, 1958; Guarino and Lioia, 1958; Troisi and Cavallazzi, 1961) and nonfatal (57 cases) outcomes (Wirtschafter and Schwartz, 1939; Jordi, 1944; Agronovich, 1948; Baader, 1950; Paparopoli and Cali, 1956; Menschick, 1957; Smirnova and Granik, 1970), but none of the reports provided exposure concentra tions. Furthermore, although EDC was usually reported to be the primary vapor to which the workers were exposed, the preponderance of exposures were poorly characterized mixtures of EDC and other solvents, or EDC of unknown purity. The occupational exposures were generally associated with maintenance and cleaning operations or the use of EDC as a paint thinner, but many (39 cases) involved exposure to Granosan, a fumigant composed of 30$ carbon tetrachloride and 70$ EDC (Domenici, 1955; Salvini and Mazzucchelli, 1958; Guarino and Lioia, 1958; Paparopoli and Cali, 1956). 9-69 CMA 051703 The effects associated with the above cited fatal and non-fatal acute inhalation exposures were very similar to those found after ingestion (NIOSH, 1976). In general, the initial symptoms appeared to be consistent with both CNS effects (headache, dizziness, lethargy, feelings of drunkenness, unconscious ness) and gastrointestional disturbances (nausea, vomiting, diarrhea), which are characteristic of chlorinated aliphatic hydrocarbon toxicity. In some cases workers were asymptomatic and were not overcome during exposure, but later became unconscious. Other signs and symptoms of inhalation exposure commonly included cyanosis, epigastric tenderness and/or pain, hepatomegaly, and jaundice. Laboratory studies were consistent with these observations and indicative of hepatic dysfunction (increased serum bilirubin and urobilinogen, hypoglycemia, abnormal function tests) and renal disturbances (oliguria, anuria, abnormal function tests). In addition, conjunctivitis, respiratory tract irritation and inflammation, rales and leukocytosis were occasionally associated with the acute vapor exposures. Death from acute inhalation exposure wa3 generally attributed to respira tory and circulatory failure (NIOSH, 1976), although it is not certain if EDC exerted a direct toxic effect on the cardiopulmonary system or precipitated a shock-like state that could have elicited many of the changes observed at autopsy. Autopsies frequently revealed pulmonary edema, congestion of internal organs (liver, kidneys, spleen, lungs, brain) and cellular degeneration and necrosis (liver and kidneys). Hyperemia and hemorrhagic lesions were also observed in the kidneys, brain, respiratory tract, lungs, and heart. 9.2.1.1.1.3. Dermal Exposure. Several reports described instances (a total of 6 cases) in which skin contact with EDC occurred concurrently with inhalation exposures (Wirtschafter and Schwartz, 1939; Anonymous, 1946; Rosenbaum, 1947; Hadengue and Martin, 1953). Severe dermatitis was a commonly 9-70 CMA 051704 reported result of the dermal exposures, as is the case with many chlorinated hydrocarbons which produce defatting of the skin. Several of these reports also suggest that dermal absorption may significantly contribute to total exposure (characteristic symptoms of intoxication were noted in workers who were wearing EDC-soaked clothing). 92.1.1.2. Experimental Investigations. 9.2.1.1.2.1. Physiologic Effects. Borisova (1957) examined eye sensi tivity to light and plethysmographic and spirographic responses in a small number of subjects (3 to 4) who were experimentally exposed to concentrations of EDC that ranged from =1 to 12.4 ppm (4 to 50 mg/m ). This range of concentrations represented subthreshold, threshold and above threshold odor perception concen trations (Section 7*1.2.2). The concentration of EDC in air was determined nephelometrically; the method is sensitive enough (0.001 mg/sample reported) to monitor EDC at levels lower than the lowest concentration tested (4 mg/ra^). In the light sensitivity experiment, baseline perception thresholds were determined prior to the exposures for each of 3 subjects (17 to 24 years old), and the subjects inhaled EDC vapor for 15 minutes prior to testing (Borisova, 1957). When tested after 40 minutes of dark adaptation, the threshold (inten sity) at which light was perceived was found to be higher during exposure to EDC, and the threshold appeared to increase with increasing concentrations of EDC (Table 9-20). No change in the light sensitivity of eyes was observed in any of the subjects at 1 ppm, and the lowering of eye sensitivity to light was most marked at 12.4 ppm. The effect of low concentrations of EDC on blood volume (vascular con striction) and pulse fluctuations was studied with a finger plethysmograph (Borisova, 1957). Four subjects were exposed to vapor concentrations of 1, 1.5, 3, 5.7 and 12.4 ppm for 30 seconds or 15 minutes. A 30-second exposure at 9-71 051705 CMA Cl TABLE 9-20 Effect of Ethylene Dichloride Exposure on Eye Sensitivity to Light Concentration ppm (mg/nr) 1 (4) 1.5 (6) 2.2 (9) 3 (12) 4.3 (17.5) 5.7 (23-2) 5.2 (25) 7.4 (30) 12.4 (50) Subject 1 no effect -0.2 -0.25 -0.25 -0.3 -0.45 -0.45 -0.5 -1.2 Perception Threshold (Units of Optical Density) Subject 2 Subject 3 no effect <0.05 -0.3 -0.25 -0.35 -0.4 -0.5 -0.5 -1.2 no effect no effect +0.15 no effect -0.45 -0.25 -0.7 -0.75 -0.6 clSource: Borisova, 1957 ^Eye sensitivity to light was determined after 40 minutes of adaptation in the dark at different EDC concentrations. 9-72 CMA 051706 1.5 ppm resulted in a temporary vasoconstriction in all 4 subjects. Complete data were not reported, but vasoconstriction appeared to be more pronounced at 3 ppm and higher concentrations. Results of tests in which EDC exposure was extended to 15 minutes reportedly showed that the above concentrations caused "characteristic changes" in the plethysmograms of all subjects that included increased pulse beat, pulse waves, and pulse amplitude. The observations at the higher concentrations in both the 30-second and 15-minute exposure experiments also suggested that the degree of response was proportional to the exposure concentration. Exposure to concentrations of 1.5, 3, 5-7, and 12.4 ppm for 1 minute produced changes in depth of breathing in 4 subjects (as indicated by increased amplitude of respiratory waves on spirograms), but concentrations of 1 ppm reportedly had no effect on respiration (Borisova, 1957). 9*2.1.1.2.2. Odor Detection and Recognition Thresholds. Heilman and Small (1973, 1974) determined an absolute odor threshold of 6.0 ppm and an odor recognition threshold of 40 ppm for EDC. A trained odor panel was used to characterize the odor properties of this compound and 101 other petrochemicals, but the number of panelists was not stated. The absolute odor threshold is the concentration at which 50$ of the odor panel observed an odor, and the recogni tion threshold is the concentration at which 100$ of the panel defined the odor as representative of EDC. Borisova (1957) determined the odor threshold of EDC with 20 subjects and a total of 1256 tests. Thirteen subjects perceived EDC vapor at a concentration of approximately 6 ppm (23.2 to 24.9 mg/m^), 6 subjects perceived EDC at 4.5 ppm (17.5 mg/m ), and 1 subject detected the compound at 3 ppm (12.2 mg/m^). When diluted in water,the odor recognition threshold of EDC was determined to be 29 ppm (v/v water) (Amoore and Venstrom, 1966). An odor panel of 13 men and 16 women was used for the determination, and the threshold defined by the 9-73 osn01 panelists was the concentration of EDC that yielded 70)f correct response (signi ficant at the 1$ level) in a paired comparison against pure water as a control. 9.2.1.2. REPEATED AND CHRONIC EXPOSURES. 9.2.1.2.1. Case Reports -- Repeated exposure to EDC vapor in the work place has resulted in effects that are consistent with those resulting from acute exposures. Symptoms and signs including anorexia, nausea, vomiting, weakness and fatigue, nervousness, epigastric pain/discomfort and irritation of the respiratory tract and eyes were described in numerous case reports of industrial exposures (McNally and Fostvedt, 1941, Siegel, 1947; Rosenbaum, 1939; Watrous, 1947; Rejsek and Rejskova, 1947; Delplace et al., 1962; Suveev and Babichenko, 1969). In one study (Suveev and Babichenko, 1969) examination of 12 symptomatic workers who were brought to a clinic revealed paleness and cold sweat (12 of 12), bradycardia (9 of 12), systolic murmur (5 of 12), diarrhea (5 of 12; 3 of 12 with blood) and enlarged livers that were soft and tender to palpitation (9 of 12); muffled heart sounds, increased rate of respiration, rales, coated and dry tongues were also observed but incidences of occurrence were not stated. Signs indicative of nervous system dysfunction have also been reported in cases of chronically exposed workers; these include nystagmus, fine tongue tremors and sluggish patellar reflex (McNally and Fostvedt, 1941), encephalic disorders (Delplace et al., 1962), and decreased muscle tone, loss of reflexes, a positive Romberg's sign, and deafness (Suveev and Babichenko, 1969)* Complaints of hand and arm eczema that appeared within the first year of exposure were recorded in 11 of 16 cases by Delplace et al. (1962). It should be noted that exposure concentrations and information on the types of exposures were not provided in any of the above cited reports. Rosenbaum (1947) discussed experiences with cases of EDC intoxication in Russian industries from 1934 to 1945. He observed over a period of 10 years that 9-74 CMA 051708 characteristic symptoms of acute poisoning could develop with repeated exposure to concentrations of 75 to 125 ppm in air. Fatalities have resulted when workers experienced these symptoms 2 or more times in a period of 2 to 3 weeks. There was no mention, however, of the number of people affected or durations of exposure. Byers (19^3) noted that a "number of persons" exposed to EDC complained of delayed toxic effects. The workers stated that the worst effects, which varied from lassitude and malaise to nausea, vomiting and abdominal pain, occurred after the evening meal. These symptoms were associated with exposure to concentrations "only slightly in excess of 100 ppm for 7.5 hours daily", and were not completely alleviated when ventilation procedures reduced the EDC concentration to 70 ppm. Exposure conditions were described in partial detail for 2 workers who were chronically exposed to EDC for 7 and 9 months during the manufacture of hexachlorophene (Guerdjikoff, 1955). EDC was used as a catalyst in this process. During one of the operations, which was repeated for 2 to 3 minutes several times a day for a total of about 30 minutes, exposures were associated with adding EDC, trlchlorophenol and sulfuric acid to the reaction vat. EDC exposure concentra tions were not measured during the filling operations, but the workers wore an air-supplied respirator and were probably only exposed to EDC occasionally due to improper fit. During another operation which lasted 10 minutes and was repeated 3 to 4 times a day, the exposure concentration of EDC was about 120 ppm. During a final operation, the workers were exposed once a day for =*10 to 15 minutes when an EDC pipe was cleaned; these exposure concentrations were not measured but were considered by Guerdjikoff (1955) to be more than 120 ppm. Both workers exhibited similar symptoms, typical of EDC exposures, of anorexia, epigastric pain, fatigue, irritability, and nervousness after about 3 weeks of exposure. Neuro logical effects such as hand tremors, hyperhidrosis and difficulty in walking were eventually experienced. 9-75 CMA 051709 9.2.1.2.2. Health Surveys -- Kozik (1957) reported the results of a health and morbidity survey of Russian aircraft industry workers. All of the workers in the study group (size not stated) were employed in a shop where glue that contained EDC as a solvent was used to bond rubber sheets to metal forms in a soft tank fabrication process. Most of these workers were gluers, but a small number worked inside the completed tanks to disassemble the forms. EDC was emitted to the air during application and glue drying. About 500 atmospheric measurements of EDC were reported to have been taken (Kozik, 1957). Although the sampling and analytical methods were not mentioned, NIOSH (1976) felt that the data were presented in sufficient detail to permit estimations of TWA exposures. NIOSH estimated that 44 to 46$ of the total exposure occurred during the gluing operations, when the TWA concentrations were =28 ppm during application and =16 ppm when the glue was drying. When other operations were performed in the same shop (during the second half of the work- shift), the EDC TWA concentrations were =11 ppm. The TWA for the total shift was estimated to be 1 5 ppm. Concentrations ranged from approximately 4 to 50 ppm; concentrations in excess of 20 ppm were associated only with the gluing and drying operations and occurred about 15$ of the time. NIOSH (1976) has noted, however, that the aforementioned TWA concentrations may be an overestimate of most of the workers' exposures for several reasons. First, the tabulation of measurements in the glue application category also contained high values (45 to 52 ppm) that were experienced only by an insignificant number of workers who disassembled the molds within the finished tanks. Second, the measurements were apparently not breathing zone measurements and third, the ventilation system was designed with the exhaust ducts on the floor. In light of these considerations, NIOSH concluded that a more realistic appraisal of the TWA exposure of the majority of the workers is 10 to 15 ppm. 9-76 CMA 051710 Workers (total number not stated) who were engaged In the production of soft tanks during the years 1951 to 19 55 experienced increased morbidity and lost workdays when compared with workers in the entire factory (Table 9-21). Disease categories examined included acute gastrointestinal disorders, neuritis and radiculitis, and other diseases. An in-depth analysis of the morbidity rate with temporary disability for 1954 to 1955 reportedly showed high rates for gastro intestinal diseases, liver and gall bladder diseases, and diseases of the muscle, tendons and ganglion (Table 9-21). The liver and gall bladder diseases were considered by Kozik (1957) to be related to a specific toxic effect of EDC (the dyspeptic symptoms it causes reportedly are often diagnosed as gastritis), but the diseases of the muscle, tendon and ganglia were associated with the numerous repetitive motions the workers had to make when applying the glue. Further examination of 83 of the gluers revealed diseases of the liver and bile ducts (19 of 83), neurotic conditions (13 of 83), autonomic dystonia (11 of 83), asthenic conditions (5 of 83), and goiter and hyperthyroidism (10 of 83). Vi3ual-motor reactions were studied at.the beginning and end of 14 workdays in 17 of the gluers and 10 control machinists (Kozik, 1957). It was stated that a device was used to determine simple and complex reaction (color differentiation) times, as well as reaction times in a modification of the complex reaction task; a total of 3700 reaction tests were conducted, but details of the tests were not given. A comparison of the mean rates for all three reactions show no signifi cant differences in the two groups either before or after work. Nervous system dysfunction was suggested, however, by the inadequately reported results of the complicated reaction tests. "Most" of the gluers made errors in the complex reaction task, while the machinists did not make any errors (additional data are not available). In the modified complex reaction test, errors were committed 9-77 CMA 051711 TABLE 9-21 Morbidity and Lost Workdays of Aircraft Industry Gluers Exposed to Ethylene Bichloride (Rates/100 Workers) Year 1951 Cases Days Total Morbidity Plant Shop Acute Gastro intestinal Disorders Neuritis and Radiculitis Plant Shop Plant Shop Other Diseases Plant Shop 120.2 159.8 5.1 995.8 1445.5 19.3 11.6 5.2 43.5 99.9 13.0 34.4 127.0 354.2 43.2 541.8 Liver and Gall Bladder Diseases0 . Acute Gastritis0 Chronic Gastritis0 Diseases of the Muscles, Tendons, and Ganglia0 __ 1952 Cases Days 12k.0 960.9 137.6 4.2 996.0 15.1 5.7 5.0 23.1 44.8 9.7 34.0 94.5 335.2 40.8 378.7 _- ** 1953 Cases Days 135.6 1040.8 163.9 14.4 1236.5 1 5.6 6.2 7.5 19.1 67.3 16.5 35.3 146.0 338.3 53.5 524.0 _ 1954 Cases 150.7 191.8 5.3 Days 1175.9 1563.2 19.3 9.6 7.9 31 .8 73.8 16.7 40.6 182.8 386.4 63.8 596.2 21 251.5 13 64.5 6 45 48 170 1955 Cases Days 127.6 176.6 3.6 978.4 1462.4 12.1 5.0 5.9 15.3 51.1 10.3 37.9 90.2 345.7 63-3 640.5 24 290 83 27 14 8 50 ^Source: Kozlk, 1957 DThe incidences are reportedly elevated, but reference values were not given. I ZTLISO both before and after work by 15 of 17 gluers; 4 of 10 machinists made errors, but only at the end of the workday. Cetnarowicz (1959) investigated the possibility of EDC poisoning in Polish oil refinery workers. The workers were employed in a mineral oil purification (dewaxing) process that involved mixing oils with a solvent that contained 80% EDC and 20$ benzene at 40C; the mixture was subsequently cooled to -24C, and precipitated paraffin was separated by centrifugation. Durations of exposure were not stated, but concentrations of EDC ranged from =10 to 200 ppm (0.04 to 0.8 mg/Z), with the highest excursions in the centrifuge room (Table 9-22). Benzene concentrations ranged from =3-1 to 7*8 ppm (0.01 to 0.025 mg/), but were not specifically reported for the different work areas detailed in Table 9-22. Nineteen workers (18 to 48 years old; 18 men, 1 woman) who had been employed for 2 to 8 months were included in the initial study group; 2 men and the woman were not examined due to medical history complications. The results of these examinations are summarized in Table 9-23 and in the text below. Ten of the 16 workers were employed in the centrifuge room, where EDC concentrations ranged from 62 to 200 ppm. All 10 of these workers complained of a burning sensation in the eyes and lacrimation, and 6 of the 10 workers stated that they had experienced dryness of the mouth, an unpleasant sweet aftertaste, dizziness, fatigue, drowsiness, nausea, occasional vomiting, constipation, and loss of appetite. Three of these workers also complained of pain in the epigastrum. All of the above-mentioned subjective complaints disappeared when the workers changed workplaces, but reappeared upon reexposure. Of the 6 workers employed in the pump and recrystallization rooms (10 to 37 ppm), only 1 complained of similar symptoms. A3 detailed in Table 9-23, clinical evaluation of the 16 workers indicated the likelihood of liver effects (tenderness to palpitation with minimal enlargement, epigastric pain, elevated urobilinogen 9-79 CMA 051713 TABLE 9-22 Concentrations of Ethylene Dichloride in Oil Refinery Mineral Oil Purification Process Air* Location ppm (repeated measurements) Centrifuge room 64 62 Pump room 1 16 10 Pump room 2 25 13 Pump room 2 30 37 Crystallization room 30 37 200 17 - Source: Derived from Cetnarowicz, 1959 by NIOSH, 1976. 9-80 r CMA 051714 051715 TABLE 9-23 Effects Observed in Polish Oil Refinery Workers* Concentration of 1,2-DCEa Effect 62, 64 and 200 ppm 10 to 37 ppm 10 to 200 ppm n 5 Dryness of the mouth; unpleasant sweet aftertaste; dizziness; lassitude; sleepiness; nausea; vomiting; poor appetite Burning sensation of the eyes that disappeared with adaptation to the atmosphere Epigastrium pain Insignificant tenderness of the epigastrium Livers tender to palpitation with minimal enlargement Normal arterial blood pressure Relaxed pulse (60 to 65/minutes) Intensified reflexes and automic neuroses Complaints similar to those associated with exposure to 62, 64 and 200 ppm Sweet aftertaste; dizziness; nausea; vomiting; lack of appetite Livers tender to palpitation with insignificant enlargement Epigastrium pain Emaciation (2 to 10 kg below expected weight) Unremarkable opthalmologic examination Unremarkable examination of upper respiratorytract, lungs and heart Augmented reflexes and vegetative neurosis Incidence 6/10b 10/10b 3/10" 7/10 4/10b 10/10 b 6/10* 3/10b (J 1/6 6/42^ 3/42d 2/42 16/16 16/16 16/16 3/16 9TLX S 0 XfWO Concentration of 1,2-DCEa 10 to 200 ppm (cont.) TABLE 9-23 (cont.) Effect Icteric skin coloration Elevated urobilinogen levels X-ray observable chronic catarrh of the stomach with atrophy of the mucous membrane Periodic spasm of the pylorus Moderate hyperchromia anemia (3,430,000 erythrocytes/cu mm and 60% hemoglogin) Slight reticulocytosis (0.1 to 0.3%) Diminished osmotic fragility of erythrocytes in NaCl Slight leukocytes (11 ,200/cu mm) Low platelet count (40,000 to 55,000 cu mm) Decreased number of neutrophils (40 to 50%) Slightly increased number of neutrophils (70 to 78%) with decreased number of lymphocytes (15 to 25%) "Abnormal" distribution of white blood cells Increased number of erythrocytes. Increased percentage of polymorphonuclear neutrophils, mild stimulation of erythropoiesis with a less significant increase of leukopoiesis in the bone marrow Elevated serum bilirubin (2.3 mg %) Incidence 1/l 6 "majority" of 16 6/1 6 3/16 1/I3e 4/13e 6/1 3e 1/I3e 2/1 3e 2/1 3e 6/1 3e 4/13e 5/1 3e 1/16 TABLE 9-23 (confc.) 9-83 Concentration of 1,2-DCEa Effect Incidence 10 to 200 ppm (cont.) Elevated blood nonprotein nitrogen (55 mg %} Normal overall quantity of serum protein Diminished serum albumin Elevated serum globulin levels Diminished blood fibrin content Positive Takata-Ara liver function test Borderline Takata-Ara liver function test Positive Cadmium turbidity test Borderline Cadmium turbidity test Negative glucose tolerance test Decreased secretion of gastric hydrochloric acid X-ray observable catarrhal changes of gastric mucosa Periodic pyloric spasms 1/16 16/16 6/1 6 8/16 3/16 4/1 6 5/1 6 6/1 6 5/16 8/16 1 2/1 6 6/1 ^ 3/6 Source: Cetnarowicz, 1999 (derived from NIOSH, 1976) Concurrent exposure to =3 to 8J benzene (see text) ^Workers employed in the centrifuge room (see Table 9-22) cWorkers employed in the pump and crystallization rooms (see Table 9-22) d42 workers initially examined; effects on these workers prompted further examinations of 16 workers from one shift (see below). 0 Hematological examinations were performed on 13 workers. f 3 of the 6 with catarrh CMA 051717 levels, positive Takata-Ara liver function tests, negative glucose tolerance tests), and changes in the gastrointestinal tract (X-ray observable chronic gastritis with mucosal inflammation, pyloric spasms, impaired hydrochloric acid secretion). The distribution of these findings by room (i.e., high or low exposure) was not, however, given. Other abnormal findings included blood and bone marrow changes that may be attributable to the concurrent exposure to benzene (Table 9-23). Rozenbaum (1947) reported the results of studies of approximately 100 Russian workers from different industries (not specified) who had experienced exposure to EDC at levels below 25 ppm (<0.1 mg/2,) in air for 6 months to 5 years. "Many" of these workers exhibited non-specific functional nervous system disorders (e.g., red dermographism, muscular torus, bradycardia, increased perspiration), and "some" of the subjects complained of fatigue, irritability, headaches and insomnia. Hematologic or functional organ changes (not elaborated) were not, however, noted. The incidence of the observed effects, average exposure concentrations, the range of exposure concentrations to which the workers were exposed and information on experimental design were not reported. Brzozowski et al. (1954) considered that absorption of EDC through the skin was largely responsible for adverse health effects among certain agricultural workers in Poland. EDC was used as a fumigant and work practices involved transport of the liquid to fields in barrels, pouring the EDC by hand into buckets, carrying the open buckets to the site of application, and pouring the EDC into a series of holes in the ground. The workers were exposed to particu larly high concentrations of EDC vapor during pouring. Significant dermal exposure is indicted by the frequent spilling of quantities of EDC on clothes and sleeves during the transport of the open buckets to the places of application 9-84 CMA 051718 (resulting in soaked clothing that was not changed), and by the fact that the workers used EDC to wash their skin. A single atmospheric sample, comprising 10 subsaraples collected from locations representative of the working zone (the collection apparatus was moved from place to place with the workers), showed a concentration of 4 ppm of EDC. Because of the practical difficulties of sample collection (the workers did not stay at one location sufficiently long to collect an entire sample), conditions were simulated in a laboratory to estimate potential exposure concentrations better. Analysis of laboratory samples showed concentration of -14.5 to 15 ppm; a sample taken during the pouring of EDC into buckets, considered to be the maximum exposure of a worker, contained 60 ppm. Medical examinations were performed on 118 of the agricultural workers (Brzozowski et al., 1954). Ninety had positive findings that included conjunc tival congestion (82/118), weakness (54/118), reddening of the pharynx (50/118), bronchial symptoms (43/118), metallic taste in the mouth (40/118), headache (39/118), dermatographism (37/118), nausea (31/118), cough (30/118), liver pain (29/118), conjunctival burning sensation (24/118), tachycardia (21/118), and dyspnea after effort (21/118). The Quick Test for hippuric acid was used to evaluate liver function and was reported to be positive in 40 of 56 investiga tions. Skin sensitization tests with pieces of gauze soaked with 0.1 $ EDC in alcohol or 50$ EDC in soybean oil were reportedly negative when scored after 40 hours of contact, but the number of workers tested was not stated. 9-85 CMA 051719 9.2.2. Effects in Animals 9.2.2.1. ACUTE EXPOSURE 9.2.2.1.1. Exposure -- The observed toxic effects of ingested EDC in laboratory animals summarized in Table 9--24 tend to confirm the findings from cases involving human oral exposures. The LDpu for a single oral exposure was reported to be 680 mg/kg in a study that employed 80 rats and 4 dosage levels (MeCollister et al., 1956) and 770 mg/kg in a range-finding study of rats (Smyth et al., 1969). These data suggest that EDC is moderately hazardous by the oral route, and indicate that the compound is more acutely toxic than 1,1-dichloro- ethane, 1,1,1-trichloroethane, pentachloroethane, and hexachloroethane (Patty, 1981). According to a World Health Organization report (1970; cited by Torkelson and Howe, 1981) rabbits and mice are as susceptible as rats to EDC. Heppel et al. (1945) observed 60% mortality (6 of 10 treated) in mice 10 days after an acute oral dose of 700 mg EDC/kg body weight, which was administered as a 5% solution in olive oil. Similar treatment with 900 mg/kg EDC in 5% olive oil produced mortality in 10/10 mice, but 6/6 survived a dose of 500 mg/kg given as a 10 percent solution in olive oil. Munson et al. (1982) recently determined that the single oral dose LD^^'s of EDC for male and female CD-I mice were 489 and 413 mg/kg, respectively. In these studies, EDC was apparently administered in water solution, and all mice died over a 48-hour period. Gross pathologic examination indicated that the lungs and liver appeared to be the target organs. Dogs and humans may tolerate larger doses. Since EDC causes vomiting, the higher tolerance in dogs and humans is likely attributed to the ability to vomit in these two species. Ristler and Luckhardt (1929) found that oral doses >500 mg/kg tended to be vomited by dogs. In 2 unanesthetized dogs (weighing 9 and 12.5 kg) administration of 0.63 g/kg EDC by gavage produced initial excitement (8 minutes after treatment) 9-86 CMA 051720 Route Oral Oral Oral Oral Oral Inhalation Inhalation TABLE 9-24 Effects of Acute Exposure to Ethylene Dichloride Species Nuaber Dose Effect rats 80 rats 6/group alee 10 10 6 alee US (CD-I, sale) aloe NS (CD-I,resale) 0.68 g/lcg 0.77 g/lcg 0.90 g/kg (In olive oil) 0.70 g/lcg (in olive oil) 0.50 g/kg 0.489 g/kg (in water) 0.413 g/kg LD50 "50 10/10 dead 6/10 dead no deaths "50 "50 rats 6 aale or feaale 1000 ppa for 4 h Sheroan Strain (100-150 weight) 2/6, 3/6 or 4/6 dead in 14 day observation period. rats (feaale) 4-6/group 12,000 ppa for 0.1 h 0.2 h 3000 ppa for 0.3 h 0.5 h 1000 ppa for 1.5 h 3.0 h 300 ppa for 3.0 h 5.5 h 200 ppa for 7.0 h Ho adverse effects Adverse effect No adverse effeot Adverse effeot Ho adverse effeot Adverse effect No adverse effeot Adverse effect No adverse effect Reference McColllater et al., 1956 Smyth et al., 1969 Heppel et al., 1945 Munson et al., 1982 Munson et al.. 1982 Carpenter et al., 1949 Spencer et al. , 1951 O 051721 CMA 0 5 1 ^ 2 2 9 - SC Route Inhalation Inhalation Species rats rats mice guinea pigs rabbits cats hogs raccoons Number 20 22 40 40 51 44 32 44 22 10 30 10 41 24 10 32 31 32 30 20 33 20 20 20 15 15 12 13 22 20 23 14 12 16 3 2 2 TABLE 9-24 (oont.) Dose 3000 ppm for 6 h 3000 ppm for 5 h 3000 ppm for 4 h 3000 ppm for 3 h 3000 ppm for 2 h 3000 ppm for 1.6 h 3000 ppm for 1.0 h 3000 ppm for 0.7 h 3000 ppm for 0.5 h 1500 ppm for 8 h 1500 ppm for 7 h 1500 ppm for 6 h 1 500 ppm for 4 h 1 500 ppm for 3 h 1 500 ppm for 2 h 1000 ppm for 8 h 1000 ppm for 7 h 1 000 ppm for 6 h 800 ppm for 7 h 600 ppm for 8 h 600 ppm for 7 h 600 ppm for 5 h 300 ppm for 7 h 3000 ppm for 7 h 3000 ppm for 3.5 h 3000 ppm for 1.5 h 1 500 ppm for 7 h 1500 ppm for 4 h 3000 ppm for 7 h 1500 ppm for 7 h 1500 ppm for 2 h 3000 ppm for 7 h 1500 ppm for 7 h 3000 ppm for 7 h 3000 ppm for 7 h 3000 ppm for 7 h 3000 ppm for 7 h Effect 20/20 dead 22/22 dead 38/40 dead 24/40 dead 6/51 dead 1/44 dead 1/32 dead 1/44 dead 0/22 dead 7/10 dead 24/30 dead 7/10 dead 2/41 dead 1/24 dead 0/10 dead 20/32 dead 17/31 dead 5/32 dead 10/30 dead 4/20 dead 3/33 dead 0/20 dead 0/20 dead 20/20 dead 1 5/1 5 dead 0/15 dead 4/20 dead O/13 dead 22/22 dead 20/20 dead 1/23 dead 14/14 dead 6/12 dead 12/16 dead 0/3 dead 2/2 dead 0/2 dead Reference Spencer et a. Heppel et al TABLE 9-2H (Cont.) Route Species Number Dose Effect Reference Intraperitioneal guinea pig (sale) 4/group 1 50 , 300 and 600 mg/kg 1 dose Subcutaneous dogs cats rabbits albino rats 6 NS NS NS 0.94 or 1.26 g/kg body weight 0.94 or 1.26 g/kg body weight 0.94 or t.26 g/kg body weight 0.94 or 1.26 g/kg body weight Subcutaneous rats Subcutaneous mice 10/group 4/group at the lowest dose 1 0/group 0.33, 0.5, 0.75, 1.0, 1.25 g/kg (in olive oil) 0.25, 0.38, 0.75 g/kg (in olive oil) No effect on liver histology 24 hours after injection. No effect on serum ornithine carbamyl transferase (OCT) activity 24 hours later at 150 or 300 rag/kg. Increased OCT activity at 600 mg/kg; death of 1 animal. Divineenzo and Krasavage, 1974 Death within 24 hours at Kuewabara et al., 1968 higher dose; histology: mild perilobular fatty degeneration in liver, swelling of tubular cells and mild hemorrhage in the kidney, lung edema his tological changes more marked in dogs. In dogs only: corneal opacity evident by 10 hours; necrosis of corneal endothelium; corneas became clear by fifth day. 0.75/kg: 50$ mortality; 0.38 g/kkg: no deaths Heppel et al., 1945 0.38 g/kg: 80$ mortality 0.25 g/kg: no deaths Heppel et al., 1945 h = hours CMA 0 5 1 7 2 3 followed by decreased excitement. Progressive incoordination and salivation was evident. In 24 minutes, sleep (from which the dogs could be aroused) supervened for another 20 minutes, at which time depression was relieved. Vomiting occurred subsequently, after which signs of recovery were evident (Kistler and Luckhardt, 1929). Larger doses of 1.26 g/kg in 2 dogs, 1.89 g/kg in one dog, and 2.51 g/kg in two dogs produced similar symptoms of greater severity. Vomiting occurred earlier after administration of the compound; little food or water was ingested and vomiting persisted in one dog that received 1.26 g/kg EDC. The corneas of the eyes in all five animals were opaque within 24 hours. 9.2.2.1.2. Dermal Exposure -- Skin absorption LD^Q values of 4.9 g/kg (Torkelson and Rowe, 1981) and 2.8 g/kg (Torkelson and Rowe, 1981) have been determined with rabbits. Strong erythema, edema, or slight necrosis was produced upon application of 0.01 mS, of undiluted EDC to the uncovered rabbit belly (Smyth et al., 1969). 9.2.2.1.3. Inhalation Exposure -- Sayers et al. (1930, cited in Browning, 1965 and NIOSH, 1976) reported that death occurred in guinea pigs within a few minutes of exposure to 100,000 ppm EDC, and on the day following exposure to 10,000 ppm EDC for 25 minutes. Progressive signs of intoxication were observed in groups of 3 or 6 guinea pigs that were exposed to EDC at concentrations ranging from 600 to 60,000 ppm for durations up to 8 hours. One- third of each group was sacrificed immediately after exposure, another one-third sacrificed after 4 days of exposure and the final one-third was sacrificed at the end of the eighth post-exposure day unless death supervened. The signs of intoxication (squinting and lacrimation of the eyes and rubbing of the nose, vertigo, static and motor ataxia, retching movements, unconsciousness, incoordi nation of extremities, and marked changes in respiration) appeared in less than 10 minutes, and death occurred in 30 minutes, after exposure to 60,000 ppm EDC. 9-90 CMA 051724 Exposure to a lower concentration (10,000 ppm) for 15 to 20 minutes produced these symptoms in 25 minutes and delayed death a day or more after exposure. No mortality or signs of intoxication occurred in guinea pigs exposed to 1200 ppm for 8 hours. Congestion and edema of the lungs and generalized passive congestion of the visceral organs were observed in animals that died during exposure (Sayers et al., 1930, cited in NI0SH, 1976). In animals sacrificed immediately after exposure, congestion of the liver, spleen, lungs, and kidneys was observed. Pulmonary congestion, pulmonary edema and renal hyperemia were seen in those animals that died 1 to 8 days after exposure, and the renal and lung effects were more pronounced in the animals sacrificed 3 to 4 days post-exposure than in those sacrificed immediately after exposure. Partial resolution of visceral effects was observed by 8 days after exposure. In these studies, the severity of pathological changes was dependent on duration and concentation of exposure. Heppel et al. (1945) reported that a single exposure to 3000 ppm EDC (12.4 mg/#,) for 7 hours produced death in all of 14 treated guinea pigs within 3 days of exposure. The guinea pigs were inactive and breathing was labored after expo sure. Microscopic examination of 11 guinea pigs revealed congestion in the liver, lung and adrenal glands. Focal necrosis of the adrenal cortex in 5 of the guinea pigs, and slight to moderate fatty degeneration of renal tubular epithe lium was noted in 8 of the animals. In another group of 8 guinea pigs similarly exposed to 3000 ppm EDC, fatty infiltration of the myocardium was observed in all of 7 animals that were examined histologically. Changes in the lung, kidney and adrenal cortex, similar to those observed in the first group of animals were also found. Heppel et al. (1945) also found that inhalation exposure to 3000 ppm EDC (12.4 mg/#,) for 7 hours was fatal to rats, mice and rabbits within 3 days of 9-91 CMA 051725 exposure. Varying degrees of narcosis were observed in the animals during exposure, and dyspnea and increasing weakness preceded death. Pulmonary conges tion, mild to moderate degeneration of renal tubular epithelium and liver, and occasional necrosis of the adrenal cortex were observed at autopsy; congestion of the spleen was additionally found in rats. Inhalation of 3000 ppm EDC for 7 hours was fatal for 2 hogs but was not fatal for 2 raccoons or 3 cats. Reduction of dose by exposure to one-half the concentration of EDC (1500 ppm) for a similar duration (7 hours) was lethal to 6 of 12 guinea pigs and to less than one-fifth of the rats (4 of 20). This lower dose was fatal, however, to all of a group of 20 mice. Reduction of the duration of exposure in rats to 4 hours and mice to 2 hours decreased mortality (Heppel et al., 1944). Spencer et al. (1951) subjected groups of 4 to 6 female rat3 to varying concentrations of EDC (200, 300, 1000, 3000 or 12,000 ppm) for varying durations to determine the doses and durations of single exposures that produced and did not produce adverse effects. No-adverse effect exposures ranged from 200 ppm (0.8 mg/Jt) for 7 hours to 12,000 ppm (48.6 mg/A) for 0.1 hours. Doubling the duration of exposure at 12,000 ppm produced adverse effects. Exposure to 3000 ppm EDC for 0.3 hours was without effect, but increasing the duration of exposure to 0.5 hours caused adverse effects. Spencer et al. (1951) also sacrificed groups of 10-54 rats after inhalation exposure to EDC at concentrations ranging from 300 to 20,000 ppm for durations of 8.0 to 0.1 hours. A decrease in body weight, increase in liver and kidney weight, increase of blood urine nitrogen, increase of plasma prothrombin clotting time, decrease in serum phosphatase, increase in liver lipids and histo logical changes in the kidneys, liver and adrenal glands were observed. The kidney changes consisted of tubular damage that ranged from slight parenchyma tous degeneration of the epithelium to complete necrosis accompanied by 9-92 051^26 CMA- interstitial edema, congestion and hemorrhage. Changes ranging from slight congestion and slight parenchymatous degeneration to marked hemorrhagic necrosis were observed in the liver. Some parenchymatous degeneration of the adrenal cortex was found in the most severely affected animals. Pulmonary congestion and edema was observed at concentrations above 3000 ppm. Carpenter et al. (1949) reported an inhalation LCj.q in rats to be 1000 ppm in air for a 4-hour exposure. 9.2.2.1.3.1 Central Nervous System and Cardiovascular System. Inhala tion of 3000 ppm EDC (12.4 mg/Jl) for 7 hours in five species (rabbits, guinea pigs, hogs, rats and mice) produced varying degrees of narcosis (Heppel et al., 1945). Spencer et al. (1951) partially attributed the inactivity or stupor and a slower response to handling in rats, at vapor concentrations of 3000 ppm and lower, to toxic injury to organs other than depression of the central nervous system. At concentrations of 12,000 ppm and lower, varying degrees of "drunken ness" were attributed to depression of the central nervous system. Considerable depression of the central nervous system was produced at 22,000 ppm causing death in rats within 0.4 hours. Animals that died from exposure to 20,000 ppm EDC were in a state of deep anesthesia during the period of exposure. At all vapor concentrations a large proportion of the rats died suddenly after leaving the exposure chamber. Marked cyanosis, reduced body temperature, stupor or coma, and failing respiration were observed. Spencer et al. (1951) hypothesized that this response is suggestive of cardiovascular collapse. Other deaths, which occurred over a period of two to seven days, were accompanied by loss in body weight and other signs of toxicity; the authors attribute these deaths to renal injury. Inhalation of EDC (0.1 to 5 cc, introduced by volatilization into a Jackson Carbon Dioxide Absorption Apparatus) produced a drop in blood pressure, depres sion of the knee jerk reflex and stimulation followed by a short cessation of respiration in sodium barbital anesthetized dogs (number unspecified) (Kistler 9-93 CMA. 051727 and Luekhardt, 1929). These dogs had been subjected to spinal cord transection prior to EDC treatment. Similar effects on blood pressure and the knee jerk were observed in sodium barbital anesthetized dogs (11-20 kg) after intravenous injection of amounts as small as 0.1 ce, while 0,3 cc caused a cessation of respiration for a duration of up to 40 seconds. 9.2.2.1.3.2. Eyes. Inhalation of EDC was observed to produce a clouding of the cornea in dogs and foxes, but not in rabbits, guinea pigs, mice, rats, hogs, raccoons, chickens or cats (Heppel et al., 1944). A single exposure to a vapor concentration of 1000 ppm for 7 hours produced turbidity in the eyes of dogs (Table 9-25). A higher concentration of 3000 ppm caused turbidity in the cornea of the red fox after a single exposure of 7 hours duration. Repeated exposure interspersed with two days of no exposure at a lower concentration of 400 ppm produced clouding in the cornea of six dogs, which cleared during the days free of exposure. Development of resistance to toxic effects in the cornea after repeated exposure was observed. Corneal opacity was also observed within 1 0 hours in 6 dogs that received a subcutaneous injection of 0.94 g/kg EDC (Kuwabara et al., 1968) (Table 9-25). Marked swelling of most of the endothelial cells of the cornea was seen at 12 hours, and the reparative process was found to occur by 24 hours. Similar corneal changes were produced in dogs, rabbits and cats when EDC (a 1$ suspension dissolved in mineral oil and homogenized in 0.5$ gelatin solution) was instilled directly into the anterior chamber of the eye. The authors suggested that the apparent vulnerability of the dog eye to EDC by routes other than direct local application was due to "a greater amount of dichloroethane coming in contact with the dog endothelium rather than an unusual susceptibility of the eye itself." 9-94 CMA 051728 Route Inhalation Inhalation Inhalation Inhalation TABLE 9-25 Effects of Ethylene Bichloride on the Cornea Speclea Huaber of Anlaals Dose Effect Reference rabbits guinea pigs ice rats hogs raccoons cats 16 14 19 20 2 2 2 3000 ppa for 7 h 1 exposure No effect on eyes Heppel et al., 1944 red fox 1 3000 ppa for 7 h 1 exposure red fox raccoon dog 1 (sane aniaal as tested above) 2 1500 and 1000 ppa tor 7 h 1 exposure at each concentration 6 1500 ppa for 7 h, 1 exposure Loss of appetite; barely detectable turbidity in eyes within 6 h; eyes were opaque 2 d later; death 6 d after exposure. No adverse effect on eyes Heppel et al., 1944 Heppel et al., 1944 No effect on cornea of 1; faint turbidity in eye of 1; intense clouding of both corneas In 4; histology; corneal edeaa, degeneration or corneal eplthellua, Infiltration of polymorpho nuclear leucocytes into sub stantia propria. Heppel et al., 1944 TABLE 9-25 (Cont.) Route Speclea Number of Animals Dose Effect Reference Inhalation dog 10 Inhalation Inhalation guineapigs rats rabbits dog US NS NS 3 1000 ppa for 7 h, 1 exposure 1500 ppa for 7 h, repeated exposure 1500 ppm for 7 h, repeated exposure Symmetric turbidity in the corneas of 8; up to 3 weeks required for partial regression. Death in most animals after 4 exposures; no adverse effect on eyes. Heppel et al.t 1944 Heppel et al., 1944 Intense bilateral corneal opacity in both eyes 48 h after first exposure; death after 5, 6 or 30 exposures. Turbidity in eyes retained throughout. Heppel et al. 1944 Jo a\ Inhalation dog 10 1000 ppm for 7 h, 5 d/wk, Increasing turbidity of Heppel et al., 1944 repeated exposure, cornea during 5 exposure days, length NS clearing during 2 non-exposure days; increased tolerance with successive bouts of exposure; complete resistance of cornea finally. Inhalation cats onkeys NS 1000 ppm for 7 h, 5 d/wk, repeated exposure, length NS No adverse effects in eyes Heppel et al., 1944 chickens rodents (species, NS) CMA 0 5 1 7 3 0 TABLE 9-25 (Cont.) Route Subcutaneous Inhalation Species Number of Animals dogs (puppies and adults) 6 cats rabbits albino rats dogs NS NS NS 6 Dose 0.9*1 g/kg 0.9*1 g/kg 0.9*1 g/kg 0.9*1 g/kg 400 ppm for 7 h, 5 d/wk, for 10 weeks Effect Reference Corneal opacity evidence within 10 hours; necrosis of corneal endothelium; clearing by fifth day. Kuwabara et al., 1968 No effect on cornea Kuwabara et al., 1968 No effect on cornea Kuwabara et al., 1968 No effect on cornea Kuwabara et al., 1968 Mild clouding during first week, cleared during 2 non exposure days; 5th week of exposure; faint opacity of cornea 10th week of exposure: no trace of turbidity. Heppel et al., 1944 9-9 7 h = hour; d = da/; wk = week 9.2.2.2. SUBCHRONIC AND CHRONIC EXPOSURE 9.2.2.2.1. Oral Exposure -- EDC has been tested for carcinogenicity in an NCI bioassay with Osborne-Mendel rats and B6C3F1 mice (NCI, 1978). The compound was administered at time-weighted average doses of 95 and 47 mg/kg (rats of both sexes), 195 and 97 mg/kg (male mice) and 299 and 149 mg/kg (female mice) for a period of 78 weeks, according to the regimen described in Section 9.5. Fifty animals of each sex were treated/dose level, and the animals were observed for an additional 28 weeks (rats) or 12-13 weeks (mice) after pretreatment. As detailed in Section 9*5, no distinct dose-related mean body weight depression was apparent in either male or female rats relative to vehicle controls. Mortality was, however, early and severe in many of the dosed rats, particularly those given the highest dose. Mean survival was -55 weeks for the high-dose males and females. Toxic rather than carcinogenic effects of EDC appeared to be responsible for the deaths. No distinct, dose-related mean body weight depression was observed in male mice or in low-dose female mice, but mean body weight depression for high-dose female mice was apparent as early as the 15th week of treatment. A significant positive association between increased dosage and elevated mortality was found for female mice; 12% (36/50) of the females died between weeks 60 and 80. The presence of one or more tumors in these mice suggests that these deaths may have been tumor related. There was no statistically significant association between dosage and mortality for male mice. Munson et al. (1982) evaluated the effect of subchronic 14-day and 90-day oral EDC exposures on the immune response of male CD-I mice. EDC was adminis tered daily by gavage in the 14-day study at levels of 4.9 and 49 mg/kg, which represent 0.01 and 0.1 times the single dose LD^q determined in a preliminary 9-98 CMA 051732 study (Section 9.2.2.1.1). For the 90-day study, EDC was administered in the drinking water at levels intended to be equivalent to those administered by gavage; in addition, a 10-fold higher dose was added because the results of the 14-day exposure indicated that doses >49 mg/kg could be tolerated. The calcu lated time-weighted average doses of EDC delivered in the 90-day study based on actual fluid consumed were reported to be 3 24 and 189 mg/kg. Results of standard toxicological analyses showed that body weight was unaltered during the 14-day study, but that EDC did elicit a dose-dependent decrease in growth rate and fluid consumption when administered over 90 days. Thirty-two mice per dose were weighed in the 90-day study but the numbers weighed in the 14-day study were not stated. EDC did not alter the weights of selected organs (liver, spleen, lungs, thymus, kidneys, brain) in either of the studies. It was further found that exposure to 49 mg/kg/day EDC caused a 30> decrease in leukocyte number in the 14-day study, although the number of leukocytes was normal after 90 days of exposure. Other hematologic parameters (hematocrit and hemoglobin evaluated after 14 or 90 days, erythrocytes and platelets evaluated after 90 days), coagu lation values (fibrinogen and prothrombin time evaluated after 14 days), and clinical chemistry parameters (lactic dehydrogenase, serum glutamic-pyruvate transaminase and blood urea nitrogen evaluated after 14 days) were unaltered by EDC exposure. The hematological/coagulation/clinical chemistry parameters were assessed in 10-12 and 16 mice/dose in the 14-day and 90-day studies, respec tively. The status of the humoral immune system was determined by measuring the number of IgM spleen antibody-forming cells (AFC) to sheep erythrocytes (sRBC) after 14 and 90 days, the serum antibody level to sRBC after 90 days, and the lymphocyte response to the B-cell mitogen LPS (lipopolysaccharide from Salmonella typhosa 0901) after 90 days (Munson et al., 1982). Results of assays 9-99 CMA 051733 with 10-12 mice (14 days) and 16 mice/dose showed that EDC produced a significant (P<0.05) reduction in AFCs at 4.9 and 49 mg/kg in the 14-day study (25 and 40$ suppression, respectively), but that EDC exposure for 90 days caused no signifi cant (P<0.05) change in the number of AFC spleen cells (although there was an indication of suppression at 189 mg/kg/day). There also appeared to be an EDC dose-dependent reduction in hemagglutination titer after 90 days, although this response was not significant at the p<0.05 level. EDC did not alter the spleen cell response to three concentrations of LPS. Cell-mediated immunity was assessed by measuring the delayed hypersensitivity response (DTH) to sRBC and the response to the T-lymphocyte mitogen, concanavalin A. EDC produced a slight but significant (P<0.05) non-dose-dependent inhibition at both 4.9 and 49 mg/kg in the 14-day study, but 90-day exposure did not alter DTH response or spleen lymphocyte response to concanavalin A. EDC did not alter the functional activity of the reticuloendothelial system, as measured by the vascular clearance rate and C1 tissue (i.e., liver, spleen, lungs, thymus or kidneys) uptake of Cr after 90 days. Dexamethasone was used as a positive control in the above studies. Results of similar tests with female mice were not presented, but reportedly were not remarkably different from the males. The results of the above experiments (Munson et al., 1982) indicate that EDC produced a suppression of both humoral and cell-mediated immunity when adminis tered daily for 14 days by stomach tube, but not when consumed in the drinking water for 90 days (although there were trends toward suppression at the high exposure level). Two explanations were offered for these results. First, the effective dose at the immunocompetent cell may be higher with the bolus presenta tion than with the semi-continuous self-administration in the drinking water. Second, EDC may induce its own metabolism over the longer exposure period, thereby effectively reducing the amount of chemical reaching the immune cells. 9-100 CMA 051734 9.2.2.2.2. Inhalation Exposure -- Four animal species (15 rats of both sexes, 8 guinea pigs of both sexes, 2 male and 1 female rabbits, and 2 male monkeys) were exposed to EDC vapor at levels of 400 ppm (1.62 rag/#) and 100 ppm (0.405 mg/#) for 7 hours daily, 5 days/week for 6 months (Spencer et al., 19 51). In addition, 15 rats of both sexes and 8 guinea pigs of both sexes were similarly exposed to 200 ppm EDC (0.81 rag/S,) for 1 51 and 180 7-hour periods, respectively. Repeated exposure at 100 ppm did not produce adverse effects iri any of the four species as determined by general appearance and behavior, mortality, growth, body and organ weights, and gross and microscopic examination of tissues (Table 9-26). No adverse effects were indicated by periodic hematological examination of the treated rats, guinea pigs, or rabbits. Exposure to 200 ppm EDC did not produce any adverse effects in the rats, but did elicit slight parenchymatous degeneration of the liver with a few diffusely distributed fat vacuoles in half of the guinea pigs. A slight increase of total lipid, phospholipid, neutral fat and free and esterified chloresterol was also found in the guinea pigs exposed to 200 ppm EDC, and the final body weights of the treated male guinea pigs were significantly different from control animals. Severe toxic effects were found in the rats, guinea pigs, and monkeys that were exposed to 400 ppm EDC (1.62 mg/#) (see Table 9-26). The three rabbits that were exposed to 400 ppm EDC were not adversely affected, although Heppel et al. (1946) reported a high mortality in rabbits exposed to this concentration of EDC. Heppel et al. (1945) exposed rats, mice, rabbits, guinea pigs, hogs and dogs to 1 500 ppm EDC (6.4 mg/#,). Almost all of these animals died before 6 daily exposures of 7 hours each were completed. Hemorrhaging in the lungs, gastro intestinal tract and adrenals; fatty degeneration of the myocardium; degenera tion within the renal tubules; and congestion of the liver and intestines were found at autopsy. 9-101 CMA 051735 CMA 0 5 1 7 3 6 Route Inhalation Inhalation Inhalation \o 1I o ho Inhalation Species Humber TABLE 9-26 Effects of Subchronic Exposure to Ethylene Bichloride Dose Effect Reference rata 26 (average weight: 198 g) 1000 ppa (3.0 mg/i) 7 h/d x 5 d/wk 20 dead within 15 exposures; Heppel et al., 1946 progressive weakness culmin ating in Inability to stand. Histology of 4 rats: degeneration and proliferation changes in renal tubular epltheliua; chronic splenitis in 20 rats; pulmonary congestion in 2 rats. rabbits 6 (average weight: 2480 g) 1000 ppm (3.9 mg/t) 7 h/d x 5 d/wk 5 dead within 43 exposures survivor exposed 64 days Heppel et al., 1946 guinea pig guinea pig guinea pig 15 (average weight: 161 g) 10 (average weight: 558 g) 16 (average weight: 900 g) 1000 ppa (3.9 mg/t) 7 h/d x 5 d/wk 1000 ppa (3.9 mg/t) 7 h/d x 5 d/wk 1000 ppa (3.9 mg/t) 7 h/d x 5 d/wk 10 dead within 2 exposures 10 dead within 2 exposures 16 dead within 4 exposures lacrlmation and marked Inactivity during exposure; congestion of lung, liver, heart, kidney, adrenal gland and spleens. Heppel et al., 1946 Heppel et al.. 1946 Heppel et al., 1946 dogs 6 (7000-8300 g) 1000 ppa (3.9 mg/l 7 h/d x 5 d/wk 1 dead after 30 exposures 1 dead after 43 exposures Heppel et al., 1946 TABLE 9-26 (Cont.) Route Inhalation Inhalation Inhalation Inhalation Inhalation Inhalation Species Humber Dose Effect Reference cats 6 (2660 g average weight) 1000 ppm (3.9 mg/E) 7 h/d x 5 d/wk 2 dead after 43 exposures congestion and fatty meta morphosis in liver of all. Heppel et al., 1946 monkeys 2 (4880 g average weight) 1000 ppm (3.9 mg/E) 7 h/d x 5 d/wk 1 dead after 2 exposures 1 dead after 43 exposures; fatty degeneration of liver and slight fatty changes in kidney; focal myocarditis in 1 monkey. Heppel et al., 1946 cats 4 500 ppm 6 h/d x 5 d/wk for 6 wks Ho deaths; dilated hearts; Hoffmann et al. , 1971 blood urea nitrogen increased to 114 mg/100 mi. rabbits 4 500 ppm 6 h/d x 5 d/wk for 6 wks 3/4 dead after 10-17 exposures; Hoffman et al. , 1971 dilated hearts. guinea pigs 10 500 ppm 6 h/d x 5 d/wk for 6 wks 9/10 dead after 4--14 exposures; apathy and weight loss, fatty degeneration and necrosis of myocardium, liver, kidney and adrenals. Hoffman et al. , 1971 rats 10 500 ppm 6 h/d x 5 d/wk for 6 wks All dead after 1-5 exposures; hyperemia of lungs; fatty degeneration and neorosis of myocardium, liver, kidney, and adrenals. Hoffman et al., 1971 Cm 051737 CMA 0 5 1 7 3 8 Route Inhalation Speclea rats Number Dose 15 aale 1 feaale (average weight: 167 g) 1(00 ppm (1.54 mg/1) for 7 h/d x 5 d/wk for 60 exposures Inha1ation 23 controls 0 guinea pigs 18 male 2 feaale (average weight: 303-737) 400 ppm (1.54 mg/1) for 7 h/d x 5 d/wk Inhalation rabbits Inhalation dogs 30 controls 0 2 male 3 female (average weight: 3110 g) 4 controls 400 ppm (1.54 ag/1) for 7 h/d x 5 d/wk 0 3 male puppies (average weight: 1330 g) 6 female adult (average weight: 9100 g) 400 ppm (1.54 ng/R) for 7 h/d x 5 d/wk for 167-177 exposures 1) controls 0 Effeot Reference Total dead: 9 (6 dead after 4 exposures) Histology In 6 survivors: no changes In 5; diffuse ayocarditis and fatty degeneration of liver, kidney and heart in 1. 1 dead. Histology: no change. 13 dead after 45 exposures 1 dead after 65 exposures Histology in 10: fatty degeneration of liver and kidney in 4 dead; fatty degeneration of heart in 2 dead; no histological change in 6 survivors of exposure. 3 deaths. All dead after 97 exposures. Ho hematological changes. Heppel et al., 1946 Heppel et al.. 1946 Heppel et al.. 1946 No deaths. Ho deaths; 6 adults: no effect on mean arterial pressure, Bromsulphaleln excretion rate, prothrombin time, plasma total protein, albumin, globulin, nonprotein nitrogen, icterus index, urine; alight fatty change in livers of 5 and kidney of 1. No deaths. Heppel et al., 1946 Bouts Inhalation Specie? rats Humber 15 sale, 15 female Inhalation rats 20 Bale, 20 female Inhalation guinea pigs 8 Bale, 8 female InhalatIon guinea pigs 2 male TABLE 9-26 (Cont.) Dose 400 ppm (1.62 mg/1) 7 h/d, 5 d/wk 400 ppm (1.62 ag/1) 7 h/d, 5 d/wk 400 ppa (1.62 mg/t) 7 h/d, 5 d/ wk 400 ppa (1.62 ag/t) 7 h/d, 5 d/wk Effect Reference Feaales died within 10 exposures. Males died within 40 exposures. Spencer et al., 1951 60( mortality after 2 or 3 exposures; rapid los3 of body weight; slight increase of liver and kidney weight; slight cloudy swelling of liver with a few large fat vacuoles. Ho significant differences In blood urea nitrogen, nonprotein nitrogen, serua phosphatase and plasaa prothroabln clotting tlae. Males died within 10 exposures. Females died within 24 exposures. Spencer et al., 1951 Sacrificed after 1, 3, 4, and Spencer et al., 1951 10 exposures; rapid loss of body weight; Increased liver and kidney weight; slight to moderate central fatty degeneration of liver; slight to aoderate cloudy swelling of renal tubular epithelium; no alteration in lungs, heart, spleen and testes; increased BUN and increased blood non protein nitrogen levels; no change in serua phosphatase or plasaa prothrombin clotting tlae. TABLE 9-26 (Cont.) Route Inhalation Inhalation Inhalation Spectra Number Dose Effect Reference rabbita ' 2 male, 1 female 400 ppm (1.62 mg/i) 7 h/d, 5 d/wk for 165 exposures Ho effect on general Spencer et al., 1951 appearance, behavior; mortality, body weight, histology of liver, kidney, lungs, heart, spleen or testes; organ weights, blood non- protein nitrogen. BUN, serum phosphatase, plasma prothrombin clotting time. monkeys 2 males 400 ppm (1.62 mg/l) 7 h/d, 5 d/wk One sacrificed in moribund condition after 8 exposures: enlarged liver with in creased neutral fat and esterified cholesterol content, marked degeneration and vacuolation of hepatic cells; moderate degeneration of renal tubular epithelium with cast formation; in creased plasma prothrombin clotting time. Second sacrificed after 12 exposures: similar, but milder, changes. No significant hematological change. Spencer et al., 1951 rats 12 male Osborn e-Mend e1 (average weight: 72 g) 12 controls 200 ppm (0.73 mg/fc) 7 h/d x 5 d/wk 0 8 dead after 6 exposures No deaths. Heppel et al. _106 CMA 0 5 1 7 4 0 TABLE 9-26 {Cont,) Route Specie3 Humber Dose Effect Reference Inhalation rats 1 male Hlstar 11 female Hlstar (average weight: 2*19 g) 200 ppm (0.73 mg/l) 7 h/d * 5 d/wk 14 controls 0 7 dead; 1 after 73 exposures, 6 after 44 exposures. Symptoms: weight loss; listless; crusting of eyes. Histology of 5 rats killed after 86 exposures: fatty degeneration of kidney In 1 rat; no other changes noted In liver, heart, lungs, kidney, adrenals and spleen; no effect on red and white blood cell count, hemoglobin and differential counts (7 treated compared with 6 control rats). 1 dead. Heppel et al., 1946 v> l Inhalation mice 20 200 ppm (0.73 mg/t) 7 h/d x 5 d/wk 16 dead after 7 exposures Heppel et al., 1946 o Inhalation guinea pigs 12 male, 2 female (average weight: 376 g) 200 ppm (0.73 mg/1) 7 h/d x 5 d/wk 18 controls 0 4 dead after 68 exposures; 1 dead after 115 exposures. Histology of 9 guinea pig3 killed after 124 exposures; pulmonary congestion in 4, liver necrosis in 1, necrosis of adrenal cortex in 1. 1 dead. Histology of 5 guinea pigs: fatty liver and myocardium of 2; no other changes noted In liver, heart, lung, kidneys, adrenals and spleen. Heppel et al., 1946 CMA 0 5 1 7 4 1 TABLE 9-26 (Cont.) Route Inhalation Inhalation Inhalation Inhalation Species Humber Dose Effect Reference rabbits 5 200 ppm (0.73 mg/1) 7 h/d x 5 d/wk (average weight: 2720) 4 controls 0 Ho deaths. No histological changes found; no effect on red and white blood cell count, hemoglobin, and differential counts compared with controls. Ho deaths. Heppel et al., 19*16 monkeys 2 males (average weight: 8740 g) 200 ppm (0.73 mg/i) 7 h/d x 5 d/wk for 125 exposures no controls No deaths. Histology of both: focal calcification in adrenal medulla of 1; fatty droplets in liver and myocardium of both. Heppel et al., 1946 rats 15 male, 15 female 200 ppm (0.81 mg/A) 7h/d, 5 d/wk for 151 exposures guinea pigs 8 male, 8 female 200 ppm (0.81 mg/1) 7 h/d, 5 d/ wk; for 180 exposures No adverse effects on general appearance, behavior, growth, mortality, final body and organ weights, hematology, gross and microscopic histology. Spencer et al., 1951 Final body weight Spencer et al., significantly different from controls in males, and growth less than controls, but not slgiflcant in females. Significant increase in male liver weights only; slight parenchymatous hepatic degeneration in half of guinea pigs of both sexes; no effect on hematology; flight increase in trial lipid, phospholipid, neutral fat, free and esterlfied cholesterol. 1951 CMA 0 5 1 7 4 2 TABLE 9-26 (Cont.) -109 CMA 0 5 1 7 4 3 Route Sped ps Number Dose Effect Reference Inhalation rats 15 male, 15 female 100 ppm (0.405 mg/l) 7 h/d, 5 d/wk males: 151 exposures females: 14? exposures No adverse effect on general Spencer et al., 1951 appearance, behavior, mortality, growth, final body and organ weights, hematology, gross and microscopic histology, BUN, blood non-protein nitrogen, serum phosphatase, plasma prothrombin clotting time, total levels lipid, phospho lipids, neutral fat in liver, free and esterified cholesterol in liver. Inhalation guinea pigs 6 male, 100 ppm (0.405 mg/t) 7 h/d, No adverse effect on Spencer et al., 19 51 8 female 5 d/wk mortality, growth, final males: 121 exposures body and organ weights, VO females: 162 exposures BUN, blood non-protein nitrogen, serum phosphatase, plasma prothrombin clotting time, gross and microscopic histology, total liver lipid phospholipids, neutral fat in liver, free and esterified cholesterol in liver. Inhalation rabbits 2 male, 1 female 100 ppm (0.405 mg/l) 7 h/d, 5 d/wk for 1 78 exposures No adverse effect on appearance, behavior, growth, final body and organ weights, gross and microscopic histology, hematology. Spencer et al., 1951 Inhalation monkeys 2 male 100 ppm (0.405 mg/t) 7 h/d, 5 d/wk for 148 exposures No adverse effect on appearance, behavior, growth, final body and organ weights, gross and microscopic histology, hematology. Spencer et al., 1951 TA$LE 9-26 (Cont.) Route Inhalation Species rata Number 16 resale 23 sale Inhalation guinea piga 10 Bale 6 resale Inhalation Ice 30 control 19 Juveniles Inhalation cats rabbits guinea pigs rata 4 4 10 10 h = hour; d = day; wk = week BUN = Blood urea nitrogen Dose Errect Reference 100 pps (0.42 g/l) 7 h/d x 5 d/wk for 4 months No deaths, no effect on rate of growth in 15 sales cospared with 15 sale controls. 15 of 16 resales becase pregnant; rat pupa were unaffected by exposure. No histological effects in liver, heart, lungs, kidney, adrenal glands and spleen of 10 rats exasined. Heppel et al., 1946 100 pps (0.42 sg/&) 7 h/d x 5 d/wk for 4 months 0 2 dead (disease of neck with enlarged caseous glands) No histological effects in liver, heart, lungs, kidney, adrenal glands and spleen of 10 anisals exasined. 3 dead (disease of neck with enlarged caseous glands). Heppel et al., 1946 100 pps (0.42 ng/1) 7 h/d x 5 d/wk No deaths; no effect on weight Heppel et al., 1946 for 19 exposures gain. 100 pps 6 h/d x 5 d/wk for 17 wks No clinical symptoms Hoffman et al., 1971 No effect on serus levels of creatinine, urea, SOOT, SGPT, on body weight. No changes in liver, kidney. and other organs (not specified). tw/tso In another study, Heppel et al. (1946) found that repeated exposure (7 hours/day, 5 days/week) to 1000 ppm EDC elicited death in most of the exposed rats, rabbits, guinea pigs, dogs, cats and monkeys (see Table 9-26). Rats, rabbits and guinea pigs appeared to be more susceptible than the other species tested. Pathological changes, which were varied, included congestion of lung, liver, heart, kidney, adrenal gland and spleen in guinea pigs; congestion and fatty metamorphosis in the liver of all exposed cats; renal changes in exposed monkeys and rats; and pulmonary congestion in a few rats. Chronic splenitis occurred in all rats exposed to repeated inhalation of 1000 ppm EDC. Focal myocarditis was found in one monkey. Similar repeated exposures to 400 ppm EDC produced high mortality in rabbits, rats, and guinea pigs (see Table 9-26), but mortality was not observed in 9 dogs subjected to from 167 to 1 77 exposures at this concentration. No changes were observed in a variety of clinical parameters that were assessed in 6 adult dogs, although slight fatty changes were noted in the livers of five and the kidney of one of the dogs. Rat and guinea pig survivors of 400 ppm EDC exposure did not exhibit histological changes with the exception of 1 of 6 rats that exhibited diffuse myocarditis and fatty degenera tion of the liver, kidney and heart. Repeated inhalation exposure to 200 ppm EDC produced mortality in mice, rats and guinea pigs, but not in rabbits or monkeys (see Table 9-26). Pathologic effects of exposure to 200 ppm EDC included a few cases of pulmonary congestion, fatty degeneration in the kidney of one rat, necrosis of the liver and adrenal cortex in one guinea pig, and fatty droplets in the liver and myocardium of both monkeys. No clinical or histopathological effects were observed in 39 rats or 16 guinea pigs exposed to 100 ppm EDC for 4 months or in 19 juvenile mice exposed to this level of EDC for 19 exposures. Hoffman et al. (1971) exposed groups of 4 cats, 4 rabbits, 10 guinea pigs and 10 rats to 500 or 100 ppm EDC for 6 hours/day, 5 days/week for 6 weeks. 9-111 CMA 051745 Mortality from exposure to 500 ppm EDC was high (see Table 9-26). Variable changes in the heart, lungs, liver, kidney, and adrenals were found at autopsy. No toxic effects were observed in the exposed animals at 100 ppm. An elevation of BUN and creatinine was noted in one fourth of the rabbits, but the significance of these changes is uncertain. The exposed cats did not grow as well as the control cats. The effect of EDC on blood elements and on renal and liver function was investigated using groups of 10 rabbits that were exposed to 3000 ppm for 4 hours, or to 3000 ppm for 2 hours/day, 5 days/week for 90 days (Lioia, 1959a,b,c,d). The only significant changes observed after the acute 4-hour exposure were granulations in about 20$ of the granulocytes. Varying degrees of anemia accompanied by leukopenia and thrombocytopenia and frequent hyperplasia of granuloblastic and erythroblastic parenchyma in the bone marrow were observed after subchronic exposure. There was also a reduction in leukolipids, but no change in polysaccharides, peroxidase, or ribonucleic acid. In tests for liver function, a decrease in albumin-globulin ratio, a slightly elevated BSP retention, slightly elevated values in colloidal tests (cadmium and cholesterol), and normal Van den Berg (indicating an absence of elevated serum bilirubin) and blood amino acid levels were observed. Measurement of creatinine clearance, portal blood flow, and glomerular filtration rate indicated altered renal function. Congestion, vascular degeneration and small necrotic areas were found in livers and kidneys, Dimitrieva and Kuleshov (1971) examined the effect of EDC on brain activity in 18 albino rats exposed to 1235 ppm EDC (5 mg/l) for 3.5 months. The duration, frequency and number of exposures were not reported. Electroencephalograms were recorded before exposure and at monthly intervals thereafter from silver and platinum electrodes implanted in the brains of the test animals. The stimulus was composed of an arhythmic photic stimulus of constant intensity and pulse 9-112 CMA 051746 duration. A maintained frequency of activity was observed in the EEG of the treated rats. A progressive diminution of amplitude of vacillations occurred, with the amplitude of delta rhythms reaching 50-70 MV (amplitude of delta rhythm before exposures was not specified), while the amplitude of beta rhythm decreased to 10 to 15 MV (amplitudes of pre-exposure beta rhythms were 30 to 80 MV). A loss of ability to assimilate an imposed rhythm wa3 observed by the authors. The results of an inhalation bioassay for carcinogenesis with Sprague-Dawley rats and Swiss mice have been published (Maltoni et al., 1981). The experimental design and results of these assays are detailed in Section 9.5, but it should be noted that exposure to 250 to 150 ppm, 50, 10 or 5 ppm EDC for 7 hours/day, 5 days/week for 78 weeks elicited no treatment-related changes in body weight or survival ability in rats of either sex or male mice. There was, however, decreased survival ability in female mice exposed to the high level of EDC. The animals exposed to 250 ppm at the onset of the study exhibited signs of toxicity (i.e., ruffling of hair, hypomotility and loss of muscle tone), prompting dose decrease to 150 ppm after several weeks. Systemic pathological examinations were performed on all the major organs of all animals with or without pathological changes, but the only non-neoplastic histologic effects reported were some "regressive" changes in the liver and adrenal glands that were not dose-related (Cox, 1982). In a related study with Sprague-Dawley rats, Spreafico et al. (1981) inves tigated the effect of inhalation exposure to identical levels of EDC (250 to 150, 50, 10 and 5 ppm) on blood clinical chemistry parameters. Histological examina tions were not performed on the rats exposed to EDC in this study. Eight to 10 animals per dose level were sacrificed after 3, 6, 12 or 18 months of 7 hours/day, 5 days/week exposure. Rats used for the 3, 6 and 18 month measure- 9-113 CMA 051947 merits were started at 3 months of age, and the 12 month determination was made with animals that were exposed from 14 months of age. The results suggest that long-term inhalation exposure of one year or longer to EDC at levels of 150 ppm does not produce marked toxicity in rats exposed from 3 months of age (Spreafico et al., 1981). There were no statistically significant changes between treated and control animals with respect to circulating levels of red blood cells, total white blood cell numbers, platelet numbers, relative percentages of lymphocytes, granulocytes and monocytes, and circulating protein levels. Percent albumin values were significantly increased in both sexes at 3 and 18 months but not at 6 months, but no clear dose-response relationship was apparent. Gamma globulin percentages were significantly lower at 3 months in the 150 ppm EDC group. Although changes were not apparent at 6 months, at 18 months there was a significant decrease observed only at 5 and 10 ppm. No treatment related effects were found on levels of y-glutamyl trans peptidase (y-GT), serum glutamic-oxalacetic transaminase (SGOT), serum glumatic-pyruvic transaminase (SGPT), serum alkaline phosphatase, bilirubin or cholesterol (Spreafico et al., 1981). A slight, but not significant, increase in creatine phosphokinase (CPK) level was seen at 18 months in males exposed to 50 and 150 ppm EDC. Lactic acid dehydrogenase (LDH) levels were significantly elevated after 3 months of exposure in both treated male and female groups, but a dose-response relationship was apparent only in males. At 6 months, there was an insignificant increase in LDH levels. At 18 months, significantly higher LDH levels were observed only in males exposed to 5, 50 and 150 ppm EDC without an apparent dose-response relationship. No clear changes were observed for BUN levels, serum glucose levels or uric acid levels (Spreafico et al., 1981). No consistent and significant differences in a battery of urinary tests (pH, proteins, bilirubin, glucose, hemoglobin, erythrocytes, leukocytes, epithelial 9-114 CMA 051748 cells, casts, cystals, mucus and microorganisms) were observed after 18 months of inhalation exposure (Spreafico et al., 1981). Changes in liver and kidney function were indicated, however, in rats exposed to EDC at 14 months of age. SGPT levels were significantly increased in rats of both sexes exposed to 50 and 1 50 ppm. y-GT levels were significantly elevated in females treated with 50 and 1 50 ppm. SGOT levels were significantly elevated at the two lower doses (5 and 10 ppm), and significant decreases were observed at the 2 higher doses (50 and 150 ppm). Cholesterol levels were signi ficantly lower at 50 and 1 50 ppm in both sexes, but there were no significant changes in bilirubin, CPK, alkaline phosphatase, LDH or glucose levels. Signifi cantly higher levels of uric acid were found at 50 and 1 50 ppm and higher blood glucose levels were observed at 1 50 ppm. No significant changes were found in blood elements or in the battery of tests conducted on urine in the rats that were initially exposed at 14 months of age (Spreafico et al., 1981). 9.2.3. Summary of Acute, Subchronic and Chronic Toxicity. 9.2.3*1. INHALATION EXPOSURE -- Information regarding the acute effects of inhaled EDC in humans is available primarily from cases of occupational exposure (see Section 9.2.1.1). Although the reported .cases had both fatal and non-fatal outcomes, many of the reports were foreign and none of the reports provided quantitative exposure data. Further, although EDC was usually reported to be the primary vapor to which the workers were exposed, the preponderance of exposures were to poorly characterized mixtures of EDC and other solvents, or to EDC of unknown purity. Symptoms and signs of acute inhalation exposure were often indicative of CNS and gastrointestinal disturbances, and clinical evidence of liver and kidney dysfunction, as well as irritation of the respiratory tract and eyes, have also been observed. Death was usually ascribed to respiratory and circulatory failure, and autopsies frequently revealed pulmonary edema and 9-11 5 CMA 051749 congestion, cellular degeneration, necrosis and hemorrhagic lesions of most internal organs (e.g., liver, kidneys, spleen, lungs and respiratory tract, brain, stomach and intestines). The results of acute experimental studies with three or four subjects suggest that the threshold of light perception (duration of exposure not stated), depth of breathing (1-minute exposure) and vasoconstriction (30-second and 15minute exposures) increased with exposure to EDC (Borisova, 1957). Spirographic and plethysmographic responses reportedly first differed from baseline values at 1.5 ppm and appeared to be dose-related to 12.4 ppm, the highest level tested; exposure to 1 ppm (the lowest level tested) reportedly had no effect on the physiologic end points. Although these Russian experiments appear to indicate a threshold of toxic action, the reliability of the data is uncertain due to the small number of subjects tested and an absence of reported baseline measurements for each subject. Some assurance of data credibility can be gleaned from the fact that the range of concentrations tested (i.e., =1-12.4 ppm) represented corroborated sub-threshold, threshold and above threshold odor perception concentrates. Borisova (1957) determined the odor perception threshold of EDC to be =6 ppm, and this value is consistent with an absolute odor threshold of 6 ppm that was determined by Heilman and Small (1973, 1974) in a Union Carbide study. Further, the sensitivity of the analytic method used by Borisova (nephelometry) appears to be more than adequate to determine EDC at the reported experimental levels. Heilman and Small (1973, 1974) also determined an odor recognition threshold of 40 ppm for EDC. Acute inhalation studies with a variety of animal species indicate that the effects of single EDC exposures are similar to those in humans. Immediate symptoms of toxicity are related to CNS depression (e.g., narcosis) and delayed histopathologic changes (e.g., congestion and degenerative effects) have been 9-116 a&T5,0 observed primarily in the liver, spleen, kidneys, lungs and adrenals. As detailed in Section 9.2.2.1.3 and Table 9-24, the severity of effects are depen dent upon duration and concentration of exposure. For rats exposed for 5-8 hours, it appears that adverse effects are not elicited by exposure at 200 ppm (Spencer et al., 1951); signs of intoxication first appear at 300 ppm (Spencer et al., 1951) and mortality at >600 ppm (Heppel et al., 1974; Carpenter, 1949). Concentrations as high as 12,000 ppm were inhaled by rats for 0.1 hours without adverse effects (Spencer et al., 1951). The dose-response relationships for acute inhalation exposure are not as thoroughly characterized for other species. Corneal opacity was observed in dogs exposed via inhalation to 1000 ppm for 7 hours (Heppel et al., 1944). Signs of intoxication were not present in guinea pigs that were exposed to 1200 ppm EDC for 8 hours (Sayers et al., 1930), but exposure to 1500 ppm for 7 hours produced mortality in mice, guinea pigs and rabbits (Heppel et al., 1945). Histopathological effects (e.g., pulmonary congestion/edema, alterations to kidneys, liver, spleen and adrenals) were generally apparent in rats, mice, guinea pigs and rabbits at concentrations of >3000 ppm (Spencer et al., 1951 ; Heppel et al., 1945; Sayers et al., 1930). Spencer et al. (1930) felt that narcosis that was elicited by concentrations <3000 ppm was attributable to organ injury rather than depression of the CNS. Limited quantitative data are available, primarily from the foreign litera ture, regarding the effects of repeated EDC exposure on humans. The studies that are available are deficient because control data are lacking and because dura tions of exposure and numbers of subjects were poorly characterized. The avail able data suggest that chronic intermittent exposure in the range of 10-37 ppm may represent a lowest-observed-adverse-effect level (L0AEL) but, as summarized 9-11 7 CMA 051751 subsequently, the data do not provide a basis for identifying a no-observedadverse-effect level (NOAEL) or a no-observed-effect-level (NOEL) for humans. Case reports (Byers, 19^3; Rosenbaum, 1947; Guerdjikoff, 1955) and a foreign health survey (Cetnarowicz, 1959) of workers who were exposed repeatedly to 1,2-EDC vapor indicate that typical symptoms and signs of acute poisoning developed with exposure to concentrations in the range of =60-200 ppm. In the Cetnarowicz (1999) survey, 16 workers who were exposed to =10-200 ppm for 2-8 months were examined; 10 of 10 who were exposed to 60-200 ppm complained of adaptable eye irritation and 6 of the 10 experienced typical symptoms of expo sure, but only 1 of 6 exposed to 10-37 ppm were symptomatic. Positive symptoms and signs of exposure, particularly those indicative of mucous membrane irrita tion, were also observed in 90 of 118 agricultural workers who were concurrently exposed to EDC vapor (=15-60 ppm) and liquid (prolonged dermal contact) (Brzozowski et al., 1954). The results of two other foreign health surveys of workers exposed to EDC in the range of the Cetnarowicz (1959) low exposure group (10-37 ppm) support Cetnarowicz's apparent finding that overt symptoms may not be the predominant effect of lower exposures; there appears, instead, to be a prevalence of neuro logic and clinical effects. Rosenbaum (1947) examined 100 workers who were exposed to <25 ppm for 6 months to 5 years and found disturbances that included "heightened lability" of the autonomic nervous system, increased hidrosis and frequent complaints of fatigue, irritability and sleeplessness, but no hemato logic or functional changes of the internal organs. In the study by Kozik (1957), which is particularly notable for its reliable exposure data, workers exposed to time-weighted average concentrations of 10-15 ppm experienced increased morbidity, particularly from gastrointestinal, liver and bile duct disorders. The number of workers surveyed in this study was not clearly stated 9-118 CMA 051752 in the available translation, but it was >83- The occurrence of overt symptoms in these workers was not discussed, but impaired performance on control reaction time tests suggested a possible effect on the nervous system. Case reports of workers who were repeatedly exposed to unknown concentra tions of EDC also suggest that neurologic effects such as nervousness, irrita bility, tremors and loss of reflexes (McNally and Fostvedt, 1941; Delplace et al., 1962; Suveev and Babichenko, 1969) and complaints of skin and mucous membrane irritation (Suveev and Babichenko, 1969; Rosenbaum, 1947; Guerdjikoff, 1955) are more prevalent in chronically exposed workers than in workers who were acutely exposed. Studies with several species of animals provide information regarding the threshold region of effects for subchronic and chronic inhalation exposure to EDC. Three studies in which rats, guinea pigs, rabbits, cats and monkeys were exposed to 100 ppm EDC for 7 hours/day, 5 days/week for 4-6 months have shown no treatment-related adverse effects on survival, growth, hematology, clinical chemistry, organ weights or histology (Heppel et al., 1946; Spencer et al., 1951 ; Hoffman et al., 1971). Adverse effects were also not noted in juvenile mice that were exposed to 100 ppm EDC for 19 exposures (Heppel et al., 1946). The apparent NOAEL of 100 ppm is supported by the results of comprehensive chronic studies with rats in which similar weekly exposures to EDC (7 hours/day, 5 days/week) at concentrations of 5, 10 or 50 ppm for 18 --19 months had no significant treatmentrelated adverse effects on histology (Maltoni et al., 1981) or blood or urine clinical chemistry indices (Spreafico et al., 1981) in rats of either sex; exposure to 150 ppm elicited decreased survival in female rats, but no clear effects on clinical chemistry parameters in either sex. There was an indication of renal and liver damage (increased SGPT, increased serum "y-GT, decreased cholesterol, increased uric acid, increased blood glucose) in mature (i.e., 1 4- 9-119 CMA 051753 months-old) rats that were similarly exposed to 50 or 150 ppm EDC for 12 months, but hematologic effects were not found and histological examinations were not conducted. Subchronic exposure to 200 ppm EDC (7 hours/day, 5 days/week) produced toxic effects in several species, but the severity of effects appeared to vary with investigation. Heppel et al. (19^6) observed mortality among rats, mice and guinea pigs exposed to 200 ppm EDC, but not among rabbits or monkeys, and histo logical lesions were not found in any of these species. Spencer et al. (1951) did not observe mortality in rats or guinea pigs similarly exposed to 200 ppm EDC, but other effects were noted in the guinea pigs (decreased body weight gain and increased liver weight in males, slight hepatic degeneration in males and females). Rabbits and monkeys were not tested by Spencer et al. (1951). An unequivocal frank-effect level (FEL) of 400 ppm is representative for EDC as indicated by production of high mortality and histopathological alterations in the liver and kidney in rats and guinea pigs after a few exposures (Heppel et al., 1946; Spencer et al., 1951). Spencer et al. (1951) reported that exposure to 400 ppm EDC had no effect on rabbits (as reported at 200 ppm), but Heppel et al. (1946) observed some mortality at this level. Exposure to 500 ppm EDC produced high mortality in rats, guinea pigs and rabbits within a few exposures (Hoffman et al., 1971). 9.2.3.2. ORAL EXPOSURE -- Limited data are available on the effects of oral exposure to EDC. Human case reports of accidental or intentional ingestion of EDC indicate that the toxic response to oral exposure is similar to that of inhalation exposure (Section 9.2.1 .1 .1 .1 ). Ingestion of quantities of EDC esti mated to have ranged from 8-200 ml have been reported to be lethal (see Table 9-18). This is equivalent to =140-3570 mg/kg if it i3 assumed that an average human weighs 70 kg. Median lethal doses in rats (McCollister et al., 1956; Smyth 9-1 20 CMA 051754 et al., 1969) and mice (Heppel et al., 1945) have been reported in the range of 700 mg/kg. Higher oral doses (up to 2.5 g/kg) were tolerated by dogs without mortality (Kistler and Luckhardt, 1929). The apparent higher tolerance in dogs and some humans may be related to the ability of these species to vomit. Dose information was not reported in human case reports of non-lethal ingestion of EDC. Administration of 189 mg/kg/day EDC in the drinking water for 90 days caused a decrease in growth rate in mice, but no significant effects on organ weights, hematology indices, clinical chemistry indices, blood coagulation or immune system function, although a trend towards immunosuppression was noted (Munson et al., 1982). Similar administration of 24 or 3 mg/kg/day EDC in the drinking water for 90 days had no effect on mice. The only dose-response data that are available for chronic oral exposure to EDC are the results of a 78-week NCI carcinogenesis bioassay with rats and mice (NCI, 1978). Administration of 95 mg/kg/day EDC via gavage (5 days/week) caused early and severe mortality in rats of both sexes, and 47 mg/kg/day caused decreased survival in male rats (after 90 days) and female rats (throughout the experiment). Non-carclnogenic toxic effects appeared to be the cause of the deaths, as indicated by the presence of a variety of lesions including broncho pneumonia and endocardial thrombosis. Body weight depression was not found in any of the treated male or female rats. Treatment-related weight depression and mortality was found in female mice that were similarly exposed to 299 mg/kg/day, but not in females exposed to 149 mg/kg/day or in males exposed to 195 or 97 mg/kg/day. The presence of tumors in the high dose female mice suggested, however, that the deaths may have been tumor-related. 9-1 21 CMA 051755 9.3* REPRODUCTIVE AND TERATOGENIC EFFECTS Murray et al. (1980, unpublished) performed a subchronic inhalation study to determine the effects of ethylene dichloride (1,2-dichloroethane, EDC) on the reproductive ability of rats and effects on their offspring. The results of this study were reported at the fifth annual Banbury Conference (Rao et al., 1980). Twenty to thirty male and female Sprague-Dawley rats were initially exposed to 25, 75, and 150 ppm EDC (lot no. TAC06228, analyzed as 99.98$ pure by Dow Chemical Laboratories) for 60 days (6 hrs/day, 5 days/week for 12 weeks), and they were successively bred to produce two litters, the F^ and The breeding protocol consisted of caging one male and one female rat for 4 consecutive days, and remating any females that had not mated (no sperm in vaginal smear) with a different male after a 3 day rest period. The results from remated animals were combined with those of the first breeding. The females were exposed to EDC throughout breeding, gestation and lactation (6 hrs/day, 7 days/week) except for day 21 of gestation to day 4 postpartum to allow for delivery and rearing of the young. A second mating, for the production of the F^ generation, took place after the F1A litter was sacrificed (day 21 of birth). During the seventh week of this study, both control and treated animals suffered from symptoms of sialoacryoadenitis (red crusty material around the eyes and nose and conjunctivitis), which was thought to have resulted from a viral infection. The authors noted that more males than females contracted the disease, but suggested that this greater incidence was related to the housing accommodations (males were caged away from females) and not to any greater susceptibility of the males for developing infection after EDC exposure. All 9-122 CMA 051T56 animals (both control and experimental) recovered one to two weeks after the onset of the disease with no recurrence for the duration of the study. Murray et al. (1980) reported that the physical well being of the parental generation was not significantly affected by EDC exposure. However, during the first and second weeks of exposure, the females consumed significantly less food than the controls, although for the rest of the study, there were no alterations in food consumptions. In the males, food consumption was both sporadically increased and decreased throughout the study with a general trend for increased food consumption in males exposed to the highest concentration of EDC (150 ppm) for longer durations of exposure (weeks 15 to 29). There was a statistical increase in the absolute liver weight at the 150 ppm level, and a statistical linear trend for increased liver weights in adult male rats exposed to EDC (Murray et al., 1980). However, this trend was not observed for liver weights in relation to body weight. All adult males displayed some degree of chronic renal disease. However, the severity of the disease did not appear to be related to treatment. Both control and experimental females displayed signs of renal disease. There was a greater incidence of inflammation and edema of the salivary glands in males exposed to 150 ppm. Three deaths occurred during the course of the study, but these deaths did not appear to be related to EDC exposure. The fertility and survival index on days 1, 7, 14, or 21 was not statistically different at any dose level. However, the greatest pup mortality was observed in groups exposed to 150 ppm between days 14 and 21. The sex ratio, average number of live pups per litter and neonatal body weight were not affected. There were sporadic, statistically significant differences in 9-123 CMA 051757 reproductive indices of treated versus control groups. However, these did not appear to be attributable to EDC exposure. A few neonates exhibited external and internal malformations. However, the incidences of these effects were sporadic and statistically Insignificant, reflecting natural variability and not the result of EDC exposure (Murray et al., 1980). Therefore, it was concluded that EDC exposure, under these conditions, did not adversely affect reproduction in rats or the development of their offspring. Lane et al. (1982) evaluated the effects of EDC on the reproductive capability of mice using a multigenerational reproductive protocol modified for screening teratogenic and dominant lethal effects. Animals were exposed to EDC for 35 days, then 10 male and 30 female mice (parental generation F0) were mated to produce the first set offspring (F1A). After the F1A were weaned, the F0 adults were remated to produce the second set offspring (F,jg). A parental stock was chosen from the F^ litter (30 females, 10 males) to produce a second generation of offspring (F2A). After weaning, the F1B adults were remated to produce offspring (Fgg) For use *n teratology and dominant lethal screening tests. In this study, the EDC (Aldrich Chemical Co., 99? pure) was dissolved in a 1? solution of Emulphor EL-620 (GAF Corp., Linden, NJ) and then further diluted with deionized water to concentrations of 0.03, 0.09, and 0.29 mg/mA for nominal doses of approximately 5, 15, and 50 mg/kg/day. The test animals were continuously maintained on EDC solutions or control solution (deionized water, or water containing 0.17 mg/mfl, p-dioxane dissolved in 1.0? Emulphor solution). 9-124 CMA 051758 Fresh drinking solutions were prepared twice weekly and placed in amber glass bottles with cork stoppers and stainless steel drinking tubes. The authors reported no decrease in fluid consumptions in either the EDC or the solvent control groups. Lane et al. (1982) reported that EDC produced no treatment-related signs of toxicity such as lowered body weights or gross pathological changes in major organs. There were no significant differences in the fertility index or gestation index observed in the F1A, or generations. There were sporadic incidences of increased mortality throughout the generations but these were not dose-related; the reason for the deaths was not apparent at necropsy. There were no differences in the litter sizes at birth, pup body weight, or survival of pups at days 4 and 21 of birth. There was a decrease in the survival indices for the F2A generation as compared to values for the F^ and F^g generation, but these decreases were not dose related. In the dominant lethal screening tests, there were no statistically significant dose-related effects. However, both increases and decreases were observed in the ratios of dead to live fetuses. In the teratology screening, continuous administration of EDC produced no apparent adverse reproductive effects or observable abnormalities. It should be noted that continuous exposure used in this study is not a recommended protocol for evaluating teratogenic effects. In teratology testing, the duration of exposures is usually limited to the period of organogenesis. Also in the study, the F^ skeletal specimens were lost and, therefore, these effects could not be evaluated. From this study (Lane et al., 1982), it is not possible to determine conclusively whether EDC has the potential to cause adverse reproductive or teratogenic effects because the doses were not high enough to produce any adverse 9-125 CMA 051T59 effects, including maternally toxic effects. However, under the conditions of the experiment, it appears that EDC given in drinking water (nominal doses of 5, 15, 50 mg/kg/day) produced no observable adverse reproductive effects in Swiss ICR mice. Schlachter et al. (1979) conducted teratology testing in 16 to 30 SpragueDawley rats and 19 to 21 New Zealand White rabbits. The animals were exposed to inhalation to either 100 or 300 ppm EDC (Lot Nos. TA0201851L and TA022851L, analyzed as >99-9% pure by the Dow Chemical Laboratories). The rats were exposed 7 hrs/day on days 6 through 15 of gestation. Two thirds of the maternal rats died when exposed to 300 ppm EDC in contrast to no deaths occurring in the control and 100 ppm groups. In the 300 ppm group, only one rat in six survivors had implantation sites; all of these sites were resorbed. Two additional rats at the 300 ppm level showed early implantation sites when the uterus was stained with sodium sulfide. In the 100 ppm group, three rats delivered early on day 21; however, there was no indication of embryo or fetotoxicity in the offspring. In addition, the rest of the rats at the 100 ppm level delivered normally with no alterations in litter size, number of resorptions or fetal body measurements (crown to rump length). At the 300 ppm level, one of the six surviving rats had a decrease in mean body weight gain, while in the 100 ppm level, there was an increase in body weight on days 6, 8, 10, 16, and 21 of gestation. At the 300 ppm level, the absolute liver weight was decreased, while the relative liver weight was increased; at the 100 ppm level there was no difference. In the 100 ppm group, there was an increase in water consumption on days 15-17 and 18-20 of gestation, 9-126 CMA 051760 but food consumption was not altered. There was no increase in the occurrence of malformations with only isolated sporadic incidents observed within the range of normal variability. In this teratology study (Schlachter et al., 1979), four out of twenty-one rabbits in the 100 ppm group, and three out of nineteen rabbits in the 300 ppm group died. Three control rabbits delivered early (days 15, 28, and 29 of gestation) as did one animal at the 300 ppm level (day 28 of gestation). Two animals that died at the 100 ppm level also aborted on days 26 and 28 of gestation. There was no effect on mean litter size, incidence of resorptions or fetal body measurements (crown to rump length) in animals delivering normally. Staining of the uterus revealed only one additional implantation site in control animals. The mean body weights of pregnant animals were generally comparable to those of controls, but there was an increase in mean body weight gains in the 300 ppm group on days 19 through 28 of gestation. A slight, statistically insignificant increase in both absolute and relative liver weights were observed in both the 100 and 300 ppm groups. There was no increase in the incidence of malformations, although sporadic occurrences within the range of normal variability were reported. It was concluded from this study that EDC exposure does not produce adverse effects on the developing conceptus. Several Russian studies by Vozovaya (1971, 1975, 1976, 1977) have reported that EDC produces a number of adverse reproductive outcomes which include lengthening the total estrous cycle, changes in the duration of various stages of the estrous cycle, increases in the number of perinatal embryonic deaths, decreases in the weight of newborn animals, and decreases in the weight gain of offspring after birth. In addition, EDC was reported to concentrate in the 9-127 CMA 051761 placenta, amniotic fluid and fetal tissue. However, it is difficult to evaluate or seriously consider these reports since they are presented with insufficient detail or critical scientific review. Inadequacies in these reports include: lack of original data, various techniques and tests mentioned but results not presented, no information on the source or purity of the chemical, statistical analysis mentioned, but the type of statistical tests not stated. The above comments apply similarly to the study by another Russian scientist, Urusova (1953), who reported that EDC accumulated in the milk of nursing mothers. This is obviously an important observation. However, it cannot be accepted as scientific evidence. This report was presented in a subjective, anecdotal manner with many inadequacies in reporting. These inadequacies include lack of details concerning the numbers of women involved in the study, the women's age or physical status, the concentrations and duration of EDC exposure after dermal exposure, the repeatability of this finding and number of samples analyzed. Because of the concern for EDC contamination in mother's milk, Skyes and Klein (1957) conducted a study to determine whether oral administration of EDC might pass into the milk of cows. Only five cows were used in this study; two cows were exposed to 100 ppm EDC for 22 days; two cows were administered 500 ppm EDC for 10 days followed by 1000 ppm for 12 days; one cow was used as the control. Two different breeds of cows, Holstein and Jersey, were used, but it was not stated which cows received which dose. Seven milk samples collected over a three week period containing no more than 0.4 ppm EDC. However, the authors stated that this amount was too low for accurate detection by the Volhard titration method used in this study. The samples collected from cows ingesting EDC had consistently higher concentrations of EDC than the controls, but because of the 9-128 CtfA 05 n62 small sample size and the lack of sophisticated analytical procedures, it is not possible to make firm conclusions as to the amount of EDC entering the milk supply. More importantly, this study was not designed to evaluate the possible health consequences of EDC ingested in the milk of nursing offspring. However, recent pharmacokinetic studies do indicate that EDC has a tendency to accumulate in fatty tissues (Spreafico et al., 1980) although the reproductive health effects of EDC in breast milk have not been evaluated. 9.3.1. Summary and Conclusions. The available scientific information on the potential of ethylene dichloride (EDC) to affect reproductive or developmental processes adversely includes a teratology test conducted in rats and rabbits (Schlachter et al., 1980), a single-generation reproductive test conducted in rats (Murray et al., 1980), a multigenerational reproductive test conducted in mice (Lane et al., 1981), several reports by Russian scientists describing a number of adverse reproductive effects observed in laboratory animals and man (Vozovaya 1971, 1975, 1976, and Urusova, 1953). and an investigation measuring the amount of EDC found in the milk of cows after ingesting EDC (Sykes and Klein, 1953). The results of reproductive and teratogenicity testing (Murray et al., 1980, Schlachter et al., 1979; and Lane et al., 1982) indicate that EDC has little potential for producing adverse reproductive affects or for adversely affecting the developing conceptus, except when the mother is exposed to doses high enough to produce maternal toxicity. The Russian studies describing a variety of adverse reproductive effects associated with EDC exposure do not provide conclusive scientific evidence because the studies were inadequately performed or reported. The study of EDC in the milk of cows is inadequate since only a few animals were used and the biological effects of EDC-contaminated milk were not investigated. 9-129 CMA 051763 In conclusion, the available studies indicate that EDC has little ability to affect the reproductive or developmental processes adversely in laboratory animals except at maternally toxic levels. However, it should be noted that these studies have inadequacies that weaken the strength of this conclusion. The multigenerational study by Lane et al., 1982 does not include a range of doses that include maternally toxic doses. The study by Murray et al. (1980) does not evaluate reproductive effects for more than a single generation. The studies by Sykes and Klein (1953) on the effects on lactation are inadequate. In addition, no properly conducted epidemiological study has been done to evaluate the effects of EDC on human reproduction. Therefore, although the studies to date indicate that EDC does not pose a specific hazard to reproductive or developmental systems, this cannot be conclusively established, especially for humans, without additional studies. The chemical similarity of EDC to ethylene dibromide (EDB) and 1,2-dibromochloropropane (DBCP) might suggest that additional testing related to testicular toxicity would be appropriate. 9-130 CMA 051764 9.4. MUTAGENICITY A review of the literature indicates that ethylene dichloride (EDC) has been tested for mutagenic activity in bacteria, plants, Drosophila, mammalian cells in vitro, and intact rodents. These studies are discussed below and are summarized in Tables 9-27 to 9-32. The reader may also refer to published reviews of the mutagenic potential of EDC (e.g., Fishbein, 1976,- 1979; Fabricant and Chalmers, 1980; Rannug, 1980b; Simmon, 1980). 9.4.1. Gene Mutation Studies 9.4.1.1. BACTERIAL TEST SYSTEMS -- Many investigators have studied the ability of EDC to cause gene mutations in bacteria (Table 9-27). Most reported marginal positive responses, approximately two-fold above background, without metabolic activation and stronger positive responses with exogenous hepatic metabolic activation, indicating that EDC is mutagenic to bacteria by itself but that metabolites, such as S-(2-chloroethyl)-L-cysteine, are more potent mutagens. Ethylene dichloride has been reported positive in four Salmonella/microsome plate incorporation assays (McCann et al., 1975; Rannug 1976; Rannug and Ramel, 1977; Rannug et al., 1978), in assays testing the mutagenicity of bile obtained from EDC-perfused rat livers or livers from EDC-treated mice (Rannug and Beije, 1979), and in two Salmonella spot tests. In one of the Salmonella spot tests (Brem et al., 1974), an analysis of duplicate experiments carried out on at least three different occasions revealed a twofold increase in revertant counts for strains TA1530 and TA1535 (mean values of 50 and 54 revertants on treated plates versus 23 and 26 in control plates for TA1530 and TA1535, respectively). No difference in revertant counts was noted for strain TA1538. This response is consistent with that expected for an alkylating agent. The authors stated that 9-131 CMA 051765 CMPt 0 5 1 7 6 6 Reference Brea et al., 1974 Principe et al., 1981 \o UJ ru Test System Salmonella (spot teat) Salmonella/ mammalian microsome assay (spot test) S. coelicolor forward mutation assay to Str A. nidulans forward mutation to 8-AGr Summary of Mutagenicity Testing of EDC: Gene Mutations In Bacteria Strain Activation System Chemical Information Results 0 1 -* TA1530 TA1535 TA1538 None on filter disk Weak positive Source: Not given Purity: Not given Salmonella revertant3 TA1530 TA1535 TA1538 EDC 50 54 19 Water .23 26 19 Chioramphen1co1 20 31 14 1. Could not perform plate incorporation tests because of volatility. TA1535 TA1537 TA1538 TA98 TA100 p<0.0la Doae ufc/plate -S9 +S9 0 100 0 100 39 46 33 103 17 8 10 8 19 12 21 27 82 83 77 84 188 188 171 169 Dose lit/plate 0 2 10 20 100 0 250 500 Survival t 100 100 100 100 100 100 100 42 Strr/plate 2.5 + 0.6 0.2 + 0.2 0.7 + 0.4 0.7 + 0.4 1.0 + 0.8 8-AG/plate 2.5 * 0.9 2.0 1.1 1.0 + 0.7 1. Positive controls indicated system working properly. 2. Positive results in Salmonella in spot te3t with TA1535. 3- No precautions taken to prevent excessive evaportion of EDC and ensure adequate exposure. 4. Toxicity results indicate exposure was minimal. CMA 051767 Reference McCann et al., 1975 Test System Salmonella/ microsome assay (plate test) Rannug, 1976 Salmonella/ microsome assay (plate test) TABLE 9-27 (cont.) Strain Activation System Chemical Information Results Comments TA100 TA1535 FCB-induced rat liver S9 mix Concentration Tested: 1.3 x lo" pg/plate (13 pmol) Source: Aldrich Chemical Co. Purity: Not given, but stated to be highest purity Negative or at best only marginal positive response. Induced 25 colonies plate above background. (0.19 revertant3/ pmol) in TA100. 1. Non-mutagenic in thi3 study. Reproducible doseresponse curves not obtained. 2. Metabolic activation did not Increase positive response. 3- Chloroethanol and chloroacetaldehyde (two putative intermediates in the metabolism of EDO in mammals) tested positive (l.e., 0.06 and 746 revertants/ umol, respectively). Liver fractions Concentration tested: from Sprague- Up to 60 pmol/plate Pauley or R strain Wlstar Source: BDH Chemicals, Ltd. rats Induced with phenobar- Purity: Not given but bltal with and reported to be without NADPH- checked by glass generating system capillary column and with and ch rooatography without gluta- using a flame thione S-trans- ionization detector ferasas A, B and C. Marginally positive without activation (two-fold increases positive response with activation (tenfold Increases). Spontaneous background 8-14 revertants/plate. 1. EDC activated by the liver cytosol fraction; mixedfunction oxygenases not Involved. 2. NADPH-independent GSH S-transferase dependent activation. 3. Strain differences noted in ability to metabolize EDC. 4. Thought that mutagenicity of EDC after activation caused by formation of highly reactive half sulfur mustard, S-(2-chloroethy1)-Lcystelne. 051768 Reference Test System Rannug and Ramel, 1977 Salmonella/ microsome assay (plate test) Rannug et al., 1978 Salmonella/ microsome assay (plate test) vO UJ *r Rannug and Belje, 1979 Salmonella/ Isolated perfused rat liver O Rannug and Belje, Salmonella/ 1979 (plate test) TABLE 9-27 (cont.) Strain Activation System TA1535 S9 mix from livers of un induced male R strain Wistar rats plus NADPHgenerating system TA1535 Rat liver microsome system with and without HA DP TA1530 TA1535 Isolated perfused liver from male R strain Wistar rats Chemical Information Results Comments Concentration tested: Up to 45 pmol/plate Source: BDH Chemicals, Ltd. Purity: Hot given Positive response (two-fold increase without activation; nearly ten-fold increase with activation.) Negative controls yielded roughly 15 revertants/ plates. .1 Compared mutagenicity of EDC tar with EDC. The level of EDC present at the highest dose tested for EDC tar would only exert a weak mutagenic effect, yet a strong response was observed. Concentration tested: 45 pmol/plate Source: EDC given BDH Chemicals, Ltd. Purity: Not given for EDC, but checked using glass capillary chroma tography using a flame lonizazation detector 2. Marginal positive 1. response without activation (two-fold increase 24.6 + 3.06 at 45 pool vs. 13.0 + 1.76 control); positive response with activation 2. (ten-fold Increase) independent of presence of NADP. Activation of EDC tar dependent on NADPH. EDC activa tion Independent Of NADPH. EDC major component of EDC tar. Nutagenicicity of tar increased with metabolic activation only with addition of HADP. Mutagenicity of EDC tar not solely due to EDC. Concentration tested: 0.1 ml (1.3 bM) for up to 4 hours. Positive. Highest response 15-60 minutes after addition of EDC (45-60 revertants compared to 7-10 in controls). 1. Positive responses consis tent with conju gation of EDC with glutathione. TA1535 Bile from male CBA mice 80 mg/kg EDC i.p.; removal of liver and collection of bile 30 and 60 minutes later. Positive. Greater than two-fold increases with bile from liver removed 30 minutes after addition of EDC (28.8 + 27 revertants compared to 11.3 + 1.1). TABLE 9-27 (cont.) Heference Teat System Strain Activation System Chemical Information Results Comments King et al., 1979 Salmonella/ microsome assay (plate test) E. coll K 12/ 343/113 (suspension test and intrasanguineous host-mediated assay) TA1535 TA100 TA1537 TA1538 TA96 PCB-induced rat liver S9 Concentration tested: 36 pool/plate 10 mH (suspension assay) 2 mmol/kg l.p. injection female NHRI mice Source: Merck Co. ' Darmstadt, PRO Hegative Negative Negative .1 Standard plate Incorporation test was conducted. No precautions were taken to prevent excessive evaporation of EDC. Nestman et al., Salmonella/ 1980 microsome assay vo (plate test and and desiccator exposure). Ol TA1535 TA100 TA1537 TA1538 TA96 PCS-induced rat liver S9 mix. Purity: Hot given. Stated that samples had correct melting point and elemental analysis. Concentration tested: Up to 9 mg/plate (91 pool) in desiccators. 10 mg/plate in plate tests. Source: Chem Service Purity: Hot given Solvent: DMSO Negative in standard teat. Positive in desiccator testing in strain TA1535. .1 Stated that maximum yield with TA100 is 20 revertants above background (l.e., negative). For TA1535 a doubling of mutant colonies observed. No other data pre sented . 2. Cannot adequately evaluate results. n 051769 TABLE 9-27 (oont.) Reference Test System Strain Activation System Chemical Information Results Comments Stolzenberg and Bine, 1980 Salmonella/ microsome assay (plate test) TA100 PCB-induced rat liver S9 mix (2 mg protein/0.5 ml) Concentration tested: Up to 10 pmol/plate Source: Aldrich Chemical Co. Purity: 99< pure Negative 1. All compounds tested in trip licate with and without S9 mix. 2. Experimental values minus background revertants. umol/plate 10 -1 1 10 Revertants -S9 +S9 0 0 15 Toxic 0 0 No growth 9L- Barber et al., 1981 Salmonella/ microsome assay (vapor exposure) WO TA1535 TA100 TA1538 TA98 PCB-induced rat liver S9 mix Concentration tested: Up to 231.8 pmol/plate as determined by GLC analysis of distilled water samples. Source: Eastman Kodak Co Purity: 99.98J Negative in standard plate te3t. Positive in desiccator testing in strains TA1535 and TA100. 1. Bacteria exposed in gas tight exposure chambers. 2. Plastic plates found to absorb dibrooomethane in parallel experiment. Thus, glass plates used for all other testing. 3- Weak positive result. 0.002 revertant3/nmol in TA1535 with or without activation. 0.001 revertants/ nmol in TA100 with or without activation. 4. Revertants selected from each experiment and tested to ensure that they were actually his . plate incorporation tests could not be performed because of the volatility of the test agent. Positive and negative control tests were conducted for these experiments and indicated the systems were working properly. Principe et al. (1981) conducted a spot test using strains TA1535, TA1537, TA1538, TA98, and TA100. A positive response was observed for TA1535 when a triangular shaped paper disc soaked with 100 uS, EDC was placed on the agar in the presence of S9 from Aroclor 1254-induced rat liver (i.e., 103 revertants on the treated plate vs. 33 revertants for the negative control). Negative responses were obtained with the other strains. A negative response was also obtained in plate incorporation tests conducted with TA100 and TA1535 at doses up to 100 pi/plate. A standard assay was conducted; no precautions were taken to prevent excessive evaporation of the EDC. Similarly in forward mutation tests conducted with Streptomyces coelicolor and Aspergillus nldulans negative responses were obtained at doses up to 100 and 500 pH/plate in plate incorporation and spot tests. There was a 100$ survival in the test conducted with S. coelicolor and a 60$ reduction in cell survival at the highest dose with A. nidulans. Thus, these tests are judged to provide less than adequate conditions for assessing the mutagenicity of EDC because insufficient exposure to the test organisms may have occurred. McCann et al. (1975) exposed Salmonella strains TA1525, TA100, and TA98 to EDC (Aldrich, stated to be highest purity available) concentrations as high as 13 mg/plate (131 pmol/plate). An equivocal response was observed in TA100 (0.19 revertants/pmol). However, reproducible dose-related response curves were not obtained. The presence of an exogenous S9 mix metabolic activation system (from rat livers induced with either phenobarbital or Aroclor 1254) did not increase the response. Chloroacetic acid, which is a known mammalian metabolite of EDC, and the putative intermediates (i.e., chloroethanol and chloroacetaldehyde; see 9-137 CMA 051771 section 9.1.3.3A), were also tested for their mutagenic potential. Chloroacetic acid was negative, but chloroethanol yielded a marginally positive and chloroacetaldehyde a strong positive result (0.06 and 746 revertants/iriraol, respectively, in TA100). The authors speculated that the mutagenic response of EDC may be due to relatively inefficient conversion of chloroethanol to chloroacetaldehyde in the in vitro system. Two reports (Rannug, 1976; Rannug and Ramel, 1977) compared the mutagenicity of EDC with that of EDC tar, a complex mixture formed during the manufacture of vinyl chloride from either acetylene, ethylene, or a mixture of the two. The mixture is called EDC tar because EDC is a major component comprising about 30$ of the mass. The sample of EDC tested in these studies was from British Drug House (BDH) Chemicals, Ltd. (purity not given). Salmonella tester strain TA1535 was dosed up to 45 pmol/plate in the presence or absence of an exogenous metabolic activation system (from livers of male strain R Wistar rats) with and without NADP. Positive (twofold) increases in revertant frequencies over background (13.0 + 1.76) were observed in the plates receiving the highest dose (24.8 + 3-06) in tests conducted without metabolic activation. However, with activation, a stronger positive response was observed. The revertant count was elevated tenfold over the spontaneous level (132.8 + 8.35 vs. 15*5 + 1.21). The response to EDC was independent of the presence of NADP; in contrast, the mutagenicity of EDC tars was increased with metabolic activation only in the presence of NADP. It was concluded that the mutagenicity of EDC tar cannot be ascribed primarily to EDC, because EDC and EDC tar have different requirements for metabolic activation. In addition, the concentration of EDC present in EDC tar at the highest dose tested would yield only a marginal positive response if tested alone, while in fact a strong response was observed for the EDC tar. 9-138 CMA 051772 Following these observations, Rannug et al. (1978) tested Salmonella strain TA1535 with EDC (BDH Chemicals Ltd.; although the purity was not reported, the material was checked by glass capillary chromatography using a flame ionization detector) at doses up to 60 praol/plate. Tests were conducted with and without an exogenous rat liver metabolic activation system. The components of the activation system were varied in order to study the mechanism of activation of EDC. Phenobarbital-induced liver fractions from Sprague-Dawley or R strain Wistar rats were prepared with and without an NADPH-generating system and with and without glutathione transferases A, B, and C. It was observed that EDC is activated by the cytosol fraction of liver homogenates; thus, mixed-function oxygenases are not involved. The activation was NADPH-independent but required glutathione (GSH) A or C S-transferase enzyme activity. The extent of activation was found to be dependent on the rat strain used (liver fractions from R strain rats were more effective than those from Sprague-Dawley rats) and the handling of the extract (e.g., storage on ice or freezing reduced the effectiveness of the activation system). The report by McCann et al. (1975) (described earlier in this section) that EDC was not metabolized to a mutagenic intermediate may be due to differences in the exogenous activation system used (S9 mix rather than cytosol fraction) in the two studies. Rannug et al. (1978) hypothesized that the mutagenicity of EDC after metabolic activation was caused by the formation of a highly reactive half sulfur mustard, S-(2-chloroethyl)-L-cysteine (see section 9.1.3.3.B). This compound (>99$ purity) was found to be more strongly mutagenic in TA1535 than is EDC. At 0.2 pmol/plate, 12.8 + 1.5 and 176.8 + 11.1 revertants were observed per plate after treatment with EDC and S-(2-chloroethyl)L-cysteine, respectively. At 5.0 pmol/plate the observed numbers of revertants per plate were 13*0 + 1.1 and 1954 (only one plate tested), respectively. Thus, S-(2-chloroethyl)-L-cysteine gives a strong mutagenic effect at low doses where no effect can be seen for EDC. 9-139 CMA 051773 In intact mammals most GSH conjugates are normally excreted in bile. Rannug and Bieje (1979) reasoned that if EDC were metabolically activated by conjugation with glutathione to form a half sulfur mustard, mutagenic products in the bile would be produced in EDC-treated mammals or perfused livers. To test this hypothesis, an EDC concentration of 0.1 mS, (1.3 mmol) were perfused through R strain Wistar rat livers for up to 4 hours. Bile was collected right after addition of EDC and at 15 and 30 minutes, as well as 1, 1.5, 1.75, 2, 3 and 4 hours later. The bile was then added directly to top agar or diluted about 5 to 10 times in sterile water and added to top agar containing Salmonella strain TA1535 for plate incorporation tests. Positive responses were obtained. The greatest response (45-60 revertants per plate, compared to 7-10 in negative controls) was reached about 15 minutes to 1 hour after addition of EDC. In a second experiment, 80 mg/kg EDC was given to male CBA mice intraperitoneally. The animals were sacrificed, their livers removed, and bile collected 30 and 60 minutes later for mutagenicity testing with TA1535 in plate incorporation tests. An increase in revertants (greater than twofold) was observed for bile collected 30 minutes after treatment compared to bile from negative control animals (28.8 + 27 revertants and 11.3 + 1*1 revertants, respectively). The positive responses obtained with bile from the perfused rat livers and intact mouse livers are consistent with the hypothesis that EDC is activated by conjugation with gluta thione. Four studies have been reported in which EDC was found to be negative in the standard Salmonella/microsome assay plate incorporation tests (King et al., 1979; Nestmann et al., 1980; Stolzenberg and Hine, 1980; Barber et al., 1981). The maximum doses employed in the first three studies were 36 pmol/plate, 91 pmol/plate, and 10 pmol/plate, respectively; the fourth study did not report the doses used. The doses reported in these studies therefore encompass a range 9-140 CMA 051774 at which positive responses have been reported in other studies. Except for the report by Stolzenberg and Hine (1980), the negative Salmonella plate incorporation studies were conducted with appropriate positive controls. Each test was conducted with and without PCB-induced rat liver S9 mix. It should be pointed out, however, that the positive controls, which require activation, were not structually similar to EDC. Therefore, these positive controls may not be able to determine the effectiveness of the components in the S9 mix necessary for EDC activation. King et al. (1979) also reported negative responses when E. coli K12/343/113 was tested in either a liquid suspension test or an intrasanguineous host-mediated assay. Two of the negative studies (i.e., Nestmann et al., 1980; Barber et al., 1981) reported that although EDC did not induce mutations in standard plate incorporation tests, positive responses were obtained when the studies were conducted in air tight exposure chambers. Nestmann et al. (1980) exposed Salmonella strains TA1535 and TA100 to doses from 3 to 9 mg/plate (30 to 91 iimol/plate) in desiccators. It was reported that this treatment yielded positive results, at least for TA1535 in which there was a doubling in the number of mutant colonies over the control. It was stated that concentrations were tested up to levels where cell-killing was observed, but no data are given and insuffi cient detail is provided to allow the results to be adequately evaluated. Barber et al. (1981) exposed Salmonella tester strains TA1535, TA98, and TA100 to four levels of EDC vapors (Eastman Kodak Co., 99.98)1 pure) in a 3-1*liter airtight exposure chamber. These exposures resulted in estimated plate concentrations ranging from 31*8 to 231.8 umol/plate as determined by gas liquid chromatography analysis of distilled water samples placed in the exposure chamber. Linear, dose-related increases in revertant counts were observed for TA1535 and TA100. The mutagenicity of EDC in these two strains was 0.002 and 9-141 CMA 051775 0.001 revertants/nmol, respectively. No difference in revertant counts or potency of EDO was noted in comparisons of tests done with and without metabolic activation. The positive results were obtained by Nestmann et al. (1980) and Barber et al. (1981) only when tests were conducted in airtight exposure chambers. This suggests that standard mutagenicity testing of EDC (bp 83-84) may not provide an adequate assessment of its mutagenic potential due to excessive evaporation. In summary, the positive responses obtained without metabolic activation indicate that EDC is a mutagen in bacteria. The positive responses obtained with metabolic activation indicate that one or more of its metabolites are more potent mutagens. The negative findings reported in bacterial tests are not considered to contradict the reported positive results, because the negative results may have been due to excessive evaporation of EDC or to inadequacy of the S9 activation system. 9.4.1.2. EUCARYOTIC TEST SYSTEMS -- In addition to causing gene mutations in bacteria, EDC also has been shown to cause gene mutations in eukaryotes. 9.4.1.2.1. Higher Plants -- Two reports were evaluated concerning the ability of EDC to induce mutations in higher plants (Table 9-28). Ehrenberg et al. (1974) treated barley seeds (variety Bonus) with 30.3 mmol EDC (Merck, purity not given) for 24 hours and scored for sterile spikelets at maturity or chlorophyll mutations in about 600 spike progenies in the subsequent generation. The dose level tested corresponds to the LD^q. EDC treated kernels had an increased incidence of chlorophyll mutations (6.8$) compared to untreated controls (0.06$). Kirichek (1974) exposed eight varieties of pea seeds (100 each) to gaseous EDC (purity and concentration not given) for 4 hours. The germination of treated 9-142 CMA 051776 TABLE 9-28 Summary of Mutagenicity Testing of EDC: Higher Plants Reference Teat System Ehrenberg et al.. 1974 Segregating chlorophyll (gene) imitations In barley Chemical Information Concentration tested: 200 seeds treated with 30.3 mmol/24 hours (LD^_) in a closed vessel. Results Comments Positive response. (6-8$ mutants from treated progeny vs. 0.06$ mutants from control progeny). .1 About 600 spike progeny were tested. Source: Merck Co. Darmstadt, PRO Purity: Hot given Kiricheck, 1974 Visible mutations In peas, 8 varieties Concentration tested: EDC (concentration not reported) or water (negative controls) vapors for 4 hours. Source: Not reported Purity: Not reported Reported postive. 5.42-28.13$ of seeds reported to be mutated. .1 English translation of Russian article. 2. Germination of the treated seed varied with the variety tested from 15$ to 58$ compared to 100$ germination of the control seeds. 3- Control mutation frequency not given. 4. Putative mutations not characterized. 5. Hot possible to adequately evaluate results. n O tn -j seeds differed with the variety tested but was reduced (15-50$ germination) compared to negative control seeds exposed to water vapor for 4 hours (100$ germination). From 5.4$ to 28.13$ of the seeds were reported to be mutated. It is reported that two times as many morphological mutants were induced as chloro phyll mutations. The author considered this a positive response, but the muta tion frequency for negative controls was not given. This limitation of the report plus a lack of information concerning the characterization of the putative mutations precludes an adequate evaluation of the results. 9.4.1.2.2. Insects -- Four studies were evaluated concerning the ability of EDC to cause mutations in Drosophila melanogaster (Table 9-29). These studies demonstrated the ability of EDC to cause sex-linked recessive lethal mutations (Shakarnis, 1969, 1970; King et al., 1979). The fourth study demonstrated the induction of somatic cell mutations by EDC (Nylander et al.f 1979). Shakarnis (1969, 1970) performed two experiments to assess the ability of EDC (purity not given) to cause sex-linked recessive lethal mutations in Drosophila. In the first study (Shakarnis 1969), adult females from a radio sensitive strain (Canton S) were exposed to 0.07$ EDC gas for 4 or 8 hours at 2425C, Immediately after treatment the females were mated to Muller-5 or In w B males. Fertility of treated females was reduced 47$ by the 4-hour treatment and 91$ by the 8-hour treatment. The F^ progeny were scored for lethality (measured as the absence of wild-type males). A statistically significant (p<0.05) timerelated increase in the incidence of sex-linked recessive lethals was observed. The frequencies of lethals were 0.3$ (negative controls), 3.2$ (4-hour treatment) and 5.9$ (8-hour treatment). In his second study, Shakarnis (1970) exposed females from a radiostable strain (D-32) of Drosophila melanogaster to 0.07$ EDC vapors (source and purity not reported) for 4 or 6 hours. Immediately 9-144 CMA 051778 TABLE 9-29 Summary of Mutagenicity Testing of EDC: Gene Mutation Tests in Insects Reference Shakarnis, 1969 Test System Drosophi la melanogaster sex-linked recessive lethal test Chemical Information Concentration tested: virgin 3-day-old Canton S females exposed to 0.07? EDC gas for 4 or 8 hours at 24-25C. Source: Hot given Purity: Not given Results Postive response Comments a 1. Muller-5 or In w B males. 2. Fertility of treated females reduced significantly (47? after 4 hour treatment and 91? after 8 hour treatment). 3. Canton S strain reported to be sensitive. Duration of Treatment (h) with Number of Lethal Mutations vo 0.07? EDC Chromosomes Scored No. ? Control 4 8 4910 2061 4750 15 0.30 67 3.22 201 5.91 p<0.05 CMA 0 5 1 7 7 9 TABLE 9-29 (cont.) Reference Shakarnls, 1970 Test System Drosophllla melanogaster sex-linked recessive lethal test Chemical Information Concentration tested: virgin 3-day-old radiostable D-32 strain females exposed to 0.07* EDC gas for 4 or 6 hours at 24-25C. Source: Not given Purity: Not given Results Positive response Comments 1. Muller-5 males. 2. Experiment conducted twice for 4 hour exposure and three times for 6 hour exposure. 3. 4 hour treatment did not significantly reduce fertility but 6 hour treatment reduced it by 50*. Duration of Treatment (h) with 0.07* EDC Number of Chromosomes Scored Lethal Mutations No. * Control 4 6 1904 2205 2362 1 0.05 46 2.00 76 3.30 (p<0.05) CMA 0 5 1 7 8 0 051781 Reference King et al 1979 yl > ^ Q TABLE 9-29 (eont.) Teat Syatern Drosophila melanogaater sex-linked recessive lethal teat Chemical Inforoation Reaults Concentration teated: 50 mM solution of EDC in 5% sucrose fed to 1- to 2-day-old Berlin K males for 3 days (near Positive reaponae Source: Merck Co. Darmstadt, FRG Purity: Not given but atated to have correct melting point (SIC) and elemental analysis Commenta 1. Base females. 2. Lethal frequency increased for all broods. Greatest effect in brood II, which corresponds to the spermatid stage of spermatogenesis. Cone. (mH) Brood Days after Treatment Number of Chromosomes Scored Lethal Mutations No. I P 0 I-III 50 I II III I-III 0-9 0-3 4-6 7-9 0-9 22,048 1,185 1,179 156 2,520 47 0.21 6 0.51 41 3.48 <0.01 2 1.28 49 1.94 <0.01 TABLE 9-29 (cont.) Reference Nylander et al., 1978 Teat System Drosophila melanogaster somatic cell mutations sc stocks. Chemical Information Results Comments Concentration tested: 0.1ft and 0.5ft EDC in food at 25"C and 75ft humidity during larval development. Source: Fisher Scientific Co. Purity: Not given Positive response 1. Mutations at z locus scored in flies of stable and unstable genotype. Both have same phenotype. Instability thought to be caused by transpo3able genetic element. 2. Survival of treated flies reduced significantly In both genetically stable (34ft reduction for 0.1ft and 77ft reduction for 0.5ft dose) and genetically unstable stocks (86ft reduction for 0.5ft dose). i 4= CD ap<0.001 Treatment Number Males Scored Number with Sectors ft t Value Differences t Value Differences Within Genotypes Between Genotypes Control Stable Unstable 0.01ft Stable Unstable 0.05ft Stable Unstable 4441 5363 6260 2689 610 201 2 4 263 274 44 50 0.045 0.075 4.20 9.28 7.21 24.86 -- -- 18.74* 24.31* 11.46' 13.12' 0.60 9.16a 5.66a CMA 0 5 1 7 8 2 after treatment they were mated to Muller 5 males and the progeny scored for sex-linked recessive lethals. The 4-hour treatment did not significantly reduce fertility but the 6-hour treatment reduced it by 50$. As in the first study, a statistically significant (p<0.05) time-related increase in sex-linked recessive lethals was observed. A frequency of 0.05$ lethals was observed in the negative controls, and frequencies of 2.00$ and 3.30$ lethals were observed in offspring from the 4-hour and 6-hour treatments, respectively. This corresponds to the mutagenic effect induced by an acute dose of two to three thousand (2000 to 3000) rad of x-rays. King et al. (1979) also studied the ability of EDC to cause sex-linked recessive lethals in Drosophila. One to two day old Berlin K males were fed a 50 mM solution of EDC (Merck and Co., purity not given but correct melting point and elemental analysis reported) in 5$ sucrose for 3 days. This dose approximated the LD,-0. The males were immediately mated to virgin Base females every 3 days for a total of 9 days to determine if EDC preferentially damaged particular stages in spermatogenesis. Progeny of the generation were scored for lethality. The lethal frequency was found to be elevated for all three broods (0.51$, 3.48$, and 1.28$, respectively, for broods I, XI, and III) compared to the negative controls (0.21$). The highest lethal frequency (3.48$) was found in brood II (p<0.01), which corresponds primarily to the spermatid stage of spermatogenesis. Nylander et al. (1979) raised flies on food containing 0.1$ and 0.5$ EDC (Fisher Scientific Co., purity not given) during larval development and scored F^ progeny for the induction of somatic cell sex-linked mutations in the eye. A genetically unstable stock (sc z w+) and a genetically stable stock (z Dp w+6 lei 9 ) both having the same phenotype were used in these studies and mated to attached X females. Mutations result in the expression of normal (dark red) eye pigment in adult treated males. The genetic instability of sc z w+ is 9-149 CMA 051783 caused by the insertion of a transposable genetic element. The survival of flies raised on the EDC-treated food was significantly reduced compared to the negative control values (e.g., 77$ reduction for the stable stock and 86$ reduction of the unstable stock at 0.5$ EDC). Statistically significant increases in somatic cell mutations occurring in both stocks were observed at both the low and high doses (p<0.001). The positive responses in Drosophila indicate that EDC is capable of causing both somatic cell and heritable germinal mutations in a multicellular eukaryote. 9.4.1.2.3. Mammals 9.4.1.2.3.1. In Vitro. Tan and Hsie (1981) found that EDC (Matheson, Coleman and Bell, purity not given) caused a dose-related increase in HGPRT mutations in cultured Chinese hamster ovary (CHO) cells (Table 9-30). CHO-K^BH^ cells were exposed in suspension culture for a period of 5 hours to EDC concentrations ranging up to 3 mM (30$ survival) in tests with exogenous rat liver S9 mix for metabolic activation and concentrations ranging up to 50 mM (50$ survival) in tests conducted without metabolic activation. Positive responses were observed both in tests with (5 mutants/10^ cells/mmol) and without (1 rautant/10^ cells/mmol) metabolic activation (p<0.01). EDC was detected as a mutagen in studies not requiring metabolic activation only at high doses, but the induced mutation frequency was increased about tenfold over control values. Only about a fourfold additional increase in mutagenicity was noted with metabolic activation but excessive toxicity precluded testing at concentrations greater than 3mM (i.e., S9 mix increased mutagenic activity by approximately fourfold and cytotoxic activity from 5 to 25-fold). The metabolic activation system was only effective in the presence of NADP. This contrasts with Rannug (1976) who found metabolic activation of EDC to be NADP-independent. 9-150 CMA 051784 TABLE 9-30 Summary of Mutagenicity Testing of EDC: Mammalian Systems Reference Tan and Hsie, 1901 Test System Chinese hamster ovary cell HGPRT gene mutation asaay Gocke et al., 1903 Mouse Spot Test C57Bl/6JHan (F) X T-stock (M) Chemical Information Results Comments Concentration tested: Up to 3 mM in tests with rat liver S9 mix and up to 50 mM in tests without metabolic activation. LD is 1 mM with activation and 6 mM without activation. One l.p. injection 10 days after conception of 300 mg/kg EDC in olive oil. Source: Merck Co. Darmstadt, FRG .Positive response , 1 Mutagenic activity of EDC without and 60 vs. 3 mutants/10" with metabolic activatiog calculated clonable cells for to be 1 and 5 mutants/10 cells/ 50 and 0 mM EDC mmol, respectively. without activation.. 28 vs. 3 mutants/10" 2. S9 mix increases mutagenicity by clonable cells for about fourfold and cytotoxicity 1.5 and 0 mM EDC 5- to 25-fold. with activation. 3. HADP required in S9 mix for metabolic activation. 7/11011 offspring had spots compared to 2/812 in the oil controls and 3/2161 in the cumulated controls. .1 Data suggestive of a positive response Purity: Hot given CMA 0 5 1 7 8 5 9.4.1.2.3.2. In Vivo. Gocke et al. ( 1983) conducted a mouse spot test to evaluate the ability of EDC to induce somatic cell mutations in developing mice. One 300 mg/kg intraperitoneal injection of EDC (Merck, purity not given) was administered to C57 Bl/6JHan females 10 days after mating with T-stock males. Seven out of the resulting 11 OH offspring had mutant coat color spots compared to 2/812 in the controls and 3/2161 in the combined untreated controls. The data show a significant (p=0.03) effect against the cumulated controls, but no significance (p=0.l8) against the oil control. These data are suggestive of a positive response. The consistency of positive results obtained in higher plants, Drosophila, and cultured mammalian cells demonstrates that EDC causes gene mutations in higher eukaryotes. 9.H.2. Chromosome Aberration Studies. Five studies were evaluated regarding the ability of EDC to cause chromosome aberrations (Table 9-31). One was an abstract of testing EDC for its ability to cause chromosome breakage in Allium root tips and cultured human lymphocytes (Kristoffersson, 197H). Two were Drosophila melanogaster X chromosome nondisjunction tests (Shakarnis, 1969, 1970), and two were micronucleus tests (King et al., 1979; Jenssen and Ramel, 1980). In the paper by Kristoffersson (197H) EDC (source, purity, and concentra tion not given) was reported to cause C-mitoses in Allium root tip cells but not to cause chromosome breaks in Allium root tip cells or human lymphocytes. Because the report was an abstract, insufficient information was available to substantiate the reported results. However, the induction of C-mitoses in Allium root tip cells suggests that EDC can interfere with the mitotic spindle apparatus. 9-152 CMA osne LQLISO V* Reference Shakarnis, 1969 ViO UU)l ap<0.05 TABLE 9-31 Summary of Mutagenicity Testing of EDC: Chromosomal Aberrations Testa Test System Drosophila melanoRaster X chromosome nondisjunction. Canton S (F) X Muller-5 (M) Chemical Information Results Comments Concentration tested: virgin Canton S females exposed to 0.07% EDC in 1.5 desiccator for 4 and 8 hours at 24-25C. Source: Not given Purity: Not given Statistically 1. The study was poorly significantly designed in that the (p<0.05) increases Canton S stock did not in exceptional possess appropriate female progeny reported genetic markers to for 4-hour exposure rule out non-virginity and male and female as the cause for the progeny for 8-hour "exceptional" progeny. exposure. However, test judged to be 2. The control rate was invalid. much lower than the historical background rate. 0.07% EDC Duration of Treatment (h) Normal Progeny No. Females No. Males Exceptional Progeny Females Males No. % No. % Control 4 8 5,848 24,125 8,437 5,727 21,297 7,773 00 00 8 0.03 3 0.01 15 0.18a 7 0.09a 88LTS0 Reference Shakarnis, 1970 vIO V-EJ1r Q TABLE 9-31 (cont.) Test System Drosophila melanomaster X chromosome nondisjunction. Canton S (F) X Muller -5 (M) Chemical Information Results Concentration tested: 3-day-old females from radiostable D-32 strain exposed to 0.07$ EDC in a 1.5 l desiccator for 4 or 6 hours at 24-25C. Source: Not given Purity: Not given Incidence of nondis junction greater in treated group than in control. Increase not statistically significant. Te3t judged to be invalid. Comments See comments for previous 3tudy. 0.07$ EDC Duration of Treatment (h) Normal Progeny No. Females No. Males Exceptional Progeny Females Males No. $ No. $ Control 4 6 2472 3584 4034 2205 3401 4090 00 1 0.03 4 0.10 1 0.03 1 0.03 3 0.07 TABLE 9-31 Ccont.) Reference Test System Chemical Information Results Comments King et al., 1979 Micronucleus test: NMRI mice Concentration tested: 2 i.p. injections of 4 mmol/kg (400 mg/kg) given 24 hours apart. 4 animals/dose sacrificed after second injection. Source: Merck Co. Darmstadt, FRG Purity: Not specified, but melting point and ele mental analysis were correct Negative 1. 1,000 polychromatic erythrocytes analyzed/animal. 2. Frequency of micro nuclei not given. Jenssen and Ramel, 1980 Micronucleus test: CBA mice Concentration tested: single i.p. injection 100 mg/kg. Source: BDH Chemicals, Ltd. Purity: Not given Negative. (0.15 + 0.14) polycromatic erythrocytes with micronuclei in controls vs. 0.17 0.10 in treated animals). CMA. 0 5 1 7 8 9 Data perhaps consistent with this possibility, but not conclusively showing non-disjunction, are found in studies by Shakarnis (1969, 1970) using Drosophila melanogaster. Females were exposed to 0.07? EDO vapors (source and purity not given). In the first study (1969) a statistically significant (p<0.05) increase in exceptional progeny, indicative of meiotic nondisjunction, was reported after a 4-hour treatment (0.03? exceptional females) and 8-hour treatment (0.18? exceptional females and 0.09? exceptional males) compared to the negative control value (0?; 0/11575 progeny). In the second study, the incidence of exceptional progeny was elevated in the treated groups but not significantly so. However, both studies were flawed by an experimental design which did not allow discrimination between progeny resulting from nondisjunction compared to those from non-virgin females. Micronucleus tests were performed by King et al. (1979) and Jenssen and Ramel (1980). Both studies reported negative results. King et al. (1979) injected NMRI mice with two intraperitoneal injections of 4 mmol/kg (400 mg/kg) EDC (Merck Co., purity not specified but melting point and chemical analysis reported to be correct). The authors state this corresponds to an "approximate lethal dose" (the L.Dl,o for intraperitoneal injections of EDC in mice is 250 mg/kg). The injections were given 24-hours apart; the animals were killed 6- hours after the second injection, and bone marrow smears were made. One thousand polychromatic erythrocytes (PCEs) were analyzed per animal. Frequencies of micronuclei were not given, but the results were evaluated to be negative by the authors. Jenssen and Ramel (1980) also reported negative results in a micronucleus test. CBA mice were given a single intraperitoneal injection of EDC (BDH Chemicals Ltds., England, purity not given) at a dosage of 100 mg/kg. The animals were sacrificed the next day and PCEs (number not given) were scored for 9-156 CMA 051790 micronuclei. The frequencies of PCEs with micronuolei were 0.15 + 0.14 in the controls and 0.17 + 0.10 in treated animals. The equivocal X-chromosome loss test in Drosophila (Shakarnis, 1969) may suggest that EDC can cause meiotic nondisjunction. A properly designed experiment should be conducted to determine this. The negative responses obtained in the micronucleus tests may indicate that EDC does not cause chromosomal damage in mice. However, because EDC has not been adequately tested for its ability to cause structural chomosomal aberrations, it would be appropriate to perform mammalian in vitro and in vivo cytogenetic tests. Such testing is required before a judgment can be made on the ability of EDC to cause chromosomal aberrations. 9.4.3. Other Evidence of DNA Damage. Three other tests have been conducted on the genotoxieity of EDC. These tests do not measure mutagenic events per se in that they do not demonstrate the induction of heritable (i.e., somatic or germinal) genetic alterations, but positive results in these test systems suggest that DNA has been damaged. Such test systems provide supporting evidence useful for assessing genetic risk. 9.4.3.1. BACTERIAL TEST SYSTEMS 9.4.3.1.1. PolA Assay -- Ethylene dichloride has been reported positive in the polA assay which measures toxicity associated with unrepaired damage in DNA (Table 9-32). Brem et al. (1974) soaked sterile filter disks with 10 pJi (80 pmol) EDC. These were centered on the agar surface of petri dishes covered with bacteria (one set with polA* strain, the other with polA~ strain). After incubation the plates were scored for differential inhibition of growth. An 8-mm zone of growth inhibition was observed in the polA* strain compared to a 9-mm zone of inhibition for the polA~ strain. These responses were said to be 9-157 CMA 051791 TABLE 9-32 Suoaary of Mutagenicity Testing of EDC: PolA Assay Reference Brem et al., 1974 Test System Strain polA differential cell killing assay E. coll polA polA* Activation System (tone Chemical Information Concentration tested: 10 jit on filter disk Results Comments Questionable positive 1. All assays carried out in duplicate on at least three different occasions. 2. Only a small difference in diameter (i.e., 1 mm) was noted between the zones of killing in the two strains. Thus, the A*/A- ratio may be misleading. Pol A Zone of Inhibition vO A+ A" a+/auooi " mm EDC 8 9 1.26 MMS 45 54 1.44 Chloramphenicol 28 28 1.00 Q O Ui -O tD K> reproducible. The ratio between the zones of inhibition (polA+/polA-) was 1.26 which is interpreted by the authors to be a positive response. However, it should be noted that 1 mm is not a big difference, and the ratio may be misleading. 9.4.3.2. EUKARYOTIC TEST SYSTEMS 9.4.3.2.I. Unscheduled DNA Synthesis -- One test has been performed to assess the ability of EDC to cause unscheduled DNA synthesis CPerocco and Prodi 1981). Although demonstration that a chemical causes unscheduled DNA synthesis does not provide a measurement of its mutagenicity per se, it does indicate that the material damages DNA. Perocco and Prodi (1981) collected blood samples from 5 healthy humans, separated the lymphocytes, and cultured 5 x 10 of them in 0.2 ml medium for 4 hours at 37C in the presence or absence of EDC (Carlo Erban, Milan, Italy or Merck-Schuchardt, Darmstadt, FRG, 97$--99% pure). The tests were conducted both in the presence and in the absence of PCB-induced rat liver S9 mix. A comparison was made between treated and untreated cells for scheduled DNA synthesis (i.e., DNA replication) and unscheduled DNA synthesis. No difference was noted between the groups with respect to scheduled DNA synthesis measured as dpm of [ H] deoxythymidylic acid (TdR) after 4 hours of culture (2661 + 57 dpm in untreated cells compared to 2287 + 60 dpm in cells treated with 5 pJt/mJl [0.06 mM] EDC). Subsequently 2.5, 5, and 10 pi/mS, (0.03, 0.06 and 0.1 mM) EDC was added to cells which were cultured in 10 mM hydroxyurea to suppress scheduled DNA synthesis. The amount of unscheduled DNA synthesis was estimated by measuring dpm from incorporated [^H]TdR 4 hours later. At 10 \il/ml EDC 483 + 37 and 532 + 21 dpm were counted without and with exogenous metabolic activation, respectively. Both values were lower than corresponding negative controls of 715 + 24 and 612 + 26 dpm, respectively. No positive controls were run to ensure that the system was working properly although testing of chloromethyl methyl ether 9-159 CMA 051793 (CMME) with activation resulted in a doubling of dpms over the corresponding negative control values (1320 + 57 at 5 CMME versus 612 + 26 untreated). The authors calculated an effective DNA repair value (r) for each chemical based on the control and experimental values with and without metabolic activation. Ethylene dichloride was evaluated by the authors as positive in the test, but they did not state their criteria for classifying a chemical as positive. It is important to note that none of the experimental values from cells treated with EDC without metabolic activation had higher dpm values than the controls. Furthermore, although two out of three experimental values were greater than the controls with metabolic activation (673 + 45 at 5 p8./mJl and 630 + 34 at 2.5 p/mS, compared to control value of 612 + 26) the increases were not statistically significant. The positive finding reported in this work is therefore judged to be inconclusive. 9.4.3.2.2. Detection of DNA Adducts -- Reitz et al. (1982) compared the pharmacokinetics of EDC administered to Osborne-Mendel rats after inhalation and after exposure by gavage. (See section 9*1.3, especially 9.1.3*5.) As part of their study, DNA alkylation was measured in bacteria at EDC cytosol concentrations corresponding to those used in the DNA binding study (Table 9--33)Two gram aliquots of TA1535 were incubated with 7.06 pmol [ 14 CJ EDC/mJl (sp. act. = 3*2 raCi/mmol) and varying amounts of cytosol. DNA alkylation values at cytosol concentrations of 2.2, 7.8, 27, and V\% were 8.65, 27, 107, and 137 dpra/mg purified DNA, respectively. This corresponds to 4, 12.5, 49, and 64 alkylations C x 10 DNA nucleotides. The corresponding reversion frequencies were 4.6 + 0.82, 23.5 + 3.0, 80.2 + 9.6, and 111 + 2.6, respectively (n = 3 in each case). A direct correlation between the degree of alkylation and an increase in mutation frequency was indicated by linear regression analysis (r = 0.9976). In the DNA alkylation studies with rats [^C] EDC (sp. act. = 0.32 mCi/mmol) was administered to groups of three animals by gavage (150 mg/kg) or 9-160 CMA 051794 TABLE 9-33 Summary of Mutagenicity Testing of EDC: DMA Binding Studies Reference Test System Strains Activation System Chemical Information Reitz et al., 1982 DNA alkylation and mutagenesis in Salmonella TA1535 PhenobarbitalInduced rat liver cytosol S Cytosol 2.2 7.8 27 71 DNA alkylation in rats OsborneMendel rat NA Route of Exposure Gavage 150 mg/kg Inhalation 150 ppm, 6 hours Revertants <1.6 + 0.82 23.5 + 3.0 80.2 + 9.6 111 + 2.6 Alkylation x 10"** Nucleotides 4 12.5 *19 69 Tissue Liver Spleen Kidney Stomach Liver Spleen Kidney Stomach Alkylation x 10~^ Nucleotides (means from 2 experiments 21.3 ; 13.9 5.8 ; 2.5 17.4 ; 14.5 14.9 ; 6.7 8.2 ; 1.8 ; 5.2 ; 2.8 ; 3.3 1.8 2.0 1.9 Comments 1. Bacteria incubated with 7*06 nmol EDC/m. 2. Significant correlation between degree of alkylation and increased reversion frequency (r = 0.9976). 3. Rats sacrificed 4 hours post gavage or immediately after inhalation. 4. Sp. act. 3.2 mCi/mmol for bacteria; 0.32 mCi/mmol for rats. CMA 0 5 1 7 9 5 inhalation (150 ppm, 6 hours). The animals were subsequently sacrificed and DNA was extracted from the liver, spleen, kidney, and stomach for measurement of DNA alkylation. Overall, three to five times more DNA alkylation was present after gavage than after inhalation. The values ranged from 5.8 + 0.7 to 23.1 + 7.4 alkylation/10^ nucleotides for gavage versus 1.8 + 0.3 to 8.2 + 3*3 alkylation/10^ nucleotides for inhalation. Under the conditions of test used in the experiments by Reitz et al. (1982) EDO exposure resulted in a-similar degree of adduct formation in both rats and bacteria. Because DNA is the genetic material in both bacteria and rats, these data predict that mutations were induced in the rat at the exposures used. This is in keeping with the positive responses in other eukaryotes including Drosophila and cultured CHO cells. DNA alkylation was not measured in rat gonads so it is not possible to estimate what the heritable genetic risk might be. 9.4.4. Summary and Conclusions. Ethylene dichloride (EDC) has been shown to cause gene mutations in bacteria, plants, Drosophila, and cultured Chinese hamster ovary (CHO) cells. Positive responses were observed in bacterial and CHO cells in tests that did not require an exogenous metabolic activation system. Stronger positive responses were found when hepatic metabolic activation systems were incorporated. Based on these positive findings in different test systems representing a wide range of organisms, EDC is judged to be capable of causing gene mutations. EDC has been reported to cause meiotic chromosomal nondisjunction in Drosophila. The induction of meiotic nondisjunction is a significant genotoxic effect; however, insufficient data are provided to be able to evaluate this test adequately. A positive response in a properly conducted test is needed to permit a judgment on the validity of this report. With respect to its ability to cause structural chromosomal aberrations, sufficient testing has also not been 9-162 CMA 051796 performed. There is only one study on the ability of EDC to cause structural chromosomal aberrations (i.e., in Allium root tip cells and human lymphocytes). Because this study was reported in an abstract, the author's conclusions are unsubstantiated. Even though negative results are reported from micronucleus tests, additional information is needed to draw conclusions on the ability of EDC to cause chromosomal aberrations. For example, it would be appropriate to test EDC in in vitro and i vivo mammalian cytogenetic assays. A sister chromatid exchange assay would also be useful in assessing the ability of EDC to cause chromosome damage. There are no available data on the ability of EDC to damage DNA in mammalian germ cells. Thus, studies on the ability of EDC to reach germinal tissue would be appropriate to determine whether EDC has the potential to cause heritable mutations which may contribute to the genetic disease burden. The finding that EDC causes heritable mutations in Drosophila and alkylates DNA in several somatic tissues in the rat reinforces the need for further germ cell studies in mammals. Based on the weight-of-evidence, EDC is judged to be a mutagen that may have the potential to cause adverse effects in humans. 9-163 CMA 051797 9.5. CARCINOGENICITY The purpose of this section is to provide an evaluation of the likelihood that ethylene dichloride (EDC) (1,2-dichloroethane) is a human carcinogen and, on the assumption that it is a human carcinogen, to provide a basis for estima ting its public health impact, including a potency evaluation in relation to other carcinogens. The evaluation of carcinogenicity depends heavily on animal bioassays and epidemiologic evidence. However, other factors, including muta genicity, metabolism (particularly in relation to interaction with DNA), and pharmacokinetic behavior, have an important bearing on both the qualitative and quantitative assessment of carcinogenicity. The available information on these subjects is reviewed in other sections of this document and used in this evalu ation as appropriate. This section presents an evaluation of the animal bio assays, the human epidemiologic evidence, the quantitative aspects of assessment, and finally, a summary and conclusions dealing with all of the relevant aspects of the carcinogenicity of EDC. 9.5.1. Animal Studies. The carcinogenic potential of EDC has been investigat ed in a number of studies in which EDC was administered to rats and mice via various routes of administration. The following studies will be discussed: one gavage study performed by the National Cancer Institute (1978) in which EDC was administered to rats and mice; two inhalation studies, including one by Spencer et al. (1951) using rats, and one bioassay by Maltoni et al. (1980) in which EDC was administered to rats and mice; one intraperitoneal study by Theiss et al. (1977) in which EDC was administered to mice; and one skin painting study by Van Duuren et al. (1979) in which EDC was administered to mice. 9.5.1.1. NATIONAL CANCER INSTITUTE (1978) RAT STUDY -- Hazleton Labora tories America Inc., Vienna, Virginia, under the sponsorship of the NCI, con- 9-164 051'798 ducted a bioassay of 1,2-dichloroethane (EDC) using Osborne-Mendel rats. The NCI published a final report of the bioassay in 1978. Their results were also reported by Weisburger (1977), the International Agency for Research on Cancer (IARC, 1979), and Ward (1980). Technical-grade EDC, obtained for this study from the Dow Chemical Company, Midland, Michigan, was tested for its purity. Gas-liquid chromatography re vealed a purity of 98% to 99% EDC with 4 to 10 minor peaks (Ward, 1980). How ever, in the NCI bioassay a purity of greater than 90% was reported, with only two unidentified minor contaminants. Recently this discrepancy was resolved when the original sample of EDC was reanalyzed, at which time the EDC was found to be greater than 99% pure, with several unidentified contaminants. This analysis was also performed by the National Institute for Occupational Safety and Health (NIOSH) after completion of the bioassay; the sample contained about 99% EDC, with chloroform as the major contaminant (Hooper et al., 1980). Solutions of EDC were prepared in corn oil and administered by oral intubation to 200 Osborne-Mendel rats starting at 8 weeks of age. The design summary of this experiment is given in Table 9-34. On the basis of a sub chronic EDC study, 50 rats of each sex were used for each of two dose levels in the chronic study. The maximum tolerated dose (MTD) was determined to be 95 mg/kg/day for both males and females; the second dose was one-half the MTD (47 mg/kg/day). Twenty animals of each sex served as untreated controls; an equal number were given the vehicle (corn oil) by gavage. Because of the inadequacies of the subchronic studies, the initial doses administered to the test animals were found to be inappropriate. Early signs of toxicity necessitated several changes in the dosages (Table 9-34), Animal weight and food consumption per cage were obtained weekly for the first 10 weeks and monthly thereafter. Animals were 9-165 CMA 051799 TABLE 9-34. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC) GAVAGE EXPERIMENT IN OSBORNE-MENDEL RATS Group Initial number of animals 1,2-dichloroethane dosage3 Observation period Treated Untreated (weeks) (weeks) Time-weighted average dosage over a 78-week period*5 Males Untreated control Vehicle-control Low-dose 20 20 50 High-dosed 50 -- 0 50 75 50 50c 0 100 150 100 100c 0 -- 78 7 10 18 34 7 10 18 34 -- 106 32 -- -- -- 9 32 -- -- -- 9 23 -- 0 47 -- -- -- 95 -- -- -- -- Females Untreated control Vehicle-control Low-dose 20 20 50 High-dosed 50 106 0 78 32 50 7 -- 75 10 -- 50 18 -- 50c 34 9 0 MW 32 100 150 100 100c 0 7-- 10 --- 18 -- 34 9 15 0 47 -- -- -- WWW 95 -- -- -- " aDosage, given in mg/kg body weight, was administered by gavage five consecutive days per week. ^Time-weighted average dosage = (dosage x weeks received)/78 weeks. cThese dosages were cyclically administered with a pattern of one dosage-free week followed by 4 weeks (5 days per week) of dosage at the level indicated. ^All animals in this group died before the bioassay was terminated. SOURCE: NCI, 1978. 9-166 CMA 051800 checked daily for mortality. Weight depression was observed in both groups exposed to EDC, By 50 weeks, the weight depression averaged 12% in high-dose rats (Figure 9-8). Mortality was early and severe in dosed animals, especial ly those given the highest doses. The mean survival was approximately 55 weeks on test for high-dose males and females (Figure 9-9 and Table 9-35). The early deaths were usually not due to cancer; rather, the toxic effects of EDC appeared to be responsible for these deaths. Rats dying early had a variety of lesions, including bronchopneumonia and endocardial thrombosis, which may have contributed to early death. The pneumonia may have been the result of a viral, bacterial, or mycoplasmal infection, and the exposure to the chemical may have increased the tendency to develop severe pulmonary lesions, which would lead to death. For the high-dose male rats, 50% (25/50) were alive at week 55 and 16% (8/50) were alive at week 75. Survival was higher in the other groups; 52% (26/50) of the rats in the low-dose group lived at least 82 weeks, and 50% (10/20) in the vehicle-control group lived at least 72 weeks. In the high-dose female rats, 50% (25/50) were alive at week 57; 20% (10/50) of the rats in the low-dose group were alive at week 85. Despite the sacrifice of five females at week 57, 65% (13/22) of the untreated control group survived until the end of the study. A gross necropsy was performed on each animal that died during the experi- . ment or was killed at the end. A total of 28 organs, plus any tissues contain ing visible lesions, were fixed in 10% buffered formalin, embedded in paraplast, and sectioned at 5 y for slides. Hematoxylin and eosin stain were used rou tinely, and other stains were employed when necessary. Diagnoses of tumors and other lesions were coded according to the Systematized Nomenclature of Pathology (SNOP) of the College of American Pathologists (1965). 9-167 CMA 051801 MEAN BODY WEIGHT (Grams) 750 750 Low-Dose MALE RATS High-Dose o-i--i--|--i--i--i--i--i--i--i--i--i--i--i--i--i--r1 0 15 30 45 60 75 90 105 120 TIME ON TEST (Weeks) MEAN BODY WEIGHT (Grams) TIME ON TEST (Weeks) Figure 9-8. Growth curves for male and female Osborne-Mendel rats administered EDC by gavage. SOURCE: NCI, 1978. 9-168 CMA 051802 PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 1.0- -n-. 0.8- 0.60.4- i____ l_____ n_ l U FEMALE RATS - 1.0 -0.8 Hi tl. a, I_____ -0.6 -0.4 Untreated Control 0.2 H--------- Vehicle-Control - 0.2 --------- Low-Dose --------- High-Dose 0.0- I 1 I 1| 1 I i r 0.0 15 30 45 60 75 90 105 120 TIME ON TEST (Weeks) Figure 9-9, Survival comparisons for male and female Osborne-Menael rats administered EDO bv gavage. SOURCE: NCI, 1978. 9-169 CMA 051803 TABLE 9-35. TERMINAL SURVIVAL OF OSBORNE-MENDEL RATS TREATED WITH 1,2-DICHLOROETHANE (EDC) Group Males Weeks in study Animals alive at end of study Females Weeks in study Animals alive at end of study Untreated control Vehicle-control Low-dose High-dose 106 110 110 101 4/20a (20%) 4/20 (20%) 1/50 (2%) 0/50b (0%) 106 13/20a (65%) no 8/20 (40%) 101 1/50 (2%) 93 0/50b (0%) aFive rats were sacrificed at 75 weeks. bAll animals in this group died before the bioassay was terminated. SOURCE: Adapted from NCI, 1978. Squamous cell carcinomas of the forestomach occurred in 3/50 (6%) low-dose males, 9/50 (18%) high-dose males, 0/50 (0%) high-dose females, and 1/49 (2%) low-dose females (Table 9-36). None of these tumors occurred in the controls. For male rats, the Cochran-Armitage Trend Test indicated a significant (p = 0.01) positive association between dosage and the incidence of squamous cell carcinoma of the stomach. The Fisher Exact Test confirmed the significance of these results with p values of 0.039 and 0.001 when comparisons were made with the matched vehicle-control group and the pooled vehicle-control* group, respectively. Squamous cell carcinomas of the forestoraach were first observed in highdose male rats at 51 weeks after oral intubation of EDC. These lesions were *The pooled vehicle-control group combined the vehicle-controls from the studies of 1,2-dic.hloroethane, 1,1,2-tric.hloroethane, and trichloroethane. 9-170 CMA 051804 characterized microscopically by acanthosis and hyperkeratosis in the super ficial area. The basal epithelial layer contained papillary cords and nests of anaplastic squamous epithelium supported by a dense band of fibrous connective tissue. These carcinomas extended through the muscularis mucosa, submucosa, muscular layers, and serosa, and in one high-dose male, metastasized to adja cent tissues. TABLE 9-36. SQUAMOUS CELL CARCINOMAS OF THE FORESTOMACH IN OSBORNE-MENDEL RATS TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Males p value3 Females p value3 Untreated control Matched vehiclecontrol Pooled vehiclecontrol Low-dose High-dose 0/20 (0%) 0/20 (0X) 0/60 (0%) 3/50 (6%) 9/50 (18%)b NS 0.001 0/20 (0%) 0/20 (0%) 0/59 (0%) 1/49 (2%) 0/50 (0%) NS NS ap values calculated using the Fisher Exact Test. Treated versus pooled vehicle-control. bA squamous cell carcinoma of the forestoraach metastasized in one male in this group. NS = not significant when p values are greater than 0.05. SOURCE: Adapted from NCI, 1978. Hemangiosarcomas were noted in some treated rats, as described in Table 9-37, but not in any of the controls. Low-dose males and females showed higher incidences of hemangiosarcoma than high-dose animals. Tumors were observed in several sites, including spleen, liver, adrenal glands, pancreas, large intes tine, subcutaneous tissue, and abdominal cavity. The Cochran-Armitage Trend 9-171 CMA 051805 Test indicated a significant (p = 0.021) positive association between dosage and the incidence of hemangiosarcomas in male rats as compared to the pooled vehicle-control rats; the trend was also positive in female rats (p = 0.042). The Fisher Exact Test confirmed these findings, with statistically significant p values for high-dose males versus pooled vehicle-controls (p = 0.016), for low-dose males versus pooled vehicle-controls (p = 0.003), and for both lowand high-dose females versus pooled vehicle-controls (p = 0.041). TABLE 9-37. HEMANGIOSARCOMAS IN OSBORNE-MENDEL RATS TREATED WITH 1,2-DICHL0R0ETHANE (EDO) Group Males p value3 Females p value3 Untreated control Matched vehiclecontrol Pooled vehiclecontrol Low-dose*1 High-dosec 0/20 (0%) 0/20 (0%) 1/60 (2%) 9/50 (18%) 7/50 (14%)b 0.003 0.016 0/20 (0%) 0/20 (0%) 0/59 (0%) 4/50 (8%) 4/50 (8%) 0.041 0.041 ap values calculated using the Fisher Exact Test (one-tailed). Treated versus pooled vehicle-control. ^Only 48 animals were examined for hemangiosarcomas of the large intestine. cOnly 49 animals were examined for hemangiosarcomas of the spleen and adrenals and 48 for hemangiosarcomas of the pancreas. SOURCE: Adapted from NCI, 1978. In addition to stomach carcinomas and hemangiosarcomas, EDC-treated female rats showed significant increases in the incidence of mammary adenocarcinomas (Table 9-38). Tumors were observed in the high-dose group as early as 20 weeks after treatment. The Cochran-Armitage Trend Test detected a significant 9-172 CMA 051806 (p < 0.001) positive association betwen the dosage and the incidence of mammary adenocarcinomas when compared with either control group. This tumor Incidence was significant when the high-dose group was compared with either the matched vehicle-control group (p < 0.001) or the pooled vehicle-control group (p = 0.002) using the Fisher Exact Test. Historically, this tumor was observed in 4/200 (2%) of the vehicle-control females. TABLE 9-38. ADENOCARCINOMAS OF THE MAMMARY GLAND IN FEMALE OSBORNE-MENDEL RATS TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Adenocarcinoma of the mammary gland p value Untreated control Matched vehicle-control Pooled vehicle-control Low-dose High-dose 2/20 (10%) 0/20 (0%) 1/59 (2%) 1/50 (2%) 18/50 (36%) NS 0.0008 ap values calculated using the Fisher Exact Test (one-tailed). pooled vehicle-control. NS = not significant. Treated versus SOURCE: Adapted from NCI, 1978. An increased incidence of fibromas of the subcutaneous tissue was reported in both high-dose (p = 0.007) and low-dose (p 0.017) male rats when compared to the pooled vehicle-control group. In summary, a statistically significant increase in the incidence of squa mous cell carcinomas of the forestomach, hemangiosarcomas of the circulatory system, and fibromas of the subcutaneous tissue occurred in male rats. There 9-173 CMA 051807 was also a statistically significant increase in the incidence of adenocarci nomas of the mammary gland and hemangiosarcomas of the circulatory system in female rats. 9.5.1.2. NATIONAL CANCER INSTITUTE (1978) MOUSE STUDY -- Hazleton Labora tories America Inc., under the sponsorship of the NCI, conducted a bioassay of 1,2-dichloroethane (EDC) using B6C3F1 mice. The results of this study were published by the NCI (1978) and were also reported by Weisburger (1977), the International Agency for Research on Cancer (IARC, 1979), and Ward (1980). Technical-grade EDC (with the same purity as that described in the NCI rat study) was administered to 200 B6C3F1 mice starting at 5 weeks of age. The design summary for this experiment is given in Table 9-39. On the basis of results of the subchronic studies, 50 mice of each sex were used at each of the two dose levels for the chronic study. The MTD was determined to be 195 mg/kg/ day for male mice and 299 mg/kg/day for female mice. The second dose, which was one-half the MTD, was determined to be 97 mg/kg/day for male mice and 149 mg/kg/day for female mice. Twenty mice of each sex served as untreated controls, and an equal number were given the vehicle (corn oil) by gavage. Because of inadequacies in the subchronic studies, the initial doses administered to the test animals were found to be inappropriate. Signs of toxicity in these animals early in the study led to changes of the dosages several times (Table 9--40). Animal weights and food consumption were recorded for the first 10 weeks and monthly thereaf ter. Animals were checked daily for mortality and signs of toxic effects. No dose-related mean body weight depression was observed in male mice or low-dose female mice (Figure 9-10). A depression in the mean body weight of the highdose female mice was apparent as early as week 15. The estimated probabilities of survival for male and female mice in the control and EDC-dosed groups are 9-174 CMA 051808 TABLE 9-39. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC) GAVAGE EXPERIMENT IN B6C3F1 MICE Group Initial number of animals 1,2-dichloro- Observation period ethane Treated Untreated dosagea (weeks) (weeks) Time-weighted average dosage*5 Males Untreated control Vehicle-control Low-dose 20 20 50 High-dose 50 -- 0 75 100 0 150 200 0V -- 78 8 70 8 70 -- 90 12 -- -- 12 -- -- 13 -- 0 97 -- 195 -- ----- Females Untreated control Vehicle-control Low-dose 20 20 50 High-dose 50 91 0 78 32 125 8 -- 200 3 -- 150 67 -- 0 13 250 8 -- 400 3 -- 300 67 -- 0 13 0 149 -- -- 299 -- -- aDosage, given in mg/kg body weight, was administered by gavage five consecutive days per week. ^Time-weighted average dosage * (dosage x weeks received) weeks receiving chemical SOURCE: Adapted from NCI, 1978. 9-175 CMA 051809 shown in Figure 9-11. Terminal survival of treated and control mice is shown in Table 9-40. For male mice, no statistically significant association between dosage and mortality was observed. In the high-dose group, 50% (25/50) of the mice were alive at 84 weeks and 42% (21/50) survived until the end of the study. In the low-dose group, however, survival was low. By 24 weeks, 52% (26/50) of the low-dose group and 55% (11/20) of the untreated control group had died. In the high-dose group, 72% (36/50) of the animals died between weeks 60 and 80. These deaths may have been tumor-related, since 69% (25/36) had one or more tumors. Survival was high in the other groups; 68% (35/50) of the low-dose and 80% (16/20) of the untreated control groups survived until the end of the study. TABLE 9-40. TERMINAL SURVIVAL OF B6C3F1 MICE TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Males Weeks in study Animals alive at end of study Untreated control Vehicle-control Low-dose High-dose 90 90 90 91 7/20 (35%) 11/20 (55%) 11/50 (22%) 21/50 (42%) SOURCE: Adapted from NCI, 1978. Females Weeks in Animals alive study at end of study 90 16/20 (80%) 90 16/20 (80%) 91 34/50 (68%) 91 1/50 (2%) A gross necropsy was performed on each animal that died during the experi ment or was killed at the end. Twenty-eight organs in all, plus any tissues containing visible lesions, were fixed in 10% buffered formalin, embedded in 9-176 CMA 051810 MEAN BODY WEIGHT (Grams) 50 50 -30 -20 10- --------- Vehicle-Control -- Low-Dose ----------High-Dose --------1--|--i--i--i------ 1--i------- 1--i--|--i--r 0 15 30 45 60 75 90 TIME ON TEST (Weeks) -10 MALE MICE !-------1------r- --0 105 120 MEAN BODY WEIGHT (Grams) Figure 9-10. Growth curves for male and female B6C3F1 mice administered EDC by gavage. SOURCE: Adapted from NCI, 1978. 9-177 CMA 051811 PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL Figure 9-11. Survival comparisons for male and female B6C3F1 mice administered EDC by gavage. SOURCE: Adapted iron NCI, 1978 9-178 CMA 051812 paraplast, and sectioned at 5 p for slides. Hematoxylin and eosin stain were used routinely, with other stains employed as necessary. Diagnoses of tumors and other lesions were coded according to the Systematized Nomenclature of Pathology (SNOP) of the College of American Pathologists. The histopathologic findings of the study concerning hepatocellular carcinomas in mice are tabulated in Table 9-41. Hepatocellular Carcinomas occurred in 2/17 (12%) untreated control males, 1/19 (5%) low-dose males, and 12/48 (25%) high-dose males. A significant number of hepatocellular carcinomas were observed in high-dose males. The Cochran-Armitage Trend Test indicated a positive dose-response association when the high-dose group was compared with either the matched vehicle-controls (p * 0.025) or the pooled vehicle-controls (p = 0.006). The Fisher Exact Test supported this finding with a significant (p * 0.009) comparison of the high-dose to the pooled control group. TABLE 9-41. HEPATOCELLULAR CARCINOMAS IN B6C3F1 MICE TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Males p valuea Females p valuea Untreated controls Matched vehiclecontrols Pooled vehiclecontrols Low-dose High-dose 2/17 (12%) 1/19 (5%) 4/59 (7%) 6/47 (13%) 12/48 (25%) NS 0.009 0/19 (0%) 1/20 (5%) 0/50 (0%) 1/47 (2%) NS NS ap values calculated using the Fisher Exact Test (one-tailed). pooled vehicle-control. NS = not significant. Treated versus SOURCE: Adapted from NCI, 1978. 9-179 CMA 051813 A large number of alveolar/bronchiolar adenomas were found in mice treated with EDC (Table 9-42); 31% in both male (15/48) and female (15/48) high-dose mice. These adenomas were not observed in untreated or vehiclecontrol males. For both sexes the Cochran-Armitage Trend Test showed a sig nificant (p - 0.005) positive dose-response association when the dosed groups were compared to either control group. The Fisher Exact Test also indicated that the high-dose group for both sexes had significantly higher (p " 0.016) incidence rates than either of the two control groups. For female mice, the Fisher Exact Test comparing the low-dose group to the pooled vehicle-control group was also statistically significant (p = 0.046). TABLE 9-42. ALVEOLAR/BRONCHIOLAR ADENOMAS IN B6C3F1 MICE TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Males p value3 Females p value3 Untreated controls Matched vehiclecontrols Pooled vehiclecontrols Low-dose High-dose^ 0/20 (0%) 0/19 (0%) 0/59 (3%) 1/47 (2%) 15/48 (31%) NS 0.0025 1/19 (5%) 1/20 (5%) 2/60 (3%) 7/50 (14%) 15/48 (31%) 0.046 0.0201 ap values calculated using the Fisher Exact Test (one-tailed). Treated versus pooled vehicle-control. ^In addition, one high-dose female mouse had an alveolar/bronchiolar carcinoma. NS not significant. SOURCE: Adapted from NCI, 1978. 9-180 CMA 051814 1 Squamous cell carcinomas of the forestomach occurred in 1/19 (5%) vehiclecontrol males, 1/46 (2%) low-dose females, and 5/48 (10%) high-dose females (Table 9-43). For female mice, the Cochran-Armitage Trend Test indicated a significant (p = 0.035) positive association between dosage and the incidence of squamous cell carcinomas of the forestomach when comparing the dosed groups to the pooled vehicle-controls. The Fisher Exact Test, however,-was not significant. The microscopic appearance of the stomach of mice was comparable to that described for rats. TABLE 9-43. SQUAMOUS CELL CARCINOMAS OF THE FORESTOMACH IN B6C3F1 MICE TREATED WITH 1,2-DICHL0R0ETHANE (EDC) Group Males p value3 Females p value3 Untreated controls Matched vehiclecontrols Pooled vehiclecontrols Low-dose High-dose 0/20 (0%) 1/19 (5%) 1/59 (2%) 1/46 (2%) 2/50 (4%) NS NS 0/20 (0%) 1/20 (5%) 1/60 (2%) 2/50 (4%) 5/48 (10%) NS NS ap values calculated using the Fisher Exact Test (one-tailed). pooled vehicle-control. NS = not significant. Treated versus SOURCE: Adapted from NCI, 1978. The incidence of adenocarcinomas of the mammary gland in female mice treated with EDC is presented in Table 9-44. The Cochran-Armitage Trend Test indicated a significant (p = 0.007) positive association between dosage and 9-181 CMA 051815 the Incidence of adenocarcinomas of the mammary gland when compared to the pooled vehicle-controls. The Fisher Exact Test confirmed these results with a significant (p 0.003) comparison of both high-dose (7/48) and low-dose (9/50) groups to the pooled vehicle-control groups (0/60). TABLE 9-44. ADENOCARCINOMAS OF THE MAMMARY GLAND IN FEMALE B6C3F1 MICE TREATED WITH 1,2-DICHLOROETHANE (EDC) Group Adenocarcinoma of the mammary gland p value3 Pooled vehicle-controls Matched vehicle-controls Low-dose High-dose 0/60 (0%) 0/20 (0%) 9/50 (18%) 7/48 (15%) 0.0005 0.0026 ap values calculated using the Fisher Exact Test (one-tailed). Treated versus pooled vehicle-control. SOURCE: Adapted from NCI, 1978. Endometrial tumors observed in female mice are described in Table 9-45. The Cochran-Armitage Trend Test indicated a significant (p = 0.017) positive association between dosage and combined incidence. The Fisher Exact Test showed a statistically significant (p * 0.014) incidence in the high-dose (5/47) group. In summary, the NCI study in B6C3F1 mice demonstrated a statistically significant increase in incidences of hepatocellular carcinomas and alveolar/ bronchiolar adenomas in male mice and a statistically significant increase in incidences of alveolar/bronchiolar adenomas, mammary carcinomas, and endometrial tumors in female mice. 9-182 CMA 051816 TABLE 9-45. ENDOMETRIAL POLYP OR ENDOMETRIAL STROMAL SARCOMAS IN FEMALE B6C3F1 MICE TREATED WITH 1,2-DICHLOROETHANE (EDC) Group Endometrial polyp or endometrial stromal sarcomas p valuea Pooled vehicle-controls Matched vehicle-controls Low-dose High-dose 0/60 (0.0%) 0/20 (0.0%) 5/49 (10.0%) 5/47 (11.0%) 0.016 0.014 ap values calculated using the Fisher Exact Test (one-tailed). Treated versus pooled vehicle-control. SOURCE: Adapted from NCI, 1978. 9.5.1.3. SPENCER ET AL. (1951) RAT STUDY -- An early inhalation study conducted by Spencer et al. (1951) found no evidence of carcinogenic activity when 15 male and 15 female Wistar rats were exposed 151 times during a 212-day period to EDC at 200 ppm for 7 hours per exposure. 9.5.1.4. MALTONI ET AL. (1980) RAT STUDY ---- A more recent inhalation study conducted by Maltoni et al. (1980) in Sprague-Dawley rats provided no evidence of carcinogenicity after lifetime exposure to EDC. The EDC used in this study was supplied by Montedison, and had a purity of 99.82%, with five contaminants (Table 9-46). The design of the experiment is given in Table 9-47. Maltoni et al. exposed four groups of 12-week-old Sprague-Dawley rats (each group consisting of 180 rats of both sexes) to EDC concentrations of 250150 ppm, 50 ppm, 10 ppm, and 5 ppm, respectively, 7 hours per day, 5 days per week, for 78 weeks. After several days of 250 ppm exposure, the rats began to exhibit severe toxic effects, and the concentration was reduced to 150 ppm. Two 9-183 CMA 051817 TABLE 9-46. CHARACTERIZATION OF 1,2-DICHLOROETHANE (EDC) INHALATION EXPERIMENT IN SPRAGUE-DAWLEY RATS Component 1,2-Dichloroethane 1,1-Dichloroethane Carbon tetrachloride Trichloroethylene Perchloroethylene Benzene SOURCE: Maltoni et al., 1980. Purity 99.82% 0.02% 0.02% 0.02% 0.03% 0.09% groups, composed of 180 rats per group, served as controls. One of the two control groups was kept In an exposure chamber under the same conditions and for the same length of time as the exposed rats. At the end of the treatment period, the animals were allowed to live until spontaneous death. Animals were weighed every 2 weeks during the treatment period and every 8 weeks there after. All detectable gross pathologic changes were recorded. A complete autopsy was performed on each animal, with histopathologic examinations conduc ted on the following: brain, Zymbal glands, retrobulbar glands, lnterscapular brown fat, salivary gland, tongue, lungs, thymus, diaphragm, liver, pancreas, kidneys, spleen, stomach, various segments of the intestine, bladder, gonads, lymph nodes (axillary, inguinal, and mesenteric), and any other organ with pathologic lesions. The extent of mortality varied with the different groups, but there appears to be no direct relationship between mortality and exposure to EDC (Table 9-48). 9-184 CMA 051818 TABLE 9-47. DESIGN SUMMARY FOR 1,2-DICHL0R0ETHANE (EDC) EXPERIMENT IN SPRAGUE-DAWLEY RATSa Group Concentration Animals*1 Sex Number I 250-150 ppmc M 90 F 90 II 50 ppm M 90 F 90 III 10 ppm M 90 F 90 IV 5 ppm M 90 F 90 V Controls in chambers M 90 F 90 VI Controls M 90 F 90 aExposed 7 hours/day, 5 days/week, for 78 weeks. bsprague-Dawley rats, 12 weeks old at start. cAfter a few weeks the dose was reduced to 150 ppm, because of high toxicity at the 250 ppm level. SOURCE: Maltoni et al., 1980. 9-185 CMA 051819 TABLE 9-48. SURVIVAL OF SPRAGUE-DAWLEY RATS EXPOSED TO EDC AT 52 AND 104 WEEKS3 Group Dose Initial (ppm) Sex numbe rs Survivors at 52 weeks of age Number Percent Survivors at 104 weeks of age Number Percent Survivors after 52 weeks from start of study Number Percent I 250-150b M 90 79 87.8 10 11.1 67 74.4 F 90 84 93.3 21 23.3 79 87.8 II 50 M 90 87 96.7 17 18.9 70 77.8 F 90 87 96.7 29 32.2 84 93.3 III 10 M 90 81 90.0 13 14.4 70 77.8 F 90 87 96.7 26 28.9 81 90.0 IV 5 M 90 89 98.9 45 50.0 75 83.3 F 90 90 100.0 48 53.3 85 94.4 V Controls M 90 80 88.9 12 13.3 64 71.1 In chambers F 90 79 87.8 22 24.4 73 81.1 VI Controls M 90 83 92.2 16 17.8 72 80.0 F 90 88 97.8 36 40.0 84 93.3 aRats were 12 weeks old at the start of the experiment. bAfter a few weeks the dose was reduced to 150 ppm because of high toxicity at the 250 ppm level. SOURCE: Maltoni et al., 1980 CMA 051820 The highest survival rate was observed in both males and females of the group exposed to EDC at 5 ppm. In females the highest mortality rate was observed in the control group in the chamber and in the group exposed to EDC at 250-150 ppm. The survival rates at 52 and 104 weeks of age are reported in Table 9-48. At these ages the overall survival rates were 93.9% and 27.3%, respectively. The survival rates after 52 weeks from the start of the experiment are also given in Table 9-48, The overall survival rate was 83.7%. The results of histopathologic analysis are shown in Table 9-49. No statistically significant increase in the incidence of any specific type of tumor was found in the treated rats when compared with controls. There was, however, an increased incidence of mammary tumors in some rats in Group IV, particularly when compared with the control group in the chamber (Table 9-50). It appears that the increase in mammary tumors in some of the treated groups, particularly when compared to controls in the chamber, was not due to malignant tumors, but to fibromas and fibroadenomas. The Increased incidence of mammary fibromas and fibroadenomas is statistically significant in groups exposed to EDC at 250-150 ppm, 50 ppm, and 5 ppm, when compared to controls in the chamber, but not significant when compared to controls outside of the chamber. Although the author stated that the onset of fibromas and fibroade nomas was age-correlated, the incidences of these tumors observed in the vari ous groups were probably due instead to the differences in survival rates within the groups. The highest difference in incidence was found between the control groups in the chamber, which had low survival rates, and the groups treated with EDC at 5 ppm, which had high survival rates. The difference in survival rates is thus seen to be related to the differences in incidence in the two control groups. 9-187 CMA 051821 VtiW.V. 9-49. TIMOR INCIIWNCK IN SPRAOOK-DAWLBY RATS BXPOSKII TO l. 2-D1CHLOROBTHAME (EDC) Animals with tumors Ma--ary tumorsCymbal gland carcinomasLeukemiasNephroblastomas {nm p ral ion Animals0_____________________ Average Average Average Average latency latency latency latency Number Corrected Tata 1 t i rue Total tine Total t lne Total time Sex at si iri number imnK' r Percent*1 (k)c number Percent^ (wk)d number Percent^ (wk)d number Percent^ (uk)<> T .'SO-iso K V Mr mo MO IHO 11 vID m 0000 SO ppm 10 ppm M K M ami K H K H ami F MO MO 1 HO 90 MO IHI1 IV ' pp M 90 F 9f) M and F 18(1 V taint roln In H 90 chambers K 90 M ,1ild K 1H0 V I t.ont rols M F N a nrl F 90 91) LHlI 89 90 179 90 40 180 89 90 1 79 90 90 IHO 90 90 180 90 90 IHO 11 12.1 92.9 0 52 57.7 7H.6 2 b1 15.2 81.1 2 10 M. 1 88.8 0 58 64.4 78.5 1 bfl 17.H 80.0 1 5 5.6 60.8 0 4 1 47.8 79,4 0 48 26.8 77.5 0 ft11 12.2 110.2 65 72.2 81.2 2 76 42.2 87.L * 8 8.9 85.5 l 18 42.2 81.3 0 14b 25.5 81.6 S 5,5 92.0 l 52 57.8 85.5 1) 57 31.7 86.1 1 ___ 2.2 l.l -- 1.1 0.6 -- -- -- -- 2.2 1.1 L.l -- 0.6 l.l -- 0.6 ___ 78.0 78.0 -- 86.0 86.0 -- -- -- -- 47.0 47.0 78.0 -- 78.0 74.0 -- 74.0 0 0 0 i 2 3 4 0 4 2 6 8 i 3 4 0 3 3 __ __ 0 -- --0 ----0 i.i 80.0 0 2.2 74.5 0 1.7 76.3 0 4.6 -- 2.2 74.0 -- 74.0 0 0 0 2.2 133.5 1 6.7 83.5 0 4.4 96.0 1 i.i 37.0 0 3.1 64.3 0 2.2 59.5 0 -- 3.3 1.7 -- 79.3 79.3 0 0 0 ___ -- -- -- -- -- -- -- -- 1. L -- 0.7 -- -- -- -- -- -- ___ -- -- -- -- -- -- -- -- 84.0 -- 84.0 -- -- -- -- -- -- Tut ,il 1080 1070 (continued on the Following page) CMA 0 5 1 8 2 2 051823 VID 0<s0D o TARLK 9-49. (cnnt Inued) Group 1 1( 11E IV V VI Concent rat Ion Sea 2MJ-IVI ppwne SO ppm in r>pm > pp Controls in chambers Controls M F H and F H H and F M F M and F M F H and F H F H and F H M and F Tot al number 0 1) n n i) 0 tl 0 0 0 0 0 0 o 0 0 Liver Perrenr^* ____ -- -- -- -- -- -- -- -- --- -- -- -- -- -- Angiosarcomas Average 1atencv t Ime (k><! _____ -- -- -- -- -- -- -- --- -- -- -- -- -- -- -- Total number 0 o tl 0 a ft 0 0 0 ft 0 0 0 ft 0 ft Animals with tumors Other Sites Percent** Average latency t Ime Ink)11 _____ -- -- -- -- -- --- -- -- -- -- -- -- -- -- -- __ -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Tntal number 0 0 a 0 ft 0 0 0 0 ft 0 ft 0 0 0 0 Angiomas and fIbroanglomas Li ver Other sites Percent** Average 1atency time {wMc Tntal number Percent*1 Average 1atency time <wk)d ... -- -- -- -- -- -- -- -- -- -- _____ -- -- -- -- -- 0 0 ------- -------- 0-- -- 0-- -- -- -- -- _____ -- -- ft -- 0 -------- -------- 0 ------- -------- 0-- , ui izs.q 1 i.i ni.o 2 UI E2A.0 _____ ---- 0 _____ _____ 0 -- -- 0-- -- ui nn.o -- 1 o.7 no.o (continued on the following page) 051824 Croup 1 11 11 [ IV V VI Consentrat Inn Hepatoma a Average latency Total time Sex number Perrent ** (wk)*1 2 5n-| V) ppm* SO ppm 10 ppm 5 ppm Control** in chanhers Controls M H and F M M and Y M K M rind F M H mid K H F M and F H F M and F ft 0 0 0 0 0 0 (1 ft 0 0 0 0 0 0 0 0 0 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- TABLE 9-49. (continued) Animals with tumors Forestomach epithelial Skin carcinomas Total minber Percent b Average latency time (wk)d Total number Percent** Average latency time (vk>d 0 ---- 0 D ---- 1 l.l 60.0 0 0.6 60.0 0 -- ---------- '' l l.l 78.0 l 0.6 78.0 l l.l 101.0 1 (1.6 101.0 2 2.2 78.5 1 LI 63.0 3 1.7 73.1 2 2.2 110.0 l l.l 102.0 3 1.7 107.1 0 ---- 0 0 ---- l L 1 56.0 l 0.6 56.0 1 i.i 94.0 ft *-- -- l 0.6 94.0 ft -- -- O -- -- 0 ---- 0 -- -- 0-- 0 -- -- 0 ---- 0" Subcutaneous sarcomas Total number Percent** Average latency time <k)d 0-- 0-- 0-- 0-- 0-- 0-- 0-- -- -- -- -- -- -- 1 l.l 30.0 0 1 0.6 30.0 0-- -- 1 1. 1 78.0 1 0.6 78.0 0-- 0-- 0 -- -- 051825 \0 kO n Group l II m IV V VI Concentration Sex 2StJ-| 50 ppme *>*J ppm 10 ppm 5 ppm Controls In chambers Contro Is M F H and F W F H and F H F H and F M F K and F H F M and K H F H and F TABLE 9-49. (continued) Animals with tumors Encephalic tumors Neurnhlaslomas Other sites Ot here Total number Percent b Average latency t ime (klk)d Total number Percent'* Total number Benign number Malignant number 0 ___ ___ 0 ---- 0 ---- 0 ---- 0 ---- 0-- 0 -- -- 0-- 0 ---- (1 -- -- 0 ---- 0 -- -- 0 ---- 0 ---- 0 ---- 0 -- -- 0 ---- 0 --- -- 0 __ 9 0-- 11 0 -- 20 1 1.1 14 1 1.1 6 2 1.1 20 0 1.1 2-- 2 2.2 4 4 8 0-- 16 0-- 9 0 -- 25 l 1.1 1 1.1 2 1.1 0 ___ 3 3.3 3 i.7 8 4 12 7 8 15 72 56 12 8 14 0 33 17 3 22 31 53 15 i 54 20 5 7L 22 93 70 53 12 3 Number 15 54 69 20 56 76 13 43 56 30 65 95 17 38 55 14 56 70 Totals* Percent** Number of dlf ferent tumors/tumor bearing animals 16.9 60.0 38.5 22.2 62.2 42.2 14.6 47.8 31.3 33.3 72.2 52.8 18.9 42.2 30,6 15.6 62.2 38.9 1.2 2.0 1.9 1.6 1.7 1.7 1.1 1.6 1. 5 1.4 1.8 1.7 1.3 1.9 1.7 1.1 1.7 1.6 Exposure by Inhalation to EDO In air at 250-150 ppm, 50 ppn, 10 pp.. and 5 ppm, 7 hours/day. 5 days/week. ftir 78 weeks. Results after 148 weeks (end of experiment). aSprague-Dawley rats, 12 weeks old at start, bfhe percentages refer to the corrected numbers. cAverage age at the onset of the first mammary tumor per animal detected at the periodic control or at autopsy. ^Average time from the start of the experiment to detection at the periodic control or at autopsy. eAfter a few weeks the dose was reduced to 150 ppm, because of the high toxicity at the 250 ppm level, fSeveral animals with two or more tumors. SdllKCK: Ha It on l et at. , 1980. TABLE 9-50. MAMMARY TUMORS IN SPRAGUE-DAWLEY RATS EXPOSED TO 1,2-DlCHLOROETKANE (BDC) Animals Nuaber Corrected Group Concentration Sex at start number8 I 250-150 ppad II 50 ppm III 10 ppa IV 5 ppa V Controls In chaabers VI Controls Total M f M and F H F M and F M F M and F M F M and F H F M and F M F M and F 90 90 180 90 90 180 90 90 180 90 90 180 90 90 180 90 90 180 1080 89 90 179 90 90 180 89 90 179 90 90 180 90 90 180 90 90 180 1078 Total nuaber Hanoary tumors Percent*' Average latency tlae (wk)c Nuaber of tumors/1 uaorbearing animals 11 12.3 92.9 52 57.7 78.6 63 35.2 81.1 10 11.1 88.8 58 64.4 78.5 68 37.8 80.0 5 5.6 60.8 43 47.8 79.4 48 26.8 77.5 11 12.2 110.2 65 72.2 83.2 76 42.2 87.1 8 8.9 85.5 38 42.2 83.3 46 25.5 83.6 5 5.5 92.0 52 57.8 85.5 57 31.7 86.1 1.1 1.9 1.7 1.4 1.5 1.5 1.0 1.4 1.4 1.4 1.6 1.5 1.0 1.8 1.6 1.0 1.5 1.5 (continued on the following page) 361- 9Z8X90 TABLE 9-50. (continued) Group ConcentratInn Sex Total number Percent^ Number Mammary tumors Average latency Percent* time (wk)c Number Mammary tumors e Histological evaluation Zymbal gland care 1nomas Average latency t ime Percent* (wk)c Number Average 1atency time Percent* (wk)^ Number Percent* Average latency t ime (vk)c 1 250-150 ppm^ M 9 81 .a 7 77.8 99.1 , 11. 1 92.0 F 50 96. t 47 94.0 81.3 8 16.0 71.5 H and F 59 91.6 54 91.5 81.6 9 15.2 73.7 , 11. L 80,0 0 1 6.0 99.3 0 4 6.8 85.5 0 ___ ____ ---- T l 50 ppm __H 9 90.0 7 77.8 IO0.1 1 11.1 60.0 , 11.1 64.0 0 F 51 91*4 49 92.4 80.7 9 1 7.0 73.8 1 L. 9 88.0 0 -- ___ M and F b2 91.2 5b 90.3 81.1 10 16.1 72.4 2 3.2 76.0 0 ---- 111 10 ppm __ __M 5 100.0 1 bO.O 72.0 0-- ___ 2 40.0 44.0 0 F 38 88.4 31 8b. 8 80.9 8 21,0 88.5 0 ---- 0 ___ ____ M and F 4) 89, 6 lb 83.7 80. 1 8 18.6 88. 5 2 4.6 44.0 0 ---- [V 5 ppm M 11 IIHJ.O 11 100.0 1 10.2 0-- ___ __1 9. 1 114.0 0 v> F 60 92.3 5b 91.3 86.4 10 16.7 87.4 2 3.1 88.0 0 ___ ____ i-- H and F 71 91.4 67 94.4 90,3 10 14. 1 87.4 3 4.2 1<J1.3 0 ---- VO __ __U> V Controls In M 8 100.0 7 87.5 85.1 0-- ___ L 14.3 88.0 0 chambers F 15 92.1 27 77.1 85.5 15 46.8 80. L 1 2.6 124.0 0 ___ ____ H and F 4) 91.5 34 79.1 85.4 15 38. 5 60. 1 2 4.6 106.0 0 ---- VI Cont rols M 5 100.0 3 60.0 105. 7 0 ___ ___ F 49 94.2 47 95.9 88.1 8 16.3 80.4 M and F 54 94.7 50 92.b 89.3 8 14.8 80.4 2 40.0 72,0 0 0 ---- 2 2 3.7 72.0 2 __ 4. | 73.0 3.7 7 3.0 Exposure by inhalation to EDC In air at 250-150 ppm. 50 ppm. 10 ppm, and 5 ppm, 7 hours/day,, 5 days /week, for 7H weeks. Results after 148 weeks (end of experiment). aAn1ma!s alive after 12 weeks, when the first tumor (a mammary carcinoma) was observed. **The percentages refer to the corrected numbers. rAverage age at the onset of the first mammary tumor per animal detected at the periodic control or at autopsy. ^After a few weeks the dose was reduced to 150 ppm, because of ttie high toxirity at the 250 ppm level. eTwo or more tumors of the same or different types (fibroadenomas, carcinomas, sarcomas, carcinosarcomas) may be present In the 9ame animal. ^The percentages refer to total number of animals hearing mammary tumors. *The percentages refer to total number of anLmals bearing mammary tumors, histologically examined. SOURCE: Maltonl et al., I9B0. CMA 0 5 1 8 2 7 9.5.1.5. MALTONI ET AL. (1980) MOUSE STUDY -- Four groups of Swiss mice (180 mice of both sexes per group) were exposed to EDC, at the level of purity previously described for the rat inhalation study, in concentrations of 250-150 ppm, 50 ppm, 10 ppm, and 5 ppm, respectively, 7 hours per day, 5 days per week, for 78 weeks. After several days of 250 ppm exposure, the mice began to exhibit severe toxic effects, and the concentration was reduced to 150 ppm. One group of 249 mice served as controls. The design of the experiment is given in Table 9-51. At the end of the treatment period, the animals were allowed to live un til spontaneous death. The remainder of the procedure was the same as that described previously for the Maltoni et al. (1980) rat study. The survival rates for female mice were slightly lower in the group treated with EDC at 250-150 ppm. The survival rates for mice at 52 and 78 weeks of age are shown in Table 9-52. At 52 and 78 weeks, the overall survival rates were 82.4% and 45.9%, respectively. The survival rates after 52 weeks from the start of the experiment are also given in Table 9-52. The overall survival rate was 67.8%. The results of histopathologic analysis of various tumors are shown in Table 9-53. These results do not indicate a statistically significant increase in the incidence of any specific type of tumor in the treated mice as compared with controls. In conclusion, although Maltoni et al. conducted extensive carcinogenicity studies in rats and mice, no significant incidences of tumors were seen in any of the target organs. Several factors may have contributed to these findings, such as differences in the strains of rats used, and pharmacokinetic differences in rates of formation and retention of reactive metabolites in the target organs between gavage and inhalation routes of administration. 9-194 051828 TABLE 9-51. DESIGN SUMMARY FOR 1,2-DICHLOROETHANE (EDC) EXPERIMENT IN SWISS MICE Group Concentration Animals3 Sex Numbe r I 250-150 ppmb M F II 50 ppm M F III 10 ppm M F IV 5 ppm M 'F V Control M F 90 90 90 90 90 90 90 90 115 134 aSwiss mice, 11 weeks old at start. Exposed 7 hours/day, 5 days/week, for 78 weeks. ^After a few weeks the dose was reduced to 150 ppm, because of high toxicity at the 250 ppm level. SOURCE: Maltoni et al., 1980. 9-195 CMA 051829 TABLE 9-52. SURVIVAL OF SWISS MICE EXPOSED TO 1,2-DICHLOROETHANE (EDC) AT 52 and 78 WEEKS8 Croup Dose Cpp> Sex I 250-150 ppab M F II 50 M F III 10 M F IV 5 M F V Control M F Initial nuabera Survivors at 52 weeks of axe Nuaber Percent Survivors at 78 weeks of axe Nuaber Percent Survivors after 52 weeks froa start of study Nuaber Percent 90 5b 62.2 26 28.9 39 43.3 90 75 83.3 44 48.9 58 64.4 90 68 75.6 30 33.3 46 51.1 90 83 92.2 49 54.4 73 81.1 90 74 82.2 34 37.8 59 65.6 90 86 95.6 50 55.6 72 80.0 90 54 60.0 26 28.9 42 46.7 90 84 93.3 68 75.6 84 93.3 115 91 79.1 42 36.6 72 62.6 134 127 94.8 76 56.8 112 83.6 aMlce were It weeks old at the start of the experlaent. bAfter a few weeks the dose was reduced to 150 ppa, because of high toxicity at the 250 ppa level. SOURCE Maltoni et al., 1980 -196 TABLE 9-53. TUMOR INCIDENCE IN SWISS MICE EXPOSED TO l,2-DICHLOROETHANE tEDC) Group Concentration Sex I 250-150 ppm* M F H and F 11 50 ppm n F M and F III 10 ppn H F M and F IV 5 ppn H F M and F V Controls M F M and F Total Animals* Nunher Corrected at start number*1 90 90 180 90 90 180 90 90 180 90 90 180 L15 134 249 969 St 84 L65 87 87 174 89 88 177 69 89 158 111 133 244 918 __________________________________ Animals with tutors Mammary tumors_________ _________ Pulmonary adenomas______________ Total number Percent0 Average latency time (vk)<l 0 ___ _ 5 6.0 69.0 5 3.0 69.0 0---- 3 3.4 76.0 3 1.7 76.0 Total number 0 3 3 3 2 5 Percent0 _ 3.6 1.8 Average latency time (wk)^ 85.7 85.7 3.4 75.3 2.3 74.5 2.9 75.0 1 t.I 32.0 4 4.5 85.7 6 6.6 73.7 2 2.3 53.5 7 4.0 67.7 6 3.4 75.0 0-- -- \ 1.4 44.0 5 5.6 82.6 4 4.5 85.0 5 3.2 82.6 5 3.2 76.S 0---- 4 3.6 78.5 7 5.3 77.4 4 3.0 51.2 7 2.9 77.4 8 3.3 64.9 _______________ Leukemias Total number Percent0 Average latency time (wk)<l 1 1.2 75.0 I L. 2 63.0 2 1.2 69.0 6 6.9 69.8 4 4.6 59.2 10 5.7 65.6 6 6.7 54.5 5 5.7 69.6 a 6.2 61.4 2 2.9 79.0 6 6.7 69.6 6 5. t 72.1 6 5.4 65.8 15 U.3 61.3 2L 8.6 62.6 (continued on the following page) CMA 0 5 1 8 3 1 TABLE 9-53. (continued) Group Concentration Sex Nephroblastomas Total number Percent0 Average latency time (wk)d AnInals with tumors Kidney adenocarcinomas Total number Percent0 Average latency time (wk)<* Angiosarcomas _______Liver _______________ Other sites Total number Percent* Average latency time (wk)<* Total number Percent* Average latency t lme (wk)d '-198 I 250-150 ppm* H P 0 0 VO H and F 0 II 50 ppm M0 P0 M and F 0 til 10 ppm H0 P0 N and F 0 IV 5 ppm H0 F0 M and P 0 V Controls In >1 0 chambers F 0 H and F 0 VI Controls H 0 F0 H and F 0 n 0 0 0 0 0 0 0 0 0 0 --- --- 1 l.l 62.0 ] 0.6 62.0 0 0 0 0 0 0 0 -- 1 00 0 -- 1 0 --- --- 0 0 --- --- 0 0 --- --- 0 0 --- --- 0 0 --- --- 0 0 --- --- 0 00 00 00 0 --- --- 0 0 --- --- 0 0 --- --- 0 01 00 01 L. 2 40.0 0.6 40.0 125.0 131.0 126.0 0.9 69.0 0.4 69.0 (continued on the following page) TABLE 9-53* (continued) Group ConcentratIon Sex Animals with tumors Angiomas and flbroanglomas Liver Other sites Total number Percent0 Average latency time (wk)1' Total number Percent0 Average latency tine Hepatomas Total number- Percent0 Average latency time (wk)d t 250-150 ppme M 0 ---- H and F 0 0. -- -- 11 50 ppm H 0 ---- F l I.t m.o M and F i 0.6 83.0 111 10 ppm H 0 ___ F 0 -- -- H And F 0 ---- IV 5 ppn H 0 ---- H and F 0 0 ---- V Controls In H 0 chambers F 0 M and F 0 0.9 -- 0.4 69,0 -- 69.0 0---- 0 0---- i 1.1 76.0 t L.L 83.0 2 1.1 79. S 0 ____ 93.0 1 1.1 93.0 l 0.6 -- 0---- 0---- l 0.9 76.0 0---- 1 0.4 76.0 0 -- -- 0 0 -- -- 0 ---- 0 ---- 0 ---- 0 ---- 0 ---- 0 ---- 0 ---- 0 ---- 4 3.5 82.0 0 ---- 4 1.6 82.0 Forestomach epithelial tumors Total number Percent0 Ave rage latency t Ime {wk)<* 0 ---- 0 0 ---- 0 ___ ___ 0 -- -- 0 ---- 0 -- ____ 0 ---- 0 ---- 0 ---- 0 0 ---- 0 ---- 0 ---- 0 -- -- (continued on the following page) Croup 1 11 111 IV V Concentration Sex 2W-ISII ppm* H F H and E 50 ppn H F H and F 10 ppm H and F 5 ppm M F M and F Controls In chambers H F M and F Skin epithelial tumors Total number Percentc Average latency time (wk)d 0 ___ 0 -- -- 0 -- -- 0 ___ ____ 1 1.1 103*0 i 0.6 lOi.O 0 ___ ___ 0 -- -- 0 -- -- 0 ___ -- 1 L.l 79*0 l 0.6 79*0 l 0*9 109.0 1 ft. 7 77.0 2 0.8 93.0 TABLE 9-53. (continued) Animals with tumors Subcutaneous sarcomas Others Total number Percentc Average latency t Ime (wV>d Total nunher Benign numhe r Malignant number 0 _ __ 0---- 0---- 0 ___ -- 0---- 0---- 000 2 20 2 20 1 01 4 31 5 32 1 1.1 77.0 l 10 l l.l 25.0 ' l L 1 2 1.1 51.0 3 2 1 0---- 0---- 0-- -- 10l 4 3l 5 32 1 0.9 106.0 1 10 0---- 422 0 0.4 106.0 5 3 2 Number Total* Percent*1 Humber of different tumors/tumorbearing animals 2 2.4 ll 12.9 13 7.7 9 10*6 15 17.2 24 14.0 ) 2 13.5 17 19.1 29 16.3 4 5.7 19 21.3 23 14.5 14 12.4 27 20.1 41 16.6 1.0 1.0 1.0 1.2 l.l 1.2 l. 1 1.0 l* 1 t.O 1.0 1,0 1*4 l.l 1.2 Exposure by Inhalation to EDC In air at 250-150 ppm, 50 ppm, 10 ppm, and 5 ppm, 7 hours/dayt 5 days/week, for 78 weeks. Results after 119 weeks (end of experiment). aSwlss nice* 11 weeks old at start, ^Animals alive after 24 weeks, when the first tumor (a leukemia) was observed* cThe percentages refer to the corrected nunhers* dAver age time from the start of the experiment to detection at the periodic control or at autopsy. eAfter a few weeks the dose was reduced to 150 ppn, because of high tnxlcity at the 250 ppm level. ^Several animals with two or more tumors* SOURCE: Mallnnl et al,, I960. CMA 0 5 1 8 3 4 9.5.1.6. THEISS ET AL. (1977) MOUSE STUDY -- Theiss et al. (1977) con ducted a pulmonary tumor bioassay with EDC and other organic contaminants of drinking water in the United States. The compounds were injected intraperitoneally into 6- to 8-week-old mice of the A/st strain. Each dose of reagentgrade EDC, with tricaprylin as the vehicle, was injected into mice in groups of 20, three times a week, for a total of 24 injections per mouse. Dose levels of 20, 40, and 100 mg/kg (the maximum tolerated dose) were used. The mice were sacrificed 24 weeks after the first injection, and their lungs were placed in Tellyesnlczky's fluid. After 48 hours, the lungs were ex amined microscopically for surface adenomas, and the frequency of lung tumors in each group was compared with that in a vehicle-treated control group by means of the Student's t Test. The incidenceof lung tumors Increased with dose, but but none of the groups had pulmonary adenoma responses that were significantly greater (p > 0.05) than that of the vehicle-treated control mice (Table 9-54). Because they found a nonsignificant elevation in pulmonary tumor response, the authors suggested further investigation of the carcinogenic potential of EDC. TABLE 9-54. PULMONARY TUMOR RESPONSE IN STRAIN A/st MICE INJECTED WITH 1,2-DICHL0R0ETHANE (EDC) Dose/injection (mg/kg) Number of injections Fraction surviving 0a 20 40 100 24 46/50 (92%) 24 14/20 (70%) 24 16/20 (80%) 24 20/20 (100%) aTric.aprylin vehicle. SOURCE: Theiss et al., 1977. 9-201 Number of tumors/mouse 0.39 + 0.06 0.21 + 0.06 0.44 + 0.11 0.75 + 0.17 CMA 051835 9.5.1.7. VAN DUUREN ET AL. (1979) MOUSE STUDY -- Van Duuren et al. (1979) conducted a bioassay of EDC and of a suspected metabolite, chloroacetaldehyde, as initiators, promoters, and complete carcinogens using two-stage skin tests. Female non-inbred ICR/Ha Swiss mice were used, with treatment beginning at 6 to 8 weeks of age. The experimental group consisted of 30 animals, exclusive of the no-treatment groups or those receiving repeated application' of phorbol myristate acetate (PMA). The results of this study are given in Table 9-55. The compounds, in 0.1 raL and 0.2 ml. acetone, were applied three times a week to the dorsal skin. Skin lesions were diagnosed as papillomas when they reached approximately 1 mm and persisted for 30 days or more. All animals were examined daily and weighed monthly; findings were recor ded monthly. Animals in poor health or with large tumor masses were killed. Animals were completely autopsied at the termination of the experiment or at death. Tissue sections were fixed in 10X formalin, processed, blocked in par affin, and stained with hematoxylin and eosin for pathologic diagnosis. The results indicate that neither EDC nor chloroacetaldehyde induced a statistical ly significant increase in the incidence of carcinomas of the skin. This result was probably due to the low dose levels used in the study. EDC was found to induce a statistically significant increase in the incidence of benign lung papillomas. 9.5.1.8. SUMMARY -- The NCI study (1978), as well as the studies by Theiss et al. (1977) and Van Duuren et al. (1979), show that, under appropriate conditions, EDC can increase the incidence of tumors. The data obtained in mutagenicity studies have also shown that, under appropriate experimental conditions, EDC can cause DNA damage (Storer et al., 1982) and mutations (Gueijgerich et al., 1980). In contrast, the lifetime inhalation study conducted by Maltoni et al. (1980) showed no increased incidence of tumors. 9-202 CMA 051836 TABLE 9-55. MOUSE SKIN BlnASSAY OK I,2-DlCHLOROKTHANK <EDC) AND CHI.OR0ACETALDEHYDE Int tt at lon-promot loir Compound Dose mg/appl1 rat Inn/ mutism EMC 12b.0 Ch1oruarcta1dehyde WA controls <UO mice) (90 mice ) Acetone <0.1 mL) No treatment (100 mice) 1.0 0.0025 0.0050 -- Days to first tumor 157 152 141 449 -- Nice with pspt1 lumas0/ total pspi1 lumas V) VI (O 9/10 (1) 6/' (2) -- Kcpeated application*1 Duse mg/app11 ration/ moose Days to first tumor Mice with papl!lomasc/ total papiLlomas 12b. f) 0 42. fl -- 0 1.0 0 -- -- 0.1 nJL -- -- -- -- -- -- 0 0 No. of mice with distant tumors^ 2b tong p<0.0(H)5 T stomach l? lung l stomach 14 lung 1 stomach -- -- 1 l lung 2 stomach V> lung 5 stomach aAll applications were to the dorsal skin by micropipette, EOT was administered once only In 0,2 ml. acetone, followed 14 days later by 5 yg of KHA In (1.2 L acetone three times weekly. Chlnroaoetaldehyde was admlnlstered onre only In 0,1 ml. acetone, followed 14 days later by 2.5 yg of PKA In 0.1 mL acetone three times weekly, kfiPC was administered in 0,2 ml. acetone. Chloroacetaldehyde was administered three times weekly in 0.1 mL acetone. cNunhers of mice with squamous cell carcinomas are given In parentheses. dALl lung tumors are benign papillomas; stomch tumors are papllLomas of the forestomach and squamous cell car cinomas of the forestomarh; p values are given only where significant, l.e., p < 0.05. SOURCE: Adapted from Van Dimrcn et al., 1979, CMA 0 5 1 8 3 7 The apparent discrepancies between inhalation and other routes of exposure in the oncogenicity studies have led to considerable discussion of the conduct and results of these studies (Maltoni et al.f 1980; Hooper et al., 1980; Reitz et al. , 1982) (see discussion in section 9.5.3.). The MCI Clearing House Committee review of the NCI bioassay report (1978) acknowledged several design features which may have adversely affected the outcome of the experiment. For example, the studies were conducted in rooms housing animals dosed with other volatile carcinogens. It was concluded, however, that these shortcomings were not significant enough to invalidate the findings. Other investigators (Hooper et al., 1980) have concluded that unless unusual and unexpected synergisms occurred, the presence of other animals in the test rooms would not have affected the results. Regardless of the explanations for the observed differences (Hooper et al., 1980; Reitz et al., 1982), the data show both an ability of EDC to produce neoplasms and an inhalation level at which no tumors are formed. This may be due to the difference in rates of formation of reactive metabolites, rates of formation of DNA-adduct, and rates of deactivation between the oral and inhala tion routes. Thus, the results indicate that EDC Is carcinogenic in rats and mice, and is also a mutagen. 9.5.2. Epidemiologic Studies. No epidemiologic studies have been published regarding the carcinogenicity of EDC. 9.5.3. Risk Estimation from Animal Data. Evidence for the carcinogenicity of EDC consists principally of positive long-term gavage studies in mice and rats (NCI, 1978), a suggestive positive pulmonary tumor bioassay in mice (Theiss et al., 1977), and a suggestive positive two-stage mouse skin painting bioassay (Van Duuren et al., 1979). The NCI bioassay reported excess incidences of forestomach squamous cell carcinomas, circulatory system hemangiosarcomas, and subcutaneous tissue fibromas in male rats, and in female rats, mammary gland 9-204 CMA 051838 adenocarcinomas and hemangiosarcomas. In male mice, an excess incidence of hepatocellular carcinomas and bronchiolar adenomas were found, and in female mice, bronchiolar adenomas, mammary carcinomas, and endometrial tumors. Theiss et al. reported that their pulmonary tumor bioassay resulted in a doserelated increased frequency of lung adenomas in mice, while Van Duuren et al. found an increased incidence of benign lung papillomas in mice treated with EDC by skin painting. In contrast to these positive reports, lifetime inhalation studies in mice and rats by Maltoni et al. (1980) showed no excess incidence of tumors. Part of the explanation for these differing results may be the differences in the pharmacokinetics and metabolism of EDC for the two routes of administration, as discussed below. It Is generally assumed that the carcinogenic potential of EDC is dependent on the metabolism of the parent compound to nucleophilic metabolites (such as chloroacetaldehyde, the half-sulfur mustard S-(2-chloroethyl)-GSH, and its episulfonium ion) that extensively covalently bind to cellular lipids and proteins with organ localization and binding intensity paralleling sites of tumor formation in liver, lung, spleen, and forestomach (Reitz et al., 1982). DNA adduct formation with EDC metabolites has also been demonstrated both in vitro (Banerjee and Van Duuren, 1978, 1979; Banerjee et al., 1980; Guengerich et al., 1980) and in vivo (although at a low level) (Reitz et al., 1982; Storer et al., 1982). 9.5.3.1. GENERAL OBSERVATIONS -- For EDC, the positive results of the NCI (1978) oral gavage lifetime studies in male Osborne-Mendel rats and male B6C3F1 mice showing a statistically significant dose-related excess of tumors (hemangiosarcomas in rats and hepatocellular carcinomas in mice) were select ed for risk assessment. Tumors were found at both "high" (maximum tolerated dose) and "low" doses (one-half maximum tolerated dose), as summarized in 9-205 CMA 051839 Table 9-56. The chronic oral dosage used in the NCI rat bioassay (95 and 47 mg/kg/ day) was well below (8- to 16-fold) the acute LD50 for rats (680 to 770 mg/kg; McCollister et al., 1956; Smyth, 1956; Heppel et al., 1945). Similarly, the chronic oral dosage in the NCI mouse bioassay (195 and 97 mg/kg/day) was well below the acute LD50 report for mice (489 and 413 mg/kg male and female, respectively; Munson et al., 1982). Nonetheless, signs of toxicity from chronic administration of EDC (less weight gain, shortened life span) were evident in the NCI bioassay for both rats and mice. Presumably EDC is sub jected to elimination processes during the 24-hour interval between doses (and on weekends), and hence the chronic dosage of the NCI assays may not be cumulative. EDC has a short half-life in rodents after acute oral administra tion (in corn oil) of 1 hour for a 150 mg/kg dose (Spreafico et al., 1979, 1980); hence, the conditions of the NCI study could be represented kinetically as repeated (multiple) acute exposures rather than chronic exposure in which the administration is repeated at intervals (24 hours in this case) that are shorter than the time required to clear all EDC from the body. Thus the toxicity of EDC evident in the NCI gavage study of rats and mice may not be due to the intact EDC molecule per se but to cumulative cellular damage from covalent binding of reactive metabolites from EDC metabolism. It is of in terest that the indices of toxicity (weight gain, depression, mortality) in the NCI gavage study were not more severe for mice than for rats, although the maximum and half-maximum "tolerated" time-weighted average daily dose was twofold greater for mice than for rats on a mg/kg basis (Table 9-56). How ever, on a proportional surface area basis for the two species (rat, 500 g; mouse, 33 g)2/3 = 6.1, the doses given to rats in terms of mg/animal were actually slightly greater than those given to mice (ratio, rat:mouse, 7.4; 9-206 CMA 051840 TABLE 9-56. INCIDENCE RATES OF HEMANGIOSARCOMAS IN THE CIRCULATORY SYSTEMS OF MALE OSBORNE-MENDEL RATS AND OF HEPATOCELLULAR CARCINOMAS IN MALE B6C3F1 MICE Species Average terminal Sex weight (g) Time-weighted average gavage dose mg/kg/day mg/animal/day Incidence Rats M 500 0 47 95b 0 23.5 47.5 0/40 9/48 7/27 (0.0%) (19%) (26%) Mice M 33 00 1/19 (5%) 97 3.2 6/47 (13%) 195 6.4 12/48 (25%) aGavage dose in corn oil administered 5 days/week with a time-weighted average dose of: dosage x weeks received divided by all weeks receiving EDC, Mg/animal based on average terminal weights of rats (500 g) and mice (33 g). ^All animals in this group died before the bioassay was terminated. SOURCE: NCI, 1978. 051841 Table 9-56). The fact that the NCI and Maltoni et al. carcinogenicity bioassays were conducted using two different routes of EDC administration (oral and inhala tion, respectively), presents substantial interpretive difficulties, pharmaco kinetic and otherwise, for the evaluation of the positive results of the NCI study and the negative findings of the Maltoni et al. study. A major reason for these difficulties is evident from a consideration of the dosages used in the two bioassays. The time-weighted average dose levels administered by gavage per day to Osborne-Mendel rats in the NCI study were 47 and 95 mg/kg (5 days/week for 78 weeks); the inhalation doses for rats (S-D) in the Maltoni study were 150, 50, 10, and 5 ppm, 7 hours/day, 5 days/week, for 78 weeks. In both assays higher doses (NCI, 150 mg/kg; Maltoni, 250 ppm, 6 hours) were initiated but were reduced after a few weeks of treatment. The data of Reitz et al. (1982) provide an approximate comparison of these doses. These inves tigators determined the assimilated dose of Osborne-Mendel rats given a single oral administration of EDC (150 mg/kg) and a single inhalation exposure (150 ppm, 6 hours). These doses represent the highest levels of EDC administered for any significant length of time in the two bioassays. The assimilated EDC from the 150 mg/kg gavage dose was observed to be completely absorbed (150 mg/kg), while the inhalation exposure was estimated to represent an average assimilated dose of 50.7 mg/kg, i.e., one-third of the oral dosage (Table 9-57). Furthermore, the assimilated dose from 150 ppm inhalation exposure to rats (the highest dose level of the 150, 50, 10, and 5 ppm Maltoni et al. bioassay), 50.7 mg/kg, is approximately equal to the lowest time-weighted dose of the NCI bioassay of rats but is less than the lowest dose given to mice (Table 9-56). Therefore, in respect to dosage levels of the NCI and Maltoni et al. bioassays, it appears that the animals in the Maltoni study were subjected 9-208 CMA 051842 TABLE 9-57. DISPOSITION OF l4C-EDC IN RATS3 AFTER SINGLE ADMINISTRATIONS OF 150 MG/KG ORALLY AND 150 PPM FOR 6 HOURS BY INHALATION Oral mg/kg Inhalation mg/kg Assimilated dose'3 Exhaled air (unchanged EDC) Metabolized C02 Urine Feces Carcass Cage wash 152.3 38.6 44.2 5.9 (29.0%) 8.2 1.2 91.6 34.4 2.6 1.5 4.6 1.5 1.2 0.6 108.2 (71.0%) 50.7 13.4 0.9 0.04 (1.8%) 3.6 0.7 42.8 11.9 0.9 0.3 2.2 0.3 2.2 0.3 49.8 (98.2%) aOsborne-Mendel rats, 150 to 250 g. ^Radioactivity recovery in exhaled air, urine and feces, 48 hours after admin istration. The values given are means SD for 4 rats for each route of exposure and are milligram equivalents of 14C-EDC radioactivity. SOURCE: Adapted from Reitz et al., 1982. 9-209 CMA 051843 to lower effective dose levels than those in the NCI mouse study and were, at most, comparable to the low-dose group in the NCI rat study. Other evidence of the effective dosage difference between the two bioassays is summarized in Table 9-58 and discussed in section 9.5.3.2.5. 9.5.3.2. INTERSPECIES DOSE CONVERSION 9.5.3.2.1. General Considerations -- This section discusses the metabolism, pharmacokinetics, covalent binding, and toxicologic findings that are relevant to the carcinogen risk assessment of EDC. Information on the metabolism and kinetics is used to the extent possible for calculating the carcinogenic risk for EDC. Scaling of toxicologic effects, including carcinogenicity, among species has been elaborated in several published papers (Freireich et al., 1966; Gillette, 1976; Krasovski, 1976; Dedrick, 1973; Dedrick and Bischoff, 1980; and Gehring et al., 1980). The major components requiring consideration in determining an appropriate extrapolation base for scaling carcinogenicity data from laboratory animals to man are: 1) toxicologic data, 2) metabolism and kinetics, and 3) covalent binding. The published literature concerning the biological basis for extrapolation of the dose-carcinogenic response relationship of laboratory animals to man has been reviewed in a paper prepared by Davidson (1984) for the Carcinogen Assessment Group and presented by Parker and Davidson (1984). 9.5.3.2.2. Toxicologic Data -- Section 9.2 of this document outlines the general features of the acute, subchronic, and chronic toxiclties of EDC in man and animals. However, comparative quantitative data across species on specific indices of EDC toxicity are experimentally lacking. Gross measures such as acute oral LD50 are similar in rats and mice (680 to 770 mg/kg, rats; 413 to 489 mg/kg, mice) and reflect overwhelming CNS and cardiac depression effects rather than specific organ toxicities (McCollister et al., 1956; Smyth 9-210 CMA 051844 TABLE 9-58. COMPARISON OF THE METABOLISM AND PHARMACOKINETICS OF THE "HIGH" DOSES IN RATS OF THE MALTONI ET AL. INHALATION AND NCI GAVAGE BIOASSAYS Observation Maltoni et al. inhalation 150 ppm, 7 hours 5 days/week NCI gavage 150 mg/kga 5 days/week Assimilated dose AUC, ug/mL x min Spreafico et al. Reitz et al. Balance study with 14C-EDC, mg/kg Reitz et al. Fraction metabolized, mg/kg Reitz et al. Peak blood levels, ug/mL Spreafico et al. Reitz et al. Time to peak blood level, hr Spreafico et al. Reitz et al. Blood clearance time, hr Spreafico et al. Reitz et al. Hepatic DNA covalent binding ymole equiv EDC/mole DNA Reitz et al. 350b 2910 50.7 49.8 llb 9.4 4 to 6 2.5 to 6 1.5b 1.5C 8.2d 3.3 7297 4500 150 108.2 66.8 30 to 40 0.6 0.25 5.6 5 21.3 13.9 aTime-weighted average for 78-week treatment period was 95 mg/kg/day. bIn terpolation from the data of Spreafico et al. (1978, 1979, 1980), Table 9-56. cEstimate from data of Reitz et al. (1982), Figure 9-16. ^Determined 4 hours after gavage and immediately at termination of inhalation exposure. 9-211 CMA 051845 1956; Munson et al., 1982). Other toxicologic end points have been measured, but not across species in comparative dose-related comprehensive studies. Differences in toxicity according to species, strain, or sex have not been reported for EDC. 9.5.3.2.3. Metabolism and Kinetics -- Few data are available on the metabolism and kinetics of EDC in man, but these kinetics have been studied in mice and rats. 9.5.3.2,3.1. Metabolism. In mice and rats, EDC is extensively metabolized after both oral and inhalation exposures. After oral and inhalation exposures in rats, at 150 mg/kg and 150 ppm for 6 hours ("high" doses in thd NCI and Maltoni et al. carcinogenicity bioassays), 71% of the oral dose and 98% of the inhalation dose was metabolized (Table 9-57). However, as outlined below, metabolism is dose-dependent, with proportionately greater percentages of the dose metabolized at low doses and less at higher doses. Metabolism occurs by multiple pathways in liver, microsomal, and cytosol functions. Microsomal P450 oxidation produces 2-chloroacetaldehyde, while cystosolic reactions of EDC with glutathione are postulated to produce the half-sulfur mustard S-(2-chloroethyl)-GSH and its episulfonium ion. Both chloroacetaldehyde and the half sulfur mustard are reactive metabolites that covalently bind to cellular macromolecules, a mechanism that is presumed to underlie the liver and other organ tissue cytopathology observed from EDC exposure. The cytosolic interaction of EDC with glutathione results in an activation to a highly reactive half-mustard metabolite as well as to nonreactive gluta thione conjugates from "detoxification reactions." Johnson (1965) acutely administered 40 mg/kg EDC in corn oil to rats by gavage and observed an average of 52% depletion of hepatic GSH within 2 hours (peak EDC blood concentration after oral dosing of 150 mg/mL in corn oil, 40 minutes; Table 9-56). Reitz et 9-212 CMA 051846 al. (1982) assayed GSH levels in rats after a 150 mg/kg oral dose in corn oil. After termination of a 150 ppm, 6-hour inhalation exposure, 4 hours after exposure, hepatic GSH was depleted by 77% and 74%, respectively. These obser vations demonstrate the involvement and importance of cellular levels of GSH to the cytotoxic potential of EDC, both for activation and for detoxification. It would appear that the "high" doses in the carcinogenicity bioassays do not completely overwhelm the availability of cellular GSH. Species Comparison. The metabolism of EDC has been studied in only two species, the rat and mouse. There is no evidence that the metabolic pathways in these two species differ qualitatively, since urinary end-metabolites are similar. In these two species, metabolism has been found to be capacitylimited, with dose-dependent kinetics. There are, however, no direct compara tive balance studies (same laboratory, same time period) of the extent of metabolism in these species, as are available, for example, for certain of the halogenated ethylene compounds. For the mouse, Yllner (1971b) provided data on the extent of metabolism in this species (Figure 9-12); 10 years later, for the rat, Reitz et al. (1982) found that for a gavage dose of 150 mg/kg (a dose approaching "saturation" for rat and mouse), 108 mg/kg was metabolized (Table 9-57). Thus, for a 200 g rat, 21.6 mg of a 30-mg dose was metabolized. For a comparable dose (150 mg/kg), Yllner found that 2.25 mg was metabolized by a 25 mg mouse. The ratio of metabolism, rat:mouse, for this particular dose (150 mg/kg) is 9.6, while the ratio of the surface areas Is (200/25)2/3 4,0. Comparison of these data suggests that metabolism of EDC in the rat and mouse is more associated with the relative body weights of these species than with surface area. However, for the reasons given, direct comparison of data from these two diverse studies cannot be fully justified on experimental grounds. 9-213 CMA 051847 AB sI) to *--1 DOSE (mg/kg) DOSE (mg/kg) Q Figure 9-12, Relationship in mice between dose of EDC (mg/kg) and the amount of EDC exhaled unchanged (Panel A) and the amount of the dose metabolized to urinary metabolites plus CO2 o (Panel B)* The female NMRI mice weighed 20-30 g. CO SOURCE: Adapted from Yllner, 1971b 00 9.5.3.2.3.2. Kinetics. Conclusive evidence exists that the pharmacoki netics of EDO in rats and mice are nonlinearJ To detect and establish the nonlinear nature of the pharmacokinetics of a compound, at least two, and preferably more, dosage levels must be studied (Wagner, 1975). For EDC, the studies of Yllner (1971b) in mice, and of Spreafico et al. (1978, 1979, 1980) in rats meet this essential requirement, while the single-dose studies of Reitz et al. (1982) do not. EDC is completely and rapidly absorbed after oral administration (Yllner, 1971b; Spreafico et al., 1978, 1979, 1980; Reitz et al., 1982) with a firstorder constant, ka, for an oral dose of 150 mg/kg of 0.11 minutes-*, indicating 95% absorption within 30 minutes. Yllner was the first to demonstrate both the nonlinear nature of the pharmacokinetics of EDC after absorption and the sub stantial first-pass effect with EDC administration. Yllner administered *^CEDC intraperitoneally in olive oil to NMRI mice at four dose levels, 50, 100, 140, and 170 mg/kg. This dosage range approximated that of the NCI bioassay in mice. Yllner quantitated *^C-radioactivity in urine and feces (as EDC metabolites), as well as *^C02 (EDC metabolite) and unchanged *^C-EDC in ex haled air for 72 hours after administration. Total recovery of l^C-EDC radioactivity ranged from 97% to 102% of the dose. The results are shown plotted in Figure 9-12. With increased dose, the portion of the dose exhaled as unchanged EDC (first-pass effect) increased in an exponential manner, so that at a dose of 170 mg/kg, 45% of the dose was excreted as EDC. The remain ing portions of the doses were metabolized. Figure 9-12B shows the nonlinear relationship between dose and metabolism, indicative of nonlinear MichaelisMenten kinetics for the metabolism of EDC, with "saturation" of metabolism occurring in the mouse between 150 to 200 rag/kg administered dose. These data can be used to estimate the fraction of the assimilated dose metabolized 9-215 CMA 051849 and contributing to tumorigenesis for the two dosage levels of the NCI mouse bioassay. Spreafico et al. (1978, 1979, 1980) investigated the pharmacokinetics of EDC in rats after oral dosing, inhalation exposure, and intravenous bolus administration at two (inhalation) or three dose levels (oral and intravenous administration). These workers determined the blood EDC concentration-time curves (C,t curve) after the various administrations and fitted the C,t data to a linear two-compartment open kinetic model, thereby testing the linearity of EDC pharmacokinetics. According to basic assumptions and definitions, linear kinetics require that kinetic parameters (T 1/2, AUC, V<j> clearance, slopes of semilog plots of blood concentration decay curves, etc.) be indepen dent of dose. Hence, these kinetic parameters, determined at two or more dose levels in accordance to a linear kinetic model, provide a rigorous test for nonlinearity (Wagner, 1975). If the magnitude of some or all of the parameters changes with dose (and especially if there are dose-related trends), then nonlinear kinetics are highly probable. If Michaelis-Menten kinetics are operative, then one expects (1) that the percentage metabolism by the Michaelis-Menten path will decrease with increase of dose (as in the data of Yllner above), (2) that the area under the blood C,t curve will increase more than proportionately with increase in dose, and (3) that semilog plots of blood levels will curve inwardly with pseudolinear terminal portions, and slopes of the pseudolinear terminal portion will decrease with increase in dose. Figure 9-13 presents the serailog plots of blood C,t curves obtained by Spreafico et al. (1980) after oral administration of three dose levels of EDC to rats. Also shown are adipose tissue, lung, and liver C,t curves. In each case, the slope of the apparently linear decline curves decreases markedly 9-216 CMA 0518^0 co 7*- Xoc E 6- E VO ho u 2.S- DOSE l-fl/ktl I * 1 I I I t 1| Tl T 1 | T T-> I | I M r | I I II || IITJTTI l'[ 1 I' I f | I I ' 1 I | ) I I I] I I t l [ 25 58 75 180 125 158 175 280 225 258 275 988 n DOSE (ppw 6 hr. j Figure 9-13. Relationship in Sprague-Dawley rats between the area under the blood EDC oCn concentration-time curve (AUC) and the gavage dose of EDC in corn oil (mg/kg) or inhaled doses of EDC (ppm for 6 hours). 00 Cn SOURCE: Adapted from Spreafico et al.T 1980 with increase in dose, in a manner inconsistent with first-order linear kinetics but providing an excellent indication of the nonlinear dose-dependent nature of the kinetics of EDC in rats. Immediately apparent also from Figure 9-13 is that, as a consequence of nonlinear kinetics, the higher the dose the longer and more disproportionate is the time of clearance of EDC from the blood and, therefore, from the body. Definitive evidence of the dose-depen dency of EDC kinetics is given by the linear kinetic parameters obtained by Spreafico et al. by fitting their data to a linear two-compartment model, as shown in Table 9-59. Both experimentally observed measures (peak blood concentration, time to peak, areas under C,t curve) and calculated kinetic parameters (half-life, volume distribution, clearance) are not independent of dose, as required by linear kinetics, but rather increase with increase in dose. Furthermore, the nonlinear dose-dependent nature of EDC pharmacoki netics is evident for all three routes of administration: oral, Inhalation, and intravenous. Spreafico et al. did not further analyze their data, al though it would have been appropriate for them to have fitted their data to a first-order absorption model with parallel Michaelis-Menten kinetics, as well as to a first-order elimination model. The area under the blood C,t curve (AUC) is an experimental measure inde pendent of its assumption of any kinetic model. The AUC can be represented by the definite integral t AUC = / C dt o The AUC is related to the amount of compound absorbed and, under certain conditions, can be used to estimate the total internal dose. The AUCs deter mined from oral administration of EDC in corn oil vehicle and from inhalation 9-218 0518^2 CMA TABLE 9-59. EVIDENCE OF NONLINEAR KINETICS FOR EDC IN RATS AFTER GAVAGE, INTRAVENOUS AND INHALATION DOSAGE Kinetic parameter Oral mg/kg dose3 25 50 150 Inhalation ppm, 6-hr dose 50 250 Intravenous mg/kg dose 25 50 150 Experimental determination*5 Peak blood cone., yg/mL 13.2 31.9 66.8 1.3 31.3 1.5 8.0 38.1 Time to peak, hr ~0.1 ~0.2 -0.6 4 6 zero time N3 AUC, jig x mL-* x min 446 1700 7297 26 1023 9 54 59 v> Calculated from kinetic model*5 Ti/2, min 24.6 44.1 56.7 12.7 22.1 7.3 9.5 14.1 Vd, mL 367 328 390 ---- 231 239 162 Clearance, mL x mln"* 10.6 5.6 3.9 ---- 22.0 17.5 8.0 aGavage dose administered in corn oil. ^These parameters were experimental observations independent of any kinetic model. The calculated parameters were obtained by fitting the blood EDC concentration-time data to a linear two-compartment open model. SOURCE: Adapted from Spreafico et al., 1978, 1979, 1980. CMA 0 5 1 8 5 3 exposure at two or three dose levels, respectively, to rats are shown plotted against administered dose in Figure 9-13. As expected from the dose-dependent nature of EDC kinetics, the AUCs increase disproportionately with the dose for both oral and inhalation exposures. Consistent also with Michaelis-Menten dose-dependent kinetics for the metabolism of EDC is the fact that when the logarithm of the AUCs is plotted against the logarithm of the oral dose, a linear relationship is obtained as shown in Figure 9-14. Since the AUCs represent the portion of the EDC dose that reaches the systemic circulation, in the case of oral administration the AUCs principally represent the absorbed dose (assumed to be complete) minus the first-pass effect (primarily elimination of unchanged EDC via the lungs). Comparison of AUCs for oral and intravenous doses (Table 9-59), a method used to estimate first-pass effects, shows that for a 25 mg/kg oral dose of EDC, the AUC (446 yg x mL-* x min) is 25% less than for an identical Intravenous dose (595 yg x mL"l x min). Hence, the AUCs after oral administration, like those after intravenous administration, approximate the internal dose subject to metabolism. Of interest, therefore, is a comparison of the AUCs (or inter nal dose) after oral and inhalation exposures to EDC, which were the condi tions of the NCI and Maltoni carcinogenicity bioassays. From Figure 9-13, it is readily apparent that the AUCs for an oral dose of EDC of 100 rag/kg (the "high" dose of the NCI study) and 150 ppm for 6 hours (the highest dose of the Maltoni study) are approximately 10:1. The disproportionate increase of AUCs, or internal dose subject to metabo lism, relative to linear increases in oral dosages of EDC, can be more clearly seen if the ratio of the AUC to dose (AUC per unit dose) is plotted against the administered dose, as shown In Figure 9-15. As the oral dose increases, the internal dose approaches an asymptotic limit similar to the Michaelis- 9-220 CMA 051854 In AUC (ug/mL x min) Figure 9-14. Linear relationship for EDC between the logarithm of the area under the blood i C,t curve (AUC) and the logarithm of gavage dose in corn oil given to Sprague-Dawley rats. The coefficient of regression, r2, is 0.989. 0 tt-n* SOURCE: Adapted from Spreafico et al., 1978, 1979, 1980. CD 01 Cn DOSE (mg/kg oral) Q Figure 9-15. Relationship in Sprague-Dawley rats between gavage doses of EDC in corn oil O and the ratio from the area under the blood EDC concentration-time curve (AUC) divided by CHP* OD the administered dose. The dashed line represents the limit approached in change of AUC per unit dose as the administered dose is increased. C<TP> SOURCE: Adapted from Spreafico et al., 1978, 1979, 1980. Menten expression. Assuming that the internal dose closely approximates or is proportional to the portion of the administered dose metabolized, then EDC metabolism in the rat approaches saturation at about 150 to 200 mg/kg, as in the mouse. This relationship can be used to estimate the internal dose and metabolism of EDC for the administered oral doses used in the NCI bioassay. Reitz et al. (1980, 1982) also carried out an investigation of the phar macokinetics of EDC. As stated in Reitz et al. (1982), one objective was to determine the absorbed dose of EDC in rats (Osborne-Mendel) and to characterize its elimination after single exposures at a single dose level from inhalation (150 ppm, 6 hours) and oral administration in corn oil (150 mg/kg). These doses were used because they represented the "highest levels of EDC adminis tered for any significant length of time in the two bioassays" (NCI and Maltoni bioassays). Actually, the oral dose exceeds the time-weighted average "high" dose for Osborne-Mendel rats of the NCI study (95 mg/kg, Table 9-56). As shown by the data of Spreafico et al. (see above), the oral dose (150 mg/kg) is close to a saturating dose, and in comparison, the inhalation dose (150 ppm, 6 hours) is considerably less (--1/10; Figure 9-13). Because Reitz et al. did not examine the kinetics of multiple dose levels, they were unable to determine the dose-dependent nonlinear nature of EDC kinetics. However, these investigators fitted their blood C,t data to kinetic models empirically. They found that their blood C,t curve, after Inhalation expo sure, could be fitted to a linear two-compartment model with first-order elim ination, while the blood C,t data after oral administration was "complex" and was fitted to a two-corapartraent, first-order absorption model with MichaelisMenten elimination kinetics. Their blood C,t data are shown in Figure 9-16. The Reitz et al. observations, because of their internal inconsistency, are of limited interest and use; there is no reason to believe that the eliraina- 9-223 CMA 051857 MINUTES \D to t4o>* Figure 9-16. Blood levels of GDC following exposure to GDC by (A) Inhalation (150 ppm, 6 hours) or (B) gavage (150 mg/kg). The Inhalation C,t data were fitted to a linear two-com partment kinetic model, and the gavage C,t data were fitted to a nonlinear two-compartment Michael!s-Menten model. The solid line Is the predicted blood concentration from the model and the data point means of 4 rats SD. A goodness-of-fit was not reported for either model. The calculated kinetic parameters are: Inhalation Linear model ______ Gavage Nonlinear model CMA. 0 5 1 8 5 8 CQ (peak blood level), 9.4 pg/mL AUC, 2910 pg/mL x min Kgi 0.091 min t1/2r 7,6 mln Vj, 425 mL/kg Peak blood level, 30-40 pg/mL AUC, 4500 pg/mL x min Vra, 0.166 pg/mL x mln Knit 1*96 pg/mL V,j, 3400 mL/kg SOURCE: Adapted from Reitz et al., 1982 tion kinetics of EDC differs with respect to the two routes of administration, inhalation and oral. The data of Spreafico et al. show that the elimination kinetics of EDC for all three routes of administration (inhalation, oral, and intravenous), are dose-dependent, nonlinear kinetics. Reitz et al. (1982), in balance studies in rats, compared the assimilated or "absorbed" dose of EDC after oral (150 mg/kg in corn oil) or inhalation (150 ppm, 6 hours) exposure to ^C-EDC. Radioactivity in exhaled air, urine, feces, and carcass was determined for 48 hours after exposure. Total recovery of EDC radioactivity averaged 101% for the oral dose; it was not possible to determine recovery after inhalation exposure. The results are given in Table 9-57. The assimilated dose (152 mg/kg) found after oral administration repre sented complete absorption of EDC from the GI tract; the assimilated dose (50.7 mg/kg) following inhalation exposure proved to be one-third of the oral dose (Table 9-57). As found by Yllner (1981b) in mice and by Spreafico et al. (1978, 1979, 1980) in rats, the data of Reitz et al. also indicate that the percentage of the administered dose excreted by pulmonary excretion increased with the dose and that the remaining percentage (metabolized) decreased, suggesting dose-dependent Michaelis-Menten kinetics for metabolism. Reitz et al. found that the major urinary metabolites of EDC in the rat appeared to be identical in the same relative amounts, i.e., independent of the route of exposure. 9.5.3.2.4. Covalent Binding -- Covalent binding from both oxidative microsomal and cytosolic metabolism of EDC has been demonstrated ^n vitro (Banerjee and Van Duuren, 1978, 1979; Banerjee et al., 1980; Sipes and Gandolfi, 1980; Guengerich et al., 1980). Metabolites of EDC covalently bind in vitro to added protein and DNA; binding from cytosolic metabolism depends on the presence of GSH and requires GSH transferases. 9-225 CMA 051859 In vivo covalent binding from EDC metabolism has also been demonstrated in the rat. However, neither a dose-binding relationship nor turnover studies are available for EDC; the studies conducted have only been at single, fixeddose levels. Theoretically, covalent binding might be expected to relate to the dose-dependent metabolism of EDC. Reitz et al. (1982) determined total tissue levels of covalent binding and specific binding to DNA in rats exposed to ^C-EDC by the oral (150 mg/ kg) and inhalation (150 ppm, 6 hours) routes, i.e., "high" doses of the carcinogenicity bioassays. No striking differences between the two routes were observed in the distribution of covalent binding and DNA binding among the various organ tissues, although the tissue levels of covalent binding were generally higher after inhalation exposure for total binding and after gavage exposure for DNA binding. DNA isolated from the organ tissues was assessed for covalent binding, and in general, higher levels of DNA alkylation (threeto fivefold) were observed after gavage exposure, with highest values for liver and kidney (Table 9-60). These DNA covalent binding studies are in good accord with the differences in metabolism and pharmacokinetics (Table 9-58) noted for the NCI gavage bioassay in rats (150 mg/kg "high" dose) and the Maltoni et al. inhalation bioassay (150 ppm, 7 hours "high" dose). For mice, Storer et al. (1982) reported that DNA damage was produced in vivo in the livers of male B6C3F1 mice following single oral doses of EDC at 200 mg/kg (the "high" dose of the NCI mouse bioassay). DNA was evaluated by differental sedimentation of liver nuclei in high-density sucrose gradient. Nuclei from EDC-treated mice isolated 4 hours after dosing sedimented more slowly, and total DNA recovery from the nuclei was decreased slightly (16%) as compared with untreated mice. 9.5.3.2.5. Comparison of Maltoni et al. and NCI Studies -- The NCI (1978) 9-226 CMA 051860 TABLE 9-60 . DNA COVALENT BINDING IN RATS EXPOSED TO 14C-EDC Gavage Inhalation 150 mg/kg 150 ppm, 6 hours praole equvalents bounds/mole DNA Mean SD (n - 3) Experiment 1 Liver Spleen Kidney Stomach 21.3 7.4 5.8 0.7 17.4 t 2.3 14.9 8.2 3.3 1.8 0.7 5.2 3.7 2.8 Experiment 2 Liver Spleen Kidney Stomach 13.9 2.1 2.5 0.3 14.5 6.2 6.7 3.3 1.2 1.8 0.5 2.0 + 0.3 1.9 aAnimals were sacrificed 4 hours post gavage or immediately following inhalation exposure. SOURCE: Reitz et al., 1982. 9-227 CMA 051861 lifetime gavage studies in rats and mice produced excess tumors, while the Maltoni et al. (1980) lifetime inhalation study did not. The kinetic studies of EDC disposition in rats after gavage and inhalation exposures, and the metabolism studies of Reitz et al. (1982) provide partial explanations for the contrasting results of the two carcinogenicity bioassays. The two EDC studies differ in (1) assimilated or effective internal dose, (2) fraction of assimilated dose metabolized to nucleophilic metabolites, and (3) peak blood levels and duration of metabolism from a single daily dose. Table 9-58 contrasts some metabolism and kinetic parameters of the "high" doses of the Maltoni inhalation (150 ppm, 7 hours) and NCI gavage (150 mg/kg) rat bioassays available from the studies of Spreafico et al. (1978, 1979, 1980) and Reitz et al. (1982). While the data from these two investigative groups are from differing study designs and differ in some measures, they are never theless in surprisingly good agreement. Table 9-58 shows that the internal dose assimilated by rats exposed to 150 ppm EDC is one-tenth to one-third less than that from a 150 mg/kg gavage dose, and that it produces less than one-half the metabolites of a 150 mg/kg gavage dose. Furthermore, peak blood levels of EDC from a 150 mg/kg gavage dose are three- to fivefold greater, and are achieved within one-half hour, as compared to 3 to 6 hours from inhalation exposure. Since the rate of metabolism is proportional to blood level, these observations suggest chat the intensity of covalent binding may be expected to be greater with the gavage dose, and this has been found to be the case; covalent binding to DNA Tn vivo is three- to fivefold higher after gavage exposure than after inhalation exposure. Additionally, it can be noted that, contrary to what might be assumed, an EDC inhalation exposure of 6 to 7 hours (150 ppm) does not produce a signifi cantly longer presence of EDC in the blood available for continuing liver and 9-228 CMA 051862 other organ metabolism than an oral gavage dose of 150 mg/kg. In both in stances, blood EDO is essentially eliminated (cleared) within 6 to 10 hours, indicating that the "chronic" dosage in these bioassays is better described as multiple single dose administration, since a given day's dose of EDC may be expected to be eliminated from the body before the next day's dose. However, the residual DNA covalent binding and general cellular covalent binding, greater for the NCI gavage doses, may be cumulative and may exceed the capacity of repair mechanisms with chronic EDC administration. In total, these observations provide cogent reasons to conclude that the dose range of the Maltoni inhalation study (5 to 150 ppm) may be insufficient to produce measurable tumorigenesis. However, as will be discussed later in section 9.5.3.4,6, reasons other than those indicated here cannot be excluded. 9.5.3.2.6. Calculation of Human Equivalent Doses -- The NCI carcinogen icity studies of EDC in mice and rats were performed by daily (5 days/week) gavage dosing of EDC in corn oil. Experimental metabolism and pharmacokinetic observations pertinent to the conditions of the NCI mouse bloassay are those of Yllner (1971b), who administered acute doses of EDC in corn oil to mice by gavage and determined at several dosage levels the fraction of the assimilated dose metabolized. Pertinent studies in relation to the NCI rat bioassay are the experimental kinetic observations of Spreafico et al. (1978, 1979, 1980) who administered acute doses of EDC in corn oil to rats at several dosage levels. The studies of both Yllner and Spreafico et al. included dose ranges comparable to those of the NCI bioassays. However, the observations were not made on "chronic" dosed animals, as in the NCI bioassays, although the experi mental evidence indicates that body clearance of EDC from rodents after a single daily dose, while dose-dependent, is less than 12 hours for the highest assay dose, i.e., less than the 24-hour dosing interval of the NCI bioassay. 9-229 CMA 051863 While there is little information on the metabolism or pharmacokinetics of EDC in man, the pertinent experimental observations in animals may be summarized as follows: 1. It is assumed that biochemical lesions precede the toxic effects of EDC. Formation of reactive metabolites (P450 oxidation, chloroacetaldehyde; GSH-mediated cytosolic reaction, S-(2-chloroethyl)-GSH) is followed by irre versible binding to proteins, lipids, and nucleic acids critical to cell func tion and resulting in toxic, mutagenic, or carcinogenic events. 2. After gavage administration in corn oil to mice or rats, EDC is rapidly and completely absorbed. 3. After gavage administration, a portion of the administered dose (increasing with the dose) is excreted via the lungs unchanged. Metabolism of EDC is less than one-half saturation at the time-weighted average dose levels of the NCI rat bioassay (47 and 95 mg/kg), but is at or near saturation for the highest dose of the NCI mouse bioassay (195 mg/kg). 4. EDC is cleared from the body well within the bioassay dosing interval, even at the highest bioassay doses. The clearance is by first-order pulmonary excretion of unchanged EDC and by parallel dose-dependent Michaelis-Menten metabolism kinetics. The end-metabolites (urinary metabolites) are more slowly cleared than EDC itself. The rapid clearance of EDC, representing a fraction of the 24-hour bioassay dosage interval, indicates that accumulation with multiple doses would not occur; there is time between doses for repair of cellu lar damage from EDC metabolism, and also for repletion of cellular glutathione. 5. There is no evidence to suggest qualitative differences in the meta bolic pathways of EDC in the two species, mouse and rat, and no difference resulting from the route of exposure (oral or inhalation) in the rat. 6. Covalent binding to cellular protein and lipids occurs _iri vivo after 9-230 CMA 051864 oral administration of EDC to the rat. DNA alkylation occurs not only at the organ sites of tumors found in the NCI bioassay but also at other sites. There is no information available on the turnover time or repair time of DNA adducts, but DNA binding was evident 4 hours after oral dosing. DNA alkylation after gavage is estimated at only 2 to 20 / 10^ nucleotides. Using this metabolic and pharmacokinetic information, the lifetime average human equivalent doses for the NCI rat and mouse carcinogenicity bioassays of EDC have been calculated as follows. 9.5.3.2.6.1. NCI Bioassay (Mouse): Liver Carcinomas. In this study, the time-weighted average gavage dose levels of EDC were 195 and 97 mg/kg/day, or, based on the average terminal weight of the male mice (33 g), 3.2 and 6.4 mg/male raouse/day (Table 9-56). Doses in this range given in corn oil have been found to be completely absorbed by rodents, although a dose-related, first-pass effect has been observed, leading to a portion of the dose being excreted in exhaled air unchanged. From the data of Yllner (1971b), a dosemetabolism curve for EDC administered to mice is available (Figure 9-12). The metabolism of EDC in mice shows dose-dependent kinetics with the "high" dose of the NCI bioassay (195 mg/kg) at saturation of metabolism (Figure 9-12); at this dose level, the amount of metabolites contributing to tumorigenesis is 95 mg/kg. At the "low" dose level of the NCI bioassay (97 mg/kg), the amount metabolized is 73 mg/kg. Hence, the fractions of the daily assim ilated dose metabolized by the mice (33 g) are: Male mice Assay dose level time-weighted average mg/kg 195 97 9-231 Amount metabolized (AM) mg/kg mg/animal 95 3.14 73 2.41 CMA 051865 The lifetime average daily exposure (LAE) of the mice is given by: LAEty * (78 weeks/90 weeks) x (5 days/7 days) x AM, mg/animal/day where AM is the amount of dose metabolized. For humans, the LAE is given by: LAEh LAEm x scaling factor The interspecies scaling is assumed to be on a surface area basis (W2/3), The scaling factor for mouse to humans is (70/0.033)^/3 155, Table 9-61 presents the lifetime average human equivalent doses for the NCI mouse car cinogenicity bioassay of EDC, expressed in units of mg/day, mg/m2 surface area/day, and mg/kg/day. TABLE 9-61. LIFETIME AVERAGE HUMAN EQUIVALENT DOSES BASED ON THE NCI MOUSE BIOASSAY Male mice gavage dose (time-weighted) mg/kg Equivalent lifetime average human exposure dosagea mg/day (mg/m2)/day (mg/kg)/day 97 246.2 133.1 3.52 195 320.7 173.4 4.58 aWhere the terminal average weight of the male mouse is 0.033 kg; the average weight for a human is assumed to be 70 kg with 1.85 in surface area. 9-232 CMA 051866 9.5.3.2.6.2. NCI Bioassay (Rat): Angiosarcomas. For this study, the time-weighted average gavage doses ("high" and "low" doses) given to male Osborne-Mendel rats in corn oil were as follows: Gavage dose male 0-M rat mg/kg (time-weighted) 47 95 mg/animal/day 23.5 47.5 and the corresponding dose per single animal/day is given as calculated from the average terminal weight of 500 g. Spreafico et al. (1978, 1979, 1980) and Reitz et al. (1982) found that gavage doses in corn oil spanning this dosage range were rapidly and completely absorbed, although a portion, depending on dose, effects a first-pass of the liver and is excreted in exhaled air un changed. The area under the EDC blood C,t curve (AUC) represents EDC that has reached the systemic circulation after oral absorption and first-pass pul monary excretion. The AUC represents the approximate internal dose available for metabolism, and thus is equivalent (or nearly so) to the fraction of the administered dose metabolized. Data from Spreafico et al. (see Figure 9-15) and Reitz et al. (1982) can be used to determine the fraction of the dose metabolized for the NCI bioassay on rats. On the basis of Figure 9-15, the rate of change (i.e., the slope to the curve) at dose levels 47, 95, and 150 mg/kg can be estimated to be 0.36, 0.17, and 0.094, respectively. Since the rate of change decreases as the dose increases, it is reasonable to assume that the relative amount of EDC exhaled unchanged is inversely proportional to these rates. Now, on the basis of Reitz et al. (1982), 29% of EDC was exhaled unchanged after an administered 9-233 CMA 051867 dose of 150 mg/kg. Thus, the percentage unchanged at NCI doses, 47 and 95 mg/kg, are, respectively, 0.29 x 0.094/0.36 = 0.08, or 8%; and 0.029 x 0.094/0.17 = 0.16, or 16%. Therefore, the fractions of EDC metabolized for the NCI study on rats are 92% at the dose 47 mg/kg and 84% at the dose 95 mg/kg. The amounts metabolized for the NCI EDC bioassay are as follows: Male rats Assay dose level time-weighted average mg/kg 47 95 Amount metabolized (AM) mg/kg mg/animal 43.24 79.80 21.62 39.90 The lifetime average daily exposure (LAE) of the rats is given by: LAEr * (78 weeks/104 weeks) x (5 days/7 days) x AM, mg/animal/day where 104 weeks is assumed to be the average life span for rats, and AM is the amount of dose metabolized. For humans, the LAE is given by: LAEfl LAEr x scaling factor The interspecies scaling is assumed to be on a surface area basis (W^/3). The scaling factor for rats to humans is (70/0.5)2/3 = 26.96. Table 9-62 presents the lifetime average human equivalent doses for the NCI rat bioassay, expressed in units of mg/day, mg/m^ surface area/day, and mg/kg/day. 9-234 CMA 051868 TABLE 9-62. LIFETIME AVERAGE HUMAN EQUIVALENT DOSES BASED ON THE NCI RAT BIOASSAY Gavage dose (time-weighted) mg/kg Equivalent lifetime average human exposure dosagea rag/day (mg/m2)/day (mg/kg)/day 47 312.25 168.78 4.46 95 576.27 311.50 8.23 aWhere the terminal average weight of the male rat is 0.5 kg; the average weight for a human is assumed to be 70 kg with 1.85 nr surface area. 9.5.3.3. CHOICE OF RISK MODEL \ 9.5.3.3.1. General Considerations -- The unit risk estimate for EDC represents an extrapolation below the dose range of experimental data. There is currently no solid scientific basis for any mathematical extrapolation model that relates exposure to cancer risk at the extremely low concentrations, including the unit concentration given above, that must be dealt with in eval uating environmental hazards. For practical reasons the correspondingly low levels of risk cannot be measured directly either by animal experiments or by epidemiologic studies. Low-dose extrapolation must, therefore, be based on current understanding of the mechanisms of carcinogenesis. At the present time the dominant view of the carcinogenic process involves the concept that most cancer-causing agents also cause irreversible damage to DNA. This position is based in part on the fact that a very large proportion of agents that cause cancer are also mutagenic. There is reason to expect that the quantal response that is characteristic of mutagenesis is associated with a linear (at low doses) nonthreshold dose-response relationship. Indeed, there is substantial 9-235 CMA 051869 evidence from mutagenicity studies with both ionizing radiation and a wide variety of chemicals that this type of dose-response model is the appropriate one to use. This is particularly true at the lower end of the dose-response curve; at high doses, there can be an upward curvature, probably reflecting the effects of multistage processes on the mutagenic response. The low-dose linear nonthreshold dose-response relationship is also consistent with the relatively few epidemiologic studies of cancer responses to specific agents that contain enough information to make the evaluation possible (e.g., radiation-induced leukemia, breast and thyroid cancer, skin cancer Induced by arsenic in drinking water, liver cancer induced by aflatoxins in the diet). Some supporting evi dence also exists from animal experiments (e.g., the initiation stage of the two-stage carcinogenesis model in rat liver and mouse skin). Because its scientific basis, although limited, is the best of any of the current mathematical extrapolation models, the nonthreshold model, which is linear at low doses, has been adopted as the primary basis for risk extrapola tion to low levels of the dose-response relationship. The risk estimates made with such a model should be regarded as conservative, representing a plausible upper limit for the risk; i.e., the true risk is not likely to be higher than the estimate, but it could be lower. For several reasons, the risk estimate based on animal bioassays is only an approximate indication of the absolute risk in populations exposed to known carcinogen concentrations. First, there are important species differences in uptake, metabolism, and organ distribution of carcinogens, as well as species differences in target site susceptibility, immunological responses, hormone function, dietary factors, and disease. Second, the concept of equivalent doses for humans compared to animals on a mg/surface area basis is virtually without experimental verification as regards carcinogenic response. Finally, 9-236 Ct$h 05181 human populations are variable with respect to genetic constitution and diet, living environment, activity patterns, and other cultural factors. The risk estimate can give a rough indication of the relative potency of a given agent as compared with other carcinogens. Such estimates are, of course, more reliable when the comparisons are based on studies in which the test species, strain, sex, and routes of exposure are similar. 9.5.3.3.2. Mathematical Description of the Low-Dose Extrapolation Model The mathematical formulation chosen to describe the linear (at low doses) non threshold dose-response relationship is the linearized multistage model. This model employs enough arbitrary constants to be able to fit almost any monotonically increasing dose-response data, and it incorporates a procedure for estimating the largest possible linear slope (in the 95% confidence limit sense) at low extrapolated doses that is consistent with the data at all dose levels of the experiment. Let P(d) represent the lifetime risk (probability) of cancer at dose d. The multistage model has the form P(d) =* 1 - exp [-(q0 + q^d + q2d2 + + qkdk)] where qi 0, i =* 0, 1, 2, ..., k Equivalently, Pt(d) " 1 - exp (-(qjd + q2d2 + .* . .# + qfcdk)] where P (d) = P(d) - P(0) t i1 _- oPf(m0) 9-237 CMA 051871 is the extra risk over background rate at dose d or the effect of treatment. The point estimate of the coefficients q^, i = 0, 1, 2, .... k, and con sequently, the extra risk function, Pt(d), at any given dose d, is calculated by maximizing the likelihood function of the data. The point estimate and the 95% upper confidence limit of the extra risk, Pt(d), are calculated by using the computer program, GL0BAL83, developed by Howe (1983). At low doses, upper 95% confidence limits on the extra risk and lower 95% confidence limits on the dose producing a given risk are determined ff from a 95% upper confidence limit, qp on parameter q^. Whenever qj >0, at low doses the extra risk Pt(d) has approximately the form Pt(d) qj x d. Therefore, q^ x d is a 95% upper confidence limit on the extra risk and R/q^ is a 95% lower confidence limit on the dose, producing an extra risk of R. Let Lq be the maximum value of the log-likelihood function. The upper-limit, q^, is calculated by increasing q^ to a value q* such that when the log-likelihood is remaximized subject to this fixed value, qp for the linear coefficient, the resulting maximum value of the log-likelihood L^ satisfies the equation 2(L0 - Li) - 2.70554 where 2.70554 is the cumulative 90% point of the chi-square distribution with one degree of freedom, which corresponds to a 95% upper-limit (one-sided). This approach of computing the upper confidence limit for the extra risk Pt(d) is an improvement on the Crump et al. (1977) model. The upper confidence limit for the extra risk calculated at low doses is always linear. This is conceptu ally consistent with the linear nonthreshold concept discussed earlier. The slope, qj, is taken as an upper bound of the potency of the chemical in inducing cancer at low doses. (In the section calculating the risk estimates, Pt(d) will be abbreviated as P.) 9-238 CMA 051872 In fitting the dose-response model, the number of terms in the polynomial is chosen equal to (h-1), where h is the number of dose groups in the experi ment, including the control group. Whenever the multistage model does not fit the data sufficiently well, data at the highest dose is deleted, and the model is refit to the rest of the data. This is continued until an acceptable fit to the data is obtained. To determine whether or not a fit is acceptable, the chi-square statistic h2 2 c (xi - Nipi> X " l NiPi (1-pp is calculated where i,L is the number of animals in the i dose group, X^ is the number of animals in the ifc^ dose group with a tumor response, P^ is the probability of a response in the ic^ dose group estimated by fitting the multi stage model to the data, and h is the number of remaining groups. The fit is determined to be unacceptable whenever X2 is larger than the cumulative 99% point of the chi-square distribution with f degrees of freedom, where f equals the number of dose groups minus the number of non-zero nultistage coefficients. 9.5.3.3.3. Adjustments for Less Than Lifespan Duration of Experiment -- If the duration of experiment Le is less than the natural life span of the test animal L, the slope q*, or more generally the exponent g(d), is increased by multiplying a factor (L/Le)^. We assume that if the average dose d is continued, the age-specific rate of cancer will continue to increase as a constant func tion of the background rate. The age-specific rates for humans increase at least by the third power of the age and often by a considerably higher power, as demonstrated by Doll (1971). Thus, it is expected that the cumulative tumor rate would increase by at least the third power of age. Using this fact, it is t/f assumed that the slope q^, or more generally the exponent g(d), would also 9-239 CMA 051873 7^ increase by at least the third power of age. As a result, if the slope q^ [or g(d)] is calculated at age Le, it is expected that if the experiment had been continued for the full life span L at the given average exposure, the slope qaj [or g(d)] would have been increased by at least (L/Le) 3 . This adjustment is conceptually consistent with the proportional hazard model proposed by Cox (1972) and the time-to-tumor model considered by Daffer et al. (1980), where the probability of cancer by age t and at dose d is given by P(d,t) " 1 - exp [--f(t) x g(d)]. 9.5.3.4. CALCULATION OF UNIT RISK 9.5.3.4.1. Definition of Unit Risk -- This section deals with the unit risk for EDC in air and water and the potency of EDC relative to other carcino gens that the CAG has evaluated. The unit risk estimate for an air or water pollutant is defined as the incremental lifetime cancer risk occurring in a hypothetical population in which all individuals are exposed continuously from birth throughout their lifetimes to a concentration of 1 pg/nr* of the agent in the air they breathe, or to 1 pg/L in the water they drink. This calculation is done to estimate in quantitative terms the impact of the agent as a carcinogen. Unit risk estimates are used for two purposes: (1) to com pare the carcinogenic potency of several agents with each other, and (2) to give a crude indication of the population risk that might be associated with air or water exposure to these agents, if the actual exposures are known. 9.5.3.4.2. Unit Risk Estimates and Interpretation -- The unit risk estimate based on animal bioassays is only an approximate indication of the absolute risk in populations exposed to known carcinogen concentrations. There may be important differences in target site susceptibility, immunological 9-240 Cm 051874 responses, hormone function, dietary factors, and disease. In addition, human populations are variable with respect to genetic constitution and diet, living environment, activity patterns, and other cultural factors. The unit risk estimate can give a rough indication of the relative potency of a given agent compared with other carcinogens. The comparative potency of different agents is more reliable when the comparison is based bn studies in the same test species, strain, and sex. The quantitative aspect of carcinogen risk assessment is prepared because of its possible value in estimating the potential magnitude of the public health hazard. However, it should be recognized that the estimation of cancer risks to humans at low levels of exposure is uncertain. At best, the linear extrapola tion model used here provides a rough but plausible estimate of the upper-limit of risk; i.e., it is not likely that the true risk would be much more than the estimated risk, but it could very well be considerably lower. The risk esti mates presented in subsequent sections should not be regarded as immutable representations of the true cancer risks; however, the estimates presented may be used to the extent that the concept of upper-risk limits is found to be useful. 9.5,3.4.3. Alternative Low-Dose Extrapolation Models -- In addition to the multistage model currently used by the CAG for low-dose extrapolation, two more models, the probit and the Weibull, are also employed for purposes of comparison. These models cover almost the entire spectrum of risk estimates that could be generated from existing mathematical extrapolation models. Generally statistical in character, these models are not derived from biolog ical arguments, except for the multistage model, which has been used to support the somatic mutation hypothesis of carcinogenesis (Armitage and Doll, 1954; Whittemore, 1978; Whittemore and KelLer, 1978). The main difference among these models is the rate at which the response function P(d) approaches zero 9-241 CMA 051875 or P(0) as dose d decreases. For instance, the probit model would usually predict a smaller risk at low doses than the multistage model because of the difference of the decreasing rate in the low-dose region. However, it should be noted that one could always artificially give the multistage model the same (or even greater) rate of decrease as the probit model by making some dose transformation and/or by assuming that some of the parameters in the multistage model are zero. This, of course, is not reasonable without knowing, a priori, what the carcinogenic process for the agent is. 9.5.3.4,A. Calculation of the EDC Carcinogenic Potency (Slope) -- The multistage model is used in connection with the most sensitive tumor sites, and for purposes of comparison, other extrapolation models and data sets are also used to estimate the carcinogenic potency of EDC. Data from both rats and mice (NCI, 1978) are used to estimate the carcino genic potency of EDC. For rats, hemangiosarcomas in the circulatory system of male rats were selected, because these tumors are the most sensitive and were not located at the site of direct contact with the agent. Because of the high mortality rate in the high-dose group, the cancer risk is calculated by using two types of data: 1. Dichotomous data. The number of animals that survived at least 50 weeks was used as the denominator of the incidence rate (Table 9-63). This data set serves as a basis for comparing multistage models with the other two extrapolation models. 2. Time-to-death data (Table B-l, Appendix B). This data set is more appropriate for use in the calculation of the carcinogenic potency of EDC. In this calculation, it is assumed that animals with heman giosarcomas were killed by the tumors. This assumption seems reason able since almost all of the animals with hemangiosarcomas died before terminal sacrifice. 9-242 CMA 051876 For mice, hepatocellular carcinomas in male mice (Table 9-64) were used. Again, this tumor site was selected because it is the most sensitive in mice. Using the incidence data in Tables 9-63 and 9-64 and the corresponding human equivalent doses, the maximum likelihood estimates of the parameters in each of three extrapolation models are calculated and presented in Table A-l of Appendix A. These models can be used to calculate point estimates of risk at given doses or point estimates of doses for given levels of risk. The 95% upper-bound estimates of the risk at 1 mg/kg/day, calculated by various models using different data sets, are presented in Table 9-65. From this table, it is observed that the multistage model predicts a comparable risk on the basis of either hemangiosarcomas in male rats or liver carcinomas in male mice, while the probit and Weibull models ace extremely unstable and predict a wide range of risk, depending on the data base used. The slope, qj, which is the 95% upper-bound estimate of the linear coefficient in the multistage model, is also comparable when different data sets are used. The values are presented in Table 9-65. it --9 The CAG recommends that the slope estimate = 9.1 x 10 ^/mg/kg/day be used to represent the carcinogenic potency of EDC by oral exposure. This value is calculated on the basis of hemangiosarcomas in male rats using the multistage model with the time-to-death factor. This value will be used to estimate the unit risk of EDC by inhalation and drinking water. 9.5.3.4.5. Risk Associated with 1 yg/L of EDC in Drinking Water -- It is assumed that 100% of EDC in drinking water can be absorbed, and that the water intake is 2 L/day. Under these assumptions, the daily dose from con sumption of water containing 1 yg/L (1 ppb) of EDC is d = 1 yg/L x 2 L/day x 10"^ mg/yg x 1/70 kg = 2.9 x 10-5 mg/kg/day 9-243 CM& 051877 TABLE 9-63. INCIDENCE RATES OF HEMANGIOSARCOMAS IN THE CIRCULATORY SYSTEM OF MALE OSBORNE-MENDEL RATS Experimental dose (mg/kg/day) Human equivalent metabolized dose (mg/kg/day)a Tumor incidence rates*5 00 0/40 47 4.46 9/48 95 8.23 7/27 aHuman equivalent doses are taken from Table 9-62. ^The number of animals that survived at least 50 weeks is used as the denominator for the EDC-treated group. SOURCE: NCI, 1978. TABLE 9-64. INCIDENCE RATES OF HEPATOCELLULAR CARCINOMAS IN MALE B6C3F1 MICE Experimental dose (mg/kg/day) Human equivalent metabolized dose (mg/kg/day)3 00 97 3.52 195 4.58 aHuman equivalent doses are taken from Table 9--61. SOURCE: NCI, 1978. 9-244 Tumor incidence rates 1/19 6/47 12/48 CMA 051878 TABLE 9-65. THE 95% UPPER-BOUND ESTIMATE OF RISK AT 1 MG/KG/DAY USING THREE DIFFERENT MODELS3 Data base Multistage (with time factor)15 Probit Heraangiosarcomas in male rats 6.0 x 10"2 (8.7 x lO"2) 2.81 x lO-1 Hepatocellular carcinomas in male mice 6.1 x 10~2 1.87 x 10"^ Weibull 2.70 x 10-1 5.04 x 10"^ aIt is assumed that, at low doses, a given dose is totally metabolized. There fore, the risk estimates presented here are corresponding to either adminis tered or metabolized dose. ^The corresponding 95% upper-bound estimates of the linear component in the multistage model are: q* =6.1 x 10_2/mg/kg/day on the basis of dichotomous data (hemangiosarcoma); q* = 9.1 x 10-2/mg/kg/day on the basis of time-to-death data (hemangiosarcoma); q* = 6.2 x 10-2/mg/kg/day on the basis of hepatocellular carcinomas. 9-245 CMA 051879 Therefore, the upper-bound estimate of the incremental risk associated with 1 jjg/L of EDC in drinking water is P 9.1 x 10~2 x 2.9 x 10-5 * 2.6 x 10"^ 9.5.3.4.6. Risk Associated with 1 yg/m^ of EDC in Air -- The only available inhalation study (Maltoni et al., 1980) did not indicate any signif icant carcinogenic effects in either rats or mice. This seems contradictory to the result of the NCI gavage study, in which EDC was shown to be carcinogenic to both rats and mice. The seemingly discrepant results between the two exposure routes might be ascribed to one or more of the following causes: 1. The strains of test animals differ in responsiveness; 2. There is a difference in duration of exposure in the peak blood levels of EDC, and therefore the target tissue levels of its intermediate reactive metabolites are different for the doses in the two studies; 3. An artifact has been introduced by the intercurrent mortality. One issue that is of interest to the risk assessment is to determine whether or not the dose used in the inhalation study is too low to induce a detectable tumor response in the number of animals tested, in view of the fact that EDC is carcinogenic by oral route of exposure. Reitz et al. (1982) indicates that rats exposed to EDC in air at 150 ppm for 6 hours had an assim ilated dose of 50.7 mg/kg and a metabolized dose of 49.8 mg/kg. On this basis, the high-dose group (150 ppm to 250 ppm, 7 hours/day) in the Maltoni et al. study on rats should have assimilated and metabolized doses greater than 50.7 mg/kg/day and 49.8 mg/kg/day, respectively. Therefore, the Maltoni et al. high-dose group should be at least as responsive as the NCI low-dose (47 mg/kg/day) group of rats in which 19% (9/48) of the male rats developed heman-- 9-246 CMA 051880 giosarcoraas. If the dose available to animals (assimilated or metabolized) is the only factor that determines tumor response, one would expect to see at least one tumor type in the Maltoni et al. study based on what was observed in the NCI study. It is unlikely that 19% (9/48) of heraangiosarcomas would be observed in one study and none in the other in which more animals (79 male rats survived at 52 weeks) were used in the experiment. The probability of observing such an event is very small (p < 0.0001) if the total dose available to animals is indeed the only factor that determines the tumor response. Therefore, the discrepant results between the two exposure routes cannot be totally explained by the difference in dose available to animals. The observation of greater (three- to fivefold) peak blood concentrations in the gavage study when compared to the inhalation study, together with dif ferences in the rates of formation, deactivation, and binding to macromolecules between the two routes of exposure, may explain the differing response pattern. The implication of the considerations just discussed is that, in addition to the EDC kinetic information (oral vs. inhalation) on assimilated and metabo lized dose, it Is important to determine whether strains of test animals, duration of exposure, and/or route of exposure make significant differences in the tumorigenic responses. If the strains of animals differ in responsiveness, then the most sensitive animal strain (i.e., the one from the NCI gavage study) should be used for potency estimation via inhalation. On the other hand, if the route of exposure makes a difference in the tumorigenic response, it would be more appropriate to use the inhalation study rather than the gavage study to estimate the risk by inhalation. Since the reason for the discrepant results between the two exposure routes is not known, both the NCI gavage study and the negative inhalation study by Maltoni et al. (1980) are used to calculate the risk for EDC by inhalation. The unit risk estimates calculated on the 9-247 CMA 051881 basis of these two data sets provide a range of risk estimates that reflect several types of uncertainty, including strains and routes of exposure used in the bioassays. 9.5.3.4.6.1. Unit Risk for Air Calculated on the Basis of Rat Gavage Data. The daily dose due to 1 pg/m^ of EDC in air is d = 1 pg/m^ x 20 m-Vday x 10"^ mg/kg/70 kg 2.86 x 10"4 mg/kg/day where 20 m^ is assumed to be the volume of daily air intake by a 70 kg person. In this calculation, the EDC in air breathed is assumed to be 100% absorbed. It is reasonable to assume 100% absorption and metabolism at low doses. Thus, the upper-bound estimate of the Incremental risk due to 1 pg/m^ of EDC in air is P - 9.1 x 10~2 x 2.9 x 10-4 2.6x 10"5 The information on EDC kinetics is not used in this derivation of unit risk. Although Reitz et al. (1982) provides some information on the compara tive metabolism orally (at 150 mg/kg) and by inhalation (at 150 ppm, for 6 hours), this Information cannot be appropriately used for route-to-route extrap olation because the relative amounts metabolized between the two routes of exposure do not appear to be linearly related to the administered dose, as evidenced by the data of Spreafico et al. on the blood concentration-time curve (AUC). Using the Spreafico et al. data in Table 9-59 and the assumption that the amount metabolized is linearly related to AUC, as the administered EDC dose decreases, the amount metabolized, when administered by inhalation, decreases in a greater proportion than the amount of EDC metabolized when administered by a single bolus by the oral route. Therefore, if the comparative metabolism 9-248 CMA 051882 observed at high doses in Reitz et al. (1982) is used for route-to-route extrap olation, the inhalation risk at low doses (e.g., 1 pg/m^) would be over estimated. 9.5. 3.4.6.2. Unit Risk for Air Calculated on the Basis of a Negative Rat Inhalation Study.A negative inhalation study by Maltoni et al. (1980) can be used to calculate an upper-bound estimate of risk. The data from male rats is used to calculatean upper-bound estimate of risk because it was found to be most sensitive in the NCI study. The smallest upper-bound estimate is calcu lated by using the information from the high-dose group in which 79 male rats survived at 52 weeks. These animals were exposed to EDC in air at 150 ppm, 7 hours/day, 5 days/week, for 78 weeks. The lifetime average exposure is cal culated to be d = (150 ppm x 7 hours/24 hours) x (5 days/7 days) x 78 weeks/104 weeks - 23.44 ppm or equivalently, d ** 23.44 ppm x 4,121 (pg/m3)/ppm 96,596.24 pg/ra^ Assuming no "tumor" is observed among the 79 animals, the 95% upper limit of the tumor probability is R = 0.037, calculated by solving the equation (1 R)= 0.05. By using the one-hit model, R * 1 - exp(-b x d), the slope, b, is calculated as b = [ - ln(1--R)]/d " 3.9 x 10_7/(ug/m3) Thus, for male rats, the risk due to 1 pg/m^ of EDC in air is 3.9 x 10-7. To convert this animal risk estimate into human risk, it is assumed that body 9-249 CMA 051883 burden per surface area is equivalent among species. For 1 yg/m^ of EDC in air, the total EDC uptake (body burden) of a rat weighing 0.35 kg is 1 yg/m^ x 0.224 m-Vday where 0.224 m^/day is the daily air intake for a 0.35 kg rat. The concentra tion for humans, C(yg/m^), which is equivalent to 1 yg/m^ for rats, is calculated to be C * 0.38 yg/m^, by solving for C from the equation 20 m^/day x C(yg/m^) m 1 yg/m^ x 0.224 m^/day (70 kg)2/3 (0.35 kg)2/3 where 20 m^/day is assumed to be the volume of air intake by a 70 kg person. Therefore, the upper-bound estimate of incremental human cancer risk due to 1 yg/m^ of EDC in air is P =* 3.9 x 10-7/0.38 - 1.0 x 10-6 This risk estimate is 26 times smaller than that calculated on the basis of the gavage study. As discussed previously, this upper-bound risk estimate is appropriate only if the discrepant results in tumor response between the oral and inhalation routes is due to the possible difference in the rates of forma tion or rates of deactivation or rates of covalent bond formation and if, in combination, these results differ between oral versus inhalation routes of exposure, but are not due to the strains of test animals. 9.5.3.5. COMPARISON OF POTENCY WITH OTHER COMPOUNDS -- One of the uses of quantitative potency estimates is to compare the potencies of various car cinogens. Figure 9-17 is a histogram representing the frequency distribution of potency indices of 54 suspect carcinogens evaluated by the CAG. The data 9-250 CMA 051884 4th QUART1LE , 3rd QUARTILE , 2nd 1st QUARTILE , QUART1LE 1 x UT1 4 * 10'2 2 x 1 o *3 1 QUENCY Figure 9-17. Histogram representing the frequency distribution of the potency indices of the 54 suspect carcinogens evaluated by the Carcinogen Assessment Group. 9-251 CMA 051885 summarized by the histogram are presented in Table 9-66. The potency index is derived from q^, the 95% upper bound of the linear component in the multistage model, and is expressed in terms of (mmol/kg/day)-^. Where human data were available for an agent, they have been used to calculate the index. Where no human data were available, animal oral studies have been used in preference to animal inhalation studies, since oral studies constitute the majority of animal studies. Based on available data concerning heraangiosarcoraas in male rats (NCI, 1978), the potency index for EDC has been calculated as 9 x 10. This figure is derived by multiplying the slope q^ - 9.1 x 10"Vmg/kg/day and the molecular weight of EDC, 98.9. This places the potency index for EDC in the fourth quartile of the 54 suspect carcinogens evaluated by the CAG. The ranking of relative potency indices is subject to the uncertainties \ involved in comparing a number of potency estimates for different chemicals based on varying routes of exposure in different species, by means of data from studies whose quality varies widely. All of the indices presented are based on estimates of low-dose risk, using a low-dose linear extrapolation model. These indices may not be appropriate for the comparison of potencies if linearity does not exist at the low-dose range, or if comparison is to be made at the high-dose range. If the latter is the case, then an index other than the one calculated above may be more appropriate. 9.5.4. Summary 9.5.4.1. QUALITATIVE -- Although seven cancer bioassay studies of EDC have been reported, only the NCI bioassay in which EDC was administered to rats and mice by gavage produced a clear positive tumorigenic response. In the NCI (1978) rat study, EDC produced a statistically significant increase in the incidence of squamous cell carcinomas of the forestomach. 9-252 CMA 051886 July 1985 TABLE 9-66. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED BY THE CARCINOGEN ASSESSMENT GROUP AS SUSPECT HUMAN CARCINOGENS Compounds CAS Number Level of evidence Humans Animals Grouping based on IARC criteria Slope*3 (mg/kg/day)_l Molecular weight Potency indexc Order of magnitude dogio index) 9-253 Acrylonitrile Aflatoxin Bj Aldrin Allyl chloride Arsenic Bla]P Benzene Benzidene Beryllium 1,3-Butadiene Cadmium Carbon tetrachloride Chlordane 107-13-1 1162-65-8 309-00-2 107-05-1 7440-38-2 50-32-8 71-43-2 92-87-5 7440-41-7 106-99-0 7440-43-9 56-23-5 57-74-9 L L I S I S S L I L I I S S L 1 S S S S s s s L oo 00 2A 0.24(W) 2A 2900 53.1 312.3 1x10+1 9xlO+5 +1 +6 3 11.4 369.4 4x10+3 +4 1.19xl0-2 76.5 9x10"* 0 1 15(H) 149.8 2xl0+3 +3 2B 11.5 252.3 3xl0+3 +3 1 2.9xl0"2(W) 78 2x10 0 1 234(W) 184.2 4xl0+4 +5 2A 2.6 9 2x10+1 +1 2B 1.0xl0_1(I) 54.1 5x10 +1 2A 6.1(W) 112.4 7xl0+2 +3 2B 1.30x10-1 153.8 2x10+1 +1 3 1.61 409.8 7x10+2 +3 (continued on the following page) TABLE 9-66. (continued) Compounds CAS Number Level of evidence Humans Animals Grouping based on IARC criteria Slope0 (mg/kg/day)-1 Molecular weight Potency indexc Order of magnitude <log10 index) 9-254 Chlorinated ethanes 1,2-Dichloroethane 107-06-2 hexachloroethane 67-72-1 1,1.2,2-Tetrachloroethane 79-34-5 1,1,2-Trichloroethane 79-00-5 Chloroform 67-66-3 Chromium VI 7440-47-3 DDT 50-29-3 Dlchlorobenzidine 91-94-1 1,1-Dichloroethylene (Vinylidene chloride) 75-35-4 Dichloromethane (Methylene chloride) 75-09-2 Dieldrin 60-57-1 2,4-Dinitrotoluene 121-14-2 Diphenylhydrazine 122-66-7 Epichlorohydrin 106-89-8 Bis(2-chloroethyl)ether 111-44-4 I I I I I S I I I I I I I I I s L L L S S S S L L S s s s s 00 CO 2B 9.1xl0-2 98.9 9x10 +1 3 1.42x10-2 236.7 3x10 0 3 0.20 167.9 3xl0+t +1 3 5.73xlO-2 133.4 8x10 +1 2B 7x10-2 1 41(W) 119.4 100 8x10 4x10+3 +1 +4 2B 0.34 354.5 lxl0+2 +2 2B 1.69 253.1 4x10+2 +3 3 1.16(1) 97 lxl0+2 +2 3 6.3xl0-4(I) 84.9 5x10-2 -1 2B 30.4 380.9 lxl0+4 +4 2B 0.31 182 6xl0+1 +2 2B 0.77 180 1x10+2 +2 2B 9.9xl0-3 92.5 9xl0_1 0 2B 1.14 143 2xl0+2 +2 (continued on the following page) TABLE 9-66. (continued) Compounds CAS Number Level of evidence3 Humans Animals Grouping based on IARC criteria Slope33 (mg/kg/day)-* Molecular weight Potency indexc Order of magnitude dogiQ index) Bis(chloromethyl)ether 542-88-1 Ethylene dibromide (EDB) 106-93-4 Ethylene oxide 75-21-8 Heptachlor 76-44-8 Hexachlorobenzene 118-74-1 Hexachlorobutadiene 87-68-3 Hexachlorocyclohexane technical grade alpha isomer beta isomer gamma isomer 319-84-6 319-85-7 58-89-9 Hexachlorodibenzodioxin 34465-46-8 Nickel refinery dust (Nickel subsnlfide) > L11 . uni mis Oi me 1 by in i t r> > unine Hi. i /1 ri i l r.i nine Di lmi y 1 illL r<i nine N-n t l rosopy ri 1 idine N-nilt oso-N-e l bylurea P2-/5-9 55-I 924-'n- i 930-'>')~-' 759-- 1-9 S I L I l I I I I I S I I l 1 1 s s s s s L S L L S s s s s s s 1 9300(1) 2B 41 115 187.9 1x10+6 8xlO+3 +6 +4 2A 3.5x10-1(1) 44.1 2x10+1 +1 2B 3.37 373.3 1x10+3 +3 2B 1.67 284.4 5xlO+2 +3 3 7.75xlO-2 261 2x10+1 +1 4.75 2B 11.12 3 1.84 3 1.33 290.9 290.9 290.9 290.9 1x10+3 3x10+3 5x10+2 4xl0+2 +3 +3 +3 +3 2B 6.2xl0+3 391 2x10+6 +6 1 l.05(W) 240.2 2.5xl0+2 +2 2B 25.9(not by qj) 74.1 2x10+3 +3 2B 43.5(not by q?) 102.1 4xlO+3 2B 5.43 158.2 9x10*2 +-1 2B 2.13 100.2 !xlO+2 +2 2B 32.9 117.1 4xl0+J +4 (continued on the following p.ige) 051889 TABLE 9-66. (continued) Compounds CAS Number Level of evidence3 Humans Animals Grouping based on 1ARC criteria Slope*5 (mg/kg/day)-1 Molecular weight Potency indexc Order of magnitude (login index) N-nitroso-N-methylurea 684-93-5 N-nitroso-diphenylamine 86-30-6 PCBs 1336-36-3 Phenols 2,4,6-Trichlorophenol 88-06-2 Tetrachlorodibenzop-dioxin (TCDD) 1746-01-6 Tetrachloroethylene 127-18-4 Toxaphene 8001-35-2 Trichloroethylene 79-01-6 Vinyl chloride 75-01-4 I I I I I I I I S s s s s s L S L/S S 2B 2B 2B 2B 2B 3 2B 3/2B 1 302.6 4.92xl0-3 4.34 1.99xl0-2 1.56x10+3 5.1x10*2 1.13 1.1x10-2 1.75x10-2(1) 103.1 198 324 197.4 322 165.8 414 131.4 62.5 3x10+4 1x10 1x10+3 4x10 5x10+7 8x10 5xl0+2 1x10 1x10 +4 0 +3 +1 +8 +1 +3 0 0 aS - Sufficient evidence; L - Limited evidence; I Inadequate evidence. bAnimal slopes are 95% upper-bound slopes based on the linearized multistage model. They are calculated based on animal oral studies, except for those Indicated by I (animal inhalation), W (human occupational exposure), and H (human drinking water exposure). Human slopes are point estimates based on the linear nonthreshold model. Not all of the carcinogenic potencies presented in this table represent the same degree of certainty.. All are subject to Q change as new evidence becomes available. The slope value is an upper bound In the sense that the true value (which Is unknown) is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero. Thus, the use of the slope estimate in risk evaluations requires an appreciation for the Implication of the upper bound concept as well as the "weight of evidence" for the likelihood that the substance Is a human carcinogen. cThe potency index is a rounded-off slope in (mmol/kg/day)-1 and Is calculated by multiplying the slopes in (mg/kg/day)-1 by the molecular weight of the compound. 051890 hemangiosarcomas of the circulatory system, and fibromas of subcutaneous tissue in male rats. There was also a statistically significant increased incidence of adenocarcinomas of the mammary gland and hemangiosarcomas of the circulatory system in female rats. In the NCI (1978) mouse study, EDC produced a statistically significant increased incidence of hepatocellular carcinomas and alveolar/bronchiolar adenomas in male mice and a statistically significant increased incidence of alveolar/bronchiolar adenomas, mammary carcinomas, and endometrial tumors in female mice. Two inhalation studies of EDC were conducted. The study by Spencer et al. (1951) in Wistar rats showed no evidence of a positive response. However, this study was inadequate to assess thte carcinogenicity of EDC because the experiment was conducted in a small number of animals for 212 days with 151 exposure days at 200 ppm. A second study by Maltoni et al. (1980), conducted in both rats and mice, did not produce a statistically significant increase in tumor incidences in any of the organ sites as compared to control animals. The study by Theiss et al. (1977), a pulmonary tumor bioassay in which EDC was administered intraperitoneally to strain A mice, produced an elevated, but not a statistically significant, increase in the incidence of lung tumors in treated animals. In a study by Van Duuren et al. (1979), EDC was applied to the skin of ICR/Ha mice to assess its carcinogenic potential as a skin tumorigen. This study did not show a statistically significant increase in skin carcinomas; however, an increased incidence of benign lung tumors in mice treated with the high dose (126 mg/mouse) was reported. No case reports or epidemiologic studies concerning EDC are available in the published literature. 9-257 CMA 051891 9.5.4.2. QUANTITATIVE -- Data from gavage studies using both rats and mice have been used to estimate the carcinogenic potency of EDC. An evaluation of the metabolism and kinetic data shows that there is no evidence to suggest qualitative differences in the metabolic pathways of EDC in the mouse and rat, and no difference resulting from the route of exposure (oral or inhalation) in the rat. Lifetime average human equivalent doses for the NCI rat and mouse carcino genicity bioassay can be calculated and are used in estimation of the cancer potency. For rats, data on hemangiosarcomas in the circulatory system are used. For mice, data on hepatocellular carcinomas are used. The carcinogenic poten cies estimated on the basis of these two data sets are comparable when the linearized multistage model is used. The upper-bound estimate of EDC potency (slope) is qj * 9.1 x 10 4/mg/kg/day. This is calculated on the basis of hemangiosarcomas using the time-to-death data and an adjusted dose derived from the metabolism/kinetic evaluation. The upper-bound estimate of the incremental cancer risk due to 1 pg/L of EDC in drinking water is 2.6 x 10"^. Because of the discrepant tumor responsiveness between oral and inhala tion assays, two upper-bound estimates of the incremental cancer risk due to I ug/m^ of EDC in air are calculated: 2.6 x 10"-* on the basis of the NCI gavage study, and 1.0 x 10"^ on the basis of a negative inhalation study by Maltoni etal. (1980). These two estimates reflect the various uncertainties associated with the data used, including the uncertainties due to the strains of test animals and routes of exposure used in the bioassays. If the strains of test animals differ in responsiveness, the unit risk estimate, 2.6 x 10"5, calculated from the positive response of the most sensitive strain of animals, should be used. On the other hand, if the discrepant results between the 9-258 CMA 051892 gavage and inhalation studies are due solely to the difference in route of exposure, than it would be more appropriate to use the unit risk estimate, 1.0 x 10"6, calculated on the basis of an inhalation study, even if it is a negative study. Given the evidence currently available, there is no certain scientific reason to explain why animals responded differently in the NCI gavage study compared with the Maltoni et al. inhalation study. 9.5.5. Conclusions. There is evidence that EDC is a probable human carcino gen. This conclusion is based on: (1) multiple tumor types in both an oral rat bioassay and an oral mouse bioassay; (2) suggestive evidence in two other bioassays, one which showed an elevated Increase in lung tumors with intraperitoneal injection and a second study which showed a significantly increased incidence of benign lung tumors with skin application of EDC; (3) demonstrated evidence of reactive metabolites of EDC and formation of DNA adduct; and (4) evidence that EDC is also a mutagen. There is direct animal evidence for carcinogenicity via the oral (gavage) route of exposure, but the only specific bioassay data for inhalation exposure are a negative rat and mouse study. Because of the presence of distant site tumors in the oral studies, it is likely that EDC is also carcinogenic via inhalation exposure. The negative findings in the rat and mouse inhalation studies do not contradict the indirect, suggestive, and ancillary evidence for the likelihood of EDC being carcinogenic via inhalation. The U.S. Environmental Protection Agency is using, on an interim basis, a proposed classification scheme for evaluating the weight-of-evidence for carcinogenicity (U.S. EPA, 1984), Using this classification, the positive findings in the oral rat and mouse studies would be considered as a sufficient level of evidence in experimental animals. The sufficient level of animal evidence, together with an absence of epidemiologic data, provides a basis for 9-259 CMA 051893 an overall weight-of-evidence ranking of Group B2, meaning chat EDO is "probably" carcinogenic in humans. Applying the International Agency for Research on Cancer (IARC) classifi cation scheme, which EPA has used in the past, the positive findings in the experimental animals and the absence of epidemiologic data provide an overall weight-of-evidence ranking of Group 2B, meaning that EDC Is "probably" carci nogenic in humans. While the "probable" carcinogenicity conclusion obviously applies to ingestion exposure since the positive evidence comes from gavage experiments, it is likely that EDC is a probable carcinogen for humans via inhalation expo sure. In view of the evidence, it Is prudent to consider EDC to be carcinogen!* via inhalation exposure, although the potency may vary from that estimated from gavage. Research investigations to verify this assessment conclusion should be implemented. Assuming that EDC is carcinogenic for humans, upper-bound incremental unit risks have been estimated for both ingestion and inhalation exposure. The development of these unit risk estimates is for the purpose of evaluating the "what if" question: If EDC is carcinogenic for humans, what is the possible magnitude of the public health impact? The upper-bound estimate of EDC's carcinogenic potency Is * 9.1 x 10~z/mg/kg/day based on the occurrence of hemangiosarcomas in the NCI gavage rat study. The upper-bound estimate of the incremental cancer risk due to 1 yg/L of EDC in drinking water is 2.6 x 10"6, based on the previously mentioned potency value. Two upper-bound estimates of the incremental cancer risk due to 1 yg/m^ of EDC in air are calculated: 2.6 x 10"^, on the basis of the gavage potency value and 1.0 x 10"6, on the basis of a negative inhalation study by Maltoni et al. (1980). The inhalation unit risks are presented as two values in recognition of the 9-260 CMA 051894 1 uncertainties associated with the differing data that were used to estimate the values. The upper-bound nature of these estimates is such that the true risk is not likely to be exceeded and may be lower. The potency index for EDC, defined as q* x molecular weight, lies in the fourth quartile among 54 carcinogens evaluated by the Carcinogen Assessment Group. 9-261 CMA 051895 APPENDIX A COMPARISON AMONG DIFFERENT EXTRAPOLATION MODELS Three models used for low-dose extrapolation, assuming the independent background, are: Multistage: P(d) " 1 - exp [-(q^d + ... + q^d^)] where q^ are non-negative parameters; Probit: P(d) - A + Bln(d) / f(x) dx -- CO where f(.) is the standard normal probability density function; Weibull: P(d) - 1 - exp [-bdk] where b and k are non-negative parameters. The maximum likelihood estimates (MLE) of the parameters in the multi stage model are calculated by means of the program GLOBAL83, which was developed by Howe (1983). The MLE estimates of the parameters in the probit and Weibull models are calculated by means of the program RISK81, which was developed by Kovar and Krewski (1981). Table A-l presents the MLE of parameters in each of the three models that are applicable to a data set. 9-262 CMA 051896 TABLE A-l. MAXIMUM LIKELIHOOD ESTIMATES OF THE PARAMETERS FOR EACH OF THE THREE EXTRAPOLATION MODELS BASED ON DIFFERENT DATA BASES Data base Hemangiosarcomas in male rats Hepatocellular carcinomas in male mice Multistage q^ = 4.15 x q2 = 0 qj = 0.0 q2 * 1.00 x 10-2 o tIo i ! Probit A - -1.48 B = 0.39 A - -4.27 B - 2.27 Weibull b = 8.46 x 10-2 k - 0.60 b - 5.70 x 10-4 k = 3.95 9-263 CMA 051897 APPENDIX B RISK CALCULATION BASED ON TIME-TO-EVENT DATA Because of the high mortality rate of rats in the high-dose group of the NCI (1978) gavage study, it is more appropriate to use time-to-event data (Table B-l) to calculate the potency of EDC. The probability of cancer by time t at dose d is given by: P(d,t) - 1 - exp [--f(t) x g(d)] where g(d) is a polynomial in dose d, and f(t) is a function of time t. The maximum likelihood estimate of the parameters and the asymptotic properties of the incremental risk estimate were investigated by Daffer et al. (1980). Their approach to estimating the parameters resembles Cox's regression-lifetable approach (Cox 1972), in which the time function f(t) need not be specified. Using the data in Table B-l, the lifetime cancer risk is estimated to be q* = 9.1 x 10-2/mg/kg/day This value is the 95% upper bound of the risk calculated at t * 90 weeks. Since the model fits the data very well up to 90 weeks but poorly beyond 90 weeks (overestimates), P(d, 90) is used to approximate the lifetime risk. This seems reasonable because the median life span for control animals is also less than 90 weeks (approximately 70% of the control animals died before 90 weeks). 9-264 CMA 051898 TABLE B-l. TIME-TO-DEATH IN WEEKS FROM HEMANGIOSARCOMAS IN MALE OSBORNE-MENDEL RATS FED EDC BY GAVAGE Control (vehicle and untreated): dose = 0 28, 37, 50, 50, 53, 53, 53, 54, 57, 57, 57, 57, 57, 61, 61, 69, 71, 76, 78, 80, 80, 83, 85, 86, 87, 89, 90, 101, 101, 104,, 106 , 106, 106, 106,, 106, 108, 110, 110,, 110, 110. Low-dose group: dose 4.46 mg/kg/day 30, 34, 36, 51, 52, 52, 55, 55, 59, 61, 63, 65, 69, 73(H)*, 74(H), 75, 75, 76, 77, 77, 77(H), 77, 80, 81, 82, 82, 82, 84, 87(H), 89(H), 89, 89, 89, 90, 92, 92, 93(H), 95(H), 96, 97, 98, 99, 99, 102(H), 103, 103, 104, 104, 109(H), 110. Hlgh-dose group; dose * 8.23 mg/kg/day 3, 8, 9, 9, 10, 10, 10, 10, 10, 11, 15, 15, 16, 20, 22, 33, 33, 33, 34, 37, 41, 48, 51, 52, 54, 57, 58, 59, 60, 61(H), 62, 63, 63, 68(H), 68(H), 71* 72, 73, 74, 74(H), 74, 74, 76(H), 76, 78(H), 83(H), 84, 89, 101. aH indicates death from hemangiosarcoma. 9-265 CMA 051899 1 10. REFERENCES Agranovich, B.Y.A. 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Health. 26: 281-284, 10-54 CMA 051953 umiea oidtes Environmental Protection Agency center tor bnvironmental Research Information Cincinnati OH 45268 Official Business H Penalty for Private Use, $300 Please make all necessary changes on the above label, detach or copy, and return to the address in the upper left-hand corner If you do not wish to receive these reports CHECK HERE . detach, or copy this cover, and return to the address in the upper left-hand corner EPA/600/8-84/006F