United States Office of Science Environmental Protection and Technology June 30, 2002 Agency Washington, D. C. EPA 822 R 03 018 DRINKING WATER CRITERIA DOCUMENT ON BROMINATED TRIHALOMETHANES DRAFT February 20, 2003 Prepared For Health and Ecological Criteria Division Office of Science and Technology Office of Water U. S. Environmental Protection Agency Washington, D. C. 20460 Under EPA Contract No. 68 C 99 206 Work assignment 0 16 by Syracuse Research Corporation 6225 Running Ridge Road North Syracuse, NY 13212 Under subcontract to The Cadmus Group, Inc. 135 Beaver Street Waltham, MA 02452 FOREWORD Section 1412 ( b) ( 3) ( A) of the Safe Drinking Water Act, as amended in 1986 requires the Administrator of the Environmental Protection Agency to publish Maximum Contaminant Level Goals ( MCLGs) and promulgate National Primary Drinking Water Regulations for each contaminant, which, in the judgment of the Administrator, may have an adverse effect on public health and which is known or anticipated to occur in public water systems. The MCLG is nonenforceable and is set at a level at which no known or anticipated adverse health effects in humans occur and which allows for an adequate margin of safety. Factors considered in setting the MCLG include health effects data and sources of exposure other than drinking water.

This document provides the health effects basis to be considered in establishing the MCLGs for brominated trihalomethanes found in chlorinated drinking water. To achieve this objective, data on pharmacokinetics, human exposure, acute and chronic toxicity to animals and humans, epidemiology and mechanisms of toxicity were evaluated. Specific emphasis is placed on literature data providing dose response information. Thus, while the literature search and evaluation performed in support of this document was comprehensive, only the reports considered most pertinent in the derivation of the MCLGs are cited in this document. The comprehensive literature search in support of this document includes information published up to July 2001, however, more recent information may have been added during the review process.

When adequate health effects data exist, Health Advisory values for less than lifetime exposure ( One day, Ten day and Longer term, approximately 10% of an individual's lifetime) are included in this document. These values are not used in setting the MCLGs, but serve as informal guidance to municipalities and other organizations when emergency spills or contamination situations occur.

Geoffrey Grubbs Director, Office of Science and Technology Office of Water Acknowledgments This document is derived and updated/ expanded of the Draft for the Drinking Water Criteria Document on Trihalomethanes ( U. S. EPA, 1994) and the Summary of New Health Effects Data on Drinking Water Disinfectants and Disinfectant Byproduct ( D/ DBPs) for the Notice of Availability ( NODA) ( U. S. EPA, 1997). This document includes an evaluation of literature on Brominated Trihalomethanes resulting from a full literature search for toxicity data conducted in July, 2001. In addition, few newer studies identified after the literature search date have been included as available at the time of document preparation.

Chemical Manager:

Nancy Chiu, Ph. D.

External Peer Reviewers:

Annette L. Bunge, Ph. D. Colorado School of Mines John Reif, M. Sc. ( Med), D. V. M., Colorado State university Judy Buelke Sam, M. A. Toxicology Services TABLE OF CONTENTS I. EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I  1 II. PHYSICAL AND CHEMICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . II  1 A. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II  1 B. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II  2 III. TOXICOKINETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  1 A. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  1 B. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  3 C. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  5 D. Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  13 E. Bioaccumulation and Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  13 F. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  13 IV. HUMAN EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  1 A. Occurrence in Drinking Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  1 1. National Surveys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  2 2. Other Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  8 3. Estimates of Tap Water Ingestion Exposure to Brominated Trihalomethanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  13 B. Exposure from Sources Other Than Drinking Water . . . . . . . . . . . . . . . . . IV  17 1. Dietary Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  17 2. Air Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  20 3. Concentrations and Exposures Associated with Swimming Pools and Hot Tubs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  27 4. Soil Concentrations and Exposure . . . . . . . . . . . . . . . . . . . . . . . . . IV  30 C. Overall Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  30 D. Body Burden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  31 1. Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  31 2. Mother's Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  35 E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  35 V. HEALTH EFFECTS IN ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  1 A. Acute Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  1 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  1 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  6 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  8 B. Short Term Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  8 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  14 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  21 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  24 C. Subchronic Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  27 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  30 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  31 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  33 D. Chronic Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  34 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  34 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  38 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  39 E. Reproductive and Developmental Effects . . . . . . . . . . . . . . . . . . . . . . . . . . V  41 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  41 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  56 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  59 F. Mutagenicity and Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  67 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  67 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  74 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  81 G. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  85 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  85 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  92 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  95 H. Other Key Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  97 1. Immunotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  97 2. Hormonal disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  99 3. Structure Activity Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . V  100 I. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  101 1. Health Effects of Acute and Short Term Exposure of Animals . . . . V  101 2. Health Effects of Longer term Exposure of Animals . . . . . . . . . . . V  101 3. Reproductive and Developmental Effects . . . . . . . . . . . . . . . . . . . V  102 4. Mutagenicity and Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . V  103 5. Carcinogenicity Studies in Animals . . . . . . . . . . . . . . . . . . . . . . . . V  103 6. Other Key effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  104 VI. HEALTH EFFECTS IN HUMANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 A. Clinical Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 B. Epidemiological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  1 1. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  7 2. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  11 3. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  15 C. High Risk Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  15 D. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  16 VII. MECHANISM OF TOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  1 A. Role of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  1 B. Biochemical Basis of Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  1 C. Mode of Action of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  2 D. Interactions and Susceptibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  5 1. Potential Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  5 2. Greater Childhood Susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . VII  6 3. Other Potentially Susceptible Populations . . . . . . . . . . . . . . . . . . VII  13 E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII  16 VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS . . . . . . . . . . . . . . . . . . VIII  1 A. Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  1 1. Noncarcinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  1 2. Carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  28 B. Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  35 1. Noncarcinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  35 2. Carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  49 C. Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  55 1. Noncarcinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  55 2. Carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  70 D. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  75 IX. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX  1 APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A  1 APPENDIX B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B  1 APPENDIX C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C  1 LIST OF FIGURES Figure III 1 Proposed Metabolic Pathways for Brominated Trihalomethanes . . . . . . . . . . . III  6 Figure V 2 Proposed Routes for GST Mediated Metabolic Activation of Trihalomethanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  70 LIST OF TABLES Table I 1 Summary of Quantification of Toxicological Effects for Brominated Trihalomethanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I  15 Table II 1 Physical and Chemical Properties of the Brominated Trihalomethanes . . . . . . . . II  1 Table III 1 Recovery of Label 8 Hours after Oral Administration of 14C Labeled Brominated Trihalomethanes to Male Sprague Dawley Rats or Male B6C3F 1 Mice . . . . . . . . . . . . . . . III  1 Table III 2 Cumulative Excretion of Label after Oral Administration of 14C Labeled Bromodichloromethane to Male F344 Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  2 Table III 3 Over view of Tissue Collection for Analysis of Bromodichloromethane in Sprague Dawley Rat Tissues and Fluids ( CCC, 2000c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III  4 Table IV 1. Brominated Trihalomethane Concentrations Measured in U. S. Public Drinking Water Systems Serving 100,000 or More Persons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  7 Table IV 2 NRWA Brominated Trihalomethane Results for Small Surface Water Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  8 Table IV 3 Bromodichloromethane Concentrations in Drinking Water from the U. S. EPA TEAM Study ( µ g/ L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  9 Table IV 4 Dibromochloromethane Concentrations in Drinking Water from the U. S. EPA TEAM Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  10 Table IV 5 Bromoform Concentrations in Drinking Water from the U. S. EPA TEAM Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  11 Table IV 6 Estimated Drinking Water Exposures to Brominated Trihalomethanes in U. S. Public Drinking Water Systems Serving More than 100,000 Persons . . . . . . . . . . . . . . . . . . . . . IV  14 Table IV 7. Estimated Distribution of Drinking Water Exposures to Brominated Trihalomethanes for Populations in U. S. EPA TEAM Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  16 Table IV 8. Selected Concentration Data for Individual Brominated Trihalomethanes ( ppt) in Outdoor Air as Summarized in Brodzinsky and Singh ( 1983) . . . . . . . . . . . . . . . . . . . . . . IV  22 Table IV 9 Mean Bromodichloromethane Concentrations in Blood Following Three Types of Water Use Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  33 Table IV 10 Median Tap Water Trihalomethane Levels ( ppb) in Cobb County and Corpus Christi Homes, Water Treatment Plants, and Distribution Systems . . . . . . . . . . . . . . . . . . . . . . . IV  34 Table IV 11 Between Site Comparison of Median Blood Levels ( ppt) and Changes in Blood Levels ( ppt) after Showering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV  35 Table V 1 Summary of LD 50 Values for Brominated Trihalomethanes . . . . . . . . . . . . . . . . . V  1 Table V 2 Summary of Acute Toxicity Studies for Brominated Trihalomethanes . . . . . . . . . V  2 Table V 3 Summary of Short Term Toxicity Studies for Brominated Trihalomethanes . . . . V  9 Table V 4 Summary of Subchronic Toxicity Studies for Brominated Trihalomethanes . . . V  28 Table V 5 Summary of Chronic Toxicity Studies for Brominated Trihalomethanes . . . . . . V  35 Table V 6 NTP ( 1998) Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  44 Table V 7 Summary of Experiments Conducted by Bielmeier et al. ( 2001) . . . . . . . . . . . . V  46 Table V 8 Mean Consumed Doses ( mg/ kg day) of Bromodichloromethane in the Range Finding Study Conducted by CCC ( 2000c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  49 Table V 9 Summary of Reproductive Studies of Brominated Trihalomethanes . . . . . . . . . V  61 Table V 10 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data for Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  72 Table V 11 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data for Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  79 Table V 12 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data for Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  84 Table V 13 Tumor Frequencies in F344/ N Rats and B6C3F 1 Mice Exposed to Bromodichloromethane in Corn Oil for 2 Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  86 Table V 14 Hepatic and Renal Tumors in Male F344/ N Rats Administered Bromodichloromethane in the Drinking Water for Two Years . . . . . . . . . . . . . . . . . . . . . . V  90 Table V 15 Frequencies of Liver Tumors in B6C3F 1 Mice Administered Dibromochloromethane in Corn Oil for 105 Weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  94 Table V 16 Tumor Frequencies in the Large Intestine of F344/ N Rats Exposed to Bromoform in Corn Oil for 2 Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V  95 Table VI 1 Epidemiological Studies Investigating an Association Between Chlorinated Drinking Water and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI  2 Table VI 2 Epidemiological Studies Investigating an Association Between Chlorinated Drinking Water and Adverse Pregnancy Outcomes or Altered Menstrual Function . . . . . . . . . . . . . . VI  3 Table VI 3 Means and Adjusted Differences in Menstrual Cycle and Follicular Phase Length by Quartile of Individual and Summed Brominated Trihalomethanes . . . . . . . . . . . . . . . . . . . VI  13 Table VIII 1 Summary of Candidate Studies for Derivation of the One day HA for Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  4 Table VIII 2 Summary of Candidate Studies for Derivation of the Ten day HA for Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  10 Table VIII 3 Summary of Candidate Studies for Derivation of the Longer term HA for Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  17 Table VIII 4 Summary of Candidate Studies for Derivation of the RfD for Bromodichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  22 Table VIII 5 Summary of Preliminary BMD Modeling Results for the Bromodichloromethane RfD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  25 Table VIII 6 Tumor Frequencies in Rats and Mice Exposed to Bromodichloromethane in Corn Oil for 2 Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  32 Table VIII 7 Summary of Cancer Risk Estimates for Bromodichloromethane . . . . . . . . VIII  34 Table VIII 8 Summary of Candidate Studies for Derivation of the One day HA for Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  36 Table VIII 9 Summary of Candidate Studies for Derivation of the Ten day HA for Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  37 Table VIII 10 Summary of Candidate Studies for Derivation of the Longer term HA for Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  41 Table VIII 11 Summary of Candidate Studies for Derivation of the RfD for Dibromochloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  45 Table VIII 12 Results of Preliminary BMD Modeling of Selected Data from NTP ( 1985) Studies VIII  47 Table VIII 13 Frequencies of Liver Tumors in Mice Administered Dibromochloromethane in Corn Oil for 105 Weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  53 Table VIII 15 Summary of Candidate Studies for Derivation of the Ten day HA for Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  57 Table VIII 16 Summary of Candidate Studies for Derivation of the Longer term HA for Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  60 Table VIII 17 Summary of Candidate Studies for Derivation of the RfD for Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  66 Table VIII 18 Tumor Frequencies in Rats Exposed to Bromoform in Corn Oil for 2 Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  73 Table VIII 19 Carcinogenic Risk Estimates for Bromoform . . . . . . . . . . . . . . . . . . . . . VIII  74 Table VIII 20 Summary of Advisory Values for Bromodichloromethane, Dibromochloromethane, and Bromoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII  75 Table A 1 Candidate Studies and Data for BMD Modeling  Bromodichloromethane . . . . . A  3 Table A 2 Candidate Studies and Data for BMD Modeling  Dibromochloromethane . . . . A  15 Table A 3 Candidate Studies and Data for BMD Modeling  Bromoform. . . . . . . . . . . . . A  24 Table A 4 Model Equations used in BMD Calculations for Health Advisories . . . . . . . . . . A  29 Table A 5 Benchmark Dose Modeling Results for Bromodichloromethane . . . . . . . . . . . . A  41 Table A 6 Benchmark Dose Modeling Results for Dibromochloromethane . . . . . . . . . . . . A  50 Table A 7 Benchmark Dose Modeling Results for Bromoform . . . . . . . . . . . . . . . . . . . . . A  56 Table C 1 DBCM Concentrations Measured in U. S. Public Drinking Water Systems Serving 100,000 or More Persons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C  4 Table C 2 Selected Concentration Data for Individual Brominated Trihalomethanes ( ppt) in Outdoor Air as Summarized in Brodzinsky and Singh ( 1983) . . . . . . . . . . . . . . . . . . . . . . . . C  5 Table C 3 Results of RSC Calculations for DBCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C  17 Draft  Do Not Cite or Quote February 20, 2003 I  1 I. EXECUTIVE SUMMARY Brominated trihalomethanes are volatile organic liquids that have a number of industrial and chemical uses. The chief reason for health concern is that they are generated as by products during the disinfection of drinking water. The brominated trihalomethanes occurring in water are bromoform, dibromochloromethane, and bromodichloromethane. These compounds are formed when hypochlorous acid oxidizes any bromide ion present in water to form hypobromous acid, which subsequently reacts with organic material to form the brominated trihalomethanes.

Toxicokinetics No human data on absorption of brominated trihalomethanes are available. Measurements in mice and rats indicate that gastrointestinal absorption of brominated trihalomethanes is rapid ( peak levels attained less than an hour after administration of a gavage dose) and extensive ( 63% to 93%). Most studies of brominated trihalomethane absorption have used oil based vehicles. A study in rats found that the initial absorption rate of bromodichloromethane was higher when the compound was administered in an aqueous vehicle than when administered in a corn oil vehicle.

Data for distribution of brominated trihalomethanes in human organs and tissues are limited. Bromoform was found primarily in the stomach and lungs of a human overdose victim, with lower levels detected in intestine, liver, kidney and brain. Dibromochloromethane was found in 1 of 42 samples of human breast milk collected from women living in urban areas. Radiolabeled brominated trihalomethanes were detected in a variety of tissues following oral dosing in rats and mice. Approximately 1 to 4% of the administered dose was recovered in body tissues when analysis was conducted 8 or 24 hours post treatment. The highest concentrations were detected in stomach, liver, blood, and kidneys when assayed 8 hours after administration of the compounds. Bromodichloromethane was detected at a concentration of 0.38 µ g/ g in the milk of one of three female rats exposed to approximately 112 mg/ kg day during a reproductive/ developmental study. Bromodichloromethane was not detected in placentas, amniotic fluid, or fetal tissue collected on gestation day 21 from rats exposed to doses up to approximately 112 mg/ kg day or in plasma collected from postpartum day 29 weanling pups. Bromodichloromethane was detected at concentrations slightly above the limit of detection in placentas from two litters born to rabbits exposed to 76 mg/ kg day. Bromodichloromethane was detected in one fetus from a rabbit exposed to 76 mg/ kg day "... at a level below the limit of detection". Bromodichloromethane was not detected in placentas from female rabbits exposed to doses of approximately 32 mg/ kg day, or in amniotic fluid or the remaining fetuses from rabbits exposed to doses of approximately 76 mg/ kg day.

Brominated trihalomethanes are extensively metabolized by animals. Metabolism of brominated trihalomethanes occurs via two pathways. One pathway predominates in the presence of oxygen ( the oxidative pathway) and the other predominates under conditions of low oxygen tension ( the reductive pathway). In the presence of oxygen, the initial reaction product is trihalomethanol ( CX 3 OH), which spontaneously decomposes to yield the corresponding Draft  Do Not Cite or Quote February 20, 2003 I  2 dihalocarbonyl ( CX 2 O). The dihalocarbonyl species are reactive and may form adducts with cellular molecules. When intracellular oxygen levels are low, the trihalomethane is metabolized via the reductive pathway, resulting in a highly reactive dihalomethyl radical (° CHX 2), which may also form covalent adducts with cellular molecules. The metabolism of brominated trihalomethanes and chloroform appear to occur via the same pathways, although in vitro and in vivo data suggest that metabolism via the reductive pathway occurs more readily for brominated trihalomethanes. Both oxidative metabolism and reductive metabolism of trihalomethanes appear to be mediated by cytochrome P450 isoforms. The identity of cytochrome P450 isoforms that metabolize brominated trihalomethanes has been investigated in several studies which used bromodichloromethane as a substrate. The available data suggest that the cytochrome P450 isoforms CYP2E1, CYP2B1/ 2, and CYP1A2 metabolize bromodichloromethane in rats. The human isoforms CYP2E1, CYP1A2, and CYP3A4 show substantial activity toward bromodichloromethane in vitro and low but measurable levels of CYP2A6 activity have also been detected. Based on the available data, CYP2E1 and CYP1A2 are the only isoforms active in both rats and humans. CYP2E1 shows the highest affinity for bromodichloromethane in both species and the metabolic parameters K m and k cat are similar for rat and human CYP2E1. In contrast, the metabolic parameters for CYP1A2 differ in rats and humans. The pattern of results for isozyme activity obtained from an inhalation study of bromodichloromethane was similar to the pattern reported for male F344 rats treated with bromodichloromethane by gavage.

The lung is the principle route of excretion in rats and mice. Studies with 14C labeled compounds indicate that up to 88% of the administered dose can be found in exhaled air as carbon dioxide, carbon monoxide, and parent compound. Excretion in the urine generally appears to be 5% or less of the administered oral dose. Data from one study suggest that fecal excretion accounts for less than 3% of the administered dose.

Human Exposure Brominated trihalomethanes are found in virtually all water treated for drinking; however, concentrations of individual forms vary widely depending on the type of water treatment, locale, time of year, sampling point in the distribution system, and source of the drinking water. Occurrence data for brominated trihalomethanes are available from 13 national surveys and 9 additional studies that are more restricted in scope. The procedures used for sampling processing and storage and calculation of summary statistics should be carefully considered when evaluating and comparing brominated trihalomethane occurrence data. Some methods restrict trihalomethane formation by refrigeration or the use of quenching agents, whereas others maximize trihalomethane formation by storage at room temperature. Approaches to data summarization vary in their treatment of data below the analytical detection level or minimum reporting level.

When all available national survey data are considered, bromodichloromethane concentrations in drinking water range from below the detection limit to 183 µ g/ L ( ppb), while dibromochloromethane and bromoform concentrations range from below the detection limit to 280 µ g/ L ( ppb). When data for the three brominated trihalomethanes are compared, the frequency of Draft  Do Not Cite or Quote February 20, 2003 I  3 detection and measured concentrations of bromodichloromethane in drinking water supplies tend to be higher than those for dibromochloromethane. Bromoform is detected less frequently and at lower concentrations than the other two brominated trihalomethanes, except in some ground waters. Concentrations of all trihalomethanes in drinking water were generally lower when the raw water is obtained from ground water sources rather than surface water sources. The most recent national survey data are those collected by the U. S. EPA under the Information Collection Rule ( ICR). Monitoring data were collected over an 18 month period between July 1997 and December 1998 from approximately 300 water systems operating 501 plants and serving at least 100,000 people. Summary occurrence data stratified by raw water source ( groundwater or surface water) are available for finished water, the distribution system ( DS) average, and the DS high values. The mean, median, and 90th percentile values for surface water DS average concentrations in the ICR survey are 8.6, 70.2, and 20.3 µ g/ L, respectively, for bromodichloromethane ( range of individual values 0  65.8 µ g/ L); 2.4, 4.72, and 13.2 µ g/ L, respectively, for dibromochloromethane ( range 0  67.3); and 0. 1.18, and 3.10, respectively, for bromoform ( range 0  3.43).

Exposure to brominated trihalomethanes via ingestion of drinking water was estimated using data obtained for disinfectants and disinfection byproducts under the Information Collection Rule ( ICR). ICR data offer several advantages over other national studies for purposes of estimating national exposure levels of adults in the United States to brominated trihalomethanes via ingestion of drinking water. First, they are recent and reflect relatively current conditions. Second, data of very similar quality and quantity were collected systematically from a large number of plants ( 501) and systems ( approximately 300), including both surface and ground water systems. Third, the mean, median, and 90th percentile value were estimated on the basis of all samples taken, not just the sample detects. Thus, these descriptive statistics are representative of the exposures of the entire populations served by those systems, not just the populations served by systems with higher concentrations of these compounds. However, this study can not be considered representative of smaller public water supplies or water supplies from the most highly industrialized or contaminated areas.

Exposure was calculated by multiplying the concentration of individual brominated trihalomethanes in drinking water by the average daily intake, assuming that each individual consumes two liters of water per day. The annual median, mean, and upper 90th percentile values are presented for both surface and ground water systems. Assuming that the DS High value actually represents the average exposure level of persons served by one plant distribution pipe with the longest water residence time, the DS High value might be used to estimate a high end exposure level.

For bromodichloromethane, the median, mean, and 90th percentile population exposures from surface water systems are estimated to be 17, 20, and 40 µ g/ person/ day, respectively. The same values for populations exposed to bromodichloromethane from ground water systems are lower   3.6, 8.1, and 22 µ g/ person/ day, respectively. For dibromochloromethane, the median, mean, and 90th percentile population exposures from surface water systems are estimated to be 4.8, 9.4, and 26 µ g/ person/ day, respectively. The corresponding values for populations exposed Draft  Do Not Cite or Quote February 20, 2003 I  4 to dibromochloromethane from groundwater system are lower, with estimates of 2.7, 6.2, and 18 µ g/ person/ day, respectively. For bromoform, the median, mean, and 90th percentile population exposures from surface water systems are estimated to be near 0, 2.4, and 6.2 µ g/ person/ day, respectively. The same values for populations exposed to bromoform from ground water systems are higher, with estimates of 0.65, 3.8, and 9.6 µ g/ person/ day, respectively.

For purposes of comparison, estimates of ingestion exposure to bromodichloromethane, dibromochloromethane, and bromoform in drinking water were also estimated from data collected in other, older studies. Ingestion from ground water supplies was estimated from the median levels found in the Ground Water Supply Survey conducted by U. S. EPA in 1980 81. Based on the range of median levels ( 1.4   2.1 µ g/ L ( ppb)) and a consumption rate of two liters per day, the median ingestion exposure to bromodichloromethane may range from 2.8 to 4.2 µ g/ day. Similarly, median exposure to dibromochloromethane may range from 4.2 to 7.8 µ g/ day, and for bromoform, median exposure may range from 4.8 to 8.4 µ g/ day. Exposure to bromodichloromethane from surface water supplies can be estimated based on the range of median values observed under different conditions in the National Organics Monitoring Survey conducted by U. S. EPA in 1976 1977, which mainly sampled surface water systems. Based on a range of 5.9   14 µ g/ L ( ppb), exposure to bromodichloromethane from surface water is estimated to be between 12 and 28 µ g/ day. Similarly, based on the range of medians reported for dibromochloromethane concentrations, the median exposure is estimated to be up to 6 µ g/ day. The median levels of bromoform in the surface water supplies have been found to be less than the EPA Drinking Water minimum reporting levels ( MRLs) of 0.5   1 µ g/ L ( ppb). An estimate of exposure based on the MRLs will be overly conservative because the actual concentration of bromoform is not detectable. Based on the range of MRLs, 0.5   1 µ g/ L ( ppb), the exposure to bromoform is estimated to range from 1 to 2 µ g/ day for surface water supplies.

Ingestion exposure to brominated trihalomethanes in drinking water can also be estimated from the concentrations found at the tap in the U. S. EPA's Total Exposure Assessment Methodology ( TEAM) study. Estimates of the average of the population intakes for ingestion of bromodichloromethane from drinking water range from 0.42 to 42

g/ person/ day. The upper 90th percentile estimates range from < 2.0 to 90

g/ person/ day. Estimates of the average population intake of dibromochloromethane from drinking water range from 0.2 to 56

g/ person/ day. The upper 90th percentile estimates range from < 0.9 to 86

g/ person/ day. Estimates of the average of the population intakes of bromoform, for those areas in which bromoform was measurable in a majority of the samples, range from 1.6 to 16.2

g/ person/ day. The upper 90th percentile estimates range from 2.4 to 26

g/ person/ day. Four of the six locations in the TEAM study, however, had a low frequency ( less than 10%) of detection of bromoform in measurable quantities.

Sources of uncertainty in these estimates of ingestion exposure include use of different analytical methods, failure to report quantitation limits, using measures near the detection limit, failure to report how nondetects are handled when averaging values ( e. g., set to zero or one half the detection limit), and failure to report sample storage method and duration. In addition, many environmental factors influence the concentrations of these compounds in drinking water at the Draft  Do Not Cite or Quote February 20, 2003 I  5 tap and in vended or bottled waters used for drinking. These factors include season and temperature, geographic location, source of water, residence time in distribution system, and others.

Relatively few studies have analyzed non beverage foods for the occurrence of brominated trihalomethanes. In the few studies available, bromodichloromethane has been detected in nonbeverage foods ( i. e., in one sample of butter at 7 ppb, in three samples of ice cream at 0.6 to 2.3 ppb, in 6 of 10 samples of bean curd at 1.2 to 5.2 ppb, and in one sample of bacon). In addition, bromodichloromethane was detected in one sample each of eleven foods out of 70 tested in 14 Market Baskets for the FDA Total Diet Study. The detected concentrations ranged from 10 to 37 ppb for individual food items. Studies that analyzed non beverage foods for dibromochloromethane and bromoform detected neither compound in any of the tested samples. Brominated trihalomethanes have been detected in up to a third or one half of the types of prepared beverages examined in some studies, being detected most frequently in colas and other carbonated soft drinks. Bromodichloromethane has been found most frequently of the three compounds and bromoform the least frequently. Bromodichloromethane was detected in approximately half of the prepared beverages examined by McNeal et al. ( 1995) in the United States and in all of 13 soft drinks that they analyzed. One sampled soft drink contained bromodichloromethane at a concentration of 12 ppb; the remainder of the samples contained less than 4 ppb. Bromodichloromethane was detected in one sample of fruit juice at 5 ppb.

Some data on the occurrence of brominated trihalomethanes in foods and beverages are available from studies conducted in Italy, Japan, and Finland. These studies were also limited in scope to examination of relatively few food or beverage items. Bromodichloromethane, dibromochloromethane, and bromoform concentrations measured in foods and beverages in Italy, Japan and Finland ranged from undetectable to 40 ppb, undetectable to 13.9 ppb, and undetectable to 10.7 ppb, respectively. Because of possible differences in water disinfection or food processing practices, these data may not be representative of concentrations in foods and beverages produced in the U. S.

Measured concentrations of brominated trihalomethanes in outdoor air are variable from site to site. When data from several urban/ suburban and source dominated sites in Texas, Louisiana, North Carolina and/ or Arkansas were combined, the resulting average outdoor air concentrations were 110 ppt ( 0.74 µ g/ m3) for bromodichloromethane, 3.8 ppt ( 0.032 µ g/ m3) for dibromochloromethane, and 3.6 ppt ( 0.037 µ g/ m3) for bromoform. A regional study conducted at several sites in southern California found bromodichloromethane, dibromochloromethane, and bromoform in 35%, 17%, and 31% of the samples, respectively. The maximum concentrations observed were 40 ppt ( 0.27 µ g/ m3) for bromodichloromethane; 290 ppt ( 2.5 µ g/ m3) for dibromochloromethane; 310 ppt ( 3.2 µ g/ m3) for bromoform. Bromodichloromethane was detected in 64% ( n= 11) and 17% ( n= 6) of personal air samples collected in Texas and North Carolina. The detected concentrations ranged from 0.12 to 4.36 µ g/ m3 ( 0.017 to 0.65 ppb). Dibromochloromethane was not detected.

Mean concentrations in indoor air ranged from 0.38 to 0.75 µ g/ m3 for bromodichloro Draft  Do Not Cite or Quote February 20, 2003 I  6 methane; 0.44 to 0.53 µ g/ m3 for dibromochloromethane, and 0.29 to 0.35 µ g/ m3 for bromoform, as determined from 15 minute samples collected in 48 New Jersey residences. It was not clear whether these values were based exclusively on detected concentrations. In a separate study, levels of brominated trihalomethanes in indoor air were locally increased ( e. g., in shower/ bath enclosures and vanity areas) during showering and bathing events. The levels of individual brominated trihalomethanes in air were reported to be consistent with the levels in tap water.

No data for occurrence of brominated trihalomethanes in soil were available in the materials reviewed for this document. The chemical and physical properties of the brominated trihalomethanes indicate that they should volatilize readily from wet or dry soil surfaces. Therefore, ingestion of soil is not expected to be a significant route of exposure.

Brominated trihalomethanes have been detected in the blood and breast milk of humans. The level of individual brominated trihalomethanes in blood increases shortly after exposure to these compounds in tap water ( by dermal contact and/ or inhalation of the volatilized compound) during bathing and showering. Dibromochloromethane was detected in one of eight samples of breast milk collected from women living in the vicinity of U. S. chemical manufacturing plants or user facilities.

The RSC ( relative source contribution) is the percentage of total daily exposure that is attributable to tap water when all potential sources are considered ( e. g., air, food, soil, and water). Ideally, the RSC is determined quantitatively using nationwide, central tendency and/ or high end estimates of exposure from each relevant medium. In the absence of such data, a default RSC ranging from 20% to 80% may be used.

The RSC used in the current and previous drinking water regulations for dibromochloromethane is 80%. This value was established by use of a screening level approach to estimate and compare exposure to dibromochloromethane from various sources. Information considered for during this process is summarized in Appendix C. There are some uncertainties in the 80% RSC that are related to the availability of adequate concentration data for dibromochloromethane in media other than water. Parallel RSC calculations were not performed for bromodichloromethane and bromoform. The EPA has set the regulatory level for these chemicals in drinking water at zero because it has been determined that they are probable human carcinogens. Therefore, determination of an RSC is not relevant for these chemicals because it is the Agency's policy to perform RSC analysis only for noncarcinogens.

The use of chlorine to disinfect swimming pools and hot tubs results in the formation of brominated trihalomethanes. Swimming pool and hot tub users are potentially exposed to brominated trihalomethanes via dermal contact, ingestion, and inhalation of compounds released to the overlying air. As a result, swimming pool and hot tub users may experience greater overall exposures to brominated trihalomethanes than the general population. One study indicated that bromodichloromethane, dibromochloromethane, and bromoform concentrations in swimming pool and hot tub water ranged from 1 to 105 µ g/ L ( ppb), from 0.1 to 48 µ g/ L ( ppb), and from less than 0.1 to 62 µ g/ L ( ppb), respectively. Concentrations of the same brominated trihalomethanes Draft  Do Not Cite or Quote February 20, 2003 I  7 in the air two meters above the pool water ranged from less than 0.1 to 14 µ g/ m3 ( 0.015   2.09 ppb), from less than 0.1   10 µ g/ m3 ( 0.011   1.2 ppb), and from less than 0.1 to 5.0 µ g/ m3 ( 0.0097   0.48 ppb), respectively. Data from several studies confirm the uptake of brominated trihalomethanes from swimming pools, hot tubs, and environs by dermal and/ or inhalation pathways.

Health Effects of Acute and Short term Exposure of Animals Large oral doses of brominated trihalomethanes are lethal to mice and rats. Reported acute LD 50 values range from 450 to 969 mg/ kg for bromodichloromethane, 800 to 1,200 mg/ kg for dibromochloromethane, and 1,388 to 1,550 mg/ kg for bromoform.

Acute oral exposure to sublethal doses of brominated trihalomethanes can also produce effects on the central nervous system, liver, kidney, and heart. Ataxia, anaesthesia, and/ or sedation were noted in mice receiving 500 mg/ kg bromodichloromethane, 500 mg/ kg dibromochloromethane, or 1,000 mg/ kg bromoform. Renal tubule degeneration, necrosis, and elevated levels of urinary markers of renal toxicity have been observed in rats receiving 200 to 400 mg/ kg bromodichloromethane. Elevated levels of serum markers for hepatotoxicity and have been observed in rats at doses of bromodichloromethane ranging from approximately 82 to 400 mg/ kg day, and hepatocellular degeneration and necrosis were observed at 400 mg/ kg. Effects on heart contractility were reported in male rats at doses of 333 and 667 mg/ kg dibromochloromethane.

Short term oral exposure of laboratory animals to brominated trihalomethanes has been observed to cause effects on the liver and kidney. Hepatic effects, including organ weight changes, elevated serum enzyme levels, and histopathological changes, became evident in mice and/ or rats administered 38 to 250 mg/ kg day bromodichloromethane, 147 to 500 mg/ kg day dibromochloromethane, or 187 to 289 mg/ kg day bromoform for 14 to 30 days. Kidney effects, characterized by decreased p aminohippurate uptake, histopathological changes, and organ weight changes, became evident in mice and/ or rats administered 148 to 300 mg/ kg day bromodichloromethane, 147 to 500 mg/ kg day dibromochloromethane, or 289 mg/ kg day bromoform for 14 days. Evidence for decreased immune function was noted at bromodichloromethane and dibromochloromethane doses of 125 mg/ kg day.

The inhalation toxicity of bromodichloromethane has been evaluated in wild type and p53 heterozygous FVB/ N and C57BL/ N mice. Dose related renal tubular degeneration, and associated regenerative cell proliferation were seen in all strains at concentrations of 10 ppm and above after one week of exposure. Dose related increases in hepatic degeneration and regenerative cell proliferation were observed at 30 ppm and above. After three weeks of exposure, macroscopic and histologic lesions in the kidney and liver were resolved and cell proliferation indices had returned to near baseline levels. Pathological changes were more severe in the FVB/ N compared to the C57BL/ N mice and were more severe in the heterozygotes than in the wild type strains. Draft  Do Not Cite or Quote February 20, 2003 I  8 Health Effects of Subchronic and Chronic Exposure of Animals The predominant effects of subchronic oral exposure occur in the liver and kidney. The effects produced in these two organs are similar in nature to those described for short term exposures, with liver appearing to be the most sensitive target organ for dibromochloromethane and bromoform exposure. Histopathological changes in the liver were reported in mice and/ or rats administered 200 mg/ kg day bromodichloromethane, 50 to 250 mg/ kg day dibromochloromethane or 50 to 283 mg/ kg day bromoform. Histopathological changes in the kidney were reported in mice and/ or rats administered 100 mg/ kg day bromodichloromethane, or 250 mg/ kgday dibromochloromethane.

As observed for shorter durations of exposure, the predominant effects of chronic oral exposure are observed in the liver and kidney. Histopathological signs of hepatic toxicity in mice and/ or rats became evident at doses of 6 to 50 mg/ kg day for bromodichloromethane, 40 to 50 mg/ kg day for dibromochloromethane, and 90 to 152 mg/ kg day for bromoform. Signs of bromodichloromethane induced renal toxicity became evident in mice and rats treated with doses of 25 and 50 mg/ kg day, respectively.

Reproductive/ Developmental Effects in Animals Reproductive and developmental studies of brominated trihalomethanes are summarized in Table V 9. Data on the developmental effects of brominated trihalomethanes suggest that these chemicals are toxic to the fetus in most cases only at doses that result in maternal toxicity. Signs of maternal toxicity ( decreased body weight, body weight gain and/ or changes in organ weight) were reported in rats administered bromodichloromethane at 25 to 200 mg/ kg day and in rabbits administered 4.9 to 35.6 mg/ kg day. Signs of maternal toxicity were observed in rats or mice administered 17 ( marginal) to 200 mg/ kg day dibromochloromethane and in mice administered 100 mg/ kg day bromoform. Maternal toxicity was not observed in female rats dosed with up to 200 mg/ kg day of bromoform. Several well conducted studies on the developmental toxicity of bromodichloromethane gave negative results at doses up to 116 mg/ kg day in rats and 76 mg/ kgday in rabbits when administered in drinking water. However, in other studies, slightly decreased numbers of ossification sites in the hindlimb and forelimb were observed in fetuses of Sprague Dawley rats administered 45 mg/ kg day in the drinking water on gestation days 6 to 21and sternebral aberrations were observed in the offspring of Sprague Dawley rats administered 200 mg/ kg day by gavage in corn oil. Reductions in mean pup weight gain and pup weight were observed when the pups were administered bromodichloromethane in the drinking water at concentrations of 150 ppm and above ( biologically meaningful estimates of intake on a mg/ kg day basis could not be calculated for this study). Full litter resorption has been noted in F344 rats, but not Sprague Dawley rats, treated with bromodichloromethane at doses of 50 to 100 mg/ kg day during gestation days 6 to 10. Chronic oral exposure of male F344 rats to bromodichloromethane resulted in reduced sperm velocities at doses of 39 mg/ kg day. This response was not accompanied by histopathological changes in any reproductive tissue examined. Adverse clinical signs and reduced body weight and body weight gain were observed in parental generation female rats and F 1 male and female rats at 150 ppm ( approximately 11.6 to 40.2 mg/ kg day) in a two Draft  Do Not Cite or Quote February 20, 2003 I  9 generation drinking water study of bromodichloromethane. In the same study, small but statistically significant delays in sexual maturation occurred in F1 males at 50 ppm ( approximately 11.6 to 40.2 mg/ kg day) and F1 females at 450 ppm ( approximately 29.5 to 109 mg/ kg day). These delays may have been secondary to dehydration caused by taste aversion to bromodichloromethane in the drinking water.

Four of five studies on the reproductive or developmental toxicity of dibromochloromethane gave negative results when tested at doses of up to 200 mg/ kg day. In the fifth study, dibromochloromethane administered at 17 mg/ kg day in a multigenerational study resulted in reduced day 14 postnatal body weight in one of two F2 generation litters. At 171 mg/ kg day, the mid dose in the study, decreased litter size, viability index, lactation index, and postnatal body weight were observed in some F1 and/ or F2 generations.

The developmental and reproductive toxicity of bromoform has been examined in two studies. Bromoform administered to Sprague Dawley rats at 100 mg/ kg day in corn oil by gavage resulted in a significant increase in sternebral aberrations in the apparent absence of maternal toxicity. In a continuous breeding toxicity protocol, gavage doses of 200 mg/ kg day in corn oil resulted decreased postnatal survival, organ weight changes, and liver histopathology in F1 ICR Swiss mice of both sexes. No effects on fertility or other reproductive endpoints were noted.

Mutagenicity and Carcinogenicity Studies In vitro and in vivo studies of the mutagenic and genotoxic potential of bromodichloromethane, dibromochloromethane, and bromoform have yielded mixed results. Synthesis of the overall weight of evidence from these studies is complicated by the use of a variety of testing protocols, different strains of test organisms, different activating systems, different dose levels, different exposure methods ( gas versus liquid), and in some cases, problems due to evaporation of the test chemical. Overall, a majority of studies yielded more positive results for bromoform and bromodichloromethane. The genotoxicity and mutagenicity data for dibromochloromethane are inconclusive. Recent studies in strains of Salmonella engineered to contain rat theta class glutathione S transferase suggest that mutagenicity of the brominated trihalomethanes may be mediated by glutathione conjugation.

Carcinogenicity Studies in Animals The carcinogenic potential of individual brominated trihalomethanes has been investigated in oral exposure studies conducted in mice and rats. Brominated trihalomethanes administered individually in drinking water induced the formation of aberrant crypt foci ( ACF), putative preneoplastic lesions, in the colons of male F344/ N rats, but not male B6C3F 1 mice. However, a cancer bioassay of bromodichloromethane administered in the drinking water did not detect colon cancer in male F344/ N rats. Ingestion of bromodichloromethane in corn oil significantly increased the incidence of liver tumors in female mice, renal tumors in male mice and in male and female rats, and tumors of the large intestine in male and female rats. A drinking water exposure study of Draft  Do Not Cite or Quote February 20, 2003 I  10 bromodichloromethane in rats reported a significant induction of liver tumors at the lowest dose tested, but not at higher doses. Administration of dibromochloromethane in corn oil significantly increased the incidence of liver tumors in male and female mice. Administration of bromoform in corn oil increased the incidence of uncommon intestinal tumors in male and female rats, with the effect reaching statistical significance in females. No data are available for the carcinogenicity of brominated trihalomethanes via the inhalation or dermal routes.

Other Key Health Effects Data from Animal Studies The immunotoxicity of brominated trihalomethanes has been investigated in mice and rats. Short term bromodichloromethane exposure resulted in decreased antibody forming cells in serum, decreased hemagglutinin titers, and/ or suppression of Con A stimulated proliferation of spleen cells at doses of 125 to 250 mg/ kg day.

Studies in pregnant F344 rats detected decreased levels of progesterone in animals administered 75 or 100 mg/ kg bromodichloromethane by aqueous gavage on gestation day 8 or 9. Increased levels of luteinizing hormone were observed two to three days after dose administration. Disruption of luteal responsiveness to luteinizing hormone has been proposed as a possible mode of action by which bromodichloromethane elicits full litter resorption in F344 rats, but additional data are required to confirm this hypothesis. The available evidence does not suggest that dibromochloromethane or bromoform affect hormonal parameters.

Limited structure activity data for brominated trihalomethanes and chloroform suggest that bromination may influence the proportion of compound metabolized via the oxidative and reductive pathways, with brominated compounds being more extensively metabolized by the reductive pathway. Additional evidence suggests that a GST mediated pathway may play an important role in metabolism of brominated trihalomethanes.

Health Effects in Humans Limited human health data are available for the brominated trihalomethanes. In the past, bromoform was used as a sedative for children with whooping cough. Doses of 50 to 100 mg/ kgday usually produced sedation without apparent adverse effects. Some rare instances of death or near death were reported, although these cases were generally attributed to accidental overdoses. No human toxicological data were available for bromodichloromethane or dibromochloromethane.

Numerous epidemiological studies have examined the association between water chlorination and increased cancer mortality rates. None of these studies has identified a strong association between cancer and exposure to any individual brominated trihalomethane.

Several epidemiological studies have examined the association of chlorinated water use with various pregnancy outcomes, including low birth weight, premature birth, intrauterine growth retardation, spontaneous abortion, stillbirth , and birth defects. An association has been Draft  Do Not Cite or Quote February 20, 2003 I  11 reported for exposure to bromodichloromethane ( or a closely associated compound) and a moderately increased risk of spontaneous abortion during the first trimester. A confirmation of this finding is pending reanalysis of the original data to correct a differential misclassification error identified in a subsequent analysis of the study data. An association has also been reported for exposure to bromodichloromethane ( or a closely associated compound) and 1) stillbirth of fetuses weighing more than 500 g and 2) increased risk of neural tube defects in women exposed to  20 µ g/ L of bromodichloromethane prior to conception through the first month of pregnancy. An association has been reported for total brominated trihalomethanes and reduced menstrual cycle and follicular phase length in women of child bearing age. Among the individual brominated trihalomethanes, dibromochloromethane displayed the strongest association with altered menstrual function. However, because chlorinated water contains many disinfection by products, it is not possible to directly conclude from these studies that bromodichloromethane or bromodichloromethane are developmental toxicants in humans. Nevertheless, these studies raise significant concern for possible human health effects. The methodology used to estimate exposure to brominated trihalomethanes in tap water has been examined with the goal of refining estimates of intake of these compounds in epidemiological studies.

Susceptible Populations There is currently no clear evidence to suggest that children or the fetus are at greater risk for adverse effects from exposure to bromoform or dibromochloromethane than are adults. Where evidence of developmental toxicity has been observed in animals exposed to these chemicals, it generally appears to occur only at doses that elicit signs of maternal toxicity. Associations between concentration of bromodichloromethane ( or a co occurring chemical) and spontaneous abortion or still birth have been observed in two epidemiological studies. Evidence in rats indicates that exposure to bromodichloromethane causes full litter resorption in F344 rats but not Sprague Dawley rats, at doses many fold higher than are likely to occur from human ingestion of drinking water. Full litter absorption appears to result from a maternally mediated mode of action, rather than from a direct effect on the embryo. A mechanism of action for bromodichloromethane related pregnancy loss is suggested for the rat ( reduced sensitivity of the corpus luteum to luteinizing hormone), but is not without alternative explanation. At present, there is insufficient information on the mode of action leading to full litter resorption in rats to fully evaluate the relevance of this outcome to potential reproductive and/ or developmental toxicity in humans. There is presently no evidence to suggest that or the fetus are at increased risk for brominated trihalomethane toxicity as a result of higher levels of metabolizing enzymes.

Subpopulations with either high levels of glutathione S transferase or low baseline levels of glutathione may potentially be more sensitive than the general population to brominated trihalomethane induced toxicity, but there are currently no epidemiological or animal data to confirm this speculation. The functional significance of polymorphisms in cytochrome P450 isoforms that metabolize brominated trihalomethanes is also unknown. Populations that have higher levels of CYP2E1 activity as a result of co exposure to ethanol or other inducers or as a result of altered health or physiological states may experience potentially higher risk. Draft  Do Not Cite or Quote February 20, 2003 I  12 Mechanism of Toxicity It is generally believed that the toxicity of the brominated trihalomethanes is related to their metabolism. This conclusion is based largely on the observation that liver and kidney, the chief target tissues for these compounds, are also the primary sites of their metabolism. In addition, treatments which increase or decrease metabolism also tend to increase or decrease trihalomethane induced toxicity in parallel.

Metabolism of brominated trihalomethanes is believed to occur via oxidative and reductive pathways. Limited structure activity data for brominated trihalomethanes and the structurallyrelated trihalomethane chloroform suggest that bromination may influence the proportion of compound metabolized via the oxidative and reductive pathways, with brominated compounds being more extensively metabolized by the reductive pathway. Additional evidence suggests that a GSH mediated pathway may play an important role in metabolism of brominated trihalomethanes. These data raise the possibility that brominated trihalomethanes may induce adverse effects ( toxicity and carcinogenicity) via several different pathways.

The precise biochemical mechanisms which link brominated trihalomethane metabolism to toxicity have not been characterized, but many researchers have proposed that toxicity results from the production of reactive intermediates. Reactive intermediates may arise from either the oxidative ( dihalocarbonyls) or the reductive ( free radicals) pathways of metabolism. Such reactive intermediates are known to form covalent adducts with various cellular molecules, and may impair the function of those molecules and cause cell injury. Free radical production may also lead to cell injury by inducing lipid peroxidation in cellular membranes. Direct evidence showing a relationship between the level of covalent binding intermediates generated by either pathway and the extent of toxicity is not available for the brominated trihalomethanes. Manipulation of cellular glutathione levels suggests that this compound may play a protective role in brominated trihalomethane induced toxicity.

Individual brominated trihalomethanes have been shown to induce tumors in laboratory animals. The mode of action by which brominated trihalomethanes induce tumors in target tissues has not been fully characterized. DNA adducts can be formed by interaction of reactive metabolites ( resulting from oxidative and reductive metabolism) with DNA. In addition, preliminary evidence suggests that DNA adducts can be formed through conjugation with glutathione and bioactivation of the resulting conjugates. In contrast to chloroform, the role of cytotoxicity and associated regenerative cell proliferation in tumorigenicity of brominated trihalomethanes is presently unclear. Comparison of dose response data for liver toxicity ( including cell proliferation) and tumorigenicity in mice suggests that tumor formation occurs at concentrations lower than those which stimulate cell proliferation. No evidence for increased cell proliferation in kidney was obtained in studies using doses up to 246 mg/ kg day for bromodichloromethane, 312 mg/ kg day for dibromochloromethane, or 379 mg/ kg day for bromoform.

Interaction with agents which increase or decrease the activity of enzymes responsible for Draft  Do Not Cite or Quote February 20, 2003 I  13 metabolism of brominated trihalomethanes may modify carcinogenicity/ toxicity. Pretreatment with inducers of CYP2E1 has been observed to increase the hepatotoxicity of bromodichloromethane and dibromochloromethane in male rats. Pretreatment with m xylene, an inducer of the CYP2B1/ CYP2B2 isoforms, increased the hepatotoxicity of dibromochloromethane in male rats. Conversely, administration of the cytochrome P450 inhibitor 1 aminobenzotriazole prevented bromodichloromethane induced hepatotoxicity in rats. Recent findings indicating possible glutathione mediated metabolism of brominated trihalomethanes suggest that treatments or agents which alter glutathione S transferase activity could potentially modify the toxicity of brominated trihalomethanes.

The severity of brominated trihalomethane toxicity is potentially affected by the vehicle of administration. In a study of vehicle effects on the acute toxicity of bromodichloromethane, a high dose ( 400 mg/ kg) of the chemical was more hepato and nephrotoxic when given in corn oil compared to aqueous administration, but this difference was not evident at a lower dose ( 200 mg/ kg).

Quantification of Noncarcinogenic Effects Candidate health effects endpoints were analyzed by benchmark dose ( BMD) modeling using a benchmark response of 10% added risk. The BMDL 10 was defined as the 95% lower bond on the BMD estimate. For bromodichloromethane, a BMDL 10 of 30 mg/ kg day identified on the basis of full litter resorption in F344 rats was used to calculate a One day Health Advisory ( HA) value of 1 mg/ L. A BMDL 10 of 18 mg/ kg day for single cell hepatic necrosis, identified in a 30 day drinking water study in rats, was used to calculate a Ten day HA value of 0.6 mg/ L. A BMDL 10 of 18 mg/ kg day for reduced maternal body weight gain on gestation days 6 9, identified in a developmental study in rats, was used to calculate a Longer term HA of 0.6 mg/ L for a 10 kg child. A Longer term HA value of 2 mg/ L was calculated for a 70 kg adult based on the same endpoint. The calculations for the Reference Dose ( RfD) of 0.003 mg/ kg day and Drinking Water Exposure Level ( DWEL) of 90 µ g/ L employed a duration adjusted BMDL 10 of 0.8 mg/ kgday for fatty degeneration of the liver, identified in a 24 month dietary study in rats. Because bromodichloromethane is classified as a probable human carcinogen, a Lifetime HA is not recommended.

For dibromochloromethane, no suitable study was located for the calculation of a One day HA value. Use of the 10 day HA value as a conservative estimate is recommended. The Ten day HA value of 0.6 mg/ L was calculated using a BMDL 10 of 5.5 mg/ kg day for hepatic cell vacuolization, identified in a 28 day feeding study in rats. A duration adjusted BMDL 10 value of 1.7 mg/ kg day for hepatic cell vacuolization, identified in a 13 week gavage study in rats, was used to calculate Longer term HA values of 0.2 and 0.6 mg/ L for a 10 kg child and a 70 kg adult, respectively. A duration adjusted BMDL 10 value of 1.6 mg/ kg day for fatty changes identified in a 2 year gavage study in mice was used to calculate a RfD of 0.02 mg/ kg day and a DWEL of 700 µ g/ L. The Lifetime HA for dibromochloromethane is 60 µ g/ L. This value was calculated using the default RSC value of 80% for exposure via ingestion of drinking water. Because this compound is classified as a possible human carcinogen, the derivation of the Lifetime HA Draft  Do Not Cite or Quote February 20, 2003 I  14 incorporated an uncertainty factor of 10.

For bromoform, an estimated dose of 54 mg/ kg day that caused sedation in children was used to calculate a One day HA value of 5 mg/ L. A BMDL 10 of 2.3 mg/ kg day for hepatic vacuolization, identified in a one month dietary study in rats, was used to calculate a value of 0.2 mg/ L for the Ten day HA for the 10 kg child. This value was also recommended for use as the Longer term HA for a 10 kg child. A duration adjusted BMDL 10 value of 2.6 mg/ kg day for hepatic vacuolization, identified in a 13 week gavage study in rats, was also used to calculate a value of 0.9 mg/ L for the Longer term HA for the 70 kg adult. The BMDL 10 value of 2.6 mg/ kgday was also used to calculate an RfD of 0.03 mg/ kg day and a DWEL of 1000 µ g/ L. Because bromoform is classified as a probable human carcinogen, a Lifetime HA is not recommended.

Quantification of Carcinogenic Effects Chronic oral exposure studies performed by the National Toxicology Program in rats and mice provide adequate data to derive quantitative cancer risk estimates for the three brominated trihalomethanes, although the chemicals were administered in a corn oil vehicle. For bromodichloromethane a unit risk of 1.0 x 10 6 ( µ g/ L) 1 was derived, based on the incidence of renal tumors in male mice. The oral slope factor and concentration for excess cancer risk of 10 6 were 3.5 x 10 2 ( mg/ kg day) 1 and 1.0 µ g/ L, respectively. For dibromochloromethane, a unit risk of 1.2 x 10 6 ( µ g/ L) 1 was derived, based on liver tumors in female mice. The oral slope factor and concentration for excess cancer risk of 10 6 were 4.3 × 10 2 ( mg/ kg day) 1 and 0.8 µ g/ L, respectively. For bromoform, a unit risk of 1.3 x 10 7 ( µ g/ L) 1 was derived, based on tumors of the large intestine in female rats. The oral slope factor and concentration for excess cancer risk of 10 6 were 4.56 × 10 3 ( mg/ kg day) 1 and 8 µ g/ L, respectively. These values were calculated using an animal to human scaling factor of body weight3/ 4 in accordance with proposed U. S. EPA guidance ( U. S. EPA, 1996; 1999).

In a previous assessment of the carcinogenicity of brominated trihalomethanes, the Carcinogenic Risk Assessment Verification Endeavor ( CRAVE) group of the U. S. EPA assigned bromodichloromethane and bromoform to Group B2: probable human carcinogen. CRAVE assigned dibromochloromethane to Group C: possible human carcinogen. Under the proposed 1999 U. S. EPA Guidelines for Cancer Assessment, bromodichloromethane and bromoform are likely to be carcinogenic to humans by all routes of exposure. This descriptor is appropriate when the available tumor data and other key data are adequate to demonstrate carcinogenic potential to humans. This finding is based on the weight of experimental evidence in animal models which shows carcinogenicity by modes of action that are relevant to humans. Dibromochloromethane shows suggestive evidence of carcinogenicity, but not sufficient to assess human carcinogenic potential. This descriptor is used when the evidence from human or animal data is suggestive of carcinogenicity, which raises a concern for carcinogenic effects but is not judged sufficient for a conclusion as to human carcinogenic potential. This finding is based on the weight of experimental evidence in animal models which indicate limited or equivocal evidence of carcinogenicity. Draft  Do Not Cite or Quote February 20, 2003 I  15 IARC has recently re evaluated the carcinogenic potential of the brominated trihalomethanes. IARC classified bromodichloromethane as a Group 2B carcinogen: possibly carcinogenic to humans. IARC classified dibromochloromethane and bromoform as Group 3: not classifiable as to carcinogenicity in humans.

Table I 1 summarizes the quantification of noncarcinogenic and carcinogenic effects for brominated trihalomethanes. Draft  Do Not Cite or Quote February 20, 2003 I  16 Table I 1 Summary of Quantification of Toxicological Effects for Brominated Trihalomethanes Advisory Value Reference Bromodichloromethane One day HA for 10 kg child 1 mg/ L Narotsky et al. ( 1997)

Ten day HA for 10 kg child 0.6 mg/ L NTP ( 1998)

Longer term HA for 10 kg child 0.6 mg/ L CCC ( 2000d)

Longer term HA for 70 kg adult 2 mg/ L CCC ( 2000d)

RfD 0.003 mg/ kg day Aida et al. ( 1992b)

DWEL 100 µ g/ L Aida et al. ( 1992b)

Lifetime HA Not applicable

Oral Slope Factor c 3.5 x 10 2 ( mg/ kg day) 1 NTP ( 1987)

Concentration for excess cancer risk ( 10 6) 1.0 µ g/ L NTP ( 1987)

Unit Risk 1x10 6 ( µ g/ L) 1 NTP ( 1987)

Dibromochloromethane One day HA for 10 kg child b 0.6 mg/ L Aida et al. ( 1992a)

Ten day HA for 10 kg child 0.6 mg/ L Aida et al. ( 1992a)

Longer term HA for 10 kg child 0.2 mg/ L NTP ( 1985)

Longer term HA for 70 kg adult 0.6 mg/ L NTP ( 1985)

RfD 0.02 mg/ kg day NTP ( 1985)

DWEL 700 µ g/ L NTP ( 1985)

Lifetime HA 60 µ g/ L NTP ( 1985)

Oral Slope Factor c 4.3 x 10 2 ( mg/ kg day) 1 NTP ( 1985)

Concentration for Excess cancer risk ( 10 6) 0.8 µ g/ L NTP ( 1985)

Unit Risk 1.2 x 10 6 ( µ g/ L) 1 NTP ( 1985)

Bromoform One day HA for 10 kg child 5 mg/ L Burton Fanning ( 1901)

Ten day HA for 10 kg child 0.2 mg/ L NTP ( 1989a)

Longer term HA for 10 kg child a 0.2 mg/ L NTP ( 1989a)

Longer term HA for 70 kg adult 0.9 mg/ L NTP ( 1989a)

RfD 0.03 mg/ kg day NTP ( 1989a) Table I 1 ( cont.)

Advisory Value Reference Draft  Do Not Cite or Quote February 20, 2003 I  17 DWEL 300 µ g/ L NTP ( 1989a)

Lifetime HA Not applicable

Oral Slope Factor c 4.6 × 10 3 ( mg/ kg day) 1 NTP ( 1989a)

Concentration for Excess cancer risk ( 10 6) 8 µ g/ L NTP ( 1989a)

Unit Risk 1.3 x 10 7 ( µ g/ L) 1 NTP ( 1989a)

a The calculated value for the Longer term HA was slightly higher than the values for the Ten day HAs. Therefore, use of the Ten day HA for a 10 kg child is recommended as an estimate of the Longer term HA for a 10 kg child. b Use of the Ten day HA recommended as an estimate of the One day HA for a 10 kg child. c The oral slope factor was calculated using the Linearized Multistage model ( extra risk) and an animal to human scaling factor of body weight3/ 4 Abbreviations: BW, Body weight; DWEL, Drinking water exposure limit; HA, Health advisory; LMS; Linearized Multistage Model Draft  Do Not Cite or Quote February 20, 2003 II  1 C H Cl Cl Br C H Br Cl Br C H Br Br Br II. PHYSICAL AND CHEMICAL PROPERTIES A. Properties and Uses Bromodichloromethane ( CHBrCl 2), dibromochloromethane ( CHBr 2 Cl) and bromoform ( CHBr 3) are clear liquids with higher densities than the structurally related compound chloroform. They have limited solubility in water but are very soluble in organic solvents ( Windholz, 1976). Some important physical and chemical properties of these bromine containing trihalomethanes are summarized in Table II 1. Brominated trihalomethanes are sufficiently volatile to evaporate from drinking water ( Jolley et al., 1978).

Table II 1 Physical and Chemical Properties of the Brominated Trihalomethanes Property Chemical Bromodichloromethane Dibromochloromethane Bromoform Structure Chemical Abstracts Registry Number 75 27 4 124 48 1 75 25 2 Registry of Toxic Effects of Chemical Substances Number PA 5310000 PA 6360000 PB 5600000 Synonyms dichlorobromomethane chlorodibromomethane tribromomethane Chemical Formula CHBrCl2 CHBr2Cl CHBr3 Molecular Weight 163.83 208.29 252.77 Boiling Point 90 ° C 116C 149  150 ° C Melting Point  57.1 ° C  6 7 ° C Specific Gravity ( 20 ° ) 1.980 2.38 2.887 Vapor Pressure 50mm ( 20 ° C) 15 mm ( 10 ° C) 5.6 mm ( 25 ° C)

Stability in Water volatile volatile volatile Water Solubility 3,032 mg/ L ( 30 ° C) 1,050 mg/ L ( 30 ° C) 3,190 mg/ L ( 30 ° C)

Log Octanol: Water Partition Coefficient ( Kow) 2.09 2.23 2.37 Draft  Do Not Cite or Quote February 20, 2003 II  2 Source: USEPA ( 1992c; 1994b)

Brominated trihalomethanes also occur in drinking water as by products of chlorination. Bromide ( Br), a common constituent of natural waters, is oxidized by hypochlorous acid ( HOCl 3) to form hypobromous acid ( HOBr) in the following reaction:

Br + HOCl 3

HOBr + Cl

Hypobromous acid reacts with naturally occurring organic substances in water ( e. g., humic and fulvic acids) to yield the bromine containing trihalomethanes bromoform, dibromochloromethane and bromodichloromethane ( in increasing order of formation rates) ( Jolley et al., 1978).

Trihalomethanes may also be produced by reaction with endogenous organic material in the gut. Mink et al. ( 1983) treated adult male Sprague Dawley rats with a single oral dose of 48 mg Cl ( as sodium chloroacetate) and 32 mg Br ( as potassium bromide). All three brominated trihalomethanes were detected in the stomach contents of nonfasted rats following treatment ( Mink et al., 1983). Bromoform and dibromochloromethane were also detected in the plasma.

In the past, bromoform, bromodichloromethane and dibromochloromethane have been used in pharmaceutical manufacturing and chemical synthesis, as ingredients in fire resistant chemicals and gauge fluids, and as solvents for waxes, greases, resins, and oils ( U. S. EPA, 1975). However, use patterns have changed over time. At present, the primary use of bromodichloromethane is as a chemical intermediate for organic synthesis and as a laboratory reagent ( ATSDR 1989). Dibromochloromethane is reportedly used in laboratory quantities only ( ATSDR 1990). Use of bromoform is limited to performance of geological tests, use as a laboratory reagent, and use in quality assurance programs in the electronics industry ( ATSDR 1990).

B. Summary Brominated trihalomethanes are volatile organic liquids that occur in drinking water as byproducts of disinfection with chlorine. The brominated trihalomethanes occurring in water are bromoform, dibromochloromethane and bromodichloromethane. These compounds are formed in water when hypochlorous acid oxidizes bromide ions to form hypobromous acid, which subsequently reacts with organic material. In the past, individual brominated trihalomethanes have been used for a variety of industrial purposes. Currently, these compounds are used as laboratory reagents and, in the case of bromodichloromethane, as an intermediate in chemical synthesis. Draft  Do Not Cite or Quote February 20, 2003 III  1 III. TOXICOKINETICS This section summarizes available information on the absorption, distribution, metabolism and excretion of brominated trihalomethanes. Because the toxicokinetic properties of brominated trihalomethanes appear to be generally similar, data in this section are presented for this class of compounds as a group, rather than by individual chemical.

A. Absorption Mink et al. ( 1986) compared the absorption of bromodichloromethane, dibromochloromethane, and bromoform in male Sprague Dawley rats and male B6C3F 1 mice. The study animals received single oral doses of 14C labeled compound in corn oil by gavage at dose levels of 100 mg/ kg ( rats) or 150 mg/ kg ( mice). Total recovery of label in exhaled air, urine, or tissues after 8 hours ranged from 62% to 93% ( Table III 1), indicating that gastrointestinal absorption was high for all three compounds. The level of radiolabeled carbon monoxide in exhaled air was not quantified in this experiment. Carbon monoxide has since been recognized as a product of brominated trihalomethane catabolism.

Mathews et al. ( 1990) administered 14C bromodichloromethane by gavage in corn oil to male Fischer 344 rats at doses of 1, 10, 32, or 100 mg/ kg, and monitored the radiolabel in exhaled air, urine, feces, and tissues. Absorption was extensive, with at least 86% of the dose recovered as expired volatiles, CO 2, or CO. Only small amounts were recovered in urine (< 5%) or in feces (< 3%) within 24 hours of administration, regardless of the size of the dose ( Table III 2).

Table III 1 Recovery of Label 8 Hours after Oral Administration of 14C Labeled Brominated Trihalomethanes to Male Sprague Dawley Rats or Male B6C3F1 Mice Chemical Percent of Label Species Expired CO2 Expired Parent Urine Organs Total Recovery Bromodichloromethane Rat 14.2 41.7 1.4 3.3 62.7 Mouse 81.2 7.2 2.2 3.2 92.7 Dibromochloromethane Rat 18.2 48.1 1.1 1.4 70.3 Mouse 71.6 12.3 1.9 5.0 91.6 Bromoform Rat 4.3 66.9 2.2 2.1 78.9 Mouse 39.7 5.7 4.6 12.2 62.2 Adapted from Mink et al. ( 1986) and U. S. EPA ( 1994b). Draft  Do Not Cite or Quote February 20, 2003 III  2 Table III 2 Cumulative Excretion of Label after Oral Administration of 14C Labeled Bromodichloromethane to Male F344 Rats Dose Time ( hrs posttreatment Percent of Dose Expired CO2 Expired CO Expired Volatiles Urine Feces Total Recovery 1 mg/ kg 1 9.5 ± 1.1 NRa 2.1 ± 1.5 NR NR 11.6 ± 1.3 4 37.0 ± 3.2 1.5 ± 0.7 2.7 ± 1.8 NR NR 41.1 ± 2.8 8 62.9 ± 2.2 2.7 ± 1.1 NR NR NR 68. ± 1.7 16 76.4 ± 3.2 NR NR NR NR 81.8 ± 2.9 24 77.5 ± 3.3 3.3 ± 1.5 3.0 ± 1.6 4.1 ± 0.2 2.7 ± 1.5 90.7 ± 1.8 10 mg/ kg 1 8.0 ± 2.0 NR 2.0 ± 0.8 NR NR 10.0 ± 1.85 4 39.9 ± 3.2 1.9 ± 0.4 2.7 ± 1.1 NR NR 44.5 ± 3.0 8 66.0 ± 4.0 3.4 ± 0.9 NR NR NR 72.1 ± 3.9 16 81.3 ± 1.7 NR NR NR NR 87.4 ± 1.5 24 82.1 ± 1.8 4.3 ± 1.0 2.8 ± 1.1 4.3 ± 0.2 0.7 ± 0.2 94.2 ± 1.6 100 mg/ kg 1 1.9 ± 0.9 0.1 ± 0 1.5 ± 1.2 NR NR 4.6 ± 1.8 4 5.5 ± 1.8 0.3 ± 0.1 4.2 ± 1.9 NR NR 10.0 ± 2.9 8 NR NR NR 0.6 ± 0.4 NR 10.6 ± 3.0 16 33.4 ± 7.4 2.3 ± 0.7 5.7 ± 2.1 NR NR 42.0 ± 8.3 24 71.0 ± 1.7 5.2 ± 0.3 6.3 ± 2.1 4.1 ± 0.2 0.7 ± 0.3 87.3 ± 1.6 Adapted from Mathews et al. ( 1990) and U. S. EPA ( 1994b). aNot reported Draft  Do Not Cite or Quote February 20, 2003 III  3 Lilly et al. ( 1998) examined the effects of vehicle on the absorption of orally administered bromodichloromethane in an experiment designed to develop and validate a physiologically based pharmacokinetic model. Male F344 rats ( 3 animals/ dose/ vehicle/ assay) were gavaged with 0, 50, or 100 mg bromodichloromethane/ kg in either corn oil or 10% Emulphor ® , and bromodichloromethane levels were monitored in blood and exhaled air. The dose levels approximated doses previously utilized in two year cancer bioassays of bromodichloromethane ( NTP, 1987). Concentrations of bromodichloromethane in blood and exhaled air peaked rapidly, reaching maximal concentrations less than one hour after administration. The vehicle of administration had significant effects on the blood and exhaled air concentrations. Delivery of bromodichloromethane in 10% Emulphor ® resulted in faster initial uptake, as inferred from higher blood, tissue and breath chamber concentrations, when compared to corn oil ( data presented graphically). At 6 hours after administration, more than 90% and 100% of the administered dose had been absorbed from the corn oil and Emulphor ® vehicles, respectively.

B. Distribution Data on the distribution of brominated trihalomethanes in exposed humans are limited. Roth ( 1904) measured the bromoform content of tissues of a man who died from an accidental oral overdose of bromoform and found levels in stomach and lung of 130 and 90 mg/ kg wet weight, respectively. Lower levels were reported in the intestine, liver, kidney, and brain. Pellizzari et al. ( 1982) measured trihalomethanes in 42 samples of human milk taken from women in urban areas. Dibromochloromethane was detected in one sample. Neither the level measured nor the detection limit were reported for this study.

Data on the distribution of brominated trihalomethanes in animals are available from studies in rats and mice. Mink et al. ( 1986) compared the distribution of bromodichloromethane, dibromochloromethane, and bromoform in male Sprague Dawley rats and male B6C3F 1 mice. Single oral doses of 14C labeled compound in corn oil were administered by gavage at dose levels of 100 mg/ kg ( rats) or 150 mg/ kg ( mice). Tissue levels of radioactivity were measured 8 hours after dose administration. The chemical form of the label measured in the tissues ( e. g. parent or metabolite, bound or free) was not determined. In the rat, the total organ content of label ranged from 1.4% to 3.6% for the various compounds. The stomach, liver, and kidneys contained higher levels than bladder, brain, lung, muscle, pancreas, and thymus. In mice, 4% to 5% of the administered compound was recovered in the organs. However, an additional 10% of the label associated with bromoform was recovered in the blood of mice, yielding total organ levels of 12% to 14%. The authors attributed this elevated recovery of label to carboxyhemoglobin formation. The levels of carboxyhemoglobin were not measured in this experiment.

Mathews et al. ( 1990) investigated the distribution of bromodichloromethane following oral exposure in male Fischer 344 rats. Animals were given a single oral gavage dose of 1, 10, 32, or 100 mg/ kg of 14C bromodichloromethane dissolved in corn oil. Approximately 3% to 4% of the administered dose was detected in tissues after 24 hours. The highest levels ( 1% to 3%) were measured in liver. Repeated doses of 10 or 100 mg/ kg day for 10 days resulted in total retention of only 0.9% to 1.1% of the administered label, and had no effect on the tissue Draft  Do Not Cite or Quote February 20, 2003 III  4 distribution of bromodichloro methane.

The Chlorine Chemistry Council ( CCC, 2000c) sponsored a study which analyzed the levels of bromodichloromethane in parental tissues and fluids and F 1 generation tissues as part of a reproductive and developmental study in Sprague Dawley rats ( see Section V. E. 1 for a full study description). Data from this study were summarized in Christian et al. ( 2001b). Bromodichloromethane was administered in the drinking water at concentrations of 0, 50, 150, 450, or 1350 ppm. The estimated dosage on a mg/ kg day basis varied with the stage of the study ( see Table V 7). Plasma and other tissue samples were collected for analysis as described in Table III 3. All samples were maintained frozen and shipped to the analytical lab ( Lancaster Laboratories, Lancaster, PA). Analysis of plasma collected from male and female rats prior to mating and from female rats during gestation and lactation did not detect quantifiable levels of bromodichloromethane ( limit of detection 0.11 µ g). Bromodichloromethane was detected at a concentration of 0.38 µ g/ g in the milk from one female in the 1350 ppm group. Bromodichloromethane was not detected in placentas, amniotic fluid, or fetal tissue collected on GD 21 or in plasma collected from postpartum day 29 weanling pups.

Table III 3 Over view of Tissue Collection for Analysis of Bromodichloromethane in Sprague Dawley Rat Tissues and Fluids ( CCC, 2000c).

Generation Sex Physiological state Tissue Day of collection No. of Samples; Collection freq. Comments P M, F Pre mating Plasma Day 1 of exposure 3 rats/ sex/ group; 3 times/ day

P M, F Pre mating Plasma Day 14 of exposure 3 rats/ sex/ group; 3 times/ day

P F Pregnant Plasma GD 20 3 rats/ group; 3 times/ day Rats continuously exposed since study day 1 P F Pregnant Placenta amniotic fluid, and fetuses GD 21 3 litters/ day Tissues pooled by litter P F Lactating Plasma LD 15 3 rats/ group 3 times/ day P F Lactating Milk LD 15 3 rats/ group 1 IU oxytocin admin. by IV approx. 5 min. before milking F1 M, F Weaning Plasma LD 29 3 pups/ sex; 3 litters; 3 times/ day

Modified from CCC ( 2000c) Draft  Do Not Cite or Quote February 20, 2003 III  5 Abbreviations: P, parental; M, male; F, female; GD, gestation day; LD, lactation day The Chlorine Chemistry Council ( CCC, 2000a) sponsored a study which analyzed the levels of bromodichloromethane in parental tissues and fluids and F 1 generation tissues as part of a reproductive and developmental study in New Zealand White rabbits ( see Section V. E. 1 for a full study description). Data from this study were summarized in Christian et al. ( 2001b). Bromodichloromethane was administered to groups of rabbits ( 4/ concentration) in the drinking water at concentrations of 0, 50, 150, 450, or 1350 ppm. The estimated doses at these concentrations were 0, 4.9, 13.9, 32.3, or 76.3 mg/ kg day, respectively. Blood samples were collected on GD 7 and 28. Amniotic fluid, and placenta samples were collected on GD 29 after collection of a third blood sample, and amniotic fluid and placental tissue were pooled by litter. Blood samples were collected from three randomly selected fetuses per litter. Bromodichloromethane was detected at concentrations of 0.15 and 0.17 µ g/ g ( limit of detection 0.11 µ g/ g) in placentas from two litters in the 1350 ppm exposure group. Bromodichloromethane was detected in one fetus from the 1350 ppm group "... at a level below the limit of detection". Bromodichloromethane was not detected in placentas from does exposed to concentrations up to 450 ppm, in amniotic fluid from does exposed to concentrations up to 1350 ppm, or in the remaining fetuses of does exposed to concentrations as high as 1350 ppm.

C. Metabolism 1. Overview The toxicity of the brominated trihalomethanes is mediated by cytochrome P450 mediated bioactivation to reactive metabolites. The pathways for brominated trihalomethane metabolism were initially inferred from studies of the structurally related trihalomethane chloroform ( U. S. EPA, 1994b). Additional details of brominated trihalomethane metabolism have subsequently been elucidated in a number of laboratories using both in vitro and in vivo approaches and are described below. Figure III 1 presents a general metabolic scheme for chloroform and the brominated trihalomethanes.

The metabolism of brominated trihalomethanes occurs via at least two pathways ( U. S. EPA 1994b). The oxidative pathway requires NADPH and oxygen, whereas the reductive pathway can utilize NADPH or NADH and is inhibited by oxygen. Both reactions are believed to be mediated by cytochrome P450 isoforms. In the presence of oxygen ( oxidative metabolism), the reaction product is trihalomethanol ( CX 3 OH), which spontaneously decomposes to yield a reactive dihalocarbonyl ( CX 2 O) such as phosgene ( CCl 2 O). Dihalocarbonyls may undergo a variety of reactions, such as adduct formation with various cellular nucleophiles, hydrolysis to yield carbon dioxide, or glutathione dependent reduction to yield carbon monoxide. When oxygen levels are low ( reductive metabolism), the metabolic reaction products appear to be free radical species such as the dihalomethyl radical (° CHX 2). These radicals are highly reactive and may also form covalent adducts with a variety of cellular molecules. Evidence supporting this metabolic scheme and information on species differences in the rate and extent of trihalomethane metabolism are presented below. Additional data derived from studies of chloroform Draft  Do Not Cite or Quote February 20, 2003 III  6 CHX3 CX3OH CX2O CHX2 P 450 P 450 X NADPH or NADH 2NADPH O2 H2O HX Oxidative Pathway Reductive Pathway CO+ GSSG CO2 OTZ RCX2OH RH 2GSH H2O Cysteine 2HX 2HX 2HX Figure III 1 Proposed Metabolic Pathways for Brominated Trihalomethanes X = halogen atom ( chlorine or bromine); R = cellular nucleophile ( protein, nucleic acid);

GSH = reduced glutathione; GSSG = oxidized glutathione;

OTZ = oxothiazolidine carboxylic acid; P 450 = cytochrome P 450 Adapted from Stevens and Anders ( 1981); Tomasi et al. ( 1985) Draft  Do Not Cite or Quote February 20, 2003 III  7 are described in U. S. EPA ( 1994b).

The metabolism of trihalomethanes ( including chloroform and the brominated trihalomethanes) has been most intensively studied using chloroform as a substrate. These studies indicate that many factors influence metabolism, including strain, species, chloroform concentration, and possibly gender. A comprehensive review of chloroform studies is beyond the scope of this document. However, because the P450 mediated metabolism of the brominated trihalomethanes is expected to be similar to that of chloroform, descriptions of a few representative studies of chloroform metabolism are provided to provide additional background information on the metabolism of trihalomethanes.

A key question in hazard characterization is the identity of the P450 isoforms responsible for bioactivation of brominated trihalomethanes to reactive metabolites. This is because individuals or subpopulations with elevated levels of these enzymes may be at greater risk for adverse effects. The identities of the cytochrome P450 isoforms responsible for trihalomethane metabolism have been investigated most intensively in studies of chloroform ( studies of brominated trihalomethanes are described in sections 2 and 3 below).

Studies by Nakajima et al. ( 1995) and Testai et al. ( 1996) indicate that chloroform concentration plays a critical role in determining the role of different isoforms and the associated effects of metabolic inducers. Nakajima et al. ( 1995) pretreated male Wistar rats with three inducers of specific P450 isoforms and subsequently administered a single dose of chloroform by gavage in corn oil. The inducers used were phenobarbital ( CYP2B1/ 2), n hexane ( CYP2E1), and 2 hexanone ( CYP2B1/ 2 and CYP2E1). Liver damage ( as determined by serum enzyme activity and histopathology) was greatest at the mid dose in the hexane treated animals. In contrast, rats pretreated with phenobarbital or 2 hexanone showed a dose related increase of liver damage at all dose levels. The pattern of damage was consistent in each case with the tissue distribution patterns of the induced cytochrome P450 isoform( s). The study authors concluded on the basis of these results that CYP2E1 catalyzes chloroform metabolism at low doses and that CYP2B1/ 2 catalyzes chloroform metabolism at higher doses.

While experimental evidence indicates that CYP2E1 and CYPB1/ 2 catalyze the oxidative pathway, the identities of the cytochrome P450 isoforms which catalyze the reductive pathway have not been established. In general, CYP2E1 protein can catalyze reductive as well as oxidative reactions ( Lieber, 1997) and this isoform has been implicated in the production of trichloromethyl radicals from carbon tetrachloride ( see Lieber et al. 1997). However, evidence for a dual role of either CYP2E1 or CYP2B1/ 2 in catalyzing the oxidative and reductive pathways for trihalomethane metabolism has been contradictory, perhaps as a result of the different concentrations of chloroform used in different experiments ( summarized in Testai et al. 1996). To address the issue of concentration, Testai et al. ( 1996) studied the role of different isoforms in chloroform using microsomes prepared from Sprague Dawley rats pretreated with a variety of cytochrome P450 inducers. The microsomes were incubated under varying conditions of chloroform concentration, oxygenation, and presence of isoform specific inhibitors or antibodies. Under the conditions utilized in this series of experiments, the authors concluded that the Draft  Do Not Cite or Quote February 20, 2003 III  8 cytochrome P450 isoforms involved in oxidative metabolism of brominated trihalomethanes do not participate in the reductive pathway.

Recent evidence from studies in Salmonella typhimurium strains engineered to express the rat glutathione S transferase theta 1 1 ( GSTT1 1) gene suggests that bioactivation of brominated trihalomethanes to mutagenic species is also mediated by one or more glutathione S transferasemediated conjugation pathways ( Pegram et al., 1997; DeMarini et al., 1997). Details of these studies are presented in Section V. E. Proposed routes for glutathione conjugation and bioactivation are illustrated in Figure V 2, located in Section V. E.

2. In Vitro Studies Ahmed et al. ( 1977) investigated the in vitro oxidative ( aerobic) metabolism of brominated trihalomethanes to carbon monoxide by the rat liver microsomal fraction. Metabolism of bromoform resulted in the highest level of carbon monoxide formation, followed by dibromochloromethane and bromodichloromethane in decreasing order. Glutathione, NADPH and oxygen were required for maximal carbon monoxide production. This activity was inducible by phenobarbital or 3 methylcholanthrene pretreatment ( agents which are known to increase cytochrome P 450 activity) and was inhibited by the cytochrome P 450 inhibitor SKF 525 A. Similar results were reported by Stevens and Anders ( 1979). In addition, Stevens and Anders ( 1979) reported the formation of 2 oxothiazolidine 4 carboxylic acid ( OTZ) when bromoform was incubated in the presence of cysteine. Dihalocarbonyls react with cysteine to form OTZ. Thus, detection of OTZ provides evidence that a dihalocarbonyl intermediate was formed during bromoform metabolism.

Wolf et al. ( 1977) studied the in vitro metabolism of bromoform and chloroform to carbon monoxide under anaerobic conditions using liver preparations from phenobarbital induced rats. Bromoform metabolism resulted in much greater levels of carbon monoxide production than did the metabolism of chloroform. Gao and Pegram ( 1992) reported that binding of reactive intermediates to rat hepatic microsomal lipid and protein under reductive ( anaerobic) conditions was more than twice as high for bromodichloromethane as for chloroform. These data suggest that reductive metabolism may be a more important pathway for metabolism of brominated trihalomethanes than for chloroform.

Tomasi et al. ( 1985) studied the anaerobic activation of bromoform, bromodichloromethane, and chloroform to free radical intermediates in vitro using rat hepatocytes isolated from phenobarbital induced male Wistar rats. The production of a free radical intermediate was measured by electron spin resonance ( ESR) spectroscopy using the spin trap compound phenyl t butylnitrone. The intensity of the ESR signal was greatest for bromoform, followed by bromodichloromethane and then chloroform. The largest ESR signal was detected when hepatocytes were incubated under anaerobic conditions. Incubation in the presence of air resulted in a reduction of the signal, as did addition of cytochrome P 450 inhibitors such as SKF 525A, metyrapone, and carbon monoxide. These data were interpreted to indicate that freeradical formation depended on reductive metabolism of the trihalomethanes mediated by the Draft  Do Not Cite or Quote February 20, 2003 III  9 cytochrome P450 system. Comparison of the ESR spectra for chloroform, deuterated chloroform, and bromodichloromethane indicated that the free radical intermediate produced by chloroform metabolism was ° CHCl 2. The authors speculated that the brominated trihalomethanes are also metabolized by transfer of an electron directly from the cytochrome to the halocompound with the successive formation of the dihalomethyl radical (° CHX 2) and a halide ion ( X).

3. In Vivo Studies Mink et al. ( 1986) compared the metabolic products of bromodichloromethane, dibromochloromethane, and bromoform in male Sprague Dawley rats and male B6C3F 1 mice ( strain not reported). Animals were given a single oral dose of 14C labeled compound by gavage in corn oil at dose levels of 100 mg/ kg for rats and 150 mg/ kg for mice. Levels of 14C were measured in exhaled carbon dioxide recovered within 8 hours after dose administration. Expired carbon dioxide accounted for 4.3% to 18.2% of the administered label in rats ( Table III 1), suggesting that the parent compound had undergone limited metabolism and oxidation. In mice, the fraction of label excreted as carbon dioxide was higher, ranging from 40% to 81%. These data indicate that oxidative metabolism of brominated trihalomethanes to carbon dioxide was more rapid and extensive ( by a factor of four to ninefold) in mice than in rats. As previously noted in Section III. A, production of carbon monoxide, a known metabolite of brominated trihalomethanes, was not measured in this study.

Anders et al. ( 1978) investigated the formation of carbon monoxide from brominated trihalomethanes in corn oil administered to Sprague Dawley rats at doses of 1 mmol/ kg ( 119 to 252 mg/ kg) by intraperitoneal injection. Bromoform produced the highest levels of blood carbon monoxide, followed by dibromochloromethane and bromodichloromethane in decreasing order. A dose response relationship was noted for bromoform following administration of 252, 506, or 1,012 mg/ kg. Carbon monoxide production was inducible by pretreatment with phenobarbital, but pretreatment with 3 methylcholanthrene had no effect. Carbon monoxide production was significantly inhibited by SKF 525 A. Administration of 3H bromoform resulted in decreased carbon monoxide formation when compared to bromodichloromethane and dibromochloromethane, indicating that the carbon hydrogen bond breakage may be the ratelimiting step under aerobic conditions. Similar results were later reported by Stevens and Anders ( 1981).

Tomasi et al. ( 1985) studied the in vivo metabolism of chloroform, bromodichloromethane and bromoform to free radical intermediates in rats. Starved, phenobarbital induced male Wistar rats ( number not stated) were given intraperitoneal injections of 1,100 mg/ kg chloroform, 820 mg/ kg bromodichloromethane, or 1,260 mg/ kg bromoform dissolved in olive oil. The animals were sacrificed and the livers were homogenized. The production of a free radical intermediate by the livers was determined by ESR spectroscopy. The authors reported detection of free radicals in the livers of all treated rats. The intensity of the ESR signal followed a ranking similar to that observed in in vitro experiments ( bromoform > bromodichloromethane > chloroform), confirming that the reductive formation of free radicals is greater for brominated trihalomethanes than for chloroform. Draft  Do Not Cite or Quote February 20, 2003 III  10 Mathews et al. ( 1990) studied the metabolism of 14C bromodichloromethane in male Fischer 344 rats. Animals ( n = 4) were given a single oral dose of 1, 10, 32, or 100 mg/ kg of bromodichloromethane dissolved in corn oil. Levels of labeled carbon dioxide and carbon monoxide in exhaled air were measured for 24 hours. Approximately 70% to 80% of the dose was metabolized and exhaled as 14CO 2 and 3% to 5% of the dose as 14CO. However, 14CO 2 production was slower following a single dose of 100 mg/ kg than after the administration of a single dose of 32 mg/ kg or lower, suggesting saturation of metabolism. Repeated doses of 100 mg/ kg day for 10 days resulted in an increased rate of 14CO 2 production, compared with the initial rate. The authors concluded on the basis of these data that bromodichloromethane may induce its own metabolism.

Thornton Manning et al. ( 1994) evaluated the effect of bromodichloromethane exposure on cytochrome P450 isozyme activity in female F344 rats ( 6/ dose). Gavage doses of 75 to 300 mg/ kg day were administered in a solution of 10% Emulphor ® ( an emulsifier) for five consecutive days. Treatment resulted in decreased activity of the CYP1A and CYP2B isozymes. In contrast, there was no effect on CYP2E1 activity.

Pankow et al. ( 1997) investigated the metabolism of dibromochloromethane in male Wistar rats following single and repeated gavage doses. Rats receiving a single dose ( 6 animals per group) were treated with 0 ( vehicle only), 0.4, 0.8, 1.6 or 3.1 mmol dibromochloromethane kg dissolved in olive oil. Rats receiving multiple doses were gavaged with 0 ( vehicle only) or 0.8 mmol dibromochloromethane/ kg once a day for 7 days. The blood or plasma concentrations of parent compound, bromide, and carbon monoxide ( as carboxyhemoglobin, COHb) were measured following dibromochloromethane administration. The level of oxidized glutathione ( GSSG) in the liver of treated animals was also assayed. Oral administration of dibromochloromethane resulted in a significant elevation of plasma bromide levels at all doses tested. Bromide did not return to baseline levels even after 10 days. Repeated administration of 0.8 mmol dibromochloromethane/ kg resulted in significantly higher plasma levels of bromide than were measured following a single dose of 0.8 mmol/ kg. COHb was also elevated in a dosedependent manner following either single or repeated administration of dibromochloromethane, but returned to baseline levels within 24 hours after treatment. GSSG levels were significantly increased at 12 and 24 hour time points following a single 0.8 mmol/ kg dose ( no other doses were examined). Levels of GSSG returned to baseline levels by 48 hours after treatment.

Pankow et al. ( 1997) conducted additional experiments to determine whether reduced glutathione ( GSH) is a requirement for in vivo dibromochloromethane metabolism and to identify P450 isozymes involved in the metabolism of dibromochloromethane. Pretreatment of rats with buthionine sulfoximine ( an agent which depletes GSH) reduced GSH concentrations as anticipated and decreased the rate of bromide and COHb production. In contrast, pretreatment with butylated hydroxyanisole ( which increases GSH levels) increased the rate of bromide and COHb production. These results suggest that GSH plays a role in dibromochloromethane metabolism. Further studies were conducted to determine which cytochrome P450 isoform( s) participate in the in vivo metabolism of dibromochloromethane. Simultaneous exposure to 0.8 Draft  Do Not Cite or Quote February 20, 2003 III  11 mmol dibromochloromethane/ kg and diethyldithiocarbamate ( a potent inhibitor of P450 isoform CYP2E1) partially inhibited the production of bromide and COHb. In contrast, pretreatment with isoniazid ( an potent inducer of CYP2E1) increased formation of bromide and COHb. These experiments indicate that CYP2E1 is at least partially responsible for dibromochloromethane metabolism. Pretreatment with phenobarbital, an inducer of cytochrome P450 isoforms CYP2B1 and 2B2, increased the concentration of bromide in plasma, suggesting that CYP2B1 and 2B2 may also participate in the catabolism of dibromochloromethane. Pretreatment with m xylene, which induces both CYP2E1 and CYP2B1/ 2, resulted in higher bromide levels than inducers of CYP2E1 ( isoniazid) or CYP2B1/ 2 ( phenobarbital) administered individually. Pankow et al. ( 1997) concluded on the basis of these multiple experiments that 1) bromide and carbon monoxide are metabolites of dibromochloromethane; 2) dibromochloromethane is metabolized via the oxidative pathway; 3) the oxidative metabolism of dibromochloromethane is catalyzed by CYP2E1 and CYP2B1/ 2; and 4) dibromochloromethane plays a role in the induction of CYP2E1.

Allis et al. ( 2001) investigated the effect of inhalation exposure to bromodichloromethane on the activity and protein levels of CYP1A2, CYP2B1, and CYP2E1 in female F344 rats ( 6/ dose). In addition, the effect of inhalation exposure on the activity level of CYP1A1 was assayed. Serum bromide ion concentration, an indicator of the total metabolism of bromodichloromethane, was measured in samples drawn from a separate set of animals ( 4/ concentration) exposed to the same concentrations. The test animals were exposed for 4 hours to measured bromodichloromethane concentrations of 0, 106, 217, 419, 812, 1620, and 3240 ppm. The microsomal isozyme activities assayed were: p nitrophenol hydrolase ( PNP), an indicator of CYP2E1 activity; pentoxyresorufin O dealkylase ( PROD), an indicator of CYP2B1/ 2 activity; ethoxsyresorufin O dealkylase ( EROD), an indicator of CYP1A1 activity; and methoxyresorufin O dealkylase ( MROD), an indicator of CYP1A2 activity. The pattern of results for isozyme activity obtained in this inhalation study was similar to that reported for male F344 rats treated with bromodichloromethane by gavage. CYP2E1 activity as measured by PNP activity was not significantly affected by treatment. MROD, EROD, and PROD activities showed modest increases at low exposure concentrations. The increases were statistically significant for EROD and MROD at the 106 ppm exposure concentration. Decreases were observed at higher exposure concentrations relative to controls. These decreases were statistically significant for PROD at 3240 ppm and for EROD and MROD at concentrations of 800 ppm and greater. The results for isozyme protein levels, as measured by Western blots, were generally consistent with the results for isozyme activity. The study authors speculated that the most dramatic reductions in isozyme activity ( PROD and MROD) were a result of suicide inhibition. In addition, they concluded that analyses of the EROD and MROD activity and protein level patterns indicates that CYP1A2 is involved in the metabolism of bromodichloromethane. The study authors noted that it is not typical for this isozyme to metabolize the small molecules ( such as chloroform) that are the usual substrates for CYP2E1, but observed that the presence of the large bromide ion may make bromodichloromethane a suitable substrate for CYP1A2. Blood bromide concentration reached a maximum at 200 ppm, indicating that metabolism was saturated a concentrations equal to or greater than 200 ppm. Draft  Do Not Cite or Quote February 20, 2003 III  12 Allis et al. ( 2002) reported additional evidence for metabolism of bromodichloromethane by CYP1A2. Induction of CYP1A2 without parallel induction of CYP2E1 or CYP2B1/ 2 ( accomplished by pretreatment with 2,3,7,8 tetrachlorodibenzodioxin, TCDD), increased hepatotoxicity in male F344 rats administered a gavage dose of 400 mg/ kg bromodichloromethane. Hepatotoxicity was assessed by measurement of alanine aminotransferase ( ALT) and sorbitol dehydrogenase ( SDH) activity. Pretreatment with TCDD increased serum bromide levels ( a measure of total bromodichloromethane metabolism) in rats treated with 200 or 400 mg/ kg when compared to uninduced controls. The apparent inconsistency between lack of hepatotoxicity and increased total metabolism at 200 mg/ kg was explained by effective detoxification at this dose, presumably by glutathione. Selective inhibition of CYP1A2 activity, by administration of isosafrole to TCDD induced animals prior to treatment with 400 mg/ kg bromodichloromethane significantly reduced the hepatotoxic response and serum bromide concentrations.

Allis and colleagues ( Allis et al., 2002; Allis and Zhao, 2002; Zhao and Allis, 2002) assessed the ability of various rat and human CYP isoenzymes to metabolize bromodichloromethane and determined kinetic parameters for those showing measurable metabolic activity. Allis and Zhao ( 2002) tested five rat and six human CYP isoenzymes in vitro for metabolism of bromodichloromethane using recombinant systems expressing single isozyme activities. The tested recombinant isoenzymes were rat CYP2E1, CYP2B1/ 2, CYP1A2, CYP2C11, AND CYP3A1 and human CYP2E1, CYP1A2, CYP2A6, CYP2B6, CYP2D6 and CYP3A4. The results of this study indicate that the principal metabolizing enzymes in rat are those identified previously, namely CYP2E1, CYP2B1/ 2, CYP1A2. Results for CYP3A1 suggest that it may have weak metabolic activity, but the level of activity was not sufficient for a quantitative assessment. CYP2C11 was not active. Human CYP2E1, CYP1A2, and CYP3A4 showed substantial metabolic activity toward bromodichloromethane. Human CYP2A6 showed lower, but measurable, levels of activity. CYP2B6 and CYP2D6 were not active. Based on these assays, only CYP2E1 and CYP1A2 metabolize bromodichloromethane in both species. CYP2E1 is the high affinity enzyme in both rats and humans, with Km values approximately 27 fold lower than those for the isoenzymes with the next lowest value ( CYP2B1 in rats, CYP1A2 in humans). The metabolic parameters K m and k cat for rat and human CYP2E1 were similar. In contrast, the metabolic parameters for CYP1A2 were not similar across species. The study authors concluded that the results of this study appear consistent with observations in vivo for the rat ( Allis et al., 2002) and with predictions of the existing PBPK model for bromodichloromethane in the rat ( Lilly et al., 1998).

Zhao and Allis ( 2002) determined kinetic constants for metabolism of bromodichloromethane by CYP2E1, CYP1A2, and CYP3A4 in human liver microsomes. Constants for individual isoenzymes were determined by addition of enzyme specific inhibitory antibodies for two isoenzymes to the microsomal preparations while measuring the activity of the third. Measurements were performed in microsomes obtained from four donors. CYP2E1 was found to have the lowest K m ( 2.9 µ M) and the highest catalytic activity. The K m values for CYP1A2 and CYP3A4 were approximately 20 fold higher ( 60 µ M) and the catalytic activity was lower. Eleven additional human microsomal preparations were characterized for activity of 10 Draft  Do Not Cite or Quote February 20, 2003 III  13 CYP isoenzymes. The initial rate of metabolism in each preparation measured at 9.7 µ M bromodichloromethane was compared to the activity of individual isoenzymes. Statistical analysis showed a significant correlation only with CYP2E1 activity at the tested concentration. The results of this suggest that CYP2E1 would dominate metabolism at the low level of exposure expected from ingestion of drinking water. , study However, the study authors have noted that humans are highly variable in the induction of CYP isoenzymes and that the contributions of the three isoenzymes to metabolism of bromodichloromethane in individuals may not be entirely predictable.

D. Excretion Mink et al. ( 1986) compared the excretion of bromodichloromethane, dibromochloromethane, and bromoform in male Sprague Dawley rats and male B6C3F 1 mice. Animals were given single oral doses of 14C labeled compound in corn oil by gavage at dose levels of 100 mg/ kg and 150 mg/ kg for rats and mice, respectively. The lung was the principal route of excretion in both species, accounting for 45% to 88% of the administered label, either as carbon dioxide or as parent compound. Small amounts of label ( 1.1% to 4.9%) were recovered in urine, but the chemical identity of labeled compounds was not investigated.

Mathews et al. ( 1990) exposed Fischer 344 rats to either a single oral dose of 1, 10, 32, or 100 mg/ kg, or 10 day repeated doses of 10 or 100 mg/ kg day bromodichloromethane dissolved in corn oil. Approximately 70% to 80% of the administered dose was excreted in exhaled air as 14Ccarbon dioxide, with 3% to 5% as 14C carbon monoxide. In general, less than 5% of the dose was excreted in the urine or feces.

E. Bioaccumulation and Retention No data were located regarding the bioaccumulation or retention of brominated trihalomethanes following repeated exposures. However, based on the rapid excretion and metabolism of the brominated trihalomethanes and the low levels of the structurally related compound chloroform detected in human post mortem tissue samples, marked accumulation and retention of these compounds are not anticipated.

F. Summary No data on absorption of brominated trihalomethanes were available for humans. Measurements in mice and rats indicate that gastrointestinal absorption of brominated trihalomethanes is rapid ( peak levels attained less than an hour after administration of a gavage dose) and extensive ( 63% to 93%). Most studies of brominated trihalomethane absorption have used oil based vehicles. A study in rats found that the initial absorption rate of bromodichloromethane was higher when the compound was administered in an aqueous vehicle when compared to administration in a corn oil vehicle.

Data for distribution of brominated trihalomethanes in human organs and tissues are Draft  Do Not Cite or Quote February 20, 2003 III  14 limited. Bromoform was found primarily in the stomach and lungs of a human overdose victim, with lower levels detected in intestine, liver, kidney and brain. Dibromochloromethane was found in 1 of 42 samples of human breast milk collected from women living in urban areas. Radiolabeled brominated trihalomethanes were detected in a variety of tissues following oral dosing in rats and mice. Approximately 1 to 4% of the administered dose was recovered in body tissues when analysis was conducted 8 or 24 hours post treatment. The highest concentrations were detected in stomach, liver, blood, and kidneys when assayed 8 hours after administration of the compounds. Analyses of placentas, amniotic fluid and fetuses from female rats and rabbits administered bromodichloromethane in drinking water indicate that this compound does not accumulate in these tissues or fluids. There are no data which are suggestive of strain specific differences in metabolism.

Brominated trihalomethanes are extensively metabolized by animals. Metabolism of brominated trihalomethanes occurs via two pathways. One pathway predominates in the presence of oxygen ( the oxidative pathway) and the other predominates under conditions of low oxygen tension ( the reductive pathway). In the presence of oxygen, the initial reaction product is trihalomethanol ( CX 3 OH), which spontaneously decomposes to yield the corresponding dihalocarbonyl ( CX 2 O). The dihalocarbonyl species are quite reactive and may form adducts with cellular molecules. When intracellular oxygen levels are low, the trihalomethane is metabolized via the reductive pathway, resulting in a highly reactive dihalomethyl radical (° CHX 2), which may also form covalent adducts with cellular molecules. The metabolism of brominated trihalomethanes and chloroform appear to occur via the same pathways, although in vitro and in vivo data suggest that metabolism via the reductive pathway occurs more readily for brominated trihalomethanes. Both oxidative metabolism and reductive metabolism of trihalomethanes appear to be mediated by cytochrome P450 isoforms. The identity of cytochrome P450 isoforms that metabolize brominated trihalomethanes has been investigated in several studies which used bromodichloromethane as a substrate. The available data suggest that the cytochrome P450 isoforms CYP2E1, CYP2B1/ 2, and CYP1A2 metabolize bromodichloromethane in rats. The human isoforms CYP2E1, CYP1A2, and CYP3A4 show substantial activity toward bromodichloromethane in vitro and low but measurable levels of CYP2A6 activity have also been detected. Based on the available data, CYP2E1 and CYP1A2 are the only isoforms active in both rats and humans. CYP2E1 shows the highest affinity for bromodichloromethane in both species and the metabolic parameters K m and k cat are similar for rat and human CYP2E1. In contrast, the metabolic parameters for CYP1A2 differ in rats and humans. The pattern of results for isozyme activity obtained from an inhalation study of bromodichloromethane was similar to the pattern reported for male F344 rats treated with bromodichloromethane by gavage.

The lung is the principle route of excretion in rats and mice. Studies with 14C labeled compounds indicate that up to 88% of the administered dose can be found in exhaled air as carbon dioxide, carbon monoxide, and parent compound. Excretion in the urine generally appears to be 5% or less of the administered oral dose. Data from one study suggests that fecal excretion is less than 3% of the administered dose. Draft  Do Not Cite or Quote February 20, 2003 III  15 Draft  Do Not Cite or Quote February 20, 2003 IV  1 IV. HUMAN EXPOSURE A. Occurrence in Drinking Water The occurrence of brominated trihalomethanes in U. S. drinking water has been determined in both national scale and localized studies. The occurrence of bromodichloromethane and bromoform has been described in eleven national surveys. Dibromochloromethane occurrence has been described in twelve national surveys. Nine localized studies on the occurrence of brominated trihalomethanes are also described below.

It is important to note that a variety of sampling and preservation techniques are used for collection of occurrence data on brominated trihalomethanes. The addition of chlorine to raw water as a disinfectant at water treatment plants results in the formation of hypochlorous acid in the processed water. The acid in turn reacts with organic materials to produce chloroform and also oxidizes available bromide ions to form hypobromous acid. Hypobromous acid reacts with organic materials in the processed water to form the brominated trihalomethanes. Because these chemical reactions occur over periods of days in treated waters, the method used to sample drinking waters can affect the measured concentrations of trihalomethanes in the water. Therefore, appropriate sampling and preservation methods must be selected to ensure that the analytical data are representative of the desired endpoint. For example, if an investigator wants to know the concentration of trihalomethanes in the water at the time of sampling, a reducing agent is added to the sample containers to " quench" or prevent further formation of trihalomethanes. If an investigator wants to know the maximum amount of trihalomethanes that could occur, no quenching is used and the reactions are allowed to run to completion at room temperature. If a concentration similar to that at a household tap is desired ( i. e., after the water spends several days in the distribution system, the samples generally are not quenched but are refrigerated to slow the reactions ( Wallace, 1997). Information on sample handling has been included in the discussion of individual studies when available in the materials reviewed for this document.

Spatial and temporal variability exist in the occurrence data reported for brominated trihalomethanes. Multiple factors contribute to this variability. With respect to spatial variability, the geographical distribution of bromide ion in soil is not uniform ( Shacklette and Boerngen, 1984). Brominated byproducts may predominate or comprise a substantial proportion of the disinfection byproduct profile in regions with high soil concentrations. Brominated trihalomethanes may continue to form within water distribution systems due to the action of free residual chlorine on remaining humic precursors, resulting in substantial intra system spatial variability ( Chen and Weisel, 1999). Temporal variability may result from seasonal variation in the concentration of brominated trihalomethanes as a result of seasonal fluctuations in precursor material ( Brett et al., 1979). Short term variability may be introduced by changes in the demand cycle to individual homes or neighborhoods. Draft  Do Not Cite or Quote February 20, 2003 IV  2 1. National Surveys The National Organics Reconnaissance Survey ( NORS), conducted by U. S. EPA, collected drinking water samples from 80 cities nationwide in 1975. The survey sampled for several organics, including brominated trihalomethanes, at the water treatment facilities. The sampling method employed was refrigeration without quenching; therefore, brominated trihalomethane concentrations may have increased following collection. Bromodichloromethane was found in 98% of the systems sampled. The median concentration was 8 µ g/ L ( ppb), and the maximum level was 116 µ g/ L ( ppb). Dibromochloromethane was found in 90% of the systems sampled at a median concentration of 2 µ g/ L ( ppb). The detection limit for dibromochloromethane and bromodichloromethane was 0.1 µ g/ L ( ppb). The median concentration for bromoform was below the detection limit of approximately 5 µ g/ L ( ppb) ( Symons et al,. 1975). NORS was performed prior to the promulgation of the total trihalomethane regulation; therefore, these results may be higher than current levels.

The National Organics Monitoring Survey ( NOMS) was conducted by the EPA from March 1976 to January 1977 ( Wallace, 1997). In NOMS, 113 community water supplies were sampled. Surface water was the major source for 92 of the systems, and ground water was the major source for the remaining 21 systems. The NOMS used three sample storage methods. During Phase 1, all samples were refrigerated. In Phase 2, the samples were allowed to stand at 20 to 25