United States Office of Science and EPA-822-R-05-011
Environmental Technology November 15,2005
Protection Agency Washington, D.C.
EPA Office of Water
DRINKING WATER CRITERIA DOCUMENT
FOR BROMINATED TRIHALOMETHANES
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-02-206
Work assignment 3-16
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
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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 January, 2005; 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.
Ephraim King
Director, Office of Science
and Technology
Office of Water
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Acknowledgments
This document is derived and updated/expanded of the Draft for the Drinking Water Criteria
Document on Trihalomethanes (U.S. EPA, 1994), Summary of New Health Effects Data on
Drinking Water Disinfectants and Disinfection Byproducts (D/DBPs) for the Notice of
Availability (NODA) (U.S.EPA, 1997), and Draft Drinking Water Criteria Document on
Brominated Trihalomethanes (U.S. EPA, 2003). This document includes and evaluation of
literature on brominated trihalomethanes resulting from full literature searches conducted up to
January 2005 for toxicity data. In addition, few newer studies identified after the literature
search date have been included as available at the time of document preparation.
Contracting Officer
Renita Tyus
Cincinnati, OH, USEPA
Project Officer
Jane Holtorf
Office of Water, USEPA
Work Assignment Manager
Nancy Chiu
Office of Science and Technology, USEPA
Authors
Lori Moilinen, Ph.D., DABT
Syracuse Research Corporation
Nancy Chiu, Ph.D.
Office of Science and Technology, USEPA
Julie Stickney, Ph.D., DABT
Syracuse Research Corporation
Other Contractor Technical Support
Frank Letkiewicz, B.S.
The Cadmus Group, Inc.
Karen Ferrante, M.S.
The Cadmus Group Inc.
MadhulikaNataraja, M.S.E
The Cadmus Group Inc.
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Reviewers
This document has been peer reviewed by both EPA scientists and by scientists external to EPA.
Internal EPA Reviewers
Bob Bellies, Ph.D.
National Center for Environmental Assessment, Office of Research & Development, USEPA
Jennifer Jinot, Ph.D.
National Center for Environmental Assessment, Office of Research & Development, USEPA
Bob Sonawane, Ph.D.
National Center for Environmental Assessment, Office of Research & Development, USEPA
External Peer Reviewers
Hudy Buelke-Sam, M.A.
Toxicology Services, Greenfield, Indiana
Annette Loch Bunge, Ph.D.
Professor, Department of Chemical Engineering, Colorado School of Mines, Golden, Colorado
JohnReif, M.Sc., D.V.M.
Department Head, Department of Environmental Health, College of Veterinary Medicine and
Biomedical Sciences, Colorado State University, Fort Collins, Colorado
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TABLE OF CONTENTS
External Peer Reviewers 4
I. EXECUTIVE SUMMARY I - 1
II. PHYSICAL AND CHEMICAL PROPERTIES II - 1
A. Properties II - 1
B. Summary II - 2
III. TOXICOKINETICS Ill - 1
A. Absorption Ill - 1
B. Distribution Ill - 3
C. Metabolism Ill - 5
D. Excretion Ill - 15
E. Bioaccumulation and Retention Ill - 15
F. Summary Ill - 15
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-l
1. Bromodichloromethane V-l
2. Dibromochloromethane V-6
3. Bromoform V - 7
B. Short-Term Exposures V - 8
1. Bromodichloromethane V - 14
2. Dibromochloromethane V - 22
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3. Bromoform V - 25
C. Subchronic Exposure V - 28
1. Bromodichloromethane V - 31
2. Dibromochloromethane V - 33
3. Bromoform V - 34
D. Chronic Exposure V - 35
1. Bromodichloromethane V - 37
2. Dibromochloromethane V - 41
3. Bromoform V - 42
E. Reproductive and Developmental Effects V - 43
1. Bromodichloromethane V - 43
2. Dibromochloromethane V - 59
3. Bromoform V - 62
F. Mutagenicity and Genotoxicity V - 70
1. Bromodichloromethane V - 70
2. Dibromochloromethane V - 78
3. Bromoform V - 84
G. Carcinogenicity V - 90
1. Bromodichloromethane V - 90
2. Dibromochloromethane V - 101
3. Bromoform V - 104
H. Other Key Health Effects V - 108
1. Immunotoxicity V - 108
2. Hormonal Disruption V - 110
3. Structure-Activity Relationships V - 117
I. Summary V- 118
1. Health Effects of Acute and Short Term Exposure of Animals ... V - 118
2. Health Effects of Longer-term Exposure of Animals V - 118
3. Reproductive and Developmental Effects V-119
4. Mutagenicity and Genotoxicity V - 120
5. Carcinogenicity and Related Studies in Animals V - 120
6. Other Key effects V - 121
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 - 12
2. Dibromochloromethane VI - 27
3. Bromoform VI - 30
C. High Risk Populations VI - 31
D. Summary VI - 32
ii
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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 - 14
E. Summary VII - 16
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII - 1
A. Bromodichloromethane VIII - 1
1. Noncarcinogenic effects VIII - 1
2. Carcinogenic Effects VIII - 27
B. Dibromochloromethane VIII - 34
1. Noncarcinogenic effects VIII - 34
2. Carcinogenic Effects VIII - 48
C. Bromoform VIII - 55
1. Noncarcinogenic effects VIII - 55
2. Carcinogenic Effects VIII - 69
D. Summary VIII - 75
IX. REFERENCES IX - 1
APPENDIX A A - 1
APPENDIX B (Electronic Format) B - 1
APPENDIX C C - 1
in
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LIST OF TABLES
Table 1-1 Summary of Quantification of Toxicological Effects for Brominated
Trihalomethanes
I- 16
Table II-1 Physical and Chemical Properties of the Brominated Trihalomethanes .... II - 1
Table III-l Recovery of Label 8 Hours after Oral Administration of 14C-Labeled
Brominated Trihalomethanes to Male Sprague-Dawley Rats or Male
B6C3FJ Mice III-l
Table III-2 Cumulative Excretion of Label after Oral Administration of 14C-Labeled
Bromodichloromethane to Male F344 Rats Ill - 2
Table III-3 Over view of Tissue Collection for Analysis of Bromodichloromethane in
Sprague-Dawley Rat Tissues and Fluids (CCC, 2000c) Ill - 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 (jig/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
IV
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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-l Summary of LD50 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 - 29
Table V-5 Summary of Chronic Toxicity Studies for Brominated Trihalomethanes . . V - 36
Table V-6 NTP (1998) Study Design V - 47
Table V-7 Summary of Experiments Conducted by Bielmeier et al. (2001) V - 49
Table V-8 Mean Consumed Doses (mg/kg-day) of Bromodichloromethane in the
Range Finding Study Conducted by CCC (2000c) V - 52
Table V-9 Summary of Reproductive Studies of Brominated Trihalomethanes V - 65
Table V-10 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation
Data for Bromodichloromethane V - 76
Table V-l 1 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation
Data for Dibromochloromethane V - 83
Table V-12 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation
Data for Bromoform V - 89
Table V-13 Tumor Frequencies in F344/N Rats and B6C3Fj Mice Exposed to
Bromodichloromethane in Corn Oil for 2 Years - Adapted from
NTP (1987)
Table V-14 Hepatic and Renal Tumors in Male F344/N Rats Administered
Bromodichloromethane in the Drinking Water for Two Years
(George et al., 2002)
V-92
V-95
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Table V-15 Frequencies of Liver Tumors in B6C3FJ Mice Administered
Dibromochloromethane in Corn Oil for 105 Weeks V - 102
Table V-16 Tumor Frequencies in the Large Intestine of F344/N Rats Exposed to
Bromoform in Corn Oil for 2 Years V - 105
Table V-17 Summary of Hormone Profile Experiments (Bielmeier et al., 2001) .... V - 111
Table V-18 Summary of Bielmeier et al. (2004) Study in Female F344 Rats V - 113
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, Altered Menstrual Function, or
Sperm Quality VI - 4
Table VI-3 Means and Adjusted Differences in Menstrual Cycle and Follicular Phase
Length by Quartile of Individual and Summed Brominated
Trihalomethanes VI - 22
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 - 35
VI
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Table VIII-9 Summary of Candidate Studies for Derivation of the Ten-day HA for
Dibromochloromethane VIII - 36
Table VIII-10 Summary of Candidate Studies for Derivation of the Longer-term HA for
Dibromochloromethane VIII - 40
Table VIII-11 Summary of Candidate Studies for Derivation of the RfD for
Dibromochloromethane VIII - 44
Table VIII-12 Results of Preliminary HMD Modeling of Selected Data from NTP (1985)
Studies VIII - 46
Table VIII-13 Frequencies of Liver Tumors in Mice Administered
Dibromochloromethane in Corn Oil for 105 Weeks VIII - 52
Table VIII-14 Carcinogenic Risk Estimates for Dibromochloromethane 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 - 61
Table VIII-17 Summary of Candidate Studies for Derivation of the RfD for
Bromoform VIII - 65
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-l 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 - 42
Table A-6 Benchmark Dose Modeling Results for Dibromochloromethane A - 50
vii
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Table A-7 Benchmark Dose Modeling Results for Bromoform A - 56
Table C-l 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
Figure III-l Proposed Oxidative and Reductive Metabolic Pathways for Brominated
Trihalomethanes Ill - 6
Figure III-2 Proposed GSTT1-1-Catalyzed Glutathione Conjugation of
Bromodichloromethane Ill - 9
Vlll
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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 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 or their metabolites 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 |ig/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 at least 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 (CX3OH), which spontaneously decomposes to yield the corresponding
dihalocarbonyl (CX2O). The dihalocarbonyl species are reactive and may form adducts with
I -1 November 15, 2005
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cellular molecules. When intracellular oxygen levels are low, the trihalomethane is metabolized
via the reductive pathway, resulting in a highly reactive dihalomethyl radical (»CHX2), 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 Km and kcat 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.
Recent studies suggest that metabolism of brominated trihalomethanes may occur via a
glutathione-^-transferase (GST) theta-mediated pathway. Based on the existing data, the related
trihalomethane chloroform is not metabolized to any significant extent via the GST theta
pathway. These data suggest that common pathways of metabolism (and mode of action for
health effects) cannot be assumed for chloroform and the brominated trihalomethanes.
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.
I - 2 November 15, 2005
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When all available national survey data are considered, bromodichloromethane concen-
trations in drinking water range from below the detection limit to 183 |ig/L (ppb), while
dibromochloromethane and bromoform concentrations range from below the detection limit to
280 i-ig/L (ppb). When data for the three brominated trihalomethanes are compared, the
frequency of 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 was 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 |ig/L, respectively, for
bromodichloromethane (range of individual values 0-65.8 i-ig/L); 2.4, 4.72, and 13.2 |ig/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 lag/person/day, respectively. The
same values for populations exposed to bromodichloromethane from ground water systems are
I - 3 November 15, 2005
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lower - 3.6, 8.1, and 22 lag/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 lag/person/day, respectively. The corresponding values for populations exposed
to dibromochloromethane from groundwater system are lower, with estimates of 2.7, 6.2, and 18
lag/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 lag/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 lag/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 |ig/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
[ig/day. Similarly, median exposure to dibromochloromethane may range from 4.2 to 7.8 |ig/day,
and for bromoform, median exposure may range from 4.8 to 8.4 |ig/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 to 14 |ig/L (ppb), exposure to bromodichloromethane from surface water is
estimated to be between 12 and 28 jig/day. Similarly, based on the range of medians reported
for dibromochloromethane concentrations, the median exposure is estimated to be up to
6 jig/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 to 1 i-ig/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 to 1 |ig/L (ppb),
the exposure to bromoform is estimated to range from 1 to 2 [ig/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 jig/person/day. The upper
90th percentile estimates range from <2.0 to 90 jig/person/day. Estimates of the average
population intake of dibromochloromethane from drinking water range from 0.2 to 56
|ig/person/day. The upper 90th percentile estimates range from < 0.9 to 86 jig/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 jig/person/day.
The upper 90th percentile estimates range from 2.4 to 26 jig/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
I - 4 November 15, 2005
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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
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 non-beverage 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 |ig/m3) for bromodichloromethane, 3.8 ppt (0.032 |ig/m3) for
dibromochloromethane, and 3.6 ppt (0.037 |ig/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 |ig/m3) for bromodichloromethane; 290 ppt (2.5 |ig/m3) for
dibromochloromethane; 310 ppt (3.2 |ig/m3) for bromoform. Bromodichloromethane was
detected in 64% (n=l 1) and 17% (n=6) of personal air samples collected in Texas and North
Carolina. The detected concentrations ranged from 0.12 to 4.36 |ig/m3 (0.017 to 0.65 ppb).
Dibromochloromethane was not detected.
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Mean concentrations in indoor air ranged from 0.38 to 0.75 |ig/m3 for bromodichloro-
methane; 0.44 to 0.53 |ig/m3 for dibromochloromethane, and 0.29 to 0.35 |ig/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.
Bromoform and dibromochloromethane have been identified in soil and sediment
samples collected at NPL hazardous waste sites. Soils and other unconsolidated surficial
materials may become contaminated with bromoform and dibromochloromethane by chemical
spills, the landfilling of halomethane-containing solid wastes, or the discharge of chlorinated
water. However, no data were located to suggest that land releases are a significant source of the
chemicals in the environment (ATSDR, 2003).
The chemical and physical properties of the brominated trihalomethanes indicate that
they should volatilize readily from wet or dry soil surfaces. Bromoform and
dibromochloromethane have only a minor tendency to be adsorbed by soils and sediments and
will tend to be highly mobile. Therefore, ingestion of soil is not expected to be a significant
route of exposure (ATSDR, 2003).
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 use 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.
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The use of chemicals containing chlorine and bromine 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 |ig/L (ppb),
from 0.1 to 48 |ig/L (ppb), and from less than 0.1 to 62 |ig/L (ppb), respectively. Concentrations
of the same brominated trihalomethanes in the air two meters above the pool water ranged from
less than 0.1 to 14 |ig/m3 (0.015 to 2.09 ppb), from less than 0.1 to 10 |ig/m3 (0.011 to 1.2 ppb),
and from less than 0.1 to 5.0 jig/m3 (0.0097 to 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 LD50 values range from 450 to 969 mg/kg for bromodichloromethane, 800 to 1200 mg/kg
for dibromochloromethane, and 1388 to 1550 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 1000 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
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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.
Health Effects of Subchronic and Chronic Exposure of Animals
The predominant effects of subchronic oral exposure to brominated trihalomethanes
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/kg-day 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 were 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. 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 dibromo-
chloromethane 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/kg-day 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 21 and 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.
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Additional studies in F344 rats that varied the timing of bromodichloromethane administration
indicate that gestation days 6-10 are a critical period for induction of full litter resorption.
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 Ft male and female
rats at 150 ppm (approximately 11.6 to 40.2 mg/kg-day) in a two generation drinking water
study of bromodichloromethane. In the same study, small but statistically significant delays in
sexual maturation occurred in Fx males at 50 ppm (approximately 11.6 to 40.2 mg/kg-day) and
Fj 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.
Two studies on the reproductive or developmental toxicity of dibromochloro-methane
gave negative results when tested at doses of up to 200 mg/kg-day. In another separate 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 Fx 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 in decreased postnatal survival, organ weight changes, and liver histopathology
in Fj 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 that
contain rat theta-class glutathione S-transferase suggest that mutagenicity of the brominated
trihalomethanes may also be mediated by glutathione conjugation.
Carcinogenicity and Related Studies in Animals
The carcinogenic potential of individual brominated trihalomethanes administered in oil
has been investigated in chronic oral exposure studies in mice and rats. Ingestion of
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bromodichloromethane caused 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. Ingestion of
dibromochloromethane caused liver tumors in male and female mice, and ingestion of
bromoform caused intestinal tumors in male and female rats.
Studies of induction of aberrant crypt foci (ACF) show that bromodichloromethane,
dibromochloromethane, and bromoform given in drinking water significantly increase the
number and focal area of ACF in the colons of male F344 rats, Eker rats, and strain A/J mice, but
not in colons of B6C3FJ mice. The biological significance of this induction is unclear, as
intestinal tumors have not been observed either in the colons of F344 rats treated with
dibromochloromethane by corn oil gavage or in the colons of rats exposed to
bromodichloromethane in the drinking water for two years. Administration of individual
brominated trihalomethanes in a high animal fat diet did not significantly increase the number of
ACF when compared to a diet containing normal levels of fat.
Exposure of male and female rats Eker rats (a rodent hereditary model of renal cancer) to
bromodichloromethane did not significantly increase the incidence of urinary bladder epithelial
hyperplasia, individual cell hypertrophy, renal tumors, hemangioma of the spleen, or
leiomyomas or mesenchymal cell proliferation in the uterus of females.
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 liters, and/or suppression of Con A-stimulated proliferation
of spleen cells at doses of 125 to 250 mg/kg-day.
No studies have been reported for hormonal effects following exposure to
dibromochloromethane or bromoform. There is evidence from studies in F344 rats and cultured
human placental trophoblasts that bromodichloromethane causes hormonal disruption. Rats
exposed to bromodichloromethane on gestation days 8 or 9 show reduced serum levels of LH
and progesterone. Serum LH reductions indicate that the mode of action for this strain-specific
effect involves altered LH secretion; however, a contributing effect on LH signal transduction
has not been ruled out.
Exposure to bromodichloromethane alters the function of human placental trophoblasts,
as shown by reduced CG secretion and by changes in morphological differentiation. The mode
of action for the observed effects is unknown. Possible mechanisms proposed by the study
authors for effects on CG secretion include disruption of CG synthesis at the translational or
post-translational level (e.g., by altering glycosylation of CG subunits or disruption of
dimerization) or indirect effects on secretion via disruption of gonadotropin releasing hormone
activity. The significance of these findings for human health is that placental trophoblasts are
the sole source of CG during normal human pregnancy and play a major role in the maintenance
of the conceptus. If the observed effect on CG secretion is substantiated in future studies, it may
help to explain apparent adverse pregnancy outcomes associated with consumption of
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chlorinated drinking water in some epidemiological studies (e.g., increased incidence of
spontaneous abortion as reported by Waller et al., 1998).
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/kg-day 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 incidence. Very few studies have examined the association
between cancer and exposure to brominated trihalomethanes, and possible increased cancer
incidence in bladder was suggested. Recent 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 reported for exposure to bromodichloromethane (or a closely associated
compound) and a moderately increased risk of spontaneous abortion during the first trimester.
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, 2) reduction in birth
weight (small for gestational age), and 3) increased risk of neural tube defects in women exposed
to >20 |ig/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. A study of semen quality in healthy men found an association
between increased exposure to bromodichloromethane in residential tap water and decreased
sperm linearity; exposure to dibromochloromethane or bromoform was not associated with
decrements in semen quality.
To directly conclude that bromodichloromethane and dibromochloromethane are
developmental or reproductive toxicants in humans can be complicated by the fact that there are
many disinfection byproducts in chlorinated water. 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.
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Susceptible Populations
There is currently no clear evidence that children or the fetus are at greater risk for
adverse effects from exposure to bromoform or dibromochloromethane than are adults.
Associations between concentration of bromodichloromethane (or a co-occurring chemical) and
spontaneous abortion or still birth have been observed in several epidemiological studies.
Evidence in rats indicates that exposure to bromodichloromethane causes full litter resorption in
F344 rats but not Sprague-Dawley rats. 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 that infants, children,
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.
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
structurally-related 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 also 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
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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. The role of cytotoxicity and associated
regenerative cell proliferation in tumorigenicity of brominated trihalomethanes is presently
unclear. Comparison of dose-response data for liver and kidney toxicity (including cell
proliferation) and tumorigenicity in mice and rats suggests that tumor formation occurs at
concentrations lower than those which stimulate cell proliferation.
Interaction with agents which increase or decrease the activity of enzymes responsible
for 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% extra risk. The BMDL10 was defined as the 95% lower
bond on the BMD estimate. For bromodichloromethane, a BMDL10 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 BMDL10 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
BMDL10 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
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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 |ig/L employed a duration adjusted BMDL10 of
0.8 mg/kg-day 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 BMDL10 of 5.5 mg/kg-day for hepatic cell
vacuolization, identified in a 28-day feeding study in rats. A duration-adjusted BMDL10 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 BMDL10 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 |ag/L. The Lifetime HA for dibromochloromethane is 60 |ig/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 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 BMDL10 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 BMDL10 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 BMDL10 value of 2.6 mg/kg-
day was also used to calculate an RfD of 0.03 mg/kg-day and a DWEL of 1000 |ig/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 bromodi-
chloromethane, a unit risk of 1.0 x 10"6 (iig/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 |ig/L, respectively. For dibromochloromethane, a unit risk of
1.2 x 10"6 ([ig/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 x 10"2 (mg/kg-day)"1 and 0.8 |ig/L,
respectively. For bromoform, a unit risk of 1.3 x 10"7 ([ig/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 4.6xlO"3 (mg/kg-day)"1 and 8 |ig/L, respectively. These values were calculated using
an animal-to-human scaling factor of body weight374 in accordance with proposed U.S. EPA
guidance (U.S. EPA, 1992b; 1999).
I - 14 November 15, 2005
-------
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, bromodichlorom ethane 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.
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 1-1 summarizes the quantification of noncarcinogenic and carcinogenic effects for
brominated trihalomethanes.
I - 15 November 15, 2005
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Table 1-1 Summary of Quantification of Toxicological Effects for Brominated
Trihalomethanes
Advisory
Value
Reference
Bromodichloromethane
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
RfD
DWEL
Lifetime HA
Oral Slope Factor c
Concentration for excess cancer risk (10"6)
Unit Risk
1 mg/L
0.6 mg/L
0.6 mg/L
2 mg/L
0.003 mg/kg-day
100 ng/L
Not applicable
3. 5 xlO'2 (mg/kg-day)-1
l.Ong/L
IxlO'6 (i-ig/L)-1
Narotsky et al. (1997)
NTP(1998)
CCC (2000d)
CCC (2000d)
Aidaetal. (1992b)
Aidaetal. (1992b)
-
NTP(1987)
NTP(1987)
NTP(1987)
Dibromochloromethane
One-day HA for 10-kg child"
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
RfD
DWEL
Lifetime HA
Oral Slope Factor c
Concentration for Excess cancer risk (10"6)
Unit Risk
0.6 mg/L
0.6 mg/L
0.2 mg/L
0.6 mg/L
0.02 mg/kg-day
700 ng/L
60 ng/L
4.3 xlO'2 (mg/kg-day)-1
0.8 ng/L
LlxlO^Gig/L)-1
Aidaetal. (1992a)
Aidaetal. (1992a)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
Bromoform
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child a
Longer-term HA for 70-kg adult
RfD
DWEL
5 mg/L
0.2 mg/L
0.2 mg/L
0.9 mg/L
0.03 mg/kg-day
1000 ng/L
Burton-Fanning (1901)
NTP(1989a)
NTP(1989a)
NTP(1989a)
NTP(1989a)
NTP(1989a)
1-16
November 15, 2005
-------
Table 1-1 (cont.)
Advisory
Lifetime HA
Oral Slope Factor c
Concentration for Excess cancer risk (10"6)
Unit Risk
Value
Not applicable
4.6X10'3 (mg/kg-day)'1
S^g/L
l.SxlQ-'djg/L)-1
Reference
--
NTP(1989a)
NTP(1989a)
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.
0 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
1-17
November 15, 2005
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II.
PHYSICAL AND CHEMICAL PROPERTIES
A.
Properties and Uses
Bromodichloromethane (CHBrCl2), dibromochloromethane (CHBr2Cl) and bromoform
(CHBr3) 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-l Physical and Chemical Properties of the Brominated Trihalomethanes
Property
Structure
Chemical Abstracts
Registry Number
Registry of Toxic Effects of
Chemical Substances
Number
Synonyms
Chemical Formula
Molecular Weight
Boiling Point
Melting Point
Specific Gravity (20°)
Vapor Pressure
Stability in Water
Water Solubility
Log OctanoLWater Partition
Coefficient (Kow)
Bromodichloromethane
H
Cl C Cl
Br
75-27-4
PA 53 10000
dichlorobromomethane
CHBrCl2
163.83
90°C
-57.TC
1.980
50mm (20°C)
volatile
3,032 mg/L
(30°C)
2.09
Chemical
Dibromochloromethane
H
L. Bi
Br
124-48-1
PA 6360000
chlorodibromomethane
CHBr2Cl
208.29
116C
~
2.38
15mm(10°C)
volatile
1,050 mg/L
(30°C)
2.23
Bromoform
H
Bi C Bi
Br
75-25-2
PB 5600000
tribromomethane
CHBr3
252.77
149 - 150°C
6-7°C
2.887
5.6mm(25°C)
volatile
3,190mg/L(30°C)
2.37
Source: U.S. EPA (1992c; 1994b)
II-l
November 15, 2005
-------
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
(HOC13) to form hypobromous acid (HOBr) in the following reaction:
Br + HOC13 -> 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
by-products 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.
II - 2 November 15, 2005
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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 B6C3FJ 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-l), 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, carbon dioxide, or carbon monoxide. 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-l Recovery of Label 8 Hours after Oral Administration of 14C-Labeled
Brominated Trihalomethanes to Male Sprague-Dawley Rats or Male B6C3Ft Mice
Chemical
Bromodichloromethane
Dibromochloromethane
Bromoform
Percent of Label
Species
Rat
Mouse
Rat
Mouse
Rat
Mouse
Expired
CO2
14.2
81.2
18.2
71.6
4.3
39.7
Expired
Parent
41.7
7.2
48.1
12.3
66.9
5.7
Urine
1.4
2.2
1.1
1.9
2.2
4.6
Organs
3.3
3.2
1.4
5.0
2.1
12.2
Total
Recovery
62.7
92.7
70.3
91.6
78.9
62.2
Adapted from Mink et al. (1986) and U.S. EPA (1994b).
III-l
November 15, 2005
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Table III-2 Cumulative Excretion of Label after Oral Administration of 14C-Labeled
Bromodichloromethane to Male F344 Rats
Dose
1 mg/kg
10 mg/kg
100
mg/kg
Time
(hrs post-
treatment)
1
4
8
16
24
1
4
8
16
24
1
4
8
16
24
Percent of Dose
Expired
CO2
9.5±1.1
37.0±3.2
62.9±2.2
76.4±3.2
77.5±3.3
8.0±2.0
39.9±3.2
66.0±4.0
81.3±1.7
82.1±1.8
1.9±0.9
5.5±1.8
NR
33.4±7.4
71.0±1.7
Expired
CO
NRa
1.5±0.7
2.7±1.1
NR
3.3±1.5
NR
1.9±0.4
3.4±0.9
NR
4.3±1.0
0.1±0
0.3±0.1
NR
2.3±0.7
5.2±0.3
Expired
Volatiles
2.1±1.5
2.7±1.8
NR
NR
3.0±1.6
2.0±0.8
2.7±1.1
NR
NR
2.8±1.1
1.5±1.2
4.2±1.9
NR
5.7±2.1
6.3±2.1
Urine
NR
NR
NR
NR
4.1±0.2
NR
NR
NR
NR
4.3±0.2
NR
NR
0.6±0.4
NR
4.1±0.2
Feces
NR
NR
NR
NR
2.7±1.5
NR
NR
NR
NR
0.7±0.2
NR
NR
NR
NR
0.7±0.3
Total
Recovery
11.6±1.3
41.1±2.8
68.±1.7
81.8±2.9
90.7±1.8
10.0±1.85
44.5±3.0
72.1±3.9
87.4±1.5
94.2±1.6
4.6±1.8
10.0±2.9
10.6±3.0
42.0±8.3
87.3±1.6
Adapted from Mathews et al. (1990) and U.S. EPA (1994b).
aNot reported
III-2
November 15, 2005
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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 B6C3FJ 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
III - 3 November 15, 2005
-------
retention of only 0.9% to 1.1% of the administered label, and had no effect on the tissue
distribution of bromodichloromethane.
The Chlorine Chemistry Council (CCC, 2000c) sponsored a study which analyzed the
levels of bromodichloromethane in parental tissues and fluids and Fx 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 |ig). Bromodichloromethane was detected at a
concentration of 0.38 |ig/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
P
P
P
P
P
P
F,
Sex
M,F
M, F
F
F
F
F
M,F
Physiological
state
Pre-mating
Pre-mating
Pregnant
Pregnant
Lactating
Lactating
Weaning
Tissue
Plasma
Plasma
Plasma
Placenta
amniotic fluid,
and fetuses
Plasma
Milk
Plasma
Day of
collection
Day 1 of
exposure
Day 14 of
exposure
GD20
GD21
LD15
LD15
LD29
No. of Samples;
Collection freq.
3 rats/sex/group;
3 times/day
3 rats/sex/group;
3 times/day
3 rats/group;
3 times/day
3 litters/day
3 rats/group
3 rats/group
3 pups/sex;
3 litters;
3 times/day
Comments
-
-
Rats
continuously
exposed since
study day 1
Tissues pooled
by litter
3 times/day
1 IU oxytocin
admin, by IV
approx. 5 min.
before milking
"
Modified from CCC (2000c)
Abbreviations: P, parental; M, male; F, female; GD, gestation day; LD, lactation day
III-4
November 15, 2005
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The Chlorine Chemistry Council (CCC, 2000a) sponsored a study which analyzed the
levels of bromodichloromethane in parental tissues and fluids and Ft 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. Bromodichloro-
methane was detected at concentrations of 0.15 and 0.17 |ig/g (limit of detection 0.11 i-ig/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-l 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 (CX3OH), which spontaneously
decomposes to yield a reactive dihalocarbonyl (CX2O) such as phosgene (CC12O).
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 (»CHX2).
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 are described in U.S. EPA (1994b).
Ill - 5 November 15, 2005
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Figure III-l Proposed Oxidative and Reductive Metabolic Pathways for Brominated Trihalomethanes
RH
Reductive Pathway Oxidative Pathway
\S
P-450 P-450
CHX2 •* /- -\ Cl IX3 s' ^ -^ ^- CX3OH ^^r oX2O ^•
X" NADPH 2NADPH O2 H2O HX
or
NADH
^^~
2GSH
I
X^ ^
2HX
H2O
2HX
Cysteine
x^_ ^^
RCX2OH
CO+GSSG
CO,
OTZ
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)
2HX
III-6
November 15, 2005
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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 cytochrome P450 isoforms involved in oxidative metabolism of brominated
trihalomethanes do not participate in the reductive pathway.
Ill - 7 November 15, 2005
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Studies conducted in Salmonella typhimurium strains that express the rat GST theta gene
(GSTT1-1) (Pegram et al., 1997, DeMarini et al., 1997; Landi et al., 1999b) and using liver
cytosols isolated from rats, mice, and humans (Ross and Pegram, 2003) provide evidence for a
third pathway of metabolism via GST theta-mediated conjugation of bromodichloromethane
with glutathione (GSH). Rat GSTT1-1 is closely related to human theta-class GSTs, suggesting
that humans are likely to have the capability to conjugate brominated trihalomethanes via this
pathway if it is active in vivo (Meyer et al., 1991).
Ross and Pegram (2003) studied the characteristics of bromodichloromethane
conjugation with GSH in liver cytosols from mice, rats, and humans. Conjugation of
bromodichloromethane with GSH in mouse liver cytosol was time- and protein-dependent, was
not affected by an inhibitor of alpha-, mu-, and pi-class GSTs, and correlated with activity
toward a GSTT1-1-specific substrate (l,2-epoxy-3-(4'-nitrophenoxy)propane). Conjugation
activity toward bromodichloromethane in hepatic cytosols of different species followed the rank
order mouse, followed by rat, then human. The initial conjugate formed was S-chloromethyl-
GSH (GSCHC12). This compound was unstable and degraded to multiple metabolites including
S-hydroxymethyl-GSH (GSCH2OH), S-formyl-GSH, and formic acid (HCOOH). These data
indicate that GSTTl-1-mediated glutathione conjugation of bromodichloromethane occurs in
mammalian liver cytosol, via reactions consistent with the metabolic scheme presented in
Figure III-2.
Ill - 8 November 15, 2005
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Figure III-2 Proposed GSTTl-1-Catalyzed Glutathione Conjugation of Bromodichloromethane"
DMA damage
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aSolid arrows represent pathways that are supported by direct experimental evidence. Dashed arrows indicate pathways that are inferred from the experimental
evidence and literature precedents. Proposed scheme is from Ross and Pegram (2003).
Ill-9
November 15, 2005
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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-525 A, metyrapone, and carbon monoxide. These data were interpreted to
indicate that free-radical formation depended on reductive metabolism of the trihalomethanes
mediated by the 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 »CHC12. 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 (»CHX2) and a
halide ion (X").
Ill -10 November 15, 2005
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As noted in IIIC1, recent evidence from studies in Salmonella typhimurium strains that
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-
transferase-mediated conjugation pathways (Pegram et al., 1997; DeMarini et al., 1997; Landi et
al., 1999b). Details of these studies are presented in Section V.F.
3. In Vivo Studies
Mink et al. (1986) compared the metabolic products of bromodichloromethane,
dibromochloromethane, and bromoform in male Sprague-Dawley rats and male B6C3FJ 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-l),
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 rate-
limiting 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, bromodichloro-
methane, 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.
Ill-11 November 15, 2005
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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 14CO2 and 3% to 5% of the dose as 14CO. However, 14CO2
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 14CO2 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 dibromochloro-
methane/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. COFIb was also elevated in a dose-
dependent 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
mmol dibromochloromethane/kg and diethyldithiocarbamate (a potent inhibitor of P450 isoform
CYP2E1) partially inhibited the production of bromide and COHb. In contrast, pretreatment
III -12 November 15, 2005
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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: />-nitrophenol hydrolase (PNP), an
indicator of CYP2E1 activity; pentoxyresorufm-0-dealkylase (PROD), an indicator of
CYP2B1/2 activity; ethoxsyresorufm-O- dealkylase (EROD), an indicator of CYP1A1 activity;
and methoxyresorufm-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.
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
III -13 November 15, 2005
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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 Km and &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 Km (2.9 |j,M) and the highest catalytic activity. The Km values for
CYP1A2 and CYP3A4 were approximately 20-fold higher (60 |j,M) and the catalytic activity was
lower. Eleven additional human microsomal preparations were characterized for activity of 10
CYP isoenzymes. The initial rate of metabolism in each preparation measured at 9.7 |j,M
bromodichloromethane was compared to the activity of individual isoenzymes. Statistical
analysis showed a significant correlation only with CYP2E1 activity at the tested concentration.
At the low concentrations expected from drinking water exposure, the results of this study
III -14 November 15, 2005
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suggest that CYP2E1 would dominate metabolism. 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 B6C3FJ 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
14C-carbon 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
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
III -15 November 15, 2005
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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 at least 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 (CX3OH), which spontaneously decomposes to yield the corresponding
dihalocarbonyl (CX2O). 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
(•CHX2), 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 Km and kcat 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.
Recent evidence suggests that brominated trihalomethanes are also metabolized by a
glutathione-S-transferase theta (GSTT)-mediated pathway. The available evidence indicates that
this pathway has a low affinity for chloroform, suggesting that the brominated trihalomethanes
could cause health effects by a different mode of action than those caused by chloroform.
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.
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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, 1998). 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.
IV -1 November 15, 2005
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1. National Surveys
The National Organics Reconnaissance Survey (NORS), conducted by U.S. EPA,
collected drinking water samples from 80 cities nationwide in 1975 (Symons et al., 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 |J.g/L (ppb), and the maximum level was 116 |ig/L (ppb). Dibromochloromethane was
found in 90% of the systems sampled at a median concentration of 2 |ig/L (ppb). The detection
limit for dibromochloromethane and bromodichloromethane was 0.1 |ig/L (ppb). The median
concentration for bromoform was below the detection limit of approximately 5 |ig/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°C for 2 to 3 weeks to maximize trihalomethane formation. Phase 3 had two parts. The
samples identified as 3T were allowed to stand an additional 2 to 3 weeks. The samples
identified as 3Q were quenched by addition of sodium thiosulfate. As expected, the highest
trihalomethane values occurred in Phases 2 and 3T. Bromodichloromethane was detected in
over 90% of the systems sampled. The median concentration under the various sample storage
conditions ranged from 5.9 to 14 |ig/L (ppb), and the maximum concentration was 183 |ig/L
(ppb). The mean concentrations of bromodichloromethane in Phases 1, 2, 3T, and 3Q were 18,
18, 17, and 9 |ig/L (ppb), respectively. Dibromochloromethane was detected in 73% of the
systems sampled. The median concentration ranged from below the detection limit to 3 |ig/L
(ppb), and the maximum value was 280 |ig/L (ppb). The mean concentrations of
dibromochloromethane in Phases 1, 2, 3T, and 3Q were 8, 12, 11, and 6 |ig/L (ppb), respectively.
The median bromoform concentration under all sampling conditions was below the detection
limit of 0.3 |ig/L (ppb); the maximum value was 280 |ig/L (ppb). The mean concentrations of
bromoform Phases 1, 2, 3T, and 3Q were 3, 4, 4, and 2 |ig/L (ppb), respectively. NOMS was
conducted before the promulgation of the total trihalomethane regulation; therefore, these results
may be higher than current levels.
The Community Water Supply Survey (CWSS) was conducted by the EPA in 1978. The
survey examined over 1,100 samples, representing over 450 water supply systems (Brass et al.,
1981). The samples were taken at the treatment plants and in the distribution systems. In the
CWSS, 94% of the surface water supplies and 33% of the ground water supplies were positive
for bromodichloromethane. For surface water supplies, the mean of the positives and the overall
median were 12 and 6.8 |ig/L (ppb), respectively. The mean of the positives for ground water
supplies was 5.8 |ig/L (ppb), and the overall median was below the minimum reporting limit of
0.5 |J.g/L (ppb). For dibromochloromethane, 67% of the surface water supplies and 34% of the
IV - 2 November 15, 2005
-------
ground water supplies were positive. For surface water supplies, the mean of the positives and
the overall median were 5.0 and 1.5 |ig/L (ppb), respectively. The mean of the positives for
ground water supplies was 6.6 |ig/L (ppb), and the overall median was below the minimum
reporting limit of 0.5 |ig/L (ppb). For bromoform, 13% of the surface water supplies and 26% of
the ground water supplies were positive. The mean concentration of the positives in surface
water supplies was 2.1 |ig/L (ppb), and the overall median was less than 1.0 |ig/L (ppb). The
mean of the positives for ground water supplies was 11 |ig/L (ppb), and the overall median was
below the minimum reporting limit of 0.5 |ig/L (ppb) (Brass et al., 1981).
The Rural Water Survey (RWS) was conducted between 1978 and 1980 by the EPA to
evaluate the status of drinking water in rural America. Samples from over 2,000 households,
representing more than 600 rural water supply systems, were examined. In the RWS, 76% of the
surface water supplies and 13% of the ground water supplies were positive for bromodichloro-
methane, 56% of the surface water supplies and 13% of the ground water supplies were positive
for dibromochloromethane, and 18% of the surface water supplies and 12% of the ground water
supplies were positive for bromoform. For the surface water supplies, the mean of the positives
and the overall median concentrations were 17 |ig/L (ppb) and 11 i-ig/L (ppb) for
bromodichloromethane, 8.5 |ig/L (ppb) and 0.8 |ig/L (ppb) for dibromochloromethane, and 8.7
|ig/L (ppb) and <0.5 |ig/L (ppb) for bromoform. For the ground water supplies, the mean of the
positives was 7.7 |ig/L (ppb) for bromodichloromethane, 9.9 |ig/L (ppb) for dibromochloro-
methane, and 12 |ig/L (ppb) for bromoform. The overall median for ground water supplies was
below the minimum reporting limit of 0.5 |ig/L (ppb) for all three brominated trihalomethanes
(Brass, 1981).
The Ground Water Supply Survey (GWSS) was conducted from December 1980 to
December 1981 by the EPA to develop data on the occurrence of volatile organic chemicals in
ground water supplies. Out of a total of 945 ground water systems that were sampled, 466
systems were chosen at random, and the remaining 479 systems were chosen on the basis of
location near industrial, commercial, and waste disposal activities. Samples were collected at or
near the entry to the distribution system, and trihalomethane formation was allowed to continue
without quenching after sample collection. For bromodichloromethane, the median of the
positives for the randomly chosen systems serving greater than 10,000 people was 1.4 |ig/L
(ppb), and the occurrence rate was 36%. For the randomly chosen smaller systems, the median
positive concentration was 1.6 |ig/L (ppb), and the occurrence rate was 54%. The nonrandomly
chosen systems had a median positive concentration of 2.1 |ig/L (ppb) and an occurrence rate of
51%. For dibromochloromethane, the median positive concentration and the occurrence rate for
the randomly chosen systems serving greater than 10,000 people were 2.1 |ig/L (ppb) and 31%,
respectively; these values for the smaller systems were 2.9 |ig/L (ppb) and 52%. The
nonrandomly chosen systems had a median positive concentration of 3.9 |ig/L (ppb) and an
occurrence rate of 46%. For bromoform, the median positive concentration was 2.4 |ig/L (ppb)
for the randomly chosen systems serving greater than 10,000 and 3.8 |ig/L (ppb) for the
randomly chosen systems serving fewer than 10,000 people, with occurrence rates of 16% and
31%, respectively. The nonrandomly chosen systems had a median positive concentration of
4.2 i-ig/L (ppb) and an occurrence rate of 31% (Westrick et al., 1983).
IV - 3 November 15, 2005
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The National Screening Program for Organics in Drinking Water (NSP), sponsored by
the EPA, was conducted from June 1977 to March 1981 and sampled 169 systems nationwide.
Samples were collected at the treatment facilities. For dibromochloromethane, the mean and
median for 130 positives were 17.2 and 10 |ig/L (ppb), respectively. The maximum
concentration found was 131 |ig/L (ppb) (Boland, 1981).
The Technical Support Center (TSC) of the Office of Ground Water and Drinking Water
(OGWDW) maintains a ground water contaminant database. For both bromodichloromethane
and dibromochloromethane, the database contains 4,439 samples taken at the treatment facilities
from nineteen states between 1984 and 1991. For bromodichloromethane, the mean
concentration was 5.6 |ig/L (ppb), and the median was 3 |ig/L (ppb). For
dibromochloromethane, the mean concentration was 3.0 |ig/L (ppb), and the median was 1.7
l-ig/L (ppb). For bromoform, the database contains 1409 samples from 19 states taken at treat-
ment facilities between 1984 and 1991. The mean and median concentrations were determined
to be 2.5 jig/L (ppb) and 1 jig/L (ppb), respectively (U.S. EPA, 1991).
Thirty-five water utilities nationwide, 10 of which were located in California, were
sampled for bromodichloromethane, dibromochloromethane, and bromoform in the clearwell
effluent. Samples were taken for four quarters (spring, summer, and fall in 1988 and winter in
1989). The median bromodichloromethane concentration for all four quarters was 6.6 |ig/L
(ppb), with the medians of the individual quarters reported as 6.9, 10, 5.5 and 4.1 |ig/L (ppb),
respectively, and with a maximum value of 82 |ig/L (ppb). For all four quarters, 75% of the
measured concentrations were less than 14 |ig/L (ppb). The median dibromochloromethane
concentration for all four quarters was 3.6 |ig/L (ppb), with the medians of the individual
quarters reported as 2.6, 4.5, 3.8 and 2.7 |ig/L (ppb), respectively, and with a maximum value of
63 |ig/L (ppb). For all four quarters, 75% of the data were below 9.1 |ig/L (ppb). The median
bromoform concentration for all four quarters was 0.57 |ig/L (ppb), with the medians of the
individual quarters reported as 0.33, 0.57, 0.88, and 0.51 |ig/L (ppb), respectively, and with a
maximum value of 72 |ig/L (ppb). For all four quarters, 75% of the bromoform concentrations
were below 2.8 jig/L (ppb) (Krasner et al., 1989; U.S. EPA 1989a; 1989b).
The EPA's Technical Support Center compiled a database from its disinfection
by-products field studies. The studies included a chlorination by-products survey, conducted
from October 1987 to March 1989. In this survey, concentrations of bromodichloromethane,
dibromochloromethane, and bromoform were determined in finished water from the treatment
plant and in the distribution system. Systems using both surface water sources and ground water
sources were analyzed.
Mean concentrations of bromodichloromethane, dibromochloromethane, and bromoform
in finished water at the treatment plants were determined for surface water systems serving both
greater than and less than 10,000 people. Forty-two samples were taken from systems serving
more than 10,000 people, and 20 samples were taken from systems serving fewer than 10,000
people. The mean concentration of bromodichloromethane was 12.7 |ig/L (ppb) in samples from
systems serving more than 10,000 people (90th percentile, 25.0 |ig/L (ppb)) and 17.0 |ig/L (ppb)
for samples from the smaller systems (90th percentile, 29.5 |ig/L (ppb)). The mean
IV - 4 November 15, 2005
-------
dibromochloromethane concentrations was 4.7 |ig/L (ppb) for samples from the larger systems
(90th percentile, 13.8 |ig/L (ppb)) and 6.9 |ig/L (ppb) for samples from the smaller systems (90th
percentile, 24.2 |ig/L (ppb)). The mean concentrations for bromoform were 0.7 |ig/L (ppb) (90th
percentile, 1.5 |ig/L (ppb)) and 0.9 |ig/L (ppb) (90th percentile, 4.9 |ig/L (ppb)) in samples from
the larger systems and samples from the smaller systems, respectively (U.S. EPA, 1992a).
Mean bromodichloromethane, dibromochloromethane, and bromoform concentrations in
distribution systems of these surface water systems also were analyzed. Thirty-nine samples
were taken from systems serving more than 10,000 people, and 11 samples were from systems
serving fewer than 10,000 people. The mean bromodichloromethane concentrations in the larger
systems and the smaller systems were 17.4 |ig/L (ppb) (90th percentile, 35.3 |ig/L (ppb)) and
24.8 |J.g/L (ppb) (90th percentile, 51.0 |ig/L (ppb)), respectively. The mean
dibromochloromethane concentrations were 6.3 |ig/L (ppb) (90th percentile, 17.3 |ig/L (ppb))
and 10.4 |ig/L (ppb) (90th percentile, 35.0 |ig/L (ppb)), respectively. Mean bromoform
concentrations were 0.8 |ig/L (ppb) (90th percentile, 3.1 |ig/L (ppb)) and 1.4 |ig/L (ppb) (90th
percentile, 5.1 |ig/L (ppb)), respectively (U.S. EPA, 1992a).
Ground water systems serving less than 10,000 people were analyzed for
bromodichloromethane, dibromochloromethane, and bromoform in seven finished water samples
and in five distribution system samples. Mean bromodichloromethane concentrations in the
finished water samples and in the distribution system samples were 1.1 i-ig/L (ppb) (90th
percentile, 2.6 |ig/L (ppb)) and 2.2 |ig/L (ppb) (90th percentile, 5.4 |ig/L (ppb)), respectively.
Mean dibromochloromethane concentrations were 0.6 |ig/L (ppb) (90th percentile, 1.0 |ig/L
(ppb)) and 1.8 |ig/L (ppb) (90th percentile, 3.6 |ig/L (ppb)), respectively. Mean bromoform
concentrations were 0.6 |ig/L (ppb) (90th percentile, 2.6 |ig/L (ppb)) and 2.3 |ig/L (ppb) (90th
percentile, 10 |ig/L (ppb)), respectively.
For ground water systems serving more than 10,000 people, dibromochloromethane and
bromoform were not detected in single samples taken at the plant or from the distribution
system, based on a detection limit of 0.2 |ig/L (ppb). Bromodichloromethane concentrations in
the plant and distribution system samples were 0.2 and 0.4 |ig/L (ppb), respectively (U.S. EPA,
1992a).
The U.S. Geological Survey conducted an assessment of volatile organic compounds in
untreated ambient groundwater of the conterminous United States based on samples collected
between 1985 and 1995 from 2948 wells. The sampled wells were located in rural and urban
areas and included wells used for drinking and non-drinking water purposes. A minimum
reporting level of 0.2 |ig/L (ppb) was used for most of the compounds, including
bromodichloromethane, dibromochloromethane, and bromoform. In samples from the 406 urban
wells assessed, bromodichloromethane, dibromochloromethane, and bromoform were detected
in 3.0%, 2.8%, and 2.8% of the wells examined, respectively. In samples from the 2542 rural
wells examined, these compounds were detected in 0.8%, 0.6%, and 0.4% of the wells,
respectively. The measured concentration of the compounds in well water were reported in
summary graphics only. Thus, the values reported here are approximate based on visual
inspection of the figures. The median concentrations measured in the positive samples from the
IV - 5 November 15, 2005
-------
urban wells was approximately 1.0 |ig/L (ppb) for all three compounds, while the maximum
concentrations of bromodichloromethane, dibromochloromethane, and bromoform in the urban
wells were approximately 11, 11, and 13 jig/L (ppb), respectively. The median concentrations
measured in the positive samples from the rural wells were approximately 0.4 to 0.5 |ig/L (ppb)
for all three compounds, while the maximum concentrations of bromodichloromethane,
dibromochloromethane, and bromoform in the rural wells were approximately 7, 10, and 18
|ig/L (ppb), respectively.
The most recent survey of the occurrence of brominated trihalomethanes in public water
supplies (PWSs) serving at least 100,000 persons resulted from the Information Collection Rule
(ICR) promulgated in May of 1996 for disinfectants and disinfection byproducts (D/DBPs). The
rule covered both surface and ground water systems. Monitoring data were collected from about
300 water systems operating 501 plants over thelS-month period between July 1997 and
December 1998. At each plant, samples were collected monthly and analyzed for a variety of
D/DBPs on a monthly or quarterly basis. Bromodichloromethane, dibromochloromethane, and
bromoform were among the analytes evaluated quarterly (U.S. EPA, 2001a). Five samples were
taken each quarter at each plant - one of the finished water and four of the water in the
distribution system. Of the four samples from the distribution system, one represented a sample
with the same residence time as a finished water sample held for a specific period of time, two
represented approximate average water residence times in the system, and one sample was taken
where water residence time in the system is the longest. For each plant and reporting period,
EPA compiled several summary statistics. The Distribution System (DS) Average value is the
average of the four distribution system samples. The DS High Value is the highest concentration
of the four distribution system samples collected by a plant in a given quarter. The DS High
Value might be from any of the four samples and could vary from quarter to quarter depending
on which sample yielded the highest concentrations in each quarter (U.S. EPA, 2001a). Table
IV-1 summarizes the results of all six of the quarterly reporting periods.
U.S. EPA set a minimum reporting level (MRL) for bromodichloromethane,
dibromochloromethane, and bromoform of 1.0 |ig/L for the ICR. The MRL is a level below
which systems were not required to report their monitoring results, even if there were detectable
results. Values below the MRL were assigned a value of zero for the purpose of calculating
averages; this assignment affects the calculation of mean values for finished water and DS high
results and calculation of all DS average values.
Recent data for concentrations of brominated trihalom ethanes are now available for 117
small surface water plants (serving < 10,000 people) from the National Rural Water Association
Survey (NWRA) (U.S. EPA 2001b). Most, but not all, plants that participated in the survey took
two samples at each of three sampling locations. One sample was taken between November,
1999, and March, 2000, and the other between July and November, 2000, for a total of 217 THM
samples. The samples were taken at the finished water location, distribution system average
residence time location, and maximum residence time location. These data are summarized in
Table IV-2 below.
IV - 6 November 15, 2005
-------
Table IV-1 Brominated Trihalomethane Concentrations Measured in U.S. Public Drinking
Water Systems Serving 100,000 or More Persons
Source
Data Type
(a)
Number of
Samples
Median (b)
Mean (b)
90th
Percentile
Range
Bromodichloromethane (jig/L)
Suface
Water
Ground
Water
Finished
DS Average
DS High
Finished
DS Average
DS High
1856
1656
1656
604
603
603
6.6
8.6
9.9
< 1.0
1.80
2.8
8.2
10.2
11.9
7.9
4.06
5.78
17.5
20.3
23.3
6.80
11.2
16.0
<1.0-49
0-65.8
<1.0-73
<1.0-27
0-35.3
<1.0- 110
Dibromochloromethane (ug/L)
Surface
Water
Ground
Water
Finished
DS Average
DS High
Finished
DS Average
DS High
1853
1655
1655
604
602
602
1.9
2.40
2.9
< 1.0
1.35
2.1
4.03
4.72
5.57
1.38
3.09
4.60
12.0
13.2
15.0
4.10
8.94
12.9
<1.0-55.1
0-67.3
< 1.0 -67.3
<1.0-33
0-37.5
<1.0-85
Bromoform (jig/L)
Surface
Water
Ground
Water
Finished
DS Average
DS High
Finished
DS Average
DS High
1853
1653
1653
602
599
599
<1.0
0
<1.0
<1.0
0.325
1.2
0.998
1.18
1.48
0.838
1.92
2.95
2.88
3.10
3.90
2.20
4.78
7.72
<1.0-34
0-34.3
<1.0-40
<1.0-21
0-28.8
<1.0-391
(a) Finished = sample location after treatment, before entering the distribution system (DS); DS Average =
average of four sample locations in the DS; DS High = the highest concentration of the four distribution system
samples collected by a plant in a given quarter. For purposes of calculations, all values below the minimum
reporting level (MRL) of 1.0 ug/L for all three compounds were assigned a value of zero.
(b) Median and mean of all samples including those below the MRL.
Source: Disinfectants and Disinfection Byproducts (D/DBPs) ICR Data, U.S. EPA (2001a).
IV-7
November 15, 2005
-------
Table IV-2 NRWA Brominated Trihalomethane Results for Small Surface Water Plants
THM
Bromodichloromethane
(ug/L)
Dibromochloromethane
(ug/L)
Bromoform (ug/L)
Sample
Location
Finished
DS Average
DSMax
Finished
DS Average
DSMax
Finished
DS Average
DSMax
Mean
11.2
14.3
15.9
5.0
6.1
6.7
4.0
4.6
4.5
Median
6.5
9.4
10.2
1.1
1.5
1.9
0
0
0
90th
Percentile
26.6
32.2
34.2
13.4
16.3
17.1
1.2
1.2
1.3
Range
0 - 84.4
0- 100.3
0- 121.1
0-83.1
0 - 99.0
0-91.6
0-333.4
0-340.5
0-349.7
Median and mean of all samples, including those below the detection limit.
Source: National Rural Water Association Survey U.S. EPA (200Ib)
2. Other Studies
Several less comprehensive surveys have analyzed drinking water for one or more of the
brominated trihalomethanes. An overview of these studies is provided below.
The EPA Region V Organics Survey sampled finished water from 83 sites in a region
that includes Illinois, Indiana, Michigan, Minnesota, Ohio, and Wisconsin. Bromoform was
found at a median concentration of the positives of 1 |ig/L (ppb) and a maximum level of 7 |ig/L
(ppb). A total of 14% of the locations sampled contained detectable levels of bromoform (U.S.
EPA, 1980). Kelley (1985) surveyed 18 drinking water plants in Iowa for trihalomethanes, and
detected bromoform in five water supplies at concentrations ranging from 1.0 to 10 |ig/L (ppb).
The EPA's Five-year Total Exposure Assessment Methodology (TEAM) study measured
the personal exposures of a probability-based sample of residents in several U.S. cities to various
organic chemicals in air and drinking water between 1981 and 1987. As part of the study,
running tap water samples were collected from residences of nearly 850 study participants
during the morning and the evening to test for brominated trihalomethane concentrations. The
samples were quenched with sodium thiosulfate at the time of collection. Tables IV-3, IV-4, and
IV-5 show bromodichloromethane, dibromochloromethane, and bromoform concentrations
found in drinking water from the six cities surveyed. Samples of water were taken from each
participating residence at the household taps and sodium thiosulfate added as a quenching agent.
IV-8
November 15, 2005
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Table IV-3 Bromodichloromethane Concentrations in Drinking Water from the U.S. EPA
TEAM Study (jig/L)
Location
Elizabeth/
Bayonne,
New Jersey
Los Angeles,
California
Antioch/
Pittsburg,
California
Devils Lake,
North
Dakota
Greensboro,
North
Dakota
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer
1982
Winter
1983
Winter
1984
Summer
1984
Winter
1987
Summer
1987
Spring
1984
Fall 1982
Fall 1982
Spring
1987
Sample
Size
340
156
49
117
52
9
7
71
24
24
10
%
Meas-
ured
99.7
99.8
100
93
96
89
100
96
73
93
100
Concentration \ig/L (ppb)
Mean
13.6
13.6
5.4
11
20
19
26
21
0.21
7.1
10
Med-
ian
13
12
5.8
12
24
24
27
17
0.18
7.8
10
Max
23
54
16
23
38
31
36
47
1.0
11
13
Percentiles
25%
~
~
5.1
7.7
2.4
—
~
~
75%
15
15
7.1
16
31
36
—
9.2
~
90%
16
18
8.3
17
33
45
—
~
~
95%
18
20
8.3
20
37
47
—
~
~
Adopted fromHartwell, (1987), Wallace et al. (1987), Wallace et al. (1988), and Wallace (1992) by U.S. EPA
(1994b). Mean and median values of all samples, including those below the quantitation limit.
IV-9
November 15, 2005
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Table IV-4 Dibromochloromethane Concentrations in Drinking Water from the U.S. EPA
TEAM Study
Location
Elizabeth/
Bayonne,
New Jersey
Los Angeles,
California
Antioch/
Pittsburg,
California
Devils Lake,
North Dakota
Greensboro,
North Dakota
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer
1982
Winter
1983
Winter
1984
Summer
1984
Winter
1987
Summer
1987
Spring
1984
Fall 1982
Fall 1982
Spring
1987
Sample
Size
340
156
49
117
52
9
7
71
24
24
10
%
Meas-
ured
99.7
99.8
93
89
85
89
100
85
18
93
100
Concentration ng/L (ppb)
2.4
2.1
1.4
9.4
28
10
24.7
8
0.09
1.2
2.7
Med-
ian
2.4
1.9
1.6
11
32
12
18
6.4
0.06
1.2
2.6
8.4
7.2
3.0
19
55
17
70
21
0.45
1.9
3.5
Percentile
25%
~
~
2.4
15
0.98
~
~
~
75%
2.7
2.4
1.8
15
42
15
0.06
1.5
~
90%
3.2
3.1
2.0
17
43
18
~
~
~
95%
3.4
3.8
2.1
18
48
19
~
~
~
Adopted fromHartwell, (1987), Wallace et al. (1987), Wallace et al. (1988), and Wallace (1992) by U.S. EPA
(1994b). Mean and median values of all samples, including those below the quantitation limit.
IV-10
November 15, 2005
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Table IV-5 Bromoform Concentrations in Drinking Water from the U.S. EPA
TEAM Study
Location
Los Angeles,
California
Antioch/
Pittsburg,
California
Date
Sampled
Winter
1984
Summer
1984
Winter
1987
Summer
1987
Spring
1984
Sample
Size
117
52
9
7
71
Meas-
ured
69
90
89
100
69
Concentration \ig/L (ppb)
Mean
0.78
8.08
3.2
25.5
0.78
Med-
ian
0.54
3.0
3.2
9.6
0.58
Max
12
78
4.7
113
2.0
Percentile
25%
0.34
2.0
0.19
75%
0.92
5.9
1.2
90%
1.2
13
1.8
95%
1.5
53
1.9
Adopted from Wallace (1992) by U.S. EPA (1994b). Mean and median values of all samples, including those below
the quantitation limit. Bromoform was measured in fewer than 10% of samples from the other four cities in the
TEAM study and are not presented here
Furlong and D'itri (1986) reported that a survey of water treatment plants in Michigan
detected bromodichloromethane in 35 of 40 plants at a median concentration of 2.7 |ig/L (ppb)
and a maximum of 54.2 |ig/L (ppb); the mean of the positive samples was 7.4 |ig/L (ppb).
Dibromochloromethane was also detected in 30 plants at a median concentration of 2.2
|ig/L (ppb) and a maximum of 39.6 |ig/L (ppb); the mean of the positives was 5.1 |ig/L (ppb).
Bromoform was detected at three of 40 plants sampled at concentrations of 0.9, 1.3, and 1.6 |ig/L
(ppb).
Fair et al. (1988) analyzed drinking water from three community water supplies for
chlorination by-products. Bromodichloromethane concentrations ranged from 7.5 to 30 |ig/L
(ppb) in finished water and from 9.9 to 36 |ig/L (ppb) in the distribution systems.
Dibromochloromethane concentrations ranged from less than 0.5 to 19 |ig/L (ppb) in finished
water at the plant and from less than 0.5 to 23 |ig/L (ppb) in the distribution systems.
Bromoform concentrations ranged from less than 0.5 to 2.5 |ig/L (ppb) in finished water and
from less than 0.5 to 3.1 |ig/L (ppb) in the distribution systems.
IV-11
November 15, 2005
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Wallace et al. (1982) analyzed tap water for bromodichloromethane as part of a study to
determine individual exposures to volatile organics during normal daily activities of students at
the University of North Carolina, Chapel Hill. Bromodichloromethane was detected in 7 of 7
samples of tap water, at concentrations ranging from 15 to 20 |ig/L (ppb), with a mean of 17
l-ig/L (ppb). The detection limit was 0.1 |ig/L (ppb).
Chang and Singer (1984) analyzed the bromoform concentration in drinking water
samples prepared by the desalination of seawater. After pretreatment using either activated
carbon or ultrafiltration, but prior to reverse osmosis treatment, bromoform concentrations were
13 ± 14 and 110 ± 59 |ig/L (ppb), respectively. After reverse osmosis was completed, the
finished water product contained bromoform concentrations ranging from 2.0 to 51 |ig/L (ppb)
(mean, 20.17 |ig/L (ppb)) when activated carbon was used as a pretreatment and 127 |ig/L (ppb)
when ultrafiltration was used. In the reverse osmosis treatment, three reverse osmosis
membranes were evaluated. Use of a cellulose triacetate filter resulted in concentrations of 51
|ig/L (ppb), while use of a poly ether/urea thin film spiral wound membrane or a polysulfone
membrane filters which resulted in final concentrations of 5.0 |ig/L (ppb) and 2.25 |ig/L (ppb),
respectively.
Bromodichloromethane, dibromochloromethane, and bromoform were detected in 9.5 to
12.8% of drinking water samples collected in 1987 in Nassau County, New York. The county
draws its drinking water from underground aquifers. Bromodichloromethane and
dibromochloromethane had similar concentration profiles, being detected in approximately 10%
and 8.5% of the samples, respectively, at concentrations less than 4.9 ppb. The detection limit
was 1 ppb for each chemical. Bromoform was detected in 8% of the samples at 2 to 4.9 ppb, in
2.5% of the samples at 5 to 9.9 ppb, and in less than 1% of the samples at 10 to 49.9 ppb. The
detection limit was 2 ppb. None of the drinking water samples contained more than 50 ppb of
any of the trihalomethanes, and less than 1% of the samples contained between 10 and 49.9 ppb
of the brominated compounds (Moon et al., 1990).
U.S. EPA conducted a study of contaminants in household water in nine residences as
part of a larger study of health risks due to environmental contamination in the Lower Rio
Grande Valley (Berry et al., 1997). Samples of water used for drinking were taken once during a
3-day period in the spring and once during a 2-day period in the summer of 1993. Water used
for drinking in the nine residences could be traced to one of three sources: the municipal water
supply of Brownsville, Texas, vended water supplies (municipal water that had undergone
further treatment), and well water. Samples were collected using U.S. EPA protocols, including
quality assurance samples and field blanks. The detection and minimum quantitation limits for
each analyte were documented in other reports. Bromodichloromethane,
dibromochloromethane, and bromoform were detected in the household water of seven of the
nine residences during the spring and in five of the nine residences during the summer (Berry et
al., 1997). During the spring, the minimum, median, and maximum concentrations of
bromodichloromethane for the seven positive samples were 3.2, 5.2, and 24.4 |ig/L (ppb),
respectively. For dibromochloromethane, the values were 3.3, 5.1, and 17.3 |ig/L (ppb),
respectively. For bromoform, the values were 1.0, 3.0, and 14.1 |ig/L (ppb), respectively.
During the summer, the minimum, median, and maximum concentrations of
IV - 12 November 15, 2005
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bromodichloromethane in the five positive samples were 2.3, 7.7, and 34.3 |ig/L (ppb),
respectively. For dibromochloromethane, the values were 1.8, 7.6, and 49.9 |ig/L (ppb),
respectively. For bromoform, the values were 1.6, 7.8, and 31.7 |ig/L (ppb), respectively.
Weisel et al. (1999) examined concentrations of trihalomethanes in the tap water of the
homes of 49 women in New Jersey. The 49 residences were selected so that approximately half
would represent the lower extreme of trihalomethane contamination and half the upper extreme
of trihalomethane contamination identified in a previous study. Samples were stored
unquenched on ice after collection and were analyzed within 24 hours. The three brominated
trihalomethanes were detected in all 49 samples. The mean (± standard deviation)
concentrations of bromodichloromethane, dibromochloromethane, and bromoform were 5.7 ±
8.6, 2.0 ± 2.1, and 0.73 ± 0.90 |ig/L (ppb), respectively. The median values for the three
compounds were 2.6, 1.4, and 0.45 |ig/L (ppb), respectively. These values are not representative
of New Jersey, because of the selection criteria for the residences. The ranges (minimum to
maximum) of concentrations measured for each compound were 0.06 to 48 |ig/L (ppb) for
bromodichloromethane, 0.14 to 9.7 |ig/L (ppb) for dibromochloromethane, and 0.03 to 4.21 |ig/L
(ppb) for bromoform.
3. Estimates of Tap Water Ingestion Exposure to Brominated Trihalomethanes
a. Estimates Based on ICR Data for Disinfection Byproducts
The data from EPA's ICR for disinfectants and disinfection byproducts (U.S. EPA
200la) offer several advantages over the 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.
Table IV-6 presents estimated drinking water exposures to brominated trihalomethanes
of the adult populations served by large public water systems (serving 100,000 or more persons)
based on the ICR Occurrence Data (U.S. EPA, 200la). 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. Thus, the 90th percentile of the DS
High values are also presented in Table IV-6.
IV - 13 November 15, 2005
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Table IV-6 Estimated Drinking Water Exposures to Brominated Trihalomethanes in U.S.
Public Drinking Water Systems Serving More than 100,000 Persons'1
Source
Medianb
Meanb
90th Percentileb
DS High 90th
Percentile0
Bromodichloromethane (ug/person/day)
Surface Water
Ground Water
17
3.6
20
8.1
40
22
47
32
Dibromochloromethane (ug/person/day)
Surface Water
Ground Water
4.8
2.7
9.4
6.2
26
18
30
26
Bromoform (jig/person/day)
Surface Water
Ground Water
0
0.65
2.4
3.8
6.2
9.6
7.8
15
a Source: U.S. EPA (2001a). Assumes that each individual consumes 2 liters of water daily. Also assumes that
concentrations at the drinking water tap are similar to concentrations in the distribution system (DS) sampled at
locations considered to be representative of average (DS Average) and highest (DS High) retention times (see
Table IV-1).
b Based on concentrations from the DS Average values.
0 Based on the 90th percentile of the DS High values to represent a plausible 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 lag/person/day, respectively. The
same values for populations exposed to bromodichloromethane from ground water systems are
lower - 3.6, 8.1, and 22 lag/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 lag/person/day, respectively. The corresponding values for populations exposed
to dibromochloromethane from groundwater system are lower - 2.7, 6.2, and 18 lag/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 lag/person/day, respectively. The
same values for populations exposed to bromoform from ground water systems are higher -
0.65, 3.8, and 9.6 lag/person/day, respectively.
Average daily intake of dibromochloromethane was also evaluated for determination of
the Relative Source Concentration. The details of this evaluation are presented in Appendix C.
Intake for ingestion was calculated using mean intake rates of 1.2 or 0.6 L/day for total and
direct intake (NRC, 1999), respectively. Direct intake includes consumption of water directly
from the tap, but does not include intake of tap water used for preparation of heated items such
tea, coffee, or soup. Based on the ICR distribution system average concentration of 4.72 |ig/L
for dibromochloromethane in surface water, the average daily total and direct and ingestion
IV-14
November 15, 2005
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intakes would be 5.7 and 2.8 |ig/day, respectively. Absorption of dibromochloromethane from
tap water was estimated using methodology described in U.S. EPA (1992c), as modified by
Vecchia and Bunge (2002). The average dermal uptake of dibromochloromethane was estimated
to be 2 |ig per shower or bathing event. Intake via inhalation of dibromochloromethane
volatilized during household activities (e.g., showering, bathing, dishwashing, toilet flushing,
etc.) was estimated using a three-compartment model based on McKone (1987). This model
estimated an average daily inhalation exposure of 7 jig/day for the volatilized compound.
Parallel calculations were not performed for bromodichloromethane or bromoform, because
these compounds are probable carcinogens. Therefore, in accordance with U.S. EPA policy,
RSC analysis was not conducted.
b. Estimates of Ingestion Exposure Based on Other National Studies
Exposure to bromodichloromethane, dibromochloromethane, and bromoform in drinking
water from ground water supplies can be estimated from the median levels found in the GWSS.
Based on the range of median levels (1.4-2.1 |ig/L (ppb)) and a consumption rate of two liters
per day, the median exposure to bromodichloromethane may range from 2.8 to 4.2 |ig/day.
Similarly, median exposure to dibromochloromethane may range from 4.2 to 7.8 jig/day, and for
bromoform, median exposure may range from 4.8 to 8.4 jig/day. Exposure to
bromodichloromethane from surface water supplies can be estimated based on the range of
median values observed under different conditions in NOMS, which mainly sampled surface
water systems. Based on a range of 5.9-14 |ig/L (ppb), exposure to bromodichloromethane from
surface water is estimated to be between 12 and 28 jig/day. Similarly, based on the range of
medians reported for dibromochloromethane concentrations, the median exposure is estimated to
be up to 6 jig/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 |ig/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 |ig/L
(ppb), the exposure to bromoform is estimated to range from 1 to 2 [ig/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 TEAM studies. Table IV-7 presents
median, mean, 90th percentile, and 95th percentile estimates of daily intakes of
bromodichloromethane, dibromochloromethane, and bromoform, based on an assumed drinking
water ingestion rate of 2 liter per day. Table IV-7 provides estimates for those locations and
seasons with a sample size of at least 50, with one exception. Devils Lake, ND, with a sample
size of only 24, is added to represent an area with low concentrations. Thus, the influence of
small sample size on distributional statistics should be minimized in Table IV-7. The median,
mean, and 90th percentile values in Table IV-7 for the TEAM study can be compared with the
corresponding values in Table IV-6 for the ICR Occurrence data.
IV - 15 November 15, 2005
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Table IV-7 Estimated Distribution of Drinking Water Exposures to Brominated
Trihalomethanes for Populations in U.S. EPA TEAM Study (a)
Location
Season
Year
Median (b)
Mean (b)
90th
Percentile (b)
95th
Percentile
Bromodichloromethane (jig/person/day)
Elizabeth/Bayone NJ
Los Angeles, CA
Antioch/Pittsburg, CA
Devils Lake, ND
summer 82
winter 83
summer 84
winter 84
spring 84
fall 82
24
12
48
24
34
0.36
27
11
40
22
42
0.42
36
17
66
34
90
<2.0
40
17
74
40
94
<2.0
Dibromochloromethane (jig/person/day)
Elizabeth/Bayone NJ
Los Angeles, CA
Antioch/Pittsburg, CA
Devils Lake, ND
summer 82
winter 83
summer 84
winter 84
spring 84
fall 82
3.8
3.2
64
22
13
0.12
4.2
2.8
56
19
16
0.2
6.2
4.0
86
34
36
<0.9
7.6
4.2
96
36
38
<0.9
Bromoform (ug/person/day)
Los Angeles, CA
Antioch/Pittsburg, CA
summer 84
winter 84
spring 84
6.0
1.1
1.2
16.2
1.6
1.6
26
2.4
3.6
100
3.0
3.8
(a) Intakes estimated from data in Tables IV-3, IV-4, and IV-5 assuming a water ingestion rate of 2 liters per day.
Selected locations and seasons with samples sizes over 50. Added Devils Lake, ND, to represent an area with
low air concentrations.
(b) Median, mean, and upper percentiles estimated for entire population of city.
Table IV-7 demonstrates that concentrations of brominated trihalomethanes are lower in
winter than in summer, as would be expected on the basis of temperature. In this sample of
geographic locations, estimates of the average of the population intakes of
bromodichloromethane from drinking water range from 0.42 to 42 jig/person/day. The upper
IV-16
November 15, 2005
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90th percentile estimates range from <2.0 to 90 jig/person/day. Estimates of the average
population intake of dibromochloromethane from drinking water range from 0.2 to 56
|ig/person/day. The upper 90th percentile estimates range from < 0.9 to 86 jig/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 jig/person/day.
The upper 90th percentile estimates range from 2.4 to 26 jig/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.
c. Sources of Uncertainly in Estimates of Exposure from Drinking Water
Sources of uncertainty in the estimates of ingestion exposure include use of different
analytical methods, failure to report quantitation limits, use of measurements near the detection
limit, failure to report how nondetects were 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 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.
B. Exposure from Sources Other Than Drinking Water
1. Dietary Intake
a. Measured Concentrations in Foods and Beverages
Information on the levels of brominated trihalomethanes in foods and beverages is
limited. Chlorine is used in food production for applications such as the disinfection of chicken
in poultry plants and the superchlorination of water at soda and beer bottling plants (Borum,
1991). Therefore, the possibility exists for contamination of foodstuffs by disinfection by-
products with resulting dietary exposure. The occurrence of bromodichloromethane in foods and
beverages is the best characterized of the three compounds. Less information is available
concerning the occurrence of dibromochloromethane or bromoform in foods and beverages in
the United States. Some information is available from international studies, but may not be
relevant to U.S. occurrence because of different water treatment and food processing practices.
The available U.S. and international studies are summarized below.
Entz et al. (1982) analyzed food samples from Elizabeth, NJ, Chapel Hill, NC, and
Washington, DC, for bromodichloromethane. A total of 39 different food items from each city
were collected according to standards set for the FDA's Total Diet Market Basket Study. The
Adult Market Basket, representing the diet of a teenage male, is divided into 12 food groups.
Individual foods are prepared as generally consumed in the home and foods from each group are
blended together in "the proper proportions" to form composites. In this study, foods were
blended into four composites representing dairy products; meat, fish and poultry; oils, fats and
shortening; and beverages. The estimated limit of quantitation for bromodichloromethane in
IV - 17 November 15, 2005
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each of these composites was 2.3, 4.5, 8.3, and 0.5 ng/g, respectively. Five sets of each
composite were tested for a total of 20 composites. Bromodichloromethane was detected in one
dairy composite at 1.2 ppb and two beverage composites at 0.3 ppb and 0.6 ppb. Analysis of
individual foods from the beverage and dairy composites found bromodichloromethane in three
samples of cola soft drinks at concentrations of 2.3 ppb, 3.4 ppb, and 3.8 ppb and in one sample
of butter at? ppb.
Uhler and Diachenko (1987) sampled 38 food and beverage products from 15 food
processing plants in nine states. Plants were chosen on a "worst-case" basis from areas where
contaminated water would most likely be used in processing. In addition, processing plants were
chosen for study only if they produced high fat content food that came in contact with water
during processing or contained a high percentage of added water. Samples containing less than 1
ng/g were considered nondetects. Bromodichloromethane was detected in 6 out of 37 tested
food tested at the following levels: two samples of clear sodas at 1.2 and 2.3 ng/g (ppb) and one
sample of dark cola at 1.2 ng/g (ppb) out of fifteen soft drinks, and three of six samples of ice
cream at 0.6 to 2.3 ng/g (ppb). Bromodichloromethane was not found in any of the eight cheese
samples analyzed.
U.S. EPA (1985) reported that bromodichloromethane was identified in bacon. No
further information on sample size, detection limit, or study methodology was provided.
Abdel-Rahman (1982) analyzed various soft drinks for bromodichloromethane and found
average levels ranging from 0.2 to 6.6 |ig/L (ppb) for colas and from 0.1 to 0.2 |ig/L (ppb) for
clear soft drinks (Abdel-Rahman, 1982). In Italy, Cocchioni et al. (1996) analyzed 61 samples
of different commercially prepared beverages and 94 samples of mineral waters for volatile
organo-halogenated compounds. In the prepared beverages, they found maximum
concentrations of bromodichloromethane, dibromochloromethane, andbromoform of 40.6, 13.9,
and 10.7 |ig/L (ppb), respectively. The frequencies of detection of these three compounds in
prepared beverages were 46% (28/61), 43% (26/61), and 11% (7/61), respectively, with
detection limits for all three compounds of less than 1 |ig/L (ppb). In contrast, the maximum
concentration of any of the halogenated organic compounds identified in mineral water,
including chloroform, was 5.79 |ig/L (ppb).
McNeal et al. (1995) examined 27 different prepared beverages and mineral waters in the
United States for bromodichloromethane, dibromochloromethane, and bromoform at detection
limits of 0.1, 0.1, and 0.2 ng/g (ppb), respectively. Bromoform was not detected in any of the
samples. Bromodichloromethane and dibromochloromethane were detected at 12 and 1 ng/g
(ppb), respectively, in only one of seven types of mineral and sparkling waters examined. The
positive sample was the only sparkling and flavored water of the group,. Bromodichloromethane
was found in 1 of 5 flavored noncarbonated beverages examined, a fruit drink, at a concentration
of 5 ng/g (ppb); dibromochloromethane was not detected in any of these five beverages.
Bromodichloromethane was found in all 13 of the types of carbonated soft drinks examined, at
concentrations ranging from 1 to 4 ng/g (ppb) for 12 of the drinks examined and at 12 ng/g (ppb)
for the thirteenth. Dibromochloromethane was detected in only 4 of the 13 carbonated soft
IV - 18 November 15, 2005
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drinks examined at levels of 0.5 to 2 ng/g (ppb). None of the brominated trihalomethanes was
detected in either of the two types of beer examined.
McNeal et al. (1995) also examined several types of prepared non-beverage foods and
water from canned vegetables in the United States for bromodichloromethane,
dibromochloromethane, and bromoform. None of these compounds was detected in any of the
samples. The foods examined included two types of canned tomato sauce, canned pizza sauce,
canned vegetable juice, vegetable waters from two types of canned green beans and one type of
sweet corn, duck sauces, beef extract, and Lite syrup product.
The U.S. Food and Drug Administration (U.S. FDA, 2000) has analyzed for 18 volatile
organic hydrocarbons (VOCs), including bromodichloromethane and bromoform, in the Total
Diet Study since 1995. Bromodichloromethane and bromoform were analyzed in a subset of 70
food items in 14 Market Baskets. During the period 1995 to 1999, bromodichloromethane was
detected in one sample each of 11 non-beverage food items (sliced bologna, fried eggs, canned
pork and beans, smooth peanut butter, homemade cornbread, raw orange, canned pineapple,
boiled collards, red tomato, green pepper, and fast-food hamburger) (U.S. FDA, 2000). The
detected concentrations ranged from 10 to 16 ppb, with the exception of fast food hamburger
which contained 37 ppb. Bromodichloromethane was detected in one sample of bottled apple
juice at a concentration of 33 ppb. The mean detected concentration of bromodichloromethane
in three samples of tap water was 18 ppb. Dibromochloromethane was not included in the list of
VOC analytes for the Total Diet Study. Bromoform was listed as an analyte, but no detections
were reported in the data summary for 1991 to 1999. The detection limits for
bromodichloromethane and bromoform were not reported.
Imaeda et al. (1994) examined bean curd commercially available in Japan for
trihalomethanes. Neither bromoform nor dibromochloromethane were detected in any of the
samples at a detection limit of 0.1 ppb. Bromodichloromethane was detected in 6 of 10 samples
of bean curd at concentrations ranging from 1.2 to 5.2 ppb and in 1 of 10 samples of the water in
the bean curd packages at 5.2 ppb.
Kroneld and Reunanen (1990) analyzed for brominated trihalomethanes in samples of
pasteurized and unpasteurized cow's milk collected in Turku, Finland. The average
concentration of bromodichloromethane measured in pasteurized milk was 0.008 |ig/L (ppb)
(range, undetectable to 0.03 |ig/L (ppb), detection limit not specified). Dibromochloromethane
was detected in only one sample of pasteurized milk at 5 |ig/L (ppb). Traces of bromoform were
detected but not quantified. Brominated trihalomethanes were not detected in unpasteurized
milk. Their presence in pasteurized milk was considered to result from use of chlorinated water
during processing.
b. Estimated Dietary Intake
Estimates for dietary intake of brominated trihalomethanes by residents of the United
States were not identified in the materials reviewed for this document. Furthermore, information
on the levels in U.S. foods is too limited to independently calculate a reliable estimate.
IV - 19 November 15, 2005
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However, the available data suggest that the concentrations of brominated trihalomethanes in
non-beverage foods are likely low. The apparently low concentrations of brominated
trihalomethanes in non-beverage foods are consistent with the physical and chemical properties
of these compounds. The levels of individual brominated trihalomethanes in beverages prepared
in the United States appear to be less than or about equal to levels measured in disinfected
surface water.
Toyoda et al. (1990) analyzed the dietary intake of bromodichloromethane,
dibromochloromethane, and bromoform for 30 Japanese housewives in Nagoya and Yokohama,
Japan. Duplicate portions of daily meals were collected for three consecutive days and sampled
for all three brominated trihalomethanes. The types of food consumed were not reported. This
omission prevents a meaningful comparison of the studied diet to that consumed by the U.S.
population. The detection limits for bromodichloromethane, dibromochloromethane, and
bromoform were reported to be 0.1, 0.2, and 0.5 ppb, respectively. The concentration of
bromodichloromethane ranged from undetectable to 1.7 ppb (average, 0.3 ± 0.3 ppb SD). The
mean daily intake of bromodichloromethane was estimated to be 0.6 ± 0.5 |ig/day. The
concentration of dibromochloromethane ranged from undetectable to 0.6 ppb (average, 0.1 ±
0.2 ppb), and the mean dietary intake was estimated to be 0.3 ± 0.3 jig/day. The concentration of
bromoform ranged from undetectable to 8.1 ppb (average, 0.5 ±1.3 ppb). The mean dietary
intake of bromoform was estimated to be 0.9 ±1.3 |ig/day.
Brominated trihalomethanes have been detected in a number of beverages. In conducting
an exposure assessment, the potential exposures from drinking prepared beverages would not be
added to the default assumption of an adult consuming 2 liters of drinking water per day.
Instead, the prepared beverages would be considered part of the 2 liters of fluid intake per person
per day.
2. Air Intake
a. Concentrations in Outdoor Air
Brominated trihalomethanes are usually found in outdoor air at low concentrations when
all data, including nondetects, are considered. Brodzinsky and Singh (1983) reviewed,
summarized, and critically evaluated existing data for brominated trihalomethane concentrations
in ambient outdoor air for several urban/suburban or source dominated locations across the
United States (Table IV-8). No concentration data were available for rural or remote areas. The
authors reported mean, median, first and third quartile values, and minimum and maximum
values by city. In addition, they reported the same measures when the data were grouped by
type of location (i.e., urban/suburban or source dominated), and when all data were combined.
Ambient air concentrations were reported for bromodichloromethane at Magnolia, AR, El
Dorado, TX, Chapel Hill, NC, and Beaumont, TX. Bromodichloromethane was detected at
mean concentrations of 0.76 ppt, 1.40 ppt, 120 ppt, and 180 ppt for those four cities,
respectively, where ppt is expressed as parts per trillion by volume. Dibromochloromethane was
detected in the air samples from Magnolia, AR, El Dorado, TX, Chapel Hill, NC, Beaumont TX,
and Lake Charles, LA at mean concentrations of 0 ppt, 0.48 ppt, 14 ppt, 14 ppt, and 19 ppt,
IV - 20 November 15, 2005
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respectively. Bromoform was detected in air samples from Magnolia, AR, El Dorado, TX, and
Lake Charles, LA, at concentrations of 1.5 ppt, 0.81 ppt, and 50 ppt, respectively. Air
concentration data from these sites were combined for additional statistical analysis. The study
authors indicated that a value of 0.0 was entered for samples below the detection limit. Mean (±
standard deviation) outdoor air concentrations in urban/suburban and source dominated
locations, respectively, were 160 ± 29 ppt and 1.2 ± 0.4 ppt for bromodichloromethane; 15 ± 4
ppt and 0.28 ± 0.67 ppt for dibromochloromethane; and 50 ± 29 ppt and 1.1 ± 2.1 ppt for
bromoform. Brodzinsky and Singh (1983) also calculated overall (grand) means based on data
from all sites. Grand mean values for bromodichloromethane, dibromochloromethane, and
bromoform were 110 ppt (n = 26, with one nondetect), 3.8 ppt (n = 89, with 63 nondetects), and
3.6 ppt (n = 78, with 60 nondetects), respectively. When expressed on a i-ig/m3 basis, the
corresponding mean values for bromodichloromethane, dibromochloromethane, and bromoform
are 0.74 |ig/m3, 0.032 |ig/m3, and 0.037 |ig/m3.
IV-21 November 15, 2005
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Table IV-8 Selected Concentration Data for Individual Brominated Trihalomethanes (ppt) in Outdoor Air as Summarized in
Brodzinsky and Singh (1983)ab
City
n
Nondetects
Mean
(Std dev.)
Median
3rd Quartile
Maximum
Reference
Bromodichloromethane
Individual Sites
Beaumont, TX
Chapel Hill, NC
El Dorado, AR
Magnolia, AR
11
6
7
2
0
0
1
0
180 (100)
120 (210)
1.4 (0.35)
0.76 (0.0)
180
120
1.6
0.0
180
120
1.6
0.0
180
120
1.6
0.76
Wallace (1981)
Wallace (1981)
Pellizzari and Bunch (1979)
Pellizzari and Bunch (1979)
Totals
Urban/Suburban
Source Areas
Grand totals
17
9
26
0
1
1
160 (29)
1.2 (0.41)
110(82)
180
1.6
120
180
1.6
180
180
1.6
180
-
-
-
Dibromochloromethane
Individual Sites
Beaumont, TX
Chapel Hill, NC
El Dorado, AR
Lake Charles, LA
Magnolia, AR
11
6
40
4
28
0
0
35
0
28
14 (0.0)
14 (0.0)
0.48 (0.82)
19 (9.6)
0.0 (0.0)
14
14
0.0
21
0.0
14
14
0.82
27
0.0
14
14
2.5
27
0.0
Wallace (1981)
Wallace (1981)
Pellizzari et al. (1978)
Pellizzari (1979)
Pellizzari etal. (1978)
IV-22
November 15, 2005
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Table IV-8 (cont.)
City
n
Nondetects
Mean
(Std dev.)
Median
3rd Quartile
Maximum
Reference
Dibromochloromethane (cont.)
Totals
Urban/Suburban
Source Areas
Grand Totals
21
68
89
0
63
63
15 (4.2)
0.28 (0.67)
3.8 (6.7)
14
0.0
0.0
14
0.0
2.5
27
2.5
27
-
-
-
Bromoform
Individual Sites
El Dorado, AR
Lake Charles, LA
Magnolia, AR
46
4
28
35
0
25
0.81 (0.95)
50 (29)
1.5 (3.2)
0.43
62
0.0
1.3
68
0.29
2.7
71
8.3
Pellizzarietal. (1978)
Pellizzari and Bunch (1979)
Pellizzari (1979)
Pellizzarietal. (1978)
Totals
Urban/Suburban
Source Areas
Grand Totals
4
74
78
0
60
60
50 (29)
1.1(2.1)
3.6(12)
62
0.0
0.0
68
1.3
1.5
71
8.3
71
-
-
-
a Includes only data considered to be of adequate, good, or excellent quality by the study authors.
b Concentrations are reported as parts per trillion by volume
IV-23
November 15, 2005
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Shikiya et al. (1984) analyzed ambient air samples collected at four urban/industrial
locations in the California South Coast Air Basin from November 1982 to December 1983 for
the presence of halogenated hydrocarbons. Data for bromodichloromethane, dibromochloro-
methane, and bromoform were included in this analysis. The sampling locations were El Monte,
downtown Los Angeles, Dominguez, and Riverside. The air samples were analyzed using gas
chromatography with detection by electron capture. The quantitation limit, defined as a level 10
times greater than the noise level, was 10 ppt by volume for all three brominated
trihalomethanes. The detection limit was defined as three times the noise level. Summary data
for each compound included monthly means and composite means. The monthly means were
calculated as the average of all data at a site that were above the quantitation limit for a single
month; samples with concentrations below the limit of detection were not included in the
calculations. The composite means were calculated as the average value of all data for each
compound above the quantitation limit at each site. Most data in this report were presented
graphically. A few additional details were presented in a short summary statement for each
chemical. Thirty-five percent of the samples had bromodichloromethane levels above the
quantitation limit of 10 ppt (0.067 |ig/m3). Peaks in the concentration of bromodichloromethane
were observed at various sites in June and July, with downtown Los Angeles and Dominguez
registering the highest monthly means of approximately 30 ppt (0.20 |ig/m3). The highest
reported concentration was 40 ppt (0.27 |ig/m3). The highest composite mean of 100 ppt (0.67
l-ig/m3) for bromodichloromethane was observed at El Monte. In comparison, the remaining
three locations had a composite mean of 20 ppt (0.08 |ig/m3). For dibromochloromethane, only
seventeen percent of the samples had levels above the quantitation limit of 10 ppt (0.085 |ig/m3).
The highest reported concentration, monthly mean, and mean composite for
dibromochloromethane were 290 ppt (2.5 |ig/m3), 280 ppt (2.4 |ig/m3), and 50 ppt (0.43 |ig/m3),
respectively; all were recorded in downtown Los Angeles in June. Only two monthly means
were above 160 ppt; the remainder of the monthly means were below 60 ppt. For bromoform,
thirty-one percent of the samples had concentrations above the quantitation limit of 10 ppt (0.10
|ig/m3). Peaks in the concentration of bromoform were observed at various sites in May and
June, with the downtown Los Angeles site registering the highest composite mean (40 ppt; 0.41
l-ig/m3) and the highest monthly mean (310 ppt; 3.2 |ig/m3) in June 1983. Only two monthly
means were greater than 160 ppt; the remainder of the monthly means were below 60 ppt.
Atlas and Schauffler (1991) collected replicate air samples at various locations on the
Island of Hawaii during a month-long field experiment to test an analytical method for
determining halocarbons in ambient air. Dibromochloromethane was found at a mean level of
0.27 ppt, and bromoform was found at a mean concentration of 1.9 ppt. Information on sample
size and detection limit were not provided in the secondary source that reported this study (U.S.
EPA 1994b).
Wallace et al. (1982) conducted a pilot study designed to field test personal air-quality
monitoring methods. Personal air samples were collected from students at two universities:
Lamar University, Texas, located near a petrochemical manufacturing area, and the University of
North Carolina (UNC), located in a nonindustrialized area. The samples were analyzed for a
number of volatile organic compounds, including brominated trihalomethanes.
Bromodichloromethane was detected in 64% of personal air samples from 11 Lamar students,
IV - 24 November 15, 2005
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with a mean of 1.23 |ig/m3 (0.18 ppb), a median of 1 i-ig/m3 (0.15 ppb), and a range of 0.12-3.72
|ig/m3 (0.018-0.56 ppb). The limit of detection was 0.24 |ig/m3 (0.036 ppb). AtUNC, 17% of
the samples from 6 students had detectable levels of bromodichloromethane. Concentrations
ranged from 0.12-4.36 |ig/m3 (0.017-0.65 ppb) (mean, 0.83 |ig/m3 (0.12 ppb); median,
0.12 i-ig/m3 (0.017 ppb)). Based on the above information, the average daily intake of
bromodichloromethane from air using an inhalation rate of 20 m3/day was estimated to be 25
l-ig/day for Lamar students and 17 jig/day for UNC students. Dibromochloromethane was not
present above 0.12 |ig/m3 (0.018 ppb) at either site.
b. Concentrations in Indoor Air
Relatively few studies have reported the concentrations of trihalomethanes in the indoor
air of homes. Kostiainen (1995) identified over 200 volatile organic compounds in indoor air of
26 houses identified by residents as causing symptoms such as headache, nausea, irritation of the
eyes, drowsiness, and fatigue. Bromoform was detected at low (unspecified) levels in 54 percent
of the homes, and no mention was made of dibromochloromethane or bromodichloromethane.
Weisel et al. (1999) measured brominated trihalomethane concentrations in indoor air in
New Jersey residences selected to examine low and high levels of drinking water contamination
with trihalomethanes. Descriptive statistics for trihalomethane concentration in water were
provided for the combined high and low concentration groups, but not for the individual
categories. One valid 15-minute air sample was collected at each of 48 residences. The indoor
air concentrations of bromodichloromethane averaged 0.38 ± 0.82 (SD) |ig/m3 (0.057 ± 0.12
ppb) and 0.75 ± 0.96 |ig/m3 (0.11 ± 0.14 ppb) from the low and high water concentration groups,
respectively. The detection frequencies were 12/25 and 16 723 in the low and high water
concentration groups, respectively. The indoor air concentrations of dibromochloromethane
averaged 0.44 ± 0.95 |ig/m3 (0.052 ±0.11 ppb) and 0.53 ± 0.84 |ig/m3 (0.062 ± 0.09 ppb) from
the low and high water concentration groups with detection frequencies of 5/25 and 7/23,
respectively. For bromoform, the average concentrations from the low and high water
concentration groups were 0.29 ± 0.93 |ig/m3 (0.028 ±0.089 ppb) and 0.35 ± 0.94 |ig/m3 (0.034 ±
0.091 ppb), with detection frequencies of 8/25 and 4/23, respectively. It was not clear whether
the averages were based on all measured samples or only those samples that were above the
detection limit for each compound.
Kerger et al (2000) evaluated the transfer of bromodichloromethane and
dibromochloromethane to indoor air in bathrooms during showering and bathing in homes
supplied with chlorinated tap water. The test sites were three urban homes containing three
bedrooms, a full bath, and approximately 1000 square feet of living space. The compounds were
simultaneously measured in hot and cold tap water (drawn from the kitchen sink) and in the
shower/bath enclosure and bathroom vanity area. Three shower protocols were examined: 6.8
min unventilated shower; 12 min unventilated shower and 6.8 min ventilated shower. Water
flow rate and temperature were monitored but not controlled. Airborne vapor samples were
captured by Summa canister and measured by gas chromatography using electron capture
detection according to U.S. EPA method TO-14. Air samples were collected before, during and
after the water use event, for a total of 16 showers and 7 baths. Data for several events were
IV - 25 November 15, 2005
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eliminated because of technical difficulties. For all shower protocols combined (n = 12), the
increase in average airborne concentration (± standard error), expressed as i-ig/m3, in shower
enclosure or bathroom air per |ig/L in water, was 1.8 ± 0.3 for bromodichloromethane and 0.5 ±
0.1 for dibromochloromethane. For baths (n = 4), the average concentration increase during the
bath was 0.59 ± 0.21 for bromodichloromethane and 0.15 ± 0.05 for dibromochloromethane.
The relative contribution of each chemical was consistent with the relative concentration in
water and its chemical and physical properties. The average exposures measured in this study
were approximately 30% lower than results reported by other investigators using EPA analytical
methods when data were normalized for water concentration, flow rate, shower volume, and
duration. This difference may have resulted from differences in the air exchange rate between
residential showers and laboratory test showers. These data are not adequate for characterizing
levels of individual brominated trihalomethanes in the home because the measurements targeted
a specific area of the residences and the sample size consisted of only three homes.
c. Estimates of Exposure from Air
The data available for occurrence of brominated trihalomethanes in air do not permit
calculation of a nationally aggregated intake estimate for the U.S. general population. To
accurately estimate total daily inhalation exposures, factors including location and season, the
fraction of time spent indoors compared with outdoors, potential exposures of individuals while
showering or bathing, potential exposure from volatilization of brominated trihalomethanes
during other household activities (e.g., use of dishwashers, toilet flushing), exposures of
individuals who spend large amounts of time at indoor pools, and potential for occupational
exposures (e.g., for laundromat or sewage treatment plant workers) require consideration.
Although the existing data do not permit such a refined analysis, they may be used to roughly
estimate intake from air. Based on the grand means calculated for multiple sampling locations
by Brodzinsky and Singh (1983), exposure to bromodichloromethane, dibromochloromethane
and bromoform resulting from inhalation of outdoor air can be roughly estimated assuming an
inhalation rate of 20 m3/day, 100% absorption, and exposure to outdoor air for a full 24 hours per
day. Using the mean ambient air concentration of 110 ppt (0.74 jig/m3) by volume for all sites
reported in Brodzinsky and Singh (1983), the daily intake of bromodichloromethane from
outdoor air would be 21 |ig/day. Assuming a mean air concentration of 3.8 ppt (0.032 |ig/m3) for
dibromochloromethane, daily intake would be 0.64 |ig/day,. Assuming a mean air concentration
of 3.6 ppt (0.037 |ag/m3) by volume or bromoform, the daily intake would be 0.74 |ig/day.
Because these estimates are based on data from urban/suburban and industrial sites only, they
may represent high end exposures.
Adequate, nationally aggregated occurrence data are not available for calculating intake
of brominated trihalomethanes from indoor air. The indoor air concentrations measured by
Weisel et al. (1999) were not used for intake calculations because it could not be determined
how the means for each compound were calculated (i.e., whether all measurements were
averaged or only those above the detection limit). In addition, the data were based on a single 15
minute air sample collected from each of 48 homes located in a single state.
IV - 26 November 15, 2005
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While brominated trihalomethane concentrations might be expected to be higher in
indoor air than in outdoor air due to confined space and additional indoor air sources (e.g.
volatilization from showering, baths, and other household activities), the available data do not
allow such a comparison.
Based on data from personal air monitors, Wallace et al. (1982) estimated daily
inhalation of bromodichloromethane to be 25 jig/day for 11 students attending a university
located near a petrochemical manufacturing area and 17 [ig/day for 6 students attending a
university in a nonindustrialized area.. The personal air monitors registered
bromodichloromethane from both indoor (with the exception of showering and bathing) and
outdoor exposures. Dibromochloromethane was not detected and no data were available for
bromoform.
3. Concentrations and Exposures Associated with Swimming Pools and Hot Tubs
Numerous studies have reported data for concentrations of brominated trihalomethanes
and exposures associated with swimming pools and hot tubs. Exposure of swimmers or hot tub
users to brominated trihalomethanes may result from dermal, ingestion, and inhalation exposure.
When evaluating these data, it is important to note that additional disinfectants are routinely
added to water contained in swimming pools and hot tubs; therefore, the levels of brominated
trihalomethanes present may not be representative of those in tap water.
Armstrong and Golden (1986) measured bromodichloromethane, dibromochloromethane,
and bromoform concentrations in the water and surrounding air of four indoor swimming pools,
five outdoor swimming pools, and four hot tubs. Concentrations in air were measured two
centimeters from the water surface. The bromodichloromethane concentrations of water in the
outdoor pools ranged from 1 to 72 |ig/L (ppb) (mean, 33 |ig/L). Levels in the indoor pools
ranged from 1 to 90 |ig/L (ppb) (mean, 16 i-ig/L). The levels of bromodichloromethane in the hot
tubs ranged from <0.1 to 105 |ig/L (ppb) (mean, 17 i-ig/L). Means and ranges of the bromo-
dichloromethane concentration two meters above the water surface for outdoor pools, indoor
pools, and hot tubs, respectively, were: <0.1 i-ig/m3 (<0.015 ppb) (range not reported), 1.7 |ig/m3
(0.25 ppb) (range <0.1-10 |ig/m3 (0.015 -1.5 ppb)), and 1.4 |ig/m3 (0.21 ppb) (range O.1-10
I-ig/m3 (0.015 -1.5 ppb)). The dibromochloromethane concentration of water in the outdoor
pools ranged from <0.1 to 8 |ig/L (ppb) (mean, 4.2 |ig/L (ppb)). Levels in the indoor pools
ranged from 0.3 to 30 |ig/L (ppb) (mean, 9.5 |ig/L (ppb)). The level of dibromochloromethane in
the hot tubs ranged from <0.1 to 48 |ig/L (ppb) (mean, 14.4 |ig/L (ppb)). Means and ranges of
the dibromochloromethane concentration two meters above the water surface for outdoor pools,
indoor pools, and hot tubs, respectively, were: <0.1 i-ig/m3 (<0.01 ppb) (range not reported), 0.9
Hg/m3 (0.11 ppb) (<0.1-5 ng/m3 (0.012-0.59 ppb)), and 0.7 ng/m3 (0.08 ppb) (<0.1- 5 ng/m3
(0.012-0.59 ppb)). The mean bromoform concentration in the outdoor pools was less than
0.1 |ag/L (ppb). Levels in the indoor pools ranged from less than 0.1 to 20 |ig/L (ppb) (mean,
6 |ag/L (ppb)). The levels of bromoform in the hot tubs ranged from less than 0.1 to 62 |ig/L
(ppb) (mean, 13 |ig/L (ppb)). Means and ranges of the bromoform concentration two meters
above the water surface for outdoor pools, indoor pools, and hot tubs, respectively, were: <0.1
IV - 27 November 15, 2005
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|ig/m3 (<9.7 ppt) ( range not reported), 9 |ig/m3 (870 ppt) (<0.1-14 |ig/m3 (9.7-1360 ppt)), and 8
3 ( 770 ppt) (0.1-14 Lig/m3 (9.7-1360 ppt)).
Cammann and Hiibner (1995) compared concentrations of trihalomethanes in swimmers'
and bath attendants' blood and urine before and after swimming or working in indoor swimming
pools. Water and air concentrations were measure in different locations in the pool environment.
The purpose was to determine whether blood levels of trihalomethanes would reflect inhalation
exposure to trihalomethanes in the pool environment and whether those compounds also would
appear in urine. Measured concentrations of bromodichloromethane, dibromochloromethane,
and bromoform in samples of swimming pool waters collected at a depth of 10 to 20 cm were
0.69 to 5.64 jig/L (ppb), 0.03 to 6.51 jig/L (ppb), and 0.14 to 2.32 jig/L (ppb) for the three
compounds, respectively. Averages (± SD) of the 10 pool water measurements presented in
Table 1 of the report were 2.12 ± 1.52 jig/L (ppb), 1.11 ± 2.07 jig/L (ppb), and 0.42 ± 0.73 jig/L
(ppb) for bromodichloromethane, dibromochloromethane, and bromoform, respectively.
Average (± 1 SD) concentrations in the four air samples taken (location of sampling not
specified) were 15.4 ± 7.36 ng/m3 (2.30 ± 1.10 ppb), 1.94 ± 1.01 ng/m3 (0.228 ±0.119 ppb), and
below the quantitation limit (QL) (not specified, although probably 0.02 ppb) for
bromodichloromethane, dibromochloromethane, and bromoform, respectively.
Measurements of bromodichloromethane in 8 bath attendants' blood before their shifts
ranged from below QL for 12/18 measurements (67%) to 0.1 |ig/L (ppb) (Camman and Hiibner,
1995). After their shifts, the concentrations ranged from below QL in 7/18 measurements (39%)
to 0.6 |ig/L (ppb). Similarly, measurements of bromodichloromethane in swimmers' blood was
higher after than before swimming. Before swimming, blood concentrations of
bromodichloromethane ranged from less than the QL in 10/20 (50%) swimmers to 0.2 |ig/L
(ppb); while after swimming, blood concentrations were above the QL in all 20 swimmers,
ranging from ~ 0.02 to 0.4 |ig/L (ppb) in 19 of the swimmers. The twentieth swimmer had a
blood concentration of ~ 1.5 |ig/L (ppb). For all but two of the swimmers, blood concentrations
of bromodichloromethane had dropped below the QL by the next day (values for the other two
swimmers were less than 0.1 |ig/L (ppb)). Dibromochloromethane and bromoform were not
detected in the blood of either the bath attendants or swimmers. None of the brominated
trihalomethanes were detected in the urine of the study subjects. Thus, only exposure to
bromodichloromethane by inhalation (bath attendants) or inhalation, dermal absorption, and
ingestion (swimmers) is reflected in increased blood levels of the compound. Blood levels of
bromodichloromethane usually returned to pre-exposure levels within 24 hours after the
exposure .
Aggazzotti et al. (1998) evaluated concentrations of trihalomethanes in the blood and
breath of five competitive swimmers regularly training in an indoor swimming pool in Italy.
The group included three males and two females between the ages of 17 and 21 years. All were
non-smokers. Concurrent sampling of blood, alveolar air, and environmental air occurred at five
times for each of four sessions: (a) at the University Department two hours before arriving at the
pool, (b) after one hour sitting near the edge of the pool, (c), after one hour of swimming, (d)
back at the University one hour after swimming ended, and (e) at the University 1.5 hr after
swimming ended. While bromodichloromethane and dibromochloromethane were always found
IV - 28 November 15, 2005
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in water and environmental air samples at the pool immediately before and after the 1-hr
swimming session, bromoform was rarely detected in the indoor pool air. None of the three
brominated trihalomethanes were detected in the air at the University Department or in the
alveolar air of the swimmers at the Department two hours before arriving at the pool. At the
pool, prior to the swimming session, the means (± SD) of the four measured ambient air
concentrations of bromodichloromethane, dibromochloromethane, and bromoform werelO.5 ±
3.1 |ig/m3 (1.6 ± 0.46 ppb; 4 detects), 5.2 ±1.5 |ig/m3 (0.61 ± 0.17 ppb; 4 detects), and 1 detect of
0.2 |ag/m3 (0.02 ppb), respectively. At the pool, just after the 1-hr swimming session, the means
(± SD) of the four measured ambient air concentrations of bromodichloromethane,
dibromochloromethane, and bromoform were 20.0 ±4.1 |ig/m3 (2.99 ±4.1 ppb; 4 detects), 11.4
±2.1 |ig/m3 (1.34 ± 0.23 ppb; 4 detects), and 1 detect of 0.2 |ig/m3 (0.02 ppb), respectively.
Concentrations of bromodichloromethane and dibromochloromethane in the alveolar air
of the swimmers before and after the swimming session indicated inhalation uptake of both
compounds (Aggazzotti et al., 1998). At the pool, prior to the swimming session, the means (±
SD) of the 20 measured alveolar air concentrations (5 swimmers assessed at each of 4 sessions)
of bromodichloromethane and dibromochloromethane were 2.7 ±1.2 |ig/m3 (0.40 ± 0.18 ppb)
and 0.8 ± 0.8 jig/m3 (0.09 ± 0.09 ppb), respectively. Bromoform was not detected in any of the
20 samples. At the pool, after the 1-hr swimming session, the means (± SD) of the alveolar air
concentrations of bromodichloromethane and dibromochloromethane were 6.5 ±1.3 |ig/m3 (0.97
±0.19 ppb) and 1.4 ± 0.9 |ig/m3 (0.16 ± 0.11 ppb), respectively. Bromoform was not detected in
any of the 20 samples. Blood levels of bromodichloromethane and dibromochloromethane
before and after swimming, on the other hand, were below detection limits in most samples, and
hence showed no trends.
Aggazzotti et al. (1998) estimated uptake of the trihalomethanes of the resting and active
swimmers using the following assumptions. At rest, the pulmonary ventilation rate of the
women was 6 liters per minute (L/min) while that of men was 7.5 L/min. During swimming, the
ventilation rate of the women was 25 L/min while that of the men 36 L/min. The estimated
uptake rates of bromodichloromethane for the five swimmers at rest ranged from 2.8 to 3.7 jig
per hour (|ig/h), with a mean value of 3.3 ± 0.41(SD) jig/h for the three males and two females
combined. The estimated uptake rates for the same individuals actively swimming were 20 to 30
|ig/h, with a mean value of 26 ± 5.1 |ig/h. The estimated uptake rates of dibromochloromethane
for the five swimmers at rest ranged from 1.5 to 2.0 |ig/h, with a mean value of 1.8 ± 0.23 |ig/h.
The estimated uptake rates during swimming increased to between 14 and 22 jig/h, with a mean
value of 18 ± 3.6 |ig/h. Occurrence of dermal uptake was acknowledged but not estimated.
Lindstrom et al. (1997) also assessed exposure of two competitive swimmers to
bromodichloromethane during training sessions at an indoor pool. The indoor pool air
concentrations of bromodichloromethane collected over 60- and 119-minute intervals were 2.76
and 3.02 |ig/m3 (0.41 and 0.45 ppb), respectively. Breath samples were collected from the
swimmers before, during, and for 3 hours after a training workout. Breath samples collected
during the workout demonstrated a rapid uptake of bromodichloromethane to maximum alveolar
concentrations of 5 to 6 |ig/m3 (0.7 to 0.9 ppb), which are higher than the ambient air
concentrations. The authors concluded that significant (80% of total exposure) dermal
IV - 29 November 15, 2005
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absorption of the related trihalomethane chloroform from water was occurring, but did not
estimate the extent of dermal uptake for bromodichloromethane.
4. Soil Concentrations and Exposure
Data on the concentration of brominated trihalomethanes in soil were not available in the
materials reviewed for this document. Based on the measured Henry's Law constant and vapor
pressure of the individual compounds, volatilization from both wet and dry soil surfaces should
be relatively rapid (U.S. EPA 1987). Therefore, exposure from soil ingestion is not considered
to be a significant route for exposure to the brominated trihalomethanes.
C. Overall Exposure
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. The use of the 80% value for
the RSC for dibromochloromethane is supported by limited use of this chemical in industrial
applications with potential for direct release to the environment. The use of the 80% value is
further supported by apparently low concentrations in foods and soils and the potential for
human exposure to dibromochloromethane in tap water via three exposure routes: 1) ingestion as
drinking water; 2) inhalation of volatilized dibromochloromethane during use of tap water for
household activities; and 3) by dermal exposure during showering, bathing, or other activities.
The available data for concentrations of outdoor air and food, although limited, suggest that
exposures via these routes are likely to be low when compared to 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.
IV - 30 November 15, 2005
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D. Body Burden
1. Blood and Breath Levels
Barkley et al. (1980) analyzed blood samples from nine residents of the old Love Canal
area in 1978 for a variety of volatile organic compounds, including all three of the brominated
trihalomethanes. Bromodichloromethane was detected in the blood of one individual; its
concentration was 14 |ig/L (ppb). Dibromochloromethane and bromoform were not detected.
Antoine et al. (1986) analyzed the blood of 250 environmentally sensitive patients for 18
volatile organic compounds. Bromoform concentrations ranged from undetectable to 3.4 |ig/L
(ppb), with a mean of 0.6 |ig/L (ppb).
Ashley et al. (1994) analyzed samples of whole blood of 600 or more people in the
United States who participated in the Third National Health and Nutrition Examination Survey
(NHANES III) for 32 volatile organic compounds using analytical methods designed to measure
extremely low concentrations. Bromodichloromethane, with a detection limit of 0.009 |ig/L
(ppb) was detected only in 14% of 1072 samples. Dibromochloromethane, with a detection limit
of 0.013 |ig/L (ppb), was detected in only 12% of 1035 samples. Using unprocessed commercial
Vacutainer Tubes, Ashley et al. (1994) initially obtained measures of bromoform concentrations
in blood similar to those reported by Antoine et al. (1986). However, using Vacutainer Tubes
that had been processed to removed VOCs prior to use, Ashley et al. (1994) detected bromoform
in less than 10% of samples analyzed at a detection limit of 0.027 |ig/L (ppb). Wallace (1997)
obtained the summary statistics for bromodichloromethane and dibromochloromethane, which
were not published in Ashley et al. (1994). The mean (± SD) of the measured blood
concentrations were 0.0077 ± 0.0178 and 0.00886 ± 0.00856 |ig/L (ppb), respectively. The
median values were below the limit of detection. The upper 90th percentile values were 0.0122
and 0.0151 |ig/L (ppb), respectively.
Weisel et al. (1999) measured brominated trihalomethanes in the exhaled breath of
female subjects after showering. The study authors recruited 49 women who had previously
participated in a case-control study on neural tube birth defects from locations throughout the
state of New Jersey (Klotz and Pyrch, 1999). The method used to select the subjects provided a
wide range of brominated trihalomethane exposures within the home, in contrast to a distribution
of exposures that might exist within a single water distribution system or within the general
population. Exposure to brominated trihalomethanes was estimated by collection of duplicate
cold tap water samples, collection of a 15-minute air sample, and responses to a 48-hour recall
questionnaire on water use in the home. Post-shower whole breath samples were collected by
having the subject blow into a Tedlar® sampling bag at the conclusion of a shower. Background
breath samples were collected at a subsequent home visit by the investigators. Valid samples
were obtained from 33 of the subjects. However, the time of post-shower sample collection as
reported by the subjects varied from immediately after the shower to 20 minutes later. As noted
by the authors, the delay in sample collection is an important determinant in breath
concentrations because trihalomethane breath concentration declines exponentially after
exposure ceases. As a result, each subject was assigned to one of three groups: 1) breath sample
IV-31 November 15, 2005
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collected within 5 minutes after completion of shower (Group A; n = 13); 2) breath sample
collected within 5 to 20 minutes after completion of shower (Group B; n=14); or 3) breath
sample collected more than 20 minutes after showering (Group C; n=6). The breath
concentrations on individual brominated trihalomethanes for each group were compared to
measured water concentrations and estimates of exposure (calculated as the product of the water
concentration and reported duration of the shower; shower duration data and calculated exposure
estimates were not reported).
The mean (± standard deviation) concentrations of bromodichloromethane,
dibromochloromethane, and bromoform were 5.7 ± 8.6, 2.0 ± 2.1, and 0.73 ± 0.90 |ig/L (ppb),
respectively. The median values for the three compounds were 2.6, 1.4, and 0.45 jig/L (ppb),
respectively. Bromodichloromethane showed significant correlations for breath and water
concentration and breath and shower exposure for Groups A and B. Significant correlations for
dibromochloromethane and bromoform were found for Group A participants. Analytical
variability related to low concentrations of dibromochloromethane and bromoform (near the
detection limit) may have obscured trends in the data for Group B. source of in the houses and
found significant correlations between the water concentration of each brominated
trihalomethane and the concentration of that trihalomethane in expired air if the air samples were
collected within 5 minutes of showering. Results of statistical analysis for Group C were not
reported because the sample size was small and the authors considered the results questionable.
The observed results were considered consistent with showering being a source of exposure to
brominated trihalomethanes.
Backer et al. (2000) examined levels of brominated trihalomethanes in whole blood
following three types of water use events by adult volunteers: showering for 10 minutes in tap
water (n=l 1); bathing for 10 minutes in a tub filled with tap water (n=10); or consumption of one
liter of tap water over a 10 minute period (n=10). Each participant provided a blood sample
immediately before exposure, 10 minutes after exposure ended, and 30 minutes (showering and
bathing) or one hour (ingestion) after exposure. Tap water and blood samples were analyzed by
purge-and-trap/gas chromatography/mass spectrometry with detection capability in the parts per
quadrillion range. Bromoform was not detected in either tap water or whole blood. Mean tap
water concentrations of bromodichloromethane and dibromochloromethane were 6 |ig/L and 1.1
l-ig/L, respectively. The highest levels of these compounds in whole blood occurred 10 minutes
after exposure had ended. The second post-exposure measurements showed that blood levels of
both compounds had decreased, but were still above the pre-exposure baseline levels in subjects
who took showers or baths. Measurement data are shown in Table IV-9 below.
IV - 32 November 15, 2005
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Table IV-9 Mean Bromodichloromethane Concentrations in Blood Following Three Types
of Water Use Events
Water Use Event
10 Minute Shower
10 Minute Bath
Ingestion of 1 L
Median Bromodichloromethane Concentration in Whole Blood (pg/mL)
Pre-exposure
3.3
2.3
2.6
10 minutes post-exposure
19.4
17.0
3.8
30 or 60 minutes post-
exposure
10.3 (30 min)
9.9 (30 min)
2.8 (60 min)
The study authors reported that similar relative findings were obtained for
dibromochloromethane (data shown graphically in the study report). These data indicate a
dramatic difference between the whole blood levels resulting from ingestion and those resulting
from bathing or showering (including dermal, inhalation, and possibly ingestion exposure).
Blood level increases observed for each compound after ingestion of one liter of water were less
than 10% of those observed after bathing or showering for 10 minutes. The blood level
increases observed for each compound 10 minutes after bathing or showering were significantly
increased (p<0.01) when compared to the post-ingestion blood levels. Measurable levels of
bromodichloromethane and dibromochloromethane in the pre-exposure whole blood samples
were attributed to recent prior exposure or to bioaccumulation after repeated exposure to tap
water.
In addition to the differences observed in whole blood levels of bromodichloromethane
and dibromochloromethane among exposure groups, the study authors observed that the blood
concentration data for each chemical occurred in two clusters within each exposure group. The
mean increases for the two clusters observed after bathing or showering were significantly
different for bromodichloromethane. The same individuals who had greater increases of
bromodichloromethane also experienced greater increases of dibromochloromethane and
chloroform in the blood after bathing or showering. The underlying basis for the observed
clustering is unknown, but was not related to gender. The study authors suggested that
polymorphic expression of a metabolizing enzyme (e.g. glutathione-S-transferase theta) or
differences in fitness level (resulting in inhalation of larger volumes of air) may have accounted
for the observed pattern. However, they noted that differences in fitness level would more likely
be expected to result in a continuous distribution.
Lynberg et al. (2001) conducted a field study in Corpus Christi, Texas, and Cobb County,
Georgia, to evaluate exposure measures for disinfection by-products, including brominated
trihalomethanes. These areas were selected for study based on the following criteria: 1)
relatively high trihalomethane concentrations relative to national averages; 2) high intrasystem
differences that would result in a potential exposure gradient across the study population; 3) one
water distribution system with predominately chlorinated species of trihalomethanes (i.e.,
chloroform) and one water system with predominately brominated trihalomethanes; and 4) a
IV-33
November 15, 2005
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water utility service population large enough to allow rapid selection of 25 mothers per
geographic area who had given birth to healthy babies from June, 1998 through May, 1999.
Exposure to individual trihalomethanes was assessed by collection of blood and water samples
and by collection of information on water use patterns and tap water characteristics. Whole
blood samples were collected before and after showering. Levels of individual trihalomethanes
were determined for samples collected in the home of participants, in the distribution system,
and at the water treatment plants. A modified version of the Total Exposure Model (TEM) was
used to estimate uptake of trihalomethanes into the bloodstream (data for chloroform exposure
were presented for one individual in Corpus Christi). The results of the study indicate that
concentration of individual trihalomethanes varied by site and location within the water system
(Table IV-10). In Corpus Christi water samples, brominated trihalomethanes accounted for 71%
of the total trihalomethane concentration by weight. In contrast, brominated trihalomethanes
accounted for only 12% of the trihalomethanes in Cobb County water samples. Significant
differences (p = 0.0001) in the blood levels of dibromochloromethane and bromoform were
observed between study locations (Table IV-11). The differences between locations were
evident both before and after showering. The study authors indicated that there was considerable
variability in blood levels of trihalomethanes among participants from a single location. For
example, pre-shower chloroform blood levels in Cobb County ranged from 130 ppt to 1100 ppt.
No data were presented for the brominated trihalomethanes. The variability was tentatively
attributed to different patterns of household water use among participants. Significant increases
(p = 0.0001) in blood levels of all brominated trihalomethanes were observed after showering.
The increases in dibromochloromethane and bromoform were significantly greater in Corpus
Christi than in Cobb County. No TEM modeling data were presented for brominated
trihalomethanes. However, TEM results presented for chloroform exposure for one study
participant who consumed bottled water indicated that inhalation exposure in the household
accounted for approximately 98% of the calculated 24-hour chloroform dose, with the remainder
attributed to the dermal route. Overall, this study demonstrates that blood levels of brominated
trihalomethanes vary significantly across populations, with water quality characteristics and
water use activities being important variables.
Table IV-10 Median Tap Water Trihalomethane Levels (ppb) in Cobb County and Corpus
Christi Homes, Water Treatment Plants, and Distribution Systems
Trihalomethane
Bromodichloromethane
Dibromochloromethane
Bromoform
Chloroform
Cobb County
Home
(n=25)
13.5
1.7
NDa
84.8
Distribution
System
(n=20)
12.5
2.4
ND
79
Water
Treatment
Plant
(n=7)
9.5
1.4
ND
49.5
Corpus Christi
Home
(n=25)
12.2
13.5
8.7
8.2
Distribution
System
(n=30)
8.3
12.6
9.7
4.6
Water
Treatment
Plant
(n=20)
9.5
14.3
11.9
6.7
' ND, not detected (detection limit < Ippb)
IV-34
November 15, 2005
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Table IV-11 Between Site Comparison of Median Blood Levels (ppt) and Changes in Blood
Levels (ppt) after Showering
Trihalomethane
Bromodichloromethane
Dibromochloromethane
Bromoform
Chloroform
Before Shower
Cobba
6.2
1.2
0.3
70
Corpus"
6.8
7.0
3.5
25
After Shower
Cobb
38
6.1
0.5
280
Corpus
43
41
17
57
Change in Blood Level after
Showering
Cobb
30
5.0
0.2
189
Corpus
34
35
12
25
a Cobb County, Georgia
b Corpus Christi, TX
2. Mother's Milk
Pellizzari et al. (1982) analyzed the milk of eight nursing mothers for various
compounds, including bromodichloromethane and dibromochloromethane. The samples were
collected from 49 lactating women living in the vicinity of chemical manufacturing plants and/or
industrial user facilities in Bridgeville, PA, Bayonne, NJ, Jersey City, NJ, and Baton Rouge LA.
Both compounds were identified in one of the eight samples. Actual concentrations and
detection limits were not reported. Kroneld and Reunanen (1990) did not detect any of the
brominated trihalomethanes in human milk in a study conducted in Turku, Finland.
E. Summary
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 by study in the treatment of data below the analytical detection level
or minimum reporting level.
When all available national survey data are considered, bromodichloromethane concen-
trations in drinking water range from below the detection limit to 183 |ig/L (ppb), while
dibromochloromethane and bromoform concentrations range from below the detection limit to
280 i-ig/L (ppb). When data for the three brominated trihalomethanes are compared, the
frequency of detection and measured concentrations of bromodichloromethane in drinking water
IV-35
November 15, 2005
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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 |ig/L, respectively, for
bromodichloromethane (range of individual values 0-65.8 i-ig/L); 2.4, 4.72, and 13.2 |ig/L,
respectively, for dibromochloromethane (range 0 - 67.3); and 0. 1.18, and 3.10, respectively, for
bromoform (range 0 - 3.43).
Relatively few studies have analyzed non-beverage foods for the occurrence of
brominated trihalomethanes. In the few studies available, bromodichloromethane has been
detected in non-beverage 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 (probably below the minimal quantitation limit)). 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 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. With the exception of one of the 13 soft drinks examined by McNeal et al. (1995)
with a concentration of 12 ppb, none of the at least 18 other measured concentrations of
bromodichloromethane in soft drinks described above (three from Entz et al. (1982), three from
Uhler and Diachenko (1987), and the remaining 12 from McNeal et al. (1995)) exceeded a value
of 4 ppb. Bromodichloromethane was detected in one sample of fruit juice at 5 ppb.
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,
IV - 36 November 15, 2005
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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 lag/person/day, respectively. The
same values for populations exposed to bromodichloromethane from ground water systems are
lower - 3.6, 8.1, and 22 lag/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 lag/person/day, respectively. The corresponding values for populations exposed
to dibromochloromethane from groundwater system are lower - 2.7, 6.2, and 18 lag/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 lag/person/day, respectively. The
same values for populations exposed to bromoform from ground water systems are higher -
0.65, 3.8, and 9.6 lag/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 |ig/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
l-ig/day. Similarly, median exposure to dibromochloromethane may range from 4.2 to 7.8 jig/day,
and for bromoform, median exposure may range from 4.8 to 8.4 |ig/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 |ig/L (ppb), exposure to bromodichloromethane from surface water is estimated
to be between 12 and 28 |ig/day. Similarly, based on the range of medians reported for
dibromochloromethane concentrations, the median exposure is estimated to be up to 6 jig/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 |ig/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 |ig/L (ppb), the exposure to
bromoform is estimated to range from 1 to 2 jig/day for surface water supplies.
IV - 37 November 15, 2005
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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 jig/person/day. The upper
90th percentile estimates range from <2.0 to 90 jig/person/day. Estimates of the average
population intake of dibromochloromethane from drinking water range from 0.2 to 56
|ig/person/day. The upper 90th percentile estimates range from < 0.9 to 86 jig/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 jig/person/day.
The upper 90th percentile estimates range from 2.4 to 26 jig/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
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.
Average daily intake of dibromochloromethane via ingestion, dermal contact, and
inhalation of compound volatilized during household use were also estimated for determination
of the relative source contribution (RSC). Intake for ingestion was calculated using mean intake
rates of 1.2 or 0.6 L/day for total and direct intake (NRC, 1999), respectively. Direct intake
includes consumption of water directly from the tap, but does not include intake of tap water
used for preparation of heated items such tea, coffee, or soup. Based on the ICR distribution
system average concentration of 4.72 |ig/L for dibromochloromethane in surface water, the
average daily total and direct and ingestion intakes would be 5.7 and 2.8 jig/day, respectively.
The average dermal uptake of dibromochloromethane was estimated to be 2 |ig per shower or
bathing event. Average daily intake via inhalation of dibromochloromethane volatilized during
showering was estimated to be 7 |ig/day. Parallel calculations were not performed for
bromodichloromethane or bromoform, because these compounds are probable carcinogens.
Therefore, in accordance with U.S. EPA policy, RSC analysis was not conducted.
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.
IV - 38 November 15, 2005
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Concentrations in outdoor air were 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 |ig/m3) for
bromodichloromethane, 3.8 ppt (0.032 |ig/m3) for dibromochloromethane, and 3.6 ppt (0.037
l-ig/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 |ig/m3) for
bromodichloromethane; 290 ppt (2.5 |ig/m3) for dibromochloromethane; 310 ppt (3.2 |ig/m3) for
bromoform. Bromodichloromethane was detected in 64% (n=l 1) and 17% (n=6) of personal air
samples collected in Texas and North Carolina. The detected concentrations ranged from 0.12 to
4.36 |ag/m3 (0.017 to 0.65 ppb). Dibromochloromethane was not detected.
Mean concentrations in indoor air ranged from 0.38 to 0.75 |ig/m3 for bromodichloro-
methane; 0.44 to 0.53 |ag/m3 for dibromochloromethane, and 0.29 to 0.35 |ig/m3 for bromoform,
as determined from 15 minute samples collected in 48 New Jersey residences. 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.
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 |ig/L (ppb), from 0.1 to 48 |ig/L (ppb),
and from less than 0.1 to 62 |ig/L (ppb), respectively. Concentrations of the same brominated
trihalomethanes in the air two meters above the pool water ranged from less than 0.1 to 14 |ig/m3
(0.015 to 2.09 ppb), from less than 0.1 to 10 |ig/m3 (0.011 to 1.2 ppb), and from less than 0.1 to
5.0 jig/m3 (0.0097 to 0.48 ppb), respectively. Data from several studies confirm the uptake of
brominated trihalomethanes from swimming pools and environs by dermal and/or inhalation
pathways.
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.
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
IV - 39 November 15, 2005
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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 lag/person/day, respectively. The
same values for populations exposed to bromodichloromethane from ground water systems are
lower - 3.6, 8.1, and 22 lag/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 lag/person/day, respectively. The corresponding values for populations exposed
to dibromochloromethane from groundwater system are lower - 2.7, 6.2, and 18 lag/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 lag/person/day, respectively. The
same values for populations exposed to bromoform from ground water systems are higher -
0.65, 3.8, and 9.6 lag/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 to 2.1 |ig/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
l-ig/day. Similarly, median exposure to dibromochloromethane may range from 4.2 to 7.8 jig/day,
and for bromoform, median exposure may range from 4.8 to 8.4 |ig/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 to 14 |ig/L (ppb), exposure to bromodichloromethane from surface water is
estimated to be between 12 and 28 |ig/day. Similarly, based on the range of medians reported
for dibromochloromethane concentrations, the median exposure is estimated to be up to
6 jig/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 |ig/L (ppb). An
estimate of exposure based on the MRLs will be overly conservative because the actual
IV - 40 November 15, 2005
-------
concentration of bromoform is not detectable. Based on the range of MRLs, 0.5-1 |ig/L (ppb),
the exposure to bromoform is estimated to range from 1 to 2 jig/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 jig/person/day. The upper
90th percentile estimates range from <2.0 to 90 jig/person/day. Estimates of the average
population intake of dibromochloromethane from drinking water range from 0.2 to 56
|ig/person/day. The upper 90th percentile estimates range from < 0.9 to 86 jig/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 jig/person/day.
The upper 90th percentile estimates range from 2.4 to 26 jig/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
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.
The RSC 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.
Brominated trihalomethanes have been detected in the blood and breast milk of humans.
A national survey of volatile organic compounds in whole blood detected bromodichloro-
methane, dibromochloromethane, and bromoform in 14%, 12%, and less than 10% of samples,
IV-41 November 15, 2005
-------
respectively, when highly sensitive analytical methods were applied. Several studies have
demonstrated that the level of individual brominated trihalomethanes in blood or breath
increases shortly after exposure to these compounds in tap water during bathing and showering.
Exposure during these events may occur by ingestion, dermal contact and/or inhalation of the
volatilized compound. In studies which examined households with differing concentrations of
brominated trihalomethanes in tap water, the levels of individual brominated trihalomethanes in
blood or exhaled breath paralleled the tap water concentration. The studies of showering and
bathing indicate that water use patterns and water quality characteristics are important variables
in determining the blood levels of brominated trihalomethanes. 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.
IV - 42 November 15, 2005
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V.
HEALTH EFFECTS IN ANIMALS
A. Acute Exposures
This section presents data on the acute effects of brominated trihalomethanes. Acute
lethality values for the brominated trihalomethanes are summarized in Table V-l. Additional
acute toxicity data are summarized in Table V-2.
1. Bromodichloromethane
Acute lethality of bromodichloromethane has been investigated in mice and rats. LD50
values for male and female ICR Swiss mice were 450 and 900 mg/kg, respectively (Bowman et
al., 1978). Chu et al. (1980) determined LD50 values of 916 and 969 mg/kg for male and female
Sprague-Dawley rats, respectively.
Bowman et al. (1978) administered bromodichloromethane in a single gavage dose in
Emulphor®:alcohol:saline (1:1:8) to ICR Swiss mice (10/sex/group). The administered doses
ranged from 500 to 4,000 mg/kg (individual doses not reported). Sedation and anesthesia
occurred at 500 mg/kg. Males were more sensitive to the lethal effects of
bromodichloromethane than females.
Table V-l Summary of LD50 Values for Brominated Trihalomethanes
Compound
Bromodichloromethane
Dibromochloromethane
Bromoform
LD50 Values (mg/kg)
ICR Swiss Mouse a
Male
450
800
1400
Female
900
1200
1550
Sprague-Dawley Rat b
Male
916
1186
1388
Female
969
848
1147
"Bowman etal. (1978)
bChuetal. (1980)
V-l
November 15, 2005
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Table V-2 Summary of Acute Toxicity Studies for Brominated Trihalomethanes
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromodichloromethane
Bowman et al.
(1978)
NTP(1987)
NTP(1987)
Lilly et al.
(1994)
Lilly et al.
(1996)
Lilly et al.
(1997)
Keegan et al.
(1998)
Mouse
ICR
Swiss
Rat
F344/N
Mouse
B6C3F;
Rat
F344
Rat
F344
Rat
F344
Rat
F344
Gavage
(aqueous)
Gavage
(com oil)
Gavage
(oil)
Gavage
(com oil)
(aqueous)
Gavage
(com oil
or water)
Gavage
(aqueous)
Gavage
(aqueous)
M,F
M,F
M,F
M
M
M
M
10
5
5
6
6
5
6
Single
dose
Single
dose
Single
dose
Single
dose
Single
dose
Single
dose
Single
dose
500 - 4000
150
300
600
1,250
2,500
150
300
600
1,250
2,500
0
200 (LOAEL)
400
0
200 (LOAEL)
400
0
123
164 (NOAEL)
246 (LOAEL)
328
492
0
21
31
41 (NOAEL)
82 (LOAEL)
123
164
246
Anesthesia, sedation at 500
mg/kg
Increased mortality at 600
mg/kg-day. Lethargy, labored
breathing at 1250 mg/kg-day and
above, 100% mortality at the
two highest dose groups
100% mortality at the two
highest dose groups
Renal tubule degeneration and
necrosis; alteration in markers of
renal function
Renal tubule necrosis; alteration
in markers of renal function
Decreased body weight; elevated
liver and renal markers
Elevated liver markers;
decreased liver weight and body
weight
Dibromochloromethane
Bowman et al.
(1978)
NTP(1985)
Mouse
ICR
Swiss
Rat
F344/N
Gavage
(aqueous)
Gavage
(com oil)
M,F
M,F
10
5
Single
dose
Single
dose
500 - 4000
160
310
630
1250
2,500
Anesthesia, sedation
Increased mortality at 630 mg/kg
and above, with 100% mortality
in high-dose group
V-2
November 15, 2005
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Table V-2 (cont.)
Reference
NTP(1985)
Milller et al.
(1997)
Species
Mouse
B6C3F,
Rat
Wistar
Route
Gavage
(com oil)
Gavage
(olive oil)
Sex
M,F
M
Number
per dose
group
5
6
Duration
Single
dose
Single
dose
Dose
(mg/kg-day)
160
310
630
1250
2500
0
83
167
333
667
Results
Increased mortality in females at
630 mg/kg-day and above and in
males at 310 mg/kg-day and
above; 100% mortality in males
in two highest dose groups and
females in two highest dose
groups
Transient decrease in blood
pressure and heart rate;
decreased activity; effects on
heart muscle contractility and
changes in some cardiac
parameters
Bromoform
Bowman et al.
(1978)
Chu et al.
(1980)
NTP(1989a)
NTP(1989a)
Mouse
ICR
Swiss
Rat
SD*
Rat
F344/N
Mouse
B6C3F;
Gavage
(aqueous)
Gavage
(com oil)
Gavage
(com oil)
Gavage
(com oil)
M,F
M,F
M,F
M,F
10
10
5
5
Single
dose
Single
dose
Single
dose
Single
dose
500 - 4000
546
765
1071
1500
2100
125
250
500
1,000
2000
125
250
500
1,000
2000
Ataxia, sedation, and anesthesia
at 500 mg/kg
Sedation, ataxia, liver and kidney
congestion
No deaths at 500 and lower;
60% mortality at 1,000; 100%
mortality at 2,000;
shallow breathing in two highest
dose groups
10% mortality at 500 mg/kg-day
* SD, Sprague-Dawley
NTP (1987) administered single gavage doses of bromodichloromethane in corn oil to
male and female F344/N rats and E6C3Fl mice (5/sex/dose) at 150, 300, 600, 1,250, or
2,500 mg/kg. Animals were observed for 14 days, and a necropsy was performed on at least one
male and one female in each dose group. All animals dosed with 1,250 or 2,500 mg/kg died
before the end of the study. At 600 mg/kg, deaths occurred in two of five male rats, one of five
female rats, five of five male mice, and two of five female mice. Clinical signs observed in rats
at 1,250 or 2,500 mg/kg included lethargy and labored breathing. Clinical signs observed in
mice at or above 600 mg/kg included lethargy, with the exception that this sign was not observed
V-3
November 15, 2005
-------
in high-dose male mice. At necropsy, the liver from animals dosed with 1,250 or 2,500 mg/kg
appeared pale. No dose-related effects were seen on body weight gain in animals that survived.
Lilly et al. (1994) examined the effect of vehicle on the toxicity of bromodichloro-
methane. Male F344 rats (6/dose) were administered a single dose of 0, 200, or 400 mg/kg
bromodichloromethane by gavage in corn oil or in an aqueous 10% Emulphor® solution. Body
weights were significantly decreased at 400 mg/kg only in the animals receiving the aqueous
gavage. Absolute and relative kidney weights were significantly increased at 400 mg/kg in both
vehicles, with a significantly greater increase observed in the animals gavaged with oil compared
to those gavaged with 10% Emulphor® solution. Serum markers of hepatotoxicity were
significantly increased at 400 mg/kg in both vehicles with one nonsignificant increase in the
aqueous vehicle. The increases were significantly greater for two of these markers in animals
receiving the oil vehicle compared to those receiving the aqueous vehicle. Clinical observations
were supported by histopathology findings. Hepatocellular degeneration and necrosis were
observed at 400 mg/kg in animals receiving either vehicle. The difference in vehicles was
reflected in more severe hepatocellular degeneration and a higher incidence of centrilobular
necrosis in animals receiving the oil gavage compared to those receiving the aqueous gavage.
Numerous increases in urinary markers of renal toxicity were observed 24 hours after dosing.
Based on the differences observed, renal toxicity at 200 mg/kg was similar or greater in the
aqueous vehicle. Renal toxicity at 400 mg/kg, however, was greater in the oil vehicle. The time
to peak toxicity was both dose- and vehicle-dependent. At 200 mg/kg, peak damage was
observed at 24 hours in animals receiving either vehicle. At 400 mg/kg, peak damage was
observed at 48 hours following oil gavage and at 24-36 hours following aqueous gavage.
Histopathology revealed both renal tubule degeneration and necrosis at both dose levels. The
incidence of renal tubule degeneration was greater in animals receiving the aqueous gavage at
the low dose; however, the severity of renal degeneration and necrosis was greater in the animals
receiving the oil gavage at the high dose. The authors attributed the vehicle differences to slower
gastrointestinal uptake of bromodichloromethane from the oil vehicle compared to the aqueous
vehicle. At the high dose, more bromodichloromethane would be converted to a reactive
metabolite following oil dosing, while saturation would occur following aqueous dosing. At the
low dose, the difference in uptake would have less of an effect. Overall, this study found that the
kidney was more sensitive than the liver to a single dose of bromodichloromethane. A LOAEL
of 200 mg/kg was identified for each vehicle based on minimal renal tubule degeneration and
changes in markers of renal function.
Lilly et al. (1996) investigated the effect of subchronic pretreatment with corn oil on the
toxicity of bromodichloromethane. Prior to initiation of dosing with bromodichloromethane,
male Fischer 344 rats (6 animals/group) were gavaged with oral doses of corn oil or water for six
weeks (5 days/week) at a constant volume of 5 mL/kg. Following pretreatment, the animals
were gavaged with a single dose of 0, 200, or 400 mg bromodichloromethane/kg in 10%
Emulphor®. Urine was collected at 24, 36, and 48 hours following bromodichloromethane
administration. The rats were sacrificed at 48 hours and necropsies were performed. Activities
of the hepatotoxicity indicators alanine aminotransferase (ALT), aspartate aminotransferase
(AST), lactate dehydrogenase (LDH), and sorbitol dehydrogenase (SDH) were measured in the
serum, and the renal toxicity indicators alkaline phosphatase (ALK), AST, and LDH, were
V - 4 November 15, 2005
-------
measured in the urine. Additional analyses included determination of serum levels of bile acids,
triglycerides, cholesterol and albumin, and urine levels of N-acetylglucosaminidase and gamma
glutamyl transpeptidase activity. Enzymatic activities of cytochrome P450 isoforms CYP2E1
and CYP2B1/B2 were measured in the microsomal fraction of the liver to investigate whether
corn oil was an inducer of bromodichloromethane metabolizing enzymes.
Liver weight was significantly reduced only in the water pretreatment group at the high
dose. Kidney weight was reduced in both pretreatment groups at the high dose. Activities of
serum AST and LDH were significantly elevated in both pretreatment groups at 400 mg/kg.
ALT levels increased in a dose-dependent manner in the water pretreatment group, but
significant elevations were noted only at the 400 mg/kg dose in animals pretreated with corn oil.
Activities of urinary AST and LDH were greater than controls in both pretreatment groups after
24, 36, and 48 hours. ALK levels were significantly increased in both pretreatment groups at 24
hours. At 36 and 48 hours, ALK levels were elevated only in water-pretreated animals. High
incidences of renal tubular necrosis occurred at 200 and 400 mg/kg in both pretreatment groups.
There were no significant differences in the histopathological lesion scores between the
pretreatment groups. No significant differences were noted in the hepatic activity of CYP2E1 or
CYP2B1/B2 in the corn oil pretreated animals compared to the water controls. Although a
number of differences between the pretreatment groups were noted in results for specific
endpoints, the overall results from this study indicate that 6 weeks of pretreatment with corn oil
did not significantly enhance the acute hepato- or nephrotoxicity of bromodichloromethane. In
addition, the reported data suggest that vehicle-related differences in toxicity observed in other
bromodichloromethane studies are most likely due to pharmacokinetic differences in absorption
rather than altered enzyme activity induced by corn oil. This study confirms the acute LOAEL
of 200 mg/kg-day previously identified by Lilly et al. (1994) for renal toxicity.
Lilly et al. (1997) administered single doses of bromodichloromethane by gavage in
aqueous 10% Emulphor® solution to male F344 rats at dose levels of 0, 123, 164, 246, 328, or
492 mg/kg. Groups of 5 animals/dose were sacrificed at 24 and 48 hours post-dosing. Body
weights were significantly decreased at or above 246 mg/kg after 48 hours. At 24 hours,
absolute and relative kidney weights were significantly increased at or above 328 mg/kg and
246 mg/kg, respectively. At 48 hours, only relative kidney weight at the high dose was
significantly increased. At 24 hours, serum markers of liver damage (ALT and AST) were
significantly increased at or above 246 mg/kg with one marker (SDH) increased at all dose
levels. Although smaller statistically significant increases were observed at the low doses at 24
hours for ALT (123 and 164 mg/kg) and AST (164 mg/kg), the biological significance of these
increases is unclear. After 48 hours, serum levels of these markers were decreased from 24-hour
levels with statistically significant changes noted only at the higher doses. No effects in urinary
markers of kidney damage were found at either 123 or 164 mg/kg. These markers, however,
were significantly elevated after 24 hours for doses at or above 246 mg/kg with few exceptions.
No histopathological examination was conducted. These results were generally consistent with
earlier results (Lilly et al., 1994), although the present study was conducted at doses low enough
to identify a NOAEL. In contrast to the earlier results of Lilly et al. (1994), this study did not
find that the kidney was more sensitive than the liver to the toxic effects of
V - 5 November 15, 2005
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bromodichloromethane. Based on hepatotoxicity and nephrotoxicity, this study identified a
NOAEL of 164 mg/kg and a LOAEL of 246 mg/kg.
Keegan et al. (1998) investigated the acute toxicity of bromodichloromethane
administered orally in an aqueous vehicle. Male Fischer 344 rats (6 animals/group) were
gavaged with a single dose of 0, 0.125, 0.1875, 0.250, 0.5, 0.75, 1.0 or 1.5 mmol/kg dissolved in
a 10% aqueous solution of Alkamuls EL-620. These doses of bromodichloromethane are
equivalent to 0, 20.5, 30.7, 41.0, 81.9, 122.9, 163.8, and 245.7 mg/kg, respectively. Control
animals were dosed with vehicle only (10% Alkamuls EL-620). Gavage volumes were kept
constant at 5 ml/kg body weight. Animals were sacrificed 24 hours after dose administration
and the liver, kidneys, and serum were harvested. Significant decreases in body weight were
observed in animals treated with 0.75, 1.0, or 1.5 mmol/kg. Decreases in absolute liver weights
were observed in the 0.5, 0.75, 1.0, or 1.5 mmol/kg animals. No change was noted in relative
liver weights. Absolute kidney weights were not affected by bromodichloromethane treatment,
but relative kidney weights were significantly increased in the two highest dose groups (1.0 and
1.5 mmol/kg). Serum levels of ALT, SDH, and AST were assessed as an indication of liver
toxicity. Dose-dependent elevations in ALT (45% to 239% increase), AST (25% to 130%
increase) and SDH (74% to 378% increase) were observed in the 0.5, 0.75, 1.0, and 1.5 mmol
dose groups. Based on these findings, 0.25 mmol/kg (41.0 mg/kg) represents the NOAEL and
0.5 mmol/kg (81.9 mg/kg) represents the LOAEL for orally administered bromodichloromethane
in an aqueous vehicle. The study authors used the NOAEL of 41.0 mg/kg to calculate One-Day
Health Advisories for drinking water of 4 mg/L for a 10-kg child and 14 mg/L for a 70-kg adult.
2. Dibromochloromethane
The acute oral lethality of dibromochloromethane has been assessed in rats and mice of
both sexes. Chu et al. (1980) reported LD50 values in male and female Sprague-Dawley rats of
1,186 and 848 mg/kg-day for males and females, respectively. Bowman et al. (1978) reported
LD50values in mice of 800 and 1,200 mg/kg-day, respectively.
Bowman et al. (1978) investigated the acute oral toxicity of dibromochloromethane in
ICR Swiss mice (10/sex/group). Doses of 500 to 4000 mg/kg (individual doses not reported)
were administered by gavage in Emulphor®:alcohol:saline (1:1:8) to fasted animals. Sedation
and anesthesia occurred at 500 mg/kg. Males were more sensitive than females to the acute
lethal effects of dibromochloromethane.
NTP (1985) evaluated the acute toxicity of dibromochloromethane in male and female
F344/N rats. The rats (5 animals/sex/dose) received single doses of 160, 310, 630, 1,250, or
2,500 mg/kg dibromochloromethane by gavage in corn oil. The observation period following
treatment was 14 days. Mortality in high-dose rats was 100% by day 3. At the 1,250 mg/kg
dose, four male rats and one female rat died. One female rat died in the 630 mg/kg group.
Doses of 310 mg/kg or greater produced lethargy in all animals for 3 hours after dosing. A gross
necropsy was conducted on one or two animals from each group. No treatment-related effects
were observed in rats selected for gross necropsy.
V - 6 November 15, 2005
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In a concurrent study, NTP (1985) evaluated the acute toxicity of dibromochloromethane
in male and female B6C3FJ mice (5/sex/dose). The mice received single doses of 160, 310, 630,
1,250, or 2,500 mg/kg dibromochloromethane by gavage in corn oil. The observation period
following treatment was 14 days. All male mice receiving the 2,500 mg/kg and 1,250 mg/kg
doses died. Three male mice receiving the 630 mg/kg dose died, while a single male mouse died
at the 310 mg/kg dose. All female mice receiving the 2500 mg/kg dose died. Four of the
female mice administered the 1,250 mg/kg dose died between days 2 and 8 post-treatment. No
female mice died at doses of 630 mg/kg or lower. A gross necropsy was conducted on one or
two animals from each group. At necropsy, aberrations of the kidney (dark red or pale
medullae) and liver (discolored foci) were reported to be more frequently observed in treated
animals than in control animals (raw data were not presented in the study).
Miiller et al. (1997) investigated the cardiotoxic effects of acute dibromochloromethane
exposure. Male Wistar rats were administered a single dose of dibromochloromethane by
gavage in olive oil at dose levels of 0, 83, 167, 333, or 667 mg/kg. Telemetric measurements of
cardiovascular parameters (heart rate, blood pressure, body temperature, and physical activity)
were recorded in conscious rats (6/group) from 24 hours prior to administration to 72 hours
following administration. Heart rate and blood pressure were also measured in urethane-
anesthetized rats (10/group) 25 minutes following administration. For these rats, contractility
parameters, such as the Krayenbiihl index, were also calculated. Treatment-related arrhythmias
were not observed in conscious rats dosed with 83 to 333 mg/kg of dibromochloromethane,
while rats in the high-dose group exhibited premature ventricular contractions one minute
following administration. Heart rate and body temperature were initially decreased in all
treatment groups following administration, but returned to control values 24 hours post-exposure
in rats administered 83 to 333 mg/kg. In the high-dose rats, heart rate remained depressed up to
48 hours post-exposure, and body temperature decreased 4.5°C below control values by 72 hours
post-exposure. Blood pressure was initially increased in all treatment groups following
administration, but began to return to control values within 48 hours post-exposure in rats
administered 83 to 333 mg/kg. Blood pressure in the high-dose group, however, decreased
below control values 72 hours post-exposure. Physical activity was decreased in conscious rats
administered 333 and 667 mg/kg during the entire observation period. In urethane-anesthetized
rats, negative effects on muscle contractility were observed at dose levels of 333 and 667 mg/kg,
negative chronotropic (rate of contraction) effects were observed at the 333 mg/kg dose level,
and negative dromotropic (defined as influencing the velocity of conduction of excitation, as in
nerve or cardiac muscle fibers) effects were observed at dose levels of 167 to 667 mg/kg. Heart
rate, blood pressure, and several contractility parameters, however, did not exhibit dose-related
trends.
3. Bromoform
Bowman et al. (1978) assessed the acute oral toxicity of bromoform in ICR Swiss mice.
Groups often male (30 to 35 g) and ten female (25 to 30 g) mice were treated with single doses
ranging from 500 to 4,000 mg/kg. Compounds were solubilized in Emulphor®:alcohol:saline
(1:1:8) and administered by gavage to fasted animals. The period of observation following
treatment was 14 days. LD50 values were 1400 and 1550 mg/kg for males and females,
V - 7 November 15, 2005
-------
respectively. Ataxia, sedation, and anesthesia occurred within 60 minutes of treatment at doses
of 1000-mg/kg and above. Sedation lasted approximately 4 hours.
Chu et al. (1980) evaluated the acute toxicity of bromoform in male and female Sprague-
Dawley rats. Fasted adult rats (10/sex/dose) received doses of 546, 765, 1071, 1500, or 2100
mg/kg bromoform dissolved in corn oil by gavage. Clinical observations were made for 14 days
after treatment. The LD50 values for male and female rats were 1388 and 1147 mg/kg,
respectively. Clinical signs observed in treated rats included sedation, flaccid muscle tone,
ataxia, piloerection, and hypothermia. Gross pathological examination revealed liver and kidney
congestion in treated animals. Chu et al. (1982a) reported results for growth, food intake, organ
weight, histopathology, hematological indices, liver microsome aniline hydroxylase activity and
serum chemistry in surviving rats. Bromoform treatment increased liver protein concentration in
the serum of male rats at doses of 765 and 1071 mg/kg. Lymphocyte counts were decreased in
male (765 and 1071 mg/kg doses) and female (765 mg/kg) rats but the effect was not dose-
dependent. Female rats at the 765 mg/kg dose had elevated aniline hydroxylase levels.
NTP (1989a) investigated the acute oral toxicity of bromoform in male and female
F344/N rats. The rats (5/sex/group) were administered a single oral dose of bromoform (by
gavage, in corn oil) at dose levels of 125, 250, 500, 1,000, or 2,000 mg/kg. Control groups were
not included in the study design. Mortality was 10/10 at 2,000 mg/kg, 6/10 at 1,000 mg/kg, and
0/10 at 500 mg/kg or lower. Shallow breathing was observed in rats that received the 1000 or
2000 mg/kg doses. No other clinical signs were reported.
NTP (1989a) investigated the acute oral toxicity of bromoform in male and female
B6C3Fj mice. The mice received a single oral dose of bromoform (by gavage, in corn oil) at
dose levels of 125, 250, 500, 1,000, or 2,000 mg/kg. There were no controls. Mortality was
0/10 at 2,000 mg/kg, 6/10 at 1,000 mg/kg, 1/10 at 500 mg/kg, and 0/10 at 250 mg/kg or lower.
The final mean body weight of mice that survived to the end of the study period was unaffected
by bromoform exposure. Male mice that received doses of 500, 1,000, or 2,000 mg/kg and
females that received 1,000 or 2,000 mg/kg were lethargic. Shallow breathing was noted in male
mice administered the 1,000 or 2,000 mg/kg dose.
B. Short-Term Exposures
This section summarizes short-term studies (less than approximately 90 days) on the
health effects of brominated trihalomethanes in animals. Details of these studies are summarized
in Table V-3.
V - 8 November 15, 2005
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Table V-3 Summary of Short Term Toxicity Studies for Brominated Trihalomethanes
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromodichloromethane
Oral Exposure
Chu et al.
(1982a)
Munson et al.
(1982)
Condie et al.
(1983)
NTP(1987)
NTP(1987)
Aida et al.
(1992a)
Aida et al.
(1992a)
Thornton-
Manning et al.
(1994)
Thornton-
Manning et al.
(1994)
Rat
SD*
Mouse
CD-I
Mouse
CD-I
Rat
F344/N
Mouse
B6C3F;
Rat
Wistar
Rat
Wistar
Rat
F344
Mouse
C57BL/
6J
Drinking
water
Gavage
(aqueous)
Gavage
(com oil)
Gavage
(com oil)
Gavage
(com oil)
Diet
Diet
Gavage
(aqueous)
Gavage
(aqueous)
M
M,F
M
M,F
M,F
M
F
F
F
10
8-12
8-16
5
5
7
7
6
6
28 days
14 days
14 days
14 days
14 days
1 month
1 month
5 days
5 days
0
0.8
8
68 (NOAEL)
0
50 (NOAEL)
125 (LOAEL)
250
0
37
74 (NOAEL)
148 (LOAEL)
0
38
75
150 (NOAEL)
300 (LOAEL)
600
0
19
38
75 (NOAEL)
150 (LOAEL)
300
0
21
62 (NOAEL)
189 (LOAEL)
0
21
66 (NOAEL)
204 (LOAEL)
0
75 (NOAEL)
150 (LOAEL)
300
0
75 (NOAEL)
150 (LOAEL)
No signs of toxicity observed.
Decreased immune function;
increased liver weight,
decreased absolute and relative
spleen wt. (females)
Liver and kidney
histopathology
Decreased body weight gain;
renal pathology
Mortality, renal histopathology
Liver histopathology
Liver histopathology
Liver histopathology, renal
histopathology; increased liver
and kidney wt.;
elevated markers of
hepatotoxicity
Increased serum markers of
hepatotoxicity
V-9
November 15, 2005
-------
Table V-3 (cont.)
Reference
Potter et al.
(1996)
Melnick et al.
(1998)
NTP(1998)
NTP(1998)
Coffin et al.
(2000)
Lock et al.
(2004)
Lock et al.
(2004)
Species
Rat
F344
Mouse
B6C3F;
Rat
SD
Rat
SD
Mouse
B6C3F;
Rat
F344
Mouse
B6C3F;
Route
Gavage
(aqueous)
Gavage
(com oil)
Drinking
water
Drinking
water
Gavage
(Corn oil)
Drinking
water
Gavage
Gavage
Sex
M
F
M,F
M,F
F
M
M
Number
per dose
group
10
6
5-13
10
5
6
Duration
1,3, or 7
days
3 weeks
(5 d/wk)
2 weeks
35 days
11 days
11 days
28 days
(5 d/wk)
28 days
(5 d/wk)
Dose
(mg/kg-day)
123
246 (NOAEL)
0
75 (NOAEL)
ISO(LOAEL)
326
0
11
45 (NOAEL)
91 (LOAEL)
124
Group A males
0
9 (NOAEL)
38 (LOAEL)
67
0
150 (LOAEL)
300
0
138 (LOAEL)
0
50 (LOAEL)
100
0
25
50 (LOAEL)
Results
No effect in hyaline droplet
formation or cell proliferation
Increased abs. and relative liver
weight; increased serum
markers of hepatotoxicity;
hepatocyte degeneration;
increased labeling index
Transient reduction in weight
gain
Single cell hepatic necrosis in
Group A males
Hydropic degeneration in liver
(com oil gavage and drinking
water); increased relative liver
weight (gavage); increased
proliferating cell nuclear
antigen labeling index (gavage)
Increased formic acid excretion
by the kidneys; decrease urinary
pH; increased labeling index in
renal cells (only at 100 mg/kg)
Increased formic acid excretion
by the kidneys. At both doses,
no increased cell proliferation
or clinical/histological evidence
of renal damage.
Inhalation Exposure
Torti et al.
(2001)
Mouse
C57BL/6
FVB/N
(wild-
type)
Vapor
M
6
1 week
(6 hr/day)
0 ppm
1 ppm
(NOAEL)
10 ppm
(LOAEL)
30 ppm
100 ppm
1 50 ppm
Dose-dependent marginal
increase in renal tubular
degeneration in C57BL/6 mice;
mild increase in renal tubular
degeneration and marginal
increase in hepatic degeneration
in FVB/N mice; sign, increased
labeling index
V-10
November 15, 2005
-------
Table V-3 (cont.)
Reference
Torti et al.
(2001)
Torti et al.
(2001)
Torti et al.
(2001)
Torti et al.
(2001)
Species
Mouse
C57BL/6
(p53
hetero-
zygous)
FVB/N
(p53
hetero-
zygous)
Mouse
C57BL/6
FVB/N
(wild-
type)
Mouse
C57BL/6
FVB/N
(p53
hetero-
zygous)
Route
Vapor
Vapor
Vapor
Vapor
Sex
M
M
M
M
Number
per dose
group
6
6
6
6
Duration
1 week
(6 hr/day)
1 week
(6 hr/day)
3 weeks
(6 hr/day)
3 weeks
(6 hr/day)
Dose
(mg/kg-day)
0
1 ppm
(NOAEL)
10 ppm
(LOAEL)
30 ppm
100 ppm
1 50 ppm
0 ppm
0.3 ppm
1 ppm
3 ppm
(NOAEL)
10 ppm
(LOAEL)
30 ppm
Oppm
0.3 ppm
1 ppm
3 ppm
(NOAEL)
10 ppm
(LOAEL)
30 ppm
0 ppm
0.3 ppm
1 ppm
3 ppm
(NOAEL)
10 ppm
(LOAEL)
30 ppm
Results
Dose-dependent marginal to
mild increase in renal tubular
degeneration; sign, increased
labeling index
Dose-dependent mild increase
in renal tubular degeneration
and marginal increase in
nephrosis; marginal increase in
hepatic degeneration; sign.
increased relative kidney wt.
and labeling index.
Marginal increase in renal
tubular degeneration
Marginal or mild increase in
renal tubular degeneration in
both strains; marginal increase
in hepatic degeneration in
FVB/N heterozygous strain
Dibromochloromethane
Munson et al.
(1982)
Chu et al.
(1982a)
Condie et al.
(1983)
Mouse
CD-I
Rat
SD
Mouse
CD-I
Gavage
(aqueous)
Drinking
water
Gavage
(com oil)
M,F
M
M
8-12
10
8-16
14 days
28 days
14 days
0
50 (NOAEL)
125 (LOAEL)
250
0
0.7
8.5
68 (NOAEL)
0
37
74 (NOAEL)
147 (LOAEL)
Decreased immune function
No effect on growth, clinical
signs, biochemical or
histopathological endpoints
Decreased PAH uptake,
moderate liver and kidney
histopathology
V-ll
November 15, 2005
-------
Table V-3 (cont.)
Reference
NTP(1985)
NTP(1985)
Aida et al.
(1992a)
Aida et al.
(1992a)
Potter et al.
(1996)
Melnick et al.
(1998)
Coffin et al.
(2000)
Species
Rat
F344/N
Mouse
B6C3F;
Rat
Wistar
Rat
Wistar
Rat
F344
Mouse
B6C3F;
Mouse
B6C3F,
Route
Gavage
(com oil)
Gavage
(com oil)
Diet
Diet
Gavage
(aqueous)
Gavage
(com oil)
Gavage
(Corn oil)
Drinking
water
Sex
M,F
M,F
M
F
M
F
F
Number
per dose
group
5
5
7
7
10
10
Duration
14 days
14 days
1 month
1 month
1,3, or 7
days
3 weeks
(5 d/wk)
11 days
11 days
Dose
(mg/kg-day)
0
60
125
250 (NOAEL)
500 (LOAEL)
1,000
0
30
60 (NOAEL)
125 (LOAEL)
250
500
0
18 (NOAEL)
56 (LOAEL)
173
0
34 (NOAEL)
101 (LOAEL)
333
156
3 12 (NOAEL)
0
50
100 (NOAEL)
192 (LOAEL)
417
0
100 (LOAEL)
300
0
171 (LOAEL)
Results
Mortality; liver and renal gross
pathology
Liver and kidney gross
pathology
Liver histopathology
Liver histopathology; increased
relative liver weight
No effect on hyaline droplet
formation or cell proliferation
Liver histopathology; increased
serum enzymes and liver
weight. Significant increase of
hepatocyte labeling index only
at the highest dose of 417
mg/kg-day
Increased proliferating cell
nuclear antigen labeling index ,
increased relative liver wt.
(gavage); Mild lobular
ballooning hepatocytes (gavage
& drinking water)
Bromoform
Munson et al.
(1982)
Chu et al.
(1982a)
Mouse
CD-I
Rat
SD
Gavage
(aqueous)
Drinking
water
M,F
M
6-12
10
14 days
28 days
0
50
125 (NOAEL)
250 (LOAEL)
0.7
8.5
80 (NOAEL)
Increased serum enzyme
activity (AST); decrease in
antibody forming cells and
delayed-type hypersensitivity
response
No effect on growth, clinical
signs, biochemical or
histopathological endpoints
V-12
November 15, 2005
-------
Table V-3 (cont.)
Reference
Condie et al.
(1983)
NTP(1989a)
NTP(1989a)
Aida et al.
(1992a)
Aida et al.
(1992a)
Potter et al.
(1996)
Melnick et al.
(1998)
Coffin et al.
(2000)
Species
Mouse
CD-I
Rat
F344/N
Mouse
B6C3FJ
Rat
Wistar
Rat
Wistar
Rat
F344
Mouse
B6C3F,
Mouse
B6C3F,
Route
Gavage
(com oil)
Gavage
(com oil)
Gavage
(com oil)
Diet
Diet
Gavage
(aqueous)
Gavage
(com oil)
Gavage
(com oil)
Drinking
Water
Sex
M
M,F
M
M
F
M
F
F
Number
per dose
group
8-16
5
5
7
7
10
10
Duration
14 days
14 days
14 days
1 month
1 month
1,3, or 7
days
3 weeks
(5 d/wk)
11 days
11 days
Dose
(mg/kg-day)
0
72
145 (NOAEL)
289 (LOAEL)
0
100
200 (NOAEL)
400 (LOAEL)
600
800
0
50
100
200 (NOAEL)
400 (LOAEL)
600
0
62 (NOAEL)
187 (LOAEL)
618
0
56 (NOAEL)
208 (LOAEL)
728
190
379 (NOAEL)
0
200 (NOAEL)
500 (LOAEL)
0
200 (LOAEL)
500
0
301 (LOAEL)
Results
Decreased PAH uptake,
moderate histopathological
changes
Decreased body weight gain;
1/5 died at 400 mg/kg-
day; 100% mortality at two
highest doses
Stomach nodules;
ataxia, lethargy; 1/5 died at
high dose
Hepatic vacuolization, serum
chemistry /biochemistry
Hepatic vacuolization, serum
chemistry /biochemistry
No effect on hyaline droplet
formation or cell proliferation
Increase in absolute and relative
liver wt; marginally significant
increase in LI at highest dose
Liver histopathology; increased
proliferating cell nuclear
antigen labeling index (gavage
and drinking water); increased
relative liver wt (gavage).
* SD, Sprague-Dawley
V-13
November 15, 2005
-------
1. Bromodichloromethane
Munson et al. (1982) administered bromodichloromethane by aqueous gavage to male
and female CD-I mice (8 to 12/sex/group) for 14 days at levels of 0, 50, 125, or 250 mg/kg-day.
Endpoints evaluated included body and organ weights, hematology, serum enzyme levels
(SGOT, SGPT), and humoral and cell-mediated immune system functions. At 250 mg/kg-day,
body weights were significantly decreased. Significant organ weight changes included increased
relative liver weight (mid- and high-dose groups), decreased absolute spleen weight (high-dose
males and mid- and high-dose females), and decreased relative spleen weight (mid- and high-
dose females).
Among the hematology endpoints, only fibrinogen levels were significantly decreased in
high- dose males and in mid- and high-dose females. Significant clinical chemistry findings
included decreased glucose levels (high-dose males), increased ALT and AST activities (high-
dose groups), and increased blood urea nitrogen (BUN) levels (high-dose groups).
Bromodichloromethane appeared to affect the humoral immune system, as judged by
significantly decreased antibody-forming cells (high-dose males and mid- and high-dose
females) and hemagglutination liters (mid- and high-dose males and high-dose females). This
study identified a NOAEL of 50 mg/kg-day and a LOAEL of 125 mg/kg-day for
bromodichloromethane on the basis of decreased immune function in females.
Chu et al. (1982a) administered bromodichloromethane to male Sprague-Dawley rats
(10/group) in drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. These levels
corresponded to doses of 0, 0.8, 8.0, or 68 mg/kg-day, as calculated by the authors based on
recorded fluid intake. The authors observed no effects on growth rate or food consumption and
no signs of toxicity throughout the exposure. No dose-related biochemical or histologic changes
were detected (no data were provided). This study identified a NOAEL of 68 mg/kg-day, but the
reported data were too limited to allow an independent verification.
Condie et al. (1983) investigated the renal and hepatic toxicity of bromodichloromethane
in male CD-I mice (8 to 16/group). Bromodichloromethane was administered by gavage in corn
oil for 14 days at dose levels of 0, 37, 74 or 148 mg/kg-day. Biochemical evidence of liver
damage (significantly elevated ALT) was observed at the high dose, while biochemical evidence
of kidney damage (significantly decreased p-aminohippurate (PAH) uptake by kidney slices)
was observed at the mid and high dose. Significantly decreased BUN levels were observed in
the low- and mid-dose groups, but not in the high-dose group. Histopathology revealed no
consistent or important changes at the low or mid-level doses, with minimal to moderate liver
and kidney injury observed in the majority of animals at the high dose. Liver lesions included
centrilobular pallor and focal inflammation. Kidney lesions included intratubular mineralization,
epithelial hyperplasia, and cytomegaly. Although the severity of these lesions was primarily
minimal to slight, a few animals in the high-dose group exhibited moderate to moderately severe
intratubular mineralization and/or epithelial hyperplasia. This study identified a NOAEL value
of 74 mg/kg-day and a LOAEL value of 148 mg/kg-day for bromodichloromethane, based on
histopathology.
V - 14 November 15, 2005
-------
NTP (1987) administered doses of 0, 38, 75, 150, 300, or 600 mg/kg-day of
bromodichloromethane in corn oil by gavage to male and female F344/N rats (5/sex/dose) for
14 days. One low-dose and one high-dose female died before study termination. All high-dose
animals were hyperactive after dosing and either lost weight or gained no weight during the
study. Final mean body weights were not significantly affected in groups given 38, 75, or
150 mg/kg-day. At 300 mg/kg, body weights of males and females were decreased by 21% and
7%, respectively, relative to vehicle controls. At 600 mg/kg-day, body weights of males and
females were decreased by 44% and 22%, respectively, relative to vehicle controls. Necropsy
was performed on all animals. Renal medullae were reddened in all high-dose males and in one
female in each of the control, low-dose, and high-dose groups. This study identified a NOAEL
of 150 mg/kg-day and a LOAEL of 300 mg/kg-day in rats, based on decreased body weight gain.
In a parallel experiment, NTP (1987) administered doses of 0, 19, 38, 75, 150, or
300 mg/kg-day bromodichloromethane in corn oil by gavage to male and female B6C3FJ mice
(5/sex/dose) for 14 days. All male mice that received 150 or 300 mg/kg-day
bromodichloromethane died before study termination. Clinical signs included lethargy,
dehydration, and hunched posture. The final mean body weights of the mice that survived were
not significantly different from the controls. The renal medullae were reddened in four males in
the 150 mg/kg-day group, all males in the 300 mg/kg-day group, and one female in the 150
mg/kg-day group. Based on behavior, appearance, gross necropsy, and mortality, this study
identified a NOAEL of 75 mg/kg-day and a frank effect level (FEL) of 150 mg/kg-day in male
mice. An interesting point to note is that this study and the study by Condie et al. (1983) were
conducted under similar conditions (mice administered bromodichloromethane by gavage in
corn oil for 14 days), but with dramatically different results. In contrast to the 100% mortality
observed in this study for male mice, Condie et al. (1983) found only moderately severe
histopathology in male CD-I mice at 148 mg/kg-day with no deaths occurring. The reason for
this difference is unclear, but may be related to strain-specific differences in sensitivity.
Aida et al. (1992a) administered bromodichloromethane to Slc:Wistar rats (7/sex/group)
for one month at dietary levels of 0%, 0.024%, 0.072%, or 0.215% for males and 0%, 0.024%,
0.076%, or 0.227% for females. The test material was microencapsulated and mixed with
powdered feed; placebo granules were used for the control groups. Based on the mean food
intakes, the study authors reported the mean compound intakes for the one-month period as 0,
20.6, 61.7, or 189.0 mg/kg-day for males and 0, 21.1, 65.8, or 203.8 mg/kg-day for females.
Clinical effects, body weight, food consumption, hematology parameters, serum chemistry, and
histopathology of all major organs were determined. Body weights were significantly decreased
in the high-dose groups relative to the controls. The high-dose animals also exhibited slight
piloerection and emaciation. Relative liver weight was increased only in high-dose females.
Significant, dose-related biochemical findings at the low dose were limited to decreased LDH
levels in males, but the biological significance of this effect is unclear. Serum LDH levels were
also significantly decreased at the low and high dose in females. Other statistically significant,
changes included decreased glucose (high-dose males), decreased serum triglycerides (high-dose
groups), decreased serum cholinesterase activity (high-dose males and mid- and high-dose
females), and increased total cholesterol (mid- and high-dose males). The changes in
cholinesterase activity and cholesterol levels in males were not dose-related. The cholesterol
V - 15 November 15, 2005
-------
levels were within normal ranges at all doses. Treatment-related histopathological lesions were
limited to the liver and were rated as very slight or slight. The lesions were mostly confined to
the high-dose groups. Vacuolization observed in mid-dose females and in a single low-dose
male was not considered an adverse effect. Other observed effects included swelling of
hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct proliferation. These
lesions were observed only in high-dose males and females with the exception of very slight to
slight changes in individual low-dose males. No effect was observed on any hematology
parameter. Based on the histopathology observed in high-dose males and females, the LOAELs
identified in this study for bromodichloromethane in rats were 189.0 mg/kg-day in males and
203.8 mg/kg-day in females; the NOAELs were 61.7 mg/kg-day in males and 65.8 mg/kg-day in
females.
Thornton-Manning et al. (1994) administered bromodichloromethane at dose levels of 0,
75, 150, or 300 mg/kg-day by gavage to female F344 rats (6 animals/dose) for five consecutive
days. The dosing vehicle consisted of an aqueous 10% Emulphor® solution. Animals were
sacrificed on day 6. Two animals in the high-dose group died on day 5. Final body weights of
the high-dose group were significantly decreased compared to the controls. Absolute and
relative kidney and liver weights were significantly increased at 150 and 300 mg/kg-day with the
exception of a nonsignificant increase in absolute liver weight at 150 mg/kg-day. Toxic effects
on the kidney and liver were reflected in significantly increased LDH, AST, SDH, creatinine,
and BUN at 300 mg/kg-day. These results were supported by the histopathology findings. In
the liver, centrilobular vacuolar degeneration was observed at both 150 and 300 mg/kg-day with
the severity of the effect increased with increasing dose. Centrilobular hepatocellular necrosis
was also observed in one high-dose animal. In the kidney, renal tubular vacuolar degeneration
and renal tubule regeneration were observed at 150 and 300 mg/kg-day with the incidence and
severity increased with increasing dose. While minimal renal tubule necrosis was observed in
only one animal at the mid dose, all animals at the high dose exhibited mild to moderate renal
tubule necrosis. Significant decreases in the hepatic activity of the CYP1A and CYP2B markers
ethoxyresorufm-O-dealkylase (EROD) and pentoxyresorufm-O-dealkylase (PROD), were
observed at all doses. The effect, however, was not dose-related. No effect on the CYP2E1
marker paranitrophenol hydroxylase was observed. Based on kidney and liver lesions observed
at the mid dose, this study identified a NOAEL of 75 mg/kg-day and a LOAEL of 150 mg/kg-
day.
Thornton-Manning et al. (1994) conducted an analogous experiment with female
C57BL/6J mice. Six mice per group were administered an aqueous solution (10% Emulphor®)
of bromodichloromethane by gavage for five consecutive days at dose levels of 0, 75 and 150
mg/kg-day. Animals were sacrificed on day 6. All mice survived to the termination of the
experiment. No effect on body, kidney, or liver weight was observed with the exception of a
significant increase in absolute liver weight at 150 mg/kg-day. No change in cytochrome P450
activity was observed, although a nonsignificant dose-related decrease in total P450 content was
observed. ALT was significantly increased at 150 mg/kg-day, and a significant dose-related
increase in SDH activity was observed. Creatinine and BUN were not significantly increased.
No kidney or liver lesions were observed at either dose. Based on increases in serum enzyme
V - 16 November 15, 2005
-------
activity, a LOAEL of 150 mg/kg-day and a NOAEL of 75 mg/kg-day were identified for this
study.
Potter et al. (1996) investigated hyaline droplet formation and cell proliferation in the
kidney of male F344 rats. Test animals (4/dose) received 0.75 or 1.5 mmol/kg of
bromodichloromethane in 4% Emulphor® by gavage for 1, 3, or 7 days. The administered doses
corresponded to 123 or 246 mg/kg-day. No exposure-related increase in hyaline droplet
formation was observed at either dose. Binding of bromodichloromethane to cc2u-globulin was
not measured. Cell proliferation in the kidney was assessed in vivo by [3H]-thymidine
incorporation. No statistically significant effect of bromodichloromethane on tubular cell
proliferation was observed following exposures of up to 7 days, although high labeling levels
were observed in 3 of 4 rats at the 246 mg/kg-day dose.
Melnick et al. (1998) exposed female B6C3FJ mice (10 animals/group) to
bromodichloromethane in corn oil via gavage for 3 weeks (5 days/week). Doses of
bromodichloromethane used in this study were 0 (vehicle only), 75, 150, or 326 mg/kg-day. The
doses were selected on the basis that high incidences of hepatocellular adenoma and carcinoma
were previously seen in female mice exposed at 75 mg/kg or 150 mg/kg-day in a dose-dependent
manner (NTP 1987). There were no treatment-related signs of overt toxicity observed during the
study. Body weight and water intake were not significantly altered at any dose tested. However,
a significant dose-related increase in absolute liver weight and liver weight/body weight ratio
was noted for the 150 and 326 mg/kg-day dose groups. Serum ALT activity was significantly
increased in the two highest dose groups and serum SDH activity was elevated at all doses
tested. At necropsy, there was clear evidence of hepatocyte hydropic degeneration in animals
treated with 150 and 326 mg/kg-day. BrdU was administered to the animals during the last 6
days of the study, and hepatocyte labeling index (LI) analysis was conducted. The two highest
(150 and 326 mg/kg-day) doses resulted in significantly elevated hepatocyte proliferation as
measured by the LI. NOAEL and LOAEL values of 75 and 150 mg/kg-day were identified on
the basis of elevated serum enzyme activity, increased liver weight, increased cell proliferation,
and histological findings.
NTP (1998) evaluated the effect of bromodichloromethane on food and water
consumption by Sprague-Dawley rats in the course of a range-finding experiment for a study of
developmental and reproductive effects. This study was conducted in compliance with Good
Laboratory Practice Regulations as described in 21 CFR 58. Bromodichloromethane was
administered to test animals (6 animals/sex/dose) at nominal concentrations of 0, 100, 500, 1000,
and 1500 ppm in the drinking water for 2 weeks. The average doses of bromodichloromethane
estimated based on water consumption were 11, 45, 91 and 124 mg/kg-day for the 100, 500,
1000 and 1500 ppm dose groups, respectively. All animals were observed twice daily for signs
of toxicity. Body weight data were obtained twice weekly and at termination of the experiment.
Feed and water consumption were measured twice weekly. Animals were euthanized at
termination of the experiment without necropsy. No mortality or treatment-related clinical signs
were observed in any dose group. Body weights and weight gains were comparable among all
dose groups, except for body weight gains on Study Day 5 (the first day of compound
administration) in the 1000 and 1500 ppm dose groups which were decreased 127.5% and
V - 17 November 15, 2005
-------
118.5%, respectively. Feed consumption was also comparable across dose groups, with the
exception of male rats dosed with 1000 and 1500 ppm. Male rats in these dose groups showed
decreases in consumption of 31% and 41% , respectively, on Study Days 1 to 5. Water
consumption was reduced in the 500, 1000, and 1500 ppm dose groups, suggesting that
bromodichloromethane is unpalatable at higher concentrations. The greatest reduction in water
intake was noted on Study Days 1 to 5 (61% and 62% for males in the 1000 ppm and 1500 ppm
dose groups, respectively, and 38%, 40% and 52% for females in the 500, 1000 and 1500 ppm
dose groups, respectively).
NTP (1998) conducted a short-term reproductive and developmental toxicity screen in
Sprague-Dawley rats to evaluate the potential toxicity of bromodichloromethane administered in
drinking water for 35 days. This study was conducted in compliance with the Good Laboratory
Practice Regulations as described in 21 CFR 58. Groups of male and female rats (5-13/sex/dose)
were exposed to drinking water concentrations of 0, 100, 700 and 1300 ppm
bromodichloromethane using the study design described in Table V-6 (Section V.E.I). Feed and
water consumption, body weight, hematology, clinical chemistry, cell proliferation, and
pathology were evaluated in addition to developmental and reproductive endpoints. Based on
water consumption and analytical measurements of bromodichloromethane in the provided
drinking water, the calculated average daily doses were 0, 9, 38, and 67 mg/kg-day for Group A
males (not treated with bromodeoxyuridine); 0, 7, 43, and 69 mg/kg-day for Group B males
(bromodeoxyuridine-treated); and 0, 14, 69, or 126 mg/kg-day for Group C females (peri-
conception exposure, bromodeoxyuridine-treated).
The results for reproductive and developmental effects are reported in Section V.E. 1.
Alterations in hematological endpoints or clinical chemistry were not observed following
bromodichloromethane exposure, with the exception of a 14% drop in creatinine in the 100 ppm
Group A males and a 43% increase in 5'-nucleotidase in the 1300 ppm Group A males when
compared to controls. An increase in 5'-nucleotidase is an indication of hepatobiliary
dysfunction in which there is interference with the secretion of bile, and should be accompanied
by a parallel change in alkaline phosphatase activity. Since alkaline phophatase activity was
unaltered in this study, the toxicological significance of the observed increase in 5'-nucleotidase
was considered uncertain. Organ weight and organ/body weight ratios reported by NTP (1998)
were comparable in all treatment groups for both males and females. Histopathological
examination identified three tissue changes that were potentially treatment-related. Cytoplasmic
vacuolization of hepatocytes and mild liver necrosis were observed in Group A males (see Table
V-6 for details of group assignment) treated with 700 and 1300 ppm bromodichloromethane and
in Group B males treated with 1300 ppm bromodichloromethane. Hepatic necrosis was dose-
dependent, with incidences of 0/10, 0/10, 4/9, and 10/10 observed at 0, 100, 700, and 1300 ppm,
respectively. These changes were not accompanied by an increase in alkaline phosphatase
activity. Hematopoietic cell proliferation in the spleen was observed in Group A males at all
doses of bromodichloromethane. However, the biological significance of this finding with
respect to bromodichloromethane treatment was unclear, since cell proliferation in the spleen
may occur as a response to general stress. Evidence of mild kidney necrosis was evident in
Group A males in the 1300 ppm dose group, but may have resulted from decreased water intake.
BrdU labeling index (LI), a measurement of cell proliferation, was unchanged in the livers and
V - 18 November 15, 2005
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kidneys of Group B males in all dose groups. A small but statistically significant increase in the
LI was noted in the livers and kidneys of Group C females in the 1300 ppm dose group.
As discussed in Sections V.E.I, results from this study indicate that bromodichloro-
methane did not result in reproductive or developmental toxicity at drinking water
concentrations up to 1300 ppm. However, exposure to concentrations of 700 ppm and 1300 ppm
produced changes in liver histopathology in male rats and resulted in decreases in body weight
and food and water consumption in both sexes. On the basis of these results, NTP (1998)
concluded that bromodichloromethane is unpalatable at these concentrations and is a possible
general toxicant in male and female rats at concentrations of 700 ppm and above. Although not
accompanied by changes in alkaline phosphatase activity, the occurrence of individual
hepatocyte cell necrosis was clearly dose-related and thus considered appropriate for
identification of NOAEL and LOAEL values. Based on calculated average daily doses for
Group A males at the 100 and 700 ppm concentrations (Table 6A in NTP, 1998), these data
identify NOAEL and LOAEL values of 9 mg/kg-day and 38 mg/kg-day, respectively, for
occurrence of hepatic cell necrosis.
Coffin et al. (2000) examined the effect of bromodichloromethane administered by corn
oil gavage or in drinking water on cell proliferation and DNA methylation in the liver of female
B6C3F1 mice. Gavage doses of 0, 0.92, or 1.83 mmol/kg (0, 150, or 300 mg/kg, respectively)
were administered to test animals (7-8 weeks old; 10/group) daily for five days, off for two days,
and then again daily for four days. It had previously been shown that bromodichloromethane
administered by corn oil gavage at either 75 mg/kg or 150 mg/kg-day caused significant
increases in the incidence of hepatocellular adenoma and adenocarcinoma, in a dose-dependent
manner, in female mice (NTP 1987). In a separate experiment, bromodichloromethane was
administered in drinking water for 11 days at approximately 75% of the saturation level,
resulting in an average daily dose of 0.85 mmol/kg (138 mg/kg). The mice were sacrificed 24
hours after the last gavage dose and the livers were removed, weighed, and processed for
histopathological examination, proliferating cell nuclear antigen - labeling index (PCNA-LI)
analysis, and determination of c-myc methylation status. A significant, dose-dependent increase
in relative liver weight was observed in animals dosed by gavage; however, relative liver weight
was unaffected in animals administered the compound in drinking water, when compared to
controls. Histopathological findings in gavage-dosed animals consisted of hydropic
degeneration at the low dose and necrosis, fibrosis, and giant cell reaction at the high dose. No
severity or incidence data were provided. The histopathology findings for animals receiving
bromodichloromethane in the drinking water were similar to those observed in the low-dose
gavage group. Bromodichloromethane administered by gavage caused a dose-dependent
increase in the PCNA-LI which was significant at each dose tested when compared to the
control. There was no significant effect when the compound was administered in drinking water.
Administration of bromodichloromethane by gavage or in drinking water decreased methylation
of the c-myc gene. A LOAEL of 150 mg/kg, the lowest dose tested, was identified on the basis
of liver toxicity (hydropic degeneration) and increased cell proliferation in animals administered
bromodichloromethane by corn oil gavage. A NOAEL was not identified. The results of the
single-dose drinking water experiment suggest a slightly lower LOAEL of 138 mg/kg-day, based
on hydropic degeneration of the liver.
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Lock et al. (2004) administered bromodichloromethane by gavage in corn oil to male
F344 rats (5/group) at doses of 0, 50, or 100 mg/kg and male B6C3F1 mice (6/group) at doses of
0, 25, or 50 mg/kg-day for 5 days per week over a 28-day period. A dose-dependent increase in
incidences of large intestine and kidney tumors has previously been shown in male rats exposed
at 50 or 100 mg/kg-day in dose-dependent manner (NTP 1987). A dose-dependent increase in
the incidence of kidney tumors has also been shown in male mice exposed at 25 or 50 mg/kg-day
(NTP 1987). Body weights were measured on each day of dosing and a 24-hour urine sample
was collected on days 4 to 5, 11 to 12, and 18 to 19. Urine samples were analyzed for creatinine,
total protein, urinary pH, and formic acid concentration. Osmotic mini-pumps containing
bromodeoxyuridine were implanted subcutaneously 5 days prior to sacrifice. Animals were
sacrificed by overdose with halothane anaesthesia on day 29 and blood was obtained by cardiac
puncture for clinical chemistry analysis, including alanine aminotransferase activity, aspartate
aminotransferase activity, and the concentration of blood urea nitrogen and creatinine. Kidneys
were removed, weighed and prepared for histopathological analysis and determination of the
labeling index.
Large increases in formic acid excretion were observed in rats at both doses, and this was
accompanied by a decrease in urinary pH. In mice, increases in formic acid excretion were
much smaller, and urinary pH was not measured. No change was observed in body weight or
clinical chemistry markers of liver or kidney injury in exposed rats and mice. Kidney
histopathology was not altered in mice, but mild renal tubule injury was observed in 2 of 5 rats
exposed to the highest dose of bromodichloromethane (100 mg/kg-day). Increased cell
proliferation was observed in all rats exposed to the highest dose, but was not seen in low-dose
rats or in mice at either dose level.
Torti et al. (2001) conducted a one week inhalation exposure study of
bromodichloromethane in male wild-type (p53+/+) and genetically engineered p53 heterozygous
(p53+/") mice. The objective of this study was to evaluate the role of genotype in the toxic
response of mice to inhalation of bromodichloromethane. Heterozygous and wild type C57BL/6
mice (6 mice/type/concentration) and wild-type FVB/N mice (6 mice/concentration) were
exposed to target exposure concentrations of 0, 1, 10, 30, 100, or 150 ppm for six hours per day
for seven days. Heterozygous FVB/N p53+/" mice (6 mice/concentration) were exposed to
concentrations of 0, 0.3, 1, 10, or 30 ppm for six hours per day for seven days. The test animals
were evaluated for clinical and pathological changes and induced regenerative cell proliferation
in kidney and liver. Osmotic pumps for delivery of bromodeoxyuridine for determination of
labeling index were implanted at 3.5 days prior to scheduled termination. Test animals were
euthanized approximately 18 hours after the last scheduled exposure. With the exception of the
highest target concentration (150 ppm), the average measured concentrations were 102 to 114%
of the target concentrations The average high dose concentration was 78.8% of the target
concentration (150 ppm) as a result of technical problems with the metering system. The effects
observed in all mouse groups (i.e., wild-type and heterozygous) exposed to concentrations of 30
ppm or greater included; mortality, clinical signs (i.e., reddened skin and eyes), reduced body
weight gain, increased liver and kidney weight, histopathological lesions in the liver and kidney,
and increased labeling index in the kidney. Clinical signs in mice surviving exposure at 100 and
150 ppm included lethargy and labored breathing. Histopathologic evaluation revealed severe
V - 20 November 15, 2005
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renal damage consisting of nephrosis, tubular degeneration, and associated regeneration.
Centrilobular degeneration and necrosis were observed in the livers of moribund mice sacrificed
before study termination and in animals surviving for 1 week of exposure. Regenerative cell-
proliferation in the kidney cortex was significantly increased in all mouse groups (i.e., wild-type
and heterozygous) exposed to concentrations of 10 ppm and above. Regenerative cell
proliferation in the liver was less pronounced than in the kidney.
A comparison of the data for each wild-type strain indicates that FVB/N mice were more
susceptible to mortality, increased liver weight, kidney degeneration and nephrosis, and hydropic
degenerationin the liver as compared to C57BL/6 mice. For C57BL/6 mice, mortality, body
weight loss, kidney degeneration and nephrosis, and the liver labeling index were greater in the
heterozygous p53+/" than in the corresponding wild-type strain. For FVB/N mice, increased
kidney weight occurred at a lower dose (10 ppm) in heterozygous p53+/" mice, while other effects
were similar that occurring in the corresponding wild-type strain. Bromodichloromethane did
not induce cellular proliferation in the transitional epithelium of the bladder. No histopathologic
lesions were observed in the bladder. These data identify NOAEL and LOAEL values of 1 and
10 ppm, respectively, based on histopathological changes in the liver and kidney of male p53
wild-type and heterozygous C57BL/6 and FVB/N mice.
Torti et al. (2001) also conducted a three week inhalation exposure study of
bromodichloromethane in wild-type (p53+/+) and genetically engineered p53 heterozygous (p53+/"
) male mice. C57BL/6, FVB/N, C57BL/6 p53+/; and FVB/N p53+A mice (6
mice/type/concentration) were exposed to target exposure concentrations of 0.3, 1, 3, 10, or 30
ppm for six hours per day, seven days per week. The test protocol and endpoints measured were
the same as those used for the one week study described above. Test animals were euthanized
approximately 18 hours after the last scheduled exposure. Average measured concentrations
were 92 to 97% of the target concentrations in all exposure groups. Mortality was observed in
all 30 ppm dose groups with the exception of wild type C57BL/6 mice. No clinical signs of
toxicity were reported. Body weight gain was significantly reduced only in C57BL/6 wild type
mice exposed at 30 ppm. Relative kidney weights in exposed groups did not differ significantly
from the control values. Significantly increased relative liver weight was observed only in
heterozygous C57BL/6 and wild type FVB/N mice exposed at 30 ppm. Histopathologic
evaluation revealed near-normal kidney architecture. Minimal to moderate degenerative tubular
change and regenerative tubules were observed in the 10 and 30 ppm groups, but the acute
tubular nephrosis observed in the one week study was not evident. Minimal hepatocyte
degeneration was observed in heterozygous C57BL/6 mice exposed at 30 ppm and in
heterozygous FVB/N mice exposed at 10 or 30 ppm. These observations suggest that the liver
and severe renal toxicity observed in the one week experiment conducted by Torti et al. (2001)
are transient and were resolving by three weeks. No histopathologic lesions were observed in
the bladder. Regenerative cell-proliferation in the kidney cortex was near baseline levels, with
only the 30 ppm groups showing small elevations. These elevations were statistically significant
in all 30 ppm groups except C57BL/N wild type mice. No increases in regenerative cell
proliferation were evident in the liver or bladder. The NOAEL and LOAEL values in this study
are 3 and 10 ppm, respectively, based on histopathologic changes in the liver and kidney of male
p53 wild type and heterozygous C57BL/6 and FVB/N mice.
V-21 November 15, 2005
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Taken together, the 1-week and 3-week inhalation studies (Torti et al., 2001) illustrate
both strain and genotypic difference in bromodichloromethane toxicity. A comparison of
wild-type strains indicates that FVB/N mice are more susceptible to kidney toxicity and
mortality following inhalation exposure. Differences between wild-type and p53+/- mice were
observed in mortality and morbidity, body weight changes, and the severity of liver and kidney
toxicity. The C57BL/6 p53+/- mice were more susceptible than wild-type mice to
bromodichloromethane toxicity as measured by mortality, histopathology, and liver labeling
index. The same relationship was not observed in FVB/N mice. In this strain the wild-type mice
were more susceptible to toxicity as evidenced by the kidney labeling index. The role of p53
gene expression in bromodichloromethane metabolism and toxicity remains to be elucidated.
2. Dibromochloromethane
Chu et al. (1982a) administered dibromochloromethane to male Sprague-Dawley rats
(10/group) in drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. Based on
recorded fluid intake, these levels corresponded to doses of 0, 0.7, 8.5, or 68 mg/kg-day, as
calculated by the authors. The authors observed no effects on growth rate or food consumption
and no signs of toxicity throughout the exposure. No dose-related biochemical or histologic
changes were detected (no data provided). This study identified a NOAEL of 68 mg/kg-day, but
the reported data were too limited to allow an independent verification.
Munson et al. (1982) administered dibromochloromethane by aqueous gavage to male
and female CD-I mice (8 to 12/sex/group) for 14 days at dose levels of 0, 50, 125, or 250 mg/kg-
day. Endpoints measured included body and organ weights, hematology, clinical chemistry, and
humoral and cell-mediated immune system function. At 250 mg/kg-day, body weights were
significantly decreased only in high-dose males. Significant organ weight changes included
increased absolute liver weight (high-dose females), increased relative liver weight (mid- and
high-dose groups), and decreased absolute and relative spleen weight (high-dose males). The
only hematology parameter significantly affected by treatment was fibrinogen concentration,
which was decreased in high-dose males and females. Significant clinical chemistry findings
were limited to the high-dose groups. Specifically, glucose levels were significantly decreased
in both males and females, and ALT and AST activities were significantly increased in both
males and females. Dibromochloromethane appeared to affect the humoral immune system, as
judged by significantly decreased antibody-forming cells (mid- and high-dose groups) and
hemagglutination liters (high-dose groups). The cell-mediated immune system also appeared to
be affected in male animals, as judged by a significant decrease in the popliteal lymph node
stimulation index at the high dose. This study identified a NOAEL of 50 mg/kg-day and a
LOAEL of 125 mg/kg-day, based on decreased immune function.
Condie et al. (1983) investigated the renal and hepatic toxicity of dibromochloromethane.
Male CD-I mice (8 to 16/group) were administered 0, 37, 74, or 147 mg/kg-day of
dibromochloromethane by gavage in corn oil for 14 days. Biochemical evidence of liver damage
(elevated ALT) and kidney damage (decreased PAH uptake by kidney slices) was observed at
the high dose, but not at the mid-level or low doses. Similarly, histopathology revealed no
consistent or important changes at the low or mid doses, with minimal to moderate liver and
V - 22 November 15, 2005
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kidney injury at the high dose. Liver lesions included mitotic figures, focal inflammation, and
cytoplasmic vacuolation, while kidney lesions included epithelial hyperplasia and mesangial
nephrosis. The NOAEL and LOAEL values in this study were 74 mg/kg-day and 147 mg/kg-
day, respectively.
NTP (1985) administered 0, 60, 125, 250, 500, or 1,000 mg/kg-day of
dibromochloromethane to male and female F344/N rats (5/sex/dose) by gavage in corn oil for 14
days. Animals were observed twice daily for mortality and were weighed once per week.
Necropsies were performed on all animals. All high-dose rats and all females that received 500
mg/kg-day died by day 6. Three males at 500 mg/kg-day died between days 5 and 8. No deaths
occurred at or below 250 mg/kg-day. At 500 or 1000 mg/kg-day, clinical observations included
lethargy, ataxia, and labored breathing. Treatment-related macroscopic findings included
mottled livers and darkened renal medullae in animals administered 500 or 1,000 mg/kg-day.
This study identified a NOAEL of 250 mg/kg-day and a LOAEL of 500 mg/kg-day based on
behavior, gross pathology, and mortality.
In a parallel study (NTP, 1985), male and female B6C3FJ mice (5/sex/dose) were
administered 0, 30, 60, 125, 250, or 500 mg/kg-day of dibromochloromethane in corn oil by
gavage for 14 days. Treatment-related deaths occurred in 80% of the males and in 60% of the
females at the high dose. Clinical signs at this dose included lethargy, ataxia, and labored
breathing. Treatment-related macroscopic findings included mottled livers and darkened renal
medullae in high-dose males and females. White papillomatous nodules in the stomach were
also observed in males at 125, 250, or 500 mg/kg-day and in female mice at 250 or 500 mg/kg-
day. The NOAEL and LOAEL in this study were 60 mg/kg-day and 125 mg/kg-day,
respectively, based on gross lesions.
Aida et al. (1992a) administered dibromochloromethane to Slc:Wistar rats (7/sex/group)
for one month at dietary levels of 0%, 0.020%, 0.062%, or 0.185% for males, and 0%, 0.038%,
0.113%, or 0.338% for females. The test material was microencapsulated and mixed with
powdered feed; placebo granules were used for the control groups. Based on the mean food
intakes, the study authors reported calculated doses of 0, 18.3, 56.2, or 173.3 mg/kg-day for
males and 0, 34.0, 101.1, or 332.5 mg/kg-day for females. Clinical effects, body weight, food
consumption, hematology parameters, serum chemistry, and histopathology of all major organs
were determined. Body weights were significantly reduced in high-dose females relative to the
controls. High-dose females also exhibited slight piloerection and emaciation. Dose-related
increases in both absolute and relative liver weights were observed in males (significant at the
high dose) and females (significant at all dose levels with the exception of a nonsignificant
increase in absolute liver weight at the low dose). Relative kidney weights were also
significantly increased in the high-dose females. Significant decreases in alkaline phosphatase
(mid- and high-dose males and all female dose groups) and LDH (all female dose groups) were
observed, but the biological significance of these changes is unclear. Significant, dose-related
changes in serum biochemistry included reduced nonesterified fatty acids in high-dose males,
reduced triglycerides in high-dose groups, and increased cholesterol in mid- and high-dose males
and in females at all dose levels. The cholesterol levels, however, were within normal ranges at
all dose levels. Serum cholinesterase activity was also significantly decreased in high-dose
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males and mid- and high-dose females with the trend clearly dose-related in females. Liver cell
vacuolization was generally noted at a similar incidence in the controls and all dosing groups,
but dose-related increases in severity were observed in mid- and high-dose males and females.
The incidence and very slight severity of the effects at the low dose were similar to those
observed in the control groups and were not considered adverse. The severity of the liver cell
vacuolization at the mid-dose was rated as very slight to slight, while the severity at the high-
dose was rated as moderate to remarkable. Swelling and single cell necrosis were also observed,
primarily in the high-dose groups. No effect was observed on any hematology parameter.
NOAELs of 18.3 (males) and 34.0 (females) mg/kg-day and LOAELs of 56.2 (males) and 101.1
(females) mg/kg-day were identified for this study based on the histopathology findings.
Potter et al. (1996) evaluated hyaline droplet formation and cell proliferation in the
kidney of male F344 rats following exposure to dibromochloromethane. The rats (4/dose)were
dosed with 0.75 or 1.5 mmol/kg (156 or 312 mg/kg-day, respectively) of dibromochloromethane
in 4% Emulphor® by gavage for 1, 3, or 7 days. No exposure-related increase in hyaline droplets
was observed in dosed rats. Binding to cc2u-globulin was not measured. Changes in kidney
tubule cell proliferation were assessed by in vivo incorporation of [3H]-thymidine. No
statistically significant effect of dibromochloromethane exposure on this endpoint was noted
following exposures of up to 7 days duration.
Melnick et al. (1998) exposed female B6C3FJ mice (10/dose) to dibromochloromethane
in corn oil via gavage for 3 weeks (5 days/week). The doses of dibromochloromethane in this
study were 0 (vehicle only), 50, 100, 192, or 417 mg/kg-day. The doses were selected on the
basis that increases in the incidence of hepatocellular adenoma and carcinoma were previously
seen in female mice exposed at 50 mg/kg or 100 mg/kg-day in a dose-dependent manner (NTP
1985). The corresponding time-weighted doses were 0, 37, 71, 137, and 298 mg/kg-day. No
treatment-related signs of overt toxicity were observed during the study. Body weight and water
intake were not significantly altered at any dose tested. However, a statistically significant and
dose-related increase in liver weight/body weight ratio was seen in the 100, 192 and 417 mg/kg-
day dose groups. Serum ALT activity was significantly increased in the two highest dose
groups. The activity of serum SDH was significantly elevated at all doses tested except 50
mg/kg-day. However, the increase in activity (shown graphically) was very small relative to the
control at the 100 and 192 mg/kg-day doses. At necropsy, there was clear evidence of
hepatocyte hydropic degeneration in the 192 and 417 mg/kg-day dose groups. BrdU was
administered to the animals during the last 6 days of the study, and hepatocyte labeling index
(LI) analysis was conducted. Only the highest dose tested (417 mg/kg-day) resulted in
significantly elevated hepatocyte proliferation as measured by the LI. Evaluation of the data in
this study suggest a LOAEL of 192 mg/kg-day, based on a consistent pattern of positive results
for indicators of hepatotoxicity at this dose.
Coffin et al. (2000) examined the effect of dibromochloromethane administered by corn
oil gavage or in drinking water on cell proliferation and DNA methylation in the liver of female
B6C3F1 mice. Gavage doses of 0, 0.48, or 1.44 mmol/kg (0, 100, or 300 mg/kg, respectively)
were administered to test animals (7-8 weeks old; 10/group) daily for five days, off for two days,
and then again daily for four days. Dose-dependent increases in the incidence of hepatocellular
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adenoma and carcinoma were previously seen in female mice exposed at 50 mg/kg or 100
mg/kg-day (NTP 1985). The high dose was selected on the basis that it had previously been
demonstrated to be carcinogenic in female mice. Dibromochloromethane was administered in
drinking water at approximately 75% of the saturation level, resulting in an average daily dose of
0.82 mmol/kg (171 mg/kg). The mice were sacrificed 24 hours after the last gavage dose and the
livers were removed, weighed, and processed for histopathological examination, proliferating
cell nuclear antigen - labeling index (PCNA-LI) analysis, and determination of c-myc
methylation status. For histopathological analysis, stained liver sections were evaluated for
toxicity using a semi-quantitative procedure using the following severity scoring system: Grade
1 consisted of mid lobular ballooning hepatocytes; Grade 2 consisted of mid lobular ballooning
hepatocytes extending to the central vein; Grade 3 consisted of centrilobular necrosis with
ballooning hepatocytes; and Grade 4 consisted of necrosis extending from the central vein to the
mid lobule zone. A significant, dose-dependent increase in relative liver weight was observed in
animals dosed by gavage; however, relative liver weight was unaffected in animals administered
the compound in drinking water, when compared to controls. At the low gavage dose, liver
toxicity consisted mainly of a Grade 1 response. At the high dose, liver toxicity consisted
mainly of a Grade 2 response. No incidence data were provided in the study report, nor was a
severity grade reported for the control group. The histopathology findings for animals receiving
bromodichloromethane in the drinking water were similar to those observed in the low-dose
gavage group. Dibromochloromethane administered by gavage caused a dose-dependent
increase in the PCNA-LI. There was no significant effect on PCNA-LI when the compound was
administered in drinking water. Administration of dibromochloromethane by gavage or in
drinking water decreased methylation of the c-myc gene. A LOAEL of 100 mg/kg, the lowest
dose tested, was identified on the basis of liver toxicity (ballooning hepatocytes) and increased
cell proliferation in gavaged animals.
3. Bromoform
Chu et al. (1982a) administered bromoform to male Sprague-Dawley rats (10/group) in
drinking water for 28 days at dose levels of 0, 5, 50, or 500 ppm. Based on recorded fluid
intake, these levels corresponded to doses of 0, 0.7, 8.5, or 80 mg/kg-day, as calculated by the
authors. The authors observed no effects on growth rate or food consumption and no signs of
toxicity throughout the exposure. No dose-related biochemical or histologic changes were
detected (no data provided). This study identified a NOAEL of 80 mg/kg-day, but the reported
data were too limited to allow an independent confirmation.
Munson et al. (1982) administered bromoform by aqueous gavage to male and female
CD-I mice (6 to 12/sex/group) for 14 days at levels of 0, 50, 125, or 250 mg/kg-day. Parameters
evaluated included body and organ weights, hematology, clinical chemistry, and humoral and
cell-mediated immune system functions. Body weights were significantly decreased in high-
dose females, while body weights in males were significantly increased at the mid and high
doses. Absolute and relative liver weights were significantly increased in males at the mid -and
high dose and in females at the high dose. Absolute spleen weight was also decreased in mid-
and high-dose females. Hematologic effects included significantly decreased fibrinogen in
males at the high dose and significantly decreased prothrombin time in all treated males. The
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changes in prothrombin time, however, were not dose-related. Significant clinical chemistry
findings included decreased glucose levels (high-dose males), increased AST activity (high-dose
males and females), and decreased BUN levels (high-dose males). Both the humoral and cell-
mediated immune systems appeared to be affected in males at the high dose with a significant
decrease in antibody-forming cells and a significant decrease in delayed-type hypersensitivity
response. The authors stated that no treatment-related effects on the immune system in females
were observed (no data were reported). Based on changes in clinical chemistry parameters, this
study identified a NOAEL of 125 mg/kg-day and a LOAEL of 250 mg/kg-day.
Condie et al. (1983) investigated the renal and hepatic toxicity of bromoform. Male CD-
1 mice (8 to 16/group) were administered 0, 72, 145, or 289 mg/kg-day of bromoform by gavage
in corn oil for 14 days. Biochemical evidence of liver damage (elevated ALT) and kidney
damage (decreased PAH uptake by kidney slices) was observed at the high dose, but not at the
mid or low dose. Histopathological examination revealed no consistent or important changes at
the low or mid doses, with minimal to moderate liver and kidney injury at the high dose.
Specific microscopic changes included intratubular mineralization, epithelial hyperplasia,
mesangial hypertrophy and mesangial nephrosis in the kidney, and centrilobular pallor, mitotic
figures, focal inflammation, and cytoplasmic vacuolation in the liver. This study identified a
NOAEL value of 145 mg/kg-day and a LOAEL value of 289 mg/kg-day based on
histopathologic changes in the liver.
NTP (1989a) investigated the short term oral toxicity of bromoform in F344/N rats and
B6C3FJ mice. Groups of male and female rats (5/sex/group) and female mice (5/group) were
administered doses of 0, 100, 200, 400, 600, or 800 mg/kg-day of bromoform in corn oil by
gavage for 14 days. Male mice were administered 0, 50, 100, 200, 400, or 600 mg/kg-day. All
rats that were dosed at 600 or 800 mg/kg-day died before the end of the study. At 400 mg/kg-
day, only one male rat died before study termination. These rats exhibited lethargy, labored
breathing, and ataxia. At 400 mg/kg-day, final body weights were decreased by 14% in male
rats and by 4% in female rats relative to controls. In mice, one male and one female
administered the high dose died before study termination. Ataxia and lethargy were noted at 600
mg/kg-day. Final body weights of mice were comparable to those of the controls. Raised
stomach nodules were observed in males at 400 and 600 mg/kg-day and in females at 600 and
800 mg/kg-day. This study identified a NOAEL of 200 mg/kg-day and a LOAEL of 400 mg/kg-
day. based on decreased body weight and mortality in rats and on stomach nodules in mice,
Aida et al. (1992a) administered bromoform to Slc:Wistar rats (7/sex/group) for one
month at dietary levels of 0%, 0.068%, 0.204%, or 0.612% for males and 0%, 0.072%, 0.217%,
or 0.651% for females. The test material was microencapsulated and mixed with powdered feed;
placebo granules were used for the control groups. Based on the mean food intakes, the study
authors reported the mean compound intakes as 0, 61.9, 187.2, or 617.9 mg/kg-day for males and
0, 56.4, 207.5, or 728.3 mg/kg-day for females. Clinical effects, body weight, food
consumption, hematology parameters, serum chemistry, and histopathology of all major organs
were determined. Body weights were significantly reduced in high-dose males relative to the
controls. High-dose animals of both sexes exhibited slight piloerection and emaciation. Relative
liver weight was significantly increased in mid- and high-dose males and females. Significant
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changes in serum chemistry were primarily observed in the mid- and high-dose animals with the
females more significantly affected. These changes included significant decreases in (a) serum
glucose in low- and high-dose males and in mid- and high-dose females, (b) triglycerides in
high-dose males and in mid- and high-dose females, (c) cholinesterase activity in high-dose
males and in all female treatment groups, (d) LDH in mid- and high-dose females, and (e) BUN
in mid- and high-dose females. All of these changes in the groups noted exhibited strong dose-
related trends with the exception of serum glucose in males. Creatinine levels and alkaline
phosphatase activity were also significantly decreased in all female treatment groups, but the
changes were not dose-related. Significant increases, although not dose-related, were observed
for phospholipids and cholesterol in mid- and high-dose animals with the exception of a
nonsignificant increase in phospholipids in high-dose females. The only change of clear
biological significance at the low dose was a decrease in cholinesterase activity in females. No
effect was observed on any hematology parameter. Microscopic and macroscopic findings were
limited to the liver. Specifically, discoloration was observed in all males and females in the
high-dose group. The incidence and severity of liver cell vacuolization and swelling were dose-
related. Severe hepatic cell vacuolization was observed in 5/7 high-dose males and in 6/7
females at the mid and high dose. Slight to moderate liver cell swelling was observed in three
high-dose males, while all high-dose females displayed slight signs of liver cell swelling.
Females appeared to be more sensitive for development of histopathological effects, but the
changes observed in low-dose females were not considered an adverse effect. Based on the
histopathology and serum chemistry changes in the mid-dose animals, this study identified
NOAELs of 61.9 mg/kg-day for males and 56.4 mg/kg-day for females, and LOAELs of 187.2
mg/kg-day for males and 207.5 mg/kg-day for females.
Potter et al. (1996) evaluated the effect of bromoform on hyaline droplet formation and
cell proliferation in the kidney of male F344 rats. Animals (4/dose) received doses of 0.75 or 1.5
mmol/kg of bromoform in 4% Emulphor® by gavage for 1, 3, or 7 days. These doses correspond
to 190 or 379 mg/kg-day, respectively. No exposure-related increase in hyaline droplet
formation was observed. Cell proliferation in the kidney following bromoform exposure was
measured in vivo by [3H]-thymidine incorporation. No statistically significant effects were
noted following exposures of up to 7 days duration. Binding of bromoform to cc2u-globulin was
not measured.
Melnick et al. (1998) exposed female B6C3FJ mice (10 animals/group) to bromoform in
corn oil via gavage for 3 weeks (5 days/week). Doses of bromoform used in this study were 0
(vehicle only), 200, or 500 mg/kg-day. There were no treatment-related signs of overt toxicity
observed during the study. Body weight and water intake were not significantly altered at any
dose tested. However, a dose-related increase in absolute liver weight and liver weight/body
weight ratio was noted in both tested doses. Neither serum ALT nor serum SDH activity were
significantly elevated at either dose of bromoform. At necropsy, there was no evidence of
hepatocyte hydropic degeneration in animals treated with either dose. BrdU was administered to
the animals during the last 6 days of the study, and hepatocyte labeling index (LI) analysis was
conducted. Only the 500 mg/kg-day dose resulted in marginally significant increase in
hepatocyte proliferation as measured by the LI. These data suggest a NOAEL of 200 mg/kg-day
and a LOAEL of 500 mg/kg-day based on increased hepatocyte proliferation.
V - 27 November 15, 2005
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Coffin et al. (2000) examined the effect of bromoform administered by corn oil gavage or
in drinking water on liver toxicity, cell proliferation and DNA methylation in female B6C3F1
mice. Gavage doses of 0, 0.79, or 1.98 mmol/kg (0, 200, or 500 mg/kg, respectively) were
administered to test animals (7-8 weeks old; 10/group) daily for five days, off for two days, and
then again daily for 5 days. Bromoform was not shown to be carcinogenic to female mice
exposed at doses up to 200 mg/kg-day (NTP 1989a). The high dose was selected on the basis
that it had previously been demonstrated to be carcinogenic in female mice. Bromoform was
administered in drinking water at approximately 75% of the saturation level, resulting in an
average daily dose of 1.19 mmol/kg (301 mg/kg). The mice were sacrificed 24 hours after the
last gavage dose and the livers were removed, weighed, and processed for histopathological
examination, proliferating cell nuclear antigen - labeling index (PCNA-LI) analysis, and
determination of c-myc methylation status. For histopathological analysis, stained liver sections
were evaluated for toxicity using a semi-quantitative procedure using the following severity
scoring system: Grade 1 consisted of mid lobular ballooning hepatocytes; Grade 2 consisted of
mid lobular ballooning hepatocytes extending to the central vein; Grade 3 consisted of
centrilobular necrosis with ballooning hepatocytes; and Grade 4 consisted of necrosis extending
from the central vein to the mid lobule zone. A significant, dose-dependent increase in relative
liver weight was observed in animals dosed by gavage; however, relative liver weight was
unaffected in animals administered the compound in drinking water, when compared to controls.
At the low gavage dose, liver toxicity consisted mainly of a Grade 1 response. At the high dose,
liver toxicity consisted mainly of a Grade 2 response. No incidence data were provided in the
study report, nor were severity data presented for the control group. The histopathology findings
for animals receiving bromoform in the drinking water were similar to those observed in the
low-dose gavage group. Bromoform administered by gavage caused a significant, dose-
dependent increase in the PCNA-LI. Bromoform also significantly enhanced cell proliferation
when the compound was administered in drinking water. Administration of bromoform by
gavage or in drinking water decreased methylation of the c-myc gene. A LOAEL of 200 mg/kg,
the lowest dose tested, was identified on the basis of liver toxicity in gavaged animals.
C. Subchronic Exposure
This section addresses studies of brominated trihalomethanes that are of approximately
90 days in duration. Table V-4 summarizes the details of these subchronic studies.
V - 28 November 15, 2005
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Table V-4 Summary of Subchronic Toxicity Studies for Brominated Trihalomethanes
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromodichloromethane
Oral Exposure
Chu et al.
(1982b)
Chu et al.
(1982b)
NTP(1987)
NTP(1987)
NTP(1987)
Rat
SD*
Rat
SD
Rat
F344/N
Mouse
B6C3F,
Mouse
B6C3F;
Drinking
water
Drinking
water
Gavage
(corn oil)
Gavage
(com oil)
Gavage
(com oil)
M
F
M,F
M
F
20
20
10
10
10
90 days
90 days
1 3 weeks
(5 d/wk)
1 3 weeks
(5 d/wk)
1 3 weeks
(5 d/wk)
0
0.57
6.5
53
212
0
0.75
6.9
57
219
0
19
38
75 (NOAEL)
ISO(LOAEL)
300
0
6.3
13
25
50 (NOAEL)
100 (LOAEL)
0
25
50
100 (NOAEL)
200 (LOAEL)
400
Non dose-dependent hepatic
and thyroid lesions
Non dose-dependent hepatic
and thyroid lesions
Reduced body weight gain
Focal necrosis of proximal renal
tubular epithelium
Hepatic microgranulomas
Inhalation Exposure
Torti et al.
2001
Mouse
C57BL/6
FVB/N
p53
(hetero-
zygous)
Vapor
M
Not
reported
1 3 weeks
(6 h/day)
0 ppm
0.5 ppm
3 ppm
10 ppm
15 ppm
Text reported minimal cortical
scarring and occasional
regenerative tubules in
C56BL/6 mice and mild renal
cortical tubular
kary ocytomegaly .
Concentrations at which these
effects occurred were not
reported.
V-29
November 15, 2005
-------
Table V-4 (cont.)
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Dibromochloromethane
Chu et al.
(1982b)
Chu et al.
(1982b)
NTP(1985)
NTP(1985)
Daniel et al.
(1990)
Rat
SD
Rat
SD
Rat
F344/N
Mouse
B6C3F;
Rat
SD
Drinking
water
Drinking
water
Gavage
(corn oil)
Gavage
(com oil)
Gavage
(corn oil)
M
F
M,F
M,F
M,F
20
20
10
10
10
90 days
90 days
1 3 weeks
(5 d/wk)
1 3 weeks
(5 d/wk)
90 days
0
0.57
6.1
49 (NOAEL)
224 (LOAEL)
0
0.64
6.9
55 (NOAEL)
236 (LOAEL)
0
15
30 (NOAEL)
60 (LOAEL)
125
250
0
15
30
60
125 (NOAEL)
250 (LOAEL)
0
50 (LOAEL)
100
200
Hepatic lesions
Hepatic lesions
Hepatic vacuolization
indicative of fatty
metamorphosis (males)
Fatty liver and toxic
nephropathy in males
Hepatic vacuolization (males);
renal lesions (females)
Bromoform
Chu et al.
(1982b)
Chu et al.
(1982b)
NTP(1989a)
Rat
SD
Rat
SD
Rat
F344/N
Drinking
water
Drinking
water
Gavage
(com oil)
M
F
M,F
20
20
10
90 days
90 days
1 3 weeks
(5 d/wk)
0
0.65
6.1
57 (NOAEL)
218 (LOAEL)
0
0.64
6.9
55 (NOAEL)
283 (LOAEL)
0
12
25 (NOAEL)
50 (LOAEL)
100
200
Hepatic lesions and vacuolation
Hepatic lesions and vacuolation
Hepatic vacuolation in males
V-30
November 15, 2005
-------
Table V-4 (cont.)
Reference
NTP(1989a)
Species
Mouse
B6C3F,
Route
Gavage
(com oil)
Sex
M,F
Number
per dose
group
10
Duration
1 3 weeks
(5 d/wk)
Dose
(mg/kg-day)
0
25
50
100 (NOAEL)
200 (LOAEL)
400
Results
Hepatic vacuolation in males
: SD, Sprague-Dawley
1. Bromodichloromethane
Chu et al. (1982b) administered bromodichloromethane to male and female weanling
Sprague-Dawley rats (20/sex/dose) in drinking water at levels of 0, 5, 50, 500, or 2,500 ppm for
90 days. Half of each group (10/sex/dose) was sacrificed at the end of the exposure period, and
the remaining animals were given tap water for another 90 days. As calculated by the authors
(using data on water consumption and the average initial and final body weights in the vehicle
controls and the high-dose groups), these levels corresponded to doses of approximately 0, 0.57,
6.5, 53, and 212 mg/kg-day for males and 0, 0.75, 6.9, 57, and 219 mg/kg-day for females. At
2,500 ppm, food consumption was significantly depressed and significant growth suppression
occurred in both males and females. Mild histologic changes were observed in the liver and
thyroid of the male animals. Neither incidence nor severity were clearly dose-related.
Specifically, the incidence of hepatic lesions was increased in males at concentrations equal to or
greater than 50 ppm, with similar statistically significant increases in the severity of these lesions
in these dose groups compared to the control. The author noted that the hepatic lesions were
mild and similar to the control following the 90-day recovery period. Increased incidence of
thyroid lesions was also observed in males at concentrations equal to or greater than 50 ppm.
The severity of these lesions was similar to that observed in the control group. These lesions
were also mild and similar in nature to those of the control after the 90-day recovery period. The
incidence of hepatic lesions in the female treatment groups (3-5/10) was slightly increased
compared to that of the control group (0/10) with the severity significantly increased in the 50
and 2,500 ppm treatment groups, but not in the 500 ppm group. No significant numbers of
females were reported as having thyroid lesions. Lack of a clear dose-response relationship for
either incidence or severity of lesions prevented identification of reliable NOAEL or LOAEL
values.
NTP (1987) administered doses of 0, 19, 38, 75, 150, or 300 mg/kg-day of
bromodichloromethane to male and female F344/N rats (10/sex/dose) by gavage in corn oil for
5 days/week for 13 weeks. The low-dose group was administered 1.9 mg/kg-day for the first
3 weeks of the study. A necropsy was performed on all animals. Before study termination, 50%
of the males and 20% of the females in the high-dose group died. Although food consumption
was not recorded, animals in the high-dose groups appeared to eat less food. These animals were
V-31
November 15, 2005
-------
also emaciated. At 300 mg/kg-day, final body weights of the males and females were decreased
by 55% and 32%, respectively, relative to the controls. At 150 mg/kg-day, final body weights of
the males and females were decreased by 30% and 12%, respectively, relative to the controls.
Treatment-related lesions were observed only at the high dose. At 300 mg/kg-day in males,
centrilobular degeneration of the liver and occasional necrotic cells were observed in 4/9
animals. Mild bile duct hyperplasia was also observed in these animals. Kidney lesions in high-
dose males consisted of degeneration of renal proximal tubular epithelial cells (4/9) and definite
foci of coagulative necrosis of the tubular epithelium (2/9). High-dose males (4/9) also exhibited
lymphoid degeneration of the thymus, spleen, and lymph nodes, and mild to moderate atrophy of
the seminal vesicles and/or prostate. Enlarged hepatocytes were observed in females (2/9) at
300 mg/kg-day. Although degeneration of the spleen, thymus, and lymph nodes was noted in
high-dose females, the extent of the atrophy was much less than that observed in males. This
study identified a NOAEL of 75 mg/kg-day and a LOAEL of 150 mg/kg-day based on reduced
body weight gain.
In a parallel experiment, NTP (1987) administered bromodichloromethane in corn oil by
gavage to male and female B6C3FJ mice (10/sex/dose) for 5 days/week for 13 weeks. Doses
were 0, 6.25, 12.5, 25, 50, or 100 mg/kg-day for males and 0, 25, 50, 100, 200, or 400 mg/kg-day
for females. All animals survived to the end of the study. The final body weights of high-dose
males were decreased by 9% relative to the controls. The final body weights of females that
received 200 and 400 mg/kg-day were decreased 5% and 6%, respectively, relative to the
controls. No treatment-related clinical signs were noted. Treatment-related lesions were
observed only at 100 mg/kg-day in males and at 200 and 400 mg/kg-day in females. Kidney
lesions in high-dose males included focal necrosis of the proximal renal tubular epithelium
(6/10) and nephrosis of minimal severity (2/10). Microgranulomas were observed in the liver of
70% of the females that received the 200 mg/kg-day dose. NOAEL and LOAEL values for
female mice were 100 and 200 mg/kg-day, respectively, based on occurrence of
microgranulomas. This study identified a NOAEL of 50 mg/kg-day and a LOAEL of
100 mg/kg-day for male mice on the basis of liver histopathology.
Torti et al. (2001) reported results from a 13-week interim sacrifice conducted as part of
an inhalation cancer bioassay in p53 heterozygous C57BL/6 and FVB/N male mice. Test
animals were exposed to vapor concentrations of 0, 0.5, 3, 10, or 15 ppm, 6 hours/day for 13
weeks. Osmotic pumps for delivery of bromodeoxyuridine for determination of labeling index
were implanted at 3.5 days prior to scheduled termination. Test animals were euthanized
approximately 18 hours after the last scheduled exposure. No exposure-related effects were
noted for mortality, morbidity, relative body weight, relative kidney or liver weight, or cell
proliferation in liver, kidney or bladder. Histopathologic lesions were limited to the kidney. The
study authors reported minimal cortical scarring and occasional regenerative tubules in the
C57BL/6 strain. The only lesion reported for the FVB/N strain was limited to mild renal cortical
tubular karyocytomegaly. No incidence data were presented for these lesions and the
concentrations at which they occurred were not stated. Cell proliferation was not increased over
baseline in the liver, kidney or bladder.
V - 32 November 15, 2005
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2. Dibromochloromethane
Chu et al. (1982b) administered dibromochloromethane to male and female weanling
Sprague-Dawley rats (20/sex/dose) in drinking water at levels of 0, 5, 50, 500, or 2,500 ppm for
90 days. Half of each group (10/sex/dose) was sacrificed at the end of the exposure period, and
the remaining animals were given tap water for another 90 days. Based on calculations by the
authors, these levels corresponded to doses of approximately 0, 0.57, 6.1, 49, and 224 mg/kg-day
for males and 0, 0.64, 6.9, 55, and 236 mg/kg-day for females. At 2,500 ppm, food consumption
was depressed in both males and females, with the decrease reaching statistical significance in
the males. Body weight gain was also decreased at the high-dose, but not significantly. Mild
histologic changes occurred in the liver and thyroid in both males and females. Neither the
incidence nor severity exhibited clear dose-response trends, with the possible exception of the
incidence and severity of hepatic lesions in the males. The severity of hepatic lesions was
significantly increased at 50 ppm in females and at 2,500 ppm in both males and females.
Hepatic lesions included increased cytoplasmic volume and vacuolation due to fatty infiltration.
Lesions of the thyroid included decreased follicular size and colloid density and occasional focal
collapse of follicles. The severity of these lesions was not significantly different from that of the
control. The authors noted that histological changes were mild and similar to controls when
evaluated after the 90-day recovery period. These data identified a NOAEL of 49 mg/kg-day
and a LOAEL of 224 mg/kg-day for males, and a NOAEL of 55 mg/kg-day and a LOAEL of
236 mg/kg-day for females.
NTP (1985) administered dibromochloromethane by gavage in corn oil to male and
female F344/N rats (10/dose/sex). Doses of 0, 15, 30, 60, 125, or 250 mg/kg were given
5 days/week for 13 weeks. Animals were weighed weekly. All animals were submitted for
gross necropsy, while histopathology was conducted on animals in the control and high-dose
groups with the exception that the liver was examined in all males and in females at 125 mg/kg-
day, and that the kidney and salivary glands were examined in males and females at 125 mg/kg-
day. Only one male and one female in the high-dose group survived, with most deaths occurring
during weeks 8 to 10. At 125 mg/kg-day, final body weights of males were decreased 7%
relative to controls. Histopathological examination revealed severe lesions and necrosis in
kidney, liver, and salivary glands, primarily at the high-dose. Males exhibited a dose-dependent
increase in the frequency of clear cytoplasmic vacuoles indicative of fatty metamorphosis in the
liver; this effect was statistically significant at doses of 60 mg/kg-day or higher. This study
identified a NOAEL of 30 mg/kg-day and a LOAEL of 60 mg/kg-day in rats on the basis of
histopathological effects in the liver.
NTP (1985) performed a similar 13-week gavage study with dibromochloromethane in
male and female B6C3FJ mice (10/sex/dose). The doses and dosing schedule were the same as
for the rat study. No treatment-related effects on body weight or histopathology were observed
at doses of 125 mg/kg-day or lower. At the high dose, final body weights of males and females
were decreased by 6% relative to controls. Fatty metamorphosis of the liver and toxic
nephropathy were observed in high-dose males, but not in high-dose females. This study
identified a NOAEL of 125 mg/kg-day and a LOAEL of 250 mg/kg-day based on
histopathological lesions in male mice.
V - 33 November 15, 2005
-------
Daniel et al. (1990) administered gavage doses (in corn oil) of 0, 50, 100, or 200 mg/kg-
day of dibromochloromethane to male and female Sprague-Dawley rats (10/sex/dose) for 90
consecutive days. Individual dosages were adjusted weekly based on individual body weights.
During the final week of the study, urinalysis was conducted following an overnight fast.
Ophthalmoscopic examinations were performed prior to treatment and during the last week of
the study. Hematology, serum clinical chemistry, and a thorough histopathologic examination
were also conducted. No deaths, clinical signs of toxicity, or treatment-related changes in the
ophthalmoscopic examinations or hematology were observed. Final body weights were
significantly reduced in the high-dose groups by 32% in males and by 13% in females. Body
weight decreases in the other groups were less than 10% of control weights. A dose-related
increase was observed in liver weight in females that reached statistical significance at the high
dose. Clinical chemistry values indicative of hepatotoxicity and suggestive of nephrotoxicity
included increased levels of alkaline phosphatase (high-dose males and females), ALT (mid- and
high-dose males), and creatinine (mid- and high-dose males and high-dose females), and
decreased potassium levels (high-dose males). Centrilobular lipidosis (vacuolization) was
observed in the liver of almost all high-dose males and females and all mid- and low-dose males
(with one exception at each level), but in only one mid-dose female. The severity of the effect
was dose-related. Centrilobular necrosis was also observed in high-dose males and females.
Slight-to-moderate degeneration within the kidney proximal tubular cells occurred in all high-
dose males and females and to a lesser extent in mid-dose males and low- and mid-dose females.
Based on the liver histopathology in males and kidney histopathology in females, the LOAEL
for dibromochloromethane in this study was 50 mg/kg-day, the lowest dose tested.
3. Bromoform
Chu et al. (1982b) administered bromoform to male and female weanling Sprague-
Dawley rats (20 rats/sex/group) for 90 days in drinking water at levels of 0, 5, 50, 500, or
2,500 ppm. Half of each group (10/sex/dose) was sacrificed at the end of the exposure period,
and the remaining animals were given tap water for a 90-day recovery period. Based on
calculations by the authors, the administered drinking water levels corresponded to doses of
approximately 0, 0.65, 6.1, 57, and 218 mg/kg-day for males and 0, 0.64, 6.9, 55, and 283
mg/kg-day for females. At 2,500 ppm, food consumption was depressed in both males and
females, with the decrease reaching statistical significance in males. Body weight gain was also
decreased at the high-dose, but not significantly. Lymphocyte counts were significantly
decreased in high-dose males and females when evaluated 90-days after cessation of treatment.
The only change in serum biochemistry was a significant decrease in LDH in both males and
females at the high dose. This effect was also noted 90-days after cessation of treatment. Mild
histologic changes occurred in the liver and thyroid of male and female animals. Although
neither incidence nor severity were clearly dose-related, these parameters tended to increase with
dose. The severity of hepatic lesions was significantly increased in high-dose males and in
females at 500 and 2,500 ppm. Hepatic lesions included increased cytoplasmic volume and
vacuolation due to fatty infiltration. Lesions of the thyroid included decreased follicular size and
colloid density and occasional focal collapse of follicles. The severity of these lesions in the
treated animals was not significantly different from that in the controls. Although the authors
V - 34 November 15, 2005
-------
noted that histologic changes were mild and similar to controls when evaluated after the 90-day
recovery period, males in the high-dose group continued to exhibit an increased incidence of
hepatic lesions with greater severity relative to the control. These data identified a NOAEL of
57 mg/kg-day and a LOAEL of 218 mg/kg-day for males, and a NOAEL of 55 mg/kg-day and a
LOAEL of 283 mg/kg-day for females.
NTP (1989a) exposed male and female F344/N rats to bromoform by gavage for
5 days/week for 13 weeks. Animals (10/sex/dose) received doses of 0, 12, 25, 50, 100, or
200 mg/kg-day. None of the rats died before the end of the study, and body weights were not
significantly affected. All high-dose animals, as well as males dosed with 100 mg/kg-day, were
lethargic. At sacrifice, tissues were examined for gross and histologic changes. A dose-
dependent increase in the frequency of hepatocellular vacuolation was observed in male rats,
which reached statistical significance at 50 mg/kg-day (IRIS, 1993b). These hepatic effects were
not observed in females. This study identified a NOAEL of 25 mg/kg-day and a LOAEL of
50 mg/kg-day, on the basis of the hepatic vacuolation seen in male rats.
In a parallel study, NTP (1989a) exposed male and female B6C3FJ mice to bromoform
by gavage for 5 days/week for 13 weeks. Animals (10/sex/dose) received doses of 0, 25, 50,
100, 200, or 400 mg/kg-day. One female died at 100 mg/kg-day, but no other deaths at any other
dose level occurred. At sacrifice, tissues were examined for gross and histologic changes. Body
weights were not significantly affected, although males receiving 400 mg/kg-day had body
weights about 8% less than controls. A dose-related increase in the number of hepatocellular
vacuoles was seen in male mice (incidence of 5/10 at 200 mg/kg and 8/10 at 400 mg/kg reported
in text; incidence in controls not explicitly stated), but not in females. This study identified a
NOAEL of 100 mg/kg-day and a LOAEL of 200 mg/kg-day in male mice, based on
hepatocellular vacuolation.
D. Chronic Exposure
This section addresses studies on the health effects of brominated trihalomethanes that
are of one to two years in duration. These studies are summarized in Table V-5.
V - 35 November 15, 2005
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Table V-5 Summary of Chronic Toxicity Studies for Brominated Trihalomethanes
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromodichloromethane
NTP(1987)
NTP(1987)
NTP(1987)
Aida et al.
(1992b); Tobe
etal. (1982)
Aida et al.
(1992b); Tobe
etal. (1982)
Klinefelter et
al. (1995)
George et al.
(2002)
George et al.
(2002)
Rat
F344/N
Mouse
B6C3F;
Mouse
B6C3F;
Rat
Wistar
Rat
Wistar
Rat
F344
Rat
F344
Mouse
B6C3F,
Gavage
(com oil)
Gavage
(corn oil)
Gavage
(corn oil)
Diet
Diet
Drinking
water
Drinking
water
Drinking
water
M,F
M
F
M
F
M
M
M
50
50
50
40
40
7
54
50
2 years
(5 d/wk)
2 years
(5 d/wk)
2 years
(5 d/wk)
2 years
2 years
1 year
2 years
2 years
0
50 (LOAEL)
100
0
25 (LOAEL)
50
0
75 (LOAEL)
150
0
6 (LOAEL)
26
138
0
8 (NOAEL)
32 (LOAEL)
168
0
22
39 (NOAEL)
0
6.4
32.6 (NOAEL)
58.9 (LOAEL)
0
8.1 (NOAEL)
27.2 (LOAEL)
43.4
Renal and hepatic histopathology
Renal and hepatic histopathology
Reduced body weight gain
Hepatic vacuolization, serum
chemistry
Hepatic vacuolization, serum
chemistry
No evidence of treatment-related
histopathological or organ
weight effects (see reproductive
effects, section V.E for
additional data from this study)
No evidence of treatment-related
histopathological effects.
Significant negative trend for
relative kidney weight;
significantly reduced kidney
weight at high dose
Decreased absolute and relative
kidney weight. Mild treatment-
related histopathological effects
in liver.
Dibromochloromethane
Tobe et al.
(1982)
Tobe et al.
(1982)
Rat
Wistar
SPF
Rat
Wistar
SPF
Diet
Diet
M
F
40
40
2 years
2 years
0
12 (NOAEL)
49 (LOAEL)
196
0
17 (NOAEL)
70 (LOAEL)
278
Serum biochemistry, liver
appearance at necropsy;
decreased body weight gain
Serum biochemistry, liver
appearance at necropsy;
decreased body weight gain
V-36
November 15, 2005
-------
Table V-5 (cont.)
Reference
NTP(1985)
NTP(1985)
Species
Rat
F344
Mouse
B6C3F,
Route
Gavage
(com oil)
Gavage
(corn oil)
Sex
M,F
M,F
Number
per dose
group
50
50
Duration
2 years
(5 d/wk)
105 weeks
(5 d/wk)
Dose
(mg/kg-day)
0
40 (LOAEL)
80
0
50 (LOAEL)
100
Results
Histologic changes in liver,
including fat accumulation and
ground glass appearance, and
altered basophilic staining
Fatty metamorphosis in liver
and follicular cell hyperplasia in
thyroid
Bromoform
Tobe et al.
(1982)
Tobe et al.
(1982)
NTP(1989a)
NTP(1989a)
NTP(1989a)
Rat
Wistar
SPF
Rat
Wistar
SPF
Rat
F344/N
Mouse
B6C3F,
Mouse
B6C3F;
Diet
Diet
Gavage
(corn oil)
Gavage
(corn oil)
Gavage
(corn oil)
M
F
M,F
M
F
40
40
50
50
50
2 years
2 years
103 weeks
(5 day/wk)
103 weeks
(5 day/wk)
103 weeks
(5 day/wk)
0
22 (NOAEL)
90 (LOAEL)
364
0
38 (NOAEL)
152 (LOAEL)
619
0
100 (LOAEL)
200
0
50
100 (NOAEL)
0
100 (LOAEL)
200
Enzyme changes and altered
liver appearance at necropsy
Enzyme changes and altered
liver appearance at necropsy
Decreased body weight, lethargy,
mild hepatotoxicity
No observed effects on body
weight or hepatotoxicity
Decreased body weight, minimal
to mild fatty changes in liver
* SD, Sprague-Dawley
1.
Bromodichloromethane
Tobe et al. (1982) evaluated the chronic effects of bromodichloromethane administered
in the diet to male and female Slc:Wistar SPF rats (40/sex/group) for 24 months. The
histopathology data for the animals exposed to bromodichloromethane in this study were
reported by Aida et al. (1992b). The animals were 5 weeks old at the start of the study and
weighed approximately 100 g. Bromodichloromethane was microencapsulated, and an
appropriate amount was mixed with powdered feed. The concentrations administered were 0.0
0.014, 0.055, or 0.22%. Control groups (70 rats/sex) received microcapsules without the test
compound. Body weight and food consumption were monitored weekly for the first 6 months,
every 2 weeks from 6 to 12 months, and every 4 weeks during the second year of the study.
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November 15, 2005
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Interim sacrifices of at least 9 animals/sex/control group and 5 animals/sex/dose group were
conducted at 6, 12, and 18 months. All surviving animals were sacrificed at 2 years.
Necropsies, hematology, and serum biochemistry were conducted at each time of sacrifice.
Based on mean food intakes, the reported average doses were approximately 0, 6, 26, or
138 mg/kg-day for males and 0, 8, 32, or 168 mg/kg-day for females (Aida et al., 1992b).
Marked suppression of body weight gain was seen in males and females of the high-dose group.
Males and females of the high-dose group exhibited mild piloerection and emaciation. Relative
liver weight was significantly increased in the mid- and high-dose groups, while relative kidney
weight was significantly increased only in the high-dose groups. At 18 months, dose-dependent
reductions in serum cholinesterase activity and increases in y-glutamyl transpeptidase (y-GTP)
activity (indicative of bile duct proliferation) were observed in males, with the changes
significant at the high dose. The mid- and high-dose males also displayed a 27% and 65%
reduction, respectively, in total serum triglycerides (T-Gly) levels when compared to the control
group. At 18 months, serum cholinesterase levels were significantly decreased, while total
cholesterol levels were significantly increased, in all dose groups for the treated females. Serum
T-Gly levels (decreased) and y-GTP activity (increased) were also reported to deviate
significantly from control values in the mid- and high-dose females. The most sensitive markers
at 24 months were T-GLY and serum cholinesterase, with significant changes seen in all of the
male treatment groups. Gross necropsy revealed dose-related yellowing and roughening of the
liver surface. Treatment-related lesions were limited to the liver. At 24 months, fatty
degeneration and granuloma were observed in all dose groups with the exception of granulomas
in low-dose females. Specifically, fatty degeneration and granulomas were observed in low-
dose males, but not control males, and fatty degeneration was observed in low-dose females at a
higher rate (8/19) than in control females (2/32). Cholangiofibrosis was also observed in the
high-dose groups. Bile duct proliferation was observed in most high-dose animals at 6 months,
and was prevalent in the controls and all dose groups by 24 months. Histopathology was
observed in all dose groups as early as 6 months with the exception of low-dose females. Based
on the results of Tobe et al. (1982) alone, the NOAEL was 6 mg/kg-day in males and 8 mg/kg-
day in females. The LOAEL was identified as 26 (males) to 32 (females) mg/kg-day, based on
serum enzyme changes and altered liver appearance. Based on the histopathology data reported
for this study by Aida et al. (1992b), however, the entire study identified a LOAEL of 6 mg/kg-
day in male rats and 8 mg/kg-day in female rats.
NTP (1987) administered doses of 0, 50, or 100 mg/kg-day of bromodichloromethane in
corn oil by gavage to male and female F344/N rats (50/sex/dose), 5 days/week for 102 weeks.
The authors observed all animals for clinical signs and recorded body weights (by cage) once per
week for the first 12 weeks of the study and once per month thereafter. A necropsy was
performed on all animals, including those found dead, unless they were excessively autolyzed or
cannibalized. During necropsy, all organs and tissues were examined for grossly visible lesions.
Complete histopathology was performed on all female rats and on high-dose and vehicle-control
male rats. Male rats in the low-dose group that died early in the study were also examined
histologically. Survival of dosed rats was comparable to that of vehicle controls. Mean body
weight of high-dose male and female rats was decreased during the last 1.5 years of the study;
body weight gain of high-dose male and female rats was 86% and 70% of the corresponding
vehicle-control values. Body weight gain of low-dose male and female rats was comparable to
V - 38 November 15, 2005
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that of the vehicle-control group. No treatment-related clinical signs were observed. In males,
treatment-related nonneoplastic effects included renal cytomegaly, tubular cell hyperplasia,
hepatic necrosis, and fatty metamorphosis. In females, changes included eosinophilic cyto-
plasmic change, clear cell change, focal cellular change, fatty metamorphosis of the liver, and
tubular cell hyperplasia of the kidney. This study identified a LOAEL of 50 mg/kg-day based on
histologic findings in the liver.
NTP (1987) administered bromodichloromethane in corn oil by gavage to male and
female B6C3FJ mice (50/sex/dose), 5 days/week for 102 weeks. For males, doses were 0, 25, or
50 mg/kg-day. For females, doses were 0, 75, or 150 mg/kg-day. Final survival of treated male
mice was comparable to that of vehicle controls. At week 84, survival of female mice was
greater than 50% in all dose groups. After week 84, survival of dosed and vehicle-control
female mice was reduced (final survival: 26/50, 13/50, and 15/50 for the 0, 5, and 50 mg/kg-day
groups, respectively), and this decreased survival was associated with ovarian abscesses (8/50,
19/47, 18/49). Body weight gain of high-dose male mice was 87% of that of the vehicle-control
group; the body weight gain of low-dose male mice was comparable to that of the vehicle-
control group. Mean body weight of high-dose female mice was decreased during the last
1.5 years of the study. The body weight gain was reduced 55% compared to the controls at the
high dose and by 25% among low-dose females. In males, treatment-related nonneoplastic
changes included fatty metamorphosis of the liver, renal cytomegaly, and follicular cell
hyperplasia of the thyroid gland. In females, hyperplasia of the thyroid gland was observed.
This study identified a LOAEL of 25 mg/kg-day, based on histopathological findings in male
mice.
Klinefelter et al. (1995) reported interim (52-week) necropsy data from a cancer bioassay
in which male F344 rats were administered average concentrations of 0, 330, or 620 mg/L
bromodichloromethane in drinking water. Corresponding doses of 0, 22, and 39 mg/kg-day were
calculated by the authors using water consumption and body weight data. For the interim
sacrifice, 7 animals per dose group were killed, and the testis, epididymis, liver, spleen, kidney,
thyroid, stomach, intestine, and bladder were evaluated histopathologically.
Bromodichloromethane had no effect on body weight or on the kidney, liver, spleen, or thyroid
weight. There was no histopathological evidence of bromodichloromethane-related noncancer
or cancer effects on any of the examined organs. High levels of nephropathy and interstitial cell
hyperplasia were observed, but these lesions were not treatment-related. The NOAEL and
LOAEL for this study are based on reproductive endpoints. These reproductive effects are
summarized in Section VE. 1.
George et al. (2002) exposed male F344/N rats (78 animals/dose) to
bromodichloromethane via drinking water for 104 weeks. Nominal concentrations of 0, 0.07,
0.35, or 0.70 g/L were administered in drinking water containing 0.25% Emulphor®. The study
authors indicated that testing of higher concentrations was prevented by refusal of the test
animals to drink solutions containing more than 0.7 g/L. Six animals per exposure concentration
were sacrificed at 13, 26, 52, and 78 weeks for gross observation and histopathological
examination of the thyroid, liver, stomach, duodenum, jejunum, ileum, colon, rectum, spleen,
kidneys, urinary bladder, and testes. A complete rodent necropsy was performed at terminal
V - 39 November 15, 2005
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sacrifice (104 weeks) and representative samples of the tissues listed above were examined
microscopically. A complete pathological examination was performed on five rats from the
high-dose group. Serum profiles of LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine,
and total antioxidant activities were determined at 26, 52, and 104 weeks. Hepatocyte and renal
tubular cell proliferation were measured at each sacrifice by bromodeoxyuridine labeling.
The measured drinking water concentrations of bromodichloromethane were 0.06, 0.38,
and 0.76 g/L. When corrected for loss of bromodichloromethane as a result of volatility,
instability, or adsorption to glass surfaces during treatment, the corresponding administered
concentrations were 0.06, 0.33, and 0.62 g/L. Based on measured water consumption, these
levels correspond to mean daily doses for the entire study of 3.9, 20.6, and 36.3 mg/kg-day as
calculated by the study authors. No significant differences were observed among groups for feed
consumption or survival. Twenty-one to 22 unscheduled deaths were observed in each treatment
group. Mononuclear cell leukemia was seen in all dose groups and was reported to be the
primary cause of morbidity and mortality prior to 104 weeks. Exposure to
bromodichloromethane did not affect the growth rate of test animals when compared to the
control. Kidney weight was significantly depressed at the high dose and a significant negative
trend was observed for relative kidney weight. No significant changes were observed in clinical
chemistry parameters. Observed nonneoplastic changes in the liver (e.g., biliary fibrosis, bile
duct inflammation, and chronic inflammation) were considered to be age-related background
changes, since neither the incidence nor severity of the lesions differed from the control values.
Bromodichloromethane had no effect on hepatocyte proliferation as measured by
bromodeoxyuridine labeling. Renal tubular cell hyperplasia was significantly decreased in the
3.9 mg/kg-day group (-75%) and significantly increased in the 36.3 mg/kg-day group (15.8%)
relative to the control value (8.7%). Renal tubule cell proliferation was decreased in the 36.3
mg/kg-day group at 52 and 78 weeks of exposure. These data identify NOAEL and LOAEL
values for this study of 20.6 mg/kg-day and 36.6 mg/kg-day based on significant reductions in
absolute and relative kidney weight. Tumor data for this study are reported in Section V.G. 1.
In a parallel study, George et al. (2002) exposed male B6C3FJ mice (78 animals/dose) to
bromodichloromethane via drinking water for 100 weeks. Nominal concentrations of 0.05, 0.25,
or 0.50 g/L were administered in drinking water containing 0.25% Emulphor®. The vehicle
control solution consisted of 0.25% Emulphor®. Seven animals per exposure concentration were
sacrificed at 13, 26, 52, and 78 weeks for gross observation and histopathological examination of
the liver, stomach, duodenum, jejunum, ileum, colon, rectum, spleen, kidneys, urinary bladder,
and testes. A complete rodent necropsy was performed at terminal sacrifice (100 weeks) and
representative samples of the tissues listed above were examined microscopically. A complete
pathological examination was performed on five rats from the high-dose group. Serum profiles
of LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine, and total antioxidant activities
were determined at 26, 52, and 100 weeks. Hepatocyte and renal tubular cell proliferation were
measured by bromodeoxyuridine labeling at each sacrifice.
The measured drinking water concentrations of bromodichloromethane were 0.06, 0.30,
and 0.55 g/L. When corrected for loss of bromodichloromethane as a result of volatility,
instability, or adsorption to glass surfaces during treatment, the corresponding administered
V - 40 November 15, 2005
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concentrations were 0.06, 0.28, and 0.49 g/L. Based on measured water consumption, these
levels correspond to mean daily doses of 8.1, 27.2, and 43.4 mg/kg-day as calculated by the
study authors. Water consumption was significantly reduced at the mid- and high doses; the
study authors attributed the reduced intake to taste aversion. No significant differences were
observed among groups for feed consumption or survival. Exposure to bromodichloromethane
did not affect the growth rate of test animals when compared to the control. Absolute and
relative kidney weights were significantly depressed at 27.2 and 43.4 mg/kg-day when compared
to the control values. No significant changes were observed in clinical chemistry parameters.
Mild, treatment-related nonneoplastic hepatic lesions were observed in the 27.2 and 43.4 mg/kg-
day dose groups (identity and prevalence not reported). Increased incidences of hepatocellular
karyomegaly and necrosis with inflammation (prevalence and severity not reported) were not
dose-related. Bromodichloromethane treatment did not alter hepatocyte or renal tubule cell
proliferation. The NOAEL and LOAEL for this study are 8.1 and 27.2 mg/kg-day, respectively,
based on significantly decreased kidney weight. Tumor data for this study are reported in
Section V.G.I.
2. Dibromochloromethane
Tobe et al. (1982) evaluated the chronic effects of dibromochloromethane administered
in the diet to male and female Slc:Wistar SPF rats (40/sex/group) at concentrations of 0.0%,
0.022%, 0.088%, or 0.35% for 24 months. The animals were 5 weeks old at the start of the test
and weighed approximately 100 g. Dibromochloromethane was microencapsulated, and an
appropriate amount was mixed with powdered feed. Control groups (70 rats/sex) received
microcapsules without test compound. Body weight and food consumption were monitored
weekly for the first 6 months, every 2 weeks from 6 to 12 months, and every 4 weeks during the
second year of the study. Data were reported from the sacrifices of 9 animal s/sex/control group
and 5/sex/dose group at 18 months; all surviving animals were sacrificed at 24 months.
Necropsies, hematology, and serum biochemistry were conducted at the time of sacrifice. No
histopathology data for dibromochloromethane have been published from this study. Based on
reported body weights (150 to 475 g for males and 100 to 215 g for females) and food
consumption (15 to 20 g/day for males and 10 to 15 g/day for females), these levels
corresponded to doses of approximately 0, 12, 49, and 196 mg/kg-day for males and 0, 17, 70,
and 278 mg/kg-day for females. Marked suppression of body weight gain was seen in males and
females at the high dose, and mild suppression of body weight gain (about 10%) was seen in
males and females at the mid dose. Decreased T-GLY and serum cholinesterase activity and
increased y-GTP were seen in the mid- and high-dose males and females. Yellowing of the liver
surface was noted in the mid- and high-dose groups, and roughening of the liver surface was
noted in high-dose males. This study suggests NOAELs of 12 mg/kg-day (males) and 17 mg/kg-
day (females), and LOAELs of 49 mg/kg-day (males) and 70 mg/kg-day (females), based on
serum biochemistry data, decreased body weight, and gross necropsy findings.
NTP (1985) investigated the chronic oral toxicity of dibromochloromethane in male and
female F344/N rats. Groups of 50 animals/sex/dose were administered doses of 0, 40, or
80 mg/kg-day by gavage in corn oil for 5 days/week for 104 weeks. Survival was comparable in
all dose groups. Body weight gain was decreased in high-dose males after week 20; final weight
V-41 November 15, 2005
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gain was 88% of the control value. Females in both dose groups gained more weight than did
the controls. Histologic lesions in the liver were observed in males and females at both dose
levels. Changes included fat accumulation, "ground glass" appearance of the cytoplasm, and
altered basophilic staining. This study identified a LOAEL of 40 mg/kg-day for
dibromochloromethane based on liver lesions.
NTP (1985) performed a similar chronic oral exposure study of dibromochloromethane
toxicity in male and female B6C3Fj mice. Groups of 50 animals/sex/dose were administered
doses of 0, 50, or 100 mg/kg-day by gavage in corn oil for 5 days/week for 105 weeks. Survival
in females was not different from controls, while survival in high-dose males was significantly
decreased. An overdosing accident at week 58 killed 35/50 male mice in the low-dose group,
and this group was not considered further. Mean body weight was decreased in high-dose males
and females, but not in low-dose females. Treatment-related hepatocytomegaly and focal
necrosis were observed in livers of high-dose males. Females showed liver calcification at the
high dose and fatty metamorphosis at both the low and high doses. An increased incidence of
follicular cell hyperplasia in the thyroid was observed in low- and high-dose females relative to
the control. Thyroid lesions were not observed in treated males. This study identified a LOAEL
of 50 mg/kg-day for dibromochloromethane in mice.
3. Bromoform
Tobe et al. (1982) evaluated the chronic effects of bromoform administered in the diet to
male and female Slc:Wistar SPF rats (40/sex/group) for 24 months. The animals were 5 weeks
old at the start of the test and weighed approximately 100 g. Bromoform was
microencapsulated, and administered at dietary levels of 0.0%, 0.04%, 0.16%, or 0.65%.
Control groups (70 rats/sex) received microcapsules without test article. Body weights and food
consumption were monitored weekly for the first 6 months, every 2 weeks from 6 to 12 months,
and every 4 weeks during the second year of the study. Data were reported from the sacrifices of
9 animals/sex in the control group and 5/sex/dose in the exposure groups at 18 months; all
surviving animals were sacrificed at 24 months. At each time of sacrifice, necropsies,
hematology, and serum biochemistry were conducted. No histopathology data for bromoform
have been published from this study. Based on reported body weights (150 to 475 g for males
and 100 to 215 g for females) and food consumption (15 to 20 g/day for males and 10 to 20
g/day for females), these levels corresponded to doses of about 0, 22, 90, and 364 mg/kg-day for
males and 0, 38, 152, and 619 mg/kg-day for females. Marked suppression of body weight gain
was seen in males and females at the high dose, and mild suppression of body weight gain (about
15%) was seen in males and females at the mid dose. Dose-related decreases in non-esterified
fatty acids were observed in all treated males and in females at the mid and high dose. Females
also exhibited a dose-related increase in levels of y-GTP with the increases significant at the mid
and high dose. Other serum biochemistry changes in the high-dose groups included decreased
serum triglyceride (T-GLY) and increased AST and ALT activity. Specifically, T-GLY levels
significantly decreased by 86% and 80% in the male and female high-dose groups, respectively,
by study termination. AST and ALT activities at study termination were significantly increased
1.6 to 2.6-fold in animals at the high dose compared to controls, with the exception that the
increase in AST activity in males was statistically nonsignificant. Yellowing and small white
V - 42 November 15, 2005
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spots, and roughening of the surface were seen in the livers of the mid- and high-dose animals.
Roughening of the liver surface was observed in the high-dose groups. Based on the necropsy
findings and the serum biochemistry data, this study indicated NOAELs of 22 mg/kg-day for
males and 38 mg/kg-day for females, and LOAELs of 90 mg/kg-day for males and 152 mg/kg-
day for females.
NTP (1989a) exposed male and female F344/N rats (50/sex/group) to bromoform by
gavage in oil for 103 weeks (5 days/week) at doses of 0, 100, or 200 mg/kg-day. Animals were
observed for clinical signs throughout the study (2 days/week). At termination, necropsy and
histopathological examination were performed on all animals. Body weight gain was decreased
by 37% in high-dose females and by 29% in high-dose males relative to the respective controls.
Survival of the high-dose males was also decreased. Both males and females were lethargic.
Hepatic fatty change and chronic inflammation were noted in both males and females at both
doses, and minimal necrosis was increased in high-dose males. Nonneoplastic changes were not
reported in other tissues. This study identified a LOAEL of 100 mg/kg-day in both male and
female rats.
NTP (1989a) exposed groups of 50 male B6C3FJ mice by gavage in oil to doses of 0, 50,
or 100 mg/kg-day of bromoform for 103 weeks (5 days/week). Groups of 50 female mice were
administered doses of 0, 100, or 200 mg/kg-day. Animals were observed for clinical signs
2 days/week throughout the study. At termination, all animals were necropsied, and a thorough
histological examination of tissues was performed. Decreased survival was observed in females,
but not males. This was at least partly due to a utero-ovarian infection. No clinical signs were
noted. Body weight gains were 82% and 72% of the control values for low- and high-dose
females, respectively, but body weight gain was not affected in males. Increased incidences of
minimal to mild fatty changes were noted in the livers of both low- and high-dose females, but
not males. Nonneoplastic changes were not detected in other tissues. This study identified a
LOAEL of 100 mg/kg-day for female mice, based on decreased body weight and fatty changes
of the liver. No NOAEL for females was identified. For males, a NOAEL of 100 mg/kg-day
was identified.
E. Reproductive and Developmental Effects
Studies that have examined the reproductive and developmental toxicity of the
brominated trihalomethanes are summarized in Table V-9 at the end of this section.
1. Bromodichloromethane
a. Studies in Rats
Ruddick et al. (1983) investigated the teratogenicity and developmental toxicity of
bromodichloromethane in Sprague-Dawley rats. Pregnant dams (15/dose group) were
administered 0, 50, 100, or 200 mg/kg-day by gavage in corn oil on gestation days (GD) 6 to 15.
Body weights were measured on GD 1, on GD 1 through GD 15, and before and after fetuses
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were removed by caesarean section on GD 22. On GD 22, females were sacrificed and body
tissues (including the uterus) were removed for pathological examination. Females were
evaluated for the number of resorption sites, and number of fetuses. Maternal blood samples
were collected and evaluated for standard hematology and clinical chemistry parameters. The
liver, heart, brain, spleen, and one kidney were weighed. Standard histopathology was
conducted on control and high-dose females (5/group). All fetuses were individually weighed,
and evaluated for viability and external malformations. Histopathologic examination was
performed on two pups per litter. Of the remaining live fetuses, approximately two-thirds were
examined for skeletal alterations and one-third for visceral abnormalities.
Although 15 inseminated females per dose group were exposed to bromodichloro-
methane, not all females became pregnant and/or delivered litters. Therefore, the number of
litters per dose group ranged from 9 to 14. One animal died in the control group, but no deaths
occurred in any of the exposed groups. In the high-dose group, maternal weight gain was
significantly depressed by 38% as compared with controls. Although maternal weight gains
were also reduced in the low- and mid-dose groups (13% and 15%, respectively, as compared
with controls), these differences were not reported as statistically significant. Relative maternal
liver weight was significantly increased in all exposed groups (110%, 110%, and 117% for the
low-, mid-, and high-dose groups, respectively as compared with control values). Relative
kidney and brain weights were also statistically increased in the high-dose group only. These
increases in relative organ weights may have been associated with the decreased body weight
gains in treated females. No treatment-related changes in hematology, clinical chemistry,
histopathology, number of resorptions, and the number of fetuses per litter were noted. No
differences between treated and control groups were reported for fetal weights, gross
malformations (terata), and visceral abnormalities. However, an increase in the incidence of
sternebral anomalies was observed in all treated groups. The number of affected fetuses/number
of affected litters were 2/2, 8/4, 9/7, 10/6 for the control, low-, mid-, and high-dose groups,
respectively. Statistical significance of fetotoxic endpoints was not reported by the study
authors. An independent statistical analysis (using the Fisher Exact test) was conducted on the
published data for development of this Criteria Document and demonstrated that none of these
increases differed significantly from control values (p>0.05). A trend test showed a statistically
significant dose-related trend (p=0.03); stepwise analysis indicated that the trend became
nonsignificant if the high-dose (200 mg/kg-day) was omitted from the analysis. These findings
suggest that the LOAEL and NOAEL for developmental toxicity are 200 and 100 mg/kg-day,
respectively. However, it should be noted that the small sample sizes (the sampling unit is the
litter) limited the statistical power of the experiment to detect possible significant differences at
lower doses. Based on significantly decreased maternal body weight gain, the LOAEL and
NOAEL for maternal toxicity are 200 and 100 mg/kg-day, respectively.
Klinefelter et al. (1995) evaluated the effects of bromodichloromethane exposure on male
reproduction during a chronic cancer bioassay study in which F344 rats were administered
bromodichloromethane in drinking water at concentrations of 0, 330, or 620 mg/L. The authors
estimated the doses to be 0, 22, and 39 mg/kg-day. At 52 weeks, the authors conducted an
interim sacrifice, which included an evaluation of epididymal sperm motion parameters and
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histopathology of the testes and epididymides. No histologic alterations were observed in any
reproductive tissue. Sperm velocities (mean straight-line, average path, and curvilinear),
however, were significantly decreased at 39 mg/kg-day. No effect on sperm motility was
observed at 22 mg/kg-day. The NOAEL and LOAEL for reproductive effects are thus 22 and 39
mg/kg-day, respectively.
The results for sperm velocity in the study by Klinefelter et al. (1995) are of interest
because personal exposure to bromodichloromethane in tap water at home showed a weak but
statistically significant inverse association with significantly decreased sperm linearity in an
epidemiological study of semen quality (Fenster et al., 2003; see Section VI.B.2.b for summary),
suggesting the possibility of similar male reproductive effects in humans and in F344 rats treated
at a higher dose than anticipated in human exposures. Treatment-related effects on sperm
characteristics were not observed in two other reproductive studies (NTP, 1998; Christian et al.,
2002) of Sprague-Dawley rats exposed to bromodichloromethane in the drinking water at
concentrations similar to or higher than those used in the Klinefelter et al. (1995) study.
However, the differences in outcome may have occurred as a result of the strain tested or
differences in methodology. In some male reproductive studies, the use of F344 rats has been
associated with considerable variability in endpoints such as epididymal sperm motility (Zenick
et al., 1994), although Klinefelter et al. (1995) reported use of techniques designed to reduce this
variability. NTP (1998) used a shorter duration of exposure (35 days) that did not span the entire
period of spermatogenesis in rats (approximately 52 days) and Christian et al. (2002) did not
measure the sensitive sperm motility parameters (mean straight-line, average path, and
curvilinear velocities) that were affected in the Klinefelter et al. (1995) study. Neither Christian
et al. (2002) nor NTP (1998) observed treatment-related effects on fertility, but fertility is
considered to be a less sensitive indicator of male reproductive function than effects on sperm
motility.
Narotsky et al. (1997) examined both the developmental toxicity and the effect of dosing
vehicle on the developmental toxicity of bromodichloromethane. F344 rats (12 to 14/group)
were administered bromodichloromethane by gavage, in either corn oil or an aqueous vehicle
containing 10% Emulphor®, at dose levels of 0, 25, 50, or 75 mg/kg-day on GD 6 to 15. Dams
were allowed to deliver naturally, and pups were evaluated postnatally. Maternal body weights
were assessed on GD 5, 6, 8, 10, 13, and 20, and all rats were observed for clinical signs of
toxicity throughout the test period. Postnatal day (PND) 1 was defined as GD 22 irrespective of
the actual time of parturition. All pups were examined externally for gross malformations and
weighed on PND 1 and 6. Skeletal and visceral anomalies in the pups were not evaluated.
Following PND 6 examination, the dams were sacrificed and the number of uterine implantation
sites per female was recorded. The uteri of females that did not deliver litters were stained and
evaluated histopathologically to detect any cases of full-litter resorption (FLR). In order to
compare the kinetics of dosing vehicles, a separate experiment was conducted in which pregnant
females (3 to 4 animals per vehicle per time point) were administered a single dose of 75 mg/kg
on GD 6 and whole blood samples were collected at 30 minutes, 90 minutes, 4.5 hours, or 24
hours postdosing. Following blood collection, the animals were sacrificed, blood concentrations
of bromodichloromethane were measured, and pregnancy status was confirmed at necropsy.
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In the developmental toxicity study, one animal that received 75 mg/kg-day in corn oil
died before study termination. In the mid- and high-dose groups, clinical signs of toxicity were
evident among animals administered bromodichloromethane in either dosing vehicle. At
75 mg/kg-day, kyphosis (humpback) was observed in animals receiving the oil vehicle, and
piloerection was observed in animals receiving either vehicle. At 50 mg/kg-day, piloerection was
observed in animals receiving the aqueous gavage, and chromodacryorrhea/lacrimation was
observed in animals receiving the oil gavage. Maternal weight gain was significantly decreased
in all dosed groups receiving the aqueous vehicle and in the 50 and 75 mg/kg-day groups in
animals receiving the oil vehicle on GD 6 to 8 (data not reported for other time periods).
Although maternal weight gain was also reduced at 25 mg/kg-day in animals given the oil
vehicle, this decrease was not statistically significant. However, a two-way analysis of variance
(ANOVA) indicated that there was no interaction between vehicle and dose for this maternal
endpoint. All control and 25 mg/kg-day litters survived the test period; however, FLR was
observed at 50 and 75 mg/kg-day with both dosing vehicles. Statistical analysis (ANOVA) of
FLR incidence showed a significant vehicle-dose interaction. For females receiving
bromodichloromethane in corn oil, FLR was reported in 8 and 83% of the litters at 50 and 75
mg/kg-day, respectively; an additional high-dose litter was carried to term but was delivered late
(GD 23), and all pups died by PND 6. For females receiving the aqueous vehicle, FLR was
observed in 17 and 21% of the litters at 50 and 75 mg/kg-day, respectively. There were no
effects on gestation length, pre- or postnatal survival, or pup morphology in surviving litters,
with the exception noted above in the 75 mg/kg-day oil vehicle group. Based on full litter
resorption, the LOAEL for developmental toxicity is 50 mg/kg-day for both vehicles, and the
corresponding NOAEL is 25 mg/kg-day. Based on significantly reduced body weight gain
during GD 6 to 8 in dams receiving the aqueous vehicle, the LOAEL for maternal toxicity is the
lowest dose tested, 25 mg/kg-day, and a NOAEL could not be determined.
Analysis of bromodichloromethane concentrations in blood indicated that circulating
levels decreased over time with both vehicles, but tended to be higher following corn oil
administration. Bromodichloromethane blood concentrations were thus vehicle-dependent and
differed statistically at both 4.5 and 24 hours postdosing (mean of 3.1 ng/mL versus 0.4 ng/mL
for oil and aqueous vehicles, respectively, at 24 hours). The elimination half-life of
bromodichloromethane was estimated to be 3.6 hours when administered in corn oil and 2.7
hours when given in the aqueous vehicle.
Narotsky et al. (1997) also calculated both an ED05 (i.e., the effective dose producing a
5% increase in response rate above background) and a benchmark dose (BMD; as defined by the
authors, the BMD is the lower confidence interval of the ED05) for each vehicle. For the corn oil
vehicle, the ED05 and BMD were 48.4 and 39.3 mg/kg-day, respectively. For the aqueous
vehicle, the ED05 and BMD were 33.3 and 11.3 mg/kg-day, respectively. The study authors
noted that the greater BMD value for the corn oil vehicle seemed counterintuitive in view of the
higher FLR response rate in the 75 mg/kg-day aqueous vehicle group (83% for aqueous vehicle
versus 21% for corn oil vehicle). However, the dose response for bromodichloromethane-
induced FLR differed markedly between vehicles, and the response rate in the 50 mg/kg-day
corn oil vehicle group (8%) closely approximated 5%, the effect level defined by the ED05.
V - 46 November 15, 2005
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According to the study authors, this resulted in a smaller confidence interval around the ED05 for
the corn oil vehicle, yielding a less conservative (i.e., higher) BMD. These findings are
consistent with the pharmacokinetic data demonstrating a slower elimination of
bromodichloromethane following a single dose of 75 mg/kg in corn oil as compared with the
same dose in aqueous vehicle, and suggest that the influence of vehicle on FLR rate is dose-
dependent.
NTP (1998) conducted a short-term reproductive and developmental toxicity screen in
Sprague-Dawley rats to evaluate the effects of bromodichloromethane administered in drinking
water. The study was designed to identify the physiological endpoints most sensitive to
bromodichloromethane exposure, and assessed development, female reproduction, male
reproduction, hematology, clinical chemistry, and pathology. In males, the reproductive
endpoints evaluated included testis and epididymis weight, sperm morphology, density and
motility. The female reproductive parameters evaluated included mating index, pregnancy
index, fertility index, gestation index, number of live births, number of resorptions, implants per
litter, corpora lutea and pre-and post- implantation loss. Concentrations of 0, 100, 700 and 1300
ppm bromodichloromethane were selected for use in this study based on decreased water
consumption observed in a preliminary 14-day range-finding study (see Section V.B.I). Two
groups of male Sprague-Dawley rats and three groups of female Sprague-Dawley rats were
assigned to treatment groups as indicated in Table V-6.
Table V-6 NTP (1998) Study Design
Gender
Male
Female
Group
A
B
A
B
C
Description
non-BrdU treated
BrdU treated
peri-conception exposure
gestational exposure
BrdU treated, peri-
conception exposure
# Animals per Dose Group
0 ppm*
10
5
10
13
5
100 ppm
10
5
10
13
5
700 ppm
10
5
10
13
5
1300 ppm
10
8
10
13
8
: Control animals received deionized water
Test animals were dosed for 25 to 30 days, with the exception of Group B females which
were dosed from GD 6 to evidence of littering/birth (total duration approximately 15 to 16 days).
Based on measured water consumption, the nominal concentrations of 0, 100, 700 and 1300 ppm
were equivalent to doses of 0, 8, 41, and 68 mg bromodichloromethane/kg-day for all male rats
and 0, 14, 72 and 116 mg bromodichloromethane/kg-day for all female rats in groups A and C.
The calculated doses for Group B females were 0, 13, 54, and 90 mg/kg-day. All animals
V-47
November 15, 2005
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survived the treatment period, with the exception of one Group A male in the 700 ppm dose
group. Body weight and food and water consumption were decreased at many time points for
animals dosed with 700 and 1300 ppm bromodichloromethane. Body weights in the dosed
groups were decreased from 5% to 13%, food consumption was decreased from 14% to 53%,
and water consumption was decreased from 7% to 86% relative to control animals. However,
bromodichloromethane exposure did not alter any reproductive parameter investigated in males
or females, with the exception of a non-dose-related increase in the number of live fetuses per
birth at the 100 ppm concentration in Group C females, and a slight decrease in the number of
corpora lutea at the 700 ppm concentration in Group A females. On the basis of these results,
NTP (1998) concluded that bromodichloromethane was not a short-term developmental or
reproductive toxicant any of the doses tested in the study. The reproductive/developmental
NOAELs are 68 and 116 mg/kg-day for male and female rats, respectively. The adult NOAEL
and LOAEL for this study were identified on the basis of hepatic effects, which are discussed in
detail in Section V.B.I.
Bielmeier et al. (2001) conducted a series of experiments to investigate the mode of
action for bromodichloromethane-induced full litter resorption (FLR) in F344 rats. This series
of experiments included a strain comparison of F344 and Sprague-Dawley (SD) rats, a critical
period study, and two hormone profile studies. The strain comparison and critical period studies
are summarized in Table V-7 and discussed below. The hormone profile studies and related
follow-on studies reported by Bielmeier et al. (2004) are discussed in Section V.H.2 (Hormonal
Disruption).
In the strain comparison experiment, female SD rats (13 to 14/dose group) were dosed
with 0, 75, or 10 mg/kg-day by aqueous gavage in 10% Emulphor® on GD 6 to 10. F344 rats
(12 to 14/dose group) were concurrently dosed with 0 or 75 mg/kg-day administered in the same
vehicle. The incidence of FLR in the bromodichloromethane-treated F344 rats was 62%, while
the incidence of FLR in SD rats treated with 75 or 100 mg/kg-day of bromodichloromethane was
0%. Both strains of rats showed similar signs of maternal toxicity, and the percent body weight
loss after the first day of dosing was comparable for SD rats (no resorption observed) and the
F344 rats that resorbed their litters. F344 rats that maintained their pregnancies generally did not
lose weight after the first dose, although they did experience significantly less weight gain than
the controls. Both strains of rats had similar incidences of piloerection. However, the strains
showed different ocular responses to compound administration. One half (7/14) of the treated
F344 rats showed lacrimation and/or excessive blinking shortly after dosing during the first two
days of compound administration. In comparison, only 1/28 of the SD rats exhibited this
response. The study authors reported that lacrimation was not predictive of FLR in F344 rats.
The rats were allowed to deliver and pups were examined on postnatal days 1 and 6. Surviving
litters appeared normal and no effect on post-natal survival, litter size, or pup weight was
observed.
V - 48 November 15, 2005
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Table V-7 Summary of Experiments Conducted by Bielmeier et al. (2001)a
Study/Strain
Dose
(mg/kg-day)
Treatment
Period
Number of animals
Treated
Pregnant
Resorbed
%FLR
Strain Comparison
F344
F344
SD
SD
SD
0
75
0
75
100
GD 6-10
GD 6-10
GD 6-10
GD 6-10
GD 6-10
12
14
13
14
14
11
13
13
14
14
0
8
0
0
0
0
62**
0
0
0
Critical Study Period
F344
F344
F344
F344
0
75
75
75
GD 6-15
GD 6-15
GD 6-10
GD 11-15
8
10
12
13
8
10
12
13
0
5
9
0
0
50*
75**
0
Source: Table 1 in Bielmeier et al. (2001)
Abbreviations: GD, gestation day; FLR, full litter resorption; SD, Sprague-Dawley
a Additional experiments to characterize profiles for selected hormones are discussed in section V.H.2
* p<0.05; ** p<0.01; *** pO.OOlfor significant differences from controls (Fisher's Exact Test)
Bielmeier et al. (2001) conducted a second experiment to identify the critical period for
bromodichloromethane-induced FLR in F344 rats. Two different five day periods during
organogenesis were compared. Pregnant rats (12 to 13/dose group) were dosed with 75 mg/kg-
day by gavage in 10% Emulphor® on GD 6 to 10 (which includes the luteinizing hormone-
dependent period of pregnancy) or GD 11 to 15 (a luteinizing hormone-independent period).
Rats (8 to 10/dose group) dosed with 0 or 75 mg/kg-day on GD 6 to 15 served as negative and
positive controls, respectively. FLR occurred only in rats treated on GD 6 to 10 or GD 6 to 15
(incidences of 75% and 50%, respectively). In contrast, all rats treated with
bromodichloromethane on GD 11 to 15 maintained their litters. Surviving litters appeared
normal and no effect on post-natal survival, litter size, or pup weight was observed. This finding
was interpreted by the study authors as evidence for an effect of bromodichloromethane on
luteinizing hormone secretion or signal transduction.
The experiments conducted by Bielmeier et al. (2001) identified a LOAEL of 75 mg/kg-
day (the lowest dose tested) based on FLR in F344 rats. A NOAEL was not identified.
V-49
November 15, 2005
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The Chlorine Chemistry Council sponsored a range finding reproductive toxicity study of
bromodichloromethane in rats (CCC, 2000c), which was conducted according to standard U.S.
EPA test guidelines (U.S. EPA, 1998c) and GLP standards. This study is summarized in
Christian (2001b). Male and female Sprague Dawley rats (10/sex/group) were randomly
assigned to five exposure groups. Additional rats (6 males/group and 15 females/group) were
assigned to satellite groups for collection of samples for analysis of bromodichloromethane
concentrations in selected tissues and fluids (see Section III.B). Bromodichloromethane was
administered to parental rats (P generation) in drinking water at concentrations of 0, 50, 150,
450, or 1350 ppm. Exposure began 14 days before cohabitation and continued until the day of
sacrifice. Female estrous cycle evaluations were performed daily, beginning 14 days before
exposure initiation and continuing for 14 days after the first day of exposure. Clinical
observations were recorded daily during the exposure period.
Male body weights were recorded weekly during the entire exposure period and at
sacrifice; female body weights were recorded weekly during precohabitation and cohabitation,
on GD 0, 7, 14, 21, and 25, and on lactation days (LD) 1, 5, 8, 11,15, 22, and 29. Lactation was
extended for one week (LD 22-29) beyond the normal 3-week period because Fx pup body
weights in the three highest dose groups were significantly reduced on LD 21 relative to control
values (results are described below). Water and feed consumption were recorded weekly and at
sacrifice for males during the entire exposure period (except for feed consumption during
cohabitation), and more frequently for females during gestation and lactation. On LD 29, two Fx
pups per sex were selected from each litter for an additional week of postweaning observation,
provided ad libitum access to water containing the same concentration of bromodichloromethane
administered to their parents (P generation), and sacrificed on Day 8 postweaning. P generation
female rats were assessed for duration of gestation, fertility index, gestation index, number and
sex of offspring per litter, number of implantation sites, and clinical signs of toxicity during the
postpartum period. During lactation, maternal behavior was observed and recorded on LD 1, 5,
8, 11, 22, and 29. Litters were externally examined following delivery to identify the number
and sex of pups, stillbirths and live births, and gross external malformations. Litters were
observed at least twice daily during the preweaning and postweaning period for pup deaths and
clinical signs of toxicity. Litter size and viability, viability indices, lactation indices, percent
survival, and sex ratios were calculated. During the postweaning period of observation, body
weights and feed consumption were recorded at weaning and on day 8 postweaning; water
consumption was recorded daily.
At the end of the parental exposure periods (64 days for males and a maximum of 74
days for females), all P generation rats were sacrificed and a gross necropsy of the thoracic,
abdominal, and pelvic viscera was performed. In addition, testes and epididymides were excised
from males and paired organ weights were measured. Fx pups exposed to
bromodichloromethane in their drinking water for one week following weaning were sacrificed
on Day 8 postweaning and examined for gross lesions. No histopathology was performed on
either the P or Fx generation.
V - 50 November 15, 2005
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The consumption of bromodichloromethane was calculated from measured water intake
and measured concentrations of the test article. Mean consumed dosages of bromodichloro-
methane for P generation male rats during the entire exposure period, P generation female rats
during different physiologic stages, and Fx postweaning rats are summarized in Table V-8.
Males and nonpregnant female rats tended to consume similar amounts of
bromodichloromethane. Progressively higher dosages were consumed by female rats in the pre-
mating, gestation, and lactation periods, respectively. The highest dosages among all groups
were consumed by Fx female rats during the one-week postweaning observation period. A
possible source of error in the estimates for lactating females was consumption of the dams'
drinking water by their pups.
In the P generation, all male rats and all females except one survived to scheduled
sacrifice. Exposure-dependent reductions in both absolute (g/day) and relative (g/kg body
weight-day) water consumption were observed in all rats of both sexes and were attributed to
taste aversion. Reduced water consumption was most pronounced during the first week of
exposure, and was evident during precohabitation and cohabitation in both sexes, and during
post-cohabitation in males and gestation in females. However, the decrease in water
consumption during these times was not as severe as that observed during the first week of
exposure. Decreased water consumption was not clearly noted in females during lactation,
presumably reflecting the physiologic demands for high fluid consumption during this period.
Exposure-related decreases in feed consumption were noted for males and females in the 150,
450, and 1350 ppm exposure groups, and persisted in the 450 and 1350 ppm females during
gestation and lactation. Treatment-related clinical signs of toxicity were observed in both sexes
in the 1350 ppm exposure groups and were considered to be generally associated with reduced
water consumption. Males exhibited dehydration, emaciation, chromorhinorrhea, and
chromodacryorrhea during the pre-mating, cohabitation and post-cohabitation periods; however,
the most severe symptoms resolved within the first 17 days of exposure. Among females, urine-
stained fur was observed in one or more animals in the three highest dose groups during lactation
and was considered to be treatment-related. Reductions in mean body weight gain and body
weight were observed in male rats in the 450 and 1350 ppm exposure groups relative to
controls. These effects were most severe during the first week of exposure. Mean body weight
gains for the 450 ppm and 1350 ppm male groups over the entire exposure period were 91.3%
and 76.3% of the control values, respectively. At study termination, mean male body weights
were 96.5% and 91.6 % for the 450 ppm and 1350 ppm, respectively, relative to control values.
In female rats, reductions in body weight gain and body weight occurred in 150, 450, and 1350
ppm groups. These effects were most severe during the first week of exposure, but also
persisted throughout gestation and lactation. During gestation, the mean reductions in female
body weight in the 150, 450, and 1350 ppm groups were 95.8%, 95.3%, and 85.3% of the control
values, respectively. Mean body weights for the entire lactation period were not presented in the
study report; however, inspection of the data, presented separately for LD 1, 8, 15, 22, and 29,
indicated that female body weights were decreased relative to controls in a dose-dependent
manner in the three highest dose groups at all time points.
V-51 November 15, 2005
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Table V-8 Mean Consumed Doses (mg/kg-day) of Bromodichloromethane in the Range
Finding Study Conducted by CCC (2000c) and Summarized in Christian et al. (2001b)
Gen.
P
P
P
P
F,
F,
Sex
M
F
F
F
M
F
Exposure Interval
Full study
Study Days 1-64
Pre-mating
Study days 1-15
Gestation days 0-21
Lactation days 1-15
Postweaning days 1-8
Postweaning days 1-8
0 ppm
0.0
0.0
0.0
0.0
0.0
0.0
50 ppm
4.2 ±0.4
4.7 ±0.8
5.4 ±0.7
11.0±1.9
13.6 ±3. 5
13. 9 ±2.6
150 ppm
11.8±1.8
13. 3 ±2.0
16.3 ±2.2
31.4 ±2.6
41.4±7.1
40.1 ±6.8
450 ppm
27.5 ±3.4
23.5 ±5.3
41.7 ±6.4
90.3 ±7.3
106.9 ±20.8
111. 9 ±42.1
1350 ppm
67.2 ±5.6
70.8 ±1.8
111.7±6.2
222.4 ±19.9
297.8 ± 113.8
333.6 ±110.6
No gross lesions attributable to bromodichloromethane were observed in the P generation
male or female rats at necropsy. The absolute paired epididymal weights were slightly reduced
(93.2% and 92.5%, respectively) in the 450 and 1350 ppm exposure groups. However, relative
paired epididymal weights were unaffected, suggesting that the decreased absolute values were
associated with the reduced terminal body weights in these groups. Absolute and relative testes
weights were not altered by exposure to bromodichloromethane. No effects of bromodichloro-
methane were observed on any of the measured reproductive parameters in P generation male or
female rats. However, bromodichloromethane exposure was associated with a concentration-
dependent reduction in Fx pup body weights in the 150, 450, and 1350 ppm exposure groups.
Pup weights were reported for postpartum days 1, 5, 8, 15, 22, and 29. The mean litter pup
weights in treated groups were comparable to the mean litter pup weight of the control group on
LD 1. Beginning on LD 5, reductions in mean pup weights in the three highest dose groups
increased with increasing dose and duration of the postpartum period. On LD 29, pup weights
averaged 7, 12, and 29% less than controls in the 150, 450, and 1350 ppm exposure groups,
respectively. Reduced body weight gain continued to occur in the Fx pups administered parental
concentrations of bromodichloromethane in drinking water for one week postweaning. No
reductions in either body weight gain or body weight were observed in Fx pup litters in the 50
ppm group during lactation or the one-week postweaning period.
Statistical analysis was not conducted in this range finding study. Based on decreased
pup weight and pup weight gain, the LOAEL for developmental toxicity is 150 ppm, and the
corresponding NOAEL is 50 ppm. Although the effect of reduced water consumption on the
decreases in feed consumption, body weight gain, and body weight observed in the P generation
adults is unclear, the LOAEL for parental toxicity is considered to be 150 ppm and the NOAEL
is 50 ppm. Due to the marked changes in drinking water consumption by P generation female
rats during different physiological stages (pre-mating, mating, gestation, and lactation), it is not
V-52
November 15, 2005
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possible to convert the administered drinking water concentrations into biologically meaningful
average daily doses.
The Chlorine Chemistry Council sponsored a developmental toxicity study of
bromodichloromethane in rats (CCC, 2000d). Data from this study are summarized in Christian
et al. (200la). This study was conducted in accordance with U.S. EPA Health Effects Test
Guidelines OPPTS 870.3700: Prenatal Developmental Toxicity Study (U.S. EPA, 1998c) and
U.S. EPA Good Laboratory Practice Standards (40 CFR Part 160/792). Female Sprague-Dawley
rats (25/exposure group) were exposed to bromodichloromethane in the drinking water at
concentrations of 0, 50, 150, 450, and 900 ppm on days 6 to 21 of gestation (GD 6 to 21). The
rats were examined daily during the exposure period for clinical signs related to exposure,
abortions, premature deliveries and deaths. Body weights, water consumption, and feed
consumption were recorded at intervals throughout the exposure period. All study animals were
sacrificed on GD 21 and caesarean-sectioned. A gross necropsy of the thoracic, abdominal, and
pelvic viscera was performed. Data was collected for gravid uterus weight (with cervix),
number of corpora lutea/per ovary, evidence of pregnancy, number and distribution of
implantation sites, live and dead fetuses, early and late resorption, and placental abnormalities
(size, color, or shape). Individual fetuses were weighed, sexed, and examined for gross external
abnormalities. Approximately one-half of the fetuses in each litter were examined for soft tissue
alterations and the heads of these fetuses were examined by free-hand sectioning. The remaining
fetuses in each litter were examined for skeletal alterations.
Consumed dosages for GD 6 to 21 were calculated from measured water consumption
and measured body weights and averaged 0, 2.2, 18.4, 45.0, and 82.0 mg/kg-day, respectively.
No abortions, premature deliveries, deaths or treatment-related clinical signs were observed
during the study and all rats survived until scheduled sacrifice. No treatment-related gross
lesions were identified at autopsy. Exposure-related decreases in maternal body weight gains
occurred in all groups administered bromodichloromethane in the drinking water on the first day
of exposure (GD 6 to 7). The reduction in maternal body weight gain reached statistical
significance in the 150, 450, and 900 ppm groups. The effect was most severe on these days and
appeared to be related to taste aversion. The effect on maternal body weight gain was persistent
in the 450 and 900 ppm exposure groups. In contrast, the effect was transient in the 50 and 150
ppm exposure groups. Average body weights were significantly reduced in the 450 and 900 ppm
exposure groups on GD 7 to 21. Average maternal body weights in the same groups were
significantly reduced at terminal sacrifice when corrected for gravid uterine weight.
Statistically significant, exposure-related decreases in absolute (g/day) and relative (g/kg-
day) water consumption were observed in all groups exposed to bromodichloromethane. This
effect was evident for the entire exposure period (GD 6 to 21) and the entire gestation period
(GD 0 to 21). Within the exposure period, the effects were most pronounced on the first two
days of exposure and gradually decreased in severity with continued exposure. Exposure-related
decreases in absolute and relative feed consumption were observed in the 150, 450, and 900 ppm
groups. In the 150 ppm group, the effects were statistically significant only on GD 12 to 15 and
thus were considered to be of little biological importance by the study authors. In the 450 ppm
V - 53 November 15, 2005
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and 900 ppm groups, absolute and relative feed consumption was significantly reduced for the
entire exposure period (GD 6 to 21), the entire gestation period (GD 0 to 21), and at many
intervals within the exposure period. The effect of bromodichloromethane on feed consumption
tended to be most severe during the first two days of compound administration.
Caesarean section and litter parameters were unaffected by exposure of the dams to
bromodichloromethane concentrations up to 900 ppm. Litter averages for corpora lutea,
implantations, litter sizes, proportion of live fetuses, early or late resorptions, fetal body weights,
percent reabsorbed conceptuses, and percent live fetuses were comparable among all study
groups and no significant differences were observed. No cases of full litter resorption were
observed and there were no dead fetuses. Late resorption occurred in one control group litter.
All placentae appeared normal. All values for the examined litter parameters were within the
historical range of the test facility (Argus Research Laboratories, Horsham, PA) or litter
incidences of any gross external or soft tissue alterations. With respect to skeletal alterations, no
skeletal malformations were observed in any fetus. The only statistically significant (p<0.01)
changes in the occurrence of skeletal variations were reversible delays in ossification. These
included an increased fetal incidence (fetal incidence: 0 ppm, 1/182; 50 ppm, 0/199; 150 ppm,
0/200; 450 ppm, 0/188; 900 ppm, 4/194; litter incidence: 0 ppm, 1/23; 50 ppm, 0/25; 150 ppm,
0/25; 450 ppm, 0/25; 900 ppm, 2/25) of wavy ribs in the 900 ppm exposure group and a
decreased number of ossification sites per fetus per litter for the forelimb phalanges (Mean
number ± SD of ossification sites: 8.14 ±0.91, 8.30 ± 0.65, 8.09 ± 0.63, 7.92 ± 0.78, 7.46 ± 0.78)
and the hindlimb metatarsals (Mean number ± SD of ossification sites: 4.81 ± 0.25, 4.86 ± 0.23,
4.78 ± 0.27, 4.71 ± 0.28, 4.53 ± 0.33) and phalanges (Mean number ± SD of ossification sites:
6.20 ±1.19, 6.20 ±1.17, 5.84 ± 0.94, 5.86 ± 0.79, 5.29 ± 0.54). The increased fetal incidence of
wavy ribs was considered unrelated to bromodichloromethane exposure by the study authors
because the litter incidence (the more relevant measure of effect) did not differ significantly
from the control and was within the historical range for this alteration at the test facility.
The concentration-based maternal NOAEL and LOAEL for this study were 150 ppm and
450 ppm, respectively, based on statistically significant, persistent reductions in maternal body
weight and body weight gains. Based on the mean consumed dosage of bromodichloromethane,
these concentrations correspond to doses of 18.4 mg/kg-day and 45.0 mg/kg-day, respectively.
The concentration-based developmental NOAEL and LOAEL were 450 ppm and 900 ppm,
respectively, based on a significantly decreased number of ossification sites per fetus for the
forelimb phalanges and the hindlimb metatarsals and phalanges. These concentrations
correspond to mean consumed doses of 45.0 mg/kg-day and 82.0 mg/kg-day, respectively.
Bielmeier et al. (2004) conducted a series of hormone profile and hormone replacement
experiments to elucidate the mode of action for full litter resorption in F344 rats. These studies
are discussed in section V.H.2 (Hormonal Disruption).
V - 54 November 15, 2005
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b. Studies in Rabbits
The Chlorine Chemistry council sponsored a range-finding developmental toxicity study
in New Zealand White rabbits (CCC, 2000a). The data from this study have been summarized in
Christian et al. (2001b). This study was conducted in accordance with U.S. EPA Health Effects
Test Guidelines OPPTS 870.3700: Prenatal Developmental Toxicity Study (U.S. EPA, 1998c)
and U.S. EPA Good Laboratory Practice Standards (40 CFR Part 160/792).
Bromodichloromethane was provided to New Zealand White presumed pregnant rabbits
(5/group) in the drinking water at concentrations of 0, 50, 150, 450, and 1350 ppm on GD 6 to
29. Additional rabbits (4/group) were similarly assigned to satellite treatment groups for use in
the collection of samples for analysis of tissue concentrations of bromodichloromethane
(discussed in Section III.B). Body weights were recorded on GDs 0 and 4, daily during the
exposure period, and on the day of sacrifice. Feed and water consumption data were recorded
daily. The rabbits were sacrificed on GD 29 and gross necropsy of the thoracic, pelvic, and
abdominal viscera were performed. The gravid uterus was excised and weighed. Examinations
were made for number and distribution of corpora lutea, implantation sites, early and late
resorptions, and live and dead fetuses. Each fetus was examined for gross external alterations
and sex (by internal examination).
The mean consumed daily doses of bromodichloromethane for GDs 6 to 29 were 0.0, 4.9,
13.9, 32.3, and 76.3 mg/kg-day, as determined from measured body weights and measured water
consumption. Absolute (g/day) and relative (g/kg-day) maternal water intake for the exposure
period was decreased in each group administered bromodichloromethane. The relative
consumption values were 92%, 87%, 67%, and 53% of the control group value, respectively.
Absolute and relative feed consumption values were reduced in a time (onset of reductions
delayed in the 50 and 150 ppm exposure groups) and exposure-dependent manner. The relative
values for feed consumption were 96%, 96%, 90%, and 82% of the control group value for the
exposure period. No deaths, abortions, or premature deliveries occurred during the study. No
treatment-related clinical signs or gross lesions were observed. Maternal body weight gains for
the exposure period were 82%, 80%, 73%, and 50%, respectively, relative to the controls. The
study authors questioned whether these reductions were associated with bromodichloromethane
exposure since similar changes did not occur in the satellite exposure group, and suggested that
the reduced body weight gains were artifacts of the small sample size used in the study. When
body weights were corrected for gravid uterus weight, all exposed groups in the main study
experienced body weight loss while body weight gain occurred in the control group. Absolute
uterine weights were reduced in the 450 and 1350 ppm groups. This finding was most likely
associated with reduced body weight in these groups, since relative gravid uterine weights in all
dosed groups were similar to that of the control.
Litter averages for corpora lutea, implantations, litter sizes, live and dead fetuses, early
and late resorptions, percent dead or resorbed conceptuses, fetal body weights, and percent live
male fetuses were comparable for the control and all exposure groups and within the historical
ranges for the test facility (Argus Laboratories, Horsham, PA). All placentas were normal in
appearance. No gross external fetal alterations were observed in the control or treatment groups.
V - 55 November 15, 2005
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In the satellite study (described in Section III.B), analytical analyses detected trace amounts of
bromodichloromethane in placental samples from two litters in the 1350 ppm group and in one
fetus from the 1350 ppm group. Bromodichloromethane was not detected in amniotic fluid or
maternal plasma. One litter in the 450 ppm satellite exposure group consisted of only early
resorptions. The concentration-based LOAEL for maternal toxicity in this study is 50 ppm, the
lowest concentration tested, based on reduced body weight gain. This concentration corresponds
to a mean daily dose of approximately 4.9 mg/kg-day. The concentration-based NOAEL for
developmental effects was 1350 ppm (the highest dose tested). This corresponds to a mean daily
intake of approximately 76.3 mg/kg-day.
The Chlorine Chemistry Council (CCC, 2000b) sponsored a developmental toxicity study
in New Zealand White rabbits. Data from this study were summarized in Christian et al.
(200la). Bromodichloromethane was provided to pregnant rabbits (25/dose group) at
concentrations of 0, 15, 150, 450, and 900 ppm in the drinking water on GD 6-29. Consumed
doses were calculated from measured water intake and measured body weights and averaged 0,
1.4, 13.4, 35.6, and 55.3 mg/kg-day, respectively, over the 14 day treatment period. Feed
consumption, water intake, and body weight were monitored daily during the exposure period.
The rabbits were sacrificed on GD 29 and examined for gross lesions of the thoracic, abdominal,
and pelvic viscera. Uterine weight, number of implantation sites, uterine contents, and number
of corpora lutea were recorded. Each fetus was examined for weight, gross external alterations,
skeletal alterations, and sex. Visceral alterations and cavitated organs were evaluated by
dissection. One rabbit in the 900 ppm dose group was sacrificed moribund with hindlimb
paralysis caused by a back injury. Another rabbit in the 900 ppm exposure group had a dead
litter as a result of a non-treatment related uterine abnormality. No treatment-related clinical
signs or necropsy results were observed. The 450 and 900 ppm exposure groups had
significantly reduced feed and water consumption rates throughout the exposure period. These
groups also had significantly reduced body weight gains and corrected (for weight of gravid
uterus) body weight gains for both the bromodichloromethane exposure period (GD 6 to 29) and
the entire gestation period (GD 0 to 29). Bromodichloromethane had no observable effect on
implantations, corpora lutea, live litter size, early or late resorptions, percentage of male fetuses,
percentage of resorbed conceptuses, or fetal body weight. The number of litters with any
alteration, the number of fetuses with any alteration, the average percentage of fetuses with any
alteration did not differ significantly from the control. Although statistically significant
increases in the number of fused sterna centra were observed in the 150 and 450 ppm groups,
this effect was not dose-related and the observed incidences were within the historical range for
the testing facility. Litter averages for ossification sites per fetus did not differ significantly
from the control and were within historical range for the testing facility. The NOAEL and
LOAEL identified for maternal toxicity in this study were 13.4 mg/kg-day (150 ppm) and 35.6
mg/kg-day (450 ppm), respectively, based on decreased body weight gain. The developmental
NOAEL was 55.3 mg/kg-day (900 ppm) based on absence of statistically significant, dose-
related effects at any tested concentration.
Christian et al. (2002) summarized the results of a two-generation reproductive toxicity
study on bromodichloromethane conducted in Sprague-Dawley rats. The study was sponsored
V - 56 November 15, 2005
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by the Chlorine Chemistry Council (CCC, 2002) and was conducted in accordance with U.S.
EPA Health Effects Test Guideline OPPTS 870.3800: Reproduction and Fertility Effects (U.S.
EPA, 1998b) and U.S. EPA Good Laboratory Practice Standards (40 CFR Part 160/792).
Bromodichloromethane was continuously provided to test animals in the drinking water at
concentrations of 0, 50, 150, or 450 ppm. Drinking water solutions were prepared at least once
weekly and precautions were taken to prevent contamination of the solutions by extraneous
sources of chlorine. Concentrations were verified analytically at the beginning and end of each
exposure period. The tested concentrations were selected on the basis of results obtained in the
developmental toxicity screening study conducted by NTP (1998) and data obtained in a
range-finding study (CCC, 2000c; Christian et al., 2001b). Exposure of the parental generation
(30 rats/sex/concentration) was initiated when the test animals were approximately 43 days of
age and continued through a 70-day pre-mating period and a cohabitation period of up to 14
days. Parental generation males were exposed for approximately 106 days prior to sacrifice.
Exposure of parental generation female rats continued through gestation and lactation for a total
exposure period of approximately 118 days. Fx generation rats were exposed to
bromodichloromethane in utero and by consumption of the dam's drinking water during the
lactation period. At weaning, Fx rats (30/sex/concentration) were selected for a
postweaning/premating exposure period of at least 64 days, followed by a cohabitation period of
up to 14 days. Exposure continued through gestation and lactation. Fx generation females
delivered litters and the F2 litters were sacrificed on lactation day 22.
During the course of the experiment, parental and Ft generation rats were evaluated for
viability, clinical signs, water and feed consumption, and body weight. Parental and Fx
generation females were evaluated for estrous cycling (premating and during cohabitation until
mating confirmed and at sacrifice), abortions, premature deliveries, duration of gestation,
gestation index, fertility index, number and sex of offspring per litter, general postpartum
condition of dam and litter, litter size, viability index, lactation index, percent survival, sex ratio,
and maternal behavior. Litters were examined for number and sex of pups, stillbirths, live births,
and gross external alterations. Fx rats selected for continued evaluation were assessed for age at
vaginal patency or preputial separation. At sacrifice, test animals were examined for gross
pathology, organ weights, and histopathology (control and high-dose groups, 10 parental
animals/sex; reproductive organs of 50 and 150 ppm rats suspected of reduced fertility). Male
rats were evaluated for sperm concentration, percent motile sperm, sperm morphology, total
number of sperm, and testicular spermatid counts. Females were evaluated for number and
distribution of implantation sites. Fx weanlings not selected for continued evaluation (3
pups/sex/litter, when available) and all F2 weanling rats were evaluated for gross lesions,
terminal body weight, and organ weights.
Key findings in the two-generation study reported by CCC (2002) and Christian et al.
(2002) include the following. The bromodichloromethane dose-equivalent for each drinking
water concentration varied by sex and reproductive status. Average daily doses estimated for the
50, 150 and 450 ppm concentrations were 4.1 to 12.6, 11.6 to 40.2, and 29.5 to 109 mg/kg-day,
respectively, as calculated by the study authors. One death in the 150 ppm group and three
deaths (including one humane sacrifice) in the 450 ppm group were associated with reduced
V - 57 November 15, 2005
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water consumption, weight loss and/or adverse clinical signs and may have been
compound-related. Adverse clinical signs occurred in parental generation female rats and Ft
male and female rats in the 150 and 450 ppm exposure groups. Compound-related signs
included chromorhinorrhea, pale extremities, urine-stained abdominal fur, and coldness to touch.
The study authors attributed these signs to reduced water consumption.
Body weight and body weight gain were significantly reduced in the 450 ppm parental
generation males and females and 150 and 450 ppm Fx generation males and females. The
significantly reduced final body weight in 450 ppm parental generation females was associated
with decreased absolute organ weights and increased relative organ weights when expressed as a
percentage of body or brain weight. Absolute and relative water consumption rates were
significantly reduced in parental and Fx generation males and females at all concentrations of
bromodichloromethane. Water intake by parental and Fx animals was generally reduced by 10 to
20 percent in the 150 and 450 ppm groups when compared to the controls. Absolute and relative
feed consumption rates were reduced in males and females of both generations at 150 and 450
ppm when compared with the controls. There were no gross pathological or histopathological
indications of compound-related toxicity.
Most indicators of reproductive or developmental toxicity examined by Christian et al.
(2002) were not significantly affected by bromodichloromethane treatment. However, Fx and F2
generation pup body weights were reduced in the 150 and 450 ppm groups during the lactation
period after the pups began to drink the water provided to the dams. The Ft generation had
statistically significant reductions in pup body weight at weaning on lactation day 22.
Reductions in F2 pup body weight did not reach statistical significance. Small (<6%), but
statistically significant, delays in Fx generation sexual maturation occurred at 150 (males) and
450 ppm (males and females) as determined by timing of vaginal patency or preputial separation.
The study authors attributed these delays to significant reductions in body weight at weaning.
The values for sexual maturation endpoints in the 150 and 450 ppm exposure groups did not
differ significantly from control values when body weight at weaning was included as a
covariate in the analysis. Females rats with vaginal patency not evident until 40 or 41 days
postpartum (i.e., the most delayed) in the 150 and 450 ppm groups had normal estrus cycles,
mated, and produced litters. Estrous cycling in parental generation females was not affected by
exposure to bromodichloromethane. A marginal effect on estrous cyclicity was observed in Fx
females in the 450 ppm exposure group. This effect was reported to be associated with a higher
incidence of rats in the 450 ppm group (5/30) with six or more consecutive days of diestrus
relative to the controls (2/30). The study authors considered this effect to be a secondary
response associated with reduced pup weights and possible inadvertent stimulation of the uterine
cervix during the performance of vaginal smears. Averages for estrous cycles per 21 days,
cohabitation, mating indices, and fertility indices were unaffected by exposure to
bromodichloromethane. Exposure to bromodichloromethane had no effect on anogenital
distances in male or female F2 pups.
The results of this study appear to identify NOAEL and LOAEL values for reproductive
effects of 50 ppm (4.1 to 12.6 mg/kg-day) and 150 ppm (11.6 to 40.2 mg/kg-day), respectively,
V - 58 November 15, 2005
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based on delayed sexual maturation. However, the study authors have questioned whether
delayed sexual maturation in Fx males associated with reduced body weight should be treated as
reproductive toxicity or general toxicity, since the root cause appears to be dehydration brought
about by taste aversion to the compound. The parental NOAEL and LOAEL are also 50 and 150
ppm, respectively, based on reduced body weight and body weight gain in F0 females and Fx
males and females.
2. Dibromochloromethane
Borzelleca and Carchman (1982) evaluated the reproductive toxicity of dibromochloro-
methane in a two-generation study with ICR Swiss mice. The authors used a modified multi-
generation study protocol for this investigation. Groups of 10 males and 30 females (F0
generation) were exposed to dibromochloromethane in drinking water at concentrations of 0, 0.1,
1.0, or 4.0 mg/mL for seven weeks. The study authors did not estimate average daily doses for
all treated groups. However, they did indicate that the highest drinking water concentration (4.0
mg/mL) corresponded to an average daily dose of 685 mg/kg-day. Using this conversion factor,
drinking water concentrations of 0.1 and 1.0 mg/mL dibromochloromethane were estimated to
correspond to average daily doses of 17 and 171 mg/kg-day, respectively. Following the initial
exposure period, the F0 mice were mated to produce the Fla litters. Each male mouse was co-
housed for seven days with three randomly selected females. Two weeks after weaning of the
Fla litters, the F0 mice were randomly re-mated to produce the Flb litters. A similar protocol was
followed for the Flc litters. After a 21-day postnatal period, the Fla and Flc litters were sacrificed
and necropsied. The Flb generation was culled. The surviving males and females (10 males and
30 females) were exposed for 11 weeks to dibromochloromethane in drinking water at
concentrations of 0, 0.1, 1.0 or 4.0 mg/mL, and then randomly mated to produce the F2a and F2b
generations. A two-week interval occurred between weaning of the F2a generation and remating
of the Flb generation to produce the F2b generation. Thus, parental generations (F0 and Flb) were
exposed continuously to dibromochloromethane in drinking water throughout the pre-mating,
mating, gestation, and lactation periods for a total of 27 and 25 weeks, respectively. Following
weaning of their final litters, both parental generations were sacrificed and necropsied. The F2a
and F2b generations were sacrificed and necropsied following a 21-day postpartum survival
period. Additionally, a selected number of pups from the final matings of each generation (i.e.,
Flc and F2b) were screened for either dominant lethal mutations or teratologic abnormalities.
Body weight gain and drinking water consumption were recorded weekly and semi-
weekly for the F0 and Flb generations, respectively. Mating, gestation, gestation survival, and
lactation survival indices were calculated for each mating. During the 21-day postpartum period,
pups were counted on days 0, 4, 7, 14, and 21, and sexed on days 7, 14, and 21. Viability and
lactation indices were calculated. After sacrifice of all litters except Flb on day 21, one male and
one female pup per litter were randomly selected for necropsy. For teratology screening, treated
females from the F0 and Flb generations were sacrificed on GD 18. The number of
implantations, resorptions, and live and dead fetuses were counted. Fetuses were individually
weighed and examined for gross malformations; a selected subset of fetuses were examined for
either skeletal or visceral anomalies. Statistical analysis was conducted on all endpoints, using
parametric or nonparametric procedures, as appropriate for different endpoints. For statistical
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analyses performed on the pups, the sampling unit was the litter. Treatment-related effects were
considered to be statistically significant if the p value< 0.05.
As compared with concurrent controls, final body weights were significantly reduced in
the high-dose males and the mid- and high-dose females of the F0 and Flb generations. Water
consumption was unaffected by treatment, indicating that taste aversion was not a factor in the
observed decreases in body weight. Animals in both the F0 and Flb generations exhibited
enlarged livers with gross morphological changes, interpreted by the authors as indicative of
hepatotoxicity. The incidence and the severity of these alterations increased with increasing
dose, with 0, 25, 70, and 100% of the F0 animals and 0, 18, 64, and 100% of the Flb animal
exhibiting hepatic discoloration, fat accumulation, and/or lesions at 0, 0.1, 1.0, and 4.0 mg/mL,
respectively. Fertility (mating index) was significantly decreased in the high-dose group (4.0
mg/mL) only for the F2a generation. The gestational index was significantly decreased in the
high-dose group for all three Fx generations. Parental ingestion of 4.0 mg/mL dibromochloro-
methane resulted in (1) decreased litter size in all generations (Fla, Flb, Flc, F2a, and F2b); (2)
decreased viability index in four of the five generations (Fla, Flb, Flc and F2a); (3) decreased
lactation index in the F2b generation; and (4) decreased postnatal body weight in the F2b
generation. Parental ingestion of 1.0 mg/mL dibromochloromethane produced (1) decreased
litter size in the Flc generation; (2) decreased viability index in the Flb generation; (3) decreased
lactation index in the Flb and F2b generations; and (4) decreased postnatal body weight in the F2b
generation. The only statistically significant effect observed at the lowest dose tested (0.1
mg/mL) was a reduction in postnatal body weight in the F2b generation on PND 14; this effect
was not noted on PND 7 or 21. No statistically significant increases in dominant lethal or
teratogenic effects were reported in either the Flc or F2b generations.
Based on decreased postnatal body weight in the F2b generation, the marginal LOAEL for
reproductive/developmental toxicity is 17 mg/kg-day and a NOAEL could not be determined.
The developmental LOAEL is considered to be marginal because (1) this effect was only noted
in one of the two litters in the F2 generation; (2) no other adverse effects were observed at this
dose level; and (3) it was unclear from the report how many litters and pups per litter were
examined for postnatal body weight. For parental toxicity, liver alterations indicative of
hepatotoxicity were clearly evident at the two higher doses in both parental generations. At the
lowest dose tested, hepatic changes were mainly limited to discoloration, presumably due to the
accumulation of fat deposits (Borzelleca and Carchman, 1982); gross morphology was normal,
and histopathologic examination was not conducted. Therefore, the adversity of this effect was
uncertain. Based on these considerations, the lowest dose, 17 mg/kg-day, is considered to be a
marginal LOAEL for parental toxicity (both F0 and Flb generations) and a NOAEL could not be
determined.
Ruddick et al. (1983) investigated the reproductive and developmental toxicity of
dibromochloromethane in Sprague-Dawley rats. Pregnant dams (10 to 12 animals per dose
group) were administered gavage doses of 0, 50, 100, or 200 mg/kg-day in corn oil on GD 6-15.
Body weights were measured on GD 1, on GD 1 through GD 15, and before and after caesarean
section on GD 22. On GD 22, females were anesthetized, exsanguinated, and viscera (including
the uteri) were examined for pathological changes. The fetuses were removed, weighed
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individually, and examined for viability and external malformations. Two pups per dam were
placed in fixative for histopathological examination. Approximately two-thirds of the remaining
live fetuses were preserved for examination for skeletal abnormalities. The remaining fetuses
were preserved for examination for visceral alterations. Maternal blood was analyzed for
standard hematological and clinical biochemistry parameters. Following gross pathological
examination of the dams, organ weights were collected for liver, heart, brain, spleen, and one
kidney. Tissues from control and high-dose dams (5 animals/group) were subject to
histopathological examination. Where chemical related effects were observed, the affected
tissues were also examined in the mid-dose group.
Maternal weight gain was depressed by 25% in the high-dose group relative to controls.
No significant effects on maternal organ weights, hematology and clinical chemistry, number of
resorption sites, number of fetuses per litter, and mean fetal body weight gain were observed in
any of the dose groups. No treatment-related histopathology was noted in either dams or fetuses.
There were no skeletal or visceral fetal anomalies attributed to dibromochloromethane treatment.
Statistical analysis of fetal endpoints was not conducted by the study authors. However,
inspection of the data indicated that there were no dose-related effects (e.g., the number of
affected fetuses/number of affected litters for sternebral aberrations was 3/2, 2/1, 1/1, and 1/1 for
control, low-, mid-, and high-dose groups, respectively). The power of this experiment was
limited by the small number of litters per dose group. In the absence of observed fetal effects,
the NOAEL for developmental toxicity was 200 mg/kg-day, the highest dose tested, and a
LOAEL could not be determined. Based on significantly decreased maternal body weight gain,
the LOAEL and NOAEL for maternal toxicity were 200 and 100 mg/kg-day, respectively.
NTP (1996) conducted a short-term reproductive and developmental toxicity screen in
Sprague-Dawley rats. Dibromochloromethane was administered in drinking water at
concentrations of 0, 50, 150, or 450 ppm during a study period of 35 days. Males (10/group)
were treated from study days 6 through 34. At study termination, males were submitted for a
thorough examination, which included hematology, clinical chemistry, gross necropsy,
histopathology, and a complete sperm evaluation (count, density, motility, and morphology).
Group A females (10/group) were treated from study days 1 through 34. These females were
mated to treated males on study days 13 through 18 and necropsied on study day 34. Group B
females (13/group) were mated on study day 1 to untreated males, treated from GD 6 through
parturition, and necropsied on postnatal day 5. No hematology, clinical chemistry, or
histopathology was conducted on the females.
Based on measured water consumption, the authors estimated dose levels for the males as
4.2, 12.4, and 28.2 mg/kg-day, for Group A females as 6.3, 17.4, and 46.0 mg/kg-day, and for
Group B females as 7.1, 20.0, and 47.8 mg/kg-day. A few changes in clinical chemistry were
noted for the males. Alkaline phosphatase and 5' nucleotidase were increased at all dose levels
in males, but reached statistical significance only at the low dose for alkaline phosphatase and at
the mid and high dose for 5' nucleotidase. Total serum proteins were also decreased at the high
dose in males. The study authors noted that these changes could reflect mild liver damage.
However, no treatment-related microscopic lesions were observed. No statistically significant
effects were observed on any sperm parameter investigated. No effect was observed on any
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reproductive or fertility measure in Group A or B females at any dose. The proportion of male
pups was significantly decreased in Group B females at the high dose compared to the control
value. The study authors did not consider this result to be treatment-related because the control
value (0.61) was unusually high compared to historical values, and the result for the high-dose
group (0.44) was within historical background. Based on these data, the authors noted that
dibromochloromethane was not a reproductive toxicant at doses up to the high dose in either
males (28.2 mg/kg-day) or females (46.0 to 47.8 mg/kg-day). Based on the clinical chemistry
changes, the authors stated that administration of dibromochloromethane may have resulted in
general toxicity at all doses in the male treatment groups. The observed changes in clinical
chemistry, however, would not be considered adverse for the following reasons: absence of clear
dose-related response, small magnitude of the changes, and absence of supporting
histopathology data. Therefore, this study identified NOAEL values of 28.2 mg/kg-day and 47.8
mg/kg-day for males and females, respectively, for reproductive and systemic effects.
3. Bromoform
Ruddick et al. (1983) investigated the reproductive and developmental toxicity of
bromoform in Sprague-Dawley rats. Pregnant dams (14 to 15 animals/dose group) were admini-
stered gavage doses of 0, 50, 100, or 200 mg/kg-day in corn oil on GD 6 to 15. Body weights
were measured on GD 1, on GD 6 through GD 15, and before and after pups were delivered by
caesarean section on GD 22. On GD 22, females were sacrificed and body tissues (including the
uterus) were removed for pathological examination. Females were evaluated for the number of
resorption sites and the number of fetuses. Maternal blood samples were collected and
evaluated for standard hematology and clinical chemistry parameters. The liver, heart, brain,
spleen, and one kidney were weighed. Standard histopathology was conducted on control and
high-dose females (5/group). All fetuses in all groups were individually weighed, and evaluated
for viability and external malformations. Histopathologic examination was performed on two
pups per litter. Of the remaining live fetuses, approximately two-thirds were examined for
skeletal alterations and one-third for visceral abnormalities.
Maternal weight gain, organ weights, hematology, and clinical chemistry were unaffected
by bromoform treatment. No significant differences between exposed and control groups were
observed for the number of resorption sites, the number of fetuses per litter, fetal weights, fetal
gross malformations, and visceral abnormalities. No treatment-related histopathological effects
were noted in either the dams or fetuses. However an elevation in the incidence of skeletal
anomalies, including the presence of a 14th rib, wavy ribs, and interparietal bone deviations was
reported in treated animals. An increase in wavy ribs was only observed in the high-dose group.
The number of affected fetuses /number of affected litters for the presence of a 14th rib was 3/3,
4/3, 4/3 and 7/5 in the 0, 50, 100, and 200 mg/kg-day groups, respectively. The incidence of
sternebral aberrations (number of affected fetuses/number of affected litters) was 1/1, 5/3, 6/5,
13/8 in the 0, 50, 100, and 200 mg/kg-day groups, respectively. Statistical significance for
fetotoxic endpoints was not reported by the study authors. A statistical analysis (using the Fisher
Exact test) was conducted on the published data and demonstrated that the increase in sternebral
anomalies was significantly different from controls at the highest dose tested (200 mg/kg-day).
A trend test showed a statistically significant dose-related trend (p< 0.002) for this endpoint;
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stepwise analysis indicated that the trend was no longer significant when the two highest doses
(i.e., 200 and 100 mg/kg-day) were omitted from the analysis. These findings suggest that the
LOAEL and NOAEL for developmental toxicity were 100 and 50 mg/kg-day, respectively. In
the absence of observed maternal effects, the NOAEL for maternal toxicity was 200 mg/kg-day,
and a LOAEL could not be determined.
NTP (1989b) investigated the effect of bromoform on fertility and reproduction in Swiss
CD-I mice using a continuous breeding protocol. Twenty male-female pairs were administered
daily doses of 50, 100, or 200 mg/kg-day by gavage in corn oil and forty male-female pairs were
dosed with the corn oil vehicle only. Dose selection was based on a 14-day range-finding study.
The 105-day dosing period included a seven-day precohabitation phase and a 98-day
cohabitation phase. The parameters evaluated for this study were fertility, litters per pair, live
pups per litter, proportion of pups born alive, sex of live pups, or pup body weights. The last
litter born (generally the fifth litter) in the control and 200 mg/kg-day groups during a holding
period following the continuous breeding phase were reared by the dams, weaned and raised to
sexual maturity (approximately 74 days) while receiving the same treatment (vehicle control of
200 mg/kg-day bromoform) as their parents. At sexual maturity, males and females from
different litters within the same treatment group were cohabited for seven days and then housed
individually until delivery. The endpoints for this mating trial were the same as for the parental
generation. At study termination, the Fx mice were weighed, necropsied and evaluated for
selected organ weights, epididymal sperm motility, and sperm morphology. Selected organs
were fixed for histopathological examination.
In the 200 mg/kg-day treatment group, the body weights of dams at delivery were
consistently less than the control group value. The reduction in body weight was statistically
significant after delivery of the first, second, fourth, and fifth litters. The fertility index for the
parental generation was 100% for the control and treated groups (a breeding pair was designated
as fertile if they produced at least one live or dead pup). There was no detectable effect of
treatment on the number of litters per pair, the number of live pups per litter, the proportion of
pups born alive, the sex of live pups, or pup body weights. The gestational period was similar
across groups. However, postnatal survival of Fj pups in the 200 mg/kg-day group was
significantly lower than in the control group. The study authors reported that this difference was
primarily attributable to three dams who lost all of their pups by postnatal day 4. One dam in the
control group also lost her litter by postnatal day 4. The study authors noted that this result is
consistent with a treatment effect on early maternal behavior, early lactational failure, and/or the
postnatal developmental processes. When Fx mice were cohabited for one week, no effect of
treatment on mating index or fertility was observed. There were no significant differences
relative to control values for the number of live pups per litter (male, female, or combined), the
proportion of live pups, the proportion of male pups, or pup weight at birth. At sacrifice, male
and female Fx mice administered 200 mg/kg-day exhibited increased relative liver weights and
decreased relative kidney weights as compared with control values. The mean body weight for
Fj males was significantly less than the mean weight of the male control group.
Histopathological evaluation revealed minimal to moderate hepatocellular degeneration in the
livers of high-dose Fx male and female mice. Bromoform treatment had no effect on epididymal
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sperm density, motility, or morphology in Fx males. No treatment-related histologic lesions were
observed in the seminal vesicles, coagulating glands, or prostate glands of males, or in the lung,
kidney, or thyroid gland of males or females. Based on liver histopathology, decreased postnatal
survival, and other signs of toxicity (e.g., increased relative liver and decreased relative kidney
weights) in Fx mice of both sexes at the highest dose tested, the LOAEL for developmental
toxicity is 200 mg/kg-day, and the NOAEL is 100 mg/kg-day. Based on consistently decreased
body weights of pregnant dams at delivery, the LOAEL for maternal toxicity is 200 mg/kg-day
and the NOAEL is 100 mg/kg-day.
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Table V-9 Summary of Reproductive Studies of Brominated Trihalomethanes
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromodichloromethane
Ruddick et al.
(1983)
Klinefelter et al.
(1995)
Narotsky et al.
(1997)
Narotsky et al.
(1997)
NTP (1998)
Rat
Sprague-
Dawley
Rat
F344
Rat
F344
Rat
F344
Rat
Sprague-
Dawley
Gavage
(Corn oil)
Drinking
Water
Gavage
(Corn oil)
Gavage
(Aqueous)
Drinking
Water
F
M
F
F
M
9-14
7
12-14
12-14
5-10
GD6-15
52 weeks
GD6-15
GD6-15
25-30 days
0
50
100 (NOAEL) '
200 (LOAEL)
0
22 (NOAEL)
39 (LOAEL)
0
25 ( NOAEL)
50 (LOAEL)
75
0
25 (NOAEL)
50 (LOAEL)
75
0
8
41
68 (NOAEL)
Developmental toxicity study.
Statistically decreased maternal wt.
gain at high dose (38%); increased
litter incidence of sternebral
aberrations. Statistical significance
not evaluated for fetotoxic endpoints
by study authors; statistical analysis
conducted on published data for fetal
effects. Trend test indicated
statistical effect for sternebral
anomalies at highest dose tested.
Maternal LOAEL and NOAEL are
200 and 100 mg/kg-day, respectively.
Reproductive toxicity study.
Decreased sperm velocity at 39
mg/kg-day. No histopathological
alterations noted in any reproductive
tissue examined.
Developmental toxicity study
comparing the use of different gavage
dosing vehicles. Reduced maternal
weight gain GD 6-8 and lacrimation
at 50 and 75 mg/kg-day. Full litter
resorption (FLR) observed at 50 and
75 mg/kg-day (8% and 83%,
respectively). No effects on postnatal
survival, pup weight, or pup survival
in surviving litters. ED05 and BMD
for FLR calculated by study authors
as 48.4 and 39.3 mg/kg-day,
respectively. Maternal LOAEL and
NOAEL are 50 and 25 mg/kg-day,
respectively.
Developmental toxicity study
comparing the use of different gavage
dosing vehicles. Reduced maternal
weight gain GD 6-8 at all dose levels.
Full litter resorption (FLR) observed
at 50 and 75 mg/kg-day (17 and 21%,
respectively). No effects observed on
postnatal survival, pup weight, or pup
survival in surviving litters. EDO 5
and BMD for FLR calculated by
study authors as 33.3 and 11.3
mg/kg-day, respectively. Maternal
LOAEL is 25 mg/kg-day; maternal
NOAEL not determined.
Reproductive/developmental toxicity
study. Decreased food and water
consumption; decreased body weight.
No dose-related changes in
reproductive/developmental
parameters
V-65
November 15, 2005
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Table V-9 (cont.)
Reference
NTP (1998)
Bielmeier et al.
(2001)
Bielmeier et al.
(2001)
Bielmeier et al.
(2001)
CCC (2000c)
Christian et al.
(200 Ib)
Species
Rat
Sprague-
Dawley
Rat
F344
Rat
F344/
Sprague-
Dawley
Rat
F344
Rat
Sprague-
Dawley
Route
Drinking
Water
Gavage
(Aqueous)
Gavage
(Aqueous)
Gavage
(Aqueous)
Drinking
Water
Sex
F
F
F
F
F, M
Number
per dose
group
5-10
8-13
12-14
8-11
10
Duration
25-30 days
GD 6-15
GD6-10
GD 11-15
GD 6-10
GD 9
64-74 days
Males2
(days 1-
64 i.e., for
14 days
premating,
during
mating,
and for ~
6 weeks
following
mating)
Females 2
(days 1-
74, i.e., for
14 days
premating,
during
mating,
GDO-21,
lactation
days 1-29)
Dose
(mg/kg-day)
0
14
72
116(NOAEL)
0
75 (LOAEL)
F344
0
75 (LOAEL)
Sprague-Dawley
0
75
100 (NOAEL)
0
75 (LOAEL)
100
0 ppm
50 ppm
(NOAEL)
150 ppm
(LOAEL)
450 ppm
1350 ppm
Results
Reproductive/developmental to xicity
study. Decreased food and water
consumption; decreased body weight.
No dose-related changes in
reproductive/developmental
parameters
Critical exposure period study. Full
litter resorption observed in animals
treated on GD 6-10, but not in
animals treated on GD 11-15.
Strain comparison study. Full litter
resorption observed in F344 rats but
not in concurrently dosed Sprague-
Dawley rats.
Hormone profile study. Full litter
resorption observed at both doses.
Decreased serum progesterone levels
in F344 rats which experienced FLR.
Range finding
reproductive/developmental toxicity
study. Decreased body weight gain
and terminal body weight (>10%) in
males at highest dost tested but no
apparent effects on reproductive
endpoints at any dose.
Maternal toxicity (reduced body
weight and body weight gain and
decreased food and water
consumption) at 150 ppm and higher.
Dose-dependent decreases in mean
pup weight gain and pup weights
beginning on lactation day 5-29 in 3
highest dose groups. Decreased pup
body weight gain and body weight
also observed in 3 highest dose
groups in pups treated for one week
postweaning at parental drinking
water concentrations.
Reproductive/developmental LOAEL
and NOAEL are 150 and 50 ppm,
respectively, based on decreased pup
wt and wt gain; parental LOAEL and
NOAEL are 150 and 50 ppm,
respectively, based on reduced body
wt gain and wt. Findings confounded
by effects of decreased water
consumption at various time points
during treatment.
V-66
November 15, 2005
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Table V-9 (cont.)
Reference
CCC (2000d)
Christian et al.
(200 la)
CCC (2000a)
Christian et al.
(200 Ib)
CCC (2000b)
Christian et al.
(200 la)
CCC (2002)
Christian et al.
(2002)
Species
Rat
Sprague-
Dawley
Rabbit
New
Zealand
White
Rabbit
New
Zealand
White
Rat
Sprague-
Dawley
Route
Drinking
Water
Drinking
Water
Drinking
Water
Drinking
Water
Sex
F
F
F
M, F
Number
per dose
group
25
5
25
30
Duration
GD6-21
GD 6-29
GD 6-29
Up to 118
days
Dose
(mg/kg-day)
0
2.2
18.4
45.0 (NOAEL)
82.0 (LOAEL)
0.0
4.9
13.9
32.3
76.3 (NOAEL)
0
1.4
13.4
35.6
55.3 (NOAEL)
Males
F0: 106 d (incl. 70
d pre-mating)
Fp 64 d post-
weaning, 14 d
cohab.
Females
F0: 1 18 d (incl.
gest, lactation
F[ 64 d post-
weaning, 14 d
cohab., gest.,
lactation
0
50 (NOAEL)
150 (LOAEL)
450
Results
Developmental toxicity study.
Decreased maternal body weight and
body weight gain at 45.0 mg/kg-day.
Developmental LOAEL based on
slightly decreased number of
ossification sites in the hindlimb
(metatarsals and phalanges) and
forelimb (phalanges). Maternal
LOAEL and NOAEL are 82.0 and
45.0 mg/kg-day, respectively.
Range finding developmental study.
Decreased maternal body weight gain
and water and feed consumption at all
tested doses. No treatment-related
changes in reproductive or
developmental endpoints. Study
authors considered maternal LOAEL
to be < 4.9 mg/kg-day, based on
significantly reduced body weight
gain.
Developmental toxicity study.
Reduced maternal weight gain at 35.3
mg/kg-day. No dose-related changes
in reproductive or developmental
parameters. Maternal LOAEL and
NOAEL are 35.3 and 13.4 mg/kg-
day, respectively.
Reproductive/developmental LOAEL
and NOAEL are 150 and 50 ppm,
respectively, based on delayed sexual
maturation in Fj males; parental
LOAEL and NOAEL are 150 and 50
ppm, respectively, based on reduced
body wt gain and wt in F0 females
and F[ males and females. Findings
confounded by effects of decreased
water consumption as a result of taste
aversion to the test compound.
V-67
November 15, 2005
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Table V-9 (cont.)
Reference
Bielmeier et al.
(2004)
Bielmeier et al.
(2004)
Species
Rat
F344
Rat
F344
Route
Gavage
(Aqueous)
Gavage
(Aqueous)
Sex
F
F
Number
per dose
group
5-13
5-13
Duration
GD 9
GD6-10
Dose
(mg/kg-day)
0
75 (LOAEL)
100
0
75 (LOAEL)
100
Results
Hormone profile study. BDCM-
induced pregnancy loss was
associated with decreased LH
followed by decreased progesterone
on GD 10. All control dams
maintained their litters, whereas 8/9
dams exposed to
bromodichloromethane had
pregnancy loss.
Hormone supplementation study.
BCDM-induced pregnancy loss was
prevented by progesterone and hCG
supplementation. Five of seven dams
(71%) treated with 100 mg/kg-day of
bromodichloromethane plus the
hormone vehicles (corn oil or saline
injected subcutaneously) on GD 6-10
lost their pregnancies. In contrast,
dams dosed with 100 mg/kg-day of
bromodichloromethane on GD 6-10
and concurrently given progesterone
had a 0/8 (0%) incidence of
pregnancy loss. Dams dosed with
100 mg/kg-day of
bromodichloromethane on GD 8-10
and concurrently given hCG by
injection had a 1/9 (1 1%) incidence
of pregnancy loss.
LH mediated mode of action was
suggested.
V-68
November 15, 2005
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Table V-9 (cont.)
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Dibromochloromethane
Borzelleca and
Carchman (1982)
Ruddick et al.
(1983)
NTP (1996)
NTP (1996)
NTP (1996)
Mouse
ICR Swiss
Rat
Sprague-
Dawley
Rat
Sprague-
Dawley
Rat
Sprague-
Dawley
Rat
Sprague-
Dawley
Drinking
Water
Gavage
(Corn oil)
Drinking
Water
Drinking
Water
Drinking
Water
M
F
F
M
F
F
10
30
10-12
10
10
13
25-27
weeks
GD6-15
29 days
35 days
-16 days
(GD6-
parturit-
ion)
0
17 (marginal
LOAEL)
171
685
0
50
100
200 (NOAEL)
4.2
12.4
28.2 (NOAEL)
6.3
17.4
46.0 (NOAEL)
7.1
20.0
47.8 (NOAEL)
Multi-generation reproductive
toxicity study. Significant high-dose
effects include decreased gestational
index in Fj generation at high dose
and decreased litter size in Fj and F2
generations. Significant mid-dose
effects include decreased litter size,
decreased viability index, decreased
lactation index, and decreased
postnatal body weight in some Fj
and/or F2 generations. Only
significant low-dose effect is reduced
postnatal body wt in F2b generation
on postnatal day 14. Hepatic effects
observed in both parental generations
at all doses; liver effects marginal at
low dose. Parental marginal LOAEL
is 17 mg/kg-day; parental NOAEL
not determined.
Developmental toxicity study.
Significantly depressed maternal wt.
gain at high dose (25%); increased
maternal relative liver wt. and kidney
Statistical significance of fetal
endpoints not evaluated by study
authors. Based on data inspection, no
dose-related skeletal or visceral
effects observed in litters. Maternal
LOAEL and NOAEL are 200 and
100 mg/kg-day, respectively.
Reproductive/developmental toxicity
study. No treatment-related effects
on measured sperm parameters
Reproductive/developmental toxicity
study. Exposure occurred during a 6-
day mating period and most/all of
gestation. No clearly adverse effect
on any reproductive or developmental
endpoint at tested doses
Reproductive/developmental toxicity
study. No clearly adverse effect on
any reproductive or developmental
endpoint at tested doses
V-69
November 15, 2005
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Table V-9 (cont.)
Reference
Species
Route
Sex
Number
per dose
group
Duration
Dose
(mg/kg-day)
Results
Bromoform
Ruddick et al.
(1983)
NTP (1989b)
Rat
Sprague-
Dawley
Mouse
ICR Swiss
Gavage
(Corn oil)
Gavage
(oil)
F
M
F
14-15
20
20
GD6-15
105 days
0
50 (NOAEL)
100 (LOAEL)
200
0
50
100 (NOAEL)
200 (LOAEL)
Developmental toxicity study. No
statistically significant maternal
effects. Apparent treatment-related
increases in sternebral aberrations
and other skeletal endpoints.
Statistical significance of fetotoxic
endpoints not evaluated by study
authors. Statistical analysis
conducted on published data
indicated significant increase in
sternebral aberrations at two highest
doses. Maternal NOAEL is 200
mg/kg-day; LOAEL not determined.
Continuous breeding reproductive
toxicity protocol. Maternal body
weights significantly decreased at
highest dose tested. Decreased
postnatal survival, organ wt. changes,
and liver histopathology observed in
F[ mice of both sexes in high-dose
group. No noted effects on fertility,
litters/pair, live pups/litter; proportion
of live births, sex of live pups, or pup
body weight. Maternal LOAEL and
NOAEL are 200 and 100 mg/kg-day,
respectively.
NOAEL and LOAEL values reported in this column are for developmental/reproductive toxicity effects. The
NOAEL and LOAEL values for parental toxicity are reported in the "Results" column.
Doses for this study are presented as ppm in drinking water; due to marked changes in adult female water
consumption during different physiologic stages (i.e., pre-mating, mating, gestation, and lactation), it is not
possible to convert administered drinking water concentrations into biologically meaningful average daily doses.
F.
Mutagenicitv and Genotoxicitv
1. Bromodichloromethane
The results of in vivo and in vitro tests conducted to evaluate the mutagenicity, genotoxi-
city, and neoplastic transformation potential of bromodichloromethane are summarized in Table
V-10 at the end of this section.
a. In Vitro Assays
Simmon and Tardiff (1978) reported that nonactivated bromodichloromethane was
mutagenic in S. typhimurium strain TA100 when assayed in a desiccator containing the test
compound in the atmosphere. The minimum amount of bromodichloromethane required to elicit
a mutagenic response following addition to the desiccator was 600 |imol.
V-70
November 15, 2005
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Ishidate et al. (1982) assayed the mutagenicity of bromodichloromethane in S.
typhimurium strain TA100 in the presence and absence of rat liver S9 fraction. Increased
mutation frequencies were observed only in the absence of S9 activation. In contrast,
chromosomal aberrations in Chinese hamster fibroblasts were observed in the presence, but not
the absence, of S9 fraction. The concentrations tested in these assays were not reported.
Nestmann and Lee (1985) investigated the mutagenicity of bromodichloromethane at 12
to 1,200 |iM in S. cerevisiae strains D7 and XV185-14C in the presence or absence of S9
activation. No clear increase in convertants or in revertants of strain XV185-14C was observed
for bromodichloromethane in the presence or absence of S9 activation.
NTP (1987) reported that bromodichloromethane was not mutagenic when tested using a
preincubation protocol in S. typhimurium strains TA1535, TA1537, TA98, or TA100 at
concentrations reaching cytotoxic levels (20 [imol/plate; 3,333 jig/plate). Testing was done in the
absence of S9 and in the presence of S9 prepared from Aroclor-induced male hamster or rat liver.
NTP concluded that the negative results may have been due to volatilization of the test compound
from the test system. Bromodichloromethane was not mutagenic in the mouse lymphoma
L5178Y/TK+/" assay in the absence of S9, but did induce dose-related increases in forward
mutations at S9-activated concentrations greater than or equal to 2,000 |j,M (300 |ig/mL).
Cytogenetic tests with Chinese hamster ovary cells (CHO) cells were reported in this study and
also by Anderson et al. (1990). There was no evidence of induction of chromosomal aberrations
following treatment with up to 30,000 |j,M (5,000 |ig/mL) in either the presence or absence of
exogenous metabolic activation. There was also no evidence of sister chromatid exchanges
induced by the nonactivated material. In the presence of S9 activation, one of three assays
resulted in a positive response at doses greater than or equal to 24,400 |j,M (4,000 |ig/mL). These
results are difficult to evaluate because cytotoxicity was observed at similar concentrations in the
other trials.
Varma et al. (1988) tested bromodichloromethane for mutagenicity in S. typhimurium
strains TA1535, TA1537, TA98, and TA100. In the absence of S9 fraction, bromodichloro-
methane at nonactivated concentrations of 2.4 to 3.2 |imol/plate induced mutations in strain
TA1537. There was no effect in the other strains.
Bromodichloromethane was positive for the induction of DNA damage in the presence
and absence of exogenous activation, based on the results of the SOS chromotest (LeCurieux et
al., 1995). Bromodichloromethane gave a negative result in the fluctuation test modification of
the S. typhimurium reverse mutation assay.
Several studies have evaluated induction of sister chromatid exchanges following
exposure to bromodichloromethane. Morimoto and Koizumi (1983) investigated the ability of
bromodichloromethane to induce sister chromatid exchanges in human lymphocytes in vitro in
the absence of S9 activation. Bromodichloromethane caused a dose-dependent increase in sister
chromatid exchanges. The increased incidence was significant (p < 0.05) at concentrations
V-71 November 15, 2005
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greater than or equal to 400 |j,M. The potential of S9-activated bromodichloromethane to induce
sister chromatid exchanges in vitro was also investigated by Sobti (1984). A dose of 100 |j,M
increased the frequency of sister chromatid exchange in rat liver cells. In human lymphocytes, a
100-fold greater concentration of bromodichloromethane was required to elicit the same effect on
sister chromatid exchange when compared to dibromochloromethane (100 |j,M vs. 1 |iM.). Fujie
et al. (1993) observed a statistically significant, dose-related increase in sister chromatid
exchange in rat erythroblastic leukemia K3D cells treated with bromodichloromethane in the
absence of exogenous activation. Bromodichloromethane also appeared to give a positive
response in the presence of exogenous metabolic activation, although the study protocol and
results with negative controls were described less clearly for this phase of testing.
Bromodichloromethane was tested in the mouse lymphoma assay as part of an
international collaborative program under the auspices of the Japanese Ministry of Health and
Welfare (Sofuni et al., 1996). The results of this assay were equivocal. One laboratory obtained
a positive result in the activated phase, but this result was not confirmed by a second laboratory.
Results in the nonactivated phase were negative or equivocal due to poor viability of the solvent
control cell cultures.
Matsuoka et al. (1996) conducted a chromosome aberration assay with Chinese hamster
lung fibroblast (CHL/IU) cells exposed to bromodichloromethane in tightly capped flasks. A
weak induction of chromosome aberrations was observed for bromodichloromethane in the
presence and absence of exogenous metabolic activation.
Geter et al. (2004a) examined the ability of bromodichloromethane to induce DNA strand
breaks (as assessed by the alkaline unwinding assay) in CCRF-CEM human lymphoblastic
leukemia cells and primary F344/N rat hepatocytes. Exposure to 5 or 10 mM significantly
induced DNA strand breaks in CCRF-CEM cells after a two-hour exposure. Cells exposed to 1 or
5 mM bromodichloromethane showed 100% DNA strand break recovery when assayed after
incubation in bromodichloromethane-free medium for 22 hours. No evidence of strand breaks
was observed in primary rat hepatocytes exposed to 1, 5, or 10 mM bromodichloromethane for 4
hours.
Landi et al. (2003) demonstrated that bromodichloromethane induced DNA damage in
primary cultures of human lung epithelial cells as measured by tail extent movement in the Comet
assay. Human lung epithelial cells were collected by scraping the large airways of four
volunteers. DNA genotyping was used to identify 2 subjects that were GSTT1-1+ and 2 subjects
that were GSTT1-T. Cells were maintained in culture and were exposed to 0, 10, 100 or 1000 |j,M
bromodichloromethane for 3 hours prior to being flash frozen for analysis. DNA damage was
observed at each concentration tested. Variation in response among subjects was not related to
the GSTT1-1 genotype.
Robbiano et al. (2004) evaluated the ability of bromodichloromethane to cause DNA
fragmentation and micronuclei formation in primary cultures of rat and human kidney cells.
Human kidney tissue was obtained from surgical patients where a portion of the kidney was
V - 72 November 15, 2005
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removed for health reasons. Cultured cells were exposed to 0, 0.5, 1, 2, or 4 mM
bromodichloromethane for 20 hours and DNA damage was measured as tail length in the Comet
assay. Statistically significant increases in DNA damage were observed in cells from rats and
humans at all exposure levels. For the micronucleus assay, cells were exposed to 0, 1, 2, or 4 mM
bromodichloromethane for 48 hours. Bromodichloromethane caused increased micronuclei
formation in primary cultures of rat kidney cells at all exposure levels and in human kidney cells
at all exposure levels except the lowest (1.0 mM).
b. In Vivo Assays
Ishidate et al. (1982) investigated the in vivo clastogenicity of bromodichloromethane in
ddY mice, MS mice, and Wistar rats. Doses of 125 to 500 mg/kg-day were administered in olive
oil by intraperitoneal injection, and the animals were sacrificed at 18, 24, 30, 48, and 72 hours
after dosing. No significant induction of micronucleus formation in bone marrow was observed
in either mice or rats.
Morimoto and Koizumi (1983) investigated the potential of bromodichloromethane to
induce sister chromatid exchanges in male ICR/SJ mice. Animals were given doses of 0, 25, 50,
100, or 200 mg/kg-day for four days by olive oil gavage. Bromodichloromethane produced a
roughly linear dose-dependent increase in sister chromatid exchange frequency. The increase was
statistically significant (p < 0.05) at 50 mg/kg-day. The authors noted that the concentrations
required to produce an increased incidence of sister chromatid exchange were on the order of
1,000 to 10,000 times higher than the concentrations typically found in drinking water.
Hayashi et al. (1988) measured induction of micronucleated polychromatic erythrocytes in
ddY mice by intraperitoneal administration of bromodichloromethane at single doses up to 500
mg/kg in corn oil. No evidence of clastogenicity was observed. There was no clear evidence of
toxicity or cytotoxicity in the target tissue.
Fujie et al. (1990) analyzed chromosome aberrations in bone marrow from Long-Evans
rats (3/sex/dose) following oral (males only) or intraperitoneal (males and females) exposure to
bromodichloromethane. Oral administration was by gavage in saline for five consecutive days,
and the animals were sacrificed 18 hours after the last dose. Bromodichloromethane induced
dose-related increases in chromatid and chromosome breaks. A more pronounced increase in
clastogenic activity was observed following a single intraperitoneal dose, with significant (p <
0.05) effects at 16.4 mg/kg.
Hayashi et al. (1992) evaluated induction of micronuclei in mouse peripheral blood
erythrocytes by bromodichloromethane. Groups of four male ddY mice received an intraperi-
toneal injection of 0, 25, 50, or 100 mg/kg bromodichloromethane in physiological saline once a
week for 5 weeks. Micronuclei were evaluated 1 week after the last dose. No evidence of
micronucleus induction was observed. One low-dose mouse died, and weight loss was observed
in all treatment groups during exposure.
V - 73 November 15, 2005
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Robbiano et al. (2004) measured DNA damage and micronuclei formation in kidney cells
of Sprague Dawley rats sacrificed 20 hours after a single oral gavage dose of 458 mg/kg
bromodichloromethane in 0.5% dimethyl sulfoxide. The frequency of micronucleated kidney
cells was increased and DNA damage was observed, as measured by increased tail length in the
Comet assay.
Potter et al. (1996) observed no significant increase in DNA strand breakage in male F344
rats one day after a single gavage doses of 0.75 or 1.5 mmol/kg (123 or 246 mg/kg-day) of
bromodichloromethane in 4% Emulphor® using the alkaline unwinding procedure. Geter et al.
(2004a) likewise found no significant induction of DNA strand breaks using the alkaline
unwinding assay in the liver, kidney, or duodenum epithelial cells of male F344/N rats when
assayed four hours after administration of a single oral 0.3 or 0.6 mmol/kg (49 or 98 mg/kg)
gavage dose of bromodichloromethane. No significant induction of DNA strand breaks was
observed in male F344/N rats exposed to 0.6, 1.2, or 2.4 g/L of bromodichloromethane in the
drinking water for up to five weeks.
Stacker et al. (1997) investigated the in vivo genotoxicity of bromodichloromethane in an
unscheduled DNA synthesis assay in the livers of bromodichloromethane treated rats. Male
Sprague-Dawley rats (4 animals per group) were administered a single dose of 0 (control), 135 or
450 mg/kg bromodichloromethane via gavage in aqueous 1% methylcellulose. These doses were
selected by the authors to correspond to 30% and 100% of the calculated maximum tolerated dose
(MTD) for this compound. Analysis of hepatocytes for unscheduled DNA synthesis was
conducted 2 and 14 hours after treatment. There was no evidence of increased DNA synthesis in
hepatocytes at any tested dose of bromodichloromethane.
c. Mechani sti c Studi e s of Genotoxi city
A potential mechanism for mutagenicity of bromodichloromethane has been studied in
strains of Salmonella typhimurium that express the rat theta-class glutathione S-transferase Tl-1
gene (GSTT1-1). These studies provide evidence for a distinctmechanism of brominated trihalo-
methane activation and thus are discussed in detail. Pegram et al. (1997) utilized two new strains
of TA1535-derived Salmonella to investigate glutathione S-transferase-mediated bioactivation of
bromodichloromethane. One strain had been transfected with the GSTT1-1 gene (+GST) and the
other strain had the same gene inserted in a non-functioning orientation (-GST). These strains
were used in base-substitution revertant colony assays following 24 hour exposures to
concentrations of bromodichloromethane ranging from 200 to 4,800 ppm. The agar concentration
resulting from a 24 hour exposure to 4,800 ppm bromodichloromethane was 0.67 mM.
Bromodichloromethane increased the number of revertant colonies in each strain of Salmonella
tested (+GST, -GST and TA1535). The frequency of the revertants in TA1535 was significantly
increased above the spontaneous level at the three highest concentrations tested (highest
concentration 4,800 ppm; intermediate concentrations not explicitly stated), while frequency was
increased in the -GST strain only at the highest concentration. In contrast, there was a dramatic,
dose-dependent increase in bromodichloromethane-induced mutations in the +GST strain when
compared to the -GST control strain (an 18-fold increase at the 4800 ppm bromodichloromethane
V - 74 November 15, 2005
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concentration). When chloroform was tested for comparative purposes, a positive response was
observed only at the two highest concentrations tested (19,200 and 25,600 ppm). These results
provide evidence that the mutagenicity of bromodichloromethane is enhanced by GST-mediated
conjugation with GSH. The comparatively low affinity of the GST-mediated pathway for
chloroform suggests that different trihalomethanes can induce mutations by different
mechanisms.
DeMarini et al. (1997) further investigated the role of glutathione S-transferase activity in
mediating the mutagenicity of bromodichloromethane in Salmonella typhimurium. Strains of
Salmonella utilized in this investigation included RSJ100, which expresses the GSTT1-1 gene and
TPT100, which has the GSTT1-1 gene inserted in a non-functioning orientation. Mutagenicity
was assayed using a Tedlar bag vaporization technique. Bromodichloromethane (3,200 ppm)
induced a 44-fold increase in revertant colonies in the RSJ100 strain of Salmonella when
compared to background revertant frequency. The spectrum of bromodichloromethane-induced
mutations at the hisG46 allele in strain RSJ100 was analyzed using the colony probe
hybridization method. This analysis revealed that 99% of the mutations were GC~^AT. A non-
brominated halomethane, dichloromethane, was used in S. typhimurium strain TA100 (which
does not contain the GSST1-1 gene) for comparison. In contrast to bromodichloromethane-
induced mutations in RSJ100, only 15% of the mutations induced by dichloromethane in the non-
GST-expressing strain TA100 were GC~>AT type mutations. This result suggests that over-
expression ofGSTTl-1 in strain RSJ100 enhanced the mutagenicity of bromodichloromethane
and induced a specific type of mutational lesion in Salmonella. The mutagenicity of
dibromochloromethane and bromoform was also markedly enhanced in the GST-expressing strain
(discussed in sections V.G.2.C and V.G.S.c below), suggesting that the brominated
trihalomethanes are bioactivated by a similar pathway. In contrast, the mutagenicity of
chloroform was not enhanced, indicating that chloroform and the brominated trihalomethanes
may be activated via different mechanisms.
Proposed metabolic routes for GST-mediated bioactivation of bromodichloromethane to
mutagenic species are illustrated in Figure III-2 in section III.C.l.
V - 75 November 15, 2005
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Table V-10 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data
for Bromodichloromethane
Endpoint
Assay System
Results
(with/without
activation)"1
References
In Vitro Studies
Gene mutation
Chromosome aberration
DNA damage
Micronuclei formation
Sister chromatic!
exchange
Salmonella typhimurium
TA100a
TA100b
TA98, TA100, TA1535,
TA1537b
TA1537
TA1535, TA98, TA100b
RSJ100
TA1535, +GST, -GST
Mouse lymphoma cellsb
Mouse lymphoma cells
Chinese hamster fibroblastsb
Chinese hamster ovary cells b
Chinese hamster lung
fibroblasts a
Saccharomyces cerevisiae a
SOS chromotest
Human lung epithelial cells
Rat and human kidney cells
Rat and human kidney cells
Human lymphocytes a
Human lymphocytes a
Rat liver cells a
Chinese hamster ovary cells b
NT/+
-/+
-/-
-/+
-/-
NT/+
NT/+
+/-
+c/-c
+/-
-/-
+/+
(weak)
-/-
+/+
NT/+
NT/+
NT/+
NT/+
+/NT
+/NT
-c/-
Simmon and Tardiff (1978)
Ishidateetal. (1982)
NTP (1987)
Varmaetal. (1988)
DeMarinietal. (1997)
Pegrametal. (1997)
NTP (1987)
Sofuni et al. (1996)
Ishidateetal. (1982)
NTP (1987); Anderson et al.
(1990)
Matsuoka et al. (1996)
Nestmann and Lee (1985)
LeCurieux et al. (1995)
Landi et al. (2003)
Robbiano et al. (2004)
Robbiano et al. (2004)
Morimoto and Koizumi (1983)
Sobti (1984)
Sobti (1984)
NTP (1987); Anderson et al.
(1990)
V-76
November 15, 2005
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Table V-10 (cont.)
Endpoint
DNA strand breaks
Assay System
CCRF-CEM human
lymphoblastic leukemia cells
F344/N primary rat
hepatocytes
Results
(with/without
activation)"1
NT/+
NT/-
References
Geter et al. (2004a)
Geter et al. (2004a)
In Vivo Studies
Micronuclei
DNA damage
Chromosome aberrations
Unscheduled DNA
synthesis
Sister chromatid
exchange
DNA strand breaks
Mouse bone marrow cells
and rat cells
Mouse bone marrow cells
Mouse peripheral blood
erythrocytes (ip)
Rat kidney cells
Rat kidney cells
Rat bone marrow cells (oral)
Rat bone marrow cells (ip)
Rat liver cells
Mouse bone marrow cells
Rat kidney cells
Rat liver, kidney, and
duodenum epithelial cells
-
-
-
+
+
+
+
-
+
-
-
Ishidate et al. (1982)
Hayashietal. (1988)
Hayashietal. (1992)
Robbiano et al. (2004)
Robbiano et al. (2004)
Fujieetal. (1990)
Fujieetal. (1990)
Stacker etal. (1997)
Morimoto and Koizumi (1983)
Potter etal. (1996)
Geter et al. (2004a)
NT = Not Tested
a Assay was conducted in a closed system.
b Authors did not specify whether or not the assay was conducted in a closed system.
0 Equivocal results were obtained.
d With/without activation applies to in vitro tests only.
V-77
November 15, 2005
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2. Dibromochloromethane
The results of in vivo and in vitro tests conducted to evaluate the mutagenicity,
genotoxicity, and neoplastic transformation potential of dibromochloromethane are summarized
in Table V-l 1 at the end of this section.
a. In Vitro Assays
Simmon and Tardiff (1978) reported that nonactivated dibromochloromethane was
mutagenic in S. typhimurium strain TA100 when assayed in a desiccator containing the test
compound in the atmosphere. The minimum amount of dibromochloromethane required to elicit
a mutagenic response following addition to the desiccator was 57 |imol.
Ishidate et al. (1982) assayed the mutagenicity of dibromochloromethane in S.
typhimurium strain TA100 in the presence and absence of rat liver S9 fraction. Increased
mutation frequencies were observed only in the absence of S9 activation. In contrast,
chromosomal aberrations in Chinese hamster fibroblasts were observed in the presence, but not
the absence, of S9 fraction. The concentrations tested in these assays were not reported.
NTP (1985) reported that dibromochloromethane (0.5 to 50 jimol/plate; 100 to 10,000
|j,g/plate) was not mutagenic in strains TA1535, TA1537, TA98, or TA100 when tested in the
presence or absence of Aroclor-induced Sprague-Dawley rat or Syrian hamster liver S9 fractions.
Volatilization of the test compound was proposed as a possible explanation for the negative
results.
Nestmann and Lee (1985) investigated the mutagenicity of dibromochloromethane at
concentrations of 11 to 5,700 |j,M in S. cerevisiae strains D7 and XV185-14C in the presence or
absence of S9 activation. No clear increase in convertants or in revertants of strain XV185-14C
were observed in the presence of S9-activated dibromochloromethane. In the absence of S9
activation, an increased incidence of gene convertants in strain D7 was observed at concentrations
greater than 1,140 |j,M. There was no effect on the incidence of revertants under the same
conditions. The high dose of dibromochloromethane was cytotoxic.
Varma et al. (1988) tested dibromochloromethane for mutagenicity in S. typhimurium
strains TA1535, TA1537, TA98, and TA100. Dibromochloromethane produced a significantly
increased mutation frequency at the lowest S9-activated concentration (0.12 |imol/plate) in all
four strains. Dibromochloromethane at the same concentration also resulted in increased
mutation frequencies in strains TA1535 and TA1537 in the absence of S9 fraction. Higher
concentrations had no clear effect on mutation frequency. This spike in mutation frequency at the
low dose with similar responses in strains that detect frameshifts and those that detect base
substitutions is very unusual. It is possible that the reported data may have resulted from
cytotoxicity, although the number of revertants at the nonmutagenic doses was comparable to
background levels.
V - 78 November 15, 2005
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Dibromochloromethane induced mutations at the tk locus of LSI 78 Y mouse lymphoma
cells when tested at concentrations greater than or equal to 480 |j,M in screw-capped tubes. The
material was tested only in the absence of S9 activation (McGregor et al., 1991).
Loveday et al. (1990) found that dibromochloromethane did not induce chromosome
aberrations in CHO cells with S9-activation at concentrations that caused cell-cycle delay
(12,200 |iM) or in the absence of S9-activation at concentrations that were cytotoxic with a
standard harvest time (6,000 |j,M). Sister chromatid exchange was induced in CHO cells by S9-
activated dibromochloromethane at 3,600 |j,M with a delayed cell harvest, while the nonactivated
test material had no effect at concentrations up to cytotoxic levels (1,200 |j,M; 247 |ig/mL).
Morimoto and Koizumi (1983) investigated the ability of dibromochloromethane to
induce sister chromatid exchanges (SCE) in human lymphocytes in vitro in the absence of S9
activation. Addition of dibromochloromethane resulted in a dose-dependent increase in SCE.
The increased incidence was significant (p < 0.05) at concentrations greater than or equal to
400
The potential of S9-activated dibromochloromethane to induce sister chromatid exchanges
in vitro was also investigated by Sobti (1984). A dose of 100 |j,M produced an increased
frequency of sister chromatid exchange in rat liver cells. In human lymphocytes, 1 |iM
dibromochloromethane produced the same effect as 100 |j,M bromodichlorom ethane.
Fujie et al. (1993) observed a statistically significant, dose-related increase in sister
chromatid exchange in rat erythroblastic leukemia K3D cells treated with dibromochloromethane
in the absence of exogenous activation. Dibromochloromethane had the weakest response among
the brominated trihalomethanes tested. Dibromochloromethane also appeared to give a positive
response in the presence of exogenous metabolic activation, although the study protocol and
results with negative controls were less clear for this phase of testing.
LeCurieux et al. (1995) evaluated the induction of DNA damage by
dibromochloromethane in the presence and absence of exogenous activation using the SOS
chromotest. Dibromochloromethane exposure gave a positive result for induction.
Dibromochloromethane gave negative results in the fluctuation test modification of the S.
typhimurium reverse mutation assay.
Matsuoka et al. (1996) conducted a chromosome aberration assay with Chinese hamster
lung fibroblast (CHL/IU) cells exposed to dibromochloromethane in tightly capped flasks.
Dibromochloromethane induced polyploidy in the absence of S9 fraction, but not in the presence
of S9. The study authors considered activated dibromochloromethane marginally positive for
chromosome aberrations. However, there was no effect under the utilized test conditions when
gaps were excluded from consideration. There was no evidence of structural chromosome
aberration induction by dibromochloromethane in the absence of exogenous metabolic activation.
V - 79 November 15, 2005
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Dibromochloromethane was tested in the mouse lymphoma assay as part of an
international collaborative program under the auspices of the Japanese Ministry of Health and
Welfare (Sofuni et al., 1996). Dibromochloromethane yielded clearly positive results with or
without exogenous metabolic activation in two laboratories.
Geter et al. (2004a) examined the ability of dibromochloromethane to induce DNA strand
breaks in CCRF-CEM human lymphoblastic leukemia cells and primary F344/N rat hepatocytes
in vitro. Strand breaks were measured using the alkaline unwinding assay. Exposure to 5 or 10
mM dibromochloromethane significantly induced DNA strand breaks in CCRF-CEM cells after a
two-hour exposure. Cells exposed to 1 mM dibromochloromethane showed 100% recovery after
incubation in dibromochloromethane-free medium for 22 hours. Cells exposed to 10 mM
dibromochloromethane showed increased levels of DNA strand breaks, indicating a lack of
recovery and possible inhibition of DNA repair. No evidence of strand breaks in the absence of
cytotoxicity was observed in primary rat hepatocytes exposed to 1, 5, or 10 mM
dibromochloromethane.
Landi et al. (2003) evaluated the ability of dibromochloromethane to cause DNA damage
in primary cultures of human lung epithelial cells as measured by tail extent movement in the
Comet assay. Human lung epithelial cells were collected by scraping the large airways of four
volunteers. Cells were maintained in culture and were exposed to 0, 10, 100 or 1000 |j,M
dibromochloromethane for 3 hours prior to being flash frozen for analysis. DNA damage was not
observed at any concentration tested.
b. In Vivo Assays
Fujie et al. (1990) analyzed chromosome aberrations in bone marrow from Long-Evans
rats (3/sex/dose) following oral (males only) or intraperitoneal (males and females) exposure to
dibromochloromethane. Oral administration was by gavage in saline for five consecutive days,
and the animals were sacrificed 18 hours after the last dose. Dibromochloromethane induced
dose-related increases in chromosome breaks. A more pronounced increase in clastogenic
activity was observed following a single intraperitoneal dose, with significant (p < 0.05) effects at
20.8 mg/kg. Regardless of the route, the predominant types of induced aberrations were
chromatid and chromosome breaks.
Hayashi et al. (1988) measured induction of micronucleated polychromatic erythrocytes in
ddY mice by intraperitoneal administration of dibromochloromethane at single doses of up to 500
mg/kg in corn oil. No evidence of clastogenicity was observed. However, the sampling time
utilized in this experiment was insufficient (U.S. EPA, 1994b). There was no clear evidence of
toxicity or cytotoxicity in the target tissue.
Ishidate et al. (1982) investigated the in vivo clastogenicity of dibromochloromethane in
ddY and MS mice and Wistar rats. Doses of 125 to 500 mg/kg-day were administered in olive oil
by intraperitoneal injection, and the animals were sacrificed at 18, 24, 30, 48, and 72 hours after
dosing. No significant induction of micronucleus formation was observed in either mice or rats.
V - 80 November 15, 2005
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Morimoto and Koizumi (1983) investigated the potential of dibromochloromethane to
induce sister chromatid exchanges in male ICR/SJ mice. Animals were given doses of 0, 25, 50,
100, or 200 mg/kg-day for four days by olive oil gavage. Dibromochloromethane produced a
roughly linear dose-dependent increase in sister chromatid exchange frequency. The increase was
statistically significant (p < 0.05) at 25 mg/kg-day. The authors noted that the concentrations
required to produce an increased incidence of sister chromatid exchange were on the order of
1,000 to 10,000 times higher than the concentrations typically found in drinking water.
Potter et al. (1996) observed no significant effect on number of DNA strand breaks in the
kidneys of male F344 rats dosed with 0.75 or 1.5 mmol/kg ( 156 or 312 mg/kg-day)of
dibromochloromethane in 4% Emulphor® when analyzed using the alkaline unwinding procedure
one day following treatment. Geter et al. (2004a) found no significant induction of DNA strand
breaks in liver, kidney, or duodenum epithelial cells of male F344/N rats when assayed using the
alkaline unwinding assay four hours after a single oral gavage dose (0.3 or 0.6 mmol, or
approximately 62 or 125 mg/kg) of dibromochloromethane in 0.25% Emulphor. No significant
induction of DNA strand breaks was observed in liver, kidney, or duodenum epithelial cells of
male F344/N rats exposed to 0.6, 1.2, or 2.4 g/L dibromochloromethane in the drinking water for
two weeks.
Stacker et al. (1997) investigated the in vivo genotoxicity of dibromochloromethane in an
unscheduled DNA synthesis assay in the livers of dibromochloromethane treated rats. Male
Sprague-Dawley rats (4 animals per group) were administered a single dose of 0 (control), 600 or
2000 mg/kg via gavage in aqueous 1% methylcellulose. These doses were selected by the authors
to correspond to 30% and 100% of the calculated maximum tolerated dose (MTD) for this
compound. Analysis of hepatocytes for unscheduled DNA synthesis was conducted 2 and 14
hours after treatment. There was no evidence of increased DNA synthesis in hepatocytes from
rats treated with any tested dose of dibromochloromethane.
Sekihashi et al. (2002) obtained positive results for dibromochloromethane genotoxicity
using the Comet assay. The authors indicated that doses were selected to avoid confounding of
the results by cytotoxicity. In Wistar rats, positive (statistically significant differences in mean
migration) results were obtained for stomach, colon, liver, kidney, bladder, or lung tissues
removed 8 or 24 hours following administration of 200 mg/kg oral dose of. using. In ddY mice,
positive results were obtained for liver and brain samples harvested 8 or 24 hours, respectively,
after administration of a 400 mg/kg oral dose. Although a statistically significant increase in
migration was also noted for the eight hour colon sample, the study authors did not identify this
finding as a positive response.
c. Mechani sti c Studi e s of Genotoxi city
DeMarini et al. (1997) investigated the role of glutathione S-transferase activity in the
mutagenicity of dibromochloromethane in Salmonella typhimurium. Strains of Salmonella
utilized in this investigation included RSJ100, which expresses the rat glutathione S-transferase
theta 1-1 (GSTT1-1) gene and TPT100, which has the GSTT1-1 gene inserted in a non-
V-81 November 15, 2005
-------
functioning orientation. Dibromochloromethane (400 ppm) induced an 85-fold increase in
revertant colonies in the RSJ100 strain of Salmonella compared to background revertant
formation. The mutational spectra for dibromochloromethane-induced mutations at the hisG46
allele in strain RSJ100 were analyzed using the colony probe hybridization method. This analysis
revealed that 100% of the mutations were GC~>AT. A non-brominated dihalomethane,
dichloromethane, was tested in TA100 (which does not express GSTT1-1) for comparison. In
contrast to dibromochloromethane-induced mutations in RSJ100, only 15% of the mutations
induced by dichloromethane in TA100 were GC~>AT type mutations. This result suggests that
over-expression ofGSTTl-1 in strain RSJ100 mediated the mutagenicity of
dibromochloromethane and induced a specific type of mutational lesion in Salmonella. Proposed
pathways of bioactivation of dibromochloromethane and other brominated trihalomethanes are
shown in Figure 4-2.
Landi et al. (1999b) investigated the role of GSST1-1 in the mutagenicity of
dibromochloromethane in Salmonella by using one strain that expressed rat GSST1-1 (RSJ100)
and one strain that did not (TPT100). Mutagenicity of dibromochloromethane was assessed by
revertant colony formation with or without S9 metabolic activation. The addition of 800 ppm
dibromochloromethane greatly increased revertant numbers in the RSJ100 but not the TPT100
strain of Salmonella. Addition of the rat liver S9 fraction had no effect on the number of
revertants induced by dibromochloromethane exposure in either strain. These data provide
further support for the hypothesis that GSST1-1 plays a role in the mutagenicity of
dibromochloromethane. Additional experiments were conducted to investigate the effects of
exogenously added GSST1-1 on the mutagenic potency of dibromochloromethane. Red blood
cells (RBC), which express GSST1-1, were added to the experimental system to address this
question. RBC had no effect on results obtained with the TPT100 strain, but completely
suppressed the mutagenicity of dibromochloromethane in the RSJ100 strain. However, the
'protective' effect of RBC did not appear to be related to GSST1-1 activity, as this suppression
occurred even with the addition of RBC from individuals who do not express GSST1-1. The
underlying mechanism of RBC suppression of dibromochloromethane mutagenicity was not
investigated. The authors of this study speculated that tissues potentially exposed to
dibromochloromethane via the blood may be at less genotoxic risk (due to protection afforded by
the RBC) than tissues which are directly exposed to oral bromodichloromethane (such as tissues
in the gastrointestinal tract).
Proposed metabolic routes for GST-mediated bioactivation of dibromochloromethane to
mutagenic species are shown in Figure III-2 in section III.C1.
V - 82 November 15, 2005
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Table V-ll Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data
for Dibromochloromethane
Endpoint
Assay System
Results
(with/without
activation)"1
References
In Vitro Studies
Gene mutation
Chromosome aberration
DNA damage
Sister chromatid
exchange
Salmonella typhimurium
TA100a
TA100b
TA98, TA100, TA1535,
TA1537b
TA1535, TA1537b
TA98, TA100b
RSJ100
RSJ100
TPT100
Mouse lymphoma cellsa
Mouse lymphoma cells
Chinese hamster fibroblastsb
Chinese hamster ovary cellsb
Chinese hamster lung
fibroblastsa
Saccharomyces cerevisiae*
SOS chromotest
S. typhimurium fluctuation
test
Human lung epithelial cells
Human lymphocytes*
Human lymphocytes3
Rat liver cellsb
Chinese hamster ovary cellsb
Rat erythroblastic leukemia
cells
NT/+
-/+
-/-
+/+
-/+
NT/+
+/+
-/-
NT/+
+/+
+/-
-/-
-/+
(see text)
-/+
+/+
NT/-
NT/+
+/NT
+/NT
+/-
-c/+
Simmon and Tardiff (1978)
Ishidateetal. (1982)
NTP (1985)
Varmaetal. (1988)
DeMarinietal. (1997)
Landietal. (1999b)
McGregor etal. (1991)
Sofunietal. (1996)
Ishidateetal. (1982)
Loveday etal. (1990)
Matsuoka et al. (1996)
Nestmann and Lee (1985)
LeCurieux etal. (1995)
Landi et al. (2003)
Morimoto and Koizumi (1983)
Sobti (1984)
Sobti (1984)
Loveday etal. (1990)
Fujie etal. (1993)
V-83
November 15, 2005
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Table V-ll (cont.)
Endpoint
DNA strand breaks
Assay System
CCRF-CEM human
lymphoblastic leukemia cells
F344/N primary rat
hepatocytes
Results
(with/without
activation)"1
NT/+
NT/-
References
Geter et al. (2004a)
Geter et al. (2004a)
In Vivo Studies
Micronuclei
Chromosome aberrations
Sister chromatid
exchange
DNA strand breaks
DNA damage
(comet assay)
Unscheduled DNA
synthesis
Mouse bone marrow cells
Mouse bone marrow cells
Rat bone marrow cells
Rat bone marrow cells
Mouse bone marrow cells
Rat kidney cells
Rat liver, kidney, duodenum
epithelial cells
Rat stomach, colon, liver,
kidney, bladder, lung tissue
Mouse liver and brain tissue
Rat hepatocytes
-
-
+
+
+
-
-
+
+
-
Ishidate et al. (1982)
Hayashietal. (1988)
Fujieetal. (1990)
Fujieetal. (1990)
Morimoto and Koizumi (1983)
Potter etal. (1996)
Geter et al. (2004a)
Sekihashi et al. (2002)
Sekihashi et al. (2002)
Stacker etal. (1997)
NT = Not Tested
a Assay was conducted in a closed system.
b Authors did not specify whether or not the assay was conducted in a closed system.
0 Equivocal results reported.
d With/without activation applies to in vitro data only.
3.
Bromoform
The results of in vivo and in vitro tests conducted to evaluate the mutagenicity,
genotoxicity, and neoplastic transformation potential of bromoform are summarized in Table V-
12 at the end of this section.
V-84
November 15, 2005
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a. In Vitro Assays
Simmon and Tardiff (1978) reported that nonactivated bromoform was mutagenic in S.
typhimurium strain TA100 when assayed as vapor in a desiccator. The minimum amount of
bromoform required to elicit a mutagenic response following addition to the desiccator was 570
[imol.
Ishidate et al. (1982) assayed the mutagenicity of bromoform in S. typhimurium strain
TA100 in the presence and absence of rat liver S9 fraction. Increased mutation frequencies were
observed only in the absence of S9 activation. In contrast, chromosomal aberrations in Chinese
hamster fibroblasts were observed in the presence, but not the absence, of S9 fraction. The
concentrations tested in these assays were not reported.
Maddock and Kelly (1980) reported that bromoform did not induce an increase in sister
chromatid exchanges when toadfish leukocytes were exposed to concentrations of 0.4 to 400 |j,M.
Herren-Freund and Pereira (1986) assessed the initiating activity of bromoform using the
rat liver GGT-foci assay. The authors reported that a 250 mg/kg (1 mmol/kg) oral dose in an
unspecified vehicle did not initiate GGT-foci in this test.
NTP (1989a) evaluated the genotoxic potential of bromoform in multiple test systems.
Concentrations of 0.04 to 13 [imol/plate (10 to 3,333 jig/plate) produced no evidence of
mutagenicity in S. typhimurium strains TA1535 or TA1537, when assayed with or without
exogenous metabolic activation by rat or hamster liver S9 fraction. Equivocal evidence of
mutagenicity was noted in strain TA100 without activation, and in strains TA97 and TA98 in the
presence of liver microsomes prepared from Aroclor-induced Syrian hamsters. Exposure of
mouse L5178Y cells to bromoform concentrations greater than or equal to 2,300 |j,M in the
absence of S9 activation or S9-activated concentrations of at least 300 |j,M with S9 activation
resulted in forward mutations at the thymidine kinase (tk) locus. One of two laboratories
conducting the assays reported increased sister chromatid exchanges (SCE) in CHO cells exposed
to 3,800 |iM bromoform in the absence of exogenous activation. Neither laboratory observed
increased incidence of SCE in the presence of S9. S9-activated bromoform did not induce
chromosome aberrations in CHO cells; results for SCE and chromosome aberrations in the
absence of exogenous activation were equivocal.
Zeiger (1990) found that bromoform was mutagenic in S. typhimurium strain TA98 when
tested as a vapor in a closed system, but not when tested in an open system using a preincubation
protocol. Positive results were observed at levels of at least 114 [imol/desiccator, in the presence
and absence of S9 prepared from rat or hamster liver. Bromoform was negative in the closed
system with strains TA100 and TA1538 with or without rat or hamster liver S9 fraction
Roldan-Arjona and Pueyo (1993) evaluated bromoform in the S. typhimurium Ara forward
mutation assay at concentrations up to 25 [imol/plate (6.3 mg/plate). A preincubation protocol
was employed for the assay. Although a clear dose-related response was observed in the absence
V - 85 November 15, 2005
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of activation, the results were classified as questionable because a doubling of background levels
was not achieved. There was no evidence of mutagenicity in the presence of exogenous
metabolic activation. Although no attempt was made to minimize volatilization of the test
compound, cytotoxicity at the high exposure level indicated that the test material reached the
cells.
Geter et al. (2004a) examined the ability of bromoform to induce DNA strand breaks (as
assessed by the alkaline unwinding assay) in CCRF-CEM human lymphoblastic leukemia cells
and primary F344/N rat hepatocytes. Exposure to 5 or 10 mM bromoform significantly induced
DNA strand breaks in CCRF-CEM cells after a two-hour incubation. Cells exposed to 1 mM
bromoform showed increased levels of DNA strand breaks when assayed after incubation in
bromoform-free medium for 22 hours, indicating a lack of recovery; recovery at higher
concentrations could not be assessed because of cytotoxicity. No evidence of strand breaks in the
absence of cytotoxicity was observed in primary rat hepatocytes exposed to 1, 5, or 10 mM
bromoform.
Landi et al. (2003) evaluated the ability of bromoform to induce DNA damage in primary
cultures of human lung epithelial cells as measured by tail extent movement in the Comet assay.
Human lung epithelial cells were collected by scraping the large airways of four volunteers. Cells
were maintained in culture and were exposed to 0, 10, 100 or 1000 |j,M bromodichloromethane
for 3 hours prior to being flash frozen for analysis. DNA damage was observed at each
concentration tested.
b. In Vivo Assays
NTP (1989a) studied the genotoxic potential of bromoform in several test systems. Adult
male Drosophila fed with a 1,000-ppm solution of bromoform exhibited increased frequency of
sex-linked recessive lethal mutations, but no significant effect on reciprocal translocations was
observed. Intraperitoneal injection of mice with 200 to 800 mg/kg bromoform caused an increase
in sister chromatid exchange but not in chromosomal aberrations in bone marrow cells.
Fujie et al. (1990) analyzed chromosome aberrations in bone marrow from Long-Evans
rats (3/sex/dose) following oral (males only) or intraperitoneal (males and females) exposure to
bromoform. Oral administration was by gavage in saline for five consecutive days, and the
animals were sacrificed 18 hours after the last dose. Bromoform induced a dose-related increase
in the incidence of aberrant cells, with a significant (p < 0.01) increase at 253 mg/kg-day. A more
pronounced increase in clastogenic activity was observed following a single intraperitoneal dose,
with a significant (p < 0.05) effect at 25.3 mg/kg. Regardless of the route, the predominant types
of induced aberrations were chromatid and chromosome breaks.
Morimoto and Koizumi (1983) investigated the ability of bromoform and other
brominated trihalomethanes to induce sister chromatid exchanges in human lymphocytes in vitro
in the absence of S9 activation. All three brominated trihalomethanes caused a dose-dependent
increase in sister chromatid exchanges. Bromoform was more potent than bromodichloromethane
V - 86 November 15, 2005
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or dibromochloromethane. The increases were significant (p < 0.05) at concentrations greater
than or equal to 400 |j,M, 400 |j,M, and 80 |j,M for bromodichloromethane, dibromochloro-
methane, and bromoform, respectively.
Potter et al. (1996) evaluated the effect of bromoform on incidence of DNA strand breaks
in the kidney. Male F344 rats received 0.75 or 1.5 mmol/kg of bromoform in 4% Emulphor® by
gavage for 1, 3, or 7 days. These doses corresponded to 190 or 379 mg/kg. No effect was
observed on strand breaks when evaluated using the alkaline unwinding procedure one day after a
single dose.
Stocker et al. (1997) investigated the in vivo genotoxicity of bromoform in the mouse
bone marrow micronuclei assay and by analysis of unscheduled DNA synthesis in the liver of
bromoform-treated rats. In the first assay, Swiss CD mice (5/sex/dose) were treated by gavage
with doses of 0, 250, 500, or 1,000 mg/kg bromoform dissolved in aqueous 1% methylcellulose.
Micronuclei analysis was conducted 24 and 48 hours after dosing, and was negative in all dose
groups. In the second assay, male Sprague-Dawley rats (4 animals/dose) received single doses of
0, 324 or 1,080 mg/kg bromoform by gavage in aqueous 1% methylcellulose. These doses were
selected by the authors to correspond to 30% and 100% of the calculated MTD for this
compound. Analysis of hepatocytes for unscheduled DNA synthesis was conducted 2 and 14
hours after treatment. There was no evidence of increased DNA synthesis in hepatocytes from
rats treated with any tested dose of bromoform.
Geter et al. (2004a) did not observe significant induction of DNA strand breaks in liver,
kidney, or duodenum epithelial cells of male F344/N rats when evaluated using the alkaline
unwinding assay four hours after administration of a single oral gavage dose (0.3 or 0.6 mmol, or
approximately 76 and 152 mg/kg) of bromoform in 0.25% Emulphor. No evidence of induction
of DNA strand breaks was observed in liver, kidney, or duodenum epithelial cells of male F344/N
rats exposed to 0.6, 1.2, or 2.4 g/L bromoform in the drinking water for two weeks.
c. Mechani sti c Studi e s of Genotoxi city
DeMarini et al. (1997) investigated the role of glutathione S-transferase activity in the
mutagenicity of bromoform in Salmonella typhimurium. Strains of Salmonella utilized for this
investigation included RSJ100, which expresses the rat glutathione S-transferase theta 1-1
(GSTT1-1) gene and TPT100, which has the GSTT1-1 gene inserted in a non-functioning
orientation. Exposure tol,600 ppm bromoform induced a 95-fold increase in revertant colonies in
the RSJ100 strain of Salmonella compared to background revertant formation. The mutational
spectra for bromoform-induced mutations at the hisG46 allele in strain RSJ100 were analyzed
using the colony probe hybridization method. This analysis revealed that 96% of the mutations
were GC~>AT transitions. Bromoform also induced a smaller percentage (2.8%) of GC~^TA
mutations. A non-brominated halomethane, dichloromethane, was used in S. typhimurium strain
TA100 (which does not express GSST1-1) for comparison. In contrast to bromoform-induced
mutations in RSJ100, only 15% of the mutations induced by dichloromethane in TA100 were
GC—^AT type mutations. This result suggests that over-expression of GSTT1-1 in strain RSJ100
V - 87 November 15, 2005
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mediated the mutagenicity of bromoform and induced a specific type of mutational lesion in
Salmonella.
Landi et al. (1999a) investigated the mutagenicity of bromoform in in vitro exposed
human lymphocytes from both glutathione-S-transferase theta positive (GSST1-1+) and negative
(GSST1-1-) individuals. Whole blood cultures were exposed to bromoform (10"2 to 10"4 M) and
assayed for DNA breaks with the cometassay. The DNA-damaging potency of bromoform was
not significantly different in lymphocytes (the target cell for the comet assay) from GSST1-1+ and
GSST1-1- individuals. However, lymphocytes do not express GSST1-1, even in GSST1-1+
individuals, so interpretation of these data is problematic. When data were combined from both
genotypic groups, there was a weak but statistically significant induction of comets observed
following treatment with bromoform.
Proposed pathways for bioactivation of bromoform to mutagenic species are shown in
Figure III-2 in section III.C.I.
V - 88 November 15, 2005
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Table V-12 Summary of Mutagenicity, Genotoxicity, and Neoplastic Transformation Data
for Bromoform
Endpoint
Assay System
Results
(with/without
activation)"1
References
In Vitro Studies
Gene mutation
Chromosome aberration
DNA damage
Sister chromatid
exchange
Initiation
DNA strand breaks
Salmonella typhimurium
TA100a, TA1535
TA1535, TA1537b
TA100
TA97, TA98
TA100b
TA98
TA100, TA1538a
S. typhimurium Ar a
RSJ100
Mouse lymphoma cells b
Chinese hamster fibroblastsb
Chinese hamster ovary cells b
Human lymphocytes
Human lung epithelial cells
Toadfish leukocytes a
Human lymphocytes b
Chinese hamster ovary cells b
Rat liver GGT-foci assay
CCRF-CEM human
lymphoblastic leukemia cells
F344/N primary rat
hepatocytes
NT/+
-/-
-/±c
±c/-
-/+
+/+
-/-
-/+c
NT/+
+/+
+/-
-/±
NT/+
NT/+
NT/-
NT/+
-/±
-
NT/+
NT/-
Simmon and Tardiff (1978)
NTP (1989a)
Ishidateetal. (1982)
Zeiger (1990)
Roldan-Arjona and Pueyo
(1993)
DeMarinietal. (1997)
NTP (1989a)
Ishidateetal. (1982)
NTP (1989a)
Landietal. (1999a)
Landi et al. (2003)
Maddock and Kelly (1980)
Morimoto and Koizumi (1983)
NTP (1989a)
Herren-Freund and Pereira
(1986)
Geter et al. (2004a)
Geter et al. (2004a)
In Vivo Studies
Micronuclei
Mouse bone marrow cells
Mouse bone marrow cells
-
-
Ishidateetal. (1982)
Hayashietal. (1988)
V-89
November 15, 2005
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Table V-12 (cont.)
Endpoint
Chromosome aberrations
DNA strand breaks
Unscheduled DNA
synthesis
Sister chromatid
exchange
Sex-linked recessive
lethal mutations
Assay System
Mouse bone marrow cells
Mouse bone marrow cells
Rat bone marrow cells (oral)
Rat bone marrow cells (ip)
Rat renal cells
Rat liver, kidney, or
duodenum epithelial cells
Rat hepatocytes
Mouse bone marrow cells
Mouse bone marrow cells
(ip)
Drosophila
Results
(with/without
activation)"1
-
-
+
+
-
-
-
+
+
+
References
Stacker etal. (1997)
NTP (1989a)
Fujie etal. (1990)
Potter etal. (1996)
Geter et al. (2004a)
Stacker etal. (1997)
Morimoto and Koizumi (1983)
NTP (1989a)
NTP (1989a)
NT = Not Tested
a Assay was conducted in a closed system.
b Authors did not specify whether or not the assay was conducted in a closed system.
0 Equivocal results obtained.
d With/without activation applies to in vitro assays only.
G. Carcinogenicity
1. Bromodichloromethane
a. Two-Year Oral Cancer Bioassays
NTP (1987) evaluated the carcinogenic potential of bromodichloromethane in F344/N rats
in a two-year oral exposure study. Additional details of this study are provided in Section V.D. 1.
Groups of male and female rats (50/sex/group) were administered bromodichloromethane in corn
oil via gavage at doses of 0, 50, or 100 mg/kg-day for 5 days/week for 102 weeks. All animals
were examined grossly and microscopically for neoplastic lesions. Survival of all dosed animals
was comparable to or greater than the corresponding control group. Mean body weights of high-
dose male and female rats were decreased during the last 1.5 years of the study. Body weight
gains of high-dose male and female rats at study termination were 86% and 70% of the
corresponding vehicle control group, respectively. Statistically significant increases in the
V-90
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incidences of neoplasms of the large intestine and kidney were observed in male and female rats
(Table V-13). The study authors noted that neoplasms of the large intestine and kidney are
uncommon tumors in F344/N rats based on historical control data for NTP studies. They
concluded that under the conditions of these 2-year gavage studies, clear evidence of
carcinogenicity existed in male and female rats.
NTP (1987) also evaluated the potential toxic and carcinogenic effects of
bromodichloromethane mice in a two-year oral exposure study. Additional details of this study
are provided in Section V.D.I. Groups of male and female B6C3FJ mice (50/sex/dose) were
administered doses of 0, 25, or 50 mg/kg-day (males) or 0, 75, or 150 mg/kg-day (females) for 5
days/week for 102 weeks. All animals were examined grossly and microscopically for neoplastic
lesions. Survival of dosed male mice was comparable to the corresponding control group.
Survival of dosed and vehicle control females was decreased after week 84 as a result of ovarian
abscesses. Body weight gain in high-dose males was decreased by 13% when compared to the
vehicle control group. Body weight gain in low- and high-dose females was reduced by 25% and
55%, respectively. Statistically significant increases were observed in the incidences of
neoplasms of the kidney in male mice and the liver in female mice (Table V-13). The study
authors noted that neoplasms of the kidney are uncommon in B6C3FJ mice based on NTP
historical control data. They concluded that under the conditions of these 2-year gavage studies,
clear evidence of carcinogenic activity existed in male and female mice.
Tumasonis et al. (1987) exposed groups of 58 male and female Wistar rats to
bromodichloromethane in drinking water from weaning until death occurred in all of the animals
(approximately 185 weeks). The exposure level was 2,400 mg/L for 72 weeks and was reduced
to 1,200 mg/L for the remaining 113 weeks. Based on a graph presented by the authors, the
average dose over the course of the experiment was probably about 150 mg/kg-day for females
and about 100 mg/kg-day for males. Exposed animals of both sexes gained significantly less
weight (approximately 30 to 40%) than control animals. There was a statistically significant (p <
0.01) increase in the incidence of hepatic neoplastic nodules in exposed females compared to
control females (32% versus 0%). Significant increases were also reported for the occurrence of
hepatic adenofibrosis (12% versus 0%) and lymphosarcoma (17% versus 11%) in females. No
statistically significant increase in the incidence of any tumor was reported in males. Two males
and one female among the treated animals were observed to have renal adenoma or carcinoma,
while no renal tumors were observed in the controls. Statistically significant decreases in the
incidence of mammary tumors and pituitary tumors in females and lymphosarcomas in males
were observed.
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Table V-13 Tumor Frequencies in F344/N Rats and B6C3Ft Mice Exposed to
Bromodichloromethane in Corn Oil for 2 Years - Adapted from NTP (1987)
Animal
Tissue/Tumor
Male Rat
Large intestine a
Kidney a
Adenomatous polyp
Adenocarcinoma
Combined
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female Rat
Large intestine °
Kidney
Adenomatous polyp
Adenocarcinoma
Combined
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Male Mouse
Kidney d
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female Mouse
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Tumor Frequency
Control
0/50
0/50
0/50
0/50
0/50
0/50
Control
0/46
0/46
0/46
0/50
0/50
0/50
Control
1/46
0/46
1/46
Control
1/50
2/50
3/50
50 mg/kg
3/49
ll/49b
13/49b
1/49
0/49
1/49
50 mg/kg
0/50
0/50
0/50
1/50
0/50
1/50
25 mg/kg
2/49
0/49
2/49
75 mg/kg
13/48b
5/48
18/48b
100 mg/kg
33/50b
38/50b
45/50b
3/50
10/50b
13/50b
100 mg/kg
7/47b
6/47b
12/47b
6/50b
9/50b
15/50b
50 mg/kg
6/50
4/50
9/50b
150 mg/kg
23/50b
10/50b
29/50b
a One rat in the low-dose group died at week 33 and was eliminated from the cancer risk calculation.
b Statistically significant at p<0.05, compared to controls.
0 Intestine not examined in four rats from control group and three rats from high-dose group.
d In the control group, two mice died during the first week, one mouse died during week nine and one escaped in
week 79. One mouse in the low-dose group died in the first week. All of these mice were eliminated from the cancer
risk calculations.
V-92
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Aida et al. (1992b) administered bromodichloromethane to Slc:Wistar rats
(40/sex/treatment group and 70/sex/controls) at dietary levels of 0%, 0.014%, 0.055%, or 0.22%
for up to 24 months. The test material was microencapsulated and mixed with powdered feed.
Based on the mean food intakes, the mean doses were 0, 6.1, 25.5, or 138.0 mg/kg-day for males
and 0, 8.0, 31.7, or 168.4 mg/kg-day for females. The only neoplastic lesions observed were
three cholangiocarcinomas and two hepatocellular adenomas in the high-dose females, one
hepatocellular adenoma in a control female, one cholangiocarcinoma in a high-dose male, and
one hepatocellular adenoma each in a low-dose male and a high-dose male. Based on these
results, the study authors concluded that there was no clear evidence that microencapsulated
bromodichloromethane administered in the diet was carcinogenic in Wistar rats.
Voronin et al. (1987) assessed the carcinogenic potential of bromodichloromethane in
male and female CBA x C57B1/6 mice. Groups of mice (50-55/sex/concentration) were exposed
to bromodichloromethane provided in drinking water at concentrations of 0.04, 4.0, or 400 mg/L
for 104 weeks. Untreated control groups of 75 male and 50 female mice were also included in the
study design. No significant differences were observed in total tumor incidence when evaluated
by Chi square analysis. The study authors concluded that, under the conditions of this bioassay,
bromodichloromethane was not carcinogenic in mice.
George et al. (2002) evaluated the carcinogenicity of bromodichloromethane in male
F344/N rats (78 animals/dose) exposed to the compound via drinking water for 104 weeks.
Nominal concentrations of 0.07, 0.35, or 0.70 g/L were administered in drinking water containing
0.25% Emulphor®. The vehicle control solution consisted of 0.25% Emulphor®. The study
authors indicated that testing of higher concentrations was prevented by refusal of the test animals
to drink solutions containing more than 0.7 g/L. Six animals per exposure concentration were
sacrificed at 13, 26, 52, and 78 weeks for gross observation and histopathological examination of
the thyroid, liver, stomach, duodenum, jejunum, ileum, colon, rectum, spleen, kidneys, urinary
bladder, and testes. A complete rodent necropsy was performed at terminal sacrifice and
representative samples of the tissues listed above were examined microscopically. A complete
pathological examination was performed on five rats from the high-dose group. Serum profiles of
LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine, and total antioxidant activities were
determined at 26, 52, and 104 weeks. Hepatocyte and renal tubular cell proliferation were
measured at each sacrifice by bromodeoxyuridine labeling.
The measured drinking water concentrations of bromodichloromethane were 0.06, 0.38,
and 0.76 g/L. When corrected for loss of bromodichloromethane as a result of volatility,
instability, or adsorption to glass surfaces during treatment, the corresponding administered
concentrations were 0.06, 0.33, and 0.62 g/L. Based on measured water consumption, these
levels correspond to mean daily doses for the entire study of 3.9, 20.6, and 36.3 mg/kg-day as
calculated by the study authors. Mean daily doses of 6.4, 32.6, and 58.9 mg/kg-day were
calculated for the first 13 weeks of the study when the growth rate of the test animals was highest.
No significant differences were observed among groups for feed consumption or survival.
Twenty-one to 22 unscheduled deaths were observed in each treatment group. Mononuclear cell
leukemia was seen in all dose groups and was reported to be the primary cause of morbidity and
V - 93 November 15, 2005
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mortality prior to 104 weeks. Exposure to bromodichloromethane did not affect the growth rate
of test animals when compared to the control. Kidney weight was significantly depressed at the
high dose and a significant negative trend was observed for relative kidney weight. No
significant changes were observed in clinical chemistry parameters. Observed nonneoplastic
changes in the liver (e.g., biliary fibrosis, bile duct inflammation, and chronic inflammation) were
considered to be age-related background changes, since neither the incidence nor severity of the
lesions differed from the control values. Bromodichloromethane had no effect on hepatocyte
proliferation as measured by bromodeoxyuridine labeling. Renal tubular cell hyperplasia was
significantly decreased in the 3.9 mg/kg-day group and significantly increased in the 36.3 mg/kg-
day group (15.8%) relative to the control value (8.7%).
The absence of effect on body weight and other examined endpoints suggests that a
maximum toxic dose may not have been achieved in this study. However, the dosing regimen
used by George et al. (2002) was sufficient to increase the incidence of hepatocellular neoplasia
(Table V-14). The data for hepatic tumors indicate a biphasic pattern of dose-response. The
prevalence and multiplicity of hepatocellular adenoma and combined hepatocellular adenoma and
carcinoma were significantly increased at 3.9 mg/kg-day, nonsignificantly increased at 20.6
mg/kg-day, and comparable to the control values at 36.3 mg/kg-day. The prevalence and
multiplicity of hepatocellular carcinoma were increased at 20.6 mg/kg-day when compared to
control values, but the response did not reach statistical significance. The underlying basis for the
biphasic response is unknown, but the study authors noted that the observed pattern of response
could be explained by inhibition of the hepatic metabolism of bromodichloromethane by the
compound itself. Exposure to bromodichloromethane decreased the prevalence of basophilic
(control, 67%; 3.6 mg/kg-day, 62.2%; 20.6 mg/kg-day, 46%; 36.6 mg/kg-day, 34.7%) and clear
cell (17.8%, 2.2%, 2.1%, 4.1%) altered foci of cells (AFC) in a dose-dependent manner, but had
no significant effect on the prevalence of eosinophilic AFCs when compared to the controls. The
decreases in prevalence were statistically significant at the mid and high doses for basophilic
AFCs and at all doses for clear cell AFCs. One renal tubular adenoma was observed in the 3.6
mg/kg-day group and two tumors were observe in the 36.3 mg/kg-day (Table V-14). . The
historical incidence of renal tubular adenomas in male F344/N rats is very low (2/327 or 0.6%),
as determined from control groups in NTP drinking water studies. Therefore, the occurrence of
these tumors in the present study may be of biological significance. No increased incidences of
neoplasia were evident in the five high-dose animals selected for a histopathological examination
of all organs.
On the basis of the increased prevalence and multiplicity of hepatocellular neoplasms in
the 3.9 and 20.6 mg/kg-day groups, the study authors concluded that bromodichloromethane was
carcinogenic in male F344/N rats under the conditions of the bioassay. A source of uncertainty in
this conclusion is lack of knowledge on the biological mechanism underlying the dose-response
relationship observed for hepatic tumors.
V - 94 November 15, 2005
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Table V-14 Hepatic and Renal Tumors in Male F344/N Rats Administered
Bromodichloromethane in the Drinking Water for Two Years (George et al., 2002)
Tumor Type
Mean Daily Dose of Bromodichloromethane (mg/kg-day)
Vehicle Control
3.9
20.6
36.3
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Hepatocellular adenoma and
carcinoma (combined)
1/45 (2.2%)a
0.02 ± 0.02b'c
1/45 (2.2%)
0.02 ± 0.02
2/45 (4.4%)
0.04 ± 0.02
7/45 (15.5%)*
0.16 ±0.04*
1/45 (2.2%)
0.02 ±0.01
8/45 (17.8%)*
0.19 ±0.00*
3/48 (6.2%)
0.06 ± 0.02
4/48 (8.3%)
0.10 ±0.03
7/48 (14.6%)
0.17 ±0.04
2/49(4.1%)
0.04 ± 0.02
2/49(4.1%)
0.04 ± 0.02
4/49 (8.2%)
0.08 ±0.28
Kidney
Tubular cell adenoma
Tubular cell carcinoma
Tubular cell adenoma or
carcinoma (combined)
0/46 (0%)
0/46 (0%)
0/46 (0%)
1/45 (2.2%)
0/45 (0%)
1/45 (2.2%)
0/51 (0%)
0/51 (0%)
0/51 (0%)
2/44 (4.5%)
0/44 (0%)
2/44 (4.5%)
Source: George et al. (2002)
* Statistically significant when compared to the control value, p<0.05
a Prevalence (percentage of animals with tumor)
b Multiplicity, number of tumors per animal
0 Mean ± standard deviation
George et al. (2002) also evaluated the carcinogenicity of bromodichloromethane in male
B6C3FJ mice (78 animals/dose) exposed via drinking water for 100 weeks. Nominal
concentrations of 0.05, 0.25, or 0.50 g/L were administered in drinking water containing 0.25%
Emulphor®. The vehicle control solution consisted of 0.25% Emulphor®. Seven animals per
exposure concentration were sacrificed at 13, 26, 52, and 78 weeks for gross observation and
histopathological examination of the liver, stomach, duodenum, jejunum, ileum, colon, rectum,
spleen, kidneys, urinary bladder, and testes. A complete rodent necropsy was performed at
terminal sacrifice and representative samples of the tissues listed above were examined
microscopically. A complete pathological examination was performed on five rats from the high-
dose group. Serum profiles of LDH, ALT, ALP, AST, SDH, BUN, total protein, creatine, and
total antioxidant activities were determined at 26, 52, and 100 weeks. Hepatocyte and renal
tubular cell proliferation were measured by bromodeoxyuridine labeling at each sacrifice.
The measured drinking water concentrations of bromodichlorom ethane were 0.06, 0.30,
and 0.55 g/L. When corrected for loss of bromodichlorom ethane as a result of volatility,
V-95
November 15, 2005
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instability, or adsorption to glass surfaces during treatment, the corresponding administered
concentrations were 0.06, 0.28, and 0.49 g/L. Based on measured water consumption, these
levels correspond to mean daily doses of 8.1, 27.2, and 43.4 mg/kg-day as calculated by the study
authors. Water consumption was significantly reduced at the mid- and high doses; the study
authors attributed the reduced intake to taste aversion. No significant differences were observed
among groups for feed consumption or survival. Exposure to bromodichloromethane did not
affect the growth rate of test animals when compared to the control. Kidney weight was
significantly depressed at 27.2 and 43.4 mg/kg-day when compared to the control values. No
significant changes were observed in clinical chemistry parameters. Mild, treatment-related
nonneoplastic hepatic lesions were observed in the 27.2 and 43.4 mg/kg-day dose groups (identity
and prevalence not reported). Increased incidences of hepatocellular karyomegaly and necrosis
with inflammation (prevalence and severity not reported) were not dose-related. The prevalence
of renal tubular hyperplasia was 3%, 0%, 6% and 0% for the vehicle control, 8.1, 27.2, and 43.4
mg/kg-day groups, respectively. Other observed preneoplastic and neoplastic lesions (identity
and prevalence not reported) were considered background events for the male B6C3FJ mouse.
BrdU labeling index in hepatocytes and renal tubular cells was not altered at any time point.
Hepatocellular adenomas and carcinomas were observed in all treatment groups. Neither the
prevalence nor multiplicity of these tumors was significantly increased by exposure to
bromodichloromethane. Renal tubular cell neoplasia was not observed in any treatment group.
No increased incidences of neoplasia were evident in the five high-dose animals subject to a full
histopathological examination. On the basis of these data, the study authors concluded that
bromodichloromethane was not carcinogenic to male mice under the conditions employed in this
study. However, it is not evident that an adequately high dose was tested in this study.
b. Studies of Induction of Aberrant Crypt Foci
DeAngelo et al., (2002) evaluated the ability of bromodichloromethane administered in
drinking water to induce aberrant crypt foci (ACF), putative early preneoplastic lesions, in the
colons of male F344/N rats. Groups of weanling rats (6 animals/group) were exposed to distilled
water, 0.25% Alkamuls EL-620®, or 0.7 g/L bromodichloromethane in 0.25% Alkamuls EL-620
for 13 weeks. A single intraperitoneal injection of 30 mg/kg azoxymethane (AOM) served as the
positive control. Body weight and water consumption were measured twice during the first week
of the study and once per week thereafter. Colons were collected at study termination, fixed,
stained with 0.2% methylene blue, divided into three equal segments, and scanned for ACF. The
measured concentration of bromodichloromethane averaged 0.64 ± 0.06 g/L (mean and standard
error) over the course of the study. When adjusted for volatilization and adherence to glass, the
corrected concentration was 0.51 g/L. Water consumption was significantly reduced (39%) in the
bromodichloromethane exposure group when compared to the 0.25% vehicle control. The
average daily dose was 45 mg/kg-day as calculated by the study authors. Average terminal body
weights of the rats exposed to bromodichloromethane were within 10% of the control values. No
ACF were observed in colons from control animals. ACF were observed in 5/6 colons from
bromodichloromethane-exposed animals. The total number of AC/focus (30), average number of
AC/focus (3.33 ± 0.47), ACF/colon (1.50 ± 0.56), and total and average focal area (550 and 61.11
|im2, respectively) were significantly increased relative to the combined deionized water and
V - 96 November 15, 2005
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vehicle controls. All observed ACF were located in the distal (rectal) segment of the colon. For
comparison, 807 ACF and 4.95 ± 0.25 crypts per focus (mean and standard error) were observed
in the AOM positive control group. Eight percent, 42% and 50% of the ACF induced by AOM
were located in the proximal, middle, and distal segment of the colon, respectively. The study
authors reported that the localization of ACF in the distal segment of the colon is consistent with
the observed sites for tumor formation in the large intestine of rats administered
bromodichloromethane in corn oil for two years (NTP, 1987). However, the study authors noted
that ACF induced by bromodichloromethane administered in drinking water at the study test dose
do not appear to progress to neoplasia, as judged by the absence of colon neoplasms in the two-
year drinking water study conducted by George et al. (2002).
De Angelo et al. (2002) evaluated the ability of bromodichloromethane administered in
drinking water to induce ACF in the colons of male B6C3FJ (6 animals/group) and A/J mice (9
animals/group; sex not specified). Mice of the A/J strain are sensitive to chemical induction of
ACF. Test animals were exposed to distilled water, 0.25% Alkamuls EL-620®, or a target
concentration of 0.7 g/L bromodichloromethane in 0.25% Alkamuls EL-620 for 13 weeks (both
strains) or 30 weeks (A/J mice only). A single intraperitoneal injection of 50 mg/kg 4-
aminobiphenyl or 10 mg/kg AOM served as the positive controls for the B6C3FJ and A/J strains,
respectively. Body weight and water consumption were measured twice during the first week of
the study and once per week thereafter. Colons were collected at study termination, fixed, stained
with 0.2% methylene blue, divided into three equal segments, and scanned for ACF. The study
report did not provide results for measured concentration of bromodichloromethane in drinking
water solutions or an estimated dose. No differences were observed in between the control and
any treatment group for body weight or water and feed consumption. ACF development was not
observed in the colons of B6C3FJ mice treated with bromodichloromethane in the drinking water
or injected with 4-aminobiphenyl. Bromodichloromethane did not induce ACF in A/J mice.
Injection of A/J mice with the positive control compound AOM induced 47.4 ± 4.9 ACF/cm2
(mean and standard error) and 7.2 ±1.1 tumors/cm2 after 13 weeks and 17.8 ± 2.6 tumors/cm2
after 30 weeks of treatment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per focus were
observed in the AOM positive control group.
Geter et al. (2004b) investigated the influence of vehicle and mode of administration on
the induction of ACF in the colons of male F344/N rats exposed to bromodichloromethane.
Twenty-eight-day-old male F344/N rats (6/treatment) received either 0 or 50 mg/kg-day of
bromodichloromethane in corn oil by gavage (five days/week) or 0 or 0.7 g/L (equivalent to 0 or
63 mg/kg-day) of bromodichloromethane in drinking water containing 0.25% Emulphor® for 26
weeks. Animals in the positive control group received a single 15 mg/kg intraperitoneal injection
of AOM. Body weight and water consumption were measured twice weekly for the first week
and biweekly for the remainder of the experiment. Details of treatment are provided in the
related publication by George et al. (2002). At sacrifice, the colon was removed from each
animal and divided into proximal, medial, and distal segments. ACF were identified by staining
with 0.2% methylene blue.
V - 97 November 15, 2005
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Significant reductions in water consumption were observed in the positive controls and
bromodichloromethane treatment groups, but body weights in these groups were similar to the
controls. Exposure to AOM alone or with corn oil resulted in significant increases in the total
number of ACF, ACF/colon, total focal area, and total number of aberrant crypts. ACF were
observed in 4/6 animals receiving bromodichloromethane by gavage or in the drinking water
compared to 0/6 controls. Exposure to bromodichloromethane in the drinking water significantly
increased the total number of ACF (control: 0; bromodichloromethane: 8), ACF/colon (0, 1.33),
total focal area 0, 670 |im2), and total number of aberrant crypts (0, 26). Gavage exposure to
bromodichloromethane in corn oil increased the total number of ACF (control: 1;
bromodichloromethane: 9), ACF/colon (0.16, 1.5), total focal area (0, 650 |im2), and total number
of aberrant crypts (11, 33), but none of these responses were statistically significant when
compared to the control. No effect was noted in the mean number of crypts per focus or the
distribution of ACF in the proximal, medial, and distal segments for any treatment when
compared to the corresponding controls. The study authors concluded that ACF formation is
independent of the route of bromodichloromethane exposure, but noted that a longer exposure
(i.e., 52 weeks) might demonstrate differences between drinking water and corn oil as vehicles for
administration. A limitation of this study is the small sample size (6 animals/treatment), which
may have limited the power of the study to detect differences in ACF induction between the
vehicles. Geter et al. (2004c) investigated the effects of a diet containing a high amount of
animal fat on the induction of ACF in the colon of male F344/N rats exposed to
bromodichloromethane. The study was conducted because a high-fat diet is regarded as an
important nutritional influence on colon cancer development. Twenty-eight-day-old male F344/N
rats (6/treatment) received 0.7 g/L (equivalent to 56 - 63 mg/kg-day) of bromodichloromethane in
drinking water containing 0.25% Emulphor® for 26 weeks. Animals in the negative control group
received water containing 0.25% Emulphor®. Animals in the positive control group received a
single 15 mg/kg intraperitoneal injection of AOM. All animals were fed a standard laboratory
diet (Purina 5001), with half receiving the normal feed containing 4.5% fat and half receiving
feed supplemented with 19% animal fat. Body weight and water consumption were measured
twice weekly for the first week and biweekly for the remainder of the experiment. At sacrifice,
the colon was removed from each animal and divided into proximal, medial, and distal segments.
ACF were identified by staining with 0.2% methylene blue.
Water consumption was significantly reduced in the positive control (normal diet only)
and bromodichloromethane (normal and high-fat diets) treatment groups, but mean body weights
in these groups were similar to or greater than the controls. The incidences of ACF (i.e., number
of colons with ACF/number of colons scored) for the control, control + high fat,
bromodichloromethane, and bromodichloromethane + high fat groups were 0/6, 3/6, 4/6, and 5/6,
respectively. The incidence of ACF in the positive control groups was 6/6. Exposure to
bromodichloromethane significantly increased the total number of ACF, ACF/colon, total and
mean focal area, and total number of aberrant crypts in animals receiving the normal diet relative
to the control group. Similar values for these endpoints were observed for animals receiving the
high-fat diet, but the incidence of ACF in the high-fat control group was higher and thus no
significant differences were observed between the high-fat control and bromodichloromethane-
exposed groups. No significant differences were noted in the number or characteristics of ACF
V - 98 November 15, 2005
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between animals fed normal or high-fat diets and exposed to either AOM or
bromodichloromethane.
The induction of ACF in the colon has been investigated in Tsc2 mutant Long-Evans
(Eker) rats, a rodent model of hereditary renal cancer. The basic details of this study (e.g., in-life
and chemical consumption data) are described in Hooth et al. (2002) and the results for induction
of ACF are reported in McDorman et al. (2003a). Male Eker rats (8-10/concentration) received
drinking water containing 0, 0.07, or 0.7 g/L of bromodichloromethane continuously for 10
months. The administered concentrations resulted in average daily doses of approximately 0, 3.5
and 35.0 mg/kg-day, respectively, as calculated by the study authors. At necropsy, the colon was
removed, fixed, and processed for histopathological evaluation. Fixed segments of the proximal,
middle, and distal colon were stained for identification of ACF and counting of the individual
crypts in each focus. Colons from control and high-dose rats were analyzed for crypt cell
proliferation using proliferating cell nuclear antigen (PCNA).
Exposure to bromodichloromethane increased the incidence of ACF (0 mg/L, 0/10; 0.07
g/L, 7/8; 0.70 g/L, 6/8), total number of ACF (0, 9, 10), mean ACF/colon (0, 1.13, 1.25), total
crypts/ACF (0, 29, 27), mean crypts/ACF (0, 3.22, 2.7), and mean size of ACF (0, 0.36, 0.26
mm2) in the colons of male rats. There were no statistically significant responses with treatment,
which may reflect in part the small number of animals tested and evaluated in this study. At the
low dose, approximately 67% of the ACF were in the proximal colon and 33% were in the distal
colon. At the high dose, 30% of the ACF were in the proximal colon, 40% were in the middle
colon, and 30% were in the distal colon. Exposure to bromodichloromethane did not significantly
increase crypt cell proliferation in the colon as evaluated by the LI or % mitoses in individual
segments of the colon or the entire colon.
c. Cancer and Cancer-Related Studies in Cancer-Susceptible Rodent Strains
Theiss et al. (1977) examined the carcinogenic potential of bromodichloromethane in
Strain A mice (6 to 8 weeks old). Male animals (20 mice/group) were injected intraperitoneally
up to three times weekly over a period of 8 weeks. Three dose levels (20, 40, or 100 mg/kg
bromodichloromethane) were used with concurrent positive and negative control groups that
contained 20 animals each. Mice were sacrificed 24 weeks after the first injection, and the
frequency of lung tumors in each test group was compared with vehicle-treated controls. No
statistically significant increase in the incidence of lung tumors/mouse was reported.
The induction of transitional cell hyperplasia, and carcinogenicity of
bromodichloromethane has been investigated in Tsc2 mutant Long-Evans (Eker) rats, a rodent
model of hereditary renal cancer. The results of this study have been reported in three
publications (McDorman et al., 2003a,b; Hooth et al., 2002). The Eker rat model is characterized
by a spontaneous germ-line insertion mutation in the tuberous sclerosis complex (Tsc2) tumor-
suppressor gene. This mutation predisposes Eker rats to develop multiple spontaneous renal cell
carcinomas, as well as splenic hemangiosarcomas and uterine leiomyosarcomas, as early as four
V - 99 November 15, 2005
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months of age. As a result, Eker rats are highly susceptible to the effects of renal carcinogens
(McDorman et al., 2003b).
The basic details of this study (e.g., in-life and chemical consumption data) are described
in Hooth et al. (2002). Male and female Eker rats (8-10/sex/concentration) received drinking
water containing 0, 0.07, or 0.7 g/L of bromodichloromethane daily for 4 or 10 months. At
sacrifice, the test animals were 6 or 12 months of age, respectively. Complete necropsies were
performed on all animals at sacrifice. Tissues collected for microscopic examination included the
adrenal glands, gross lesions, kidneys, large intestine, liver, spleen, testicles (including
surrounding membranes), thyroid gland, urinary bladder, and uterus.
The average daily doses of bromodichloromethane were 3.5 and 35.0 mg/kg-day for males
and 6.5 and 55.6 mg/kg-day for females, as estimated by the study authors. Survival, mean body
weight, and water consumption of male and female rats exposed to bromodichloromethane were
similar to the controls. Centrilobular hypertrophy was observed in the livers of 5/8 high-dose
males. Clear cell foci of cellular atypia were observed in 3/8 high-dose males and 1/8 high-dose
males had basophilic foci. None of these lesions were present in control rats. After four months
of treatment, nearly all males in the study had at least one renal tumor. More adenomas and total
renal tumors were observed in the high-dose males than in the low dose males (adenomas: 0 g/L,
1.9 ± 1.9; 0.07 g/L, 2.1 ± 1.1; 0.7 g/L, 2.9 ± 2.0; total tumors: 0 g/L, 2.1 ± 2.0; 0.07 g/L, 2.3 ± 1.0;
0.7 g/L, 3.0 ± 1.9), but the differences were not statistically significant. The same pattern was
evident after 10 months of treatment (adenomas: 0 g/L, 5.6 ± 3.2; 0.07 g/L, 8.6 ± 7.2; 0.7 g/L, 9.6
± 5.9; total tumors: 0 g/L, 5.6 ± 3.2; 0.07 g/L, 8.8 ± 7.2; 0.7 g/L, 9.8 ± 6.1), but again the
differences were not statistically significant. The numbers of adenomas and total renal tumors in
female rats did not show a dose-related trend after 4 or 10 months of treatment. There were no
dose-related trends for incidence or lesion burden (total number of proliferative lesions/total
number of rats in the group) of hemangioma of the spleen in male or females or for leiomyomas
or mesenchymal cell proliferation in the uterus of females.
McDorman et al. (2003b) reported the analysis of preneoplastic and neoplastic renal
lesions in Eker rats exposed to 0, 0.07 or 0.7 g/L bromodichloromethane in the study described by
Hooth et al. (2002). At necropsy, a midsagittal section of each kidney was collected, preserved,
and processed for histopathological evaluation. Two sagittal sections (one per kidney) from each
animal were subsequently examined for the presence and number of atypical renal tubules,
atypical tubular hyperplasia, and renal epithelial tumors (adenomas and carcinomas) and the
severity of chronic progressive neuropathy. Characterization of renal lesions in the Eker rat is
based on the morphology and size of the lesions. Atypical tubules are normal sized tubules with a
patent lumen lined by a single layer of one or more altered cells, characterized by combinations
of cell swelling with an increase in cytoplasm, basophilia, altered nucleus to cytoplasm ratio, and
megalocytosis. Atypical hyperplasias are tubules filled with multiple layers of altered cells that
occlude the lumen and expand the normal tubule size but remain smaller than three average
tubules. Adenomas are solid, cystic, or cystopapillary foci of altered tubular epithelial cells
greater than or equal to the size of three average tubules and less than 10 mm in diameter. They
may or may not breach the basement membrane. Renal carcinomas are solid, cystic, or
V - 100 November 15, 2005
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cystopapillary foci of altered tubular epithelial cells greater than or equal to 10 mm in diameter.
Atypical tubules are the first recognizable preneoplastic lesion in this series. Some atypical
tubules may progress to the more advanced lesions of atypical hyperplasia, adenoma, or
carcinoma. Once an atypical tubule has progressed to atypical hyperplasia, it is assumed that
(given sufficient time) it will progress to an adenoma.
After 4 months of continuous exposure, the mean number of atypical tubules per rat was
significantly increased in low- and high-dose males (0 g/L, 13.5 ± 4.1; 0.07 g/L, 24.8 ± 7.8; 0.7
g/L, 35.9 ±11.5) and high-dose females (0 g/L, 49.3 ± 13.9; 0.07 g/L, 71.0 ± 21.9; 0.7 g/L, 77.3 ±
19.9). High-dose males (0 g/L, 5.1 ± 2.5; 0.07 g/L, 3.9 ± 1.8; 0.7 g/L, 9.8 ± 2.8) and low- and
high-dose females (0 g/L, 7.6 ±3.4; 0.07 g/L, 13.1 ±5.5; 0.7 g/L, 11.0 ± 4.3) had higher mean
numbers of atypical hyperplasias per rat, but the response was not statistically significant when
compared to the controls. There were no significant increases in the number of tumors or
combined hyperplasias and tumors per rat for either sex. After 10 months of exposure, no
significant differences were observed in male or female rats for atypical tumors, atypical
hyperplasias, adenomas and carcinomas (combined), or hyperplasias and tumors, although the
mean values for each category of lesion were consistently greater than the control values. There
were no significant changes in the severity of chronic progressive nephropathy in males or
females after 4 months or 10 months of continuous treatment.
McDorman et al. (2003 a) examined induction of transitional cell hyperplasia in the
urinary bladder of Eker rats continuously exposed to 0, 0.07 or 0.7 g/L bromodichloromethane for
10 months in the study described by Hooth et al. (2002). Changes in the urinary bladder were
examined to determine whether bromodichloromethane would contribute to effects on this organ
as one component of a mixture of disinfection byproducts. At necropsy, the bladder was
removed, fixed, and processed for histopathological evaluation. Midsagittal sections of fixed
bladder from male and female rats were used for preparation of slides. No evidence of urinary
bladder epithelial hyperplasia or individual cell hypertrophy was observed in male or female rats
exposed to bromodichloromethane.
2. Dibromochloromethane
a. Two-Year Oral Cancer Bioassays
NTP (1985) administered dibromochloromethane at doses of 0, 40, or 80 mg/kg-day (in
corn oil) to groups of 50 male and 50 female F344/N rats via gavage 5 times/week for 104 to 105
weeks. Details on the protocol of this study are provided in section V.D.2. Survival of dosed
male and female rats was comparable to that of the vehicle-control groups. High-dose males had
lower body weights when compared with the vehicle control. Compound-related nonneoplastic
lesions (fatty metamorphosis and ground-glass cytoplasmic changes) were found in the livers of
both sexes (See section V.D.2). Nephrosis was observed in female rats. No statistically
significant increase in the incidence of any neoplastic lesion was observed. Based on the results
of this study, the authors concluded that there was no evidence of carcinogenicity in rats
administered dibromochloromethane.
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NTP (1985) administered dibromochloromethane to groups of 50 male and 50 female
B6C3FJ mice via gavage in corn oil 5 times/week for 104 to 105 weeks. Details on the protocol
of this study are provided in section V.D.2. The administered doses were 0, 50, or 100 mg/kg-
day. Survival of female mice was comparable to that of the vehicle-control group. High-dose
male mice, however, had lower survival rates than the vehicle controls. At week 82, nine high-
dose male mice died of an unknown cause. An inadvertent overdose of dibromochloromethane
given to low-dose male and female mice at week 58 killed 35 male mice, but apparently did not
affect the females. The low-dose male mouse group was, therefore, considered to be unsuitable
for analysis of neoplasms. Compound-related nonneoplastic lesions were found primarily in the
livers of male mice (hepatocytomegaly, necrosis, fatty metamorphosis) and female mice (calcifi-
cation and fatty metamorphosis). Nephrosis was observed in male mice. In females, a
statistically significant increase in the incidence of hepatocellular adenomas and adenomas and
carcinomas combined was observed in the high-dose group. In male mice, a statistically
significant increase in the incidence of hepatocellular carcinomas and adenomas and carcinomas
combined was observed in the high-dose group. A summary of the incidence of these tumors is
presented in Table V-15. A negative trend in the incidence of malignant lymphomas was evident
in dibromochloromethane-exposed male mice when compared to the vehicle control. The study
authors concluded that the results of this study provided equivocal evidence of
dibromochloromethane carcinogenicity in male B6C3FJ mice and some evidence of
carcinogenicity in female B6C3FJ mice.
In other bioassays, Voronin et al. (1987) observed no significant tumor increases in
CBAxC57Bl/6 mice (50/sex/dose) treated with dibromochloromethane in the drinking water at
concentrations of 0, 0.04, 4.0, or 400 mg/L (approximately 0, 0.008, 0.76, or 76 mg/kg-day) for
104 weeks. In an unpublished report of a two-year dietary study, Tobe et al. (1982) reported no
increase in gross tumors in male rats dosed with up to 210 mg/kg-day or female rats treated with
up to 350 mg/kg-day.
Table V-15 Frequencies of Liver Tumors in B6C3Ft Mice Administered
Dibromochloromethane in Corn Oil for 105 Weeks - Adapted from NTP (1985)
Treatment
(mg/kg-day)
Vehicle Control
50
100
Sex
M
F
M
F
M
F
Adenoma
14/50
2/50
a
4/49
10/50
ll/50b
Carcinoma
10/50
4/50
6/49
19/50b
8/50
Adenoma or Carcinoma
(combined)
23/50
6/50
10/49
27/50c
19/50d
a Male low-dose group was inadequate for statistical analysis.
b p < 0.05 relative to controls.
0 p < 0.01 (life table analysis); p = 0.065 (incidental tumor test) relative to controls.
d p < 0.01 relative to controls.
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b. Studies of Induction of Aberrant Crypt Foci
De Angelo et al. (2002) evaluated induction ACF in the colons of male F344/N rats
exposed to dibromochloromethane in drinking water. Groups of weanling rats (6 animals/group)
were exposed to distilled water, 0.25% Alkamuls EL-620®, or 0.9 g/L dibromochloromethane in
0.25% Alkamuls EL-620 for 13 weeks. A single intraperitoneal injection of 30 mg/kg
azoxymethane (AOM) served as the positive control. Body weight and water consumption were
measured twice during the first week of the study and once per week thereafter. Colons were
collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal
segments, and examined for ACF.
The measured concentration of dibromochloromethane averaged 0.80 ± 0.05 g/L (mean
and standard error) over the course of the study. When adjusted for volatilization and adherence
to glass, the corrected concentration was 0.63 g/L. Water consumption was significantly reduced
(34%) in the dibromochloromethane exposure group when compared to the 0.25% vehicle
control. The average daily dose of dibromochloromethane was 60 mg/kg-day as calculated by the
study authors. Average terminal body weight of the rats exposed to dibromochloromethane was
within 10% of the control values. No ACF were observed in colons from control animals. ACF
were observed in three of six colons from dibromochloromethane-exposed animals. The total
number of ACF (17) and number of aberrant crypts per focus (2.43 ± 0.61) were significantly
increased relative to the combined deionized water and vehicle controls. Fourteen percent of the
observed ACF were located in the middle segment of the colon and 86% were located in the distal
(rectal) segment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per focus were observed in the
AOM positive control group. The total and average focal areas were 10,390 and 63.74 ± 3.06
|im2 respectively. Eight percent, 42% and 50% of the ACF induced by AOM were located in the
proximal, middle, and distal segment of the colon, respectively. The biological significance of
the observed increase in ACF in the colon of dosed rats is unclear, as treatment with
dibromochloromethane did not induce tumors in this site in two-year bioassays conducted in rats
or mice (Tobe et al., 1982; NTP, 1985; Voronin et al., 1987).
De Angelo et al. (2002) tested the ability of dibromochloromethane administered in
drinking water to induce ACF in the colons of male B6C3Fj (6 animals/group). Test animals
were given distilled water, 0.25% Alkamuls EL-620®, or a target concentration of 0.9 g/L
dibromochloromethane in 0.25% Alkamuls EL-620 for 13 weeks. Animals in the positive control
group received a single 50 mg/kg intraperitoneal injection of 4-aminobiphenyl. Body weight and
water consumption were measured twice during the first week of the study and once per week
thereafter. Colons were collected at study termination, fixed, stained with 0.2% methylene blue,
divided into three equal segments, and scanned for ACF. The study report did not provide results
for measured concentration of dibromochloromethane in drinking water solutions or an estimated
average daily dose. No differences were reported between the control and treatment group for
body weight or water and feed consumption (data not shown). Development of ACF was not
observed in the colons of mice exposed to dibromochloromethane in the drinking water or
injected with the positive control 4-aminobiphenyl.
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Geter et al. (2004c) investigated the effects of a diet containing a high amount of animal
fat on the induction of ACF in the colon of male F344/N rats exposed to dibromochloromethane.
The study was conducted because a high-fat diet is regarded as an important nutritional influence
on colon cancer development in humans. Twenty-eight-day-old male F344/N rats (6/treatment)
received 0.9 g/L (equivalent to 54-65 mg/kg-day) of dibromochloromethane in drinking water
containing 0.25% Emulphor® for 26 weeks. Animals in the negative control group received water
containing 0.25% Emulphor®. Animals in the normal and high fat-diet positive control groups
received a single 15 mg/kg intraperitoneal injection of AOM. All animals were fed a standard
laboratory diet (Purina 5001), with half receiving the normal feed containing 4.5% fat and half
receiving feed supplemented with 19% animal fat. Body weight and water consumption were
measured twice weekly for the first week and biweekly for the remainder of the experiment. At
sacrifice, the colon was removed from each animal and divided into proximal, medial, and distal
segments. ACF were identified by staining with 0.2% methylene blue.
Water consumption was significantly reduced in the positive control (normal diet only)
and dibromochloromethane (normal and high fat diets) treatment groups, but mean body weights
in these groups were similar to or greater than the controls. The incidences of ACF (i.e., number
of colons with ACF/number of colons scored) for the control, control + high fat,
dibromochloromethane, and dibromochloromethane + high fat groups were 0/6, 3/6, 5/6, and 5/6,
respectively. The incidence of ACF in the positive control groups was 6/6. Exposure to
dibromochloromethane resulted in significantly increased total numbers of ACF and ACF/colon
(normal diet only), increased total and mean focal area (normal and high fat diets), and total
number of aberrant crypts (normal and high fat diets). However, no significant differences were
noted in the number or characteristics of ACF between animals fed the normal or high-fat diets
and exposed to either AOM or dibromochloromethane. The ability of this study to detect
differences in induction of ACF related to diet may have been limited by the small sample size
and short duration of exposure.
3. Bromoform
a. Two-Year Oral Cancer Bioassays
NTP (1989a) exposed male and female F344/N rats (50/sex/dose) to bromoform doses of
0, 100, or 200 mg/kg-day via gavage in oil for 103 weeks (5 days/week). At study termination,
all animals were necropsied, and a thorough histological examination of tissues was performed.
Adenomatous polyps or adenocarcinomas of the large intestine were noted in three high-dose
male rats, eight high-dose female rats, and one low-dose female rat (Table V-16). Although the
number of tumors found was small, the incidence was considered to be significant because these
intestinal tumors are very rare in the rat. The NTP concluded that there was some evidence for
carcinogenic activity in male rats and clear evidence in female rats. Additional details of this
study are provided in Section V.D.3.
In a parallel study, NTP (1989a) exposed male B6C3Fj mice (50/dose) to bromoform via
gavage in corn oil at doses of 0, 50, or 100 mg/kg-day for 103 weeks (5 days/week). Female
V - 104 November 15, 2005
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mice (50 dose) received doses of 0, 100, or 200 mg/kg-day by the same protocol. At termination,
all animals underwent gross necropsy and thorough histological examinations of tissues. Survival
in both treated female groups was reduced; however, the authors attributed this reduction in
survival partly to utero-ovarian infection. A statistically significant increase in the incidence of
thyroid follicular cell hyperplasia was noted in high-dose females; however, there were no
statistically significant increases in the incidence of any neoplastic lesion in any dose group
compared to controls.
Table V-16 Tumor Frequencies in the Large Intestine of F344/N Rats Exposed to
Bromoform in Corn Oil for 2 Years - Adapted from NTP (1989a)
Tumor
Male rat
Adenocarcinoma
Polyp (adenomatous)
Female rat
Adenocarcinoma
Polyp (adenomatous)
Tumor Frequency
Control
0/50
0/50
Control
0/48
0/48
100 mg/kg
0/50
0/50
100 mg/kg
0/50
1/50
200 mg/kg
1/50
2/50
200 mg/kg
2/50
6/50
Based on the results of this study, the study authors concluded there was no evidence for
carcinogenicity of bromoform in male or female mice. Additional details of this study are
provided in Section V.D.3.
Kurokawa (1987) observed no evidence of carcinogenicity in male or female rats exposed
to microencapsulated bromoform at concentrations of 400, 1600, or 6500 ppm in the diet for 24
months.
b. Studies of Induction of Aberrant Crypt Foci
De Angelo et al. (2002) evaluated induction of ACF in the colons of male F344/N rats
exposed to bromoform in drinking water. Groups of weanling rats (6 animals/group) were
exposed to distilled water, 0.25% Alkamuls EL-620®, or a target concentration of 1.10 g/L
bromoform in 0.25% Alkamuls EL-620 for 13 weeks. A single intraperitoneal injection of 30
mg/kg azoxymethane (AOM) served as the positive control. Body weight and water consumption
were measured twice during the first week of the study and once per week thereafter. Colons
were collected at study termination, fixed, stained with 0.2% methylene blue, divided into three
equal segments, and examined for ACF.
The measured concentration of bromoform averaged 0.98 ± 0.08 g/L (mean and standard
error) over the course of the study. When adjusted for volatilization and adherence to glass, the
V-105
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corrected concentration was 0.77 g/L. Water consumption was significantly reduced (32%) in the
bromoform exposure group when compared to the vehicle control. The average daily dose of
bromoform was 76 mg/kg-day, as calculated by the study authors. Average terminal body
weights of the rats exposed to bromoform were within 10% of the vehicle control value. No ACF
were observed in colons from control animals. ACF were observed in 4/6 bromoform-exposed
animals. The number of ACF/colon (1.17 ± 0.40), total focal area (470 |im2), average focal area
(67.14 ± 8.57 |im2), total number of ACF (26), and number of aberrant crypts per focus (3.71 ±
0.36) were significantly increased relative to the combined deionized water and vehicle controls.
Fourteen percent of the observed ACF were located in the middle segment of the colon and 86%
were located in the distal (rectal) segment. In comparison, 807 ACF and 4.95 ± 0.25 crypts per
focus were observed in the AOM positive control group. The total and average focal areas were
10,390 and 63.74 ± 3.06 |im2 respectively. Eight percent, 42% and 50% of the ACF induced by
AOM were located in the proximal, middle, and distal segment of the colon, respectively.
De Angelo et al. (2002) tested the ability of bromoform administered in drinking water to
induce ACF in the colons of male B6C3Fj (6 animals/group). Test animals were given distilled
water, 0.25% Alkamuls EL-620®, or a target concentration of 1.10 g/L bromoform in 0.25%
Alkamuls EL-620 for 13 weeks. Animals in the positive control group received a single 50
mg/kg intraperitoneal injection of 4-aminobiphenyl. Body weight and water consumption were
measured twice during the first week of the study and once per week thereafter. Colons were
collected at study termination, fixed, stained with 0.2% methylene blue, divided into three equal
segments, and scanned for ACF. The study report did not provide results for the measured
concentration of bromoform in drinking water solutions or an estimated average daily dose. No
differences were reported between the control and treatment group for body weight or water and
feed consumption (data not shown). Development of ACF was not observed in the colons of
mice exposed to bromoform in the drinking water or injected with the positive control 4-
aminobiphenyl.
Geter et al. (2004c) investigated the effect of a diet containing a high amount of animal fat
on the induction of ACF in the colon of male F344/N rats exposed to bromoform. The study was
conducted because a high-fat diet is regarded as an important nutritional influence on colon
cancer development in humans. Twenty-eight-day-old male F344/N rats (6/treatment) received
1.0 g/L (equivalent to 71-73 mg/kg-day) of bromoform in drinking water containing 0.25%
Emulphor® for 26 weeks. Animals in the negative control group received water containing 0.25%
Emulphor®. Animals in the positive control group received a single 15 mg/kg intraperitoneal
injection of AOM. All animals were fed a standard laboratory diet (Purina 5001), with half
receiving the normal feed containing 4.5% fat and half receiving feed supplemented with 19%
animal fat. Body weight and water consumption were measured twice weekly for the first week
and biweekly for the remainder of the experiment. Additional details of treatment are provided in
the related publication by George et al. (2002). At sacrifice, the colon was removed from each
animal and divided into proximal, medial, and distal segments. ACF were identified by staining
with 0.2% methylene blue.
V - 106 November 15, 2005
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Water consumption was significantly reduced in the positive control (normal diet only)
and bromoform (normal and high-fat diets) treatment groups, but mean body weights in these
groups were similar to or greater than the controls. The incidences of ACF (i.e., number of
colons with ACF/number of colons scored) for the control, control + high fat, bromoform, and
bromoform + high fat groups were 0/6, 3/6, 6/6, and 6/6, respectively. The incidence of ACF in
the positive control groups was 6/6. Exposure to bromoform significantly increased the total
number of ACF, ACF/colon, total and mean focal area, and total number of aberrant crypts in
animals receiving the normal diet or high-fat diets relative to the respective control groups.
Concurrent exposure to the high-fat diet and bromoform resulted in a statistically significant
increase in the number of ACF/colon (normal diet: 2.83 ± 1.05, high-fat diet 5.33 ± 1.17).
Geter et al. (2005) investigated the effect of folate deficiency on the bromoform-induced
formation of aberrant crypt foci (ACF) in the colon of the male F344 rat. Male F344 rats(28 days
old) were give bromoform in the drinking water at a concentration of 500 mg/L for 26 weeks. A
single i.p. dose of azoxymethane was used as a positive control. Two groups of rats (control and
bromoform exposed) were fed an amino acid deficient diet containing either 0 or 2 mg folic
acid/kg. Food and water were given ad libitum. Water consumption was measured weekly
throughout the study and body weights were determined weekly through week 16 and then
biweekly until the end of the study. After 26 weeks of exposure, rats were sacrificed and colons
were removed and prepared for analysis of ACF. For each ACF found, the size, location and
number of individual crypts within the focus was noted. Blood samples were analyzed for serum
folate and homocysteine levels.
Water consumption and weight gain were not affected by bromoform administration in
either control or folate-deficient rats. Bromoform dose levels were calculated to be
approximately 96 mg/kg-day for rats given the standard diet and 99 mg/kg-day for rats given the
folate deficient diet. Folate deficient rats experienced a decrease in serum folate levels and an
increase in serum homocysteine levels. Bromoform administration in drinking water increased
the number of ACF and aberrant crypts in rats given a standard diet and in rats fed a diet deficient
in folic acid, when compared to controls. For bromoform-exposed rats, folate deficiency resulted
in a larger increase in ACF formation, when compared to rats given the standard diet.
Azoxymethane also induced the formation of ACF; however folate deficiency reduced the
formation of ACF for rats exposed to this chemical. The results of this study suggest that dietary
factors may play a modulatory role in the bromoform-induced formation of ACF in the colon of
male F344 rats.
c. Cancer and Cancer-Related Studies in Cancer-Susceptible Rodent Strains
Theiss et al. (1977) examined the carcinogenic activity of bromoform in Strain A mice.
Twenty mice per group (6 to 8 weeks old) were injected intraperitoneally up to three times
weekly over a period of 8 weeks with 4, 48, or 100 mg/kg bromoform. A positive and a negative
control group were included in the study design and each contained 20 animals. Mice were sacri-
ficed 24 weeks after the first injection and the frequency of lung tumors in each test group was
compared with vehicle-treated controls. Bromoform produced a significant increase (p = 0.041)
V - 107 November 15, 2005
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in tumor frequency only at the intermediate dose; this increase was considered indicative of a
carcinogenic response by the study authors.
H. Other Key Health Effects
1. Immunotoxicity
a. Bromodichloromethane
Munson et al. (1982) administered bromodichloromethane by gavage to CD-I male and
female mice (8-12/sex/dose) for 14 days at levels of 0, 50, 125, or 250 mg/kg-day.
Bromodichloromethane appeared to affect the humoral immune system, as judged by decreased
antibody-forming (ABF) cells in serum and by decreased hemagglutination liters. These changes
were clearly significant (p < 0.05) at the high dose in both males and females, and decreased ABF
cells were also noted at the mid dose (125 mg/kg-day) in females. This study identified a
NOAEL of 50 mg/kg-day and a LOAEL of 125 mg/kg-day for bromodichloromethane on the
basis of decreased immune function in females. Additional information on other endpoints
measured in this study is provided in Section V.B.I.
French et al. (1999) investigated the immunotoxicity of bromodichloromethane in a series
of four experiments conducted in mice and rats. Immunotoxicity in mice was examined
following exposure via ingestion of drinking water or by gavage. The immunological parameters
examined were antibody response to injected sheep red blood cells and T and B lymphocyte
proliferation. Mitogens used in the proliferation assay were concanavalin A (Con A) or phyto-
hemagglutinin-p (PHA) for T cells and lipopolysaccharide (LPS) for B cells. Female C57BL/6
mice (6 animals per group) were treated for 14 or 28 days with drinking water containing 0, 0.05,
0.25 or 0.5 g/L bromodichloromethane. All drinking water (including controls) contained 0.25%
Emulphor® to reduce volatilization of bromodichloromethane. Based on measured water
consumption, these concentrations were estimated by the authors to be equivalent to 0, 10, 37 or
62 mg/kg-day. There were no significant differences in the number of antibody forming cells,
antibody production, or spleen weights in any treatment group. Likewise, splenic and mesenteric
lymph node cell proliferative responses to T and B cell mitogens were similar in all groups.
Continuation of this study for an additional 2 weeks did not affect any measured parameter.
These data identify a NOAEL of 62 mg/kg-day for short-term exposure.
French et al. (1999) conducted a second experiment in which female C57BL/6 mice were
dosed by gavage with bromodichloromethane in 10% Emulphor® once a day for 16 days.
Treatment groups (6 animals per group) included controls (deionized water or 10% Emulphor®),
50, 125 or 250 mg/kg-day bromodichloromethane. As in the previous experiment, there were no
differences in ABF cells, antibody liters or mitogen-induced proliferation in any treatment
groups. A decrease in spleen weight and spleen-to-weight ratio was observed in the 125 mg/kg-
day group when compared to the Emulphor® control. However, spleen weights in the Emulphor®
control were significantly higher than those in the deionized water control group, making this
finding difficult to interpret.
V - 108 November 15, 2005
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French et al. (1999) investigated the immunotoxicity of bromodichloromethane in male
Fisher 344 rats following two different in vivo exposure regimens: ingestion of drinking water
containing bromodichloromethane and gavage. The immunological parameters examined were
antibody response to injected sheep red blood cells and T and B lymphocyte proliferation. The
mitogens used in the proliferation assay were concanavalin A (Con A) or phyto-hemagglutin-p
(PHA) for T cells and S. typhimurium mitogen (STM) for B cells. Six rats per treatment group
were exposed for 26 weeks to drinking water containing 0, 0.07 or 0.7 g/L bromodichloro-
methane and 0.25% Emulphor®. Based on water consumption measurements, these
concentrations were estimated by the authors to be equivalent to average daily doses of 0, 5 or 49
mg/kg-day. There was a significant suppression of Con A-stimulated proliferation of spleen cells
observed in the 49 mg/kg-day dose group. No effect on other immunological parameters was
reported. These data suggest NOAEL and LOAEL values of 5 and 49 mg/kg-day, respectively,
for immunotoxic effects.
French et al. (1999) also examined the effect of short-term exposure to relatively large
doses of bromodichloromethane on immune function. Female F344 rats (6 animals/group)
received gavage doses of deionized water, 10% Emulphor®, or 75, 150, or 300 mg
bromodichloromethane/kg in 10% Emulphor® for 5 days. Surviving high-dose animals had
decreased body, spleen, and thymus weights. Con A and PHA responses were depressed in
spleen cells isolated from high-dose animals. Two of the six rats in the 300 mg/kg-day group
died during the exposure period. The remaining high-dose animals had significantly decreased
body, spleen and thymus weights compared to both control groups. Thymus weight, but not
spleen or body weight, was also decreased in the 150 mg/kg-day group. Con A responses were
significantly depressed in both spleen and mesenteric lymph node (MLN) cells in the 300 mg/kg-
day treatment group. All three (75, 150 and 300 mg/kg-day) dose groups exhibited suppression
of PHA stimulated MLN cells when compared to the vehicle (but not the water) controls. This
discrepancy was due to the fact that Emulphor® alone significantly elevated the proliferative
response to PHA in MLN cells relative to the deionized water group. In contrast to the T cell
responses, there was a significant increase in antibody production and proliferative responses to
STM (B cells) from spleen cells at the highest dose tested (300 mg/kg-day dose group). These
data suggest a marginal NOAEL of 150 mg/kg-day and a LOAEL of 300 mg/kg-day for acute
exposure based on depression of immune response.
b. Dibromochloromethane
Munson et al. (1982) administered dibromochloromethane by gavage to CD-I male and
female mice (8 to 12/sex/dose) for 14 days at levels of 0, 50, 125, or 250 mg/kg-day and
evaluated humoral and cell-mediated immune system functions. Dibromochloromethane
appeared to affect the humoral immune system, as judged by decreased antibody-forming (ABF)
cells in serum and by decreased hemagglutination liters. These changes were significant
(p < 0.05) at the high dose in both males and females. Decreased ABF cells were also noted at the
mid dose (125 mg/kg-day) in females. This study identified a NOAEL of 50 mg/kg-day and a
LOAEL of 125 mg/kg-day for dibromochloromethane on the basis of decreased immune function
in females. Additional information on this study is provided in Section V.B.2.
V - 109 November 15, 2005
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c. Bromoform
Munson et al. (1982) administered bromoform (aqueous) by gavage to CD-I male and
female mice (6 to 12/sex/dose) for 14 days at levels of 0, 50, 125, or 250 mg/kg-day. Endpoints
evaluated included humoral immune system function. The authors judged that the humoral
immune system was not significantly affected by bromoform, although a decrease in antibody
forming (ABF) cells was reported for high-dose males. These data suggest a NOAEL of 250
mg/kg-day for effects of bromoform on the immune system. Additional information on this study
is provided in Section V.B.3.
2. Hormonal Disruption
No studies or case reports were identified that described hormonal disruption by
dibromochloromethane or bromoform.
a. Bromodichloromethane - In Vivo Studies
Oral exposure to 50 - 100 mg/kg-day of bromodichloromethane causes full litter
resorption (FLR) in F344 rats, but not in Sprague-Dawley rats (Narotsky et al., 1997; Bielmeier et
al. 2001; see Section V.E.I.a for study details). Bielmeier et al. (2001) characterized luteinizing
hormone (LH) and progesterone serum profiles in F344 rats exposed to bromodichloromethane
during gestation in two experiments (Table V-17) as part of a larger study on FLR. The objective
of these experiments was to determine whether changes in the LH and progesterone profiles were
associated with the occurrence of FLR. These hormones were selected for study because
progesterone is necessary for the maintenance of pregnancy and LH participates in the
maintenance of the corpora lutea which secrete progesterone.
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Table V-17 Summary of Hormone Profile Experiments (Bielmeier et al., 2001)
Study/Strain
Dose
(mg/kg-day)
Treatment
Period
Number of animals
Treated
Pregnant
Resorbed
% FLR
Hormone Profile P
F344
F344
F344
0
100
100
GD8-9
GD8
GD9
8
10
10
7
10
9
0
6
9
0
60*
100***
Hormone Profile IIb
F344
F344
F344
0
75
100
GD9
GD9
GD9
8
11
10
8
11
10
0
7
9
0
64*
90***
Source: Table 1 in Bielmeier et al. (2001)
Abbreviations: GD, gestation day; FLR, full litter resorption
a Tail blood collected once daily on GD 9 to 12.
b Tail blood collected at 0, 6, 12, and 24 hours after dosing.
* p<0.05; ** pO.Ol; *** pO.OOlfor significant differences from controls (Fisher's Exact Test).
In the first experiment conducted by Bielmeier et al. (2001), F344 rats (7 to 10/treatment
group) received a single 100 mg/kg dose by aqueous gavage on gestation day 8 or 9. Hormone
levels in samples of tail blood were determined on GD 9 through 12. FLR was observed in 0, 60
and 100% of the control, GD 8-dosed, and GD 9-dosed animals, respectively. A marked
reduction in progesterone levels was noted 24 hours after dosing in all rats that resorbed their
litters when compared to controls and to bromodichloromethane-treated animals that retained
their litters. The mean progesterone levels in animals dosed on GD 9 decreased from 137.94
ng/mL ± 11.44 ng/mL to 48.45 ± 23.57 ng/mL within 24 hours (n = 9). For animals treated on
GD 8, the mean progesterone level 24 hours after bromodichloromethane treatment was 67.01 ±
16.22 ng/mL in animals that resorbed litters (n = 6) and 127.19 ±14.89 in controls (n=7). The
resorbed groups had reduced progesterone levels comparable to the progesterone levels in non-
pregnant animals (n = 2) when assayed three days after compound administration. In contrast to
the effect noted on progesterone levels, administration of bromodichloromethane had no apparent
effect on LH when measured 24 hours after dosing. However, elevated LH concentrations were
observed on GD 11 to 12 in animals experiencing resorption. LH levels in these groups increased
from approximately 0.20 ng/mL on GD 10 to approximately 0.80 ng/mL on GD 11 and remained
elevated through GD 12. In contrast, LH levels in the controls decreased from 0.31 to 0.14
ng/mL over the same time period.
In the second experiment conducted by Bielmeier et al. (2001), F344 rats (8-11/treatment
group) were dosed with 0, 75, or 100 mg/kg by aqueous gavage on GD 9. Blood samples were
V-lll
November 15, 2005
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collected at 0, 6, 12, and 24 hours after dosing. The incidence of FLR was 0, 64%, and 90% in
the 0, 75, and 100 mg/kg dose groups, respectively. The progesterone levels peaked in all dose
groups (including controls) at 6 hours. At 12 and 24 hours, the progesterone levels in
bromodichloromethane-treated animals that resorbed their litters were progressively reduced.
Progesterone levels in bromodichloromethane-treated animals that retained their litters remained
comparable to levels observed in the control group. No significant differences in LH
concentration were noted among dose groups at any time point.
In a follow-on study, Bielmeier et al. (2004) conducted additional experiments to
investigate the mode of action for bromodichloromethane-induced pregnancy loss observed in the
F344 rat (Narotsky et al., 1997; Bielmeier et al., 2001). The experiments conducted by Bielmeier
et al. (2004) were designed to 1) re-examine maternal LH profiles during exposure to levels of
bromodichloromethane known to cause pregnancy loss, using a more sensitive assay for LH than
used by Bielmeier et al. (2001); 2) assess the temporal pattern of serum LH and progesterone
decreases that occur during bromodichloromethane administration at a critical time period during
gestation; and 3) test the ability of exogenously administered progesterone and hCG to prevent
pregnancy loss induced by bromodichloromethane. The experiments performed are summarized
in Table V-18.
In the first experiment, F344 rats received a single dose of 0, 75, or 100 mg/kg
bromodichloromethane via oral gavage in 10% Alkamuls EL-620 in the morning of assigned
treatment days. Maternal body weights were measured on GD 5-15 and GD 20. Pups were
individually examined and weighed on PD 1 and 6. PD 1 was defined as GD 22, independent of
the actual time of parturition, so that all pups were examined at the same time postcoitus. The
dams and pups were euthanized after PD 6. The number of uterine implantation sites was
recorded; the uteri of females that did not deliver were stained with 2% ammonium sulfide to
enhance detection of resorption sites. Blood samples for hormone analysis were collected once
daily on GD 6-11 from rats in the control and 75 mg/kg groups.
Two additional experiments using the same treatment regimen, but with more frequent
blood collection, were conducted to further characterize the changes in hormone levels on GD 9
and 10. In experiment A, tail blood samples were collected on GD 9 once before dosing and then
1.5, 3, 4.5, and 6 hours after treatment. Experiment B continued the collection schedule from
experiment A. The first blood samples were collected approximately 8 hours after the dose on
GD 9 and four additional samples were collected at 4-hour intervals. This study design was
utilized to minimize the impacts of repeated blood collection on a single animal. Progesterone
and estradiol concentrations were measured using a direct solid-phase enzyme-linked
immunosorbent assay. Serum LH levels were determined using the rat dissociation enhanced
lanthanide fluorometric immunoassay (DELPHIA). This method is reported to be 10- to 50-fold
more sensitive for detection of LH than the traditional radiolabeled immunoassay used by
Bielmeier et al. (2001), with a limit of detection of 0.014 |ig for a 25 |j,L sample. In the hormone
replacement experiments, bromodichloromethane (100 mg/kg-day by gavage) and progesterone
(10 mg/kg, twice daily by subcutaneous injection in corn oil) were administered on GD 6-10. A
V-112 November 15, 2005
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Table V-18 Summary of Bielmeier et al. (2004) Study in Female F344 Rats
Experiment
Dose
(mg/kg-day)
Number of Animals
Treated
Pregnant
Fully
Resorbed
With Live
Litters
Hormone Profiles
Daily Sampling
Frequent Sampling A
Frequent Sampling B
0
75
0
75
0
75
12
13
6
11
5
10
9
9
6
10
5
9
0
g***
1
8*
0
3
9
1
5
2
5
6
Hormone Supplementation
Control
BDCM + hormone vehicles
BDCM + Progesterone
BDCM + hCG
0
100
100
100
12
9
12
12
8
7
8
9
0
C###
0
1
8
2
8
8
Source: Bielmeier et al. (2004)
* significantly different from control, p<0.05; *** significantly different from control, p<0.001
Abbreviations: BDCM, bromodichloromethane; hCG, human chorionic gonadotropin
second group of animals received bromodichloromethane (100 mg/kg-day by gavage) and hCG
(0.5 lU/rat by subcutaneous injection in saline) on GD 8-10. Control groups received the gavage
and injection vehicles via the same route as the experimental groups.
In the daily sampling experiment, bromodichloromethane-induced pregnancy loss was
associated with marked reductions in serum progesterone and LH on GD 10. All control dams
maintained their litters, whereas 8/9 dams exposed to bromodichloromethane had pregnancy loss.
Serum progesterone levels in the control group were greater than 100 ng/mL throughout the
study. Serum LH levels in control dams generally ranged from 0.11 to 0.34 ng/mL through GD 9,
then fell to mean ± SE values of 0.061 ± 0.03 ng/mL by GD 11. Progesterone levels in dams
treated with bromodichloromethane that lost their litters were comparable to those of the controls
on GD 6-9. However, these dams had significantly reduced serum progesterone levels on GD10
when compared to the control and all measured concentrations were less than 40 ng/mL. Serum
LH levels in treated dams were less than the controls from GD 7 to GD 10, but the response was
statistically significant only on GD 7 and 10. The daily sampling protocol used in this experiment
did not distinguish which hormone was affected first. No differences between groups were
V-113
November 15, 2005
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reported for serum estradiol levels, number of implantations, postnatal loss, or number or weight
of live pups on PND 1 or 6.
In frequent sampling experiment A, 8/10 (80%) of the dosed animals had pregnancy loss
compared to 1/6 (17%) in the control group. The control animal with pregnancy loss maintained
LH levels that were consistent with the other control dams; progesterone levels were also
comparable until a sharp decline was observed in the animal with pregnancy loss on GD 10. In
contrast to typical bromodichloromethane-induced resorptions, the resorption sites in the control
animals were not visible without staining (a possible indication of earlier resorption). In frequent
sampling experiment B, 3/9 (33%) of the dosed animals had pregnancy loss compared to 0/5 (0%)
of the controls. In both experiments, animals that resorbed their litters displayed a decrease in
serum LH concentration (relative to the controls) prior to a reduction in progesterone levels. In
experiment A, serum LH levels were already significantly reduced in dosed animals in the first
samples collected on GD 9, but serum progesterone levels were not significantly reduced until
two hours after administration of the GD 9 dose of bromodichloromethane. In experiment B, the
six bromodichloromethane-treated animals that retained their litters also had significantly
decreased levels of LH and progesterone. No differences between groups were reported for
number of implantations, postnatal loss, or number or weight of live pups on PND 1 or 6 in either
experiment.
In the hormone supplementation experiments, administration of either progesterone or
hCG significantly reduced the incidence of bromodichloromethane-induced pregnancy loss. Five
of seven dams (71%) treated with 100 mg/kg-day of bromodichloromethane plus the hormone
vehicles (corn oil or saline injected subcutaneously) on GD 6-10 lost their pregnancies. In
contrast, dams dosed with 100 mg/kg-day of bromodichloromethane on GD 6-10 and
concurrently given progesterone had a 0/8 (0%) incidence of pregnancy loss. Dams dosed with
100 mg/kg-day of bromodichloromethane on GD 8-10 and concurrently given hCG by injection
had a 1/9 (11%) incidence of pregnancy loss.
The study authors had previously hypothesized that bromodichloromethane induces
pregnancy loss in F344 rats by altering luteal responsiveness to LH (Bielmeier et al., 2001). The
reduction in serum LH level with a corresponding reduction in progesterone concentration
detected in the current study suggests that bromodichloromethane alters LH secretion rather than
altering luteal responsiveness alone. The timing of the decreases in LH and progesterone levels
support the hypothesis that reduction in serum LH is a prerequisite for decreased progesterone
concentration, which subsequently results in pregnancy loss. However, other data obtained by
Bielmeier et al. (2004) indicate that a significant decrease in serum LH concentration cannot be
the sole determinant in pregnancy loss. Specifically, exposure to bromodichloromethane caused
decreases in serum LH levels in all dosed animals, but some dosed rats maintained their
pregnancies despite serum LH levels that were lower than observed in some animals that lost their
pregnancies. In discussing these results, the study authors noted that LH is released in an hourly
pulsatile pattern during pregnancy and that the severity and disruption of this pulsatility may be a
better predictor of pregnancy loss than single daily values of serum LH.
V-114 November 15, 2005
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Administration of exogenous progesterone or hCG reduced the incidence of
bromodichloromethane-induced pregnancy loss. Prevention of pregnancy loss by exogenous
progesterone supports the conclusion that the mode of action for bromodichloromethane is
maternally mediated rather than the result of direct effects on the embryo. The ability of hCG, an
LH agonist, to prevent pregnancy loss suggests that full litter resorption is mediated (at least in
part) by an effect of bromodichloromethane on maternal LH secretion. These data do not rule out
a possible effect of bromodichloromethane on luteal responsiveness to progesterone as previously
suggested by Bielmeier et al. (2001).
b. Bromodichloromethane -In Vitro Studies
Chen et al. (2003) studied the effect of bromodichloromethane on chorionic gonadotropin
(CG) secretion by human placental trophoblast cultures. This in vitro model was used to evaluate
possible effects of bromodichloromethane on the placenta. Cytotrophoblast cells were isolated
from term human placentas, plated in 24-well cluster dishes or chamber slides, and stimulated to
differentiate and produce syncytiotrophoblast-like colonies by culture for 48 hours in
Keratinocyte Growth Medium (KGM) containing 10% calf serum. The culture medium was
removed and replaced with KGM containing nominal concentrations of 0, 0.020, 20, or 2000 |j,M
bromodichloromethane (actual concentrations were not measured). The culture containers were
sealed and incubated for an additional 24 hours, after which levels of immunoreactive and
bioactive chorionic gonadotropin were determined in the culture medium. Replicate cultures
were processed for morphological evaluation or determination of lactate dehydrogenase.
Cultured adherent cells were fixed in methanol and stained for immunocytochemical
determination of CG.
Exposure to bromodichloromethane caused a significant dose-dependent decrease in the
secretion of immunoreactive and bioactive CG. Decreased CG secretion was observed at each
concentration tested, with maximum reductions of 37% (immunoreactive CG) and53%
(bioreactive CG) at the highest concentration of 2000 |j,M. This lowest concentration of 0.020
|iM is approximately 100-fold greater than maximum baseline blood levels and 35-fold higher
than the maximum peak level of bromodichloromethane measured by Miles et al. (2002) in the
blood of human subjects after showering. An effect on intracellular CG levels in
syncytiotrophoblast cultures was not detected by immunocytochemical staining. According to the
study authors, the failure to detect intracellular changes might have been related to use of
different antibodies for detection of extracellular and intracellular CG, resolution of the image
analysis procedure, or the fact that the effect on CG secretion was modest. There was no effect of
bromodichloromethane on the ratio of bioactive to immunoreactive CG, cellular protein, levels of
LDH in culture supernatants, or morphological features of the trophoblast cultures.
The results from this study suggest that bromodichloromethane affects the function of
human placental trophoblasts, as shown by reduced CG secretion in primary cultures after 24
hours of exposure to the compound. The mode of action for the effect on CG secretion is
unknown. Possible mechanisms proposed by the study authors include disruption of CG
synthesis at the translational or post-translational level (e.g., by altering glycosylation of CG
V-115 November 15, 2005
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subunits or disruption of dimerization) or indirect effects on secretion via disruption of
gonadotropin releasing hormone activity.
Chen et al. (2004) evaluated the effect of bromodichloromethane on the morphological
differentiation of human mononucleated cytotrophoblast cells to multinucleated
syncytiotrophoblast-like colonies. The objective of this study was to evaluate the mechanism of
reduced CG secretion in human placental trophoblast cultures observed in the previous study
(Chen et al., 2003). Addition of 20 to 2000 |j,M bromodichloromethane to primary
cytotrophoblast cultures during the differentiation process inhibited the subsequent formation of
multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical
staining for desmosomes and nuclei. Quantitative image analysis indicated that the number of
multinucleated colonies was significantly reduced at bromodichloromethane concentrations of
200 |iM and above, with a reduction of 80% observed at the highest tested concentration of 2000
|j,M. Secretion of immunoreactive and bioreactive CG was significantly reduced in a dose-
dependent manner under the same culture conditions, but showed a different dose-response
pattern than observed for differentiation (i.e., was effected at lower concentrations). Secretion of
immunoreactive CG was significantly reduced (by 30%) at 5 x 10"4 |j,M, the lowest dose tested.
This concentration is within the range of bromodichloromethane concentration observed in
human blood following showering with disinfected tap water (1.3 xlO"6 to 5.7 x 10"4 |j,M).
Secretion of immunoreactive and bioreactive CG was near completeat 2000 |j,M (90% reduction
for immunoreactive CG, 95% reduction for bioreactive CG) Intracellular levels of CG were
significantly reduced in a dose-dependent manner at concentrations of 20 |j,M and above, as
determined by quantitative immunocytochemical staining. This lack of CG accumulation within
the trophoblast, suggests that secretion is blocked at an earlier stage (i.e., transcription or
translatin) rather than at later stages (i.e., exocytosis). Trophoblast viability was unaffected by
bromodichloromethane at the concentrations tested in this study, as determined by cellular protein
levels and by LDH activity in culture supernatants. These findings support the idea that
bromodichloromethane affects the placenta and reduces CG production by preventing formation
of syncytiotrophoblasts, the major CG-producing cell type. The differences in the dose-response
curves observed for CG secretion and differentiation may indicate a dual effect of
bromodichloromethane on these processes. The mechanisms of action for the observed effects of
bromodichloromethane are unknown. The observation of reduced intracellular CG suggests
disruption of production at an earlier (e.g., transcription or translation) rather than a later (e.g.,
exocytosis) stage of CG production. Other possible mechanisms include an effect of
bromodichloromethane on post-translational processing (glycosylation or subunit dimerization) or
interference with GnRH activity. An effect on GnRH activity, if substantiated, would parallel one
proposed mechanism for BDCM-induced pregnancy loss in F344 rats (Bielmeier et al., 2004).
The significance of the findings reported by Chen et al. (2003, 2004) for human health is
that placental trophoblasts are the sole source of CG during normal human pregnancy and play a
major role in the maintenance of the conceptus. If the observed effect on CG secretion is
substantiated in future studies, it may help to explain adverse pregnancy outcomes that appear to
be associated with consumption of chlorinated drinking water in some epidemiological studies
(e.g., increased incidence of spontaneous abortion as reported by Waller et al., 1998).
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3. Structure-Activity Relationships
Although the mechanism of brominated trihalomethane toxicity is not known with
certainty, there is abundant evidence to indicate that adverse effects are secondary to metabolism.
Bromine is generally a better leaving group than chlorine, suggesting that bromine substitution
could potentially influence the pathway and rate of trihalomethane metabolism. Multiple studies
(described in Section III.C) indicate that metabolism of chloroform and the brominated
trihalomethanes can occur through one or both of two cytochrome P450-mediated pathways:
reductive metabolism to free radical intermediates or oxidative metabolism to dihalocarbonyls
(Figure 4-1). Although comparative data are limited, there is some evidence to indicate that
chloroform and the brominated trihalomethanes are metabolized to a different extent by these
pathways. Tomasi et al. (1985) examined the reductive metabolism of chloroform,
bromodichloromethane, and bromoform in rats and obtained the following rank order for free
radical formation: bromoform>bromodichloromethane>chloroform. Wolf et al. (1977) reported
that bromoform was more extensively metabolized under anaerobic conditions in vitro than was
chloroform. Gao and Pegram (1992) observed that binding of reactive intermediates to rat
hepatic microsomal lipids and proteins was more than twice as high for bromodichloromethane as
for chloroform when assayed under anaerobic conditions. These results collectively suggest that
reductive metabolism may be a more important metabolic pathway for brominated
trihalomethanes than for chloroform. At present, this apparent difference in metabolism has not
been linked to specific differences in toxicity.
Two mutagenicity studies provide additional information on structure-activity
relationships among the trihalomethanes. Additional details of these studies are presented in
Section V.F. Examination of mutagenicity in a strain of Salmonella typhimurium that expresses
rat theta-class glutathione-S-transferase (GST) indicated the following order for mutagenic
potency (number of revertants/ppm) of the brominated trihalomethanes:
bromoform=dibromochloromethane>bromodichloromethane (DeMarini et al., 1997). The
potency of the first two compounds was several times greater than that observed for
bromodichloromethane. Analysis of the mutational spectra of the brominated trihalomethanes
indicated that all three compounds have similar mutational spectra (predominately GC~>AT
transitions) and site specificity (middle C of a CCC sequence in target DNA). These observations
suggest that a common reactive intermediate or class of intermediates is likely to mediate the
mutagenicity of these compounds.
In the second study, Pegram et al.(1997) compared the glutathi one ^-transferase-mediated
mutagenicity of bromodichloromethane and chloroform in a GST+ strain of S. typhimurium (See
section V.F. 1). Revertants were produced in a dose-related manner in the presence of low as well
as high concentrations of bromodichloromethane. In contrast, chloroform induced a doubling of
the number of revertants only at high concentrations. This result provides evidence that bromine
substitution of trihalomethanes confers the capability for GST-catalyzed transformation to
mutagenic intermediates at low substrate concentrations. These data further suggest that
chloroform and the brominated trihalomethanes may induce adverse effects via different modes
V-117 November 15, 2005
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of action, and indicate the need for care in extrapolating the characteristics of chloroform
metabolism and toxicity to brominated trihalomethanes.
I. Summary
1. Health Effects of Acute and Short Term Exposure of Animals
Large oral doses of brominated trihalomethanes are lethal to laboratory animals. Reported
acute LD50 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 lethality values are
summarized in Table V-l.
Acute oral exposure to sublethal doses of brominated trihalomethanes can also produce
effects on the central nervous system, liver, kidney, and heart. Acute duration studies
investigating endpoints other than death are summarized in Table V-2. 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 studies of brominated trihalomethanes are summarized in Table V-3.
Short-term 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 or dibromochloromethane doses of 125 mg/kg-day. Studies examining
strain differences in response to short-term brominated trihalomethane exposure have not been
reported.
2. Health Effects of Longer-term Exposure of Animals
Subchronic studies of brominated trihalomethanes are summarized in Table V-4. 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
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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 dibromochloro-
methane, 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/kg-
day dibromochloromethane. Studies examining strain differences in response to subchronic
brominated trihalomethane exposure have not been reported.
Chronic toxicity studies of brominated trihalomethanes are summarized in Table V-5. As
observed for exposure for shorter durations, 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. Studies examining strain differences in response to
chronic brominated trihalomethane exposure have not been reported.
3. Reproductive and Developmental Effects
Reproductive and developmental studies of brominated trihalomethanes are summarized
in Table V-9. 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 dibromo-
chloromethane 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/kg-day 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 rats administered 45 mg/kg-day in the drinking water on
gestation days 6 to 2land sternebral aberrations were observed in the offspring of 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. Additional studies in F344 rats that varied the timing
of bromodichloromethane administration indicate that gestation days 6-10 are a critical period for
induction of full litter resorption. Chronic oral exposure 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
Fj male and female rats at 150 ppm (approximately 11.6 to 40.2 mg/kg-day) in a two generation
study of bromodichloromethane administered in drinking water. In the same study, small but
statistically significant delays in sexual maturation occurred in Fx males at 50 ppm
V-119 November 15, 2005
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(approximately 11.6 to 40.2 mg/kg-day) and Fx 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 dibromochloro-
methane 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 body weight on postnatal day 14 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 Ft and/or F2 generation. The developmental and reproductive
toxicity of bromoform was examined in two studies.
Bromoform administered to 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 in
decreased postnatal survival, organ weight changes, and liver histopathology in Fx mice of both
sexes. No effects on fertility or other reproductive endpoints were noted.
4. Mutagenicity and Genotoxicity
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 variable. Recent mutagenicity studies in strains of Salmonella that
contain rat theta-class glutathione S-transferase suggest that mutagenicity of the brominated
trihalomethanes may be mediated by glutathione conjugation.
5. Carcinogenicity and Related Studies in Animals
The carcinogenic potential of individual brominated trihalomethanes administered in oil
has been investigated in chronic oral exposure studies in mice and rats. Ingestion of
bromodichloromethane caused 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. Ingestion of
dibromochloromethane caused liver tumors in male and female mice, and ingestion of bromoform
caused intestinal tumors in male and female rats.
Studies of induction of aberrant crypt foci (ACF) show that bromodichloromethane,
dibromochloromethane, and bromoform given in drinking water significantly increase the number
and focal area of ACF in the colons of male F344 rats, Eker rats, and strain A/J mice, but not in
colons of B6C3FJ mice. The biological significance of this induction is unclear, as intestinal
V - 120 November 15, 2005
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tumors have not been observed either in the colons of F344 rats treated with
dibromochloromethane by corn oil gavage or in the colons of rats exposed to
bromodichloromethane in the drinking water for two years. Administration of individual
brominated trihalomethanes in a high animal fat diet did not significantly increase the number of
ACF when compared to a diet containing normal levels of fat.
Exposure of male and female Eker rats (a rodent hereditary model of renal cancer) to
bromodichloromethane at drinking water concentrations up to 0.7 g/L did not significantly induce
urinary bladder epithelial hyperplasia, individual cell hypertrophy, renal tumors, hemangioma of
the spleen, or leiomyomas or mesenchymal cell proliferation in the uterus of females.
6. Other Key effects
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 liters, and/or suppression of Con A-stimulated proliferation of
spleen cells at doses of 125 to 250 mg/kg-day.
No studies have been reported for hormonal effects following exposure to
dibromochloromethane or bromoform. There is evidence from studies in F344 rats and cultured
human placental trophoblasts that bromodichloromethane causes hormonal disruption. Rats
exposed to bromodichloromethane on gestation days 8 or 9 show reduced serum levels of LH and
progesterone. Serum LH reductions indicate that the mode of action for this strain-specific effect
involves altered LH secretion; however, a contributing effect on LH signal transduction has not
been ruled out.
Exposure to bromodichloromethane alters the function of human placental trophoblasts, as
shown by reduced CG secretion and by changes in morphological differentiation. The mode of
action for the observed effects is unknown. Possible mechanisms proposed by the study authors
for effects on CG secretion include disruption of CG synthesis at the translational or post-
translational level (e.g., by altering glycosylation of CG subunits or disruption of dimerization) or
indirect effects on secretion via disruption of gonadotropin releasing hormone activity. The
significance of these findings for human health is that placental trophoblasts are the sole source of
CG during normal human pregnancy and play a major role in the maintenance of the conceptus.
If the observed effect on CG secretion is substantiated in future studies, it may help to explain
apparent adverse pregnancy outcomes associated with consumption of chlorinated drinking water
in some epidemiological studies (e.g., increased incidence of spontaneous abortion as reported by
Waller et al., 1998).
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 GSH-mediated pathway may play an
important role in metabolism of brominated trihalomethanes.
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VI. HEALTH EFFECTS IN HUMANS
A. Clinical Case Studies
1. Bromodichloromethane
No clinical reports or short term studies were located on the effects in humans from
ingestion of bromodichloromethane.
2. Dibromochloromethane
No clinical case reports or short term studies were located on the effects in humans from
ingestion of dibromochloromethane.
3. Bromoform
In the past, bromoform was used as a sedative for children with whooping cough. Typical
doses were approximately one drop (about 180 mg), given three to six times/day (Burton-
Fanning, 1901). This dosing usually resulted in mild sedation in children, although a few rare
instances of death or near-death were reported (e.g., Dwelle, 1903; Benson, 1907). These cases
were believed to be due to accidental overdoses. Based on these clinical observations, the
estimated lethal dose for a 10- to 20-kg child is approximately 300 mg/kg, and the LOAEL for
mild sedation is approximately 54 mg/kg-day.
B. Epidemiological Studies
The brominated trihalomethanes occur as disinfection byproducts in water disinfected
with chlorine for the prevention of disease. The primary routes of human exposure to brominated
trihalomethanes are via ingestion of disinfected tap water; dermal contact with disinfected tap
water during bathing, showering, and other activities; and inhalation of brominated
trihalomethanes released during showering, bathing or household activities using disinfected tap
water. Multiple epidemiological studies have investigated the relationship between exposure to
disinfection byproducts in chlorinated drinking water and adverse health effects. These studies
fall into two basic categories: studies of association with cancer (Table VI-1) and studies of
association with adverse pregnancy or birth outcomes or alteration of reproductive function
(Table VI-2). Because the purpose of this document is to isolate the health effects of individual
brominated trihalomethanes, a detailed examination of all available studies on disinfection
byproducts is beyond the scope of this report. Epidemiologic studies published prior to 1994 are
discussed in greater detail in the Drinking Water Criteria on Chlorine (U.S. EPA, 1994a). A
number of recent publications have reviewed the association between chlorination disinfection
byproducts and cancer and adverse reproductive or developmental outcomes (e.g., Reif et al.,
1996; Mills et al., 1998; Nieuwenhuijsen et al., 2000; Bove et al., 2002).
VI - 1 November 15, 2005
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Very few studies have examined the association between cancer and exposure to
brominated trihalomethanes. A possible increased cancer incidence in bladder was suggested
(Cantor et al., 1978), while negative findings were reported for childhood acute lymphoblastic
leukemia (Infante-Rivard et al., 2001, 2002).
Table VI-1 Epidemiological Studies Investigating an Association Between Chlorinated
Drinking Water and Cancer
Reference
Study Description
Observation
Alavanja et al. (1978)
Case control study in seven New York
State counties.
Greater risk of gastrointestinal and
urinary tract cancer mortality, both
sexes, in chlorinated water areas of the
counties.
Cantor etal. (1978)
Ecological study using age-standardized
cancer mortality rates, 1968-1971; and
halomethane levels from U.S. EPA
surveys.
Strongest correlation between bromine-
containing trihalomethanes and bladder
cancer.
Struba (1979)
Case-control study of mortality in North
Carolina, 1975-1978.
Small but significant odds ratios for
rectum, colon and bladder cancers in
rural areas but not in urban areas.
Brenniman et al. (1980)
Case-control study in 70 Illinois
communities, 1973-1976. Questionnaires
sent to water treatment plants to verify
1963 inventory data on chlorine levels.
Statistically significant relative risks of
cancer of gall bladder, large intestine,
and total gastrointestinal and urinary
tract in females served by systems with
chlorinated versus nonchlorinated
ground water. Due to many uncontrolled
confounding factors, authors concluded
that chlorination was not a major factor
in the etiology of gastrointestinal and
urinary tract cancers.
Gottlieb et al. (1981)
Case-control study using mortality data in
Louisiana and estimations of exposure.
Rectal cancer significantly elevated with
respect to surface or Mississippi River
water consumption.
Young etal. (1981)
Case-control study in Wisconsin, 1972-
1977. Questionnaires sent to waterworks
superintendents on chlorine content.
Colon cancer showed significant
(p<0.05) association with chlorine intake
in all three dosage categories.
Cragleetal. (1985)
Case-control study using colon cancer
cases from seven hospitals in North
Carolina.
Consumption of chlorinated water
strongly associated with colon cancer,
above age 60.
Young etal. (1987)
Case-control study of colon cancer cases
in Wisconsin. Water consumption was
determined by interview, and chloroform
levels by historical records and
measurement.
No association found between
trihalomethane exposure and colon
cancer incidence.
VI-2
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Table VI-1 (cont.)
Reference
Study Description
Observation
Morris etal. (1992)
Meta-analysis of nine case-control studies
and one cohort study analyzing cancer
and consumption of chlorinated water or
water containing high chloroform levels.
Statistically significant relative risk of
rectal cancer and bladder cancer in
exposed groups. No colon cancer.
McGheehin et al. (1993)
Population-based case-control study
Association between bladder cancer risk
and exposure to chlorinated water and
trihalomethanes.
King and Marret (1996)
Case-control study conducted by Health
Canada
Increased risk of bladder cancer
associated with total trihalomethane
exposure.
Hildesheim et al. (1998)
Population-based case-control study of
colon and rectal cancer risk. Iowa, 1986-
1989.
Rectal cancer risk associated with
duration of chlorinated water use. No
association of colon cancer risk with
duration of chlorinated water use or
trihalomethane estimates.
Cantor etal. (1998)
Population-based case-control study of
bladder cancer risk. Iowa, 1986-1989.
Positive findings for risk restricted to
men and to current or former smokers.
In men, smoking and exposure to
chlorinated water enhanced the risk of
bladder cancer.
Marcus et al. (1998)
Ecologic study of association between
TTHM in 71 North Carolina public water
supplies and incidence of histologically
confirmed female invasive breast cancer
obtained from cancer registry data.
TTHM levels not associated with breast
cancer risk when adjusted for potential
confounding factors. Data were
consistent with TTHMs being unrelated
or weakly related to breast cancer risk.
Infante-Rivard et al.
(2001, 2002)
Population-based case-control study
comparing 491 cases of childhood acute
lymphoblastic leukemia (ages 0-9 yrs)
with 491 age, sex, and region of
residence-matched population-based
controls. Quebec, 1980-1993.
Odds ratios for exposure to bromoform,
chlorodibromomethane and
dibromochloromethane were generally
less than one (range from 0.42 to 1.02).
Postnatal exposure to bromoform at
concentrations exceeding the 95%
percentile was associated with an OR of
1.3 (95% CI = 0.71 to 2.71). The most
important limitation of this study is the
potential for exposure misclassification.
Villanueva et al. (2003)
Meta-analysis of epidemiologic study data
extracted from six case-control and two
cohort studies that examined the
relationship between exposure to
chlorinated water and bladder cancer.
Consumption of chlorinated water was
associated with sex-specific combined
risk estimates for bladder cancer of 1.4
(95% CI 1.1 to 1.9) for men and 1.2
(95% CI 0.7 to 1.8) for women. The
authors suggest that this indicates a
moderately increased risk for bladder
cancer in men.
VI-3
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Table VI-1 (cont.)
Reference
Study Description
Observation
Villanueva et al. (2004)
Pooled data from 6 case-control studies of
bladder cancer in subjects exposed to
disinfection byproducts in the United
States, Canada, France, Italy, and Finland.
Detailed data on water consumption and
THM exposure were required for
inclusion.
Consumption of chlorinated water was
associated with sex-specific combined
risk estimates for bladder cancer of 1.4
(95% CI 1.2 to 1.7) for men and 1 (95%
CI 0.8 to 1.2) for women.
Table VI-2 Epidemiological Studies Investigating an Association Between Chlorinated
Drinking Water and Adverse Pregnancy, Altered Menstrual Function, or Sperm Quality
Reference
Study Description
Observation
Aschengrau et al. 1989
Hospital-based case-control study of
spontaneous abortion and multiple water
quality parameters in Boston, MA area.
After adjustment for potential
confounders and chemical constituents,
frequency of spontaneous abortion was
increased for consumption of surface
water when compared to use of mixed
surface and ground water (OR 2.2, 95%
C.I. 1.3 - 3.6) The association between
surface water and increased risk of
spontaneous abortion was not confirmed
by a comparison of chlorinated vs.
chloraminated surface water.
Chloraminated water was used as a
surrogate for low exposure to
disinfection byproducts.
Kramer etal. (1992)
Population-based case-control study that
examined potential associations between
pregnancy outcome and exposure to
trihalomethanes in tap water. Data on
pregnancy outcomes for cases and
controls were collected from Iowa birth
certificates for non-Hispanic white
singleton births during the period January
1, 1989 to June 30, 1990.
A possible association was noted
between exposure to
bromodichloromethane concentrations
> 10 ng/L and intrauterine growth
retardation (OR = 1.7; 95% C.I. 0.9, 2.9)
when compared to drinking water
sources without detectable levels.
Aschengrau et al. 1993
Case-control study of drinking water
quality and occurrence of late adverse
effects among women who delivered
infants during August 1977 - March 1980
in Massachusetts
After adjustment for confounding,
frequency of stillbirths was increased for
women exposed to chlorinated surface
water (OR 2.6, 95% CI 0.9-7.5).
VI-4
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Table VI-2 (cont.)
Reference
Study Description
Observation
Nuckolsetal. (1995)
Cross-sectional study in Colorado of
populations drinking chlorinated and
chloraminated water
No statistically significant effects of
exposure, although odds ratio was
elevated for risk of low birth weight
infants.
Boveetal. (1995)
Cross-sectional study in New Jersey
An association was reported between
total trihalomethane levels and "small
for gestational age."
Savitzetal. (1995)
Population based case-control study in
North Carolina
Statistically significant association of
miscarriage with increasing
concentration of TTHM and with the
highest sextile of exposure(OR=2.8, 95%
C.I. 1.1, 2.7), but no relationship with
ingested dose or water source. Small
increase in risk of low birth rate.
Gallagher et al. (1998)
Retrospective cohort study of relationship
between THM exposure during third
trimester of pregnancy and low
birthweight, low term birth weight, and
preterm delivery. Colorado birth
certificate data matched to historical
water data based on census block groups.
1990-1993.
Possible association of trihalomethane
concentration in tap water at maternal
residence during third trimester and risk
of term low birth weight deliveries.
Little association with preterm delivery.
Weak association with low birth weight.
Waller etal. (1998)
Prospective study of association between
total and individual THM exposure and
spontaneous abortion. Concurrent THM
data obtained from public water supplies.
Women who drank > 5 glasses/day of
cold tap water containing > 75 \igfL
TTHMs had an adjusted odds ratio of 1.8
for spontaneous abortion. Of individual
THMs, only consumption of >5 glasses
of water containing > 18 ng/L
bromodichloromethane (or a compound
co-occurring with
bromodichloromethane) was associated
with spontaneous abortion.
Klotz etal. (1998), Klotz
and Pyrch (1999)
Case-control study of association between
drinking water contaminants (including
disinfection byproducts) and neural tube
defects. Births with neural tube defects
reported to New Jersey's Birth Defects
Registry in 1993 and!994 were matched
against control births chosen randomly
from across the State.
Elevated odds ratios, generally between
1.5 and 2.1, for the association of neural
tube defects with total THMs (TTHMs).
The only statistically significant results
were seen when the analysis was isolated
to those subjects with the highest THM
exposures (greater than 40 ppb) and was
limited to those subjects with neural tube
defects in which there were no other
malformations (OR = 2.1, 95% CI =
VI-5
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Table VI-2 (cont.)
Reference
Study Description
Observation
Doddsetal. (1999)
Retrospective cohort study in Nova Scotia
women with singleton births, 1988-1995.
Little association between TTHM level
and fetal weight- or gestational age-
related outcomes. Elevated relative risk
for stillbirths for exposure to > 100 ng/L
TTHM levels during pregnancy. Little
evidence for increased prevalence or
dose-response for congenital
abnormalities with possible exception of
chromosome aberrations for exposure
>100
Magnus etal. (1999)
Ecologic study in Norway of chlorinated
water consumption and birth defects
observed in births during period 1993-
1995. 1994 data on water quality and
disinfection practice. Water color used as
an indicator for natural organic matter
content.
Among 141,077 births, 1.8% had birth
defects. Adjusted odds ratios (high
color, chlorination vs. low color, no
chlorination) of 1.14 (0.99-1.31) for any
malformation; 1.26 (0.61-2.62) for
neural tube defects; and 1.9 (1.10-3.57)
for urinary tract defects.
Yang et al. (2000)
Study in Taiwan of association between
chlorination of drinking water and low
birth weight.
Examination of 18,025 births showed no
association between consumption of
chlorinated drinking water and low birth
weight.
King et al. (2000)
Population-based retrospective cohort
study in Nova Scotia, Canada to examine
the relationship between TTHM or
individual THMs and risk for stillbirth of
fetuses greater than 500 grams. Study
cohort assembled from a perinatal
database and consisted of 49,756
singleton births that occurred between
1988 and 1995.
Risk doubled for women exposed to a
bromodichloromethane level > 20 [ig/L
when compared to women consuming
concentrations of less than 5 \ig/L
(relative risk = 1.98, 95% confidence
interval of 1.23 - 3.49). When categories
of stillbirth (unexplained deaths and
asphyxia-related deaths) were examined,
relative risk estimates for asphyxia-
related deaths increased by 32% for each
10 [ig/L increase in
bromodichloromethane concentration.
Dodds and King (2001)
Retrospective cohort study conducted
using data from a population-based
perinatal database in Nova Scotia, Canada
and routine water monitoring data. The
cohort consisted of women who had a
singleton birth in Nova Scotia between
1988 and 1995 and who lived in an area
with a municipal water supply.
Exposure to bromodichloromethane at
concentrations of 20 [ig/L and over was
associated with increased risk of neural
tube defects (adjusted relative risk = 2.5;
95% confidence interval 1.2 to 5.1) and
decreased risk of cardiovascular
anomalies (adjusted relative risk = 0.3;
95% confidence interval 0.2 to 0.7). No
association observed for
bromodichloromethane and cleft defects.
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Table VI-2 (cont.)
Reference
Study Description
Observation
Waller etal. (2001)
Reanalysis of total trihalomethane
exposure data reported in Waller et al.
(1998).
The study authors reported no apparent
advantage in using a closest-site (vs.
utility-wide) measurement approach for
estimation of exposure to total
trihalomethanes.
Windhametal. (2003)
Prospective study of association between
total and individual THM exposure and
menstrual cycle function. Concurrent
THM data obtained from public water
supplies.
Exposure to dibromochloromethane and
sum of brominated trihalomethanes was
associated with a reductions in length of
the menstrual cycle and follicular phase
of the menstrual cycle, suggesting
possible effects on ovarian function.
Concentrations of >20 \igfL for
dibromochloromethane and >45 \igfL for
total brominated trihalomethanes were
associated with reductions in cycle and
follicular phase lengths of approximately
one day. No effect was noted on length
of luteal phase or duration of menses.
Fenster et al. (2003)
Prospective study of association between
total and individual THM exposure and
sperm quality in healthy men. Concurrent
data obtained from public water supplies.
Exposure to TTHM was not associated
with decrements in semen quality.
Individual THM levels not strongly
associated with any semen parameter.
Inverse relationship between exposure to
bromodichloromethane and sperm
linearity (linearity decreased by -0.09 ±
0.04 per unit increase in
bromodichloromethane).
Shaw et al. (2003)
Case control study of association between
total and individual THM exposure and
birth defects. Exposure data were
obtained from municipal water supplies.
Effects were estimated using both
continuous and categorical measures of
THM exposure.
No association was found between THM
as a continuous variable and neural tube
defects or other malformations. When
exposure to brominated THMs was
evaluated, either no effect or reduced
odds ratios were observed for all of the
malformations considered in the study
(ORs ranged from 0.59 to 1.2).
VI-7
November 15, 2005
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Table VI-2 (cont.)
Reference
Study Description
Observation
King et al. (2004)
An exposure assessment was performed
as part of a case-control study of stillbirth
and abruptio placentae in Nova Scotia and
Eastern Ontario. Residential water
samples were analyzed for specific and
total THMs and haloacetic acids.
Temporal and spatial variation within the
water distribution systems was examined
and the impact of water use behaviors on
the total exposure metric was determined
There was variability in the composition
of THM in the two geographic areas
under study. Significant spatial variation
was observed in large water distribution
systems and water use behaviors were
shown to significantly affect the total
exposure metric with showering
accounting for approximately 60% of the
total THM exposure. Recommendations
include the direct measurement of
different species of byproducts, the
sampling of individual households rather
than distribution systems and
incorporation of water use behaviors in
estimating the exposure of subjects in
epidemiological investigations.
Dodds et al. (2004)
Case-control study conducted in Nova
Scotia and Eastern Ontario, Canada, to
evaluate the relationship between
exposure to chloroform and BDCM and
stillbirth. Stillbirths occurring between
July 1999 and December 2001 were
identified through a population-based
perinatal database (112 stillbirth cases and
398 live birth controls).
Women with a residential THM
concentration >80 ug/L had elevated risk
of stillbirth as compared to women with
no exposure (OR = 2.2, 95% C.I. 1.1-
4.4). Similar results were seen for
chloroform and BDCM concentrations.
Infante-Rivard (2004)
Case-control study to evaluate exposure
to total and individual THMs and
potential effects on fetal growth. Cases
include newborns with low birth weight
born at a medical center in Montreal
between May 1998 and June 2000.
Distribution system data was used to
calculate THM concentrations. Mothers
and newborns were evaluated for genetic
polymorphisms in metabolic enzyme
systems.
Exposure to >30 ug/L THM was shown
to affect fetal growth, but only in
newborns with a genetic polymorphism
in the CYP2E1 gene (OR =13.2, 95%
C.I. 1.19-146.72). Exposure information
on showering and water consumption
were used to derive risk estimates.
VI-8
November 15, 2005
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Table VI-2 (cont.)
Reference
Study Description
Observation
Wright et al. (2004)
Retrospective cohort study which linked
birth certificate data from 1995-1998 with
measurement of THM and mutagenicity
for all towns in Massachusetts with a
population >10,000. Studied the effect of
3rd trimester exposure on birth weight,
mean gestational age, SGA, and preterm
delivery.
Reductions in mean birth weight were
associated with elevated exposure to
individual THMs, MX, and mutagenic
activity. Dose-response trends were
observed for THM concentrations and
risk of small for gestational age (SGA),
which is defined as a birth weight below
the 10th percentile of birth weight per
gestational age, sex, and race. Increased
risks of SGA were observed for total
THM at >40 ug/L (OR range 1.02 to
1.2), chloroform at >20 ug/L (OR range
1.02 to 1.17), and BDCM at >5 ug/L
(OR range 1.07 to 1.22). Exposure to
BDCM and total THM was associated
with an increase in mean gestational age
and a decrease in pre-term delivery.
Savitz et al. (2005)
Population-based, prospective cohort of
2,413 pregnant women from 3 water
systems in the U.S., 2000-2004.
Estimated TTHM, HAA9, and TOX (total
organic halide) exposures during
pregnancy were considered. Individual
brominated THMs and HAA species were
examined. Weekly or biweekly
distribution system DBF concentration
data were collected and linked with
maternal residence and water
consumption data (during first and second
trimesters). Outcomes examined were
early (<12 wks) and late (>= 12 wks)
pregnancy fetal loss, preterm birth, small-
for-gestational-age birth, and term birth
weight.
No association with pregnancy loss was
seen when high TTHM exposures were
compared to low exposures. An
association was found between
bromodichloromethane and pregnancy
loss. Some increased risks were seen for
losses at greater than 12 weeks' gestation
for TTHM, bromodichloromethane, and
TOX, but most results generally did not
provide support for an association.
TTHM exposure of 80 ug/L was
significantly associated with twice the
risk for small-for-gestational-age (SGA)
births during the third trimester.
VI-9
November 15, 2005
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Table VI-2 (cont.)
Reference
Study Description
Observation
Toledano et al. (2005)
National registries were used to identify
stillbirths and low or very low birth
weight deliveries between 1992 and 1998.
Postal code from the registry was used to
link each birth with a location in each
water zone. Concentration data were
derived from water supply company
sampling programs.
The adjusted odds ratios for stillbirths
and low and very low birthweight
increased slightly with exposure to total
THM in one of three water zones. Odds
ratios for this zone ranged from 1.09 (for
low exposure) to 1.21 (for high
exposure). When the three zones were
considered together, the odds ratio for
stillbirths was increased with high
exposure to total THM (OR =1.11, 95%
CI = 1.00 -1.23). Odds ratios for low or
very low birth weight were not
increased. The authors reported that
concentrations of bromodichloromethane
and total brominated THM were not
associated with an increased risk of
stillbirths or low or very low
birthweight, but did not provide the data
from this analysis.
VI-10
November 15, 2005
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A number of studies have examined the association between consumption of chlorinated
water and incidence of cancer in the intestine, rectum, bladder, brain, and/or pancreas. Based on
the evaluation of the entire cancer epidemiology database, U.S. EPA has concluded that bladder
cancer studies provide the strongest evidence for an association between exposure to chlorinated
surface water and cancer (U.S. EPA, 1998d). The association between exposure to chlorinated
surface water and cancer at other sites cannot be determined at this time because the available
data are limited.
A number of epidemiological studies have examined the potential association of
reproductive or developmental outcomes with consumption of tap water containing
trihalomethanes (reviewed in Bove et al., 1995, 2002; Reif et al., 1996; Mills et al. 1998;
Nieuwenhuijsen et al. 2000). These studies have examined three general categories of
reproductive and developmental outcomes: 1) spontaneous abortion, stillbirth, and pre-term
delivery; 2) low birth weight and growth retardation; and 3) birth defects (neural tube defects,
oral cleft, and cardiac effects). In addition, recent studies have examined the potential association
between tap water consumption and reproductive toxicity manifested as alterations in menstrual
cycle function or semen quality. Studies which found an association with one or more individual
brominated trihalomethanes are discussed by chemical below.
In their assessment of available data on the available data for reproductive and
developmental effects of disinfection byproducts, Reif et al. (1996) stress that interpretation of
epidemiologic findings for these contaminants are potentially complicated by unmeasured
confounding variables and misclassification errors. Smoking, socioeconomic status, alcohol
consumption, other environmental exposures, and reproductive history are examples of
confounding variables that have the potential to bias estimates of risk in studies of disinfection
byproducts if not measured. Misclassification errors can arise from failure to account for spatial
and temporal variability in contaminant measurements, migration of study participants, incorrect
assumptions related to water source or use, and use of water treatment data as a surrogate for tap
water concentrations. These factors may result in under- or over-classification of health risks
associated with the consumption of disinfected water. Of greatest concern are variables or errors
which might lead to underestimation of the true public health risks associated with exposure to
tap water containing brominated trihalomethanes. The positive findings in studies of brominated
trihalomethanes thus form a foundation for further studies, but should be interpreted cautiously.
It is also important to recognize that detection of an association between an increased
incidence of a reproductive effect and the concentration of an individual compound in chlorinated
water can not be interpreted as proof that the compound caused the effect. This is because
chlorinated drinking water contains many different disinfection byproducts in addition to
brominated trihalomethanes, and the occurrence and concentration of many of these products tend
to be correlated.
VI-11 November 15, 2005
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1. Bromodichloromethane
a. Cancer
No workplace or other epidemiological cancer studies were located in which humans were
exposed exclusively or primarily to bromodichloromethane.
b. Pregnancy, Birth Defects, and Reproductive Function
Ten studies were located that reported examination of associations between
bromodichloromethane and reproductive or developmental endpoints (Kramer et al., 1992; Waller
et al., 1998; King et al., 2000; Dodds and King, 2001; Fenster et al., 2003, Shaw et al., 2003;
Windham et al., 2003; Dodds et al., 2004; Savitz et al., 2005; Toledano et al., 2005).
Kramer et al. (1992) conducted a population-based case-control study to determine if
exposure to trihalomethanes in drinking water is associated with low birthweight (159 cases, 795
controls), prematurity (342 cases, 1710 controls), or intrauterine growth retardation (defined as
being lower than the 5th percentile of weight for gestational age; 187 cases, 935 controls). A
separate analysis was conducted for each endpoint, using five randomly selected controls for each
affected newborn. Data were collected from Iowa birth certificates from January 1, 1989, to June
30, 1990; the study population was restricted to residents of small towns where all of the drinking
water was derived from a single source (surface water, shallow wells, or deep wells). Exposure
data were based on a 1987 municipal water survey; birth certificate data from 1987 were not used
because data on maternal smoking status first became available in 1989. The study authors
adjusted for maternal age, number of previous children, marital status, education, adequacy of
prenatal care, and maternal smoking. Prematurity (OR = 1.0, 95% C.I. 0.6, 1.5) and low birth
weight (OR = 1.0, 95% C.I. 0.7, 1.5) did not show an association with exposure to drinking water
containing bromodichloromethane when compared to sources without detectable levels of the
compound. A possible association was noted between exposure to drinking water concentrations
of bromodichloromethane >10 |ig/L and intrauterine growth retardation (OR= 1.7; 95% C.I. 0.9,
2.9) when compared to drinking water sources without detectable levels. However, the
confidence interval included 1, indicating that the increase was not statistically significant.
Waller et al. (1998) conducted a prospective study in pregnant women to examine the
association between trihalomethanes in drinking water and spontaneous abortion (pregnancy loss
at 20 or less completed weeks of gestation). The study participants were recruited from three
facilities of a large managed health care organization which were located in regions of California
that primarily received either mixed, surface, or groundwater. Recruitment occurred when the
women scheduled their first prenatal exam after confirmation of pregnancy. A group of 5,342
subjects completed a telephone interview that obtained information on demographics, previous
pregnancy history, employment status, consumption of tap and bottled water, use of alcohol,
tobacco, and caffeine, and other factors. At the time of enrollment in the study, each woman was
at least 18 years of age, at 13 or less weeks of gestation, spoke English or Spanish, and could
provide with certainty the date of her last menstrual period. Following adjustment for elective
VI -12 November 15, 2005
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termination of pregnancy, ectopic or molar pregnancies, and multiple gestations, a total of 5,144
pregnancies remained for analysis.
Waller et al. (1998) quantified exposure to trihalomethanes by estimating the subject's
daily tap water intake at 8 weeks gestation. Concentration of total trihalomethanes and any
available data on individual trihalomethanes were obtained directly from the utility supplying
drinking water to a subject's address or zip code. Total trihalomethane levels were calculated by
averaging all measurements taken by the utility supplying a participant's home. Each participant
was assigned a personal exposure classification (high or low) to total trihalomethanes (TTHM)
and individual trihalomethanes (THM) based on the following criteria. A high personal exposure
to TTHM was defined as drinking 5 or more glasses of cold tap water per day and having a
TTHM level of 75 |ig/L or higher. Low personal exposure to TTHM was defined as either 1)
drinking less than 5 glasses of cold tap water per day, 2) having a TTHM level of less than 75
l-ig/L, or 3) receiving water from a utility that provided 95% or greater groundwater. Personal
exposures to the individual THMs (bromoform, bromodichloromethane and
dibromochloromethane) were defined in a similar manner, with a high personal exposure being
defined as drinking 5 or more glasses of cold tap water per day with an individual brominated
THM level of 16 |ig/L or higher for bromoform, 18 |ig/L or higher for bromodichloromethane, or
31 |J.g/L or higher for dibromochloromethane. Low personal exposures to the individual THMs
were defined as either 1) drinking less than 5 glasses of cold tap water per day, 2) having an
individual THM level below the cutoff, or 3) having a TTHM level less than 72 |ig/L if individual
THM levels were not reported.
The authors found a modest association between consumption of trihalomethane-
containing water and incidence of spontaneous abortion. Increased risk of spontaneous abortion
was noted starting at approximately 75 |ig/L. The adjusted odds ratio (OR) for women who drank
5 or more glasses of cold tap water per day containing an average TTHM level of 75 |ig/L or
higher during their first trimester was 1.8 (95% C.I. 1.1, 3.0). An estimated 18.4% of the study
participants were exposed to TTHM at or above this level. Because heating can volatilize and
thus reduce TTHM levels in water, the study authors recalculated personal TTHM consumption
using total tap water consumption (i.e., hot plus cold). This recalculation resulted in an OR of 1.2
(C.I. 0.8, 1.9) for high personal exposure that was substantially lower than the OR of 1.8 for high
personal exposure based on consumption of cold tap water alone. This result implicates volatile
agents in the association between tap water consumption and risk of spontaneous abortion.
Of the four individual THMs, only high bromodichloromethane exposure was associated
with spontaneous abortion alone (adjusted OR = 2.0, 95% C.I. 1.2, 3.5) and after adjustment for
other THMs in a logistic regression model (adjusted OR = 3.0, C.I. 1.4, 6.6). Waller et al. (1998)
noted that there was no additive or other effect from showering or swimming. Therefore, no
adjustment was required for these variables. Potential misclassification of exposure was
identified as the primary limitation of this study. Concentration levels for most subjects were
based on test results for a single day, and thus do not reflect potential variation in trihalomethane
levels over time. In addition, the exposure to THMs from sources other than ingestion could not
be fully characterized.
VI -13 November 15, 2005
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Because exposure misclassification appeared to be a limitation of the Waller et al. (1998)
study, Waller et al. (2001) reported a reanalysis of exposure data from that study. The objective
of the new analysis was to examine how use of alternative methods for estimation of exposure
would affect associations between TTHM exposure and risk of spontaneous abortion. This
reanalysis did not address dose-response relationships between individual brominated
trihalomethanes and occurrence of spontaneous abortion. Two exposure analyses were tested.
The first method used the utility-wide average concentration (the metric used in Waller et al.,
1998). The second method used THM measurements taken from the water system sampling site
nearest the subject's home. For each method, the authors performed 1) an unweighted analysis;
2) an analysis weighted by a factor intended to reduce exposure misclassification (see below); and
3) an analysis within a subset of the cohort that possibly had less exposure misclassification than
the entire cohort. The weighted and subset analyses were performed in an effort to reduce
exposure misclassification.
The utility-wide average method estimated the concentration of total trihalomethanes by
averaging all distribution measurements taken by the subject's utility during the first trimester of
pregnancy. In contrast to the method used in Waller et al. (1998) the time interval was not
expanded in order to capture a measurement and thus reduce missing data in cases where no
measurements were available in the first trimester. For weighted analyses of the utility-wide
average data, the study authors calculated a weighting factor that reflected the variance of the
utility-wide average. This factor approached 1 if the variance (as estimated using the standard
deviation, SD) was small and approached 0 if the variance was large:
weightuti]ily^ideavenige = 1 - (SDutmty.wideaverage/mean TTHM level across sampling database)
The mean TTHM level across the sampling database was 50 |ig/L for the years 1990-
1991. The weighting factor was set to 0 for women whose SD was greater than 50 |ig/L. The
weighting factor for women served by groundwater utilities was set to 1, because (at the time that
the study was conducted) groundwater was often not chlorinated, trihalomethane levels in these
utilities were assumed to be negligible, and the utilities were exempt from quarterly TTHM
measurements.
The closest-site method took the average of all measurements taken during the first
trimester of pregnancy at the water distribution site nearest to the subject's home. The closest-
site approach used TTHM measurements taken from the water system sampling site nearest the
subject's home and adjusted for distance between the subject's home and the sampling site. For
the weighted analyses, a factor was created that would give a high weight to women living near a
water utility sampling site and low weight to women living at a distance from a sampling site:
weightdosest.site = 1 - [(miles to closest sampling site)2/10]
If a subject lived more than 3.16 miles (the square root of 10) from a sampling site, the weight
was set to 0. Weighting factors for women served by groundwater utilities were set to 1 as
previously described.
VI -14 November 15, 2005
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For the subset approach, analyses were restricted to groups of women for whom the
exposure assessment was likely to be more accurate. Subset analyses using the utility-wide
average TTHM concentration included women whose utility measurements were all within 20
l-ig/L of each other and women served by groundwater utilities. Subset analyses using the closest-
site average concentrations used women who lived within 0.5 miles of the utility sampling site
and all women served by groundwater utilities. An ingestion metric was calculated using
individual daily cold tap water intake at eight weeks gestation as determined in Waller et al.
(1998). A categorical ingestion exposure metric was created using the first trimester THM
concentration dichotomized at 75 |ig/L and cold tap water ingestion dichotomized at 5 glasses per
day. Ingestion was also estimated by multiplying the TTHM concentration by cold tap water
consumption. A metric to capture exposure to trihalomethanes during showering was created by
multiplying THM concentration by typical shower duration and the frequency of showering.
Use of the utility-wide approach generally resulted in odds ratios equivalent to or slightly
higher than the closest-site approach. Odds ratios obtained using the utility-wide average method
for estimating TTHM (but not the closest-site method) became progressively stronger in the
weighted and subset analyses. The study authors reported a positive, monotonic dose-response
relationship between spontaneous abortion rate and an exposure metric incorporating TTHM and
personal ingestion. The study authors noted that a major limitation of this reanalysis is the lack of
a "gold standard" with which to compare the estimated TTHM ingestion of subjects in the study.
In the absence of such a standard, it is not possible to determine whether the reanalysis actually
reduced exposure misclassification. The conclusions reached by the study authors were 1) there
was no advantage in using the closest-site method over the utility-wide method for exposure
analysis; 2) use of variance-based weighting factors and subset analyses is defensible and resulted
in some increases of odds ratio, but resulting loss of sample size may limit the utility of these
techniques; and 3) use of a variety of exposure assessment techniques may lessen the impact of
bias resulting from utility-specific factors such as inconsistencies in sampling density or
unrecognized contamination problems.
The reanalysis conducted by Waller et al. (2001) identified evidence for differential
misclassification in the prior analysis of an area predominantly served by ground water ("Zone
A") reported in Waller et al. (1998). The effect of this misclassification was to bias the original
estimate of the relationship between TTHM ingestion and spontaneous abortion away from the
null. Over 400 of the women in the study cohort resided in Zone A, an area within a large mixed-
source utility that received predominantly groundwater. Zone A was not sampled for THMs
during the study period. Because other areas within the utility frequently had high TTHM
concentrations, use of a utility-wide approach for estimating TTHM concentration probably
resulted in an overestimation of exposure for Zone A residents. An investigation by the study
authors revealed that although the spontaneous abortion rate of women in Zone A was low
overall, women who drank at least 5 glasses of water per day had a spontaneous abortion rate of
14.6%. The reason for the high spontaneous abortion rate among women consuming large
amounts of Zone A water was unclear, but was reported to be consistent with other
epidemiological studies that found high rates of spontaneous abortion among women ingesting
large amounts of unchlorinated water in Region 1 of the original study. Exclusion of Zone A
residents or receding them to a level determined by later testing within the zone decreased the
VI -15 November 15, 2005
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adjusted OR for high exposure to TTHM (TTHM >75 |ig/L and intake > 5 glasses per day) to 1.5
(95% C.I. 0.8, 2.8) as compared to the adjusted OR of 1.8 (95% C.I. 1.1, 3.0) identified in the
original analysis (Waller et al., 1998). The impact of this finding on the adjusted OR calculated
for individual brominated THMs is currently unknown, but is expected to be addressed in a future
publication by Waller et al.
King et al. (2000) conducted a population-based retrospective cohort study to examine the
relationship between TTHM or individual THMs and risk for stillbirth of fetuses greater than 500
grams. The study cohort was assembled from a perinatal database in Nova Scotia, Canada and
consisted of 49,756 singleton births that occurred between 1988 and 1995. Exposure was
assigned by relating the mother's residence at the time of delivery to the levels of total and
individual THMs measured in public water supplies. Maternal age, parity, smoking during
pregnancy, infant's sex, and neighborhood family income were evaluated as potential
confounders. Relative risks (RR) were adjusted for smoking and maternal age. Exposure to
TTHMs, chloroform, and bromodichloromethane were associated with increased risk of stillbirth.
Analysis of the results suggested that exposure to bromodichloromethane was a stronger predictor
of risk than exposure to chloroform. Risk doubled for women exposed to a
bromodichloromethane level of greater than or equal to 20 |ig/L when compared to women
consuming concentrations of less than 5 |ig/L (adjusted RR = 1.98, 95% C.I. 1.23, 3.49). When
subcategories of stillbirth (unexplained deaths and asphyxia-related deaths) were examined,
relative risk estimates for asphyxia-related deaths increased by 32% for each 10 |ig/L increase in
concentration of bromodichloromethane. As noted by the authors, a potential limitation of this
study was misclassification of exposure as a result of mobility within the study population
(estimated to affect less than 10% of study subjects). This study did not examine early fetal death
(e.g. spontaneous abortion) because the perinatal database employed in this investigation
contained information only on fetuses that weighed 500 grams or more.
Dodds and King (2001) conducted a retrospective cohort study of singleton births among
49,842 residents of Nova Scotia, Canada between 1988 and 1995 to assess the relationship
between exposure to THMs and birth defects. Information on exposure concentrations consisted
of routine water monitoring data obtained from the Nova Scotia Department of the Environment
and reflected samples collected from within the water distribution system. The birth defects
examined had previously been reported in other epidemiological studies, and included neural tube
defects, cardiovascular defects, oral clefts, and chromosomal abnormalities. The perinatal
information used in the study was obtained from the Nova Scotia Atlee perinatal database. This
database contains information abstracted from medical records and includes infant diagnoses
among stillborn and live born infants up to the time of discharge from the hospital after birth. In
addition, information on prenatally diagnosed congential anomalies was obtained from pregnancy
terminations. Inclusion of these data was deemed important because, in Nova Scotia,
approximately 80% of neural tube defects are detected antenatally and the pregnancy is electively
terminated. Exposure windows were selected to target the period before or during gestation when
exposure to a potential developmental toxicant or mutagen might have the most profound effect
on a particular developmental or genotoxic endpoint. The selected windows were as follows:
average bromodichloromethane concentrations from one month before and one month after
conception were used for analysis of neural tube defects; concentrations during the first two
VI -16 November 15, 2005
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months of pregnancy were used for analysis of cardiac defects and oral clefts; and the average
concentrations three months before pregnancy were used for the analysis of chromosomal
abnormalities. Estimates of relative risks and 95% confidence intervals were obtained from
Poisson regression models. Maternal age, parity, maternal smoking, and neighborhood family
income were assessed as potential confounders. The categories used for bromodichloromethane
concentration were <5 |ig/L; 5-9 |ig/L; 10-19 |ig/L; and > 20 |ig/L.
Exposure to bromodichloromethane at concentrations > 20 |ig/L was associated with
increased risk of neural tube defects (adjusted RR = 2.5; 95% C.I. 1.2, 5.1) when adjusted for
maternal age and income level. However, there was no evidence of a dose-response trend with
increasing concentration of bromodichloromethane. In addition, the study authors noted that this
point estimate was "fairly unstable" as a result of the low number of cases (n=10) in the >20 |ig/L
exposure category. For cardiac defects, a significant reduction in risk was associated with
exposure to concentrations of >20 |ig/L (RR = 0.3; 95% C.I. 0.2, 0.7) and there was a trend of
decreasing risk with increasing exposure. The study authors considered it unlikely that exposure
above >20 |ig/L was actually protective. They suggested that this may be a chance finding or a
reflection of a negative association of bromodichloromethane with other disinfection byproducts
in this region which may increase cardiac risks. There was no apparent trend or significant
association for exposure to bromodichloromethane and occurrence of cleft defects or
chromosomal aberrations.
Fenster et al. (2003) examined the relationship between semen quality and exposure to
trihalomethanes in home tap water, using data from the California Men's Reproductive Health
Study. The participants were 157 healthy men from couples without known factors for infertility,
recruited from among 324 men after their wives met eligibility criteria for a prospective study of
menstrual function and fecundity. All participants completed an extensive baseline interview that
included questions on demographic factors, reproductive history, medical history related to risk of
infertility, occupational exposures, consumption of alcohol, caffeine and tobacco, and daily
consumption of cold tap water (or beverages made from cold tap water) at home. Cotinine was
measured in saliva samples as an indicator of exposure to tobacco smoke. Semen specimens (two
samples from each participant in most cases) were obtained between May 1990 and September
1991. Semen parameters assessed included semen volume, sperm concentration, and percent
sperm motility. The percentage of sperm with normal morphology was estimated by two methods
(the strict method and a modified form of the World Health Organization method of 1987).
Motile sperm were analyzed for straight line velocity, curvilinear velocity, linearity (the ratio of
straight line velocity to curvilinear velocity), amplitude of head displacement, and percentage of
progressively motile sperm. Total trihalomethane levels were assigned based on water utility
measurements taken during the 90 days preceding semen collection (an interval corresponding to
the period of spermatogenesis). Semen parameters were analyzed as continuous variables with
statistics accounting for repeated measures from the same man. Analyses were performed using
the following exposure variables: 1) utility-wide TTHM levels; 2) utility-wide individual THM
levels; 3) a TTHM ingestion metric obtained by multiplying TTHM concentration (i-ig/L) by cold
home tap water consumption (glasses/day); and 4) self-reported usual daily consumption of cold
water at home (glasses/day). All final models were adjusted for duration of abstinence before
VI -17 November 15, 2005
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sample collection, age, smoking status as determined by cotinine level, work in temperatures
greater than 80°F, education, income, and race.
TTHM level was not associated with decrements in semen quality. Data on individual
THMs were not included in the report; however, the authors noted that individual THM levels
were not strongly associated with any semen parameter, with the exception of an inverse
association between exposure to bromodichloromethane and linearity of sperm motion. Use of an
adjusted continuous model resulted in a decrease in linearity of - 0.09 ± 0.04 per unit increase in
bromodichloromethane concentration. Monotonic, dose-related trends were not evident for the
various measures of TTHM exposure and sperm motility, concentration, or count. The highest
level of ingestion metric (concentration multiplied by cold tap water ingestion) was associated
with a strong decrease in percent normal morphology and an increase in head defects. A
difference of-7.1 (95% C.I. -12.7, -1.6) was observed for percent morphologically normal sperm
as determined by the modified WHO method at the highest level (>160 |ig/L x glasses/day) when
compared to the lowest level (<40 |ig/L x glasses/day). However, there was no appreciable
decrement in percent normal morphology when assessed using the strict criteria. Consumption of
cold home tap water appeared to be weakly related to decreases in percent normal morphology.
For every cup of cold home tap water consumed, percent normal morphology (modified WHO
method) decreased -0.48 (standard error = 0.43) and percent normal (strict method) decreased
byO.31 (standard error = 0.21). Consumption of cold home tap water was not associated with any
other semen parameter.
The study authors noted the following strengths and weaknesses of this investigation.
Strengths of the study included a prospective study design, a healthy population, data on potential
confounders, and state-of-the-art semen analysis techniques. Potential limitations include 1)
possible exposure misclassification resulting from use of quarterly utility measurements for
estimation of exposure in the home; 2) incomplete characterization of personal exposure (e.g., no
information was collected on potential inhalation and dermal exposure via showering, bathing, or
swimming, or on factors such as storage or filtration of tap water that might modify THM levels);
and 3) lack of data on exposure to other disinfection byproducts in tap water that are known
spermatotoxicants in animals, such as halogenated acetic acids. An additional limitation may be
the lack of consistency in sperm morphology results for the two methods employed. However, as
noted by the study authors, there was no information available that directly compared the methods
utilized in this study. Because the selection criteria used in this investigation resulted in a study
population more representative of fertile men, the results can not necessarily be generalized to a
larger population. The results of this study suggest the need for further study of the effects of
disinfection byproducts (including THMs and haloacetic acids) on semen quality.
Shaw et al. (2003) evaluated the relationship between congenital malformations and
trihalomethane exposure using data from two previous case-control studies. In the first study, the
study population consisted of all livebirths and fetal deaths (after 20 weeks of gestation)
occurring among residents of 55 California counties between June 1989 and May 1991. Cases of
neural tube defects (anencephaly, spina bifida cystica, craniorachischisis, or iniencephaly) among
live births, fetal deaths, or electively terminated fetuses were compared with control liveborn
singleton infants randomly selected from each hospital. In the second study, the study population
VI -18 November 15, 2005
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consisted of all deliveries (live or stillborn) between January 1987 and December 1988 among
California residents. Cases were defined as infants or fetuses with orofacial clefts, conotruncal
heart defects, or neural tube defects that had not been included in the first study. Controls were
randomly selected from births in the same area and time period.
In both studies, mothers were interviewed to obtain medical and reproductive history and
to determine all locations where they had resided for at least 2 weeks during the periconceptional
period. Exposure to THM was estimated by matching each address to a municipal water supply
and requesting data from the company on measured or estimated concentrations of total THM,
chloroform, bromodichloromethane, dibromochloromethane, and bromoform associated with
each address and time period. Effects were estimated using a continuous measure of THM and
also using categorical measures (0, 1-24 ppb, 25-49 ppb, 50-74 ppb, >75 ppb). In addition,
effects associated with exposure to chloroform, bromodichloromethane, and
dibromochloromethane (data on bromoform were inadequate for the assessment) above or below
the 80th percentile concentration were estimated. For study 1, neural tube defects were inversely
associated with total THM both as a continuous and a categorical variable (adjusted odds ratios
were from 0.16 to 0.9). For study 2, there was no association between THM as a continuous
variable and neural tube defects or other malformations. An increased incidence of neural tube
defects was observed for the lowest category of THM (adjusted OR of 2.2), but an exposure-
response relationship was not observed. When exposure to brominated THMs was evaluated,
either no effect or reduced odds ratios were observed for all of the malformations considered in
the study (ORs ranged from 0.59 to 1.2). The authors concluded that these results did not provide
evidence of an association between exposure to THM and the occurrences of congenital
malformations, but noted that the potential for exposure misclassification may have caused
associations to be underestimated
Windham et al. (2003) examined menstrual cycle characteristics in relation to the
presence of brominated trihalomethanes in tap water in a prospective study of women living in
northern California. Data were also reported for chloroform and total trihalomethanes. The
relationships examined included: 1) cycle characteristics and concentration of individual
trihalomethanes, total trihalomethanes, and total brominated trihalomethanes in tap water; 2)
cycle characteristics and estimated water consumption (total trihalomethanes); and 3) cycle
characteristics and duration of showering (total trihalomethanes). The target population was
married women of reproductive age (18-39 years old) who were members of the Kaiser
Permanente Medical Care Program. Participants in the study were enlisted between May 1990
and June 1991. Participants were selected from among nearly 6500 women using a short
screening interview to identify women who were more likely to become pregnant (i.e., those who
reported a menstrual period within the last six weeks, were not surgically sterilized, did not use
birth control pills or lUDs, and were non-contracepting for less than 3 months). Out of 1092
eligible women, a total of 403 women collected first morning urine samples daily for 2-9
menstrual cycles (average 5.6 cycles) for measurement of steroid metabolites. The participants
filled out a daily diary during the urine collection phase and recorded vaginal bleeding as number
of pads or tampons. These data (diary and urinary hormone metabolites) were used to estimate
menstrual parameters such as cycle and phase length. Cycle length was calculated from the first
day of menses to the day before onset of the next menses. When the available data permitted, the
VI -19 November 15, 2005
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cycle was divided into the follicular phase (first day of menses through estimated day of
ovulation) and the subsequent luteal phase. Between 1424 and 1714 cycles were available for
evaluation of each parameter.
Information on water consumption (as unheated tap water or drinks made from unheated
tap water at home, drinks made from heated tap water at home, and bottled water) and other
variables (age, race, education, employment, income, pregnancy history, exercise type and
frequency, smoking, alcohol and caffeine consumption) was collected in a baseline telephone
interview prior to urine collection. Information on the number of showers taken at home per week
and their duration was also collected. Showering was examined as minutes per week and by
combining the duration and cycle-specific trihalomethane level to create combinations of high
and low exposure. Exposure to trihalomethanes was estimated from quarterly utility monitoring
data and information on drinking water and other tap water use collected during the baseline
interview. A 90-day exposure time period was assigned for each cycle because trihalomethane
monitoring was conducted by the utilities on a quarterly (i.e., about 90 days) basis. A period of
60 days before and 30 days after each cycle start date was selected for the 90-day window.
Cycle-specific exposures to total trihalomethanes and individual trihalomethanes were calculated
by averaging all trihalomethane measurements taken by a participant's utility at various points in
the distribution system (i.e., the "utility-wide average" described by Waller et al., 1998, 2001)
during the that 90-day period. Because the brominated trihalomethanes were highly correlated
and thus difficult to examine independently, the study authors also examined the sum of the levels
of the three brominated compounds. Exposure levels for the brominated trihalomethanes were
examined as categorical variables (quartiles). The cycle-specific total trihalomethane
concentration was examined as a categorical and continuous variable, as well as combined with
amount of unheated tap water and the sum of heated and unheated tap water consumed, to
calculate ingestion metrics. Statistical analyses were conducted using the menstrual cycle as the
unit of observation. Menstrual parameters were analyzed as continuous or categorical variables
in relation to primarily categorical exposure indices and the methods used accounted for repeated
measures from the same individual. Numerous covariates reflecting demographic, reproductive
history, and lifestyle factors were examined in relation to categorical trihalomethane levels and
ingestion to identify potential confounders. Age, pregnancy history, body mass index, caffeine
consumption, and alcohol consumption, as well as race and smoking, were included as variables
in all adjusted models.
Increased intake of individual brominated compounds or total brominated trihalomethanes
was associated with significantly shorter cycles when examined by quartile (Table VI-3). Similar
decrements were observed in follicular, but not luteal, phase length. Dose-response patterns were
evident for both individual and total brominated trihalomethanes. The strongest association for
an individual compound was observed for dibromochloromethane, with adjusted decrements of
1.2 days (95% C.I. -2.0, -0.38) for mean cycle length and 1.1 days (95% C.I. -1.9, -0.25) for mean
follicular phase length at the highest quartile (>20 i-ig/L). Adjusted decrements for
bromodichloromethane at the highest quartile of exposure (> 16 |ig/L) were -0.74 (95% C.I. -1.5,
-0.02) for cycle length and -0.80 (95% C.I. -1.5, -0.08) for follicular phase length. In contrast to
the brominated trihalomethanes, a clear association with reduced cycle length was not observed
for chloroform even at the highest quartile (>17 |ig/L) (difference -0.3 days; 95% C.I. -1.0, 0.40).
VI - 20 November 15, 2005
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Menses length was slightly increased at the highest quartile for bromodichloromethane exposure.
For categorical variables, the odds of having a long cycle (OR = -1.2; 95% C.I. -2.0, -0.40) or
long follicular phase (OR = -1.1; 95% C.I. -1.9, -0.29) were significantly reduced at the highest
quartile for summed brominated trihalomethane concentration (>45 i-ig/L). These data suggest
that exposure to brominated trihalomethanes (or other disinfection by-products that are associated
with brominated trihalomethanes in these waters) may affect ovarian function.
Windham et al. (2003) also examined relationships between total trihalomethane
(brominated compounds plus chloroform) exposure and menstrual parameters. A monotonic
decrease in mean cycle length was observed with increasing total trihalomethane exposure
category. At concentrations greater than 60 |ig/L, the adjusted decrement was 1.1 day (95% C.I.
-1.8, -0.40) when compared to concentrations of 40 |ig/L or less. The decrease in follicular phase
length was similar (-0.94 day; 95% C.I. -1.6, -0.24). Cycles with total trihalomethane
concentrations above the current MCL of 80 |ig/L were shorter by about one day as well (0.99
days; 95% C.I. -2.2, 0.17). A unit decrement in mean cycle and follicular phase length of 0.18
days per 10 |ig/L increase in total trihalomethane concentration (95% C.I. -0.29, -0.07) was
determined when the cycle-specific total trihalomethane level was examined as a continuous
variable. When ingestion patterns were examined, mean cycle and phase lengths showed little
variation in relation to consumption of unheated tap water at home. In contrast, increased
consumption of heated tap water was associated with significantly decreased cycle and follicular
phase lengths. The observed decrements were greater than one day with daily consumption of
three or more drinks made from heated tap water. These decrements were reduced by adjustment,
particularly when caffeine was included in the model; the adjusted decrement in cycle length was
0.68 days (95% C.I. -2.1, 0.72).
VI - 21 November 15, 2005
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Table VI-3 Means and Adjusted Differences in Menstrual Cycle and Follicular Phase
Length by Quartile of Individual and Summed Brominated Trihalomethanes
Compound
Quartile of Exposure"
lb
Mean ± SE
(days)
2-3
Adjusted Difference0
(95% CI)
4
Adjusted Difference0
(95% CI)
Cycle Length
Bromodichloromethane
Dibromochloromethane
Bromoform
Sum of Brominated
Compounds
29.8 (0.30)
30.0(0.33)
29.7 (0.26)
30.0(0.34)
-0.59 (-1.2, -0.02)
-0.69 (-1.4, -0.02)
-0.42 (-0.96, 0.13)
-0.72 (-1.4, -0.04)
-0.74 (-1.5, -0.02)
-1.2 (-2.0, -0.38)
-0.79 (-1.4, -0.14)
-1.2 (-2.0, -0.40)
Follicular Phase
Bromodichloromethane
Dibromochloromethane
Bromoform
Sum of Brominated
Compounds
17.0(0.31)
17.1 (0.34)
16.9 (0.27)
17.2(0.35)
-0.54 (-1.1, 0.01)
-0.62 (-1.3, 0.05)
-0.30 (-0.83, 0.23)
-0.66 (-1.3, 0.02)
-0.80 (-1.5, -0.08)
-1.1 (-1.9, -0.25)
-0.78 (-1.4, -0.14)
-1.1 (-1.9, -0.29)
Source: Windham et al. (2003)
a Top quartiles for bromodichloromethane, dibromochloromethane, bromoform, and the summed
brominated compounds are > 16, >20, > 12, and >45 ng/L, respectively.
b Difference from reference group; the mean provided is unadjusted with standard error (SE)
0 Adjusted for age, race, body mass index, income, pregnancy history, caffeine and alcohol
consumption, and smoking.
A non-monotonic relationship was observed for mean cycle length and an ingestion metric
combining total trihalomethane concentration and consumption of unheated tap water, with the
highest category (>60 |ig/day) showing a decrement of 0.4 days and the third category (>40-60
jig/day) showing a decrement of one day. Use of an ingestion metric based on total home tap
water consumption (i.e., heated and unheated tap water) revealed a more consistent pattern of
reduced cycle length, with adjusted decrements of greater than one day for cycle (-1.1 day; 95%
C.I. -2.2, -0.06) and follicular phase (-1.1 day; 95% C.I. -2.2, 0.03) lengths at >40 jig/day.
Examination of time spent showering did not reveal additional risks with longer showers. The
unadjusted mean cycle length varied little by time spent showering. Following adjustment, there
was a tendency toward decreased length with any category of showering above 35 minutes/week.
This relationship was stronger for follicular phase duration than for cycle length. For example,
VI-22
November 15, 2005
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the adjusted mean decrements at the longest duration (> 105 minutes) were -0.68 days (95% C.I.
-2.0, 0.68) for cycle length and -1.2 days (95% C.I. -2.6, 0.26) for follicular phase length. When
combined with TTHM concentration, decrements in cycle and follicular phase length were seen at
the higher TTHM (>60 |ig/L) and longer showers (> 70 minutes) categories (-1.2 and -1.6 days
respectively). However, the confidence intervals were wide for all duration categories and a clear
dose response pattern (i.e., shorter lengths at higher durations) was not evident.
The study authors noted several strengths and potential limitations of this study. Strengths
include the use of a prospective study design, use of biologic measures to determine menstrual
parameters, and consideration of many potential confounders. Potential limitations include a
study sample that may not be representative of the general population, and exposure
misclassification due to a lack of information on other sources of exposure such as washing,
cooking and cleaning, as well as exposures outside the home.
There are two observations in this study that might suggest involvement of compounds
other than trihalomethanes in the reduction of cycle length. First, the more consistent association
of decreased cycle length reported for heated compared to unheated tap water is unexpected if
trihalomethanes alone are the causative agent. This is because trihalomethanes volatilize from
heated water and exposure to these compounds should therefore be lower for heated tap water,
unless the volatilized compound is inhaled. It is not known if the women stayed in the room
while heating water and inhaled the trihalomethanes. Second, examination of time spent
showering did not reveal additional risks with longer showers. This is also counter to the
expected trend, as elevated blood levels of trihalomethanes have been documented after
showering (Backer et al., 2000; Miles et al., 2002) as a result of dermal and inhalation exposure.
However, information on shower duration was collected by interview and the reported lengths
may not have accurately reflected actual shower duration. Other factors that influence exposure
could explain the failure to observe an increased risk with longer showers (e.g., shower water
temperature, ventilation, etc.), but these data were not available.
Because the study by Windham et al. (2003) is the first to examine changes in menstrual
cycle function in relation to tap water exposure, there are no supporting data on the association of
disinfection by-products other than the trihalomethanes with changes in menstrual cycle function.
Although this study suggests that bromodichloromethane and other brominated trihalomethanes
(or other disinfection byproducts associated with them) have effects on ovarian function, no
definitive conclusions can be drawn regarding the identity of the compounds responsible for these
effects based on the available data.
Dodds et al., (2004) performed a population-based case-control study to examine the
relationship between THM exposure during pregnancy and stillbirth in Nova Scotia and Eastern
Ontario, Canada. Population-based perinatal databases were used to identify all stillbirths
occurring between July 1999 and December 2001. Cases included women who had stillborn
infants (i.e., a fetus weighing more than 500 g at delivery, not including pregnancy termination
for fetal anomaly) and controls were women who delivered a live born infant during the same
time period. A large number of controls was selected to maximize the statistical power of the
study. The water use behavior of study participants was evaluated through use of a telephone
VI - 23 November 15, 2005
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interview and residential tap water samples were collected. A total exposure metric was
calculated from various exposure sources including bathing and showering. Odds ratios were
adjusted for potential confounding factors (i.e., age, province of residence, household income)
and multi-variate analyses were conducted separately for total THM, chloroform and
bromodichloromethane and for subgroups of cause of death. Asphyxia-related deaths and
unexplained deaths were considered of primary interest in this study.
The study results were based on 112 cases and 398 controls. The mean total THM level
was 57 ug/L among cases and 55 ug/L among controls. The maximum concentration of total
THM, chloroform, and bromodichloromethane among study participants was 318, 315, and 21
ug/L, respectively. Subjects with a total THM level of greater than 80 ug/L had a 2-fold higher
risk of stillbirth (OR = 2.2, 95% C.I. = 1.1-4.4) relative to women that were not exposed to THM.
Similar results were seen for chloroform (OR = 2.2, 95% CI = 1.0-4.8) and
bromodichloromethane (OR = 2.2, 95% CI = 1.0-4.5). When considering the total dose metric,
women with the highest exposure to bromodichloromethane had 2.5 times the risk of stillbirth
compared with those with no exposure (OR = 2.5, 95% C.I. = 1.3-4.9). The largest odds ratio
(OR = 4.0, 95% C.I. = 1.4-11) was observed in women consuming more than 5 glasses of water
each day at a total THM concentration of greater than 50 ug/L. Bathing and showering were
considered to contribute to increased risk of stillbirth at high concentrations of THM. Although
the highest risks for stillbirth were consistently observed in the highest exposure category, no
dose-response relationship could be demonstrated for risks related to either the total THM
concentration or the total THM exposure metric.
King et al. (2004) performed an exposure assessment as part of the case-control study of
stillbirth and abruptio placentae conducted in Nova Scotia and Eastern Ontario (Dodds et al.,
2004). Residential water samples were analyzed for specific and total THMs and haloacetic
acids. Temporal and spatial variation within the water distribution systems was examined and the
impact of water use behaviors on the total exposure metric was determined. A similar level of
total THM in the two provinces was associated with a different mixture of specific disinfection
byproducts. The correlation between total THMs and bromodichloromethane was high in Nova
Scotia (r=0.63), but low in Ontario (r=0.26). Significant spatial variation was observed in large
water distribution systems and water use behaviors were shown to significantly affect the total
exposure metric with showering accounting for approximately 60% of the total THM exposure.
This study highlighted the importance of the direct measurement of different species of
byproducts, the sampling of individual households rather than distribution systems and
incorporation of water use behaviors in estimating the exposure of subjects in epidemiological
investigations.
Infante-Rivard (2004) performed a hospital-based case control study in Canada to evaluate
the relationship between THM exposure, gene polymorphism, and intrauterine growth restriction.
Cases were defined as newborns whose birth weight was below the 10th percentile for gestational
age and sex, based on Canadian standards. Controls were newborns born at the same hospital
with a birth weight above the 10th percentile. Participants in the study included 493 cases and 472
controls born at the same medical center between May 1998 and June 2000. Mothers of
newborns were interviewed to gather information on demographics, water use behavior, and
VI - 24 November 15, 2005
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medical history. Water concentrations of total THM, chloroform, bromoform,
bromodichloromethane, and dibromochloromethane were derived from regulatory data for 189
distribution systems. Exposure estimates were determined for both drinking water and
showering. Mothers and newborns were characterized for genetic polymorphism in the CYP2E1
gene and in the 5,10-methylenetetrahydrofolate reductase gene.
For the overall study population, no association was demonstrated between THM
exposure (total THM or constituent compounds) and intrauterine growth restriction (odds ratios
ranged from 0.62 to 2.44). Both average and cumulative exposure via drinking water and
showering were considered. Odds ratios were adjusted to account for confounding factors
including, gestational age, sex, race, maternal weight gain, prepregnancy body mass index,
smoking during the third trimester, primiparity, preeclampsia and previous intrauterine growth
restriction. An association was observed between intrauterine growth restriction and exposure to
total THM concentrations above the 90th percentile (29.4 ug/L) in those newborns with 1 or 2
variant alleles in the gene for CYP2E1 (OR = 13.2, 95% C.I. 1.19-146.72). This finding suggests
that exposure to THM may affect fetal growth in genetically susceptible newborns.
In a retrospective cohort study, Wright et al. (2004) used birth certificate data on 196,000
infants to evaluate the effect of total THM exposure on various indices of fetal development.
Birth certificate data from 1995 to 1998 were derived from towns in Massachusetts with
populations greater than 10,000. Information on birth weight and gestational age was linked with
town-specific aggregate data on THM concentrations. Concentration data included water
concentrations of total THM, chloroform, bromodichloromethane, total haloacetic acids,
dichloroacetic acid, trichloroacetic acid, and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-
furanone (MX). Mutagenicity of water samples was also considered in the analysis.
Reductions in mean birth weight were associated with elevated exposure to individual
THMs, MX, and mutagenic activity. For total THM, exposures in the 50th-90th percentile (33 to
74 ug/L) were associated with a 12 g reduction in mean birth weight (95% C.I. = -7 to -16 grams),
while exposure to concentrations greater than 74 ug/L (>90th percentile) was associated with an
18 g reduction in mean birth weight (95% C.I. = -10 to -26 g). Similar associations were
observed for chloroform and bromodichloromethane. Exposure to haloacetic acids was not
associated with decreases in mean birth weight. An association was observed between increased
THM exposure, mutagenicity and increased gestational age/reduced risk of preterm delivery.
Dose-response trends were observed for THM concentrations and risk of small for gestational age
(SGA), which is defined as a birth weight below the 10th percentile of birth weight matched on
gestational age, sex, and race. Statistically significant increased risks of SGA were observed for
total THM at >33 ug/L (ORs of 1.06 to 1.13 for mid and high concentration categories),
chloroform at >26 ug/L (ORs of 1.05 to 1.11 for mid and high concentration categories), and
bromodichloromethane at >5 ug/L (ORs of 1.1 to 1.15 for mid and high concentration categories).
The primary limitation of this study was the potential for exposure misclassification due to the
use of town average data for exposure estimates.
Savitz et al. (2005) was a population-based, prospective cohort of 2,413 pregnant women
from 3 water systems in the U.S., 2000-2004. Estimated TTHM, HAA9, and TOX (total organic
VI - 25 November 15, 2005
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halide) exposures during pregnancy were considered. Individual brominated THMs and HAA
species were examined. Indices examined included concentration, ingested amount, exposure
from showering and bathing, and an integration of all exposures combined. Weekly or biweekly
distribution system DBF concentration data were collected and linked with maternal residence
and water consumption data (during first and second trimesters). Periconceptual, early and late
gestational exposure windows were examined. Outcomes examined were early (<12 wks) and
late (>= 12 wks) pregnancy fetal loss, preterm birth, small for gestational age, and term birth
weight. Potential confounding factors considered were maternal age, tobacco use, race, ethnicity,
education, marital status, income, alcohol use, caffeine consumption, body mass index, age at
menarche, employment, diabetes, pregnancy history, prior fetal loss, induced abortion history,
vitamin use.
No association with pregnancy loss was seen when high TTHM exposures were compared
to low exposures. When examining individual THMs, a statistically significant association was
found between bromodichloromethane and pregnancy loss. Although non-statistically significant,
an increased risk similar in magnitude was seen between dibromochloromethane and pregnancy
loss. Some increased risks were seen for losses at greater than 12 weeks' gestation for TTHM,
bromodichloromethane, and TOX, but most results generally did not provide support for an
association. Preterm birth showed a small inverse relationship with DBF exposure (i.e. higher
exposures were less likely to have a preterm birth), but this association was weak.TTHM
exposure of 80 ug/L was significantly associated with twice the risk for small-for-gestational-age
(SGA) births during the third trimester.
Toledano et al. (2005) evaluated the association between exposure to total THM in public
water supplies and stillbirths and birthweight. Three water zones in the UK were selected for
study. National registries were used to identify stillbirths and low or very low birth weight
deliveries between 1992 and 1998. Postal code from the registry was used to link each birth with
a location in each water zone. The weighted average total THM concentrations for the 3-month
period prior to birth (corresponding to the last trimester for full-term births) was estimated using
data from mandatory sampling conducted by the water supply companies and modeling to
estimate missing data. Exposure to total THM was categorized as low, medium, or high; ranges
in each category varied between water zones. Odds ratios for stillbirth were adjusted for maternal
age and an index of socioeconomic deprivation (the Carstairs index). Odds ratios for low and
very low birth weight were adjusted for these variables as well as for sex of infant (low birth
weight only) and year of study. The adjusted odds ratios for stillbirths and low and very low
birthweight increased slightly with exposure to total THM in one of the three water zones (United
Utilities) but not the others. Odds ratios for this zone ranged from 1.09 (for low exposure) to 1.21
(for high exposure). Lower 95% confidence limits for all of the odds ratios were close to unity
(values were from 0.98 to 1.14). When the three zones were considered together, the odds ratio
for stillbirths was increased with high exposure to total THM (OR =1.11, 95% CI = 1.00 -1.23).
Odds ratios for other comparisons were not increased. The authors reported that concentrations
of bromodichloromethane and total brominated THM were not associated with an increased risk
of stillbirths or low or very low birthweight, but did not provide the data from this analysis.
VI - 26 November 15, 2005
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2. Dibromochloromethane
a. Cancer
No workplace or other epidemiological cancer studies were located in which humans were
exposed exclusively or primarily to dibromochloromethane.
b. Pregnancy, Birth Defects, and Reproductive Function
Nine studies were located that specifically sought to investigate associations between
dibromochloromethane and reproductive or developmental endpoints (Kramer et al., 1992; Waller
et al., 1998; King et al., 2000; Dodds and King, 2001; Fenster et al., 2003, Shaw et al., 2003;
Windham et al., 2003; Infante-Rivard et al., 2004; Savitz et al., 2005). The studies of Kramer et
al. (1992), Waller et al. (1998), Fenster et al. (2003), Shaw et al. (2003); Windham et al. (2003);
Infante-Rivard et al. (2004), and Savitz et al., (2005) are summarized below. The studies by King
et al. (2000) and Dodds and King (2001) are not presented because dibromochloromethane
concentrations in water were too low to allow quantitative assessment of exposure-response
relationships.
Kramer et al. (1992) reported the results of a population-based case-control study that
examined potential associations between exposure to trihalomethanes in tap water and occurrence
of low birth weight (159 cases, 795 controls), preterm delivery (342 cases, 1710 controls), and
intrauterine growth retardation (187 cases, 935 controls). Data on pregnancy outcomes for cases
and controls were collected from Iowa birth certificates for non-Hispanic white singleton births
during the period January 1, 1989 to June 30, 1990. Pregnancy outcomes were defined as
follows: low birth weight was defined as body weight less than 2,500 grams; prematurity was
defined as delivery at less than 37 weeks of gestation; and intrauterine growth retardation was
defined as body weight less than the 5th percentile of weight for gestational age. The study
population was restricted to residents of small towns (population 1,000 to 5,000) where all tap
water was derived from a single source (surface water, shallow wells, or deep wells). The
concentration of dibromochloromethane in tap water was estimated from a municipal water
survey of tap water conducted in 1986 and 1987. Exposure was assigned based on the mother's
town of residence at delivery, as reported on the baby's birth certificate. A separate analysis was
conducted for each of the three endpoints, with five randomly selected controls used for each
affected newborn. The study data were adjusted for maternal age, number of previous children,
marital status, education, number of prenatal visits, and maternal smoking. This study did not
identify any association between dibromochloromethane concentration (>4 |ig/L in the highest
exposure category) in tap water and occurrence of low birth weight, prematurity, or intrauterine
growth retardation (OR=0.8, 1.1, and 0.9, respectively).
Waller et al. (1998) conducted a prospective study in pregnant women to examine the
association between consumption of trihalomethanes in drinking water and spontaneous abortion
(defined as pregnancy loss at 20 or less weeks of gestation). The study participants were
recruited from three branches of a large managed health care organization that were located in
regions of California that received either mixed, surface, or groundwater. The protocol used for
VI - 27 November 15, 2005
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this study is provided in the summary for bromodichloromethane in section VI.B.l.b. Exposure
to dibromochloromethane exposure was quantified by estimating the participant's daily tap water
intake at eight weeks gestation. Any available data on dibromochloromethane concentration were
obtained directly from the utility supplying drinking water to a subject's address or zip code.
Exposure classifications (high or low) for ingestion of dibromochloromethane were assigned to
each participant based on the following criteria. A high personal exposure to
dibromochloromethane was defined as drinking five or more glasses of cold tap water per day and
having a dibromochloromethane concentration of 31 |ig/L or higher. Low personal exposure to
dibromochloromethane was defined as either 1) drinking less than five glasses of cold tap water
per day, 2) having a dibromochloromethane level below the cutoff, or 3) having a total
trihalomethanes level of less than 72 |ig/L if dibromochloromethane concentration was not
reported.
High personal exposure to dibromochloromethane was not associated with increased risk
of spontaneous abortion, when data were adjusted for gestational or maternal age at interview,
smoking, history of pregnancy loss, maternal race, and employment during pregnancy (adjusted
OR for all regions = 1.3, 95 percent C.I. = 0.7 - 2.4) or when other trihalomethanes were included
as covariates (adjusted OR for all regions = 0.8, C.I. = 0.2 - 2.8). The primary limitation of this
study is potential misclassification of exposure. Concentration levels for most subjects were
based on test results for a single day, and thus do not reflect potential variation in trihalomethane
levels over time. In addition, the exposure to trihalomethanes from sources other than ingestion
could not be fully characterized.
Windham et al. (2003) examined menstrual cycle characteristics in relation to the
presence of brominated trihalomethanes in tap water in a prospective study of women living in
northern California. Data were also reported for chloroform and total trihalomethanes. The
relationships examined included: 1) cycle characteristics and concentration of individual
trihalomethanes, total trihalomethanes, and total brominated trihalomethanes in tap water; 2)
cycle characteristics and estimated water consumption (total trihalomethanes); and 3) cycle
characteristics and duration of showering (total trihalomethanes). The target population was
married women of reproductive age (18-39 years old) who were members of the Kaiser
Permanente Medical Care Program. Details of the protocol used for this study and a complete
review of the results for total brominated compounds are provided in the summary of results for
bromodichloromethane in Section VI.B.l.b.
Increasing levels of individual brominated compounds or total brominated
trihalomethanes were associated with significantly shorter menstrual cycles when examined by
quartile (Table VI-2 above). Similar decrements were observed in follicular, but not luteal, phase
length. Dose-response patterns were evident for both individual and total brominated
trihalomethanes. Dibromochloromethane showed the strongest association for an individual
compound with adjusted decrements of 1.2 days (95% C.I. -2.0, -0.38) for mean cycle length and
1.1 days (95% C.I. -1.9, -0.25) for mean follicular phase length at the highest quartile (>20 |ig/L).
These data suggest that exposure to dibromochloromethane and other brominated trihalomethanes
(or other disinfection by-products that are associated with brominated trihalomethanes in these
waters) could affect ovarian function. However, the physiological impacts of the relatively small
VI - 28 November 15, 2005
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(i.e., approximately one day) decreases in menstrual cycle and follicular phase lengths,
respectively, associated with ingestion of tap water are currently unknown.
Fenster et al. (2003) examined the relationship between semen quality and exposure to
trihalomethanes in home tap water, using data from the California Men's Reproductive Health
Study. Details of the protocol used for this study and a complete review of the results for total
brominated compounds are provided in the summary of results for bromodichloromethane in
Section VLB. 1 .b. The level of dibromochloromethane in home tap water was not strongly
associated with any semen parameter.
Shaw et al. (2003) evaluated the relationship between congenital malformations and
trihalomethane exposure using data from two previous case-control studies. In the first study, the
study population consisted of all livebirths and fetal deaths (after 20 weeks of gestation)
occurring among residents of 55 California counties between June 1989 and May 1991. Cases of
neural tube defects (anencephaly, spina bifida cystica, craniorachischisis, or iniencephaly) among
live births, fetal deaths, or electively terminated fetuses were compared with 644 control liveborn
singleton infants randomly selected from each hospital. In the second study, the study population
consisted of all deliveries (live or stillborn) between January 1987 and December 1988 among
California residents. Cases were defined as infants or fetuses with orofacial clefts, conotruncal
heart defects, or neural tube defects that had not been included in the first study. Controls were
randomly selected from births in the same area and time period. Details of the protocol used for
this study and a complete review of the results for total brominated compounds are provided in
the summary of results for bromodichloromethane in Section VLB. 1 .b. Exposure to
dibromochloromethane was not associated with increases in the incidence of any measure of
congenital malformations in either study.
Infante-Rivard (2004) performed a hospital-based case control study in Canada to evaluate
the relationship between THM exposure, gene polymorphism, and intrauterine growth restriction.
Cases were defined as newborns whose birth weight was below the 10th percentile for gestational
age and sex, based on Canadian standards. Controls were newborns born at the same hospital
with a birth weight above the 10th percentile. Participants in the study included 493 cases and 472
controls born at the same medical center between may 1998 and June 2000. Details of the
protocol used for this study and a complete review of the results for total brominated compounds
are provided in the summary of results for bromodichloromethane in Section VLB. 1 .b. No
association was demonstrated between exposure to dibromochloromethane and intrauterine
growth restriction (OR = 0.62, 95% CI = 0.27-1.44).
Savitz et al. (2005) was a population-based, prospective cohort of 2,413 pregnant women
from 3 water systems in the U.S., 2000-2004. Estimated TTHM, HAA9, and TOX (total organic
halide) exposures during pregnancy were considered. Individual brominated THMs and HAA
species were examined. Details of the protocol used for this study and a complete review of the
results for total brominated compounds are provided in the summary of results for
bromodichloromethane in Section VI.B.l.b. Although non-statistically significant, an increased
risk similar in magnitude was seen between dibromochloromethane and pregnancy loss.
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3. Bromoform
a. Cancer
No workplace or other epidemiological cancer studies were located in which humans were
exposed exclusively or primarily to bromodichloromethane.
b. Pregnancy, Birth Defects, and Reproductive Function
Four studies were located that examined associations between bromoform consumption or
concentration in tap water and reproductive endpoints (Waller et al., 1998; Windham et al., 2003;
Fenster et al., 2003; Infante-Rivard et al., 2004).
Waller et al. (1998) conducted a prospective study in pregnant women to examine the
association between trihalomethanes in drinking water and spontaneous abortion (pregnancy loss
before reaching the 21st week of gestation). The study participants were recruited from three
branches of a large managed health care organization that were located in regions of California
that received either mixed, surface, or groundwater. A detailed description of the study protocol
is provided in the summary for bromodichloromethane in section VI.B.l.b.
The authors quantified exposure to trihalomethanes by estimating the participant's daily
tap water intake at eight weeks gestation. Data on concentration of total trihalomethanes and any
available data on bromoform were obtained directly from the utility supplying drinking water to a
subject's address or zip code. Each participant was assigned a personal exposure classification
(high or low) to bromoform based on the following criteria. High personal exposure was defined
as drinking five or more glasses of cold tap water per day containing a bromoform concentration
of 16 |ag/L or higher. Low personal exposure to bromoform was defined as either 1) drinking less
than five glasses of cold tap water per day, 2) having a bromoform level below the cutoff, or 3)
having a total trihalomethanes level of less than 72 |ig/L if bromoform concentration was not
reported. No association with incidence of spontaneous abortion was observed for bromoform
when data were adjusted for gestation or maternal age at interview, smoking, history of
pregnancy loss, maternal race and employment during pregnancy (AOR for all regions = 1.0, 95%
C.I. = 0.5 - 2.0) or when other trihalomethanes were included as covariates (AOR for all regions =
0.7, 95% C.I. = 0.2 - 2.1). The primary limitation of this study is potential misclassification of
exposure. Bromoform concentrations for most subjects were based on test results for a single
day, and thus do not reflect potential variation in tap water levels over time. In addition, the
exposure to bromoform from sources other than ingestion could not be fully characterized.
Windham et al. (2003) examined menstrual cycle characteristics in relation to the
presence of trihalomethanes in home tap water in a prospective study of women living in northern
California. The relationships examined included: 1) cycle characteristics and concentration of
individual trihalomethanes, total trihalomethanes, and total brominated trihalomethanes in tap
water; 2) cycle characteristics and estimated water consumption (total trihalomethanes); and 3)
cycle characteristics and duration of showering (total trihalomethanes). The target population
was married women of reproductive age (18-39 years old) who were members of the Kaiser
VI - 30 November 15, 2005
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Permanente Medical Care Program. A detailed description of this study is provided in the
summary for bromodichloromethane in section VI.B.l.b.
Increasing levels of individual brominated compounds or total brominated
trihalomethanes in home tap water were associated with significantly shorter cycles when
examined by quartile (Table VI-3 above). Similar decrements were observed in follicular, but not
luteal, phase length. Dose-response patterns were evident for both individual and total
brominated trihalomethanes. Bromoform had adjusted decrements of 0.79 days (95% C.I. -1.4,
-0.14) for mean cycle length and 0.78 days (95% C.I. -1.4, -0.14) for mean follicular phase length
at the highest quartile (> 12 |ig/L). These data suggest that bromoform and other brominated
trihalomethanes (or other disinfection by-products that are associated with brominated
trihalomethanes in these waters) may affect ovarian function. Because the study by Windham et
al. (2003) is the first to examine changes in menstrual cycle function in relation to tap water
exposure, there are no supporting data on the association of disinfection by-products other than
the trihalomethanes with changes in menstrual cycle function. Thus, although this study indicates
an association of bromoform exposure with effects on ovarian function, no definitive conclusions
can be drawn from the current data regarding the identity of the compounds in tap water
responsible for the observed effects. The physiological impacts of the relatively small (i.e., less
than one day) decreases in menstrual cycle and follicular phase lengths observed in this study are
unknown.
Fenster et al. (2003) examined the relationship between semen quality and exposure to
trihalomethanes in home tap water, using data from the California Men's Reproductive Health
Study. The participants were 157 healthy men from couples without known risk factors for
infertility, recruited from among 324 men after their wives met eligibility criteria for a
prospective study of menstrual function and fecundity. A detailed description of this study is
provided in the summary for bromodichloromethane in section VLB. 1 .b. Bromoform level in
home tap water was not strongly associated with decrements in any semen parameter. Details of
the analysis of individual THMs were not included in the report.
Infante-Rivard (2004) performed a hospital-based case control study in Canada to evaluate
the relationship between THM exposure, gene polymorphism, and intrauterine growth restriction.
Cases were defined as newborns whose birth weight was below the 10th percentile for gestational
age and sex, based on Canadian standards. Controls were newborns born at the same hospital
with a birth weight above the 10th percentile. Participants in the study included 493 cases and 472
controls born at the same medical center between may 1998 and June 2000. Details of the
protocol used for this study and a complete review of the results for total brominated compounds
are provided in the summary of results for bromodichloromethane in Section VLB. 1 .b. No
association was demonstrated between exposure to bromoform and intrauterine growth
restriction.
C. High Risk Populations
High risk (or susceptible) populations are those which experience more adverse effects at
comparable levels of exposure, which experience adverse effects at lower exposure levels than
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the general population, or which experience a higher than average exposure because they live or
work in settings with elevated environmental concentrations of the chemical of interest. The
enhanced response of these susceptible subpopulations may result from intrinsic or extrinsic
factors. Factors that may be important include, but are not limited to: impaired function of
detoxification, excretory, or compensatory processes that protect against or reduce toxicity;
differences in physiological protective mechanisms; genetic differences in metabolism;
developmental stage; health status; gender; or age of the individual. For brominated
trihalomethanes, high risk populations may potentially include those with elevated levels of
CYP2E1 (via exposure to inducing substances or because of altered physiological or health states)
or elevated levels of glutathione-S-transferase theta. These factors are discussed in greater detail
in Section VII.D.3 of this document.
A growing body of scientific evidence indicates that children may suffer
disproportionately from some environmental health risks. These risks may arise because the
neurological, immunological, and digestive systems of children are still developing (U.S. EPA,
(1998a). In addition, children may incur greater exposure because they eat more food, consume
more fluids, and breathe more air in proportion to their body weight when compared to adults
(U.S. EPA, 1998a). Factors contributing to potentially greater risk in children are discussed in
Section VII.D.2 of this document.
D. Summary
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/kg-
day 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. Very few studies have examined the
association between cancer and exposure to brominated trihalomethanes, and possible increased
cancer incidence in bladder was have been suggested. Recent 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 reported for exposure to bromodichloromethane (or a
closely associated compound) and a moderately increased risk of spontaneous abortion during the
first trimester. 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, 2) reduction
in birth weight (small for gestational age), and 3) increased risk of neural tube defects in women
exposed to >20 |ig/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. A study of semen quality in healthy men found an association
VI - 32 November 15, 2005
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between increased exposure to bromodichloromethane in residential tap water and decreased
sperm linearity; exposure to dibromochloromethane or bromoform was not associated with
decrements in semen quality.
To directly conclude from these studies that bromodichloromethane and
dibromochloromethane are developmental or reproductive toxicants in humans can be
complicated by the fact that there are many disinfection byproducts in chlorinated water.
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.
For the brominated trihalomethanes, populations at high risk may potentially include those
with elevated levels of CYP2E1 (via exposure to inducing substances or because of altered
physiological or health states) or elevated levels of glutathione-S-transferase theta. In addition,
users of hot tubs and swimming pools may experience additional exposure to brominated
trihalomethanes.
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VII. MECHANISM OF TOXICITY
A. Role of Metabolism
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. Pankow et al. (1997), for example, examined the relationship between metabolism of
dibromochloromethane and hepatotoxicity. Serum leucine aminopeptidase (LAP) activity (an
indicator of hepatotoxicity) increased in a dose-dependant fashion with any treatment that
increased the metabolism of dibromochloromethane (e.g. pretreatment with isoniazid or m-
xylene).
B. Biochemical Basis of Toxicity
The precise biochemical mechanisms which link brominated trihalomethane metabolism
to toxicity are not certain, but many researchers have proposed that toxicity results from the
production of reactive intermediates. These reactive intermediates are believed to form covalent
adducts with various cellular molecules and to impair the function of those molecules, resulting in
cell injury. Reactive intermediates may arise from the oxidative (dihalocarbonyls) or the
reductive (free radicals) pathways of metabolism as discussed in Section III.C. Support for this
mode of action has been obtained from in vitro studies of bromodichloromethane. Under both
aerobic and anoxic conditions, bromodichloromethane is metabolized to intermediates that
covalently bind to rat microsome proteins and lipids. Direct evidence showing a relationship
between the levels of covalent binding intermediates generated by the oxidative or reductive
pathways and the extent of toxicity is not currently available for brominated trihalomethanes.
Free radical generation by the reductive pathway for brominated trihalomethane
metabolism may result in lipid peroxidation. Although evidence demonstrating that lipid
peroxidation actually accounts for the observed cellular toxicity associated with brominated
trihalomethanes is lacking, at least one study has established that lipid peroxidation does occur in
conjunction with brominated trihalomethane metabolism. De Groot and Noll (1989) reported that
all three brominated trihalomethanes induced lipid peroxidation in rat liver microsomes in vitro,
and that this was maximal at low oxygen levels (between 1 and 10 mm Hg of O2). The authors
interpreted these data to support the concept that lipid peroxidation is caused by free radical
metabolites generated by the reductive metabolism of trihalomethanes.
Glutathione has been implicated in both defense against toxicity induced by brominated
trihalomethanes and in generation of mutagenic metabolites. Gao et al. (1996) examined the
effect of glutathione on the toxicity of bromodichloromethane in vivo and in vitro. Depletion of
glutathione by pretreatment of male F344 rats with the glutathione synthesis inhibitor buthionine
sulfoximine increased the hepatotoxicity of a single gavage dose of 400 mg/kg
bromodichloromethane administered in 10% Emulphor®. Biochemical indicators of
hepatotoxicity (e.g. AST, ALT, LDH) were increased approximately 11-fold and the severity of
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morphological changes (centrilobular vacuolar degeneration and hepatocellular necrosis) was
greater in the glutathione-depleted animals. Serum and urinary markers of renal damage were
also significantly increased by glutathione depletion. Renal tubule necrosis was observed only in
the glutathione-depleted group. Overall, glutathione depletion appeared to enhance hepatotoxicity
more than nephrotoxicity, an effect that was attributed to organ-specific differences in
bromodichloromethane metabolism. The addition of glutathione to reaction mixtures of rat
hepatic or renal microsomal fraction and radiolabeled bromodichloromethane resulted in 92% and
20% reductions in protein binding to bromodichloromethane, respectively. The difference in
response to glutathione addition was interpreted as evidence for existence of different metabolic
pathways in liver and kidney. Bromodichloromethane binding to lipid in liver microsomes under
anaerobic conditions was decreased in the presence of glutathione, suggesting that glutathione can
react with the dihalomethyl radical.
In contrast to the apparent protective role of glutathione described above, studies in strains
of S. typhimurium that express rat theta class glutathione S-transferase suggest that conjugation
with glutathione leads to formation of mutagenic metabolites (Pegram et al., 1997; DeMarini et
al., 1997, Landi et al., 1999b). These studies are described in greater detail in Section V.F.I.
Proposed pathways for generation of the mutagenic species are outlined in Figure III-2, also
located in Section III.C.I. Briefly, similar mutational specificity, site specificity, and mutation
spectra for the three brominated trihalomethanes support the conclusion that they are activated by
one or more common pathways. In contrast, the data do not support a glutathione S-transferase
mediated pathway for the structurally-related trihalomethane chloroform. This finding suggests
that chloroform and the brominated trihalomethanes may in some instances be metabolized by
different pathways.
C. Mode of Action of Carcinogenesis
Administration of individual brominated trihalomethanes has been associated with
formation of liver tumors (bromodichloromethane, dibromochloromethane), kidney tumors
(bromodichloromethane), and tumors of the large intestine (bromodichloromethane, bromoform)
in some experimental animals. The mode of action by which brominated trihalomethanes induce
tumors in laboratory animals is not known. However, two general modes of action have been
proposed: 1) formation of DNA adducts resulting from interaction with one or more classes of
reactive metabolites and 2) production of cytotoxicity coupled with regenerative hyperplasia.
The production of reactive metabolites from trihalomethanes is well-established. Classes
of reactive metabolites produced include dihalocarbonyls produced by oxidative metabolism and
and dihalomethyl radicals produced by reductive metabolism. Additional evidence suggests that
reactive species can be also formed via glutathione conjugation (DeMarini et al., 1997; Pegram et
al., 1997). Detection of adduct formation and consistent evidence of DNA reactivity in standard
assays are two lines of experimental evidence that would strongly support the adduct formation
hypothesis. At present there are no in vivo data available on DNA adducts resulting from
metabolism of brominated trihalomethanes. DNA reactivity can be inferred from test results of
mutagenic and genotoxic potential. As noted previously (U.S. EPA, 1994b), synthesis of the
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overall weight of evidence for genotoxicity of individual brominated trihalomethanes 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, and evidence of
mutagenicity is considered adequate for these chemicals. Study results for the mutagenicity of
dibromochloromethane are mixed, and the overall evidence for mutagenicity of this chemical is
judged to be inconclusive (U.S. EPA, 1994b).
Alternatively, the induction of tumors by individual brominated trihalomethanes could
involve an epigenetic mode of action. Induction of tumors in animal studies has been noted to
occur primarily at sites where cytotoxicity was observed (i.e., liver and kidney), and there appears
to be a correlation between hepatotoxicity and liver tumorigenicity of brominated trihalomethanes
in mice (bromodichloromethane > dibromochloromethane > bromoform) (U.S. EPA, 1994b).
This raises the possibility that regenerative hyperplasia caused by the cytotoxic effects of
brominated trihalomethanes may contribute to the tumorigenic potential of these chemicals. A
brief review of studies that have evaluated regenerative hyperplasia following exposure to
brominated trihalomethanes is provided below.
A number of studies have measured cell proliferation in liver and/or kidney following
exposure to brominated trihalomethanes. Miyagawa et al. (1995) observed evidence for the
induction of hepatocyte proliferation in male B6C3Flmice following a single oral gavage dose of
dibromochloromethane in corn oil at the maximum tolerated dose (MTD) or at one half the MTD
(200 or 400 mg/kg). Proliferation was assessed by incorporation of [3H]- thymidine using the in
vivo-in vitro replicative DNA synthesis assay at 24, 39, and 48 hours postexposure.
Potter et al. (1996) investigated cell proliferation in the kidney of male F344 rats. Test
animals received 0.75 or 1.5 mmol/kg of bromodichloromethane in 4% Emulphor® by gavage for
1, 3, or 7 days. The administered doses corresponded to 123 or 246 mg/kg-day for
bromodichloromethane, 156 or 312 mg/kg-day for dibromochloromethane, and 190 or 379
mg/kg-day for bromoform. Cell proliferation in the kidney was assessed in vivo by [3H]-
thymidine incorporation. No statistically significant effect of bromodichloromethane on tubular
cell proliferation was observed following exposures of up to 7 days, although high labeling levels
were observed in 3 of 4 rats at the 246 mg/kg-day dose of bromodichloromethane.
NTP (1998) evaluated cell proliferation in the kidney and liver of Sprague-Dawley rats as
part of a short-term reproductive and developmental toxicity screen of bromodichloromethane.
The compound was administered in drinking water for 35 days. Groups of male and female rats
were exposed to drinking water concentrations of 0, 100, 700 and 1300 ppm
bromodichloromethane using the study design described in Table V-6 (Section V.D.I). BrdU
labeling index (LI) was unchanged in the livers and kidneys of Group B males at doses up to 69
mg/kg-day. A small but statistically significant increase in the LI was noted in the livers and
kidneys of Group C females in the 1300 ppm dose group (126 mg/kg-day). The study authors
noted that the result in females was probably biologically insignificant.
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Melnick et al. (1998) exposed female B6C3FJ mice (10/dose) to bromodichloromethane,
dibromochloromethane, or bromoform in corn oil via gavage for 3 weeks (5 days/week). BrdU
was administered to the animals during the last 6 days of the study, and hepatocyte labeling index
(LI) analysis was conducted. Time-adjusted doses of 107, 336, and 357 mg/kg of
bromodichloromethane, dibromochloromethane, and bromoform, respectively, resulted in
significantly elevated hepatocyte proliferation as measured by the LI. The authors compared the
dose response for liver toxicity (including hepatic enzyme activity and LI data) and
tumorigenicity (using data from previously published NTP bioassays) for the brominated
trihalomethanes using the Hill equation model. This analysis indicated that the shape of the dose
response as well as the Hill exponents were different for liver toxicity and tumorigenicity. The
authors concluded that these data do not support a causal relationship between liver
toxicity/reparative hyperplasia and tumor development.
Coffin et al. (2000) examined the effect of bromodichloromethane,
dibromochloromethane, or bromoform administered by corn oil gavage or in drinking water on
cell proliferation and DNA methylation in the liver of female B6C3F1 mice. Administration of
all three brominated trihalomethanes by gavage or in drinking water decreased methylation of the
c-myc gene. The LOAELs identified for liver toxicity and increased cell proliferation in animals
administered in corn oil were 150-,, 100-, and 200 mg/kg , all the lowest doses tested, for
bromodichloromethane, dibromochloromethane, and bromoform, respectively. The
histopathology findings for animals receiving bromoform in the drinking water were similar to
those observed in the lowest-dose gavage group. The results of the single-dose drinking water
experiment suggest slightly lower LOAELs for liver toxicity of 138, 171- and 301 mg/kg-day for
bromodichloromethane, dibromochloromethane, and bromoform, respectively. It had previously
been shown that bromodichloromethane and dibrmomchloromethane administered by corn oil
gavage at 75- or 150 mg/kg-day and 50- and 100 mg/kg-day, respectively, caused significant
dose-dependent increases of helpatocellular adenoma and adenocarcinoma female mice,(NTP
1987, NTP 1985). Bromoform administered in corn oil at 100- or 200 mg/kg-day is not
carcinogenic to female B6C3F1 mice (NTP 1989a).
Torti et al. (2001) conducted 1-week and 3-week inhalation exposure studies of
bromodichloromethane in wild type and transgenic mice. Bromodichloromethane toxicity was
transient. Regenerative lesions and increased labeling index were evident in the kidney cortex of
mice exposed to concentrations of 10 ppm and above for one week. After three weeks of
bromodichloromethane exposure, damaged areas of kidney cortex were entirely regenerated
(residual scarring was present) and labeling index measurements had returned to near baseline
levels. The study authors noted that these results are in contrast to those observed in similar
experiments performed with chloroform, where treatment of F344 rats and B6C3FJ mice resulted
in continued cytotoxicity and elevated cell turnover for up to 90 days (Larson et al., 1996;
Templin et al., 1996). The mechanistic basis for these different responses to structurally similar
compound is unclear, but may reflect an induced change in metabolism or emergence of a
resistant cell population in animals treated with bromodichloromethane.
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George et al. (2002) reported that exposure of male F344/N rats to bromodichloro-
methane in drinking water for two years at a level that significantly enhanced the prevalence and
multiplicity of hepatocellular adenomas and carcinomas had no effect on hepatocellular
proliferation. In the same study, the prevalence of renal tubular hyperplasia, but not tumor
incidence, was significantly increased at the high dose.
Lock et al. (2004) administered bromodichloromethane by gavage in corn oil to male
F344 rats (5/group) at doses of 0, 50, or 100 mg/kg and male B6C3F1 mice (6/group) at doses of
0, 25, or 50 mg/kg-day for 5 days per week over a 28-day period. No change was observed in
body weight or clinical chemistry markers of liver or kidney injury in exposed rats and mice.
Kidney histopathology was not altered in mice, but mild renal tubule injury was observed in 2 of
5 rats exposed to the highest dose of bromodichloromethane (100 mg/kg-day). Increased cell
proliferation was observed in all rats exposed to the highest dose, but was not seen in low-dose
rats or in mice at either dose level. It had previously been shown does-dependent increase in
incidences of large intestine and kidney tumors in male rats exposed at 50 or 100 mg/kg-day in
dose-dependent manner (NTP 1987). It also had previously been shown does-dependent increase
in incidences of kidney tumors in male mice exposed at 25 or 50 mg/kg-day in dose-dependent
manner (NTP 1987).
Based on an extensive evaluation of carcinogenicity data, cytotoxicity coupled with
regenerative hyperplasia is considered the primary mode of action for tumor formation following
exposure to high concentrations of chloroform, a structurally-related trihalomethane which has
low genotoxic potential (U.S. EPA, 2000c). However, two lines of evidence suggest that
chloroform is not a prototypical trihalomethane. First, the weight-of-evidence for at least two of
the brominated trihalomethanes indicates that they are genotoxic. This contrasts with the
negative weight of evidence evaluation for chloroform. Second, there is evidence that the
brominated trihalomethanes are readily bioactivated to mutagenic products via a glutathione S-
transferase mediated pathway, while chloroform is bioactivated only at very high concentrations.
Therefore, a common mode of action for carcinogenicity of chloroform and brominated
trihalomethanes cannot be assumed on the basis of current experimental evidence. Data to
support a nonlinear primary mode of action for tumor development in liver, kidney, and large
intestine are currently lacking for the brominated trihalomethanes. In the absence of such
information, combined with a positive weight-of-evidence evaluation for genotoxicity, the mode
of action for tumor development is assumed to be a linear process.
D. Interactions and Susceptibilities
1. Potential Interactions
The toxicity of the brominated trihalomethanes appears to result from cytochrome P450-
mediated metabolism to reactive metabolites (U.S. EPA, 1994b). Therefore, agents which
increase or decrease the activity of enzymes responsible for metabolism of brominated
trihalomethanes may modify toxicity. Pankow et al. (1997) observed that pretreatment with
isoniazid or m-xylene (inducers of CYP2E1 and CYP2B1/CYP2B2, respectively) increased the
VII - 5 November 15, 2005
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hepatotoxicity of dibromochloromethane in male rats, as measured by elevated serum leucine
aminopeptidase activity. Hewitt et al. (1983) observed that pretreatment with acetone, a CYP2E1
inducer, potentiated the acute toxicity of bromodichloromethane and dibromochloromethane in
male rats. Thornton-Manning et al. (1993) also found that pretreatment with acetone potentiated
the acute hepatotoxicity of bromodichloromethane in male rats. Conversely, the cytochrome
P450 inhibitor 1-aminobenzotriazole prevented bromodichloromethane-induced hepatotoxicity in
rats (Thornton-Manning et al. 1993). Current findings regarding the existence of glutathione-
mediated pathways for brominated trihalomethane metabolism (see sectionV.E.l) suggest that
treatments or agents which alter glutathione-^-transferase activity may potentially modify the
toxicity of brominated trihalomethanes.
The severity of brominated trihalomethane toxicity is potentially affected by the vehicle of
administration. Vehicle effects are well-documented in the toxicity of chloroform (e.g., Bull et
al. 1986; Jorgenson et al. 1985) and there is some evidence that similar effects occur with
brominated trihalomethanes. 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) (Lilly et al. 1994).
2. Greater Childhood Susceptibility
A growing body of scientific evidence indicates that children may suffer
disproportionately from some environmental health risks. These risks may arise because the
neurological, immunological, and digestive systems of children are still developing (U.S. EPA,
(1998a). In addition, children may incur greater exposure because they eat more food, consume
more fluids, and breathe more air in proportion to their body weight when compared to adults
(U.S. EPA, 1998a).
U.S. EPA (1998a) recently identified three key questions to consider when evaluating
health risks in children from exposure to drinking water disinfection byproducts (DBF) such as
the brominated trihalomethanes:
• Is there information which shows that the disinfectant or DBF causes effects in the
developing fetus or impairs ability to conceive and bear children? If it causes this
type of problem will it occur at a lower dose than that which will cause other types
of effects?
If the disinfectant or DBF causes cancer, are children more likely to be affected by
it than are adults?
• If the disinfectant or DBF causes some noncancer toxic effect, are children more
likely to be affected by it than are adults?
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The data available for evaluation of these issues as they relate to brominated trihalomethanes are
addressed below.
a. Effects on the fetus and ability to conceive and bear children
General Results from Animal Studies
Animal studies on the reproductive and developmental effects of brominated
trihalomethanes are summarized in Section V.E. At the present time, there are limited studies on
dibromochloromethane with one reported developmental effects at doses that were comparable to
those of maternal toxicity. There are two studies reported developmental toxicity of bromoform.
One study reported developmental effects at dose lower than the level that caused maternal
toxicity. There is no data that indicates that either of these chemicals impairs ability to conceive
and bear offsprings. Studies of these chemicals in animals indicate that reproductive and
developmental effects occur at doses slightly higher than those observed to cause liver and renal
effects.
Bromodichloromethane has the most extensive database for developmental and
reproductive effect among the brominated trihalomethanes. Study results for the reproductive
and developmental effects of bromodichloromethane are mixed. No reproductive or
developmental effects were observed at doses up to approximately 116 mg/kg-day in females or
68 mg/kg-day in males in studies conducted in Sprague-Dawley rats (NTP, 1998). Adverse
reproductive or developmental effects were not observed in rabbits exposed to doses as high as 55
or 76 mg/kg-day in drinking water on gestation days 6 to 29 (CCC, 2000c,d; Christian et al.,
2001a, b). Increased incidences of sternebral aberrations (Ruddick et. al., 1983) and decreased
ossification sites in the forelimb and hindlimb (CCC, 2000b; Christian et al., 2001a) have been
observed in Sprague-Dawley rats administered bromodichloromethane in corn oil and drinking
water, respectively, at doses which induced maternal toxicity.
Reproductive effects of bromodichloromethane have been noted in rodent assays.
Klinefelter et al. (1995) observed effects on sperm motility in rats administered 39 mg/kg-day in
drinking water for 52 weeks, but these effects were not accompanied by histopathological
changes in male reproductive tissues. Narotsky et al. (1997) observed a significantly increased
incidence of full litter resorption (FLR) in F344 rats treated with 50 or 75 mg/kg-day
bromodichloromethane by aqueous gavage throughout the period of organogenesis. This effect
was described as an all-or-nothing phenomenon, in that the litter was either fully resorbed or
appeared normal at parturition. This pattern was interpreted by the study authors as evidence for a
maternally-mediated mechanism, rather than a direct effect of bromodichloromethane on the
developing embryo. Bielmeier et al. (2001) observed increased incidence of FLR in F344 rats
treated with 75 or 100 mg/kg-day bromodichloromethane by aqueous gavage on one or more days
during the interval from gestation day 6 to 10. This response was strain-specific and FLR was not
observed in Sprague-Dawley rats treated with up to 100 mg/kg-day on gestation days 6 tolO.
Analysis of hormone profiles reported by Bielmeier et al. (2001, 2004) suggest that
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bromodichloromethane disrupts normal endocrine function in F344 rats, either by inhibiting LH
secretion or by decreasing luteal responsiveness to LH.
Data from Epidemiological Studies
No information on developmental and reproductive effects is available from human
studies that examined populations exposed exclusively or primarily to brominated
trihalomethanes. Several studies of exposure to chlorinated water have identified potential
associations between bromodichloromethane intake or concentration in tap water and adverse
health effects. These studies have examined associations between exposure to
bromodichloromethane in drinking water and adverse pregnancy outcomes (low birth weight,
prematurity, intrauterine growth retardation, spontaneous abortion, still birth) and reproductive
function (sperm quality, menstrual cycle function). Summaries of these studies are provided in
Section VI.B.l. Weak, but significant, associations have been noted for 1) ingestion of >18 |ig/L
during the first trimester of pregnancy and increased risk spontaneous abortion (adjusted OR 2.0,
95% C.I. 1.2, 3.5; adjusted OR with adjustment for other trihalomethanes 3.0, 95% C.I. 1.4, 6.6)
(Waller et al., 1998); 2) exposure to concentrations>20 |ig/L and increased risk of stillbirth
(adjusted OR 1.98, 95% C.I. 01.23, 3.49) (King et al., 2000); and 3) exposure to
concentrations>20 |ig/L and increased risk of neural tube defects (Dodds and King, 2001).
In epidemiological studies of reproductive function, exposure to bromodichloromethane in
tap water was associated with decreased menstrual cycle length (Windham et al., 2003). The
observed decrease at the highest quartile of exposure (> 16 |ig/L) was less than one day (-0.74
days, 95% C.I. -1.5, -0.02). The decrease in overall cycle length appeared to result from
decreased follicular phase length (-0.80, 95% C.I. -1.5, -0.08), suggesting that the
bromodichloromethane has an effect on ovarian function. In a study of semen quality, exposure
to bromodichloromethane was associated with significantly decreased sperm linearity (- 0.09 ±
0.04 per unit increase in bromodichloromethane concentration), but associations with other semen
quality or sperm motility parameters were not observed (Fenster et al, 2003).
Modes of action have not been proposed to explain the responses attributed to
bromodichloromethane in the epidemiological studies described in Section VI.B.l. The results of
in vitro studies (see Section V.H.2) using differentiated human placental trophoblasts in primary
culture suggest that the placenta is a target tissue for adverse effects of bromodichloromethane in
humans and that this could be related to adverse effects on human pregnancy (Chen et al., 2003,
2004). Exposure of primary cultured trophoblasts to 0.02-2000 |j,M bromodichloromethane in the
culture medium significantly decreased secretion of immunoreactive and bioactive chorionic
gonadotropin (CG), a glycoprotein that plays a major role in maintenance of the human conceptus
during early pregnancy. The events at the molecular and biochemical level that cause decreased
secretion are unknown, but could include direct effects on synthesis (e.g., glycosylation or
dimerization) or indirect effects via disruption of gonadotropin releasing hormone (GnRH)
activity (a putative major regulator of placental CG synthesis and secretion). If substantiated, an
effect on GnRH would parallel one of the proposed mechanisms for pregnancy loss in F344 rats
(Bielmeier et al. 2001; 2004).
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Full Litter Resorption in F344 Rats
The results of the Narotsky et al. (1997) and Bielmeier et al. (2001) studies identify FLR
as the most sensitive female reproductive response among the currently available animal studies.
Strain-specific FLR observed in F344 rats exposed to bromodichloromethane is of concern
because associations have been noted between exposure to bromodichloromethane in tap water
and adverse pregnancy outcomes (spontaneous abortion, stillbirth) in epidemiological studies.
There are two issues to consider regarding the relevance of FLR observed in F344 rats to human
health. The first issue is whether the mechanism responsible for this effect in rats is applicable in
humans. Although the exact mechanism of FLR in F344 rats is unknown, the high incidence of
FLR during the LH-dependent period of pregnancy and the lack of response thereafter suggests
an LH-mediated mechanism. Serum progesterone levels were reduced at 12 and 24 hours after
dosing in all rats that experienced FLR. Because LH is required to stimulate progesterone
secretion by the corpus luteum during the period of pregnancy examined by Bielmeier et al.
(2001), these data are consistent with an effect of bromodichloromethane on LH secretion and/or
single transduction. Although the hormone profile data collected by Bielmeier et al. (2001) did
not detect an effect on LH levels, subsequent work with a more sensitive assay demonstrated
reduced LH levels following bromodichloromethane exposure (Bielmeier et al., 2004). This
result supports an effect on LH secretion. Although species differences exist, rats and humans
are similar in that either LH (in rats) or chorionic gonadotropin (hCG; in humans) is required at
specific periods for maintenance of pregnancy (Bielmeier et al., 2001; 2004). LH and hCG act
via the same receptor in rats and humans. Thus, if bromodichloromethane reduces luteal response
to LH in F344 rats by inhibition or down-regulation of the LH/hCG receptor, then it might also be
expected to have effects on hCG signal transduction in humans. If this were the case,
bromodichloromethane would be expected to increase the risk of early termination of pregnancy
in humans.
An alternative hypothesis is that bromodichloromethane causes FLR by a mechanism that
involves alteration of maternal LH secretion. Bielmeier et al. (2004) demonstrated reduced LH
serum levels in pregnant F344 rats treated with bromodichloromethane, indicating an effect on
LH secretion. Gonadotropin releasing hormone (GnRH) secreted by the hypothalamus regulates
LH secretion in the rat. GnRH from the cytotrophoblast is thought to regulate hCG secretion in
the human placenta. An effect of bromodichloromethane on hCG secretion by human
cytotrophoblast cells has been demonstrated in vitro (Chen et al., 2003, 2004). Although the
mode of action underlying this in vitro effect is unknown, it is possible that
bromodichloromethane could inhibit hCG secretion in a manner analogous to LH secretion. If so,
bromodichloromethane would be expected to increase the risk of early termination of pregnancy.
Even if this were not the case, interference of bromodichloromethane with maternal LH secretion
in humans would be expected to have other adverse effects on reproductive function in the
female, whether or not it was able to cause an event similar to full-litter resorption. It should be
noted that a possible contributing effect of bromodichloromethane on luteal responsiveness to LH
has not been ruled out.
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The second issue to consider in evaluating these data concerns the use of the F344 strain
as a model for reproductive effects in humans. The Sprague-Dawley rat is often identified as the
preferred rodent model in reproductive and developmental toxicity testing. However, this
preference is based on pragmatic considerations (high fecundity, favorable maternal behavior,
availability of extensive historical control data for reproductive and developmental endpoints),
and is not based on any evidence that there are important physiological or biochemical
differences between young adults of these strains or that one is strain is inherently a more relevant
biological model for human reproductive health than the other. Thus, the fact that this response
has been observed in F344 rats but not in Sprague Dawley rats is not a reason to consider that the
effect may not be applicable to humans. Based on these considerations, in accordance with U.S.
EPA policy, the results of this study are considered relevant to human health in the absence of
data that demonstrate otherwise.
b. Childhood Cancer and Noncancer Effects
Bioactivation to reactive metabolites is an apparent prerequisite for toxicity and
carcinogenicity of the brominated trihalomethanes. Therefore, an important issue in the
assessment of childhood risk of cancer and other adverse effects is whether the enzymes
responsible for metabolism are more active in fetuses, neonates, and or children than in adults.
This section evaluates the available data for developmental expression and/or activity of three key
metabolizing enzymes that are known or anticipated to bioactivate the brominated
trihalomethanes: CYP2E1, CYP2B1/2 (in rodents only), and glutathione-S-transferase theta.
CYP2E1
Carcinogenicity of brominated trihalomethanes has been shown to be at least partly
related to bioactivation by the cytochrome P450 isoform CYP2E1 (U.S. EPA, 1994b). Thus, a
higher level of CYP2E1 activity in children relative to adults might predispose children to greater
toxicity. Studies of human fetal liver have produced contradictory results, but suggest that
CYP2E1 protein is either not expressed or is expressed at levels lower than in adults (Hakkola et
al., 1998). Carpenter et al. (1996) detected immunoreactive CYP2E1 protein in liver samples
from fetuses ranging from 16 to 24 weeks in gestational age. The samples obtained were from
fetuses whose mothers did not have a history of alcohol use. The immunoreactive protein
exhibited a slightly lower molecular weight than observed for CYP2E1 from adult liver samples.
Expression of the corresponding mRNA was confirmed in a fetal liver sample of 19 weeks
gestational age by reverse transcriptase-polymerase chain reaction (RT-PCR). However,
CYP2E1 mRNA was not detectable in a fetal liver sample of 10 weeks gestational age,
suggesting (in the opinion of the study authors) that CYP2E1 expression may be related to
specific stages of fetal development. The catalytic capability of CYP2E1 protein in human fetal
microsomes was demonstrated by measuring the rate of ethanol oxidation to acetaldehyde. The
rate of conversion varied from 12 to 27% of that measured in adult microsomes. Treatment of
fetal hepatocytes in primary culture with ethanol or clofibrate indicated that fetal CYP2E1 protein
is inducible (approximately two-fold compared to untreated cells).
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Vieira et al. (1996) detected small amounts of CYP2E1 mRNA in fetal liver samples
(approximately 5 to 10% of the levels in adult liver) collected from fetuses aged 14 to 40 weeks.
However, these authors could not detect immunoreactive CYP2E1 protein in any of 27 fetal liver
samples Other studies have failed to detect either CYP2E1 protein or mRNA in fetal liver
samples. Cresteil et al. (1985) and Komori et al. (1989) did not detect immunoreactive protein or
mRNA in fetal liver samples of less than 16 weeks gestational age. Jones et al. (1992) did not
detect CYP2E1 mRNA or protein in liver samples that were of similar gestational age (16 to 18
weeks) to the samples examined by Carpenter et al. (1996). Juchau and Yang (1996) did not
detect CYP2E1 mRNA by RT-PCR in human embryonic tissues between days 45 and 60 of
gestation. The factors contributing to the different results are unknown, but may include inter-
individual variability, gestational age of the tissue examined (for the samples less than 16 weeks
gestational age), or the existence of factors other than developmental stage that control
expression.
Information on the presence of CYP2E1 in human fetal tissues other than the liver is
limited. Vieira et al. (1998) examined the mRNA content of human fetal lung and kidney.
CYP2E1 mRNA was expressed at a very low level in both tissues and the levels remained stable
after birth. Studies of human fetal brain tissue indicate that CYP2E1 is expressed in human
embryonic brain tissue (see Juchau et al., 1998) and that relatively low levels of CYP2E1 mRNA,
immunoreactive protein, and catalytically active protein are present during the early fetal period
of development (Brzezinski et al., 1999). In one study, a dramatic increase in CYP2E1 was
observed at approximately gestation day 50, and a fairly constant level was maintained until at
least day 113 (Brzezinski et al., 1999). The relevance of the data for lung and brain is uncertain,
since these organs are not known to be targets for brominated trihalomethane toxicity.
Vieira et al. (1996) investigated age-related variations in human CYP2E1 protein levels
and catalytic activity from birth through adulthood. These authors observed a rapid increase in
the immunoreactive CYP2E1 microsomal content within 24 hours after birth that was
independent of the gestational age of the newborn. This activation was accompanied by a
demethylation of cytosine residues in the 5'-regulatory region of the gene, suggesting that
methylation of specific residues prevents transcription in the fetal liver. The CYP2E1 protein
level gradually increased during the first year and reached the adult level in children aged 1 to 10
years. CYP2E1 catalytic activity was assessed by determination of in vitro hydroxylation of
chlorzoxazone in 89 microsomal preparations. Chlorzoxazone hydroxylation activity increased
within 24 hours after birth and steadily increased during the first year. Catalytic activity reached
adult levels at age 1 to 10 years.
Animal studies of CYP2E1 expression during development have given variable results.
One study indicated that CYP2E1 is expressed in the fetal rat liver and placenta and that levels
are increased in rat pups exposed to ethanol in utero or via lactation (Carpenter et al., 1997).
Liver samples from rat fetuses exposed to ethanol in utero showed a 2.4-fold increase in protein
levels and 1.5-fold increase in catalytic activity (Carpenter et al., 1997). Other authors have
reported that hepatic CYP2E1 gene transcription in rats is activated at birth and that the amount
VII-11 November 15, 2005
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of CYP2E1 reaches a peak prior to weaning (see Ronis et al., 1996). The protein level then falls
to approximately 25% of the peak level and remains stable into adulthood (Ronis et al., 1996).
The regulation of CYP2E1 is complex when examined at both the molecular (Lieber
1997) and physiological (Ronis et al. 1996) levels. The factors and processes responsible for the
increase in CYP2E1 protein levels and activity at birth have not been clearly identified. At the
physiological level, there is some evidence from rodent studies to suggest that growth hormone
regulates the constitutive expression of CYP2E1 (Ronis et al., 1996). The reduction of CYP2E1
from peak levels before weaning is reported to coincide with the increased levels of growth
hormone and with development of adult levels of growth hormone receptors (Ronis et al., 1996).
The occurrence of peak expression after birth has been attributed to a role of CYP2E1 in
gluconeogenesis, since there is a very high demand for energy production from glucose at this
developmental stage (see Ronis et al. 1996; Vieira et al. 1996).
CYP2B1/2 (Rodents^
Research conducted by Pankow et al. (1997) suggests that the closely-related CYP
isoforms 2B1 and 2B2 participate in the catabolism of dibromochloromethane in rats. These
isoforms show greater than 97% homology of amino acid sequence and have highly similar
genomic organization. To date, these isoforms have not been reported in adult or fetal human
tissues (Nelson et al., 1996; Juchau et al., 1998). Omiecinski et al. (1990) detected low levels of
CYP2B isoform mRNA in fetal rat liver on gestation day 15 (the earliest day in development
when the authors were able to macroscopically recognize and dissect the fetal liver) using the
polymerase chain reaction (PCR). Although the levels of mRNA expression were "substantially
lower" lower at day 15 than observed later in development, expression was clearly inducible by
pretreatment of pregnant rats with phenobarbital. Both constitutive and phenobarbital-induced
levels of mRNA increased with developmental age, reaching maximal levels at approximately
three weeks postpartum. No measurements of CYP2B activity were made in this study, so it is
not known whether changes in mRNA levels were paralleled by changes in catalytic activity.
Juchau et al. (1998) reviewed a series of experiments that employed the selective substrate
probe pentoxyresorufin to test for CYP2B1/2 catalytic activity in fetal rat tissues. The overall
conclusion upon examination of all results was that if CYP2B isoforms are expressed in fetal rats,
they occur at biologically insignificant levels. Asoh et al. (1999) examined the catalytic activity
of CYP2B isoforms in fetal rat liver and found very low activity, a finding consistent with the
conclusion of Juchau et al. (1998).
Gebremichael et al. (1995) investigated the postnatal developmental profile of CYP2B1 in
Sprague-Dawley rats. CYP2B1 activity was detectable as early as seven days postnatally and
exhibited a variable pattern of expression (no clear trend evident) when assayed at days 14, 21,
50, and 100. Asoh et al. (1999) examined the induction of CYP2B isoforms in neonatal rats. The
level of CYP2B catalytic activity was markedly higher at five days after birth relative to levels
observed in fetal hepatic tissue. Oral or intraperitoneal administration of phenobarbital to
pregnant rats increased the level of CYP2B expression and activity in neonates. Overall, these
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findings suggest that CYP2B isoform activity is likely to be lower in fetuses than in neonates or
adults and that increased levels of activity may be observed in fetuses and neonates exposed to
inducing xenobiotics. The significance of this information for risk of cancer in human fetuses,
neonates, and children is uncertain since, as noted above, the CYP2B1/2 isoforms have not been
identified in humans.
Glutathione S-Transferase Theta
Recent mutagenicity studies suggest that brominated trihalomethanes can also be activated
to mutagens by the product of the glutathione S-transf erase (GST) theta gene GSTT1-1 (DeMarini
et al.,1997; Landi et al., 1999b). Children and the fetus could potentially experience increased
risk of adverse effects if the activity of this enzyme was higher at these life stages than in adults.
Information on the developmental expression of GST genes is currently limited. Although other
classes of GSTs (alpha, mu, and pi) are expressed in fetal liver, Mera et al. (1994) reported that
theta-class GSTs were expressed in only adult liver. This finding suggests that the fetus does not
experience increased risk as a result of GST theta-mediated mutagenicity. The occurrence of
increased risk in children cannot be evaluated, since postnatal developmental pattern of GST theta
is unknown.
c. Childhood Cancers: Other Considerations
There are no studies that have examined the carcinogenicity of brominated
trihalomethanes in immature animals. Examination of childhood cancer data compiled by the
National Cancer Institute (Ries et al. 1999) indicates that the incidence of hepatic, renal, and
intestinal cancer (the types of cancer observed in animal studies of bromodichloromethane) from
causes other than genetic predisposition are low. Primary neoplasms of the liver are rare in
children younger than 15 years of age. The incidence of hepatocellular carcinoma (the type of
neoplasm observed in mice treated with bromodichloromethane or dibromochloromethane)
decreased in children younger than 15 years of age during the period 1975 to 1995. The
incidence rate of renal carcinoma remained very low (well under one case per million) in children
younger than 15 years during the period 1975 to 1995. Trend data were not available in Ries et
al. (1999) for intestinal cancer. These data do not address cancers that may be initiated in
childhood and manifested in adults.
d. Conclusion
The available evidence for developmental expression of enzymes known to metabolize
brominated trihalomethanes supports the conclusion that children do not experience greater risk
from exposure to these compounds than do adults. At present there are no cancer incidence data
from humans to suggest that brominated trihalomethanes contribute to increased risk of cancer in
children.
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3. Other Potentially Susceptible Populations
a. Subpopulations with altered levels of CYP2E1
CYP2E1 catalyzes the metabolism of brominated trihalomethanes to reactive
intermediates that mediate toxicity. Individuals with higher levels of CYP2E1 activity may
therefore be at greater risk for adverse health effects. This section describes factors associated
with increased levels of CYP2E1 activity and subpopulations who may be at increased risk as a
result of these factors.
Genetic Polymorphisms
Significant inter-ethnic differences exist in CYP2E1 polymorphism (Ronis et al., 1996;
Lieber, 1997) and it is possible that these differences could influence susceptibility to toxic
effects. The CYP2E1 polymorphisms currently reported in the literature are located in the 5'-
flanking (noncoding) regions of the gene, while the coding regions of the gene which specify
sequence appear to be well conserved among various ethnic groups (Ronis et al., 1996).
Mutations in the 5'-region of a gene can affect the regulation of gene expression. The rare mutant
c2 polymorphism of CYP2E1 is reported to be associated with higher transcriptional activity,
protein levels, and catalytic activity than the more common wild type allele (Lieber et al., 1997).
As reported by Lieber et al. (1997), the highest frequency of the c2 allele occurs in the Taiwanese
(0.28) and Japanese (0.19 to 0.27) populations. The frequencies in African-Americans,
European-Americans, and Scandinavians are much lower, generally in the range 0.01 and 0.05.
Efforts to link the occurrence of the c2 allele to higher rates of CYP2E1-mediated liver disease
have yielded inconsistent results. Thus, the functional significance of CYP2E1 polymorphism is
presently uncertain, and no conclusion can as yet be drawn about the relative risk for different
ethnic populations exposed to brominated trihalomethanes.
Altered Physiological or Health States
The physiological functions of CYP2E1 include lipid metabolism and ketone utilization
(Lieber, 1997). Induction of CYP2E1 is observed in many conditions that elevate circulating
levels of lipids, including consumption of a high-fat or low-carbohydrate diet, starvation, obesity,
and insulin-dependent diabetes. Among the individuals likely to be affected by such conditions,
diabetics constitute the most clearly-defined susceptible population. Induction of CYP2E1 in
uncontrolled insulin-dependent diabetes is well-studied. In animals, this induction results in
elevated levels of CYP2E1 in the liver, kidney, and lung (loannides et al., 1996). Acetone (a
substrate of CYP2E1) is thought to be the inducing compound (Ronis et al., 1996). As a result of
induction, diabetic animals are more susceptible to the toxicity of some chemicals metabolized by
CYP2E1. While there are no specific data for the brominated trihalomethanes, this phenomenon
has been demonstrated for other halogenated compounds including chloroform, carbon
tetrachloride, trichloroethylene, and bromobenzene (loannides et al., 1996). Because the animal
and human orthologues of CYP2E1 show similar substrate specificity and bioactivation potential,
it is possible that diabetic humans may also be more susceptible to CYP2E1-mediated toxicity.
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As CYP2E1 levels are reduced by insulin treatment, increased toxicity would be anticipated only
in poorly controlled or uncontrolled diabetics (loannides et al., 1996).
Alcohol consumption
CYP2E1 contributes to the metabolism of ethanol in humans and animals. Consumption
of ethanol induces CYP2E1 and chronic alcohol consumption is reported to result in as much as a
10-fold induction (Lieber, 1997). Hence, concurrent exposure to ethanol and brominated
trihalomethanes may increase susceptibility to adverse health effects. This interaction is of
concern because concurrent exposure to brominated trihalomethanes and ethanol is likely to occur
in a significant number of people. At present, there are no human or animal studies which
examine this interaction for brominated trihalomethanes. However, Wang et al. (1994) reported
that a single 100 mg/kg oral dose of ethanol administered to rats significantly increased the
toxicity of the structurally-related trihalomethane chloroform (also metabolized by CYP2E1).
Lieber (1997) noted that the hepatotoxicity of commonly used industrial solvents (e.g. carbon
tetrachloride, bromobenzene, and vinylidene chloride) and anesthetics (enflurane and halothane)
was increased in heavy drinkers, with a pattern of damage that was consistent with the selective
expression and induction of CYP2E1 in certain regions of the liver.
Concurrent exposure to other CYP2E1 inducers including pharmaceuticals
Because CYP2E1 is highly inducible by a wide range of xenobiotic compounds, prior
exposure to such inducers may potentially play a significant role in brominated trihalomethane
toxicity. Known inducers of CYP2E1 include certain therapeutic agents (acetaminophen,
isoniazid), volatile anaesthetics (halothane, isoflurane), and solvents (acetone, benzene, carbon
tetrachl oride, trichloroethylene) (Raucy, 1995). Individuals exposed to or consuming these
inducers on a regular basis may therefore be at greater risk for brominated trihalomethane
toxicity.
b. Subpopulations with altered levels of glutathione S-transferase theta
Individuals with Genetic Polymorphisms
Genotoxicity studies in bacteria (discussed in section V.F. 1) indicate that brominated
trihalomethanes can be activated to mutagens by the product of the glutathione S-transferase theta
gene GSTT1-1 (DeMarini et al.,1997; Landi et al., 1999b). If similar pathways for bioactivation
exist in humans, GSTT1-1 polymorphism may influence susceptibility to brominated
trihalomethane-mediated toxicity. GSTT1-1 is characterized by a deletion polymorphism which
results in total loss of glutathione S-transferase-6 activity in individuals (10 to 60% of the
population depending upon ethnicity and race) homozygous for the null genotype (GSTT1-1'1').
Individuals who are heterozygous for GSTT-1 (GSTT1-T1') have intermediate levels of enzyme
activity, while individuals homozygous for GSTT-1 (GSTT1-1+I+) have the highest levels. Landi
et al. (1999b) have suggested that GSTT1-1+/+ individuals may experience excess genotoxic risk
when exposed to brominated trihalomethanes, particularly in organs which express glutathione-^-
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transferase-theta and come in direct contact with brominated trihalomethanes. Potential target
sites would include the gastrointestinal tract and the bladder.
Concurrent Exposure to Inducers
If G-STr-7-mediated pathways for bioactivation of brominated trihalomethanes exist in
humans, factors which induce this enzyme may increase the risk of adverse health effects from
exposure. Although GSTT-1 is constitutively expressed, the level of its expression can be altered
by exposure to exogenous chemicals. Landi (2000) has summarized information on factors which
increase expression of the enzyme. In rats, aspirin increased GSTT-1 levels in the colon. Alpha-
tocopherol, coumarin; and other anticarcinogenic drugs increased gastric and esophageal levels;
and indole-3-carbinol and coumarin increased GSTT-1 levels in the liver. In mice, phenobarbital
induced hepatic GSTT-1 levels. Data for humans are limited, but there are indications that the
dietary intake of cruciferous vegetables enhances the expression of GSTT-1. It is possible that
consumption of these substances by GSTT-1 positive individuals could result in increased risk of
adverse effects. However, there are presently no data available for evaluation of this hypothesis.
c. Subpopulations with altered levels of putative protective compounds
Glutathione depletion has been observed to increase the hepatotoxicity of
bromodichloromethane (Gao et al., 1996). On the basis of these data, Gao et al. (1996) proposed
that populations with low baseline levels of glutathione (e.g., due to dietary deficiencies of
glutathione precursors such as cysteine and selenium) may be more sensitive to
bromodichloromethane-induced toxicity.
d. Possible gender differences
Apparent gender-related differences in the toxicity of brominated trihalomethanes have
been noted in studies where male and female animals were exposed concurrently (e.g. Aida,
1992a; Daniel, 1990; NTP,1987, 1989a; Tobe et al., 1982). In general, male rats and mice appear
to be somewhat more sensitive to the hepatic and renal toxicity induced by brominated
trihalomethanes than are females, although there are exceptions to this pattern (eg. the chronic
oral exposure study of bromoform conducted by NTP, 1989a in mice and the short-term study of
bromoform conducted by Aida et al., 1992a). While the basis for the apparent greater sensitivity
of males is unknown, the difference may be related to gender-specific differences in the level of
enzymes responsible for bioactivation of brominated trihalomethanes to toxic metabolites, or to
gender-specific differences in cellular protective mechanisms. It is important to note that at
present there is no evidence for gender-related differences in the activity levels of CYP2E1 or
GSTT-1 in humans.
E. Summary
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
VII -16 November 15, 2005
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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
structurally-related 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 mechanism 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 suggested that DNA adducts can be formed through conjugation with
glutathione and bioactivation of the resulting conjugates. Comparison of dose-response data for
liver and kidney toxicity (including cell proliferation) and tumorigenicity in mice and rats
suggests that tumor formation occurs at concentrations lower than those which stimulate cell
proliferation.
Interaction with agents which increase or decrease the activity of enzymes responsible for
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
VII -17 November 15, 2005
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trihalomethanes suggest that treatments or agents which alter glutathione-^-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).
A number of potentially sensitive subpopulations have been identified for health effects of
brominated trihalomethanes. A growing body of scientific evidence indicates that children may
suffer disproportionately from some environmental health risks. These risks may arise because
the neurological, immunological, and digestive systems of children are still developing. In
addition, children may incur greater exposure because they eat more food, consume more fluids,
and breathe more air in proportion to their body weight when compared to adults. U.S. EPA has
identified three key questions to consider when evaluating health risks to children from drinking
water disinfection byproducts (DBF), including the brominated trihalomethanes: 1) Is there
information which shows that the DBF causes effects in the developing fetus or impairs ability to
conceive and bear children? 2) If the DBF causes cancer, are children more likely to be affected
by it than are adults? and 3) If the DBF causes a noncancer toxic effect, are children more likely
to be affected by it than are adults? There are limited available animal studies reported
developmental effects from oral exposure to dibromochloromethane and bromoform. These
developmental effects in animals occurred at doses slightly higher than those which induce
histopathological effects in the liver and kidney. There is no evidence that these compounds
impair the ability to conceive or have offsprings.
Epidemiological studies have found an association between exposure to
bromodichloromethane in drinking water and increased spontaneous abortion and increased
stillbirth. These studies raise concern for human health effects, although the occurrence of
multiple disinfection byproducts in drinking water is a significant source of uncertainty with
resect to the causative agent. The results of in vitro studies using cultured human placental
trophoblasts show that bromodichloromethane can affect hormone secretion and affect the
morphological differentiation, suggesting that the placenta is a possible target of
bromodichloromethane toxicity in humans.
Exposure to bromodichloromethane has been linked to reproductive effects in animals. In
rats, exposure to bromodichloromethane resulted in reduced sperm motility; this effect was not
accompanied by histopathologic changes in the male reproductive system. Exposure of pregnant
F344 rats to bromodichloromethane on one or more days during the luteinizing hormone-
dependent period of gestation causes full litter resorption. This response is not observed in
similarly exposed pregnant Sprague-Dawley rats. The pregnancy loss observed in F344 rats may
result from perturbation of LH secretion or signaling processes.
VII -18 November 15, 2005
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At present, there are no cancer data which indicate that brominated trihalomethanes
contribute to increased risk of cancer in children. No studies were located which examined pre-
or post-pubertal cancer rates in humans in relation to brominated trihalomethane exposure.
Cancer bioassays of brominated trihalomethanes conducted in mice and rats have not used study
designs that included perinatal exposure.
The available evidence suggests that the toxic effects of brominated trihalomethanes are
mediated by the enzymes CYP2E1, CYP2B1/2 (in rodents), and glutathione S-transferase theta
(GSTT-1). The weight of evidence from studies of the developmental expression of these
enzymes supports the conclusion that children do not experience greater risk from brominated
trihalomethane exposure as a result of higher metabolic activity.
In addition to children, other potentially sensitive populations include those with altered
levels or activity of CYP2E1 or GSTT-1 and those with altered levels of glutathione. Factors
contributing to increases in CYP2E1 activity potentially include genetic polymorphisms; altered
physiological or health states; alcohol consumption; and concurrent exposure to other inducers,
including some pharmaceuticals and solvents. Factors contributing to increased GSTT-1 activity
include genetic polymorphisms and concurrent exposure to inducers. Based on observations in
animals, human populations with reduced levels of glutathione as a result of dietary deficiency or
other factors may experience increased sensitivity to the toxic effects of bromodichloromethane.
Apparent gender-related differences in the toxicity of brominated trihalomethanes have
been noted in studies where male and female animals were exposed concurrently. In general,
male rats and mice appeared to be more sensitive than females to liver and renal toxicity,
although some exceptions to this pattern have been noted. There is no evidence for a similar
pattern of gender response in humans.
VII -19 November 15, 2005
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VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
This section quantifies the toxicological effects of brominated trihalomethanes based on
health effects information presented in Sections V and VI. At present, there are two basic
approaches to quantification of toxicological effects: the conventional NOAEL/LOAEL
approach and benchmark dose modeling. Benchmark dose (BMD) modeling (U.S. EPA, 1995;
2000b) was chosen as the preferred approach for quantifying toxicological effects of the
brominated trihalomethanes. BMD modeling avoids several limitations of the NOAEL/LOAEL
approach, including: 1) the slope of the dose-response plays little role in determining the
NOAEL; 2) the NOAEL (or LOAEL) is limited to the doses tested experimentally; 3) the
determination of the NOAEL is based on scientific judgement, and is subject to inconsistency;
and 4) experiments using fewer animals tend to produce larger NOAELs, and as a result may
produce larger health advisories (HAs) or reference doses (RfDs) (U.S. EPA, 1995) that may not
be sufficiently protective of human health. In contrast, benchmark doses (BMDs) are not limited
to the experimental doses, appropriately reflect the sample size, and can be defined in a
statistically consistent manner. The BMD approach was therefore selected for quantification of
the toxicological effects of the brominated trihalomethanes. Values for HAs and RfDs derived
using the conventional NOAEL/LOAEL approach are presented in the text for comparison with
those obtained using the BMD approach.
The methods employed for BMD modeling are described in Appendix A. The modeling
was performed using the BMDS software (Version 1.2) developed by the U.S. EPA National
Center for Environmental Assessment. The BMDs and BMDLs were calculated based on a BMR
of 10% extra risk for all quantal endpoints analyzed. For continuous data, the BMR was defined
as 1.1 standard deviations, which corresponds to an additional risk of approximately 10% when
the background response rate is assumed to be 1% with normal variation around the mean and
constant standard deviation (Crump, 1995). The BMDL10 was defined as the 95% lower bound
on the corresponding BMD estimate. Confidence bounds were automatically calculated by the
BMDS software using a likelihood profile method.
A. Bromodichloromethane
1. Noncarcinogenic effects
a. One-day Health Advisory
Studies of the acute toxicity of bromodi chl oromethane are summarized in Table VIII-1.
Lilly et al. (1994) administered single doses of bromodi chl oromethane by either oil or aqueous
gavage to male F344 rats at dose levels of 200 or 400 mg/kg. This study identified a LOAEL of
200 mg/kg-day based on histologic lesions in the kidney and changes in urinary parameters. A
NOAEL value was not identified for either vehicle. Data for hepatic vacuolar degeneration and
renal tubular degeneration obtained using the aqueous vehicle were modeled using the BMDS
software. BMD and BMDL10 values of 263 and 182 mg/kg-day, respectively, were calculated
using the hepatic data. BMD and BMDL10 values of 131 and 8.9 mg/kg-day, respectively, were
VIII -1 November 15, 2005
-------
obtained using the renal data. The BMDL10 for renal tubular degeneration is the lowest calculated
across studies, but is not considered a reliable estimate because there is insufficient information to
accurately characterize the shape of the dose-response curve in the region of interest.
Thornton-Manning et al. (1994) administered bromodichloromethane to female F344 rats
by aqueous gavage for five consecutive days at dose levels ranging from 75 to 300 mg/kg-day.
This study identified a NOAEL of 75 mg/kg-day and a LOAEL of 150 mg/kg-day based on
increased liver and kidney weights and histologic lesions in the liver (mild centrilobular
hepatocellular vacuolar degeneration) and in the kidney (mild renal tubule vacuolar
degeneration). An analogous study (Thornton-Manning et al., 1994) conducted in female
C57BL/6J mice indicated that the mice were less sensitive to bromodichloromethane than the
rats, as no treatment-related histologic lesions were observed in the liver or kidney. However,
similar NOAEL and LOAEL values were identified based on increased liver weight and changes
in serum chemistry parameters. Data for renal tubular degeneration in rats were analyzed using
the BMD approach. BMD and BMDL10 values of 133 and 65 mg/kg-day, respectively, were
calculated for this endpoint. The BMD is in close agreement with the BMD value calculated for
the same endpoint using the data of Lilly et al. (1994).
Two reproductive studies which examined full litter resorption were also considered for
derivation of the One-day HA. Bielmeier et al. (2001) examined the occurrence of full litter
resorption in F344 rats treated with 0, 75 or 100 mg/kg-day bromodichloromethane by aqueous
gavage on gestation day 9. The LOAEL for this effect was 75 mg/kg-day. Narotsky et al. (1997)
evaluated the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/kg-day on gestation
days 6 through 15. Full litter resorption was observed at 50 and 75 mg/kg-day. The NOAEL and
LOAEL in this study were thus identified as 25 and 50 mg/kg-day, respectively. When data from
these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/kg-day were
obtained for the Narotsky et al. (1997) and Bielmeier et al. (2001) studies, respectively. The
higher value from the Narotsky et al. (1997) study was considered the more reliable estimate of
the BMD because it was based on response data that included lower doses, one of which was an
apparent NOAEL. The BMDL10 calculated from the Narotsky et al. (1997) data was 30 mg/kg-
day.
Three additional studies were considered as candidates for derivation of the One-day HA.
Lilly et al. (1997) administered single doses of bromodichloromethane by aqueous gavage to
male F344 rats at dose levels ranging from 123 to 492 mg/kg. Based on changes in urinary
parameters, this study identified a NOAEL of 164 mg/kg-day and a LOAEL of 246 mg/kg-day.
No histopathological examination was conducted in this study. The study by French et al. (1999),
which investigated immune system response, identified a similar NOAEL value. However, the
database for immune response to bromodichloromethane is limited when compared to
information on hepatic and renal toxicity. Adverse effects were noted only at the highest dose
and frank effect level, and evidence for vehicle effects on immunotoxicity endpoints was
observed. Keegan et al. (1998) administered single doses of bromodichloromethane to male F344
rats by gavage at dose levels ranging from 21 to 246 mg/kg. The study authors identified a
NOAEL of 41.0 mg/kg-day and a LOAEL of 81.9 mg/kg-day based on elevations in serum
markers of hepatotoxicity (ALT, AST, and SDH). Histopathological examination was not
VIII - 2 November 15, 2005
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conducted and this was considered to be a limitation of the investigation. These three studies
were not considered further for derivation of the One-day Health Advisory (HA), and thus data
reported in them were not analyzed using the BMD approach.
The study conducted by Narotsky et al. (1997) was selected for derivation of the One-day
HA. The critical effect in this study was full litter resorption observed in pregnant F344 rats
treated with bromodichloromethane. The BMDL10 value calculated for this endpoint was 30
mg/kg-day, which is roughly half of the most reliable BMDL10 value calculated for
histopathological changes in kidney (Thornton-Manning et al., 1994). Although dosing in the
Narotsky et al. (1997) study lasted from gestation days 6 through 15, a subsequent study by
Bielmeier et al. (2001) indicated that a single dose (75 mg/kg) of bromodichloromethane on
gestation day 9 was sufficient to elicit full litter resorption in the same strain of rats. Since there
is presently insufficient information available to fully assess the occurrence of reproductive
effects in humans exposed to bromodichloromethane, use of data for full litter resorption was
adopted as a conservative approach to derivation of the One-day HA. The One-day HA for a 10-
kg child is calculated using the following equation:
(30 mg/kg-day) (10 kg)
One-day HA = = l.Omg/L
(300) (1 L/day)
where:
30 mg/kg-day = BMDL10 based on incidence of full litter resorption in F344
rats treated with bromodichloromethane on gestation days 6
to 15.
10 kg = Assumed body weight of a child
300 = Uncertainty factor based on NAS/OW guidelines. This
value includes a factor of 10 to protect sensitive human
populations; a factor of 10 for extrapolation from animals
to humans; and a factor of 3 to account for database
limitations and uncertainty regarding possible reproductive
effects of bromodichloromethane in humans
1 L/day = Assumed water consumption of a 10-kg child
For comparative purposes, the One-day HA was derived using the conventional
NOAEL/LOAEL approach would also be based on data from the Narotsky et al. (1997) study.
This study identified a NOAEL of 25 mg/kg-day based on FLR, which was the lowest value
among the candidate studies. Using this NOAEL and an uncertainty factor of 300 as described
VIII - 3 November 15, 2005
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Table VIII-1 Summary of Candidate Studies for Derivation of the One-day HA for Bromodichloromethane
Reference
Lilly et al.
(1994)
Lilly et al.
(1994)
Lilly et al.
(1997)
Keegan et al.
(1998)
Species
Sex
Rat
F344
M
Rat
F344
M
Rat
F344
M
Rat
F344
M
M
6
6
5
6
Dose
0
200
400
0
200
400
0
123
164
246
328
492
0
21
31
41
82
123
164
246
Route
Gavage
(oil)
Gavage
(aqueous)
Gavage
(aqueous)
Gavage
(aqueous)
Exposure
Duration
Single Dose
Single Dose
Single Dose
Single Dose
1 ml points
Body, liver, and
kidney weights,
serum and urine
chemistry, liver and
kidney histology
Body, liver, and
kidney weights,
serum and urine
chemistry, liver and
kidney histology
Body, liver, and
kidney weights,
serum and urine
chemistry
Body, liver, and
kidney weights,
serum chemistry
NOAEL
nig/kg-day
--
164
41
LOAEL
nig/kg-day
200
(minimal renal
tubule
degeneration and
necrosis, changes
in urinary
parameters)
200
(minimal renal
tubule
degeneration,
changes in
urinary
parameters)
246
(changes in
urinary
parameters)
82
(elevated ALT,
AST, and SDH
activities)
BMD
nig/kg-day
Not modeled
263
131
Not modeled
Not modeled
BMDL10
nig/kg-day
182
(Hepatic
vacuolar
degeneration in
males)
8.9
(Renal tubule
degeneration in
males)
..
_
VIII-4
November 15, 2005
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Table VIII-1 (cont.)
Reference
French et al.
(1999)
Thornton-
Manning et
al. (1994)
Thornton-
Manning et
al. (1994)
Narotsky et
al.
(1997) *
Bielmeier et
al. (2001)*
Species
Sex
Rat
C57BL/6
F
Rat
F344
F
Mouse
C57BL/
6J
F
Rat
F344
F
Rat
F344
F
n
6
6
6
12-
14
8-
11
Dose
0
75
150
300
0
75
150
300
0
75
100
0
25
50
75
0
75
100
Route
Gavage
(aqueous)
Gavage
(aqueous)
Gavage
(aqueous)
Gavage
(oil)
(water)**
Gavage
(aq)
Exposure
Duration
5 days
5 days
5 days
Gestation
days 6-15
Gestation day
9
1 ml points
Body, spleen, and
thymus weights,
immune function
Body, liver, and
kidney weights,
serum chemistry,
liver and kidney
histology
Body, liver, and
kidney weights,
serum chemistry,
liver and kidney
histology
Body weight,
clinical signs,
developmental
parameters
Full litter resorption;
hormone profiles
NOAEL
nig/kg-day
150
75
75
25
..
LOAEL
nig/kg-day
300 (FEL)
(mortality,
decreased body
weight, altered
immune
response)
150
(increased liver
and kidney
weights, mild
centrilobular
hepatocellular
vacuolar
degeneration,
mild renal tubule
vacuolar
degeneration)
150
(increased liver
weight, elevated
ALT and SDH
activities)
50
(full-litter
resorption)
75
(full- litter
resorption)
BMD
nig/kg-day
Not modeled
133
Not modeled
48
23
BMDL10
nig/kg-day
..
65
(renal tubular
degeneration)
_
30
(full-litter
resorption)
4.2
(full- litter
resorption)
* The NOAEL and LOAEL values listed are for reproductive or developmental effects.
** The NOAEL and LOAEL values were the same in either vehicle. BMD modeling was performed on aqueous vehicle data only.
VIII-5
November 15, 2005
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above, the One-day HA for a 10-kg child calculated using the conventional approach would be
0.8 mg/L (rounded from 0.83 mg/L).
b. Ten-day Health Advisory
Sixteen studies were considered for derivation of the Ten-day HA for
bromodichloromethane. These studies are summarized in Table VIII-2 below. Aida et al.
(1992a) administered microencapsulated bromodichloromethane in the diet to Wistar rats for one
month at dose levels ranging from 20.6 to 203.8 mg/kg-day. This study identified aNOAEL of
61.7 mg/kg-day and a LOAEL of 189.0 mg/kg-day in male rats based on histologic changes in the
liver (swelling of hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct
proliferation). Analysis using the BMD approach calculated BMD and BMDL10 values of 34 and
17 mg/kg-day, respectively, based on data for liver cell vacuolization in females.
Data from four of the other candidate studies are consistent with the histopathological
results obtained by Aida et al. (1992a). Melnick et al. (1998) administered
bromodichloromethane by gavage to female B6C3FJ mice for 5 days/week for 3 weeks and
identified a NOAEL of 75 mg/kg-day (duration-adjusted NOAEL of 54 mg/kg-day) and a
LOAEL of 150 mg/kg-day (duration-adjusted LOAEL of 107 mg/kg-day) based on histologic
changes in the liver (hepatocyte hydropic degeneration). Analysis using the BMD approach
calculated duration-adjusted BMD and BMDL10 values of 31 and 8.4 mg/kg-day, respectively, for
this endpoint. Condie et al. (1983) administered bromodichloromethane by gavage to male CD-I
mice for 14 days and identified a NOAEL of 74 mg/kg-day and a LOAEL of 148 mg/kg-day
based on minimal to moderate liver and kidney lesions. Analysis using the BMD approach
calculated BMD and BMDL10 values of 24 and 7.5 mg/kg-day, respectively, based on data for
histopathological changes in the liver. NTP (1998) conducted histopathologic examinations in
conjunction with a study of reproductive and developmental toxicity in Sprague-Dawley rats.
Although no reproductive or developmental toxicity was observed at the dose levels investigated,
histopathological changes were noted in the liver of males rats treated with the compound for 35
days. The NOAEL and LOAEL for this effect were 9 and 38 mg/kg-day, respectively. Analysis
of data for single cell hepatic necrosis using the BMD approach calculated BMD and BMDL10
values of 35 and 18 mg/kg-day, respectively, which are virtually identical to the values calculated
using the liver cell vacuolization data for females from the Aida et al. (1992b) study. Coffin et al.
(2000) observed hydropic degeneration in female mice treated with 150 mg/kg-day
bromodichloromethane in corn oil for 11 days. These data were not modeled because other
studies utilized doses lower than 150 mg/kg-day, which allowed better characterization of
response in the low-dose region of the dose-response curve.
In contrast to the studies described above, Chu et al. (1982a) did not observe any
microscopic lesions in the liver when Sprague-Dawley rats were administered
bromodichloromethane at doses up to 68 mg/kg-day in the drinking water for 28 days. The
studies of Munson et al. (1982) and NTP (1987) did not conduct histopathological examinations.
These studies identified NOAELs ranging from 50 to 150 mg/kg-day and LOAELs ranging from
125 to 300 mg/kg-day for other endpoints, including depressed humoral immunity (Munson et al.,
VIII - 6 November 15, 2005
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1982), decreased weight gain (NTP, 1987; rats), and increased mortality and gross renal
pathology (NTP, 1987; mice). These data were not analyzed by the BMD approach.
Seven studies that examined developmental and/or reproductive endpoints were evaluated.
Two studies reported the incidence of full litter resorption in F344 rats following treatment with
bromodichloromethane. Bielmeier et al. (2001) examined the occurrence of full litter resorption
in F344 rats treated with 0, 75 or 100 mg/kg-day bromodichloromethane by aqueous gavage on
gestation day 9. The LOAEL for this effect was 75 mg/kg-day. Narotsky et al. (1997) evaluated
the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/kg-day on gestation days 6
through 15. Full litter resorption was observed at 50 and 75 mg/kg-day. The NOAEL and
LOAEL in this study were thus identified as 25 and 50 mg/kg-day, respectively. When data from
these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/kg-day were
obtained for the Narotsky et al. (1997) and Bielmeier et al. (2001) studies, respectively. The
higher value from the Narotsky et al. (1997) study was considered the more reliable estimate of
the BMD because it was based on response data that included lower doses, one of which was an
apparent NOAEL. The BMDL10 calculated from the Narotsky et al. (1997) data was 30 mg/kg-
day. Ruddick et al. (1983) observed an increased incidence of sternebral aberrations in the pups
of Sprague-Dawley rats administered bromodichloromethane in corn oil by gavage. Statistical
analysis of the published data indicated that the NOAEL and LOAEL for this effect were 100
mg/kg-day and 200 mg/kg-day, respectively. The BMD and BMDL10 obtained for this study
were 27 and 15 mg/kg-day, respectively.
The remaining reproductive/developmental studies sponsored by the Chlorine Chemistry
Council (CCC) were also evaluated. These studies were summarized by Christian et al. (2001a,
b). CCC (2000a, b) examined developmental toxicity in New Zealand rabbits and identified
developmental NOAELs of 76 and 55 mg/kg-day (the highest doses tested in each study). The
CCC (CCC, 2000c,d) also examined reproductive and developmental toxicity in Sprague Dawley
rats. In a range-finding study (CCC, 2000c), Fx generation pups exposed to
bromodichloromethane via lactation and possibly by consumption of water supplied to the dams
exhibited reduced body weights and body weight gains. These effects occurred at exposure levels
which also resulted in maternal toxicity. Biologically meaningful average daily doses could not
be established in this experiment; therefore, the concentration-based NOAEL and LOAEL values
for developmental effects were 50 and 150 ppm based on changes in Fx pup body weight and
body weight gain. In a subsequent developmental study, CCC (2000d) identified NOAEL and
LOAEL values of 45 and 82 mg/kg-day, respectively, based on decreased number of ossification
sites per fetus for the forelimb phalanges and hindlimb metatarsals and phalanges. This effect
was observed at doses associated with maternal toxicity. Endpoints from these studies were not
modeled because other studies identified adverse effects at lower doses.
Data for maternal toxicity from three reproductive/developmental studies were also
considered for derivation of the 10-day HA for bromodichloromethane. Narotsky et al. (1997)
observed decreased maternal body weight gain on gestation days 6 to 8 in female rats
administered 25 mg/kg-day (the lowest dose tested) by aqueous gavage in 10% Emulphor. BMD
modeling identified BMD and BMDL10 values of 18 and 10 mg/kg-day, respectively, for this
VIII - 7 November 15, 2005
-------
endpoint. The data reported in this study did not permit evaluation of body weight or body
weight gain at other time points during the treatment period. CCC (2000d) reported decreased
maternal body weight gain at several time points in pregnant rats administered
bromodichloromethane in drinking water, with the most severe effect observed immediately after
initiation of treatment on gestation days 6 to 7. The NOAEL and LOAEL for decreased body
weight on gestation days 6 to 7 were 18.4 and 45 mg/kg-day, respectively. When body weight
gain for this interval was modeled, the resulting BMD and BMDL10 values were approximately
18 and 15 mg/kg-day, respectively. However, the modeled fits to the data were poor, and the
results were not considered sufficiently reliable for derivation of a health advisory. To address
this problem, body weight gain data for gestation days 6 to 9 were also modeled. Reliable values
of 23 and 18 mg/kg-day were obtained for the BMD and BMDL10, respectively. The CCC
(2000b) study observed decreased maternal body weight gain at several time points in pregnant
rabbits administered bromodichloromethane in the drinking water on gestation days 6 to 29. The
NOAEL and LOAEL for decreased maternal body weight gain (corrected for gravid uterine
weight) on gestation days 6 to 21 were 13.4 and 35.3 mg/kg-day, respectively. A BMD value of
50 mg/kg-day was obtained for this data set, but the BMDS software failed to identify the
corresponding BMDL10. No further modeling was attempted since this value was well above the
lowest BMDs obtained in some other candidate studies.
As evident from the data in Table VIII-2, the four studies that examined histopathological
changes in the liver are in close agreement, having identified BMD values ranging from 24 to 35
mg/kg-day. The corresponding BMDL10 values ranged from 7.5 to 18 mg/kg-day. Maternal
toxicity occurred in rats at similar levels in two developmental studies. These studies identified
BMD values of 18 and 23 mg/kg-day, with corresponding BMDL10 values of 10 and 18 mg/kg-
day. The NTP (1998) and CCC (2000d) drinking water studies were selected to derive the Ten-
day HA. Selection of these studies was based on the administration of bromodichloromethane in
drinking water, the most relevant route of exposure. In addition, these studies utilized a lower
range of doses, which provided information on the shape of the dose-response curve in the region
of interest. The Ten-day HA is calculated according to the following equation:
(18 mg/kg-day) (10 kg)
Ten-day HA = =0.60 mg/L (rounded to 0.6 mg/L)
(300) (1 L/day)
where:
18 mg/kg-day = BMDL10 based on single cell hepatic necrosis in rats
administered bromodichloromethane in the drinking water
for 35 days or decreased maternal body weight gain on
gestation days 6-9 in pregnant female rats administered
bromodichloromethane in the drinking water.
10 kg = Assumed body weight of a child
VIII - 8 November 15, 2005
-------
300 = Uncertainty factor based on NAS/OW guidelines. This
value includes a factor of 10 to protect sensitive human
populations and a factor of 10 for extrapolation from
animals to humans, and a factor of 3 to account for
database limitations and uncertainty regarding possible
reproductive effects of bromodichloromethane in humans
1 L/day = Assumed water consumption of a 10-kg child
For comparative purposes, the Ten-day HA derived using the conventional NOAEL/LOAEL
approach would be based on data from the CCC (2000d) study. This study identified a NOAEL
of 18 mg/kg-day based on reduced maternal body weight gain in pregnant rabbits. Using this
NOAEL and an uncertainty factor of 300 as described above, the Ten-day HA for a 10-kg child
calculated using the conventional approach would be 0.60 mg/L (rounded to 0.6 mg/L).
VIII - 9 November 15, 2005
-------
Table VIII-2 Summary of Candidate Studies for Derivation of the Ten-day HA for Bromodichloromethane
Reference
Aida et al.
(1992a)
Chu et al.
(1982a)
Condie et al.
(1983)
Melnick et
al.
(1998)
Munson et
al.
(1982)
Species
Sex
Rat
Wistar
M, F
Rat
SD
M
Mouse
CD-I
M
Mouse
B6C3FJ
F
Mouse
CD-I
M, F
M
7
10
8-
16
10
8-
12
Dose
Male
0
21
62
189
Females
0
21
66
204
0
0.8
8
68
0
37
74
148
0
75
150
326
Males
0
50
125
250
Route
Feed
Drinking
water
Gavage
(oil)
Gavage
(oil)
Gavage
(aq.)
Exposure
Duration
1 month
28 days
14 days
3 weeks
(5 d/wk)
14 days
1 ml points
Clinical signs, body
weight, serum
chemistry,
hematology, histology
Clinical signs, serum
chemistry, histology
Serum enzymes, PAH
uptake in vitro,
histology
Body and liver
weights, serum
chemistry, liver
histology
Body and organ
weights, serum
chemistry,
hematology, and
immune function
NOAEL
(mg/kg-day)
62
68
74
75
50
LOAEL
(mg/kg-day)
189
(liver
histopathology
in males)
148
(elevated ALT,
decreased PAH
uptake, liver and
kidney
histopathology)
150
(liver
histopathology)
125
(depressed
humoral
immunity)
BMD
(mg/kg-day)
34
No data to model
24
125
31f
Not modeled
BMDL10
(mg/kg-day)
17
(liver cell
vacuolation in
females)
7.5
(hepatic
centrilobular
pallor)
53
(Renal epithelial
hyperplasia)
8.4
(Hepatocyte
hydropic
degeneration)
_
VIII - 10
November 15, 2005
-------
Table VIII-2 (cont.)
Reference
NTP
(1987)
NTP
(1987)
Coffin et al.
(2000)
NTP
(1998)
Ruddick et
al. (1983)*
Species
Sex
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Mouse
B6C3FJ
F
Rat
SD
M
(group A)
Rat
SD
F
n
5
5
10
5-
13
9-
14
Dose
0
38
75
150
300
600
0
19
38
75
150
300
0
150
300
0
9
38
67
0
50
100
200
Route
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Drinking
water
Gavage
(oil)
Exposure
Duration
14 days
14 days
1 1 days
35 days
Gestation
days 6-15
1 ml points
Body weight, clinical
signs, gross necropsy
Body weight, clinical
signs, gross necropsy
Relative liver wt,
liver histopathology;
labeling index
Body and organ
weights, serum
chemistry,
hematology, gross
necropsy, histology,
sperm evaluation
Body and organ
weights; maternal
serum chemistry;
hematology, and
histopathology;
developmental
parameters
NOAEL
(mg/kg-day)
150
75
9
100
LOAEL
(mg/kg-day)
300
(decreased
weight gain)
150 (EEL)*
(mortality,
lethargy, gross
renal pathology)
150
38
(liver
histopathology)
200
(sternebral
aberrations)
BMD
(mg/kg-day)
Not modeled
Not modeled
Not modeled
35
27
BMDL10
(mg/kg-day)
..
..
18
(liver cell
necrosis)
15
(sternebral
aberrations)
VIII- 11
November 15, 2005
-------
Table VIII-2 (cont.)
Reference
Narotsky et
al. (1997)*
Bielmeier et
al. (2001)*
ccc
(2000c)*
CCC
(2000d)*
Species
Sex
Rat
F344
F
Rat
F344
F
Rat
SD
M, F
Rat
SD
F
n
12-
14
8-
11
10
25
Dose
0
25
50
75
0
75
100
0 ppm
50 ppm
150 ppm
450 ppm
13 50 ppm
0.0
2.2
18.4
45.0
82.0
Route
Gavage
(oil)
(Emul-
phor)**
Gavage
(aq)
Drinking
water
Drinking
water
Exposure
Duration
Gestation
days 6-15
Gestation
day 9
Males 64
days
Females 74
days
Gestation
days 6-21
1 ml points
Body weight, clinical
signs, developmental
parameters
Full litter resorption;
hormone profiles
Reproductive and
developmental
parameters
Reproductive and
developmental
parameters
NOAEL
(mg/kg-day)
25
(developmental )
-
(maternal)
50 ppm
45.3
(developmental)
18.4
(maternal)
LOAEL
(mg/kg-day)
50
(full-litter
resorption)
25
(reduced
matermal body
weight gain
gestation days
6-8, aqueous
vehicle only)
75
(full-litter
resorption)
150 ppm
(reduced F[ pup
weight and
weight gain)
82.0
(reduced
number of
ossification sites
in phalanges or
metatarsals
occurring with
maternal
toxicity)
45
(reduced
maternal body
weight gain
gestation days
6-7)
BMD
(mg/kg-day)
48
18
23
Not modeled
Not modeled
23
BMDL10
(mg/kg-day)
30
(full-litter
resorption)
10
(reduced
matermal body
weight gain
gestation days 6-
8, aqueous
vehicle only)
4.2
(full-litter
resorption)
..
18
(reduced
maternal body
weight gain
gestation days 6-
9; see text for
comment)
VIII - 12
November 15, 2005
-------
Table VIII-2 (cont.)
Reference
ccc
(2000a)*
CCC
(2000b)*
Species
Sex
Rabbit
NZW
F
Rabbit
NZW
F
n
5
25
Dose
0.0
13.9
32.3
76.3
0
1.4
13.4
35.6
55.3
Route
Drinking
water
Drinking
Water
Exposure
Duration
Gestation
days 6-29
Gestation
days 6-29
1 ml points
Reproductive and
developmental
parameters
Clinical sign, gross
lesions, reproductive
and developmental
endpoints
NOAEL
(mg/kg-day)
76.3
(developmental)
55
(developmental)
13.4
(maternal)
LOAEL
(mg/kg-day)
.
_
35.3
(reduced
corrected
maternal body
weight gain
gestation days
6-29)
BMD
(mg/kg-day)
Not modeled
Not modeled
50
BMDL10
(mg/kg-day)
BMDS software
failed
* The NOAEL and LOAEL values listed are for reproductive or developmental effects.
** The NOAEL and LOAEL values were the same for developmental effects in either vehicle. The LOAEL for maternal toxicity was 25 mg/kg-day for the
aqueous vehicle (10% Emulphor). The NOAEL and LOAEL for maternal toxicity using the corn oil vehicle were 25 mg/kg-day and 50 mg/kg-day,
respectively. BMD modeling was performed on aqueous vehicle data only.
^ BMD and BMDL10 calculated using duration adjusted doses
Abbreviations: PEL, Frank effect level; SD, Sprague-Dawley; NZW, New Zealand White
VIII - 13
November 15, 2005
-------
c. Longer-term Health Advisory
Two rodent oral exposure studies conducted by NTP (1987) were considered for
derivation of the Longer-term HA for bromodichloromethane. In addition, eight reproductive
studies were considered. These studies are summarized in Table VIII-3 below.
NTP (1987) administered bromodichloromethane by gavage to F344/N rats for 5
days/week for 13 weeks at dose levels ranging from 19 to 300 mg/kg-day. Based on decreased
weight gain, this study identified a NOAEL of 75 mg/kg-day and a LOAEL of 150 mg/kg-day.
Treatment-related hepatic and renal lesions were observed only at the high dose. In a similar
study, NTP (1987) administered bromodichloromethane by gavage to B6C3FJ mice for 5
days/week for 13 weeks at dose levels ranging from 6.25 to 100 mg/kg-day for males and from 25
to 400 mg/kg-day for females. This study identified a NOAEL of 50 mg/kg-day and a LOAEL of
100 mg/kg-day based on histologic alterations in the kidney (focal necrosis of the proximal renal
tubular epithelium and nephrosis) of male mice. BMD analysis using the BMDS program
identified duration-adjusted BMD values of 63 and 75 mg/kg-day for focal necrosis of renal
tubular epithelium in males and vacuolated cytoplasm in the liver of females, respectively. The
corresponding BMDL10 values for these renal and hepatic effects were 35 and 47 mg/kg-day,
respectively.
Eight reproductive or developmental studies (Ruddick et al., 1983; Narotsky et al., 1997;
Bielmeier et al. 2001; CCC, 2000a,b,c,d; CCC, 2002) were also considered for derivation of the
Longer-term HA. CCC (2002) identified a LOAEL of 150 ppm (approximately 11.6 to 40.2
mg/kg-day) for delayed sexual maturation in Fx male rats in a two-generation study of
bromodichloromethane administered in drinking water. The LOAEL for parental effects in the
study was also 150 ppm, based on decreased body weight and body weight gain in F0 females and
Fj males and females. Bielmeier et al. (2001) examined the occurrence of full litter resorption in
F344 rats treated with 0, 75 or 100 mg/kg-day bromodichloromethane by aqueous gavage on
gestation day 9. The LOAEL for this effect was 75 mg/kg-day. Narotsky et al. (1997) evaluated
the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/kg-day on gestation days 6
through 15. Full litter resorption was observed at 50 and 75 mg/kg-day. The NOAEL and
LOAEL in this study were thus identified as 25 and 50 mg/kg-day, respectively. When data from
these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/kg-day were
obtained for the Narotsky et al. (1997) and Bielmeier et al. (2001) studies, respectively. The
higher value from the Narotsky et al. (1997) study was considered the more reliable estimate of
the BMD because it was based on response data that included lower doses, one of which was an
apparent NOAEL. The BMDL10 calculated from the Narotsky et al. (1997) data was 30 mg/kg-
day. Studies conducted by NTP (1998) did not detect reproductive or developmental toxicity at
doses up to 116 mg/kg-day. Two studies conducted in New Zealand White rabbits did not detect
developmental effects at doses up to 55 and 76 mg/kg-day, respectively (CCC, 2000a,b).
Three additional studies in rats identified developmental effects that occurred at dose
levels that also resulted in maternal toxicity. In a range-finding study (CCC, 2000c), Fx
generation pups exposed to bromodichloromethane via lactation and possibly by consumption of
VIII - 14 November 15, 2005
-------
water supplied to the dams exhibited reduced body weights and body weight gains. Biologically
meaningful daily doses could not be established in this experiment; therefore, the concentration-
based NOAEL and LOAEL values are 50 ppm and 150 ppm. based on reduced body weight and
body weight gain in the Fx pups. In a subsequent developmental study, CCC (2000d) identified
NOAEL and LOAEL values of 45 and 82 mg/kg-day, respectively, based on decreased number of
ossification sites per fetus for the forelimb phalanges and hindlimb metatarsals and phalanges.
These reversible developmental delays occurred at doses which also resulted in maternal.
Endpoints from these studies were not modeled because other effects were observed at lower
doses. Ruddick et al. (1983) observed an increased incidence of sternebral aberrations in the pups
of Sprague-Dawley rats administered bromodichloromethane in corn oil by gavage. Statistical
analysis of the published data indicated that the NOAEL and LOAEL for this effect were 100
mg/kg-day and 200 mg/kg-day, respectively. The lowest dose tested was 50 mg/kg-day. The
BMD and BMDL10 obtained for this study were 27 and 15 mg/kg-day, respectively. However,
examination of the modeling output indicated that none of the available models fit the data well in
the low-dose region of the curve. Therefore, the reliability of these values is questionable.
The Narotsky et al. (1997) and CCC (2000d) studies identified BMDL10 values of 10 and
18 mg/kg-day based on reduced maternal body weight gain. The CCC data were considered the
most relevant since they were obtained from a drinking water study which utilized concentrations
that resulted in daily doses well below those used in the Narotsky study. The NTP (1987) and the
Narotsky et al. (1997) studies provided similar, but higher, BMDL10 values, based on
reproductive and histopathological endpoints. The NTP (1987) study utilized
bromodichloromethane doses as low as 6.3 mg/kg-day, in contrast to the reproductive study
conducted by Narotsky et al. (1997) in which the lowest dose was 25 mg/kg-day. The NTP
(1987) data thus provide more information about the shape of the dose-response curve in the
region of interest. The BMD data for focal necrosis of renal tubular epithelium and reduced body
weight gain in pregnant female rats were thus selected as the most reliable basis for determining
the Longer-term HA. Using the lower of the two values, the duration-adjusted BMDL10 of 18
mg/kg-day for reduced maternal body weight gain, the Longer-term HA for a 10 kg child is
calculated according to the following equation:
(18 mg/kg-day) (10 kg)
Longer-term HA = =0.60 mg/L (rounded to 0.6 mg/L)
(300) (1 L/day)
where:
18 mg/kg-day = BMDL10 based on decreased maternal body weight gain on
gestation days 6-9 in pregnant female rats administered
bromodichloromethane in the drinking water.
10 kg = Assumed body weight of a child
VIII - 15 November 15, 2005
-------
300 = Uncertainty factor based on NAS/OW guidelines. This
value includes a factor of 10 to protect sensitive human
populations and a factor of 10 for extrapolation from
animals to humans, and a factor of 3 to account for
uncertainty regarding possible reproductive effects of
bromodichloromethane in humans
1 L/day = Assumed water consumption of a 10-kg child
The Longer-term HA for adults is calculated as follows:
(18mg/kg-day)(70kg)
Longer-term HA = =2.1 mg/L (rounded to 2 mg/L)
(300) (2 L/day)
where:
18 mg/kg-day = BMDL10 based on reduced body weight gain in female rats
administered bromodichloromethane in the drinking water
70 kg = Assumed body weight of an adult
300 = Uncertainty factor based on NAS/OW guidelines. This
value includes a factor of 10 to protect sensitive human
populations and a factor of 10 for extrapolation from
animals to humans, and a factor of 3 to account for
database limitations and uncertainty related to potential
reproductive effects of bromodichloromethane in humans
2 L/day = Assumed water consumption of a 70-kg adult
For purposes of comparison , a Longer-term HA derived using the conventional
NOAEL/LOAEL approach would be based on the study conducted by CCC (2000d). This study
identified a NOAEL of 18.4 mg/kg-day and a LOAEL of 45 mg/kg-day based on reduced body
weight gain on gestation days 6 to 7. Using the NOAEL of 18.4 mg/kg-day, and assuming
drinking water ingestion of 1 L/day and an uncertainty factor of 300 (including factors of 10 for
interspecies extrapolation and protection of susceptible populations and a factor of 3 for database
limitations and uncertainty regarding potential reproductive effects in humans), the Longer-term
HA for a 10 kg child would be 0.6 mg/L. The Longer-term HA for a 70 kg adult consuming 2
L/day would be 2 mg/kg-day.
VIII - 16 November 15, 2005
-------
Table VIII-3 Summary of Candidate Studies for Derivation of the Longer-term HA for Bromodichloromethane
Reference
NTP
(1987)
NTP (1987)
Ruddick et
al. (1983)*
Narotsky et
al.
(1997) *
Species
Sex
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Rat
SD
F
Rat
F344
F
M
10
10
9-
14
12-
14
Dose
0
19
38
75
150
300
Male
0
6.3
13
25
50
100
Female
0
25
50
100
200
400
0
50
100
200
0
25
50
75
Route
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
(aq)**
Exposure
Duration
13 weeks
(5 d/wk)
13 weeks
(5 d/wk)
Gestation days
6-15
Gestation days
6-15
1 ml points
Body weight,
clinical signs,
histology
Body weight,
clinical signs,
histology
Body and organ
weights;
maternal serum
chemistry;
hematology, and
histopathology;
developmental
parameters
Body weight,
clinical signs,
developmental
parameters
NOAEL
(mg/kg-day)
75
50
100
25
(developmental)
-
(maternal)
LOAEL
(mg/kg-day)
150
(decreased
weight gain)
(hepatic and
renal lesions at
300)
100
(renal lesions)
200
(increased
incidence of
sternebral
variations)
50
(full-litter
resorption)
25
(reduced
maternal body
weight gain
gestation days 6-
8, aq. vehicle)
BMD
(mg/kg-day)
Not modeled
63
75
27
48
18
BMDL10
(mg/kg-day) }
-
35
(focal necrosis of
renal tubular
epithelium in
males)
47
(Hepatic
vacuolated
cytoplasm in
females)
15
(increased
incidence of
sternebral
variations)
30
(full-litter
resorption)
10
(reduced
maternal body
weight gain,
gestation days 6-
8, aq. vehicle)
VIII - 17
November 15, 2005
-------
Table VIII-3 (cont.)
Reference
Bielmeier et
al. (2001)*
ccc
(2000c)*
CCC
(2000d)*
CCC (2002)*
Species
Sex
Rat
F344
F
Rat
SD
M, F
Rat
SD
F
Rat
SD
M, F
n
8-
11
10
25
30
Dose
0
75
100
0 ppm
50 ppm
150 ppm
450 ppm
1350 ppm
0.0
2.2
18.4
45.0
82.0
0 ppm
50 ppm
150 ppm
450 ppm
Route
Gavage
(aq)
Drinking
water
Drinking
water
Drinking
water
Exposure
Duration
Gestation day
9
Males
64 days
Females
74 days
Gestation days
6-21
Two
generations
1 ml points
Full litter
resorption;
hormone profiles
Reproductive/
developmental
parameters
Reproductive/
developmental
parameters
Reproductive
parameters
NOAEL
(mg/kg-day)
—
Developmental
50 ppm
Parental
50 ppm
45.3
(developmental)
18.4
(maternal)
50 ppm
(offspring)
50 ppm
(parental)
LOAEL
(mg/kg-day)
75
(full-litter
resorption)
Developmental
150 ppm-
Parental
50 ppm
82.0
(reduced number
of ossification
sites in phalanges
or metatarsals
occurring with
maternal
toxicity)
45
(reduced
maternal body
weight gain
gestation days 6-
7)
150 ppm
(delayed sexual
maturation in Fj
males)
150 ppm
(Reduced body
wt and body wt
gain in F0
females and Fj
males and
females)
BMD
(mg/kg-day)
23
Not modeled
Not modeled
23
Not modeled
BMDL10
(mg/kg-day) }
4.2
(full-litter
resorption)
—
18
(reduced
maternal body
weight gain
gestation days 6-
9; see comments
in text)
-
VIII - 18
November 15, 2005
-------
Table VIII-3 (cont.)
Reference
ccc
(2000a)*
CCC
(2000b)*
Species
Sex
Rabbit
NZW
F
Rabbit
NZW
F
n
5
25
Dose
0
4.9
13.9
32.3
76.3
0
1.4
13.4
35.6
55.3
Route
Drinking
Water
Drinking
Water
Exposure
Duration
Gestation day 6
to 29
Gestation days
6-29
1 ml points
Body weight,
clinical signs,
reproductive and
developmental
parameters
Clinical sign,
gross lesions,
reproductive and
developmental
endpoints
NOAEL
(mg/kg-day)
76.3
(developmental)
55.3
(developmental)
13.4
(maternal)
LOAEL
(mg/kg-day)
-
-
35.3
(reduced
maternal body
weight gain
BMD
(mg/kg-day)
Not modeled
Not modeled
50
(developmental)
BMDL10
(mg/kg-day) }
-
BMD software
failed
BMDL10 value was derived using duration-adjusted doses.
Modeled using Crump Benchmark Dose Software
The NOAEL and LOAEL values listed are for reproductive/developmental effects.
The developmental NOAEL and LOAEL values were the same in either vehicle. The LOAELs for maternal toxicity were 25 mg/kg-day and 50 mg/kg-day
for the aqueous and corn oil vehicles respectively. BMD modeling was performed on aqueous vehicle data only.
Abbreviations: NA, Not available; SD, Sprague-Dawley; NZW, New Zealand White
**
VIII -19
November 15, 2005
-------
d. Reference Dose, Drinking Water Equivalent Level and Lifetime Health Advisory
This section reports the existing RfD value for bromodichloromethane and describes the
derivation of the RfD for this compound. This section also describes the calculation of Drinking
Water Equivalent Level and Lifetime Health Advisory values which require the RfD as input.
For this document, new and existing studies were reviewed and appropriate candidate data were
selected for benchmark dose (BMD) modeling. The results of BMD modeling were used in
conjunction with appropriate uncertainty factors to calculate the RfD. A comparison of the RfD
derived using the BMD approach to the results obtained using the conventional NOAEL/LOAEL
approach is also provided.
Description of the Existing RfD
The existing RfD for bromodichloromethane is 0.02 mg/kg-day (IRIS, 1993a). This value
was derived using a duration-adjusted LOAEL of 17.9 mg/kg-day identified for renal cytomegaly
in B6C3FJ mice administered bromodichloromethane by corn oil gavage for 5 days/week for 102
weeks (NTP, 1987). An uncertainty factor of 100 was used to account for extrapolation from
animal data and for protection of sensitive human subpopulations. An additional factor of 10 was
used because the RfD was based on a LOAEL (although it was considered minimally adverse)
and to account for lack of reproductive data.
Identification of Candidate Studies for Derivation of the RfD
Several studies of chronic duration were considered for derivation of the RfD for
bromodichloromethane. These studies are summarized in Table VIII-4 below. NTP (1987)
administered bromodichloromethane to F344/N rats by gavage in corn oil at doses of 50 or 100
mg/kg-day for 5 day/week for 102 weeks. This study identified a LOAEL of 50 mg/kg-day based
on histologic alterations in the liver and kidney. In a similar study, NTP (1987) administered
bromodichloromethane by gavage in corn oil to B6C3FJ mice for 5 days/week for 102 weeks at
dose levels of 25 or 50 mg/kg-day for males and 75 or 150 mg/kg-day for females. Based on
histologic alterations in the liver, kidney, and thyroid of male mice, this study identified a
LOAEL of 25 mg/kg-day, which is consistent with the value identified in the rat study. In a third
study, Tobe et al. (1982) administered microencapsulated bromodichloromethane to Wistar rats in
the diet at dose levels ranging from 6 to 168 mg/kg-day. Histologic data for the animals exposed
to bromodichloromethane were reported by Aida et al. (1992b). This study identified a LOAEL
for male rats of 6 mg/kg-day on the basis of histopathologic changes in the liver.
Ten reproductive and/or developmental toxicity studies (Ruddick et al., 1983; Klinefelter
et al., 1995; Narotsky et al., 1997; NTP, 1998; Bielmeier et al. 2001; CCC, 2000a,b,c,d; CCC,
2002) were considered for derivation of the RfD in addition to the chronic studies. The
investigations of Ruddick et al. (1983), Klinefelter et al. (1995), and Narotsky et al. (1997)
identified NOAELs or LOAELs in rats that were substantially higher than the LOAEL identified
by Aida et al. (1992b) (Table VIII-4 below). The studies conducted by NTP (1998) did not
observe developmental or reproductive effects at doses up to 116 mg/kg-day. The study by
Bielmeier et al. (2001) identified a free-standing LOAEL of 75 mg/kg-day in F344 rats. The
studies conducted by CCC (2000a,b) identified NOAELs of 55 and 76 mg/kg-day, respectively,
VIII - 20 November 15, 2005
-------
for developmental effects in New Zealand White rabbits. The study conducted in rats by CCC
(2000c) identified concentration-based NOAEL and LOAEL values of 50 ppm and 150 ppm for
reduced body weight and body weight gain in Fx pups. The study conducted in rats by CCC
(2000d) identified NOAEL and LOAEL values of 45 mg/kg-day and 82 mg/kg-day, respectively
on the basis of decreased ossification sites per fetus per litter in the forelimb and hindlimb. Low-
range LOAEL values for maternal toxicity ranged from 13 to 25 mg/kg-day (CCC, 2000b; CCC
2000d; Narotsky et al., 1997). CCC (2002) identified a LOAEL of 150 ppm (approximately 11.6
to 40.2 mg/kg-day) for delayed sexual maturation in Fx male rats in a two-generation study of
bromodichloromethane administered in drinking water. The LOAEL for parental effects in the
study was also 150 ppm, based on decreased body weight and body weight gain in F0 females and
Fj males and females. Since these studies identified NOAEL and/or LOAEL values substantially
higher than that identified by Aida et al. (1992b), they were not further considered for derivation
oftheRfD.
Method of Analysis
Selected data from the candidate studies were analyzed using the benchmark dose (BMD)
modeling approach. Initially, data sets for potentially sensitive endpoints were selected as
described in U.S. EPA (1998b) and analyzed using the Crump Benchmark Dose Modeling
Software (K. S. Crump, Inc.). Results of this analysis are summarized in Table VIII-5.
Following the release of Version 1.2 of the BMDS program (U.S. EPA, 2000a), a subset of the
most sensitive endpoints identified using the Crump software was reanalyzed in accordance with
proposed U.S. EPA (2000b) recommendations. An advantage of analysis with the BMDS
software is that several additional models are available to fit the data. The results of the analysis
with the BMDS software are included in Table VIII-4.
Choice of Principal Study and Critical Effect for the RfD
Three data sets for histopathological effects in liver were analyzed using the BMDS
software (Table VIII-4). BMD modeling identified several endpoints with BMD values lower
than the conventionally determined LOAEL of 6 mg/kg-day (Aida, 1992b). The lowest BMD
values were obtained for fatty degeneration (1.9 mg/kg-day) in male rats (Aida et al., 1992b) and
for kidney cytomegaly (2.0 mg/kg-day) in male mice (NTP, 1987). Comparably low BMD values
were also obtained for granulomas observed in the liver of male rats (2.1 mg/kg-day) and for fatty
degeneration in the liver of female rats (3.1 mg/kg-day) in the study conducted by Aida et al.
(1992b). In contrast, the BMD values calculated for endpoints examined in other studies were
approximately 10- to 20-fold higher.
VIII - 21 November 15, 2005
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Table VIII-4 Summary of Candidate Studies for Derivation of the RfD for Bromodichloromethane
Reference
NTP
(1987)
NTP
(1987)
Aida et al.
(1992b)
Ruddick et
al. (1983)b
Narotsky et
al. (1997)b
Species
Sex
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Rat
Wistar
M, F
Rat
SD
F
Rat
F344
n
50
50
40
9-
14
13-
14
Dose
0
50
100
0
25
50
Male
0
6
26
138
Female
0
8
O/1)
jL
168
0 ppm
50 ppm
150 ppm
450 ppm
1350
ppm
0
75
100
Route
Gavage
(oil)
Gavage
(oil)
Diet
Gavage
(oil)
Gavage
(oil)
(water)
Exposure
Duration
102 weeks
(5 d/wk)
102 weeks
(5 d/wk)
24 months
Gestation days
6-15
Gestation days
6-15
1 ml points
Body weight,
clinical signs,
gross necropsy,
histology
Body weight,
clinical signs,
gross necropsy,
histology
Body weight,
clinical signs,
serum
biochemistry,
gross necropsy,
histology
Body and organ
weights;
maternal serum
chemistry;
hematology, and
histopathology;
developmental
parameters
Body weight,
clinical signs,
developmental
parameters
NOAEL
(mg/kg-day)
..
..
100
25
(developmental)
LOAEL
(mg/kg-day)
50
(lesions of
kidney and liver)
25
(lesions of liver,
kidney, and
thyroid)
6
(liver fatty
degeneration and
granuloma)
200
(sternebral
variations)
50
(full-litter
resorption)
BMD
(mg/kg-day)
..
2.0
3.1
1.9
2.1
27
48
BMDL10
(mg/kg-day) '
36.5C
(liver necrosis in
male rats)
1.5
(kidney
cytomegaly in
male mice)
2.1
(fatty
degeneration in
liver of females )
0.8
(fatty
degeneration in
liver of males)
1.4
(Granulomas in
livers of males)
15
(sternebral
variations)
30
(full litter
resorption)
VIII - 22
November 15, 2005
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Table VIII-4 (cont.)
Klinefelter
et al. (1995)b
Bielmeier et
al. (200 l)b
ccc
(2000a)b
CCC
(2000b)b
CCC
(2000c)b
Rat
F344
M
Rat
F344
F
Rabbit
NZW
F
Rabbit
NZW
F
Rat
SD
M,F
7
8-
11
5
25
10
0
22
39
0
75
100
0
4.9
13.9
32.3
76.3
0
1.4
13
36
55
0 ppm
50 ppm
150 ppm
450 ppm
1350
ppm
Drinking
water
Gavage
(aq)
Drinking
Water
Drinking
water
Drinking
water
52 weeks
Gestation day
9
Gestation days
6-29
Gestation days
6-29
Males
64 days
Females
74 days
Body and organ
weights, gross
necropsy,
histology, sperm
motion
parameters
Full litter
resorption,
hormone
profiles, body
weight
Body wt,
clinical signs,
reproductive
developmental
parameters
Maternal feed
and water
intake, body
wt.; gross
lesions; uterine
weight, no.
implantation
sites, uterine
contents, and no.
corpora; Fetal
wt., gross ext.
alterations, skel.
alterations, sex,
visceral
alterations
Reproductive/
developmental
parameters
22
76
55
(developmental)
13.4
(maternal)
50 ppm
39
(decreased sperm
velocities)
75
(full-litter
resorption)
..
35.3
(reduced
maternal body
weight gain)
150 ppm
23
..
50
(maternal)
__cl
4.2
(full-litter
resorption)
e
e
BMDS software
failed
_
VIII - 23
November 15, 2005
-------
Table VIII-4 (cont.)
CCC
(2000d)b
CCC
(2002)
Rat
SD
F
Rat
SD
M, F
25
30
0.0
2.2
18.4
45.0
82.0
0 ppm
50 ppm
150 ppm
450 ppm
Drinking
water
Drinking
water
Gestation days
6-21
Two
generations
Reproductive/
developmental
parameters
Reproductive/
developmental
parameters
45
(developmental)
18.4
(maternal)
50
(offspring)
50
(parental)
82
(reduced no. of
ossification sites
in phalanges or
metatarsals
occurring with
maternal
toxicity)
45
(reduced
maternal body
weight gain)
150
(delayed sexual
maturation in Fj
males)
150
(decrecreased
body weight and
body weight gain
in F0 females and
F[ males and
females)
(developmental)
23
(maternal)
Not modeled
Not modeled
_e
18
(reduced
maternal body
weight gain)
a BMDL10 values were derived using duration-adjusted doses.
b Ruddick et al. (1983); Klinefelter et al. (1995), Narotsky et al. (1997), Bielmeier et al. (2001), and CCC (2000a-d)are included in this table because they are
reproductive and/or developmental studies. The NOAEL, LOAEL, BMD, and BMDL10 values listed are for reproductive and/or developmental endpoints.
0 Data modeled using Crump BMD software
d No histopathological abnormalities were noted in this study, and similar effects on sperm velocity were not observed in NTP (1998); therefore, data for sperm
velocity were not modeled
e Data were not modeled since effects occurred at higher doses than other candidate endpoints
- Indicates that data were not modeled
Abbreviations: NZW, New Zealand White; SD, Sprague-Dawley
NOTE: The short-term reproductive and developmental toxicity study conducted by NTP (1998) was not included in this table because no developmental or
reproductive effects were noted at dose levels ranging from 67 to 126 mg/kg-day.
VIII - 24
November 15, 2005
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Table VIII-5 Summary of Preliminary BMD Modeling Results for the
Bromodichloromethane RfD
Study
Subchronic NTP (1987)
mouse study
Chronic NTP (1987) rat
study
Chronic NTP (1987) mouse
study
Chronic Aida et al. (1992b)
rat study
Endpoint Modeled
Focal necrosis of renal tubular epithelium in males
Vacuolated hepatocytes in females
Kidney cytomegaly in males
Liver necrosis in males
Liver fatty metamorphosis in males
Clear cell changes in liver of females
Kidney cytomegaly in males
Liver fatty metamorphosis in males
Thyroid follicular cell hyperplasia in females
Fatty degeneration in liver of males
Granulomas in liver of males
Fatty degeneration in liver of females
Granulomas in liver of females
BMDL10 (mg/kg-day) *
34
64
No acceptable fit
36.5
No acceptable fit
No acceptable fit
0.96
7.5
15
2.38
4.5
1.20
No acceptable fit
* BMD modeling conducted on duration-adjusted doses using the Crump BMD Software (K. S. Crump, Inc.).
The chronic study conducted by Aida et al. (1992b) was selected for derivation of the
RfD. The lowest dose utilized in this study was 6 mg/kg-day (in contrast to low doses of 22 to 75
mg/kg-day utilized in other candidate studies), which provides some information on the shape of
the dose-response curve in the region of interest. The lowest BMD (1.9 mg/kg-day) was obtained
for fatty degeneration in the liver of male mice. The corresponding BMDL10 for this endpoint
was 0.8 mg/kg-day. The incidence of this lesion was strongly dose-dependent, with incidences of
0/24, 5/14, 12/13, and 19/19 observed at the doses of 0, 6, 25, and 138 mg/kg-day, respectively.
The occurrence of this lesion in rats treated with bromodichloromethane is consistent with current
understanding of the mode of action of brominated trihalomethanes. This endpoint was therefore
selected to derive the RfD for bromodichloromethane.
Derivation of the RfD
The BMDL10 calculated for fatty degeneration in the liver of male rats in the chronic rat
study conducted by Aida et al. (1992b) was selected as the most appropriate basis for derivation
of the RfD for bromodichloromethane. The RfD is calculated according to the following
equation:
VIII - 25
November 15, 2005
-------
(0.8 mg/kg-day)
RfD = = 0.003 mg/kg-day (3 jig/L)
(300)
where:
0.8 mg/kg-day = Duration-adjusted BMDL10 based on fatty degeneration of the
liver in male rats
300 = Uncertainty factor based on NAS/OW guidelines. This value
includes a factor of 10 to account for intrahuman variability,
and a factor of 10 for interspecies variability, and a factor of
3 to account for uncertainty related to possible human
reproductive effects suggested (causality can not be
established from available data) by epidemiological studies.
A composite UF of 300 was used. The standard factors of 10 were used for interspecies
extrapolation and for protection of sensitive subpopulations. An additional factor of 3 was used
to account for database deficiencies related to possible reproductive or developmental effects in
humans. Use of an additional uncertainty factor of 3 is supported by findings in epidemiological
studies (Waller et al., 1998; King et al., 2000) which suggest potential associations between
bromodichloromethane exposure via drinking water and adverse pregnancy outcomes and
changes in semen quality. Although the results of the epidemiological studies can not establish
that bromodichloromethane caused the observed effects, they do raise significant concern for
potential reproductive effects in exposed human populations, and the inclusion of an additional
uncertainty factor is thus considered appropriate for protection of human health.
The DWEL for bromodichloromethane is calculated as follows:
(0.003 mg/kg-day) (70 kg)
DWEL = = 0.100 mg/L (100 jig/L)
2 L/day
where:
0.003 mg/kg-day = RfD for bromodichloromethane
70 kg = Assumed weight of an adult
2 L/day = Assumed water consumption by a 70-kg adult
Lifetime Health Advisory
VIII - 26 November 15, 2005
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The Lifetime Health Advisory (HA) represents that portion of an individual's total
exposure that is attributed to drinking water and is considered protective of noncarcinogenic
health effects over a lifetime of exposure. Bromodichloromethane has been categorized with
respect to carcinogenic potential as Group B2: Probable human carcinogen (IRIS, 1993a).
Therefore, in accordance with U.S. EPA Policy, a Lifetime HA is not recommended.
Alternative Approach for Derivation of the RfD
Use of the conventional NOAEL/LOAEL approach represents an alternative means for
deriving the RfD and DWEL. Aida et al. (1992b) identified a LOAEL of 6 mg/kg-day in male
rats on the basis of histopathological changes in the liver. Using this value and a composite
uncertainty factor of 3,000 (including factors of 10 for interspecies extrapolation, protection of
sensitive subpopulations, and use of a LOAEL, and a factor of 3 for database limitations and
uncertainty regarding potential reproductive effects in humans), the RfD derived using the
conventional approach is 0.002 mg/kg-day. Assuming a body weight of 70 kg and drinking water
ingestion of 2 L/day, the corresponding DWEL is 0.07 mg/L (70 i-ig/L).
2. Carcinogenic Effects
a. Categorization of Carcinogenic Potential
Previous Evaluations
The Carcinogenic Risk Assessment Verification Endeavor (CRAVE) group of the
U.S. EPA reviewed the available evidence on the carcinogenicity of bromodichloromethane and
assigned it to Group B2: probable human carcinogen (IRIS, 1993a). Assignment to this category
is appropriate for chemicals where there are no or inadequate human data, but which have
sufficient animal data to indicate carcinogenic potential.
IARC has recently re-evaluated the carcinogenic potential of bromodichlorom ethane
(IARC 1999a). IARC concluded that there is sufficient evidence of carcinogenicity for
bromodichloromethane in experimental animals, but inadequate evidence in humans. On this
basis, IARC classified bromodichloromethane as a Group 2B carcinogen: possibly carcinogenic
to humans.
Categorization of Carcinogenic Potential Under the Proposed 1999 Cancer Guidelines
Cancer Hazard Summary
Under the proposed guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
bromodichloromethane is likely to be carcinogenic to humans by all routes. 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.
VIII - 27 November 15, 2005
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Supporting Information for Cancer Hazard Assessment
Human Data
The information on the carcinogenicity of bromodichloromethane from human studies is
inadequate. There are no epidemiological data specifically relating increased incidence of cancer
to exposure to bromodichloromethane. There are equivocal epidemiological data describing a
weak association of chlorinated drinking water exposures with increased incidences of bladder,
rectal, and colon cancer. U.S. EPA has determined that these studies cannot attribute the
observed effects to a single compound, as chlorinated water contains numerous other disinfection
byproducts that are potentially carcinogenic.
Animal Data
The carcinogenicity of bromodichloromethane in male and female animals has been
investigated in a well-designed and conducted corn oil gavage study conducted in rats and mice, a
dietary exposure study in rats, and two drinking water studies in rats. Additional data are
available from a study in which male Strain A mice were exposed to bromodichloromethane by
intraperitoneal injection. No data are available on the carcinogenic potential of
bromodichloromethane administered via the inhalation or dermal routes.
In the corn oil gavage study (NTP, 1987), statistically significant increases were observed
in the incidences of neoplasms of the large intestine and kidney in male and female rats, the
kidney in male mice, and liver in female mice. The neoplasms observed in the large intestine and
kidney are considered rare neoplasms based on historical control data for the tested strains. In the
feeding study (Aida et al., 1992b), exposure to microencapsulated bromodichloromethane did not
result in statistically significant increases in any tumor type. Observed neoplastic lesions
included three cholangiocarcinomas and two hepatocellular adenomas in high-dose females, one
hepatocellular adenoma in a control female, one cholangiosarcoma in a high-dose male, and one
hepatocellular carcinoma each in a low- and a high-dose male. In the drinking water study
conducted by Tumasonis et al. (1985), hepatic neoplastic nodules, hepatic adenofibrosis, and
lymphosarcoma were significantly increased in female rats. No significant increase in the
occurrence of any tumor type was observed in male rats. Renal adenoma or adenocarcinoma
were noted in two males and one female treated with bromodichloromethane, while neither tumor
type was reported in the control group. In the drinking water study conducted by George et al.
(2002), the prevalence of neoplastic lesions in the liver was significantly increased only at the
lowest administered dose. Intraperitoneal injection of Strain A mice with bromodichloromethane
resulted in an apparent increase in the number of pulmonary adenomas per animals, although the
response did not reach statistical significance in any dose group.
Structural Analogue Data
Trihalomethanes structurally related to bromodichloromethane have shown varying
degrees of carcinogenic potential in rodents. Chloroform, the most extensively characterized
trihalomethane, is reported to be carcinogenic at high doses in several chronic animal bioassays,
with significant increases in the incidence of liver tumors in male and female mice and significant
VIII - 28 November 15, 2005
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increases in the incidence of kidney tumors in male rats and mice (U.S. EPA, 2001a). The
occurrence of tumors in animals exposed to chloroform is demonstrably species-, strain-, and
gender-specific, and has only been observed under dose conditions that caused cytotoxicity and
regenerative cell proliferation in the target organ. The cancer database for structurally-related
brominated trihalomethanes is more limited, but includes well-conducted studies performed by
the National Toxicology Program. In a two-year corn oil gavage study of bromoform, NTP
(1989a) found clear evidence for carcinogenicity in female rats and some evidence of
carcinogenicity in male rats based on occurrence of tumors of the large intestine (adenomatous
polyps or adenocarcinoma). In a two-year corn oil gavage study of dibromochloromethane, NTP
(1985) determined that there was some evidence of carcinogenicity in female mice and equivocal
evidence of carcinogenicity in male mice, based on the occurrence of hepatocellular adenomas
and carcinomas. Other, less well-documented, oral exposure studies (Tobe et al., 1982;
Kurokawa, 1987; Voronin et al., 1987) found no evidence for carcinogenicity of bromoform or
dibromochloromethane.
Other Key Data
Bromodichloromethane is formed as a byproduct of drinking water disinfection with
chlorine. Exposure to bromodichloromethane may occur via ingestion of tap water, via dermal
contact during showering or bathing, or by inhalation of bromodichloromethane volatilized
during household activities. Absorption of single oral doses appears to be extensive.
Bromodichloromethane is rapidly metabolized and eliminated predominately as expired volatiles,
carbon dioxide, or carbon monoxide. Only a small amount (less than 10%) is eliminated in urine
or in feces. No comprehensive tissue data are available regarding the bioaccumulation or
retention of bromodichloromethane following repeated exposure. However, because of the rapid
metabolism and excretion of bromodichloromethane, marked accumulation and retention is not
expected.
Bromodichloromethane itself is not directly reactive with DNA. Metabolism to reactive
species is a prerequisite for toxicity, as inferred from metabolic induction and inhibition studies.
In vitro and in vivo studies of the mutagenic and genotoxic potential of bromodichloromethane
have yielded both positive and negative 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. However,
because a majority of studies yielded positive results, bromodichloromethane is considered to be
at least weakly mutagenic and genotoxic. Recent studies conducted with strains of Salmonella
that express rat theta-class glutathione ^-transferase suggest that mutagenicity of the brominated
trihalomethanes may be mediated by glutathione conjugation.
Mode of Action
The mode of action for tumor induction by bromodichloromethane has not been clearly
elucidated and may involve contributions from multiple bioactivation pathways. In each case,
toxicity is believed to result from interaction of reactive metabolites with cellular
macromolecules. Proposed bioactivation pathways for bromodichloromethane include: 1)
VIII - 29 November 15, 2005
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production of reactive dihalocarbonyls by oxidative metabolism; 2) production of reactive
dihalomethyl radicals by oxidative metabolism; and 3) formation of DNA-reactive species via a
glutathione-dependent pathway. The relative contribution of each pathway to tumor induction by
bromodichloromethane has not been characterized. It is possible that only the latter two
processes lead to DNA damage in vivo, because the highly reactive dihalocarbonyl intermediate
may not survive long enough to enter the nucleus and react with DNA. For this reason,
cytotoxicity may be the primary consequence of the oxidative pathway. Cytotoxicity coupled
with regenerative hyperplasia is considered the primary mode of action for tumor formation
following exposure to high concentrations of chloroform, a structurally-related trihalomethane
which has low genotoxic potential. Data to support a similar primary mode of action for tumor
development in liver, kidney, and large intestine are currently lacking for bromodichloromethane.
In the absence of such information, combined with a positive weight-of-evidence evaluation for
genotoxicity, the mode of action for tumor development is assumed to be a linear process. The
processes leading to tumor formation in animals are expected to be relevant to humans.
Conclusion
Under the proposed guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
bromodichloromethane is likely to be carcinogenic to humans by the oral route. This weight-of-
evidence evaluation is based on 1) observations of tumors in animals treated by oral pathways; 2)
lack of epidemiological data specific to bromodichloromethane and equivocal data for drinking
water drinking water exposures that cannot reliably be attributed to bromodichloromethane
among multiple other disinfection byproducts; 3) positive results for a majority of the available
genotoxicity and mutagenicity tests; and 4) metabolism and mode of action that are reasonably
expected to be comparable across species. Although no cancer data exist for exposures via the
dermal or inhalation pathways, the weight-of-evidence conclusion is considered to be applicable
to these pathways as well. The finding for inhalation is based on the observation that patterns of
metabolizing enzyme activity in male rats are similar for exposure via the inhalation and gavage
routes. Bromodichloromethane absorbed through the skin is expected to be metabolized and
cause toxicity in much the same way as bromodichloromethane absorbed by the oral and
inhalation routes.
b. Choice of Study for Quantification of Carcinogenic Risk
In accordance with the Proposed 1999 Cancer Guidelines (U.S. EPA, 1999),
quantification of cancer risk is appropriate for compounds categorized as likely to be
carcinogenic to humans. Five oral exposure studies were available for quantification of the
carcinogenic risk associated with exposure to bromodichloromethane. Detailed summaries of
these studies are provided in Section V.G. 1. The two-year study conducted by NTP (1987) in rats
and mice was selected for quantification of carcinogenic effects associated with exposure to
bromodichloromethane. Selection of this study was based on significantly increased incidence of
several tumor types, monotonic dose response curves, and comprehensive documentation of study
design and results.
In the NTP (1987) study, groups of male and female F344/N rats (50/sex/dose) received 0,
50, or 100 mg/kg-day gavage doses of bromodichloromethane in corn oil. The doses were
VIII - 30 November 15, 2005
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administered 5 days/week for 102 weeks. In a similar experiment, groups of male and female
B6C3FJ mice (50/sex/dose) were administered doses of 0, 25, or 50 mg/kg-day (males) or 0, 75,
or 150 mg/kg-day (females) for 5 days/week for 102 weeks. All animals were subjected to gross
and microscopic examinations for neoplastic lesions. Survival of all dosed animals was
comparable to or greater than the corresponding control group. Statistically significant increases
were observed in the incidences of neoplasms of the large intestine and kidney in male and
female rats, the kidney in male mice, and the liver in female mice (Table VIII-6). The authors
noted that neoplasms of the large intestine and kidney are uncommon tumors in F344/N rats and
B6C3FJ mice. They concluded that under the conditions of these 2-year gavage studies, clear
evidence of carcinogenic activity existed for male and female rats and mice.
VIII-31 November 15, 2005
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Table VIII-6 Tumor Frequencies in Rats and Mice Exposed to Bromodichloromethane in
Corn Oil for 2 Years - Adapted from NTP (1987)
Animal Tissue/Tumor
Male rat
Large Adenomatous polyp
intestine8
Adenocarcinoma
Combined
Kidney a Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female rat
Large Adenomatous polyp
intestine °
Adenocarcinoma
Combined
Kidney Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Male mouse
Kidney d Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female mouse
Liver Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Tumor Frequency
Control
0/50
0/50
0/50
0/50
0/50
0/50
Control
0/46
0/46
0/46
0/50
0/50
0/50
Control
1/46
0/46
1/46
Control
1/50
2/50
3/50
50 mg/kg
3/49
ll/49b
13/49b
1/49
0/49
1/49
50 mg/kg
0/50
0/50
0/50
1/50
0/50
1/50
25 mg/kg
2/49
0/49
2/49
75 mg/kg
13/48b
5/48
18/48b
100 mg/kg
33/50b
38/50b
45/50b
3/50
10/50b
13/50b
100 mg/kg
7/47b
6/47b
12/47b
6/50b
9/50b
15/50b
50 mg/kg
6/50
4/50
9/50b
150 mg/kg
23/50b
10/50b
29/50b
a One rat died at week 33 in the low-dose group and was eliminated from the cancer risk calculation.
b Statistically significant at p<0.05, compared to controls.
c Intestine not examined in four rats from control group and three rats from high-dose group.
d In the control group, two mice died during the first week, one mouse died during week, nine and one escaped in week 79. One
mouse in the low-dose group died in the first week. All of these mice were eliminated from the cancer risk calculations.
VIII - 32
November 15, 2005
-------
Use of the NTP rodent studies (NTP,1987) for derivation of cancer risk estimates for
bromodichloromethane is complicated by the use of corn oil as a dosing vehicle. Although a
vehicle effect has not been reported for brominated trihalomethanes, it can be inferred from
studies of chloroform carcinogenicity that such an effect might exist, at least for hepatic tumors in
mice. Therefore, in the case of bromodichloromethane, the U.S. EPA believes that the most
appropriate basis of the cancer risk estimate is the incidence of renal tumors in male mice. Renal
tumors are considered to be appropriate because these tumors were increased in a dose-dependent
manner in both mice (male) and rats (both sexes).
c. Choice of Approach and Rationale
The LMS model (U.S. EPA, 1986) and the default linear approach described by the
Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996; 1999) were used to
quantify the cancer risk associated with exposure to bromodichloromethane. Although data are
mixed, the weight of evidence indicates that bromodichloromethane is mutagenic (see Section
V.F.I). Furthermore, recent studies (Melnick, et al. 1998; George et al., 2002) suggest that
induction of hepatic tumors occurs at doses of bromodichloromethane that have marginal or no
effect on hepatocyte labeling index, indicating that regenerative hyperplasia is not required for
tumor induction. Thus, use of a linear approach was considered appropriate for quantification of
cancer risk associated with exposure to bromodichloromethane.
d. Cancer Potency and Risk Estimates
The available estimates for cancer risk associated with bromodichloromethane are
summarized in Table VIII-7. U.S. EPA (1994b) recommended use of a cancer potency estimate
of 6.2 x 10"2 (mg/kg-day)"1 as reported in IRIS (1993a). This value was derived in accordance
with the 1986 Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 1986), based on the
occurrence of renal tumors in male mice. A unit risk of 1.8 x 10"6 was estimated using an
assumed body weight of 70 kg and a drinking water ingestion rate of 2 L. This estimate was used
to calculate a drinking water concentration of approximately 6 |ig/L associated with a 10"5 risk
(0.6 ng/L for 10"6 risk).
A cancer potency value of 3.5 x 10"2 (mg/kg-day)"1 (U.S. EPA, 1998b) was derived using
the LMS method and an animal-to-human conversion factor of body weight374 (Table VIII-7). The
use of body weight374 is consistent with recommendations in U.S. EPA (1992b). This potency
factor is also based on the occurrence of renal tumors in male mice. A unit risk of 1 x 10"6 (i-ig/L)"
1 was estimated for bromodichloromethane using an assumed body weight of 70 kg and a
drinking water ingestion rate of 2 L. This estimate was used to calculate a drinking water
concentration of approximately 10 |ig/L associated with a 10"5 risk (1 |ig/L for 10"6 risk).
Cancer risk estimates were also obtained using the LED10 (the lower 95% confidence limit
on a dose associated with 10% extra risk) of 3.0 x 103 |ig/kg-day for renal tumors in mice and
assuming a linear mode of action for the carcinogenicity of bromodichloromethane (Table VIII-
7). A cancer potency value of 3.4 x 10"2 (mg/kg-day)"1 was derived using this approach. A unit
risk of 9.6 x 10"7 (jig/L)"1 was estimated for bromodichloromethane using an assumed body
weight of 70 kg and a drinking water ingestion rate of 2 L. This estimate was used to calculate a
VIII - 33 November 15, 2005
-------
drinking water concentration of approximately 10 |ig/L associated with a 10"5 risk (1 |ig/L for 10"6
risk). These values are closely similar to corresponding values derived using the LMS approach
with body weight scaling to the 3/4 power.
Table VIII-7 Summary of Cancer Risk Estimates for Bromodichloromethane
Method of Estimation
LMS Method Using
BW3/4Conversion
(U.S. EPA, 1998b)
LMS Method Using
BW2/3Conversion
U.S. EPA(1994b)*
LED10/Linear Method
(U.S. EPA, 1998b)
Tumor Site
Liver
Kidney
Large
intestine
Liver
Kidney
Large
intestine
Liver
Kidney
Large
intestine
Species
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Sex
F
M
F
M
M
F
F
M
F
M
M
F
F
M
F
M
M
F
Slope Factor
(mg/kg-day)1
6.9xlO"2
5.5xlO"3
6.1xlO"3
3.5xlO"2
1.7xlO"2
6.1 xio"3
1.3X10'1
8.7xlO"3
9.5xlO"3
6.2xlO"2
2.5xlO"2
4.9xlO"3
6.5xlO"2
8.1 xlO"3
8.8xlO"3
3.4 xlO"2
2.8xlO"2
l.OxlO"2
Unit Risk
Glg/L)1
2.0x10-'
1.6X10'7
1.7x10-'
l.OxlO'6
4.9x10"'
1.7x10"'
3.7xlO"6
2.5x10"'
2.7x10"'
1.8xlO"6
7.1x10"'
1.4x10"'
1.9x10"'
2.3x10"'
2.5x10"'
9.6 xlO"'
8x10"'
3x10"'
LED10
Glg/kg-
day)
-
-
-
-
-
-
-
-
l.SxlO3
1.2x10"
l.lxlO4
3.0 xlO3
3.5xl03
9.6xl03
10 5 Risk
Concentration
Glg/L)
5
64
57
10
20
57
3
40
37
6
14
72
5
43
40
10
12
34
* Based on information adapted from IRIS (1993a)
B. Dibromochloromethane
1. Noncarcinogenic effects
a. One-day Health Advisory
Four candidate studies that investigated the acute oral toxicity of dibromochloromethane
were available. These studies are summarized in Table VIII-8 (below). Bowman et al. (1978)
administered dibromochloromethane by gavage to ICR Swiss mice at doses ranging from 500 to
4,000 mg/kg and found that sedation and anesthesia resulted at doses of 500 mg/kg or higher.
NTP (1985) conducted a preliminary range-finding study in which F344/N rats and B6C3FJ mice
were administered dibromochloromethane by gavage at doses ranging from 160 to 2,500 mg/kg
and found that death may result from doses at 310 mg/kg or higher in mice or rats. More
recently, Miiller et al. (1997) investigated the cardiotoxic effects of acute oral
VIII - 34
November 15, 2005
-------
dibromochloromethane exposure in male Wistar rats. In this study, rats administered doses of 83
or 167 mg/kg exhibited transient changes in cardiovascular parameters, while rats administered
doses of 333 or 667 mg/kg exhibited persistent alterations in at least one of the cardiovascular
parameters that lasted throughout the postexposure observation period. Finally, Korz and
Gatterman (1997) investigated the behavioral toxicity of acute oral dibromochloromethane
exposure in male golden hamsters and observed only transient effects on the behavioral
parameters investigated.
These studies were not considered adequate for deriving the One-day HA, since more
sensitive endpoints such as histopathology were not evaluated. Therefore, the Ten-day HA for
dibromochloromethane (0.6 mg/L) calculated below is recommended for use as the One-day HA.
b. Ten-day Health Advisory
Studies considered for derivation of the Ten-day HA for dibromochloromethane are
summarized in Table VIII-9 below. The key studies in this group are those of Aida et al. (1992a),
Condie et al. (1983), and Melnick et al. (1998). These studies reported effects on sensitive
endpoints and had data suitable for BMD analysis. Use of the remaining studies was limited by a
variety of considerations, including lack of data suitable for BMD analysis (Chu et al., 1982a;
NTP, 1996; Coffin et al., 2000), toxicological relevance or difficulty in interpretation of the most
sensitive endpoint (NTP, 1985; Munson et al. 1982), and occurrence of effects only at the frank
toxicity level (NTP, 1985).
Melnick et al. (1998) administered dibromochloromethane to female B6C3FJ mice by
gavage for 5 days/week for 3 weeks and identified a NOAEL of 100 mg/kg-day (duration-
adjusted NOAEL of 71 mg/kg-day) and a LOAEL of 192 mg/kg-day (duration-adjusted LOAEL
of 137 mg/kg-day) based on histologic changes in the liver (hepatocyte hydropic degeneration).
BMD analysis calculated BMD and BMDL10values of 112 and 68 mg/kg-day, respectively.
Table VIII-8 Summary of Candidate Studies for Derivation of the One-day HA for
Dibromochloromethane
Reference
Bowman et al.
(1978)
NTP (1985)
Mtiller et al.
(1997)
Korz and
Gatterman
(1997)
Species
Mouse
ICR Swiss
Rat
F344/N
Mouse
B6C3FJ
Rat
Wistar
Hamster
Route
Gavage
(aqueous)
Gavage
(corn oil)
Gavage
(olive oil)
Gavage
(olive oil)
Exposure
Duration
Single
dose
Single
dose
Single
dose
Single
dose
Dose
(mg/kg-day)
500 - 4000
160 - 2500
83 - 667
50
Result
Sedation; anesthesia; increased
mortality
Lethargy; death
Transient changes in blood pressure;
effects on cardiac muscle contractility
Transient changes in post-treatment
behavior
VIII - 35
November 15, 2005
-------
Table VIII-9 Summary of Candidate Studies for Derivation of the Ten-day HA for
Dibromochloromethane
Reference
Aida et al.
(1992a)
Chu et al.
(1982a)
Condie et
al.
(1983)
Melnick et
al.
(1998)
Munson et
al.
(1982)
Species
Sex
Rat
Wistar
M, F
Rat
SD
M
Mouse
CD-I
M
Mouse
B6C3FJ
F
Mouse
CD-I
M, F
n
7
10
8-
16
10
8-
12
Dose
Males
0
18
56
173
Females
0
34
101
332
0
0.7
8.5
68
0
37
74
147
0
50/37
100/71
192/137
417/298
0
50
125
250
Route
Feed
Drinking
water
Gavage
(oil)
Gavage
(oil)
Gavage
(aqueous)
Exposure
Duration
1 month
28 days
14 days
3 weeks
(5 d/wk)
14 days
1 ml points
Body weight, clinical
signs, serum
biochemistry,
hematology, histology
Clinical signs, serum
biochemistry,
histology
Serum enzymes, PAH
uptake in vitro,
histology
Body and liver
weights, serum
chemistry, liver
histology
Body and organ
weights, serum
chemistry,
hematology, immune
function
NOAEL
(mg/kg-day)
18.3
68
74
100 (marginal)
50
LOAEL
(mg/kg-day)
56
(liver
histopathology)
147
(elevated ALT,
decreased PAH,
liver and kidney
histopathology)
192
(liver
histopathology)
125
(decreased
humoral
immunity)
BMD
(mg/kg-day)
29
14
Not modeled
3.5
11
112*
Not modeled
BMDL10
(mg/kg-day)
6.7
(Liver cell
vacuolation in
females)
5.5
(Liver cell
vacuolation in
males)
1.6
(Renal mesangial
hypertrophy)
6.9
(hepatic
cytoplasmic
vacuolation)
68
(hepatic hydropic
degeneration)
_
VIII - 36
November 15, 2005
-------
Table VIII-9 (cont.)
Reference
NTP(1985)
NTP(1985)
NTP (1996)
Coffin et al.
(2000)
Species
Sex
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Rat
F344/N
M, F
Mouse
B6C3FJ
F
n
5
5
10
10
Dose
0
60
125
250
500
1000
0
30
60
125
250
500
Males
0
4
12
28
Females
0
6-7
17-20
48-48
0
100
300
Route
Gavage
(oil)
Gavage
(oil)
Drinking
water
Gavage
(oil)
Exposure
Duration
14 days
14 days
29 days
11 days
1 ml points
Body weight, clinical
signs, gross necropsy
Body weight, clinical
signs, gross necropsy
Body weight, serum
chemistry,
hematology, gross
necropsy, histology,
sperm evaluation
Relative liver wt;
liver histopathology;
labeling index
NOAEL
(mg/kg-day)
250
60
28
..
LOAEL
(mg/kg-day)
500 (FEL)
(mortality,
lethargy, gross
pathology)
125
(stomach lesions)
..
100
BMD
(mg/kg-day)
Not modeled
143
218
Not modeled
Not modeled
BMDL10
(mg/kg-day)
..
54
(stomach nodules
- males)
77
(stomach nodules
- females)
_
..
*BMD and BMDL10 calculated using duration-adjusted doses
Abbreviations :FEL, Frank Effect Level; SD, Sprague-Dawley
No data
VIII - 37
November 15, 2005
-------
These values are considerably higher (approximately 5- to 10-fold) than BMD and BMDL10
values calculated for hepatic effects using data from Condie et al. (1983) or Aida et al. (1992a).
Condie et al. (1983) administered dibromochloromethane by gavage to male CD-I mice
for 14 days and identified aNOAEL of 74 mg/kg-day and a LOAEL of 147 mg/kg-day based on
minimal to moderate liver and kidney injury. Histologic changes in the liver included focal
inflammation and cytoplasmic vacuolization similar to that observed in the study by Aida et al.
(1992a). Effects in the kidney included minimal to slight epithelial hyperplasia at the high dose
and minimal to slight mesangial hypertrophy at all (non-control) doses. Data for cytoplasmic
vacuolization and renal mesangial hypertrophy were analyzed by BMD modeling. The lowest
BMD and BMDL10 (3.5 and 1.6 mg/kg-day, respectively) among all candidate studies were
identified for mesangial hypertrophy. However, the pattern of dose-response for this endpoint
(0/16, 4/5, 7/10, 7/10 at doses of 0, 37, 74, and 147 mg/kg-day, respectively) resulted in generally
poor curve fits (0.10.49) and a high degree of model-dependence (See
summary of modeling results in Appendix A). Thus, confidence in the reliability of the BMD for
renal effects was low. The BMD and BMDL10 values for hepatic cytoplasmic vacuolization were
higher (11 and 6.9 mg/kg-day, respectively). These results were based on incidences of 1/16, 3/5,
4/10, and 8/10 at doses of 0, 37, 74, and 147 mg/kg-day, respectively.
The study by Aida et al. (1992a) was selected as the basis for derivation of the Ten-day
HA. In this study, Wistar rats of both sexes were administered microencapsulated
dibromochloromethane in the diet for one month. The dose levels ranged from 18.3 to 173.3
mg/kg-day for males and from 34.0 to 332.5 mg/kg-day for females. A NOAEL of 18.3 mg/kg-
day and a LOAEL of 56.2 mg/kg-day were identified based on histologic changes (cell
vacuolization, swelling, and single cell necrosis) in the livers of male rats. BMD analysis of data
for hepatic cell vacuolization calculated BMD and BMDL10 values of 29 and 6.7 mg/kg-day in
females and 14 and 5.5 mg/kg-day in males, respectively. The BDML10 for hepatic cell
vacuolization in male rats was selected for calculation of the 10-day HA because it was
considered the lowest reliable value based on examination of the raw data and modeling results.
The incidence of this lesion was 0/7, 1/7, 3/7, and 7/7 at doses of 0, 18, 56, and 173 mg/kg-day,
respectively.
Based on the BMDL10 calculated from the data of Aida et al. (1992a), the Ten-day HA is
derived as follows:
(5.5 mg/kg-day) (10 kg)
Ten-day HA = =0.55 mg/L (rounded to 0.6 mg/L)
(100) (1 L/day)
5.5 mg/kg-day = BMDL10 based on hepatic cell vacuolization in rats fed
dibromochloromethane for one month
10 kg = Assumed body weight of a child
VIII - 38 November 15, 2005
-------
100 = Composite uncertainty factor based on NAS/OW
guidelines; includes a factor of 10 for interspecies
variation and a factor of 10 for protection of sensitive
human populations
1 L/day = Assumed water consumption of a 10-kg child
The Ten-day HA was calculated using the conventional NOAEL/LOAEL approach for
comparison with the value obtained using the BMD approach. The Aida et al. (1992a) study
identified a NOAEL of 18.3 mg/kg-day based on the absence of hepatic effects in rats. Using this
value and the assumptions described above, the Ten-day HA would be 1.8 mg/L (rounded to 2
mg/L).
c. Longer-term Health Advisory
Four candidate studies were considered for derivation of the Longer-term HA for
dibromochloromethane. These studies are summarized in Table VIII-10 (below). Selected data
from three of these studies were modeled using the BMD approach. The results of BMD analysis
are included in Table VIII-11.
Chu et al. (1982b) administered dibromochloromethane to Sprague-Dawley rats in the
drinking water at doses ranging from 0.57 to 236 mg/kg-day. This study identified a NOAEL of
49 mg/kg-day, and a LOAEL of 224 mg/kg-day based on mild hepatic lesions (increased
cytoplasmic volume and vacuolation due to fatty infiltration) observed in males. BMD and
BMDL10 values of 18 and 5.3 mg/kg-day, respectively, were calculated for hepatic vacuolization
using the BMDS software. Daniel et al. (1990) identified a LOAEL of 50 mg/kg-day based on
hepatic lesions (centrilobular lipidosis) observed in male Sprague-Dawley rats and on kidney
lesions (tubular degeneration) observed in female Sprague-Dawley rats administered
dibromochloromethane by gavage for 90 consecutive days. BMD and BMDL10 values of 20 and
4.2 mg/kg-day, respectively, were calculated for renal tubular degeneration using the Crump
BMD software (K. S. Crump, Inc.). NTP (1985) administered doses of dibromochloromethane
ranging from 15 to 250 mg/kg-day to male and female mice. The compound was administered by
gavage in corn oil, five days per week for 13 weeks. NOAEL and LOAEL values of 125 and 250
mg/kg-day were identified on the basis of renal and hepatic lesions. BMD and BMDL10 values
were not calculated since lesions occurred only at the high dose of 250 mg/kg-day.
The NTP (1985) study conducted in rats was selected as the basis for derivation of the
Longer-term HA. In this study, F344/N rats were administered dibromochloromethane by gavage
at dose levels ranging from 15 to 250 mg/kg for 5 days/week for 13 weeks. Severe lesions and
necrosis of the kidney, liver, and salivary glands were observed primarily at the high dose.
However, males exhibited a dose-dependent increase in the frequency of clear cytoplasmic
vacuoles indicative of fatty metamorphosis in the liver. This effect reached statistical
VIII - 39 November 15, 2005
-------
Table VIII-10 Summary of Candidate Studies for Derivation of the Longer-term HA for Dibromochloromethane
Reference
Chu et al.
(1982b)
Daniel et al.
(1990)
NTP(1985)
NTP(1985)
Species
Rat
SD
M, F
Rat
SD
M, F
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
n
20
10
10
10
Dose
Males
0
0.57
6.1
49
224
Females
0
0.64
6.9
55
236
0
50
100
200
0
15
30
60
125
250
0
15
30
60
125
250
Route
Drinking
water
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Exposure
Duration
90 days
90 days
13 weeks
(5 d/wk)
13 weeks
(5 d/wk)
1 ml points
Body weight, serum
chemistry, histology
Body weight, clinical
signs, serum
biochemistry, gross
necropsy, histology
Body weight, clinical
signs, histology
Body weight, clinical
signs, histology
NOAEL
(mg/kg-day)
49
..
30
125
LOAEL
(mg/kg-day)
224
(decreased weight
gain, mild hepatic
lesions)
50
(hepatic and renal
lesions)
60
(hepatic lesions)
250
(renal and hepatic
lesions)
BMD
(mg/kg-day)*
18
20f
2.5
Not modeled
BMDL10
(mg/kg-day) *
5.3
(Liver lesions in
males)
4.2f
(kidney cortex
degeneration in
females)
1.7
(liver fatty
metamorphosis in
males)
--
* BMDL10 values were derived using duration-adjusted doses.
f Modeled using Crump benchmark dose software
Abbreviations: SD, Sprague-Dawley
VIII - 40
November 15, 2005
-------
significance at 60 mg/kg-day, and this dose was designated the LOAEL. The next lower dose (30
mg/kg-day) was designated as the NOAEL. BMD analysis using the BMDS program obtained
duration-adjusted BMD and BMDL10 values of 2.5 and 1.7 mg/kg-day, respectively. These
values were the lowest calculated among the three studies for which BMD analysis was
conducted.
Using the NTP (1985) rat study, the Longer-term HA for the 10-kg child is calculated as
follows:
(1.7mg/kg-day)(10kg)
Longer-term HA = =0.17 mg/L (rounded to 0.2 mg/L)
(100) (1 L/day)
where:
1.7 mg/kg-day = Duration-adjusted BMDL10 based on hepatic cell
vacuolization in rats exposed to dibromochloromethane by oil
gavage for 13 weeks
10 kg = Assumed body weight of a child
100 = Composite uncertainty factor based on NAS/OW guidelines;
includes a factor of 10 for interspecies variation and a factor
of 10 for protection of sensitive human populations
1 L/day = Assumed water consumption of a 10-kg child
The Longer-term HA for a 70-kg adult consuming 2 liters of water per day is calculated
according to the following equation:
(1.7mg/kg-day)(70kg)
Longer-term HA = =0.60 mg/L (rounded to 0.6 mg/L)
(100) (2 L/day)
For purposes of comparison, the Longer-term HAs were also calculated using the
conventional NOAEL/LOAEL approach. NTP (1985) identified a NOAEL of 30 mg/kg-day by
based on the absence of clinical signs or histologic alterations in rats exposed to
dibromochloromethane by corn oil gavage for 13 weeks. Using a duration adjusted dose of 21
mg/kg-day (obtained by multiplying the nominal dose by 5/7) and the assumptions for body
weight and drinking water ingestion described above, the Longer-term HAs would be 2.1 and 7.5
mg/kg-day for a 10 kg child and 70 kg adult, respectively.
VIII - 41 November 15, 2005
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d. Reference Dose, Drinking Water Equivalent Level and Lifetime Health Advisory
This section reports the existing RfD value for dibromochloromethane and describes the
derivation of the RfD for this compound. This section also describes the calculation of Drinking
Water Equivalent Level and Lifetime Health Advisory values which require the RfD as input.
For this document, new and existing studies were reviewed and appropriate candidate data were
selected for benchmark dose (BMD) modeling. The results of BMD modeling were used in
conjunction with appropriate uncertainty factors to calculate the RfD. A comparison of the RfD
derived using the BMD approach to the results obtained using the conventional NOAEL/LOAEL
approach is also provided.
Description of the Existing RfD
The existing RfD for dibromochloromethane is 0.02 mg/kg-day (IRIS, 1992). This value
was derived using a duration-adjusted NOAEL of 21.4 mg/kg-day identified for the occurrence of
hepatic lesions in F344/N rats administered dibromochloromethane by corn oil gavage, 5
days/week for 13 weeks . An uncertainty factor of 1000 was used to account for extrapolation
from animal data, for protection of sensitive human subpopulations, and for use of a subchronic
study.
Identification of Candidate Studies for Derivation of the RfD
Candidate studies considered for derivation of the RfD for dibromochloromethane are
summarized in Table VIII-11 (below). Tobe et al. (1982) administered microencapsulated
dibromochloromethane in the diet to Wistar rats for 24 months at dose levels that ranged from 12
to 278 mg/kg-day. Although the study identified a NOAEL and a LOAEL of 12 and 49 mg/kg-
day, respectively, based on decreased body weight, changes in clinical chemistry parameters, and
gross liver appearance in males, a histopathological examination was not conducted. NTP (1985)
investigated the chronic oral toxicity of dibromochloromethane in F344/N rats and B6C3FJ mice.
Only LOAEL values were identified in these studies. Specifically, the rat study identified a
LOAEL of 40 mg/kg-day based on histologic lesions in both male and female rats (e.g., fatty
change), and the mouse study identified a LOAEL of 50 mg/kg-day based on lesions in the liver
(fatty metamorphosis) and the thyroid (follicular cell hyperplasia) in the female mice. A thirteen-
week oral exposure study in rats (NTP, 1985) examined toxicity at a wider range of doses than
the chronic studies and identified NOAEL and LOAEL values of 30 and 60 mg/kg-day,
respectively, for histopathological changes in the liver.
Method of Analysis
Selected data from the candidate studies were analyzed using the benchmark dose (BMD)
modeling approach. Initially, data sets for potentially sensitive endpoints were selected as
described in U.S. EPA (1998b) and analyzed using the Crump Benchmark Dose Modeling
Software (K. S. Crump, Inc.). Results of this preliminary analysis are summarized in Table VIII-
12. Following the release of Version 1.2 of the BMDS program (U.S. EPA, 2000a), a subset of
the most sensitive endpoints identified using the Crump software was reanalyzed in accordance
with proposed U.S. EPA (2000b) recommendations. An advantage of analysis with the BMDS
VIII - 42 November 15, 2005
-------
software is that several additional models are available to fit the data. The results of the analysis
using the BMDS software are included in Table VIII-11.
Choice of Principal Study and Critical Effect for the RfD
Two studies were identified as strong candidates for selection as the principal study. The
NTP (1985) subchronic study evaluated toxicological effects in male and female rats at five
concentrations of dibromochloromethane (15, 30, 60, 125, and 250 mg/kg-day) in addition to the
control. The chemical was administered by gavage in corn oil on five days per week for 13
weeks. A relatively small sample size of 10 animals/treatment group was utilized. The endpoints
evaluated included clinical signs, body weight, serum biochemistry, and histopathological
changes in organs. NOAEL and LOAEL values of 30 and 60 mg/kg-day, respectively, were
identified on the basis of hepatic lesions using the conventional approach. Analysis of the data
for fatty metamorphosis in the liver using the BMDS program resulted in a duration-adjusted
BMD of 2.7 mg/kg-day for this endpoint, with a corresponding duration-adjusted BMDL10 of 1.7
mg/kg-day. A strength of this study with respect to BMD modeling was the use of additional
doses at the lower end of the dose-response range. Inclusion of these doses permits more accurate
characterization of the shape of the dose-response curve and thus less uncertainty in the range of
interest.
The second candidate for selection as the principal study for derivation of the RfD was the
chronic study conducted by NTP (1985). This study evaluated dibromochloromethane effects at
administered doses of 0, 40 and 80 mg/kg-day. The chemical was administered by gavage in oil
on five days per week for 104 weeks. The endpoints evaluated included clinical signs, body
weight, serum biochemistry, and histopathological changes in organs, and a LOAEL of 40 mg/kg-
day based on hepatic lesions was identified using the conventional approach. Analysis of the data
for fatty metamorphosis in the liver using the BMDS program resulted in a duration-adjusted
BMD of 2.5 mg/kg-day for this endpoint, with a corresponding duration-adjusted BMDL10 of 1.6
mg/kg-day based on duration adjusted doses.
A potential weakness of the NTP (1985) chronic study is the lack of dose-response
information at administered doses less than 40 mg/kg-day. A priori., the lack of information
regarding the shape of the curve at low doses would be expected to result in greater uncertainty
(and thus wider confidence limits) in the estimate of the chronic BMD. However, the BMD and
BMDL10 values calculated for fatty metamorphosis in the subchronic and chronic studies are
closely similar. This observation suggests that there is little potential for cumulative effects on
the occurrence of this lesion. The slight differences in the values may reflect both experimental
uncertainty and uncertainty in modeling.
VIII - 43 November 15, 2005
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Table VIII-11 Summary of Candidate Studies for Derivation of the RfD for Dibromochloromethane
Reference
Tobe et al.
(1982)
NTP (1985)
NTP (1985)
NTP (1985)
Borzelleca
and
Carchman
(1982)**
Species
Sex
Rat
Wistar
M,F
Rat
F344/N
M,F
Rat
F344/N
M,F
Mouse
B6C3FJ
M, F
Mouse
ICR
Swiss
-
n
40
10
50
50
10M
30F
Dose
(mg/kg-day)
Males
0
12
49
196
Females
0
17
70
278
0
15
30
60
125
250
0
40
80
0
50
100
0
17
171
685
Route
Diet
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Drinking
water
Exposure
Duration
24 months
13 weeks
(5 d/wk)
104 weeks
(5 d/wk)
105 weeks
(5 d/wk)
27 weeks
1 ml points
Body weight, serum
biochemistry, gross
pathology
Body weight, clinical
signs, serum
biochemistry, gross
necropsy, histology
Body weight, clinical
signs, gross necropsy,
histology
Body weight, clinical
signs, gross necropsy,
histology
Maternal body
weight, gross
pathology, fetal
weight, survival,
teratogenicity
NOAEL
(mg/kg-
day)
12
30
17
(marginal)
LOAEL
(mg/kg-day)
49
(serum enzyme
changes and
altered liver
appearance)
60
(hepatic lesions)
40
(hepatic lesions)
50
(hepatic lesions)
171
(maternal toxicity,
possible
fetotoxicity)
BMD
(mg/kg-day)
Not modeled
2.5
2.7
9.1f
Not modeled
(insuff. data
provided in
publication)
BMDL10
(mg/kg-day) *
..
1.7
(fatty
metamorphosis
in liver of
males)
1.6
(fatty changes in
liver of males)
7.1f
(thyroid
follicular cell
hyperplasia in
females)
VIII - 44
November 15, 2005
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Table VIII-11 (cont.)
Reference
NTP (1996)
**
Species
Sex
Rat
SD
M, F
n
10
Dose
(mg/kg-day)
males
0
4.2
12.4
28.2
Group A
females
0
6.3
17.4
46.0
Group B
females
0
7.1
20.0
47.8
Route
Drinking
water
Exposure
Duration
29 days
1 ml points
Body weight, serum
chemistry,
hematology, gross
necropsy, histology,
sperm evaluation
NOAEL
(mg/kg-
day)
28
LOAEL
(mg/kg-day)
BMD
(mg/kg-day)
Not modeled
BMDL10
(mg/kg-day) *
**
BMDL10 values were derived using duration-adjusted doses.
These studies have been included in this table because they are reproductive/developmental studies and would be considered relevant for derivation of the
RfD. However, Borzelleca and Carchman (1982) found only marginal evidence for developmental toxicity at the low-dose level and the NTP (1996) study
did not observe any reproductive or developmental effects at the dose levels evaluated.
f Modeled using Crump BMD software
Abbreviations: SD, Sprague-Dawley
VIII - 45
November 15, 2005
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Table VIII-12 Results of Preliminary BMD Modeling of Selected Data from NTP (1985)
Studies
Study
Subchronic NTP (1985) rat study
Chronic NTP (1985) rat study
Chronic NTP (1985) mouse study
Endpoint Modeled
Fatty metamorphosis in liver of male rats
Fatty metamorphosis in liver of male rats
Fatty metamorphosis in liver of female rats
"Ground glass" cytoplasm in liver of male rats
Nephrosis in liver of female rats
Fatty metamorphosis in liver of female mice
Thyroid follicular cell hyperplasia in female
mice
BMDL10 (mg/kg-day) *
0.93
1.16
No acceptable fit
4.93
17
7.68
7.09
BMD modeling was conducted on duration-adjusted values using the Crump BMD software.
Both studies were considered appropriate for derivation of the RfD. The NTP (1985)
investigation of chronic toxicity in rats was selected as the principal study on the basis of its
longer duration. The critical endpoint is hepatotoxicity, as evidenced by the occurrence of fatty
metamorphosis in the livers of dibromochloromethane-treated animals. This effect was dose-
dependent, with incidences of 27/50, 47/50, and 49/50 at the duration-adjusted doses of 0, 29,
and 57 mg/kg-day. Selection of this study is strongly supported by the similar BMD calculated
for the same effect in the NTP (1985) subchronic study.
Derivation of the RfD
The duration-adjusted BMDL10 value from the chronic NTP (1985) rat study was selected
as the most appropriate basis for derivation of the RfD for dibromochloromethane. The RfD is
calculated using the following equation:
RfD
(1.6 mg/kg-day)
(100)
= 0.016 mg/kg-day (rounded to 0.02 mg/kg-day)
where:
1.6 mg/kg-day
Duration-adjusted BMDL10 based on fatty changes in the
liver of male rats
VIII - 46
November 15, 2005
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100 = Composite uncertainty factor based on NAS/OW guidelines;
includes a factor of 10 interspecies extrapolation and a factor
of 10 for protection of sensitive human populations
A composite UF of 100 was used. The standard factors of 10 were used for interspecies
extrapolation and for protection of sensitive subpopulations. Furthermore, no additional
uncertainty factor was needed to account for an incomplete database. The database for
dibromochloromethane includes a two-generation study in ICR Swiss mice (Borzelleca and
Carchman 1982), a developmental toxicity study in Sprague-Dawley rats (Ruddick et al., 1983),
and a short-term reproductive and developmental toxicity study in rats (NTP, 1996). Therefore,
the database is considered nearly complete despite the lack of a developmental toxicity study in a
second species.
The DWEL for dibromochloromethane is calculated as follows:
(0.02 mg/kg-day) (70 kg)
DWEL = = 0.7 mg/L (700
2 L/day
where:
0.02 mg/kg-day = RfD
70 kg = Assumed weight of an adult
2 L/day = Assumed water consumption by a 70-kg adult
Lifetime Health Advisory
The Lifetime Health Advisory (HA) represents that portion of an individual's total
exposure that is attributed to drinking water and is considered protective of noncarcinogenic
health effects over a lifetime of exposure. Dibromochloromethane is classified with respect to
carcinogenic potential as Group C: Possible human carcinogen. The Lifetime Health Advisory
(HA) is therefore calculated as follows:
(0.7 mg/L) (0.8)
Lifetime HA = = 0.06 mg/L (60 |ig/L)
10
where:
0.7 mg/kg-day = DWEL
VIII - 47 November 15, 2005
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0.8 = Relative Source Contribution (RSC), the
proportion of the total daily exposure
contributed by the dibromochloromethane in
drinking water
10 = Uncertainty factor used in accordance with
U.S. EPA policy for Group C contaminants to
account for possible carcinogenicity
Alternative Approach for Derivation of the RfD
An alternative approach to the derivation of the RfD is use of the conventional
NOAEL/LOAEL method. The subchronic oral exposure study conducted by NTP (1985)
identified a NOAEL of 30 mg/kg-day. Using this value, a duration adjustment factor of 5/7, and
a composite uncertainty factor of 1000 (includes factors of 10 for interspecies extrapolation,
protection of sensitive subpopulations, and use of a subchronic study), the resulting RfD is 0.02
mg/kg-day (the same value as derived using the BMD approach). The corresponding DWEL is
0.7 mg/L, assuming an adult body weight of 70 kg and a drinking water ingestion rate of 2
L/day.
2. Carcinogenic Effects
a. Categorization of Carcinogenic Potential
Previous Evaluations of Carcinogenic Potential
The Carcinogenic Risk Assessment Verification Endeavor (CRAVE) group of the
U.S. EPA reviewed the available evidence on the carcinogenicity of the brominated
trihalomethanes and assigned dibromochloromethane to Group C: possible human carcinogen
(IRIS, 1992). This classification reflects inadequate human data and limited evidence of
carcinogenicity in animals.
Based on the 1996 Proposed Guidelines for Carcinogen Risk Assessment published in
1996 (U.S. EPA, 1996), dibromochloromethane is classified as cannot be determined. This
descriptor is considered appropriate when there are no or inadequate data in humans, and limited
evidence for carcinogenicity in animals.
IARC (1999c) has recently re-evaluated the carcinogenic potential of
dibromochloromethane. IARC concluded that there is limited evidence of carcinogenicity in
experimental animals and inadequate evidence in humans for dibromochloromethane.
Dibromochloromethane is therefore classified as Group 3: not classifiable as to carcinogenicity
in humans.
VIII - 48 November 15, 2005
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Categorization of Carcinogenic Potential Under the Proposed 1999 Cancer Guidelines
Cancer Hazard Summary
Under the proposed guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
dibromochloromethane shows suggestive evidence ofcarcinogenicity, but not sufficient to assess
human carcinogenic potential. This descriptor is appropriate when the evidence from human or
animal data is suggestive ofcarcinogenicity, 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 ofcarcinogenicity.
Supporting Information for Cancer Hazard Assessment
Human Data
The information on the carcinogenicity of dibromochloromethane from human studies is
inadequate. There are no epidemiological data specifically relating increased incidence of
cancer to exposure to dibromochloromethane. There are equivocal epidemiological data
describing a weak association of chlorinated drinking water exposures with increased incidences
of bladder, rectal, and colon cancer. U.S. EPA has determined that these studies cannot attribute
the observed effects to a single compound, as chlorinated water contains numerous other
disinfection byproducts that are potentially carcinogenic.
Animal Data
The carcinogenicity of dibromochloromethane in male and female animals has been
investigated in a well-designed and conducted corn oil gavage study conducted in rats and mice,
a dietary exposure study in rats, and a drinking water study in mice. No data are available on the
carcinogenic potential of dibromochloromethane administered via the inhalation or dermal
routes.
In the corn oil gavage study (NTP, 1985), the incidence of hepatocellular adenomas and
carcinomas and combined adenomas and carcinomas was significantly increased in high-dose
female mice and the incidence of hepatocellular adenomas was significantly increased in high-
dose male mice. No evidence was observed for carcinogenicity in male or female rats under the
experimental conditions employed. Voronin (1987) did not observe significant increases in mice
treated with dibromochloromethane in drinking water for 104 weeks. Tobe et al. (1982) reported
no increase in gross tumors in rats treated exposed to dibromochloromethane in the diet for two
years.
Structural Analogue Data
Dibromochloromethane is structurally related to trihalomethanes that have shown
varying degrees of carcinogenic potential in rodents. Chloroform, the most extensively
characterized trihalomethane, is reported to be carcinogenic at high doses in several chronic
VIII - 49 November 15, 2005
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animal bioassays, with significant increases in the incidence of liver tumors in male and female
mice and significant increases in the incidence of kidney tumors in male rats and mice (U.S.
EPA, 200 la). The occurrence of tumors in animals exposed to chloroform is demonstrably
species-, strain-, and gender-specific, and has only been observed under dose conditions that
caused cytotoxicity and regenerative cell proliferation in the target organ. The cancer database
for structurally-related brominated trihalomethanes is more limited, but includes well-conducted
studies performed by the National Toxicology Program. In a two-year corn oil gavage study of
bromoform, NTP (1989a) found clear evidence for carcinogenicity in female rats and some
evidence of carcinogenicity based on occurrence of tumors of the large intestine. In a two-year
corn oil gavage study of bromodichloromethane, NTP (1987) found clear evidence of
carcinogenicity in male and female rats (tumors of the large intestine), male mice (kidney
tumors), and female mice (liver tumors). In other bioassays, George et al. (2002) observed a
significantly increased prevalence of neoplastic lesions in the liver of male rats at the lowest
dose of bromodichloromethane administered in drinking water, but not at higher doses.
Tumasonis et al. (1985) reported significantly increased incidences of hepatic neoplastic
nodules, hepatic adenofibrosis, and lymphosarcoma in female rats exposed to
bromodichloromethane in drinking water.
Other Key Data
Dibromochloromethane is formed as a byproduct of drinking water disinfection with
chlorine. Exposure to dibromochloromethane may occur via ingestion of tap water, via dermal
contact during showering or bathing, or by inhalation of dibromochloromethane volatilized
during household activities. Absorption of single oral doses appears to be extensive.
Dibromochloromethane is rapidly metabolized and eliminated predominately as expired
volatiles, carbon dioxide, or carbon monoxide. Only a small amount (less than 10%) is
eliminated in urine or in feces. No comprehensive tissue data are available regarding the
bioaccumulation or retention of dibromochloromethane following repeated exposure. However,
because of the rapid metabolism and excretion of dibromochloromethane, marked accumulation
and retention is not expected.
Dibromochloromethane itself is not directly reactive with DNA. Metabolism to reactive
species is a prerequisite for toxicity, as inferred from metabolic induction and inhibition studies.
In vitro and in vivo studies of the mutagenic and genotoxic potential of bromodichloromethane
have yielded both positive and negative 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. Study
results for the mutagenicity of dibromochloromethane are mixed, and the overall evidence for
mutagenicity of this chemical is judged to be inconclusive (U.S. EPA, 1994b). Recent studies
conducted with strains of Salmonella that express rat theta-class glutathione S-transf erase
indicate that dibromochloromethane is mutagenic in this test system and suggest that
mutagenicity is mediated by formation of a reactive glutathione conjugate.
VIII - 50 November 15, 2005
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Mode of Action
Limited or equivocal evidence has been obtained for the carcinogenic potential of
dibromochloromethane. Data to support a primary mode of action for tumor development in the
liver of mice exposed to dibromochloromethane are lacking. In the absence of such information,
combined with an inconclusive weight-of-evidence evaluation for genotoxicity, the mode of
action for tumor development is assumed to be a linear process. The processes leading to tumor
formation in animals are expected to be relevant to humans.
Conclusion
Under the proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
dibromochloromethane shows suggestive evidence ofcarcinogenicity, but not sufficient to assess
human carcinogenic potentially the oral route. This weight-of-evidence evaluation is based on
1) limited or equivocal evidence of carcinogenicity in mice, but not rats, treated by oral
pathways; 2) lack of epidemiological data specific to dibromochloromethane and equivocal data
for drinking water drinking water exposures that cannot reliably be attributed to
dibromochloromethane among multiple other disinfection byproducts; 3) inconclusive results for
many of the available genotoxicity and mutagenicity tests; and 4) metabolism and mode of
action that are reasonably expected to be similar to those of structurally-related compounds that
induce tumors in experimental animals. Although no cancer data exist for exposures via the
dermal or inhalation pathways, the weight-of-evidence conclusion is considered to be applicable
to these pathways as well. The finding for inhalation is based on the observation that patterns of
metabolizing enzyme activity in male rats for the related trihalomethane bromodichloromethane
are similar for exposure via the inhalation and gavage routes. Dibromochloromethane absorbed
through the skin is expected to be metabolized and cause toxicity in much the same way as
dibromochloromethane absorbed by the oral and inhalation routes.
b. Choice of Study for Quantification of Carcinogenic Risk
The proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999) do not
indicate dose-response assessment for chemicals for which there is suggestive evidence of
carcinogenicity, but not sufficient to assess human carcinogenic potential. However, the single
oral exposure study with positive tumor data for dibromochloromethane suggests significant
cancer potency for this compound in mice. A quantitative assessment of potency was therefore
considered appropriate.
In the absence of other carcinogenicity data, hepatic tumor incidence in female mice was
selected for estimation of carcinogenic risks associated with dibromochloromethane. These data
were obtained in an NTP (1985) study in which dibromochloromethane was administered in corn
oil to male and female B6C3FJ mice (50 mice/sex/dose) by gavage 5 times/week for 104 to 105
weeks. The administered doses were 0, 50, or 100 mg/kg-day. Survival of dosed female mice
was comparable to that of the corresponding vehicle-control groups. High-dose male mice had
lower survival rates than the vehicle controls. At week 82, nine high-dose male mice died of an
unknown cause. An inadvertent overdose of dibromochloromethane given to low-dose male and
female mice at week 58 killed 35 male mice, but apparently did not affect the female mice. The
VIII-51 November 15, 2005
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low-dose male mouse group was, therefore, considered to be unsuitable for analysis of
neoplasms. Compound-related nonneoplastic lesions were found in primarily in the livers of
males (hepatocytomegaly, necrosis, fatty metamorphosis) and females (calcification and fatty
metamorphosis). Nephrosis was also observed in male mice. Statistically significant increases
in the incidence of hepatocellular adenomas and in the combined incidence of adenomas and
carcinomas were observed in high-dose female mice. In male mice, a statistically significant
increase in the incidence of hepatocellular carcinomas and combined adenomas and carcinomas
was observed in the high-dose group; however, due to the overdose of dibromochloromethane in
the mid-dose group, the authors considered the tumor incidence data inadequate for tumor
analysis. Tumor incidence data from this study are presented in Table VIII-13.
c. Extrapolation model
The LMS model (U.S. EPA, 1986) and the default linear approach described by Proposed
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996; 1999) were used to quantify the
risk associated with exposure to dibromochloromethane. Data for the mutagenicity and
genotoxicity of dibromochloromethane are mixed (see Section V.F.2). U.S. EPA (1994b) has
previously determined that the weight of evidence for dibromochloromethane mutagenicity and
genotoxicity is inconclusive. At the present time there is insufficient evidence to establish with
certainty that dibromochloromethane exerts its carcinogenic effects via a non-genotoxic
mechanism. Thus, use of linear approaches was considered appropriate for quantification of
cancer risk associated with exposure to this compound.
Table VIII-13 Frequencies of Liver Tumors in Mice Administered Dibromochloromethane
in Corn Oil for 105 Weeks - Adapted from NTP (1985)
Treatment
(mg/kg-day)
Vehicle Control
50
100
Sex
M
F
M
F
M
F
Adenoma
14/50
2/50
a
4/49
10/50
ll/50b
Carcinoma
10/50
4/50
6/49
19/50b
8/50
Adenoma or Carcinoma
(combined)
23/50
6/50
10/49
27/50c
19/50d
a Male low-dose group was inadequate for statistical analysis.
b p < 0.05 relative to controls.
0 p < 0.01 (life table analysis); p = 0.065 (incidental tumor test) relative to controls.
d p < 0.01 relative to controls.
d. Cancer Potency and Unit Risk
The only tumor data available for dibromochloromethane are for liver tumors in female
B6C3FJ mice (NTP, 1985). NAS (1987) previously utilized the tumor frequency data reported
by NTP (1985) to calculate an excess lifetime cancer unit risk of 8.3 x 10"7. The linearized
VIII - 52
November 15, 2005
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multistage model was utilized, with the assumption that 1 L of water per day containing 1 |ig/L
of dibromochloromethane was ingested. Based on this calculation, the concentration associated
with a risk of 10"6 is 0.6 |ig/L, assuming consumption of 2 L of water per day.
Other available estimates of cancer risks are summarized in Table VIII-14. U.S. EPA
(1994b) reported a slope factor of 8.4 x 10'2 (mg/kg-day)'1 calculated from the NTP (1985) data
in the absence of other appropriate tumorigenicity data for dibromochloromethane (IRIS, 1992).
This value was derived using the LMS model (extra risk) and a scaling factor of body weight273,
as specified in the 1986 Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 1986). The
reported unit risk and 10"5 risk concentration were 2.4 x 10"6 (jig/L)"1 and 4 |ig/L, respectively.
A slope factor of 4.3 x 1CT2 (mg/kg-day)"1 (U.S. EPA, 1998b) was derived using the LMS
model and a scaling factor of body weight374. The use of body weight374 as the scaling factor is
consistent with recommendations in U.S. EPA. (1992b). A unit risk value of 1.2 x 10"6 (jig/L)"1
was estimated using an assumed body weight of 70 kg and a drinking water ingestion rate of 2 L.
This estimate was used to calculate a drinking water 10"5 risk concentration of 8 |ig/L (0.8 |ig/L
at 10-6risk).
Table VIII-14 Carcinogenic Risk Estimates for Dibromochloromethane
Method of
Estimation
LMS Method Using
BW3'4 Conversion
U.S. EPA(1998b)
LMS Method Using
BW273 Conversion
U.S. EPA(1994b)*
LED10/Linear Method
U.S. EPA(1998b)
Tumor
Site
Liver
Liver
Liver
Species
Mouse
Mouse
Mouse
Sex
F
F
F
Slope Factor
(mg/kg-day)1
4.3xlQ-2
8.4xlQ-2
4.0xlO'2
Unit Risk
(lig/L)-1
1.2x10-'
2.4x10"'
1.2x10"'
10 5 Risk
Cone. (|lg/L)
8
4
9
LED10
([ig/kg-day)
2.5xl03
* Adapted from IRIS (1992)
Cancer risk estimates were also obtained using the LED10 (the lower 95% confidence
limit on a dose associated with 10% extra risk) of 2.5 x 103 |ig/kg-day for hepatic tumors and
assuming a linear mode of action for the carcinogenicity of dibromochloromethane (Table VIII-
14). A cancer potency value of 4.0 x 10"2 (mg/kg-day)"1 was derived using this approach. A unit
risk of 1.2 x 10"6 (jig/L)"1 was calculated using an assumed body weight of 70 kg and a drinking
water ingestion rate of 2 L. This estimate was used to calculate a drinking water concentration
of 9 i-ig/L associated with a 10"5 risk (0.9 |ig/L for 10"6 risk). These values are similar to values
derived using the LMS approach with body weight scaling to the 3/4 power.
VIII - 53
November 15, 2005
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The use of a corn oil vehicle in the NTP (1985) study from which these data are derived
contributes uncertainty regarding the relevance of this value to exposure via drinking water. The
U.S. EPA plans to seek data on the tumorigenicity of dibromochloromethane in water in order to
clarify this issue.
VIII - 54 November 15, 2005
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C. Bromoform
1. Noncarcinogenic effects
a. One-day Health Advisory
Acute toxicity information on bromoform is limited and no data suitable for BMD
modeling were identified. Some information is available on the former medicinal use of
bromoform in humans. In the past, oral doses of bromoform were used as a sedative for children
with whooping cough. Doses were typically one drop (approximately 180 mg) given three to six
times per day (Burton-Fanning, 1901). This treatment usually resulted in mild sedation in
children, although a few rare cases of death or near-death (believed to be due to accidental
overdoses) have been reported (e.g., Dwelle, 1903; Benson, 1907). Based on a dose of
540 mg/day given to a 10-kg child, the LOAEL for mild sedation is about 54 mg/kg-day. Using
these data, the one day-HA for bromoform is calculated according to the following equation:
(54mg/kg-day)(10kg)
One-day HA = = 5.4 mg/L (rounded to 5 mg/L)
(100) (1 L/day)
where:
54 mg/kg-day = LOAEL based on sedation in children given oral doses of
bromoform
10 kg = Assumed weight of a child
100 = Composite uncertainty factor based on NAS/OW
guidelines. Includes a factor of 10 for interspecies variation
and a factor of 10 for protection of sensitive human
populations
1 L/day = Assumed water consumption of a 10-kg child
b. Ten-day health Advisory
Candidate studies considered for derivation of the Ten-day HA are summarized in Table
VIII-15 (below). Condie et al. (1983) administered bromoform by gavage to male CD-I mice at
doses ranging from 72 to 289 mg/kg-day for 14 days and identified a NOAEL of 145 mg/kg-day
and a LOAEL of 289 mg/kg-day. The LOAEL is based on changes in clinical chemistry and on
minimal to moderate histologic changes in the kidney (intratubular mineralization, epithelial
hyperplasia, and mesangial hypertrophy and nephrosis) and in the liver (centrilobular pallor,
mitotic figures, focal inflammation, and cytoplasmic vacuolization). BMD modeling of data for
renal mesangial hypertrophy calculated BMD and BMDL10 values of 73 and 34 mg/kg-day,
respectively.
VIII - 55 November 15, 2005
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Melnick et al. (1998) administered bromoform to female B6C3FJ mice by gavage 5
days/week for 3 weeks and identified a NOAEL of 200 mg/kg-day (the lowest dose tested) and a
LOAEL of 500 mg/kg-day based on histologic changes in the liver (hepatocyte hydropic
degeneration). The duration-adjusted BMD and BMDL10 values for this endpoint were 146 and
104 mg/kg-day, respectively.
Munson et al. (1982) identified NOAEL and LOAEL values of 125 and 250 mg/kg-day,
respectively, based on elevated serum enzyme activity in mice. BMD modeling was not
conducted for this endpoint, since it was not considered a reliable basis for the Ten-day HA in
the absence of histopathological data. NTP (1989a) identified NOAEL and LOAEL values of
200 and 400 mg/kg-day, respectively, based on the occurrence of stomach nodules in rats and
mice. A BMD of 167 mg/kg-day was calculated for this endpoint in mice, with a corresponding
BMDL10 of 66 mg/kg-day. However, occurrence of these nodules may represent a portal of
entry effect. Chu et al. (1982a) identified a freestanding NOAEL of 80 mg/kg-day in a drinking
water study conducted in rats. Coffin et al. (2000) identified a LOAEL of 200 mg/kg-day based
on the occurrence of liver histopathology and increased labeling index. The data of Coffin et al.
were not modeled because other studies used lower doses and were thus able to better
characterize the low-dose portion of the dose response curve.
The study conducted by Aida et al. (1992a) assessed toxicity in Wistar rats administered
bromoform microencapsulated in the diet at doses ranging from 56 to 728 mg/kg-day. The
duration of the study was one month. This study identified a NOAEL of 56 mg/kg-day and a
LOAEL of 208 mg/kg-day based on clinical chemistry changes and histologic changes in the
liver (cell vacuolization and swelling) of females. BMD modeling of results for liver cell
vacuolization in female rats calculated BMD and BMDL10 values of 16 and 2.3 mg/kg-day,
respectively. These were the lowest values observed among modeling results for candidate
studies for the 10-day HA. On this basis, and because histopathological changes in the liver are
considered a sensitive indicator of brominated trihalomethane toxicity, the study conducted by
Aida et al. (1992a) was considered the best choice for derivation of the Ten-day HA.
VIII - 56 November 15, 2005
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Table VIII-15 Summary of Candidate Studies for Derivation of the Ten-day HA for Bromoform
Reference
Aida et al.
(1992a)
Chu et al.
(1982a)
Condie et al.
(1983)
Melnick et
al.
(1998)
Munson et
al.
(1982)
NTP (1989a)
Species
Sex
Rat
Wistar
M, F
Rat
SD
M
Mouse
CD-I
M
Mouse
B6C3FJ
F
Mouse
CD-I
M, F
Mouse
B6C3FJ
M
M
7
10
8-
16
10
6-
12
5
Route
Feed
Drinking
water
Gavage
(oil)
Gavage
(oil)
Gavage
(aqueous)
Gavage
(oil)
Dose
Males
0
62
187
618
Females
0
56
208
728
0.7
8.5
80
0
72
145
289
0
200
500
0
50
125
250
0
50
100
200
400
600
Exposure
Duration
1 month
28 days
14 days
3 weeks
(5 d/wk)
14 days
14 days
1 ml points
Body weight,
clinical signs,
serum
biochemistry,
hematology,
histology
Clinical signs,
serum
biochemistry,
histology
Serum enzymes,
PAH uptake in
vitro, histology
Body and liver
weights, serum
chemistry, liver
histology
Body and organ
weights, serum
chemistry,
hematology,
immune function
Body weight,
clinical signs,
gross pathology
NOAEL
(mg/kg-day)
62 males
56 females
80
145
200
125
200
LOAEL
(mg/kg-day)
187 males
208 females
(serum chemistry
changes, liver
histopathology)
289
(elevated ALT,
decreased PAH,
liver and kidney
histopathology)
500
(liver
histopathology)
250
(elevated serum
enzymes)
400
(stomach nodules)
BMD
(mg/kg-day)
Males
140
Females
16
Not modeled
73
146*
Not modeled
167
BMDL10
(mg/kg-day)
Males
51
(Liver cell
vacuolization)
Females
2.3
(Liver cell
vacuolization)
34
(Renal mesangial
nephrosis)
104*
(Liver hydropic
degeneration)
__
66
(stomach
nodules)
VIII - 57
November 15, 2005
-------
Table VIII-15 (cont.)
Reference
Coffin et al.
(2000)
NTP (1989a)
Ruddick et
al. (1983)**
Species
Sex
Mouse
B6C3FJ
F
Rat
F344/N
M, F
Rat
SD
F
n
10
5
14-
15
Route
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Dose
0
200
500
0
100
200
400
600
800
0
50
100
200
Exposure
Duration
11 days
14 days
Gestation
days 6- 15
1 ml points
Relative liver
weight; liver
histopathology;
labeling index
Body weight,
clinical signs,
gross pathology
Body and organ
weights; maternal
serum chemistry;
hematology, and
histopathology;
developmental
parameters
NOAEL
(mg/kg-day)
..
200
50
LOAEL
(mg/kg-day)
200
(liver
histopathology;
labeling index)
400
(decreased body
weight)
100
(sternebral
aberrations)
BMD
(mg/kg-day)
Not modeled
Not modeled
50
BMDL10
(mg/kg-day)
..
33
(sternebral
aberrations)
* Duration-adjusted dose used to calculate BMD and BMDL10
** Ruddick et al (1983) is included because it is a reproductive study.
Abbreviations: SD, Sprague-Dawley
VIII - 58
November 15, 2005
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Based on the BMDL10 identified in the Aida et al. (1992a) study, the Ten-day HA for a
10-kg child is calculated according to the following equation:
(2.3 mg/kg-day)(10 kg)
Ten-day HA = = 0.23 mg/L (rounded to 0.2 mg/L)
(100) (1 L/day)
where:
2.3 mg/kg-day = BMDL10based on the occurrence of hepatic vacuolization
in female rats exposed to bromoform in the diet for one
month
10 kg = Assumed body weight of a child
100 = Uncertainty factor based on NAS/OW guidelines. Includes
a factor of 10 for interspecies variation and a factor of 10
for protection of sensitive human populations
1 L/day = Assumed water consumption of a 10-kg child
When the BMDL10 value for liver cell vacuolization in the Aida et al. (1992a) study is
used, the Ten-day HA for a 10-kg child is calculated to be 0.2 mg/L, assuming a drinking water
ingestion rate of 1 L/day and use of a composite uncertainty factor of 100. This value is slightly
lower than the Longer-term HA for a 10 kg child of 0.3 mg/L derived using subchronic data for
the same histopathological endpoint. This small difference may reflect experimental or BMD
modeling uncertainty.
For purposes of comparison, the Ten-day HA may also be derived using the conventional
NOAEL/LOAEL approach. The lowest LOAEL among the candidate studies was 100 mg/kg-
day for developmental effects in rats (Ruddick et al., 1983). The NOAEL in this study was 50
mg/kg-day. Aida et al. (1992a) identified NOAEL values of 56 and 62 mg/kg-day and LOAEL
values of 187 and 208 mg/kg-day for histopathological changes in male and female rats
administered bromoform in the diet. Chu et al. (1982a) identified a freestanding NOAEL of 80
mg/kg-day in rats. The data of Aida et al. (1992a) were selected for calculation of the Ten-day
HA because the study tested both male and female rats, incorporated more dose levels, and
identified both NOAEL and LOAEL values and because the NOAEL identified by Chu et al.
(1982a) is close to the lowest LOAEL of 100 mg/kg-day. Using the NOAEL of 62 mg/kg-day
for male rats and assuming the default body weight for a child (10 kg), the default drinking water
intake for a child (1 L/day), and a composite uncertainty factor of 100, the Ten-day HA would be
6.2 mg/L (rounded to 6 mg/L).
VIII - 59 November 15, 2005
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c. Longer-term Health Advisory
Candidate studies for derivation of the Longer-term HA are summarized in Table VIII-16
below. All studies identified histopathological changes in liver tissue as the critical toxicological
effect. In one NTP (1989a) study, F344/N rats were administered bromoform by gavage at doses
ranging from 12 to 200 mg/kg-day for 5 days/week for 13 weeks. This study identified a
NOAEL of 25 mg/kg-day and a LOAEL of 50 mg/kg-day based on hepatic vacuolization
observed in male rats. BMD modeling with the BMDS program calculated a duration-adjusted
BMD of 4.4 mg/kg-day (based on duration-adjusted doses), with a corresponding BMDL10 of 2.6
mg/kg-day. These values were the lowest among the candidate studies.
In an analogous subchronic oral exposure study, NTP (1989a) exposed mice of both
sexes to doses of bromoform ranging from 25 to 400 mg/kg-day in addition to the control. This
study identified NOAEL and LOAEL values of 100 and 200 mg/kg-day, respectively, based on
hepatic vacuolization. BMD modeling with the BMDS program calculated a BMD of 88 mg/kg-
day (based on duration-adjusted doses), with a corresponding BMDL10 of 55 mg/kg-day. These
values were approximately 20-fold higher than the BMD and BMDL10 calculated for the NTP
(1989a) oral exposure study in rats.
Chu et al. (1982b) exposed rats of both sexes to bromoform in the drinking water for 90
days. The doses of bromoform ranged from 0.64 to 283 mg/kg-day in addition to the control.
This study identified NOAEL and LOAEL values of 57 and 218 mg/kg-day , respectively, based
on decreased weight gain and mild hepatic lesions in male mice. BMD modeling identified
BMD and BMDL10 values of 10 and 5.9 mg/kg-day using data for occurrence of hepatic lesions
in male mice. These values were approximately two-fold higher than the BMD and BMDL10
values derived using data from the NTP (1989a) oral exposure study in rats. Strengths of this
study include exposure via drinking water, larger sample size (20 animals/treatment group), and
the administration of lower doses than used in the NTP (1989a) subchronic studies. Liver
histopathology data from this study were reported as combined lesions, with the types of lesions
described in the text.
The NTP (1989a) oral exposure study conducted in rats was selected for the derivation of
the Longer-term HA on the basis of the low values obtained for the BMD and BMDL10.
Selection of this study is strongly supported by the results of Chu et al. (1982b), which identified
slightly higher values in a drinking water study.
VIII - 60 November 15, 2005
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Table VIII-16 Summary of Candidate Studies for Derivation of the Longer-term HA for Bromoform
Reference
Chu et al.
(1982b)
NTP (1989a)
NTP (1989a)
Ruddick et
al. (1983)**
NTP (1989b)
Species
Sex
Rat
SD
M, F
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Rat
SD
F
Mouse
ICR
Swiss
M, F
n
20
10
10
9-
14
20
Route
Drinking
water
Gavage
(corn oil)
Gavage
(corn oil)
Gavage
(corn oil)
Gavage
(corn oil)
Dose
Male
0
0.65
6.1
57
218
Females
0
0.64
6.9
55
283
0
12
25
50
100
200
0
25
50
100
200
400
0
50
100
200
0
50
100
200
Exposure
duration
90 days
13 weeks
(5 d/wk)
13 weeks
(5 d/wk)
Gestation
days 6-15
105 days
1 ml points
Body weight, serum chemistry,
histology
Body weight, clinical signs, gross
necropsy, histology
Body weight, clinical signs, gross
necropsy, histology
Body and organ weights; maternal
serum chemistry; hematology, and
histopathology; developmental
parameters
Continuous breeding reprod. study.
Body and organ weights,
histopathology, reproductive
parameters
NOAE
L
(mg/kg-
day)
57
25
100
50
100
LOAEL
(mg/kg-day)
218
(decreased
weight gain,
mild hepatic
lesions)
50
(hepatic
vacuolization)
200
(hepatic
vacuolization)
100
(sternebral
aberrations)
200
(decreased
maternal body
weight)
BMD
(mg/kg-day)*
Male
10
Females
No fit
4.4
88
50
_
(not modeled)
BMDL10
(mg/kg-day) *
Male
5.9
(Hepatic lesions)
Females
--
2.6
(hepatic
vacuolization in
male rats)
55
(hepatic
vacuolization in
male mice)
33
(sternebral
aberrations)
_
* BMD and BMDL10 calculated using duration-adjusted doses
** Ruddick et al (1983) is included because it is a reproductive study.
- Not modeled
Abbreviations: SD, Sprague-Dawley
VIII-61
November 15, 2005
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Based on the BMDL10 identified in the NTP (1989a) rat study, the Longer-term HA for a
10-kg child is calculated according to the following equation:
(2.6 mg/kg-day)(10 kg)
Longer-term HA = = 0.26 mg/L (rounded to 0.3 mg/L)
(100) (1 L/day)
where:
2.6 mg/kg-day = Duration-adjusted BMDL10based on the occurrence of
hepatic vacuolization in male rats exposed to bromoform by
gavage for 13 weeks
10 kg = Assumed body weight of a child
100 = Uncertainty factor based on NAS/OW guidelines; includes
a factor of 10 for interspecies variation and a factor of 10
for protection of sensitive human populations
1 L/day = Assumed water consumption of a 10-kg child
The Longer-term HA for an adult consuming 2 liters of water per day is calculated
according to the following equation:
(2.6 mg/kg-day)(70kg)
Longer-term HA = = 0.91 mg/L (rounded to 0.9 mg/L)
(100) (2 L/day)
where:
2.6 mg/kg-day = Duration-adjusted BMDL10based on the occurrence of
hepatic vacuolization in male rats exposed to bromoform by
gavage for 13 weeks
70 kg = Assumed body weight of an adult
100 = Composite uncertainty factor based on NAS/OW
guidelines; includes a factor of 10 for interspecies variation
and a factor of 10 for protection of sensitive human
populations
2 L/day = Assumed water consumption of a 70-kg adult
For purposes of comparison, the Longer-term Health Advisories may also be derived
using the conventional NOAEL/LOAEL approach. The chronic oral exposure study conducted
by NTP (1989a) identified a NOAEL of 25 mg/kg-day based on the absence of clinical signs or
VIII - 62 November 15, 2005
-------
histological alterations in rats exposed to bromoform for 13 weeks. Using this value and
assuming default body weights (10 and 70 mg/kg-day, for adults and children, respectively),
default drinking water intake rates (1 and 10 L/day for children and adults, respectively), a
composite uncertainty factor of 100, and an exposure duration factor of 5/7, the Longer-term
HAs for a child and an adult would be 2 mg/L and 6 mg/L, respectively.
d. Reference Dose, Drinking Water Equivalent Level and Lifetime Health Advisory
This section reports the existing RfD value for bromoform and describes the derivation of
the RfD for this compound. This section also describes the calculation of Drinking Water
Equivalent Level and Lifetime Health Advisory values which require the RfD as input. For this
document, new and existing studies were reviewed and appropriate candidate data were selected
for benchmark (BMD) dose modeling. The results of BMD modeling were used in conjunction
with appropriate uncertainty factors to calculate the RfD. A comparison of the RfD derived
using the BMD approach to the results obtained using the conventional NOAEL/LOAEL
approach is also provided.
Description of the Existing RfD
The existing RfD for bromoform is 0.02 mg/kg-day (IRIS, 1993b). This value was
derived using a duration-adjusted NOAEL of 17.9 mg/kg-day identified for the occurrence of
hepatic lesions in F344 rats administered bromoform by corn oil gavage 5 days/week for 13
weeks (NTP, 1989a). An uncertainty factor of 1000 was used to account for extrapolation from
animal data, for protection of sensitive human subpopulations, and for use of a subchronic study.
Identification of Candidate Studies for the Derivation of the RfD
Three chronic exposure studies, a subchronic exposure study, a prenatal developmental
toxicity study, and a reproductive toxicity study were considered for derivation of the RfD for
bromoform. These studies are summarized in Table VIII-17 (below).
Tobe et al. (1982) administered bromoform microencapsulated in the diet to Wistar rats
at dose levels ranging from 22 to 619 mg/kg-day for 24 months. A NOAEL of 22 mg/kg-day
and a LOAEL of 90 mg/kg-day were identified based on gross liver lesions and changes in
clinical chemistry parameters in male rats.
NTP (1989a) conducted chronic oral exposure studies in rats and mice. In the rat study,
animals were administered bromoform by gavage in oil for 5 days/week for 103 weeks at doses
of 100 or 200 mg/kg-day. This study identified the low dose as the LOAEL based on histologic
lesions in the liver (fatty change and chronic inflammation). In the mouse study, animals were
administered bromoform by gavage in corn oil, 5 days/week for 103 weeks at doses of 50 or 100
mg/kg-day for male mice and 100 or 200 mg/kg-day for female mice. Although no treatment-
related effects were observed in the male mice at the dose levels tested, treatment-related
histologic lesions in the liver (fatty changes) were observed in both low- and high-dose females.
Accordingly, this study identified a LOAEL of 100 mg/kg-day in female mice.
VIII - 63 November 15, 2005
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A subchronic oral exposure study conducted in rats (NTP, 1989a) was also considered as
a candidate for derivation of the RfD. This study utilized five doses of bromoform ranging from
12 to 200 mg/kg-day in addition to the control. The compound was administered to ten animals
per treatment group by gavage in corn oil, 5 days per week for 13 weeks. The endpoints
evaluated included body weight, clinical signs, gross necropsy, and histological changes. This
study identified a NOAEL and LOAEL of 25 and 50 mg/kg-day, respectively, on the basis of
histopathological changes (vacuolization) in the liver. The LOAEL identified in this study was
the lowest among all candidate studies.
The developmental study conducted by Ruddick et al. (1983) identified NOAEL and
LOAEL values of 50 and 100 mg/kg-day, respectively, for sternebral variations in the offspring
of female rats dosed with bromoform on gestation days 6-15. The reproductive toxicity study
reported by NTP (1989b) identified NOAEL and LOAEL values of 100 and 200 mg/kg-day for
reduced maternal body weight and decreased postnatal survival and liver histopathology in Fx
mice of both sexes.
VIII - 64 November 15, 2005
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Table VIII-17 Summary of Candidate Studies for Derivation of the RfD for Bromoform
Reference
Tobe et al.
(1982)
NTP (1989a)
NTP (1989a)
NTP (1989a)
Ruddick et al.
(1983)*
Species
Sex
Rat
Wistar
M, F
Rat
F344/N
M, F
Rat
F344/N
M, F
Mouse
B6C3FJ
M, F
Rat
SD
F
n
40
10
50
50
9-
14
Dose
Male
0
22
90
364
Female
0
38
152
619
0
12
25
50
100
200
0
100
200
Male
0
50
100
Female
0
100
200
0
50
100
200
Route
Diet
Gavage
(corn oil)
Gavage
(corn oil)
Gavage
(corn oil)
Gavage
(con oil)
Exposure
Duration
24 months
13 weeks
(5 days/wk)
103 weeks
(5 d/wk)
103 weeks
(5 d/wk)
Gestation
days 6-15
1 ml points
Body weight, serum
chemistry, gross
pathology
Body weight, clinical
signs, gross necropsy,
histology
Body weight, clinical
signs, gross necropsy,
histology
Body weight, clinical
signs, gross necropsy,
histology
Developmental toxicity
study; body and organ
weights
NOAEL
(mg/kg-day)
22
25
100 (male)
50
(developmental)
200
(maternal)
LOAEL
(mg/kg-day)
90
(serum chemistry
changes, gross liver
lesions)
50
(hepatic
vacuolization)
100
(decreased body
weight, lethargy,
mild liver
histopathology)
100 (female)
(decreased body
weight, mild liver
histopathology)
100
(sternebral
variations)
BMD
(mg/kg-day)
Not modeled
4.4
13
14.2*
50
BMDL10
(mg/kg-day) *
2.6
(Hepatic
vacuolization in
male rats)
1.4
(fatty changes in
liver of males)
10.6*
(fatty changes in
liver of females)
33
(sternebral
variations)
VIII - 65
November 15, 2005
-------
Reference
NTP (1989b)t
Species
Sex
Mouse
B6C3FJ
M,F
n
50
Dose
0
50
100
200
Route
Gavage
(corn oil)
Exposure
Duration
105 days
1 ml points
Continuous breeding
reprod. study. Body and
organ weights,
histopathology,
reproductive parameters
NOAEL
(mg/kg-day)
100
LOAEL
(mg/kg-day)
200
(decreased maternal
body weight;
reduced postnatal
survival and liver
histopathology in Fj
generation males
and females)
BMD
(mg/kg-day)
-
(not modeled)
BMDL10
(mg/kg-day) *
-
* BMD and BMDL10 values were derived using duration-adjusted doses.
f BMD modeled using the Crump BMD software (K. S. Crump, Inc.)
* These studies are included because they are reproductive/developmental studies.
Abbreviations: SD, Sprague-Dawley
VIII - 66
November 15, 2005
-------
Method of Analysis
Selected data from the candidate studies were analyzed using the benchmark dose (BMD)
modeling approach. Initially, data sets for potentially sensitive endpoints were selected as
described in U.S. EPA (1998b) and analyzed using the Crump Benchmark Dose Modeling
Software (K. S. Crump, Inc.). Following the release of Version 1.2 of the BMDS program (U.S.
EPA, 2000a), data from the NTP (1989a) subchronic and chronic studies conducted in rats were
reanalyzed in accordance with proposed U.S. EPA (2000b) recommendations. An advantage of
analysis with the BMDS software is that several additional models are available to fit the data.
The results of the analysis using the BMDS software are included in Table VIII-17.
Choice of Principal Study and Critical Effect for the RfD
The subchronic study conducted by NTP (1989a) was selected for derivation of the RfD.
Two factors supported selection of this study. First, the critical effect identified in the subchronic
study was consistent with the critical effects identified in the chronic NTP studies (fatty changes
in liver of male rats and female mice). Second, BMD modeling of both data sets supported
selection of the subchronic study. BMD analysis using the BMDS program calculated a BMD of
13 mg/kg-day for fatty changes in the liver of males in the chronic study, with a corresponding
BMDL10 of 1.4 mg/kg-day. The lowest duration-adjusted dose in this study was 71 mg/kg-day
and the response at this dose was high (49/50). The magnitude of the difference between the
BMD and BMDL10 values thus reflects considerable uncertainty about the shape of the curve in
the low-dose region.
Duration-adjusted BMD and BMDL10 values for hepatic vacuolization in male rats of 4.4
and 2.6 mg/kg-day, respectively, were obtained using data for the subchronic study. This BMD
is approximately three-fold less than the BMD calculated from chronic data (above). The
availability of response data for three doses below 71 mg/kg-day (duration-adjusted dose) in the
subchronic study provided additional information about the shape of the dose-response curve in
the region of interest, and thus a more reliable estimate of the BMD. Although the BMDL10
value for the subchronic study is higher than the value for the chronic study, this observation
reflects less uncertainty (smaller confidence interval) in the estimate of the subchronic BMD
when the results for the two studies are compared. The BMDL10 value calculated from the
subchronic NTP (1987) data was therefore selected for derivation of the RfD for bromoform.
The remaining studies were eliminated from consideration for the following reasons.
The study conducted by Tobe et al. (1982) did not identify a suitably sensitive endpoint
(histopathological examination was not conducted) and the data were never formally published
or submitted for peer review. The chronic study conducted by NTP (1989a) in mice reported
mild histopathological changes in female mice at a duration-adjusted dose of 71 mg/kg-day, the
lowest tested in this gender. However, both the BMD and BMDL10 were higher than those
identified in the subchronic study in rats, and these values were considered less reliable in the
absence of data at lower doses. The LOAELs identified in the developmental (Ruddick et al.,
1983) and reproductive (NTP, 1989a) studies were higher than the LOAEL values observed for
histopathology effects in the NTP subchronic study. The BMD and BMDL10 calculated for
sternebral variations in the Ruddick et al. (1983) were approximately 10-fold higher than those
identified in the subchronic study.
VIII - 67 November 15, 2005
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Derivation of the RfD
The duration-adjusted BMDL10 from the subchronic NTP (1989a) rat study was selected
for derivation of the RfD for bromoform. The RfD is calculated using the following equation:
(2.6 mg/kg-day)
RfD = = 0.03 mg/kg-day
100
where:
2.6 mg/kg-day = Duration-adjusted BMDL10 based on hepatocellular
vacuolization in the liver of male rats
100 = Composite uncertainty factor based on NAS/OW
guidelines; includes a factor of 10 for interspecies
variation, a factor of 10 for protection of sensitive human
populations
A composite uncertainty factor of 100 was used in the calculation of the bromoform RfD.
The standard factors of 10 were used for interspecies extrapolation and for protection of
sensitive subpopulations. No uncertainty factor was added for extrapolation from a subchronic
to a chronic study because the BMD and BMDL10 for the subchronic study was either
comparable to or lower than the corresponding values from the chronic study. This observation
suggests that a cumulative effect on the liver does not occur for the endpoints examined. The
database for bromoform includes subchronic and chronic bioassays conducted in rats and mice
(e.g. NTP 1989a), a two-generation reproductive toxicity study in mice (NTP 1989b), and a
developmental toxicity study in rats (Ruddick et al. 1983). Therefore, the database for
bromoform was considered sufficient and an uncertainty factor for database deficiencies was not
included in the calculation.
The DWEL for bromoform is calculated as follows:
(0.03 mg/kg-day) (70 kg)
DWEL = = 1.0mg/L(1000ng/L)
2 L/day
where:
0.03 mg/kg-day = RfD
70 kg = assumed weight of an adult
2 L/day = assumed water consumption by a 70-kg adult
VIII - 68 November 15, 2005
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Lifetime Health Advisory
The Lifetime Health Advisory (HA) represents that portion of an individual's total
exposure that is attributed to drinking water and is considered protective of noncarcinogenic
health effects over a lifetime of exposure. Bromoform has been categorized with respect to
carcinogenic potential as Group B2: Probable human carcinogen (IRIS, 1993b). Therefore, in
accordance with U.S. EPA Policy, a Lifetime Health Advisory is not recommended.
Alternative Approach for Derivation of the RfD
For comparison, the RfD can be calculated using the conventional NOAEL/LOAEL
approach. The subchronic NTP (1989a) oral exposure study identified a NOAEL of 25 mg/kg-
day based on absence of histopathological effects in rats exposed to bromoform by gavage in
corn oil for 13 weeks. Using this value, a duration adjustment factor of 5/7, and an uncertainty
factor of 1000 (including factors of 10 for interspecies extrapolation, protection of sensitive
subpopulations, and use of a subchronic study), the RfD would be 0.02 mg/kg-day. The
corresponding DWEL would be 0.7 mg/L assuming an adult body weight of 70 kg and a
drinking water ingestion rate of 2 L/day.
2. Carcinogenic Effects
a. Categorization of Carcinogenic Potential
Previous Evaluations of Carcinogenic Potential
The Carcinogenic Risk Assessment Verification Endeavor (CRAVE) group of the
U.S. EPA has reviewed the available evidence on the carcinogenicity of bromoform and has
assigned it to Group B2: probable human carcinogen (IRIS, 1993b). Assignment to this category
is appropriate for chemicals where there are no or inadequate human data, but which have
sufficient animal data to indicate carcinogenic potential.
IARC (1999b) has recently re-evaluated the carcinogenic potential of bromoform. IARC
concluded that there is limited evidence of carcinogenicity in experimental animals and
inadequate evidence in humans for bromoform. Bromoform is therefore categorized as Group 3:
not classifiable as to carcinogenicity in humans.
Categorization of Carcinogenic Potential Under the Proposed 1999 Cancer Guidelines
Cancer Hazard Summary
Under the proposed guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
bromoform is 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.
VIII - 69 November 15, 2005
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Supporting Information for Cancer Hazard Assessment
Human Data
The information on the carcinogenicity of bromoform from human studies is inadequate.
There are no epidemiological data specifically relating increased incidence of cancer to exposure
to bromoform. There are equivocal epidemiological data describing a weak association of
chlorinated drinking water exposures with increased incidences of bladder, rectal, and colon
cancer. U.S. EPA has determined that these studies cannot attribute the observed effects to a
single compound, as chlorinated water contains numerous other disinfection byproducts that are
potentially carcinogenic.
Animal Data
The carcinogenicity of bromoform has been investigated in two species. These studies
include a well-designed and conducted corn oil gavage study conducted in rats and mice and a
study in which male Strain A mice were administered bromoform by intraperitoneal injection.
No data are available on the carcinogenic potential of bromoform administered via the inhalation
or dermal routes.
In the corn oil gavage study (NTP, 1989a), neoplasms of the large intestine (adenomatous
polyps or adenocarcinoma) were observed in male and female rats. The response for combined
adenoma and carcinoma reached statistical significance in female rats. The occurrence of
tumors of the large intestine in this study was considered biologically significant because they
are historically rare in rats. NTP (1989a) concluded that there was clear evidence for
carcinogenicity in females and some evidence of carcinogenicity in males. No evidence of
bromoform carcinogenicity was observed in male or female mice. Intraperitoneal injection of
Strain A mice with three concentrations of bromoform resulted in significantly increased tumor
incidence only at the middle dose tested.
Structural Analogue Data
Trihalomethanes structurally related to bromoform have shown varying degrees of
carcinogenic potential in rodents. Chloroform, the most extensively characterized
trihalomethane, is reported to be carcinogenic at high doses in several chronic animal bioassays,
with significant increases in the incidence of liver tumors in male and female mice and
significant increases in the incidence of kidney tumors in male rats and mice (U.S. EPA, 2001).
The occurrence of tumors in animals exposed to chloroform is demonstrably species-, strain-,
and gender-specific, and has only been observed under dose conditions that caused cytotoxicity
and regenerative cell proliferation in the target organ. The cancer database for structurally-
related brominated trihalomethanes is more limited, but includes well-conducted studies
performed by the National Toxicology Program. In a two-year corn oil gavage study of
bromodichloromethane, NTP (1987) found clear evidence for carcinogenicity in male and
female rats (large intestine and kidney) and male (kidney) and female (liver) mice. Tumasonis et
al. (1987) reported a statistically significant increase in the incidence of hepatic neoplastic
nodules in rats exposed to bromodichloromethane in the drinking water. In a two-year corn oil
gavage study of dibromochloromethane, NTP (1985) determined that there was some evidence
VIII - 70 November 15, 2005
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of carcinogenicity in female mice and equivocal evidence of carcinogenicity in male mice, based
on the occurrence of hepatocellular adenomas and carcinomas. George et al. (2002) observed a
significantly increased prevalence of hepatocellular tumors only at the lowest dose administered
in a drinking water study. Other oral exposure studies found no evidence for carcinogenicity of
bromodichloromethane (Aida et al., 1992b) or dibromochloromethane (Tobe et al., 1982;
Voronin et al., 1987).
Other Key Data
Bromoform is formed as a byproduct of drinking water disinfection with chlorine.
Exposure to bromoform may occur via ingestion of tap water, via dermal contact during
showering or bathing, or by inhalation of bromoform volatilized during household activities.
Absorption of single oral doses appears to be extensive. Bromoform is rapidly metabolized and
eliminated predominately as expired volatiles, carbon monoxide, or carbon dioxide. Only a
small amount (less than 10%) is eliminated in urine or in feces. No comprehensive tissue data
are available regarding the bioaccumulation or retention of bromoform following repeated
exposure. However, because of the rapid metabolism and excretion of bromoform, marked
accumulation and retention is not expected.
Bromoform itself is not directly reactive with DNA. Metabolism to reactive species is a
prerequisite for toxicity, as inferred from metabolic induction and inhibition studies. In vitro and
in vivo studies of the mutagenic and genotoxic potential of bromoform have yielded both
positive and negative 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. However, because a
majority of studies yielded positive results, bromoform is considered to be at least weakly
mutagenic and genotoxic. Recent studies conducted with strains of Salmonella thatexpress rat
theta-class glutathione S-transf erase suggest that mutagenicity of the brominated trihalomethanes
may be mediated by glutathione conjugation.
Mode of Action
The mode of action for tumor induction by bromoform has not been clearly elucidated
and may involve contributions from multiple bioactivation pathways. In each case, toxicity is
believed to result from interaction of reactive metabolites with cellular macromolecules.
Proposed bioactivation pathways for bromoform include: 1) production of reactive
dihalocarbonyls by oxidative metabolism; 2) production of reactive dihalomethyl radicals by
oxidative metabolism; and 3) formation of DNA-reactive species via a glutathione-dependent
pathway. The relative contribution of each pathway to tumor induction by bromoform has not
been characterized. It is possible that only the latter two processes lead to DNA damage in vivo,
because the highly reactive dihalocarbonyl intermediate may not survive long enough to enter
the nucleus and react with DNA. For this reason, cytotoxicity may be the primary consequence
of the oxidative pathway. Cytotoxicity coupled with regenerative hyperplasia is considered the
primary mode of action for tumor formation following exposure to high concentrations of
chloroform, a structurally-related trihalomethane which has low genotoxic potential. Data to
support a similar primary mode of action for tumor development in liver, kidney, and large
VIII-71 November 15, 2005
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intestine are currently lacking for bromoform. In the absence of such information, combined
with a positive weight-of-evidence evaluation for genotoxicity, the mode of action for tumor
development is assumed to be a linear process. The processes leading to tumor formation in
animals are expected to be relevant to humans.
Conclusion
Under the proposed guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999)
bromoform is likely to be carcinogenic to humans by the oral route. This weight-of-evidence
evaluation is based on 1) observations of tumors in rats treated by oral pathways; 2) lack of
epidemiological data specific to bromoform and equivocal data for drinking water drinking
water exposures that cannot reliably be attributed to bromoform among multiple other
disinfection byproducts; 3) positive results for a majority of the available genotoxicity and
mutagenicity tests; and 4) metabolism and mode of action that are reasonably expected to be
comparable across species. Although no cancer data exist for exposures via the dermal or
inhalation pathways, the weight-of-evidence conclusion is considered to be applicable to these
pathways as well. The finding for inhalation is based on the observation that patterns of
metabolizing enzyme activity in male rats are similar following exposure to a structurally-related
compound (bromodichloromethane) via the inhalation and gavage routes. Bromoform absorbed
through the skin is expected to be metabolized and cause toxicity in much the same way as
bromoform absorbed by the oral and inhalation routes.
b. Choice of Study for Quantification of Carcinogenic Risk
A single oral exposure study was available for the quantification of carcinogenic risk
associated with oral exposure to bromoform. NTP (1989a) conducted an oral exposure study in
B6C3FJ mice and F344/N rats. No evidence of carcinogenicity was observed in male B6C3FJ
mice exposed to bromoform via gavage (corn oil) at doses up to 100 mg/kg-day, or in female
mice exposed at doses up to 200 mg/kg-day for 5 days/week. Male and female F344/N rats (50
rats/sex/dose) were administered bromoform via gavage at doses of 0, 100, or 200 mg/kg-day for
5 days/week for 103 weeks (NTP 1989a). At termination, all animals were necropsied, and a
thorough histological examination of tissues was performed. Adenomatous polyps or
adenocarcinomas of the large intestine were noted in three high-dose male rats, eight high-dose
female rats, and one low-dose female rat (Table VIII-18). Despite the small number of tumors
found, the increase was considered biologically significant because these tumors are historically
rare in the rat. The study authors concluded that there was some evidence for carcinogenic
activity in male rats and clear evidence in female rats.
c. Extrapolation model
The LMS model (U.S. EPA, 1986) and the default linear approach described by the
Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996; 1999) were used to
quantify the risk associated with exposure to bromoform. Although data are mixed , U.S. EPA
has previously concluded that the weight of evidence suggests that bromoform is mutagenic (see
Section V.F.3). At the present time, there are no data which indicate that bromoform-induced
VIII - 72 November 15, 2005
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tumorigenesis occurs as a consequence of cytotoxicity followed by regenerative hyperplasia.
Thus, use of a linear approach was considered appropriate for quantification of cancer risk
associated with exposure to bromodichloromethane.
Table VIII-18 Tumor Frequencies in Rats Exposed to Bromoform in Corn Oil for 2 Years
- Adapted from NTP (1989a)
Animal
Male rat
Female rat
Tissue/Tumor
Large intestine
Large intestine
Adenocarcinoma
Polyp (adenomatous)
Adenocarcinoma
Polyp (adenomatous)
Tumor Frequency
Control
0/50
0/50
0/48
0/48
100 mg/kg
0/50
0/50
0/50
1/50
200 mg/kg
1/50
2/50
2/50
6/50
d. Cancer Potency and Unit Risk
Estimates of cancer risk associated with exposure to bromoform are summarized in Table
VIII-19. U.S. EPA (1994b) reported a cancer potency estimate of 7.9 x 10'3 (mg/kg-day)'1 for
bromoform based on the incidence of intestinal tumors in rats and derived using
recommendations in the 1986 Cancer Guidelines (U.S. EPA, 1986). The calculated value for
unit risk is 2.3 x 10"7 ([ig/L)"1. This estimate was used to calculate a drinking water concentration
of 40 |ig/L associated with a 10"5 risk.
A cancer potency estimate of 4.6 x 10"3 (mg/kg-day)"1 (U.S. EPA, 1998b) based on the
incidence of intestinal tumors in rats was calculated using the LMS model and a scaling factor of
body weight374. Use of this scaling factor is consistent with recommendations in U.S. EPA
(1992b). Unit risk was estimated for bromoform using an assumed body weight of 70 kg and a
drinking water ingestion rate of 2 L. The calculated value for unit risk is 1.30 x 10"7 ([ig/L)"1.
This estimate was used to calculate a drinking water concentration of 77|ig/L associated with a
10'5 risk (8 jig/L for a risk of 10'6).
Cancer risk estimates were also obtained using the LED10 (the lower 95% confidence
limit on a dose associated with 10% extra risk) of 2.2 x 104 [ig/kg-day for intestinal tumors and
assuming a linear mode of action for the carcinogenicity of bromoform (Table VIII-19). A
cancer potency value of 4.5 x 10"3 (mg/kg-day)"1 was derived using this approach. A unit risk of
1.3 x 10"7 ([ig/L)"1 was calculated using an assumed body weight of 70 kg and a drinking water
ingestion rate of 2 L. This estimate was used to calculate a drinking water concentration of 78
|ig/L associated with a 10"5 risk (8 |ig/L for 10"6 risk). These values are similar to values derived
using the LMS approach with body weight scaling to the 3/4 power.
VIII - 73
November 15, 2005
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There are no data to suggest that tumor incidence in the large intestine is influenced by
the use of an oil vehicle. Therefore, the risk estimates reported above are believed to be
applicable to drinking water exposures.
Table VIII-19 Carcinogenic Risk Estimates for Bromoform
Method of
Estimation
LMS Method Using
BW3'4 Conversion
U.S. EPA(1998b)
U.S. EPA(1994b)*
LED10/Linear Method
U.S. EPA(1998b)
Tumor Site
Large intestine
Large intestine
Large intestine
Species
Rat
Rat
Rat
Sex
F
F
F
Slope Factor
(mg/kg-day) '
4.6X10'3
7.9xlO'3
4.5xlO'3
Unit Risk
1.3x10-'
2.3x10"'
1.3x10-'
LED,,
(|ig/kg-day)
-
2.2xl04
10 5 Risk
Concentration
(Hg/L)
77
40
78
Adapted from IRIS (1993b)
VIII - 74
November 15, 2005
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D. Summary
Table VIII-20 Summary of Advisory Values for Bromodichloromethane,
Dibromochloromethane, and Bromoform
Advisory
Value
Reference
Bromodichloromethane
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
RfD
DWEL
Lifetime HA
Oral Slope Factor c
Concentration for excess cancer risk (10"6)
Unit Risk
Img/L
0.6 mg/L
0.6 mg/L
2 mg/L
0.003 mg/kg-day
100 ng/L
Not applicable
3. 5 xlO'2 (mg/kg-day)-1
l.Ong/L
l.OxlO^Cng/L)-1
Narotskyetal. (1997)
NTP(1998)
CCC (2000d)
CCC (2000d)
Aidaetal. (1992b)
Aidaetal. (1992b)
-
NTP(1987)
NTP(1987)
NTP(1987)
Dibromochloromethane
One-day HA for 10-kg child b
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
RfD
DWEL
Lifetime HA
Oral Slope Factor c
Concentration for Excess cancer risk (10"6)
Unit Risk
0.6 mg/L
0.6 mg/L
0.2 mg/L
0.6 mg/L
0.02 mg/kg-day
700 ng/L
60ng/L
4.3xlQ-2 (mg/kg-day)-1
0.8 ng/L
l.lxlO^Cng/L)-1
Aidaetal. (1992a)
Aidaetal. (1992a)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
NTP(1985)
Bromoform
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child a
Longer-term HA for 70-kg adult
5 mg/L
0.2 mg/L
0.2 mg/L
0.9 mg/L
Burton-Fanning (1901)
NTP(1989a)
NTP(1989a)
NTP(1989a)
VIII - 75
November 15, 2005
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Advisory
RfD
DWEL
Lifetime HA
Oral Slope Factor c
Concentration for Excess cancer risk (10"6)
Unit Risk
Value
0.03 mg/kg-day
1000 ng/L
Not applicable
4.56X10'3 (mg/kg-day)-1
S^g/L
l.SxlQ-'djg/L)-1
Reference
NTP(1989a)
NTP(1989a)
--
NTP(1989a)
NTP(1989a)
NTP(1989a)
a The calculated value for the Longer-term HA was slightly higher than the values for the Ten-day HA. 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 a conservative estimate of the One-day HA for a 10-kg child.
0 Use of the Longer-term HA recommended as a conservative estimate of the Ten-day HA for a 10-kg child.
d The oral slope factor was calculated using the Linearized Multistage model 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
VIII - 76
November 15, 2005
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APPENDIX A
BENCHMARK DOSE MODELING OF HEALTH EFFECTS ENDPOINTS FOR THE
BROMINATED TRIHALOMETHANES: BROMODICHLOROMETHANE,
DffiROMOCHLOROMETHANE, AND BROMOFORM
A -1 November 15, 2005
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A. INTRODUCTION
The limitations of the NOAEL/LOAEL approach as the basis for estimating thresholds of
toxic effect are well-documented (e.g., U.S. EPA, 1995, 2001b). These limitations include:
1) the slope of the dose-response plays little role in determining the NOAEL;
2) the NOAEL (or LOAEL) is limited to the doses tested experimentally;
3) the determination of the NOAEL is based on scientific judgement, and is subject to
inconsistency;
4) experiments using fewer animals tend to produce larger NOAELs, and as a result may
produce larger health advisories (HAs) or reference doses (RfDs) (U.S. EPA, 1995,
200 Ib) that may not be sufficiently protective of human health.
In contrast, benchmark doses (BMDs) are not limited to the experimental doses,
appropriately reflect the sample size, and can be defined in a statistically consistent manner. In
light of these considerations, it is becoming common practice to conduct assessments by
performing BMD modeling for key endpoints, in addition to identification of NOAELs and
LOAELs.
This document describes the analysis of the data relevant to the development of the One-
day, Ten-day, and Longer-term Health Advisories (HAs) for |