FINAL DRAFT FOR THE DRINKING WATER
CRITERIA DOCUMENT ON TRIHALOMETHANES
April 8, 1994
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-C2-0139
by
Clement International Corporation
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3. REPORT TYPE AND DATES COVERED
4. TITLE AND SUBTITLE
Final Draft for the Drinking Water
Criteria Document on Trihalomethanes
6. AUTHOR(S)
7. PERFORMING ORGANIZATION NAME(S) AND AOORESS(ES)
Clement International Corporation
9. SPONSORING /MONITORING AGENCY NAME(S) AND ADDRESS(ES)
U.S. EPA
Office of Water
401 M St., SW
Washington, DC 20460
5. FUNDING NUMBERS
68-C2-0139
8. PERFORMING ORGANIZATION
REPORT NUMBER
10. SPONSORING /MONITORING
AGENCY REPORT NUMBER
11. SUPPLEMENTARY NOTES
12a. DISTRIBUTION /AVAILABILITY STATEMENT
12b. DISTRIBUTION CODE
13. ABSTRACT (Maximum 200 words)
This document provides the health effects basis to be considered
art establishing the maximum contaminant level goals for four
trihalomethanes found in chlorinated drinking water.
y
3
14. SUBJECT TERMS
health effects, trihalomethanes
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION
OF REPORT OF THIS PAGE
15. NUMBER OF PAGES
309
16. PRICE CODE
#N . A03
19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT
OF ABSTRACT
NSN 7540-01-280-5500
Standard Form 298 (Rev. 2-89)
Prescribed by ANSI Std. Z39-18
298-102
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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) arid 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 four 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 1989, 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.
Tudor T. Davies
Director, Office of Science
and Technology
Office of Water
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TABLE OF CONTENTS
1. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-l
A. Chloroform II-l
B. Brominated Trihalomethanes II-l
C. Occurrence II-3
D. Summary II-5
III. TOXICOKINETICS III-l
A. Absorption III-l
B. Distribution .-. III-6
C. Metabolism ' Ill-9
D. Excretion _ 111-23
E. Bioaccumulation and Retention 111-24
F. Summary Ill-25
IV. HUMAN EXPOSURE VI-1
A. Drinking Water Exposure VI-1
1. Total Trihalomethanes VI-1
2. Chloroform . . VI-4
3. Brominated Trihalomethanes VI-12
B. Exposure to Sources Other Than Drinking Water . VI-26
1. Dietary Intake VI-26
a. Chloroform VI-26
b. Brominated Trihalomethanes VI-29
2. Air Intake VI-31
a. Chloroform VI-31
b. Brominated Trihalomethanes VI-38
C. Overall Exposure VI-42
1. Chloroform VI-42
2. Brominated Trihalomethanes VI-42
D. Body Burden VI-43
1. Chloroform VI-43
a. Breath VI-43
b. Blood VI-44
c. Mother's Milk VI-45
d. Adipose Tissue VI-45
2. Brominated Trihalomrthanes VI-46
a. Blood VI-46
b. Mother's Milk VI-46
E. Summary VI-46
V. HEALTH EFFECTS IN ANIMALS V-l
A. Short-Term Exposure V-l
1. Lethality V-l
2. Other Effects V-l
a. Chloroform V-l
b. Brominated Trihalomethanes V-9
B. Longer-Term Exposure V-24
1. Chloroform V-24
2. Brominated Trihalomethanes V-41
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TABLE OF CONTENTS (cont.)
C. Reproductive and Developmental Effects V-60
1. Chloroform V-60
2. Brominated Trihalomethanes V-63
D. Mutagenicity and Genotoxicity V-65
1. Chloroform V-65
a. In Vitro Studies V-66
b. .In Vivo Studies V-69
2. Brominated Trihalomethanes V-73
a. .In Vitro Studies V-73
b. In Vivo Studies V-85
E. Carcinogenicity V-87
1. Chloroform V-87
2. Brominated Trihalomethanes V-102
F. Summary . V-110
1. Health Effects of Acute and Short-Term Exposure of
Animals V-110
2. Health Effects of Longer-Term Exposure of Animals . . . V-lll
3. Reproductive/Developmental Effects in Animals V-112
4. Mutagenicity and Genotoxicity Studies V-113
5. Carcinogenicity Studies in Animals V-113
VI. HEALTH EFFECTS IN HUMANS VI-1
A. Clinical Case Studies VI-1
B. Epideraiological Studies VI-2
C. High-Risk Populations VI-6
D. Summary VI-9
VII. MECHANISM OF TOXICITY VII-1
A. Role of Metabolism VII-1
B. Biochemical Basis of Toxicity VII-3
C. Mechanism of Carcinogenesis : VII-5
D. Interactions VII-6
E. Summary VII-8
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
A. Noncarcinogenic Effects VIII-6
1. Chloroform VIII-6
a. One-day Health Advisory VIII-6
b. Ten-day Health Advisory for Chloroform VIII-9
c. Longer-term Health Advisory for Chloroform .... VIII-12
d. Reference Dose and Drinking Water Equivalent
Level for Chloroform VIII-14
2. Bromodichloromethane .... VIII-17
a. One-day Health Advisory for
Bromodichloromethane VI11-17
b. Ten-day Health Advisory for
Bromodichloromethane VIII-18
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TABLE OF CONTENTS (cont.)
c. Longer-term Health Advisory for
Bromodichloromethane VIII-20
d. Reference Dose and Drinking Water Equivalent
Level for Bromodichloromethane VIII-23
3. Dibromochloromethane ' VIII-26
a. One-day Health Advisory for
Dibromochloromethane VIII-26
b. Ten-day Health Advisory for
Dibromochloromethane VIII-26
c . Longer-'term Health Advisory for
Dibromochloromethane VI11-29
d. Reference Dose and Drinking Water Equivalent
Level for Dibromochloromethane VIII-31
4. Bromoform VIII-34
a. One-day Health Advisory for Bromoform VIII-34
b. Ten-day Health Advisory for Bromoform VIII-35
c. Longer-term Health Advisory for Bromoform .... VIII-37
d. Reference Dose and Drinking Water Equivalent
Level for Bromoform VIII-40
B. Carcinogenic Effects VIII-43
1. Categorization of Carcinogenic Potential VIII-43
a. Chloroform VIII-43
b. Brominated Trihalomethanes VIII-43
2. Quantitative Carcinogenic Risk Estimates VIII-44
a. Chloroform VIII-44
b. Brominated Trihalomethanes VIII-46
C. Summary VIII-50
IX. REFERENCES IX-1
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LIST OF FIGURES
Page
III-l Metabolic Pathways of Trihalomethane Biotrar.sforaation . . . 111-10
IV-1 THMs in Water: Means--Results From National Surveys .... IV-3
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LIST OF TABLES
Page
1-1 Summary of Quantification of Toxicologicai Effects for
Trihalomethanes 1-13
II-1 Physical and Chemical Properties of Chloroform and
Brominated Trihalomethanes II-2
III-l Recovery of Label 8 Hours after Oral Administration of
14C-Labeled Trihalomethanes to Male Rats or Male Mice .... Ill-3
III-2 Cumulative Excretion of Label after Oral Administration of
KC-Labeled Bromndichloromethane to Male Rats Ill-4
III-3 In Vitro Binding of 14C-Chloroform Metabolites to
Microsomes of Male Mice Ill-11
IV-1 Comparison of Results of National Surveys IV-2
IV-2 Chloroform in Drinking Water from the EPA TEAM study (jig/D . . IV-12
IV-3 Bromodichloromethane in Drinking water from the EPA TEAM
Study (/ig/L) IV-20
IV-4 Dibromochloromethane in Drinking water from the EPA TEAM
Study (jug/L) IV-21
IV-5 Bromofrom in Drinking water from the EPA TEAM Study (/xg/L) . . IV-22
IV-6 Chloroform in Personal Air Samples: EPA TEAM Study (jig/m3) . . IV-33
IV-7 Chloroform in Outdoor Air Samples From the EPA TEAM
Study (jug/m3) IV-34.
V-l Summary of Oral Lethality of Trihalomethanes V-2
V-2 Summary of Short-Term Health Effects Data on Chloroform .... V-10
V-3 Effects of Brominated Trihalomethanes in Mice Dosed by
Gavage for 14 Days V-14
V-4 Incidence and Severity of Microscopic Changes in Kidney
and Liver of Mice Dosed with Brominated Trihalomethanes .... V-15
V-5 Serum Biochemical Levels in Rats Fed Brominated
Trihalomethanes For One Month V-21
V-6 Incidence and Severity of Microscopic Changes in the Liver
of Rats Dosed with Brominated Trihalomethanes for
One Month .' V-22
V-7 Summary of Short-term Health Effects Data on
Bromodichloromethane V-25
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LIST OF TABLES (Continued)
Page
V-8 Summary of Short-term Health Effects Data on
Dibromochloromethane V-26
V-9 Summary of Short-term Health Effects Data on Bromoform .... V-27
V-10 Effects of Vehicle on the Subchronic Toxicity of Chloroform
in B6C3F1 Mice V-29
V-ll Comparison of Clinical Chemistry Parameters of Mice Treated
with Chloroform in Corn Oil Versus Emulphor for 90 Days .... V-30
V-12 Effect of Vehicle on Chloroform-Induced Accumulation of
V-13
V-14
V-15
V-16
V-17
V-18
V-19
V-20
V-21
V-22
V-23
V-24
V-25
V-26
V-27
Hepatic Changes Observed in Dogs Administered Chloroform
Orally for 7.5 Years
Summary of Longer-term Studies of Chloroform
Incidence and Severity of Liver and Thyroid Lesions in Rats
Fed Brominated Trihalomethanes for 90 Days
Serum Biochemical Levels in Rats Fed Brominated
Trihalomethanes for 18 to 24 Months .
Liver Lesions in Rats Fed Bromodichlorome thane for 2 Years . .
Nonneoplastic Lesions in Rats and Mice Exposed to
Bromodichlorome thane for 2 Years
Summary of Longer-term Studies of Bromodichloromethane ....
Summary of Longer-term Studies of Dibromochloromethane ....
Summary of Longer-tern1 Sfurfiec of BromofoTi . ......
S'munary of Tn vitro Gpnor^xiciry Data on Chloroform
Summary of In Vivo Genotoxicity Data on Chloroform
Summary of Genotoxicity Data on Bromodichloromethane
Summary of Genotoxicity Data on Dibromochloromethane
Summary of Genotoxicity Data on Bromoform ....
Liver and Kidney Necrosis and Hepatoraas in Strain A Mice
Following Repeated Oral Administration of Chloroform in
Olive Oil
V-35
V-39
V-42
V-49
V-50
V-54
V-57
V-58
V-59
V-70
V-74
V-75
V-77
V-79
V-88
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LIST OF TABLES (Continued)
Page
V-28 Summary of Tumor Frequencies in Rats Administered
Chloroform for 78 Weeks V-91
V-29 Summary of Tumor Frequencies in Mice Administered
Chloroform for 78 Weeks . V-92
V-30 Kidney Tumor Incidence in Male ICI Mice Treated with
Chloroform V-94
V-31 Compound-Related'"Increased Incidences of Neoplasms in Rats
and Mice Exposed to Chloroform in the Drinking Water for
Two Years " V-.96
V-32 Effects of Chloroform Exposure on Liver Tumors Initiated
by Ethylnitrosourea Exposure in Male CD-I Mice V-99
V-33 Effect of Chloroform Exposure on Liver and Lung Tumors in
Male B6C3F1 Mice V-101
V-34 Tumor Frequencies in Rats and Mice Exposed to
Bromodichloromethane in Corn Oil for 2 Years V-103
V-35 Frequencies of Liver Tumors in Mice Administered
Dibromochloromethane in Corn Oil for 104 Weeks V-107
V-36 Tumor Frequencies in Rats Exposed to Bromoform in Corn Oil
for 2 Years V-109
VI-1 Epidemiological Studies Investigating an Association Between
Cancer and Chlorinated Water VI-7
VIII-1 Summary of Candidate Studies for Derivation of the One-day
Health Advisory for Chloroform VIII-7
VIII-2 Summary of Candidate Studies for Derivation of the Ten-day
Health Advisory for Chloroform VIII-10
VIII-3 Summary of Candidate Studies for Derivation of the
Longer-term Health Advisory for Chloroform VIII-13
VIII-4 Summary of Candidate Studies for Derivation of the DWEL
for Chloroform VIII-15
VIII-5 Summary of Candidate Studies for Derivation of the Ten-day
Health Advisory for Bromodichloromethane VIII-19
VIII-6 Summary of Candidate Studies for Derivation of the
Longer-term Health Advisory for Bromodichloromethane .... VIII-21
VIII-7 Summary of Candidate Studies for Derivation of the DWEL
for Bromodichloromethane VIII-24
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LIST OF TABLES (Continued)
Paee
VIII-8 Summary of Candidate Studies for Derivation of the Ten-
day Health Advisory for Dibromochloromethane VIII-27
VIII-9 Summary of Candidate Studies for Derivation of the.
Longer-term Health Advisory for Dibromochloromethane .... VIII-30
VIII-10 Summary of Candidate Studies for Derivation of the DUEL
for Dibromochloromethane VIII-32
VIII-11 Summary of Candidate Studies for Derivation of the Ten-
day Health Advisory for Bromoform VIII-36
VIII-12 Summary of Candidate Studies for Derivation of the
Longer-term Health Advisory for Bromoform VIII-38
VIII-13 Summary of Candidate Studies for Derivation of DWEL
for Bromoform VIII-41
VIII-14 Upper-Bound Estimates of Cancer Risk of 1 mg/kg/day of
Chloroform, Calculated by Four Models on the Basis of
Various Data Sets VIII-45
VIII-15 Carcinogenic Risk Estimates for Chloroform Calculated
by NAS VIII-47
VIII-16 Carcinogenic Risk Estimates for Brominated
Trihalomethanes VIII-49
VIII-17 Summary of Quantification of Toxicological Effects for
Trihalomethanes VIII-51
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I. SUMMARY
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 chlorination of drinking water.
The principal trihalomethanes that have been observed in water are chloroform
(CHClj), bromodichloromethane (CHBrCl2), dibromochloromethane (CHBr2Cl), and
bromoform (CHBr3) . Chloroform is formed by the action of hypochlorous acid on
endogenous organic molecules (e.g., humic or fulvic acids) present in the
water. Hypochlorous acid also oxidizes any bromide ion present to form
hypobromous acid, which reacts with the organic material to form the bromi-
nated trihalomethanes. Chloroform has been detected in finished drinking
water samples at. mean concentrations typically ranging from 9 to 100 jjg/L,
while average values of brominated trihalomethanes typically range from about
0.2 to 25 ^g/L. Chloroform and bromodichloromethane levels tend to be higher
in. systems using surface water sources than those using groundwater sources,
whereas dibromochloromethane and bromoform levels are comparable in systems
using either source.
Toxicokinetics
Measurements of gastrointestinal absorption of trihalomethanes in
mice, rats and monkeys indicate that absorption is rapid (peak blood levels at
1 hour) and extensive (64% to 98%). Limited data indicate that gastrointes-
tinal absorption of chloroform (and presumably other trihalomethanes) is also
rapid and extensive (at least 90%) in humans. Most studies of trihalomethane
absorption have used oil-based vehicles and gavage dosing. One study in rats
found higher chloroform blood levels following oral gavage administration of
1-1
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chloroform in water than after administration of chloroform in an oil vehicle.
This was interpreted as being due to higher absorption from water than from
oil, but the possible influence of first-pass metabolism was not taken into
account. Dermal absorption of chloroform in water by rats and hairless guinea
pigs is rapid and extensive. Absorption has also been reported in humans
dermally exposed to chloroform in water.
Absorbed trihalomethanes appear to distribute widely throughout the
body. Chloroform was detected in a number of postmortem tissues from humans,
with highest levels (5 to 68 /ig/kg) in body fat and lower levels (1 to
10 jig/kg) in kidney, liver, and brain. Radiolabeled trihalomethanes were
detected in a variety of tissues following oral dosing in rats and mice, with
somewhat higher J.evels in stomach, liver, blood, and kidney than in lung,
muscle, or brain. Chloroform crosses the placenta and may be detected in
fetal tissues following inhalation exposure of pregnant rats.
Trihalomethanes are extensively metabolized by both humans and
animals. The main site of metabolism is the liver, but metabolism also occurs
in the kidney. Both the oxidative and reductive metabolism of trihalomethanes
are mediated by cytochrome P-450. The oxidative pathway requires NADPH and
oxygen, whereas the reductive pathway can utilize NADPH or NADH and is
inhibited by oxygen. In the presence of oxygen (oxidative metabolism), the
reaction product is trihalomethanol (CX3OH), which then decomposes to yield a
reactive dihalocarbonyl (CX20) such as phosgene (CC120). Dihalocarbonyls are
relatively reactive species, and may undergo a variety of reactions including
adduct formation with various cellular nucleophiles, hydrolysis to yield
carbon dioxide, or glutathione-dependent reduction to yield carbon monoxide.
If oxygen is lacking (reductive metabolism), the metabolic reaction products
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appear to be free radical species such as the dihalomethyl radical (CHX2-).
These radicals are extremely reactive and also may form covalent adducts with
a "ariety of cellular molecules. Metabolism via the reductive pathway appears
to occur more readily for brominated trihalomethanes than for chloroform.
Both in vivo and in vitro studies indicate that the pattern of
trihalomethane metabolism may differ between animal species and sexes. In
vivo, one study reported that mice metabolize trihalomethanes to carbon
dioxide more extensively than do rats (40% to 80% versus 4% to 18%), although
another study indicated that rats metabolized bromodichloromethane to carbon
dioxide to an extent that was comparable to that reported for mice. In vitro.
the capacity for reductive metabolism of trihalomethanes has been found to be
greater in hepat.ic microsomes from mice than rats, and the incorporation of
label into- covalent adducts in renal microsomes has been found to be greater
in male mice than female mice. These metabolic differences may underlie some
of the important toxicological differences that have been noted between sexes
and species.
Excretion of trihalomethanes occurs primarily via the lungs. In
humans, approximately 90% of an oral dose of radiolabeled chloroform was
exhaled as the end metabolite, carbon dioxide, or as the parent compound,
chloroform. Levels in the urine were below the limit of detection (0.1%). In
mice and rats, 45% to 88% of an oral dose of chloroform or brominated trihalo-
methane was excreted from the lungs either as the parent trihalomethane or as
carbon dioxide, with 1% to 5% excreted in the urine. Intraperitoneal injec-
tion of rats with 36C1-chloroform resulted in the appearance of both inorganic
and organic forms of chloride in the urine, but the total amount was not
quantified.
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No data were located regarding the bioaccumulation and retention of
the trihalomethanes following chronic exposure. However, based on the rapid
metabolism and excretion of chloroform and the brcminsted trihalomethanes,
along with the low levels of chloroform in human autopsy samples, marked
accumulation and retention of these compounds are not anticipated.
Health Effects of Acute and Short-term Exposure of Animals
Large oral doses of trihalomethanes are lethal to laboratory animals.
Acute LD50 values range from 119 to 2,000 mg/kg for chloroform, 450 to
969 mg/kg for bromodichloromethane, 800 to 1,200 mg/kg for dibromochloro-
methane, and 1,388 to 1,550 mg/kg for bromoform. Death from acute high-dose
trihalomethane exposure was usually found to be due to central nervous system
depression and cardiac effects, and was usually accompanied by histopatholo-
gical changes in the liver and kidney.
Acute oral exposure to sublethal doses of trihalomethanes can also
produce effects on the liver, kidney, and central nervous system. In mice,
single oral doses of 60 to 89 mg/kg chloroform produced kidney damage, with
doses of 140 to 250 mg/kg producing liver damage. Organ damage was character-
ized by fatty infiltration, cellular necrosis, vacuolization, enzyme level
changes, and/or organ weight changes. Ataxia and sedation were noted in mice
receiving 500 mg/kg chloroform, 500 mg/kg bromodichloromethane, 500 mg/kg
dibromochloromethane, or 1,000 mg/kg bromoform.
Short-term exposures of laboratory animals to trihalomethanes has been
observed to cause effects on the liver, kidney, central nervous system, and
immune system. Hepatic effects, including organ weight changes, elevated
1-4
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serum enzyme levels, and histopathological changes were reported in mice
and/or rats administered 37 to 290 rag/kg/day chloroform, 148 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 (PAH) uptake, histopathological changes and organ
weight changes, were reported in mice and/or rats administered 37 to
148 mg/kg/day chloroform, 148 to 600 mg/kg/day bromodichloromethane, 147 to
500 mg/kg/day dibromochloromethane, or 289 mg/kg/day bromoform for 14 days.
Hyperactivity and/or a decreased operant response were observed in mice and/or
rats after ingesting 100 to 600 mg/kg/day bromodichloromethane, 400 mg/kg/day
dibromochloromethane, or 100 mg/kg/day bromoform for 14 to 60 days.
Health Effects of Longer-term Exposure of Animals
The predominant effects of longer-term oral exposure to trihalo-
methanes occur in the liver and kidney. The effects on these two organs are
similar to those described for short-term exposures, with liver appearing to
be the most sensitive target organ. Hepatic effects were reported in mice
and/or rats administered 15 to 180 mg/kg/day chloroform, 24 to 300 mg/kg/day
bromodichloromethane, 39 to 250 mg/kg/day dibromochloromethane, or 50 to
250 mg/kg/day bromoform. In general, these dose ranges are slightly lower
than those reported to cause effects following short-term exposures. Renal
effects were reported in mice and/or rats administered 25 to 300 mg/kg/day
bromodichloromethane or 250 mg/kg/day dibromochloromethane.
1-5
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Reproductive/Developmental Effects in Animals
Data concerning the developmental effects of trihalomethanes indicate
that these chemicals are toxic to the mother and fetus at high doses and
suggest that reproductive and developmental toxicity may occur as well. Signs
of maternal toxicity (decreased body weight and changes in organ weight) were
reported in rats, rabbits and/or mice administered 50 to 100 mg/kg/day
chloroform, 200 mg/kg/day bromodichloromethane, or 171 to 200 mg/kg/day
dibromochloromethane. Fetotoxicity, as indicated by decreased fetal body
weights, was evident in the offspring of rats administered 121 to
400 mg/kg/day chloroform or in mice administered 685 mg/kg/day dibromochloro-
methane. Oral exposure of pregnant animals to trihalomethanes has caused
variations in the offspring, mainly in the skeletal system. Delayed
ossification and sternebral aberrations have been reported in rats and/or
rabbits administered 20 to 200 mg/kg/day chloroform, 50 to 200 mg/kg/day
br.omodichloromethane, or 50 to 200 mg/kg/day bromoform. However, malforma-
tions and variations (cleft plate, imperforate anus, acaudia, delayed
ossification) have been observed in inhalation studies in which mice and/or
rats were exposed to 30 to 100 ppm chloroform.
Two studies were located which investigated the effects of trihalo-
methanes on reproduction. In one study, oral doses of 685 mg/kg/day of
dibromochloromethane administered to mice for two generations led to decreased
fertility and gestational indices. In the second study, bromoform adminis-
tered at 200 mg/kg/day had no effect on the fertility of mice.
1-6
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Mutagenicity and Genotoxicitv Studies
In vitro and in vivo studies on the mutagenic and genotoxic potential
of the trichloromethanes have yielded mixed results. Interpretation 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, and evidence of mutagenicity is considered
adequate for these chemicals. Studies on the mutagenicity of dibromochloro-
methane and chloroform were mixed, and the overall evidence for mutagenicity
of these two chemicals is judged to be inconclusive.
Carcinogenicity Studies in Animals
The carcinogenic potential of each of the four trihalomethanes has
been investigated in chronic oral exposure studies in animals. Ingestion of
chloroform in oil has been found to cause liver tumors in male and female
mice, but hepatic tumors were not detected in mice exposed to chloroform in
drinking water. Renal tumors were detected in male rats exposed to chloroform
in either oil or water, and renal tumors have been reported in male mice
exposed to chloroform in a toothpaste base.
Ingestion of bromodichloromethane in oil has been found to cause 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 in oil has been found to cause liver tumors in male and
1-7
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female mice, and ingestion of bromoform in oil has been found to cause tumors
in the large intestines of exposed male and female rats.
Health Effects in Humans
In a case study of a young man who ingested 4 ounces of chloroform (a
dose of about 2,500 mg/kg), prominent clinical findings included jaundice, an
enlarged liver, increased serum levels of bilirubin, alkaline phosphatase
(AP), and serum glutamic oxaloacetic transferase (SCOT) along with albumi-
nuria, glucosuria, ketonuria and the presence of red cells and granular casts
in the urine. These observations indicated that in humans, as in animals, the
liver and kidneys are the organs most affected by chloroform ingestion.
In the past, bromoform was given orally as a sedative to children
suffering from whooping cough. Doses of 50 to 100 mg/kg/day usually produced
sedation without any apparent adverse effects. Some cases of severe toxicity
or death were reported, but these were generally attributed to accidental
overdoses. No data were located on human exposure to either bromodichloro-
methane or dibromochloromethane.
Workers exposed to chloroform by inhalation at levels of 112 to
1,158 mg/m3 for 1 or more years complained of nausea, lassitude, dry mouth,
flatulence, thirst, depression, irritability, and "scalding" micturition, but
clinical examination and tests of liver function (serum enzyme levels) failed
to detect any abnormalities. Inhalation exposure of workers to chloroform at
levels of about 10 to 1,000 mg/m3 for 1 to 4 years was reported to be
associated with an increased incidence of viral hepatitis and enlarged liver.
1-8
-------
Some epidemiological studies suggest there may be an association
between water chlorination and increased cancer mortality rates. An associ-
ation has also been reported between exposure to water chloroform levels of at
least 10 /ig/L and intrauterine growth retardation. However, since chlorinated
water contains many by-products, it cannot be directly concluded from such
studies that trihalomethanes are human carcinogens or developmental toxicants.
No data were located regarding whether any human subpopulation may be
at greater risk to trihalomethane exposure than the general population. Data
from studies on mice suggest males may be more sensitive than females to
kidney effects, that alcohol consumption may increase toxicity in the liver.
and that diabetics may be more sensitive to liver effects.
Mechanism of Toxicity
Three lines of evidence indicate that trihalomethane metabolism is
essential for toxicity: (1) the tissues that most actively metabolize the
trihalomethanes (liver, kidney) are also the chief target tissues;
(2) chemical treatments that increase or decrease metabolism also tend to
increase or decrease toxicity in parallel; and (3) species- and sex-related
differences in metabolism are paralleled by similar differences in toxicity.
The detailed biochemical mechanisms by which trihalomethane metabolism leads
to toxicity are not certain, but covalent binding of reactive metabolites to
cellular macromolecules is one likely mechanism. Such metabolites are
produced both by oxidative metabolism to dihalocarbonyls and reductive
metabolism to free radicals. Free radical production may also lead to cell
injury by inducing lipid peroxidation in cellular membranes.
1-9
-------
Formation of DNA adduces might also account for the genotoxic and
carcinogenic potential of the trihalomethanes. Alternatively, carcinogenesis
may be related, at least in part, to increased cell proliferation following
direct tissue injury. However, neither of these potential mechanisms have
been definitively linked to trihalomethane carcinogenesis.
Several chemicals, including various ketones,. dichloroacetic acid, and
carbon tetrachloride, potentiate the toxic effects of chloroform. The
raechanism(s) of the potentiation by ketones is not known but appears to
include a process other than induction of microsomal enzymes. The vehicle
(corn oil versus aqueous) used for oral dosing also affects toxicity, with
toxicity generally being more severe following administration in oil.
Quantification of Noncarcinogenic Effects
For chloroform, a One-day Health Advisory (HA) value of 4 mg/L was
calculated from an acute oral No-Observed-Adverse-Effect Level (NOAEL) value
of 35 mg/kg in mice. A NOAEL value of 35 mg/kg/day, identified in pregnant
rabbits dosed by gavage on days 6 to 15 of gestation, was used to calculate a
Ten-day HA value of 4 mg/L. No adequate data were located for calculating
Longer-term HA values for chloroform, so the DWEL (0.4 mg/L) may be taken as a
conservative Longer-term HA for adults, and the adjusted DWEL (0.1 mg/L) as a
conservative Longer-term HA for children. A Reference Dose (RfD) of
0.01 mg/kg/day and a Drinking Water Equivalent Level (DWEL) of 0.4 mg/L wer-e
derived based on a Lowest-Observed-Adverse-Effect Level (LOAEL) value of
15 mg/kg/day identified in a 7.5-year study in dogs.
I-10
-------
For bromodichloromethane, no suitable study was located for calcu-
lating a One-day HA value. A NOAEL of 62 mg/kg/day, identified in a 28-day
feeding study in rats, was used to calculate a Ten-day HA value of 7 mg/L. A
NOAEL of 50 mg/kg/day, identified in a 13-week gavage study in male mice, was
used to calculate Longer-term HA values of 4 and 13 mg/L for a 10-kg child and
a 70-kg adult, respectively. The calculations for the RfD of 0.02 mg/kg/day
and DWEL of 0.7 mg/L employed a LOAEL of 25 mg/kg/day, identified in a 102-
week gavage study in mice. The RfD is currently under review in light of
recent data.
For dibromochloromethane, no suitable study was located for the
calculation of a One-day HA value. The Ten-day HA value of 6 mg/L was
calculated using, a NOAEL of 56 mg/kg/day, identified in a 28-day feeding study
in rats. A NOAEL value of 30 mg/kg/day, identified in a 13-week gavage study
in rats, was used to calculate Longer-term HA values of 2 and 8 mg/L for a
lOrkg child and a 70-kg adult, respectively. The NOAEL value of 30 mg/kg/day
identified in the 13-week gavage study in mice was also used to calculate an
RfD of 0.02 mg/kg/day and a DWEL of 0.7 mg/L.
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 NOAEL of
25 mg/kg/day, identified in a 13-week study in rats, was used to calculate a
value of 2 mg/L for both the Ten-day HA and the Longer-term HA for the
10-kg child. This value was also used to calculate a value .of 6 mg/L for the
Longer-term HA for the 70-kg adult. The NOAEL value of 25 mg/kg/day was also
used to calculate an RfD of 0.02 mg/kg/day and a DWEL of 0.7 mg/L.
1-11
-------
Quantification of Carcinogenic Effaces
Chloroform has been reported tc be carcinogenic in several different
chronic animal studies, increasing the frequency of liver tumors in male and
female mice and kidney tumors in male rats. Because the formation of liver
tumors in mice appears to be dependent upon the use of an oil vehicle, the
U.S. EPA has calculated the cancer risk estimate for chloroform based on the
incidence of renal tumors in male rats exposed to chloroform in drinking
water. The resulting unit risk value is 1.7xlO~7
Long-term oral studies in rats and mice performed by the National
Toxicology Program provide adequate data to derive quantitative cancer risk
estimates for the three brominated t rihalome thane s , although the chemicals
were administered in a corn oil vehicle. For bromodichloromethane , a unit
risk of l.SxlO"6 (/ig/L)"1 was derived, based on the incidence of renal tumors
in male mice. For dibromochloromethane , a unit risk of 2.4xlO"6 (^g/L)"1 was
derived, based on liver tumors in female mice. For bromoform, a unit risk of
2.3xlO"7 (/xg/L)"1 was derived, based on tumors of the large intestine in
female rats.
Table 1-1 summarizes the quantification of noncarcinogenic and
carcinogenic effects for trihaloraethanes .
1-12
-------
TABLE 1-1 Summary of Quantification of Toxicological
Effects for Trihalomethanes
Bromodi- Dibromo-
Value Chloroform chloromethane chloromethane Bromoform
One-day HA for 4 6a - 6a 5
10-kg child
(mg/L)
Ten-day HA for 4 6 6 2b
10-kg child
(mg/L)
Longer-term HA for 0.1 4 2 2
10-kg child
(mg/L)
Longer-term HA for 0.4 13 8 6
70-kg adult
(mg/L)
DUEL (70-kg adult) 0.4 0.7 0.7 0.7
(mg/L)
Concentration (jjg/L) 6 0.6 0.4 4
equivalent to 10"6
risk level
aData are insufficient or inappropriate for calculation of a One-day HA. The
value for the Ten-day HA is used as a conservative estimate.
bData are insufficient or inappropriate for calculation of a Ten-day HA. The
value for the Longer-term HA is used as a conservative estimate.
1-13
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II. PHYSICAL AND CHEMICAL PROPERTIES
A. Chloroform
Chloroform (CHC13) is a clear, colorless volatile liquid with a
nonirritating odor and a sweet taste (Hardie 1964; Windholz 1976). Some
important physical and chemical properties of chloroform are summarized in
Table II-l.
Chloroform was used as an anesthetic as early as 1847 but is no longer
employed for this purpose. It is manufactured and used as a solvent and as an
intermediate in the production of refrigerants, plastics and other solvents
(U.S. EPA 1980a).. Approximately 120,000 tons of chloroform were produced in
1977 (U.S. EPA 1977).
Because of its volatility, chloroform has the potential for
evaporation from water or other sources. Chloroform is stable in water, but
light, aeration or the presence of metals such as iron promote degradation
(Hardie 1964).
B. Brominated Trihalomethanes
Bromodichloromethane (CHBrCl2) , dibromochloromethane (CHBr2Cl) and
bromoform (CHBr3) are clear liquids with higher densities than 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-l.
II-l
-------
Table II-1 Physical and Chemical Properties of Chloroform and Brominated Trihalomethanes
Information
Property
Chemical Abstracts Registry No.
Registry of Toxic Effects of
Chemical Substances Number
Synonyms
Chemical formula
Molecular weight
Boiling point
Melting point
Specific gravity (20°C)
Vapor pressure
Stability in water
Water solubility
Chloroform
67-66-3
FS9100000
trichloro-
me thane
CHC13
119.39
61-62°C
-63.5°C
1.485
160 mm (20°C)
200 mm (25°C)
245 mm (30°C)
volatile
8,000 mg/L
(20°C) .
Bromodichloro-
ine thane
75-27-4
PA 5310000
dichlorobromo-
me thane
CHBrCl2
163.83
90°C
-57.1°C
1.980
50 mm (20°C)
volatile
3,032 mg/L.
(30°C)
Dibromochloro-
me thane
124-48-1
PA 6360000
i
chlorodibromo-
me thane
CHBr2Cl
208.29
116°C
--
2.38
15 mm (10°C)
volatile
1,050 mg/L
(30°C)
Bromoform
75-25-2
PB 5600000
tribromo-
me thane
CHBrj
252.77
149-150"C
6-7°C
2.887
5.6 mm (25°C)
vol. at i le
3,190 mg/L
(30°C)
Adapted from Windholz 1976; Verschueren 1977; Hawley 1981; U.S. EPA 1980b; and U.S. EPA (1985a).
-------
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 1975a).
Brominated trihalomethanes, although less volatile than chloroform,
are considered to be volatile enough to evaporate from drinking water (Jolley
et al. 1978) .
C. Occurrence
Chloroform occurs in the drinking water mainly as a by-product of the
chlorination process. The hypochlorite ion (OC1~) reacts with naturally
occurring organic substances in water (e.g., humic and fulvic acids) to form a
variety of by-products, including chloroform (Jolley et al. 1978). A number
of national drinking water surveys performed in the United States between 1975
and 1981 revealed that chloroform was detectable in a majority of systems
using a surface water source. Average levels usually ranged from 20 to
90 Mg/L (U.S. EPA 1975a, 1975b; Brass et al. 1977; U.S. EPA 1985a). Chloro-
form was also detectable in systems using groundwater as a source, but usually
at lower levels (1 to 10 jig/L) . This is presumably because surface water
typically contains higher levels of organic precursors than groundwater, and
may also require more extensive chlorination than groundwater. Concentration
values have also been noted to vary as a function of season, being higher in
warm weather and lower in cold weather (Wallace et al. 1987, 1988).
Brominated trihalomethanes also occur in drinking water as by-products
of chlorination. Bromide (Br~), a common constituent of natural waters, is
II-3
-------
oxidized by hypochlorous acid to form hypobromous acid (HOBr) in the following
reaction:
Br' + HOC1 ' HOBr + Cl"
Hypobromous acid then reacts with organic precursors to yield bromine -
containing trihalome thanes such as bromoform, dibromochlorome thane and bromo-
dichlorome thane (in increasing order of formation rates) (Jolley et al.,
1978) . A number of national surveys of brominated trihalome thane levels in
drinking water systems have revealed average levels typically ranging from 1
to 17 Mg/L (Boland 1981; Brass 1981; Brass et al. 1981; Bull and Kopfler 1990;
Symons et al. 1975; U.S. EPA 1991; Westrick et al . 1983). As noted above for
chloroform, mean levels of bromodichlorome thane tend to be somewhat higher for
systems using surface water sources (6 to 17 jig/L) than those using ground-
water sources (1 to 8 jzg/L) . Mean levels of dibromochlorome thane and
bromoform were comparable in systems using waters from either source (2 to
12
Trihalome thanes may be produced not only by reaction of chlorine with
humic materials in water, but also by reaction of sodium hypochlorite (NaOCl)
with endogenous organic material in the gut. Mink et al. (1983) dosed adult
male Sprague-Dawley rats (three fasted and three nonfasted per dose group)
with distilled water or 56 mg of NaOCl (8 mg/mL, pH 7.9) by gavage. One hour
after dosing, the researchers sacrificed all rats and collected blood and
stomach samples. Chloroform was detected in the plasma of one nonfasted,
treated rat, and in the stomachs of. all treated rats. In a second experiment,
rats were given a single oral dose of 100 mg NaOCl (48 mg Cl) along with 32 mg
Br" (KBr) . This led to the detection of all three brominated trihalome thanes
II-4
-------
as well as chloroform in the stomach contents of nonfasted rats (Mink et al.
1983). Bromoform and dibromochloromethane were also detected in the plasma.
D. Summary
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 chlorination of drinking water.
The principal trihalomethanes that have been observed in water are chloroform,
bromoform, dibromochloromethane and bromodichloromethane. Chloroform is
formed by the action of hypochlorous acid on endogenous organic molecules
(e.g., humic or fulvic acids) present in the water. Hypochlorous acid also
oxidizes any bro.mide ion present to form hypobromous acid, which reacts with
the organic material to form the brominated trihalomethanes. Chloroform has
been detected in finished drinking water samples at concentrations typically
ranging from 20 to 90 /xg/L, while average values of brominated trihalomethanes
typically range from about 1 to 20 jig/L. Chloroform and bromodichloromethane
levels tend to be higher in systems using surface water sources than those
using groundwater sources, whereas dibromochloromethane and bromoform levels
are comparable in systems using either source.
II-5
-------
III. TOXICOKINETICS
A. Absorption
Fry et al. (1972) administered chloroform orally to male and female
human subjects (18 to 50 years of age, 58 to 80 kg). In eight subjects who
ingested 500 mg of 'C-chloroform in olive oil in gelatin capsules, pulmonary
excretion of unchanged chloroform at 8 hours ranged from 18% to 67% of the
administered dose (mean value = 40.3%). In a separate experiment with two
subjects administered a gelatin capsule containing 500 mg of 13C-chloroform in
olive oil, 49% and 51% of the total dose was expired as 13C-carbon dioxide. •
Taken together, the data on carbon dioxide and chloroform excretion suggest
that at least 9Q% of an oral dose of chloroform is absorbed from the human
gastrointestinal tract.
Jo et al. (1990b) investigated chloroform absorption from inhalation
and dermal exposure during showers. Chloroform breath levels were measured in
one female and five male volunteers pre- and post-exposure in a normal shower
(using a defined set of parameters) and following inhalation-only shower
exposure. Breath levels measured 5 minutes after completion of the shower
correlated with tap water levels in both the normal shower and inhalation-only
shower protocols. The inhalation-only exposure resulted in breath levels that
were about half those following a normal shower. These data indicate that
humans absorb chloroform by both the dermal and inhalation routes when
showering.
Brown et al. (1974a) and Taylor et al. (1974) observed that UC-
chloroform (60 mg/kg) administered orally in olive oil to mice, rats, and
III-l
-------
monkeys was 93% to 98% absorbed, as measured by the recovery of radioactivity
in exhaled air, urine, and the carcass. Absorption was rapid, with peak blood
levels (3 to 10 ^g chloroform equivalent/mL) reached ac about 1 hour in both
mice and monkeys.
Mink et al. (1986) compared the absorption of chloroform, bromo-
dichloromethane, dibromochloromethane, and bromoform in male rats and male
mice. The rats and mice_were given single oral doses of 14C-labeled compound
in corn oil by gavage at dose levels of 100 rag/kg (rats) or 150 mg/kg (mice).
Total recovery of label in breath, urine, or tissues after 8 hours ranged from
62% to 94% (Table III-l), indicating that gastrointestinal absorption was high
for all four compounds. However, this study has been criticized since the
level of labeled, carbon monoxide in exhaled air was not determined.
Mathews et al. (1990) administered 14C-bromodichloromethane by gavage
in corn oil to male F344 rats at doses of 1, 10, 32, or 100 mg/kg, and
monitored the radiolabel in exhaled air, urine, f eces, and tissues..
Absorption was extensive--at least 86% of the dose was recovered in expired
volatiles, C02, or CO. Only small amounts were recovered in urine (<5%) or in
feces (<3%) within 24 hours of dosing in all dosing groups (Table III-2).
Withey et al. (1983) compared the rate and extent of gastrointestinal
absorption of chloroform given orally in either aqueous or corn oil vehicles.
Twelve male Wistar rats (approximately 400 g, six rats per vehicle) were given
single oral doses of 75 mg/kg 'by gastric intubation. The peak chloroform
concentration in blood was reached at approximately the same time for each
vehicle (5.6 and 6.0 minutes for water and corn oil, respectively). However,
the concentration levels differed by a factor of approximately 6.7 (39.3
III-2
-------
TABLE III-l Recovery of Label 8 Hours after Oral Administration
of 14C-Labeled Trihalomethanes to Male Rats or Male Mice
Chloroform
Percent of Label Recovered
Chemical
Expired Expired
Species C02 Parent
Urine
Total
Organs
Recovery
Rat
Mouse
6.5
49.5
64.8
26.1
2.6
4.9
3.6
13.5
78.2
94.5
Bromodichloromethane Rat • 14.2 41.7 1.4 3.3 62.7
Mouse 81.2 7.2 2.2 3.2 92.7
Dibromochloromethane
Rat
Mouse
18.2
71.6
48.1
12.3
1.1
1.9
1.4
5.0
70.3
91.6
Bromoform
Rat
Mouse
4.3
39.7
66.9
5.7
2.2
4.6
2.1
12.2
78.9
62.2
Adapted from Mink et al. (1986).
III-3
-------
TABLE III-2 Cumulative Excretion of Label after Oral Administration
of 14C-Labeled Bromodichloromethane to Male Rats
Percent of Dose
Dose
1 mg/kg
10 mg/kg
100 mg/kg
Time to
Sampling
1 hr
4 hr
8 hr
16 hr
24 hr
1 hr
4 hr
8 hr
16 hr
24 hr
1 hr
2 hr
6 hr
8 hr
24 hr
Expired
9
37
62
76
77
8
39
66
81
82
1
5
co2
.5+1.1
.0+3.2
.9±2.2
. 4^-3 . 2
.5±3.3
.0+2.0
.9+3.2
. 0+4 . 0
.3+1.7
.1±1.8
.9+0.9
.5±1.8
NR
33
71
.4+7.4
.0+1.7
Expired
CO
NRb
1.5+0
2.7+1
NR
3.3+1
NR
1.9+0
3.4+0
NR "
4.3+1
0.1+0
0.3+0
NR
2.3+0
5.2+0
Expired
Volatiles
.7
.1
.5
.4
.9
.0
.1
.7
.3
2.1+1.
2.7±1.
NR
NR
3.0+1.
2.0+0.
2.7+1.
NR
NR
2.8+1.
1.5+1.
4.2±1.
NR
5.7+2.
6.3+2.
5
8
6
8
1
1
2
9
1
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
41
68
81
90
10
44
72
87
94
4.
10
10
42
87
.6+1.3
.1+2.8
.+1.7
.8+2.9
.7+1.8
.0+1.5
.5+3.0
.1+3.9
.4+1.5
.2+1.6
6+1.8
.6+2.9
.6+3.0
.0+8.3
.3+1.6
aResults from 4 rats/dose.
''Not reported
Adapted from Mathews et al.
(1990).
III-4
-------
versus 5.9 /ig/mL for water and corn oil, respectively). At all times, the
concentration of chloroform in blood was lower when given in an oil vehicle
than when given in vatsr. The chloroform blood levels fell below the limit of
detection after approximately 200 minutes when given in oil and 240 minutes
when given in water. These data suggest that administration of chloroform in
corn oil might not be a good model for oral exposure in water. The authors
noted that the majority of toxicity studies using the oral route of dosing
have been carried out using oil-based vehicles. The study did not determine
whether the vehicle-related difference was due to differences in absorption or
in first-pass metabolism.
Staats et al. (1991) found that a two-compartment model of gastro-
intestinal absorption was better than a one-compartment absorption model at
predicting blood levels following oral administration of chloroform. Using
this model, they also found that blood levels of chloroform were higher
following gavage administration in water than in corn oil. Peak levels were
about sevenfold higher following administration in water.
Morgan et al. (1991) measured chloroform blood levels in male F344
rats during 24-hour dermal exposures of a 3.1 cm2 shaved region of the back to
2 mL of neat chloroform, or aqueous chloroform solutions that were saturated
(4,709 /ig/mL), two-thirds saturated (2,733 /xg/mL) , or one-third saturated
(1,180 /ig/mL). The chloroform blood level peaked at 51 ng/mL after exposure
to the neat chemical for 4 to 8 hours, and remained about constant for the .
duration of the exposure period. The plateau was attributed to the attainment
of an equilibrium between the toxicokinetics and the depletion of the chemical
from the exposure cell. Exposure to the aqueous solutions resulted in peak
chloroform blood levels after about 2 hours, followed by a rapid return to
III-5
-------
control levels by 24 hours of exposure. The decreasing blood levels were
attributed to depletion of chemical from the exposure cell. The study authors
stated that hydration of the skin could have increased the rate of absorption.
Bogen et al. (1992) investigated the dermal absorption of dilute
aqueous C-chloroform solutions by hairless guinea pigs. Animals were
exposed by immersion to concentrations of 10 to 100 ppb chloroform in water
for 70 minutes. Dermal uptake was estimated by comparing the rate of
radiolabel loss from water in systems with or without animals. Dermal uptake
was confirmed by measuring the radiolabel content of the urine and feces. A
pe.rme ability coefficient of 0.13 cm/hr was obtained for chloroform. The
authors concluded that dermal absorption of chloroform from water may be an
important route .of human exposure.
B. Distribution
McConnell et al. (1975) analyzed chloroform levels in postmortem
tissue from eight persons (four males and four females, 48 to 82 years old)
living in nonindustrial areas of the United Kingdom. Chloroform levels (jtg/kg
wet tissue weight) observed were: body fat, 5 to 68 (average of 51); liver, 1
to 10 (average of 7.2); kidney, 2 to 5; and brain, 2 to 4. The source of the
chloroform in these tissues was noc discussed.
Phillips and Birchard (1991) reported on a nationwide survey of the
general population by EPA's National Human Adipose Tissue Survey (NHATS).
Several hundred fat samples were pooled into 46 composite samples by age and
geographic region, and analyzed. Chloroform was measured at levels ranging
III-6
-------
from 5 co 580 ng/g in 29 of the composite samples, and the other three
trihalomethanes were not detected in any samples (Wallace 1992).
Pellizzari et al. (1982) measured trihalomethanes in 42 samples of
human milk taken from women in urban areas. Chloroform was detected in seven
samples, and dibromochloromethane was detected in one sample. Neither the
levels measured nor the detection limit were reported.
Roth (1904) measured the bromoform content of tissues of a man who
died from an accidental oral overdose of bromoform. Levels in stomach and
lung (rag bromoform/kg wet weight) were 130 and 90 mg/kg, respectively. Lower
levels were reported in the intestine, liver, kidney, and brain.
Mink et al. (1986) compared the distribution of chloroform, bromo-
dichloromethane, dibromochloromethane, and bromoform in male rats and male
mice. The rats and mice were given single oral doses of uC-labeled compound
in corn oil by gavage at dose levels of 100 mg/kg (rats) or 150 mg/kg (mice).
After 8 hours, the researchers measured the tissue levels of radioactivity.
Interpretation of these data is limited because the chemical form of the label
measured in the tissues (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 other tissues analyzed (bladder, brain, lung, muscle,
pancreas, and thymus). Similar levels of label (4% to 5% of total dose) were
recovered in the organs of mice. However, for bromoform and chloroform, an
additional 10% of the label was recovered in the blood of mice, yielding total
organ levels of 12% to 14%. The authors suspected that this was due to
carboxyhemoglobin formation, but this was not measured.
III-7
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Taylor et al. (1974.) administered an oral gavage dose of 60 mg/kg of
UC-chloroform in olive oil to mice and studied distribution by whole-body
autoradiography. The highest radioactivity levels were detected in the liver
and kidney. The radioactivity level in the kidneys of males was 3.5 times
greater than that in the kidneys of females. Castration of the males or
testosterone administration to female mice abolished this difference. This
observation may provide insight as to why male mice are more sensitive to the
nephrotoxic effects of chloroform than females (see Section V). Sex
differences in distribution were not noted by Brown et al. (1974a) in rats or
squirrel monkeys.
Mathews et al. (1990) investigated the distribution of bromodichloro-
methane following oral exposure in 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 (10 or 100 mg/kg/day for 10 days) showed no bioaccumulation
(0.9% to 1.1% total retention of label) and had no effect on the tissue
distribution of bromodichloromethane.
Withey and Karpinski (1985) exposed pregnant rats to 100 to 2,000 ppm
chloroform in air for 5 hours on day 17 of gestation. A good linear relation-
ship was observed between the exposure level and maternal and fetal blood
concentrations of chloroform. Fetal blood levels of chloroform were less than
maternal levels. This study indicates that chloroform can cross the placental
barrier and distribute to the fetus.
III-8
-------
C. Metabolism
The metabolism of trihalomethanes has been studied in a number
of laboratories, both in vitro and in vivo. Figure III-l presents a. metabolic
scheme based on these studies. In brief, metabolism of trihalomethanes occurs
via two pathways, one in the presence of oxygen (the oxidative pathway) and
the other in the absence of oxygen (the reductive pathway). Both pathways are
mediated by cytochrome P-450 and NADPH (or NADH). In the presence of oxygen,
the initial reaction product is trihalomethanol (CXjOH), which spontaneously
decomposes to yield the corresponding dihalocarbonyl (CXgO) . These dihalo-
carbonyl species (e.g., phosgene) are quite reactive and may undergo a variety
of reactions with cellular molecules. Under conditions when intracellular
oxygen levels are low, the trihalomethane is metabolized via the reductive
pathway, resulting in a highly reactive dihalomethyl radical ('CHXg), which
may also form covalent adducts with cellular molecules. Evidence supporting
this metabolic scheme and information on species differences in the rate and
extent of trihalomethane metabolism are presented below.
In Vitro Studies
The differences between oxidative and reductive pathways for
trihalomethane metabolism have been studied mainly in vitro, where oxygen
levels can be controlled.
Ilett et al. (1973) investigated the in vitro metabolic activation of
14C-chloroform using microsomal preparations from the livers and kidneys of
male mice. As shown in Table III-3, incorporation of label into microsome
protein was much more extensive in liver than in kidney. Uptake of label into
III-9
-------
RH
REDUCTIVE PATHWAY
•CHXr
OXIDATIVE PATHWAY
P-450
CHXa
P-450
f " ^\ v»n/vj f f ^\"^
X' NADPH 2 NADPH O2 H2O
or
NADH
>
V
HX
CX2Q
RCX2OH
2GSH
V
>
2HX
CO+GSSG
H2O
v
2HX
Cysteine
\
2HX
OTZ
X - halogen atom (chlorine, bromine); R » cellular nucleophile (protein, nucleic acid);
GSH - reduced glutathtone; GSSG - oxidized glulalhione; OTZ • oxolhiazolidine
carboxylic acid; P-450 - cytochrome P-450
(a) Adapted from Stevens and Anders (1981). Tomasi el al. (1985)
FIGURE I1I-1. Metabolic Pathways of Trihalomethane Biotransformntlon
-------
TABLE III-3 In Vitro Binding of 14C-Chloroform Metabolites
to Microsomes of Male Mice
Label Incorporation
(pmol/mg/5 min)
Assay Conditions Liver Kidney
Complete
-------
liver microsomes was almost entirely blocked by removal of the NADPH
generating system, supporting the concept that the reaction was mediated by
cytochrome P-450-dependent metabolism. This is further supported by the
finding that pretreatment of animals with phenobarbital (a known inducer of
cytochrome P-450) in three daily intraperitoneal injections of 80 mg/kg/day
led to a threefold increase in binding of label. Under anaerobic conditions
(produced either by flushing with nitrogen or by adding carbon monoxide to
block oxygen binding to cytochrome P-450), incorporation of label into
microsomal protein was reduced but was not eliminated. This observation is
consistent with the concept that metabolism may proceed by both reductive and
oxidative pathways. Removal of oxygen or NADPH affected binding of label to
renal microsomes less than it did binding to liver microsomes. Phenobarbital
pretreatment did. not increase binding to renal microsomes. This suggested to
the authors that a different metabolic pathway may play a significant role in
the kidney.
Evidence for cytochrome P-450-dependent oxidative metabolism of
chloroform to dichlorocarbonyl (phosgene) was reported by Pohl et al. (1977).
Cysteine was used to inhibit covalent binding of radiolabeled chloroform to
microsomal proteins by trapping a reactive intermediate as 2-oxothiazolidine-
4-carboxylic acid (OTZ). These authors suggested that unstable trichloro-
methanol forms initially via the cytochrome P-450 system, and hydrogen
chloride is eliminated spontaneously to yield the reactive phosgene, which
then binds with cysteine or protein thiol groups. Pohl and Krishna (1978) and
Pohl et al. (1979) reported that deuterium-labeled chloroform was less toxic
and less readily metabolized than unlabeled chloroform, suggesting that the
enzymic cleavage of the carbon-hydrogen bond might be the rate-limiting step
in the metabolism of chloroform to a hepatotoxic product.
111-12
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Ahmed et al. (1977) investigated the in vitro oxidative (aerobic)
metabolism of 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, bromodichloromethane,
and chloroform, 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 later 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 can react with
cysteine to form OTZ; therefore, detection of OTZ provides evidence that a
dihalocarbonyl intermediate was formed during bromoform metabolism.
Smith and Hook (1984) reported that microsomal preparations from the
kidneys of male mice metabolized chloroform to carbon dioxide and reactive
intermediates, while microsomal preparations from the kidneys of female mice
showed little or no activity for chloroform metabolism. This enzymic
difference in metabolism between sexes is in agreement with the greater
nephrotoxicity of chloroform in male mice than in female mice (Smith et al.
1983). The metabolism of chloroform by male kidney preparations required
oxygen and an NADPH regenerating system for maximal activity. Metabolism was
shown to depend on the concentration of microsomal protein, the concentration
of chloroform, and the time of incubation. Addition of carbon monoxide (an
inhibitor of cytochrome P-450) resulted in decreased chloroform metabolism.
The metabolism of chloroform to carbon dioxide obeyed Michaelis-Menten
111-13
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kinetics, and the authors reported values of 2.78 ^M for Km and a Vmax of
0.391 fjmol/mg microsomal protein/min.
Uehleke and Werner (1975) investigated the extent of irreversible
label binding to proteins and lipids resulting from the in vitro'oxidative
(aerobic) metabolism of C-chloroform in hepatic microsomes prepared from
rabbits, rats, mice, and humans. The level of irreversible reactive inter-
mediate binding to protein was highest for the rabbit and human liver
preparations. The mouse and rat had considerably lower capacities. Similar
results were reported for the extent of label bound to lipids for the animal
liver preparations, although data were not presented on label binding to
lipids from human microsomes. These results indicate that there are consider-
able species differences in the production of reactive intermediates from
chloroform. Binding under anaerobic conditions to protein and lipids of
hepatic microsomes prepared from phenobarbital-pretreated rabbits was about
50% that observed in the presence of oxygen, suggesting that the reductive
metabolism of chloroform can also generate reactive intermediates.
Corley et al. (1990) compared the in vitro oxidative metabolism of
14C-chloroform in hamsters, mice, rats, and humans. Microsomal preparations
from the liver and kidney of these species were incubated with 0.049 to
0.058 mM chloroform, and the generation of carbon dioxide was measured. In
the liver, the relative levels of metabolic activity (nmol oxidized/min/mg
protein) were as follows: hamster > mouse > rat > human. .In the kidney the
activities were lower, and the following order was observed: mouse > hamster
> rat > human. The authors used these data to develop a pharmacokinetic model
for describing the disposition of chloroform in mice, rats, and humans.
-------
Reitz et al. (1990) used the pharmacokinetic model of Corley et al.
(1990) to develop a model for the cytotoxic effects of chloroform. The model
for oral, exposure was verified by administering com oil gavage doses of 100,
200, or 500 mg/kg to male B6C3F1 mice. Cytotoxicity was determined by
directly measuring hepatocyte death histologically. For inhalation exposure,
the model was verified by exposing male B6C3F1 mice to 10, 30, or 100 ppm
chloroform in air for 5 to 6 hours and measuring stimulation of compensatory
cell regeneration as an index of cell killing. The model was then used to
compare predicted macromolecular binding and cytotoxicity for various long-
term chloroform bioassays with the observed liver tumor incidence. The
bioassays used were those of NCI (1976) (oil gavage), Jorgenson et al. (1985)
(drinking water), and Roe et al. (1979) (gavage in toothpaste). The model
predicted that tumor incidence was correlated with the instantaneous rate of
chloroform metabolism and with cytotoxicity, but not with the total amount of
chloroform metabolized in the liver. Thus the predicted peak rate of
chloroform metabolism following bolus gavage dosing was much higher than the
rate predicted for continuous exposure in drinking water, and liver tumors
were observed only under the former dosing protocol. Conolly and Andersen
(1991) extended these results to predict that, corn oil gavage administration
of chloroform results in a much higher instantaneous rate of chloroform
metabolism in male B6C3F1 mice (in which liver tumors are observed--see
Chapter 5) than in nonsensitive male Osborne-Mendel rats.
Testai and Vittozzi (1986) studied the in vitro metabolism of 14C- .
chloroform by rat liver microsomal preparations under aerobic and anaerobic
conditions. The production of a reactive intermediate was quantitated by
measuring the binding of radiolabel to microsomal proteins and lipids. Under
aerobic conditions, pretreatment with phenobarbital produced a two- to three-
111-15
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fold increase in binding, whereas p-naphchoflavone pretreatment had little
effect. While binding to microsomes prepared from untreated rats was
relatively low under anaerobic, conditions, pretreatment with p-naphthoflavor.e
or phenobarbital resulted in markedly increased binding (approximately 9- to
15-fold and 50- to 90-fold, respectively). These results indicate that
cytochrome P-450 also plays a role in the anaerobic metabolism of chloroform.
Under both aerobic and anaerobic conditions, the addition of glutathione or
cysteine reduced binding to both proteins and lipids, although glutathione
decreased binding to proteins more than it decreased binding to lipids. The
aerobic metabolism of chloroform concentrations of 1 to 15 mM decreased the
cytochrome P-450 activity by about 5% to 30%, but glutathione or cysteine
reduced this effect. The anaerobic metabolism of chloroform produced a loss
of cytochrome P-450 activity somewhat greater than that observed under aerobic
conditions, and the reduced activity was not affected by the addition of
glutathione or cysteine. This study indicates that chloroform is metabolized
via a cytochrome P-450-mediated process to a reactive intermediate that can be
scavenged by glutathione or cysteine. Since binding to uninduced microsomes
under anaerobic conditions was negligible, the study authors were able to rule
out significant residual oxygen contamination. Therefore, they concluded that
most of the covalent binding to induced microsomes under anaerobic conditions
was produced by reductive metabolism of chloroform.
Testai et al. (1987) investigated the in vitro anaerobic metabolism of
chloroform by liver microsomes from untreated and phenobarbital or p-naphtho-
flavone-pretreated B6C3F1 mice. The authors reported that, unlike untreated
rats, untreated mice had a significant capacity to produce covalent binding of
14C-labelled intermediates under anaerobic conditions. Both NADPH and NADH
could provide the reducing equivalents needed for the metabolic .-activation of
111-16
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chloroform. This activity was not significantly increased by pretreatment
with either phenobarbital or p-naphthoflavone. These differences in baseline
metabolic activity and enzyme inducibility may underlie some of the toxicclo-
gical differences observed between rats and mice (see Chapter 5). As with rat
microsomes, the loss of cytochrome P-450 in mouse microsomes exposed to
chloroform was greater under anaerobic conditions than under aerobic
conditions.
Species differences were also observed by Vittozzi et al. (1991).
Radiolabeled chloroform was found to bind under hypoxic conditions to the
lipids of liver microsomes prepared from B6C3F1 mice but essentially not to
microsomes prepared from Sprague-Dawley rats. Binding under aerobic condi-
tions was also lower in rat microsomes than in mouse microsomes. Minimal
binding was seen with human liver microsomes under aerobic conditions, while
binding was observed in 2/4 samples of microsomes prepared from human colon
mucosa biopsies, but only under reducing conditions.
Wolf et al. (1977) studied the in vitro metabolism of chloroform and
bromoform to carbon monoxide under anaerobic conditions using liver prepara-
tions from phenobarbital-induced rats. Sromoform metabolism resulted in much
greater levels of carbon monoxide production than did the metabolism of
chloroform. Similarly, 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 bromo-
dichloromethane as for chloroform. These data suggest that reductive
metabolism may be more important for brominated trihalomethanes than
chloroform.
111-17
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Tomasi et al. (1985) studied the anaerobic activation of chloroform.
bromodichloromethane, and bromoform to free radical intermediates .in vitro
using rat hepatocytes isolated from phenobarbital-induced male rats. The
production of a free radical intermediate was measured by electron spin
resonance (ESR) spectroscopy using the spin trap compound phenyl-t-butyl-
nitrone. The intensity of the ESR signal was greatest for bromoform, followed
by bromodichloromethane and. then chloroform. The largest ESR signal was
detected when hepatocytes.were incubated under anaerobic conditions.
Incubation in the presence of air resulted in a reduction of the signal, as
did addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone, and
carbon monoxide. These data were interpreted to indicate that free-radical
formation depended on reductive metabolism of the trihalomethanes mediated by
the cytochrome P.-450 system. Comparison of the ESR spectra for chloroform,
deuterated chloroform, and bromodichloromethane indicated that the free
radical intermediate produced by chloroform metabolism was -CHClj. The
authors speculated that the other trihalomethanes are also metabolized by
transfer of an electron directly from the cytochrome to the halocompound with
the successive formation of the dihalomethyl radical ('CHXj) and a halide ion
(x-).
Testai et al. (1990) investigated the chloroform and oxygen dependence
of covalent binding of label from UC-chloroform into mouse liver microsomal
proteins and lipids. Label was incorporated into protein and lipid in both
the presence and absence of oxygen, confirming the finding described above
that both oxidative and reductive pathways were involved. However, protein
binding in the presence of oxygen appeared to occur by two processes. The
first process was saturated at a low chloroform concentration (0.1 mM) and was
strongly inhibited by the reduction of the oxygen level from 20% (air) to 1%.
Ill-'18
-------
The second process occurred at higher chloroform concentrations (0.1 to 5 mM)
and was inhibited only by complete anoxia. The authors interpreted these
observations as indicating that oxidative incorporation of label occurred by
two pathways: the first by an enzyme system with high affinity for chloroform
and low affinity for oxygen, and the second by an enzyme system with low
affinity for chloroform but high affinity for oxygen. They did not speculate
on the enzymatic or biochemical mechanisms underlying these findings. Both
oxidative processes produced reactive metabolites that bound mainly to
proteins, in a process that was strongly inhibited by reduced glutathione.
Metabolites formed by the reductive pathway mainly bound to lipid, and their
production was not blocked by reduced glutathione. The authors concluded that
phosgene is the chief reactive intermediate of both of the oxidative pathways,
while free radicals are the chief reactive product of the reductive pathway.
They noted that hypoxic conditions are generally present in the liver, with
oxygen levels of about 1% to 5%, and that reduced glutathione is usually
present. These conditions could favor the formation of adducts via the
reductive pathway.
Testai et al. (1991) measured the in vitro binding of radiolabeled
chloroform to microsomes prepared from human colon or ileum mucosa recovered
from surgical biopsies. Covalent binding to lipid was observed with.about
half the colon and ileum specimens, but only under anoxic conditions. No
binding to protein was observed in the presence or absence of oxygen. By
contrast, colonic microsomes from rats did not metabolize chloroform in the
presence or absence of oxygen to a form that reacted with lipid or protein.
The study authors noted that low oxygen tension is present in the outer layers
of the colonic mucosa, and suggested that their observations support the
111-19
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claims in some epidemiological studies of an association between chloroform
exposure and colonic cancer (see Chapter 6).
In Vivo Studies
In vivo studies on trihalomethane metabolism demonstrate that both the
oxidative production of carbon dioxide and the reductive production of free
radicals occur in animals. The production of carbon monoxide has been
reported and can result from either pathway. In addition, considerable
species differences exist in the extent of trihalomethane metabolism to carbon
dioxide.
Fry et a1. (1972) reported that the extent of oxidative chloroform
metabolism, to carbon dioxide in humans was dependent on the oral dose
administered. The subjects were healthy male and female volunteers, 18 to
50 years of age (a total of 11 subjects), with body weights ranging from 60 to
80 kg. Chloroform was administered in gelatin capsules in an olive oil
vehicle. The dose levels were 0.5 g (7.5 mg/kg) for eight subjects and 0.1 g
(1.5 mg/kg), 0.25 g (3.8 mg/kg), and 1.0 g (15.4 mg/kg) for one subject each.
Based on repeated gas chromatographic analyses of exhaled breath over 8 hours,
the authors estimated that expired chloroform accounted for 0%, 12%, 40% and
65% of the dose in the 1.5-, 3.8-, 7.5- and 15.4-mg/kg dose groups, respec-
tively. Based on the authors' observation that nearly all of the administered
chloroform could be accounted for in the expired air as either chloroform or
carbon dioxide, it was estimated that approximately 100%, 88%, 60% and 35% of
the dose was metabolized to carbon dioxide, respectively. These data indicate
that metabolism of chloroform to carbon dioxide occurs by a saturable
(presumably enzymic) system in humans.
111-20
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Mink et al. (1986) compared the metabolic products of chloroform,
bromodichloromethane, dibromochloromethane, and bromoform in male rats and
male mice (strain not reported). Animals were given a single oral dose of
KC-labeled compound in corn oil by gavage at dose levels of 100 mg/kg for
rats and 150 mg/kg for mice. In rats, expired carbon dioxide accounted for
4.5% to 18.2% of the label (Table III-l), indicating 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 trihalomethanes to carbon dioxide
was more rapid and extensive (by a factor of four- to ninefold) in mice than
in-rats.
Mathews et al. (1990) studied the metabolism of 14C-bromodichloro-
methane in male Fischer rats. Animals 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
UC02 and 3% to 5% of the dose as 14CO. However, KC02 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 '^COj
production, compared with the initial rate. The authors concluded that
bromodichloromethane may induce its own metabolism.
Anders et al. (1978) investigated the in vivo formation of carbon
monoxide from trihalomethanes administered to rats by intraperitoneal
injection in corn oil. At a dose of 1 mmol/kg (119 to 252 mg/kg), bromoform
administration produced the highest levels of blood carbon monoxide, followed
111-21
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by dibromochloromechane, chloroform, and bromodichloromechane in decreasing
order. A dose-response relationship was noted for bromoform. Carbon monoxide
production was inducible by phenobarbital pretreacment but not by 3-methyi-
cholanthrene pretreatment, and was significantly inhibited by SKF-525-A.
Administration of 3H-bromoform resulted in decreased carbon monoxide
formation, 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 (19.81). In addition, Stevens and Anders (1981) reported
that the co-administration of chloroform and cysteine (which reacts with
dihalocarbonyls) to rats resulted in decreased carbon monoxide formation.
These data suggest that trihalomethanes are metabolized oxidatively to carbon
monoxide via a dihalocarbonyl intermediate, such as in the mechanism proposed
on the basis of j.n vitro data.
Ilett et al. (1973) investigated the covalent binding of metabolites
to proteins in the liver and. kidney in mice after in vivo exposure to
chloroform by intraperitoneal injection. The animals were pretreated with
phenobarbital and then given 300 to 740 mg/kg 14C-chloroform in sesame oil.
The authors reported centrilobular hepatic necrosis in both sexes and renal
necrosis in male mice. Autoradiographic analysis of tissue slices indicated
that the amount of covalent binding of radiolabel paralleled the extent of
renal and hepatic necrosis. These data suggest a possible causal relationship
between tissue necrosis and covalent binding.
Tomasi et al. (1985) studied the in vivo metabolism of chloroform,
bromodichloromethane, and bromoform.to free radical intermediates in rats.
Phenobarbital-induced rats were given intraperitoneal injections of
1,100 mg/kg chloroform, 820 mg/kg bromodichloromethane, or 1,260 mg/kg
111-22
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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. . Free radicals were detected 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 > bromodichloro-
methane > chloroform), confirming that the reductive formation of free
radicals is greater for brominated trihalomethanes than for chloroform.
D. Excretion
Fry et al. (1972) administered a single oral dose of chloroform
(500 mg) in gelatin capsules to male and female human subjects (18 to 50 years
of age, 60 to 80. kg). Pulmonary excretion of unchanged chloroform at 8 hours
ranged from 18% to 67% in eight subjects (mean value = 40.3%). Two subjects
who were administered a gelatin capsule containing 500 mg 13C-chloroform in
olive oil excreted 48.5% and 50.6% of 13C-carbon dioxide in 7.5 hours. Taken
together, these data suggest that nearly all of an oral dose is excreted via
the lungs, either as chloroform or as carbon dioxide. A linear relationship
was noted between the rate of pulmonary excretion of chloroform and the
chloroform concentration in the blood (levels of up to 5 jig/mL within 1 hour
after exposure). Urinary levels of chloroform were below 0.01% (the level of
detection). Fecal chloroform content was not measured.
Mink et al. (1986) compared the excretion of chloroform, bromodichloro-
methane, dibromochloromethane, and bromoform in male rats and male 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, respec-
tively. The lung was the principal route of excretion in both species,
111-23
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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 form of the label, was not investigated.
Mathews et al. (1990) exposed 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 bromo-
dichloromethane 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 UC-carbon monoxide. In general, less than 5% of the dose was
excreted in the urine or feces.
Van Dyke et al. (1964) administered 0.1 mL of 14C-chloroform
(1.06xl08 dpm/mL_) by intraperitoneal injection to 30 rats (200 g, strain and
sex not specified). This corresponded to a dose of about 740 mg/kg. After
12 hours, a total of 0% to 2% of the injected 14C-label was recovered as
urinary metabolites and 4% to 5% was recovered as expired COj. The
disposition of the other 93% to 96% of the dose was not determined. Adminis-
tration of a comparable dose of 36C1-chloroform (specific activity not
reported) resulted in excretion of 36C1-urinary metabolites, but the
percentage of the dose excreted in the urine was not reported. Of the total
urinary label, 73% appeared as 36C1", with 27% as organic forms.
E. Bioaccumulation and Retention
No data were located regarding the bioaccumulation or retention of
chloroform or brominated trihalomethanes following repeated exposures.
However, based on the rapid excretion and metabolism of chloroform and the
brominated trihalomethanes, along with the low levels of chloroform detected
111-24
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in human post-mortem tissue samples, marked accumulation and retention of
these compounds are not anticipated.
F. Summary
Measurements of gastrointestinal absorption of trihalomethanes in
mice, rats, and monkeys indicate that absorption is rapid (peak blood levels
at 1 hour) and extensive.-(64% to 98%). Limited data indicate that gastro-
intestinal absorption of chloroform (and presumably other trihalomethanes) is
also rapid and extensive (at least 90%) in humans. Most studies of trihalo-
methane absorption have used oil-based vehicles. One study in rats found
higher chloroform blood levels following oral gavage administration of
chloroform in wa.ter than after administration of chloroform in an oil vehicle.
This was interpreted as being due to higher absorption from water than from
oil, but the possible influence of first-pass metabolism was not taken into
account. Dermal absorption of chloroform in water by rats and hairless guinea
pigs is rapid and extensive. Dermal absorption by humans of chloroform in
water has also been demonstrated.
Absorbed trihalomethanes appear to distribute widely throughout the
body. Chloroform was detected in a number of postmortem tissues from, humans,
with the highest levels (5 to 68 jig/kg) in body fat and lower levels (1 to
10 jig/kg) in kidney, liver, and brain. Radiolabeled trihalomethanes were
detected in a variety of tissues following oral dosing in rats and mice, with
somewhat higher levels in stomach, liver, blood, and kidney than in lung,
muscle, or brain. Chloroform crosses the placenta and may be detected in
fetal tissues following inhalation exposure of pregnant rats.
111-25
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Trihalomethanes are extensively metabolized by both humans and
animals. The main site of metabolism is the liver, but metabolism also occurs
in the kidney. Beth oxidative metabolism and reductive metabolism of trihalo-
methanes are mediated by cytochrome P-450. The oxidative pathway requires
NADPH and oxygen, whereas the reductive pathway can utilize NADPH or NADH and
is inhibited by oxygen. In the presence of oxygen (oxidative metabolism), the
reaction product is trihalomethanol (CXjOH), which decomposes to yield a
reactive dihalocarbonyl £.CX20) such as phosgene (CC120). Dihalocarbonyls are
relatively reactive species, and may undergo a variety of reactions, such as
the formation of adducts with various cellular nucleophiles, hydrolysis to
yield carbon dioxide, or glutathione-dependent reduction to yield carbon
monoxide. If oxygen is lacking (reductive metabolism), the metabolic reaction
products appear .to be free radical species such as the dihalomethyl radical
(CHXj')- These radicals are extremely reactive and may also form covalent
adducts with a variety of cellular molecules. Metabolism via the reductive
pathway appears to occur more readily for brominated trihalomethanes than for
chloroform.
Both in vivo and in vitro studies indicate that the pattern of
trihalomethane metabolism may differ between animal species and sexes. In
vivo, a single study reported that mice metabolize trihalomethanes to carbon
dioxide more extensively than do racs (40% to 80% versus 4% to 18%). However,
data from another study indicate that another strain of rat was capable of
oxidizing bromodichloromethane to carbon dioxide to an extent that was
comparable to that reported for mice. I_n vitro, the capacity for reductive
metabolism of trihalomethanes has been found to be greater in hepatic
microsomes from mice than rats, and the incorporation of label into covalent
adducts in renal microsomes has been found to be greater in male mice than
111-26
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female mice. These metabolic differences may underlie some of the important;
toxicological differences that have been noted between sexes and species.
Reductive metabolism of chloroform to reactive intermediates has also been
observed with microsomes prepared from human ileum and colon samples.
Excretion of trihalomethanes occurs primarily via the lungs. In
humans, approximately 90% of an oral dose of radiolabeled chloroform was
exhaled as the end metabolite, carbon dioxide, or the parent compound,
chloroform. Levels in the urine were below the limit of detection (0.1%).
In mice and rats, 45% to 88% of an oral dose of chloroform or brominated
trihalomethane was excreted from the lungs either as parent trihalomethane or
as carbon dioxide, with 1% to 5% excreted in the urine. Intraperitoneal
injection of rats with 36C1-chloroform resulted in variable levels of labeled
chloride in the urine. The total amount of label excreted in the urine was
not reported, but 73% of the urinary label appeared as ionic chloride (36C1~)
and 27% was present in organic compounds.
No data were located regarding the bioaccumulation and retention of
the trihalomethanes following chronic exposure. However, based on the rapid
metabolism and excretion of chloroform and the brominated trihalomethanes,
along with the low levels of chloroform in human autopsy samples, marked
accumulation and retention of these compounds are not anticipated.
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IV. HUMAN EXPOSURE
A. Drinking Water Exposure
SamplinE Methodology
Several national surveys have measured THMs in drinking water at the
treatment plant or in the distribution system. ' These surveys had different
purposes; therefore, the sampling methods and location of sample collection
varied. Three different ^sampling methods were used in the national surveys,
and each method resulted in different outcomes. The three methods are as
follows: (1) if the actual concentration of THMs at the time of sample
collection is needed, then sodium thiosulfate, a reducing agent, is added to
the sample to prevent or "quench" further THM formation; (2) if the maximum
level of THM concentration in water is required, then the quenching process is
not employed, and THM formation is allowed to'continue unrestricted after
collection; and (3) if the predicted level of THMs in the water at the tap
(after spending time in the distribution system) is needed then the samples
are refrigerated to slow the THM formation until analysis is performed.
Comparison of the results of each survey should take into consideration the
sampling method employed (Wallace 1992). Table IV-1 summarizes the results of
the national THM studies. Figure 1 graphically displays how different sampling
techniques affect mean THM concentrations in the national surveys.
1. Total Trihalomethanes
The American Water Works Association Research Foundation Study
(AWWARF) conducted a nationwide survey of 727 utilities, representing more
than 105 million customers, that collected total trihalomethane data between
1984 and 1986. Concentration data for the specific trihalomethane components
IV-1
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TABLE IV-1 Comparison of Results of National Surveys
Survey
NORS*
NOMS -
NOMS -
NOMS -
NOMS -
NOMS -
AWWARFC
Phase 1«
Phase 2C
Phase 3Qd
Phase 3TC
All phases
Number of
Cities
80
111
113
106
105
105/113
727
THM
Mean
68
68
117
53
100
84
42
Concentration
Median
41
45
• 87
37
74
55
39
.ue/L
Range
NDb-482
ND-457
ND-784
ND-295
ND-695
ND-784
ND-360
Modified from Wallace, 1992.
"Samples shipped and stored at 2-8°C for one to two weeks prior to analysis;
sample method 3
b Not detected (detection limits varied among the three surveys).
c Samples stored at 20-25°C for three to six weeks prior to analysis; sample
method 2
d Sodium thiosulfate added; sample method 1
IV-2
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THMs in Water: Means
Results from National Surveys
ug/L
NORS NOMS 1 MOMS 2 NOMS 3T MOMS 3Q AWWARF
Max THM Formation
Moderate
Minimum (Quenched)
Mean values of THMs from three nationwide surveys. Different storage techniques (refrigerated; not
refrigerated; refrigerated with a reducing agent) resulted in widely varying THM concentrations.
-------
analyzed were not provided. A quenching agent, sodium thiosulface, was used
prior to analysis. The median trihalomethane values ranged from 30 jig/L (ppb)
in uhe winter to 44 jjg/L (ppb) in the summer. The overall median value wa;,
39 M§/L (PPb). similar to the results of the NOMS Survey (Wallace 1992).
2. Chloroform
A large number of. surveys have analyzed drinking water for chloroform
~ / .
content; nine describe the occurrence of chloroform in drinking water on a
national level. In 1975, the National Organics Reconnaissance Survey (NORS),
conducted by the EPA, collected drinking water samples from 80 cities nation-.
wide. The survey sampled for several organics, including trihalomethanes, at
the water treatment facilities. Eighty percent of the systems had surface
water sources, and the remaining 20% had ground water sources. The median
concentration for chloroform was 23 ^g/L (ppb), and the maximum level found
was 311 pg/L (ppb) (Symons et al. 1975). The detection limit was 0.05 pig/L
(ppb). The sampling method employed in the NORS survey was refrigeration
without quenching; therefore, chloroform concentrations may have increased
following collection. 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. For NOMS, 113 community water supplies
were sampled at the treatment plants. 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 all three sampling methods. During Phase 1, all
samples were refrigerated at 2-8°C for 1-2 weeks prior to analysis. In
IV-4
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Phase 2, the samples were allowed to stand at 20-25°C for two to three weeks
to maximize THM formation. In Phase 3T, one set of samples was allowed to
stand 3-5 weeks. In Phase 3Q, a' quenching agent, sodium thiosulfate, was
added. As expected, the highest chloroform values occurred in Phases 2 and
3T. Chloroform was detected in 92-100% of the systems sampled in all three
phases. The median chloroform concentrations of the three phases ranged from
22 to 54.5 jjg/L (ppb). The maximum value found was 540 fig/L (ppb) . Like
NORS, 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. , Samples were taken at the treatment plants and in the
distribution systems. Concentration data were combined; therefore, results
can be separated by treatment plant or distribution system. In the CWSS, 97%
of the surface water supplies and 34% of the ground water supplies were
positive for chloroform. For surface water supplies, the mean of the
positives and the overall median were 90 ^g/L (ppb) and 60 fig/L (ppb),
respectively. The mean of the positives for ground water supplies was
8.9 /ig/L (ppb). and the overall median was below the minimum reporting limit
(MRL) of 0.5 ftg/L (ppb) (Brass et al. 1981). The MRL is the lowest value of a
contaminant that can be detected based on sampling methodology, analytical
method, and laboratory and environmental conditions at the time of sampling.
It may or may not be identical to the detection limit.
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
IV-5
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systems, were examined. In Che RWS, 82% of the surface water supplies and 17%
of the ground water supplies were positive for chloroform. For the surface
vatsr supplies, the mean of the positives and the overall median
concentrations were 84 jig/L (ppb) and 57 jtg/L (ppb) , respectively. For the
ground water supplies, the mean of the positives was 8.9 /zg/L (ppb), and the
overall median was below the minimum reporting limit of 0.5 ^g/L (ppb) (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 945 ground water systems
that were sampled, 466 systems were chosen at random, while 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 chloroform formation was allowed to proceed
unrestricted. For chloroform, the median concentration of the positives for
the randomly chosen systems serving greater than 10,000 people was 1.4 jig/L
(ppb), and the occurrence rate was 37%. For randomly chosen systems serving
less than 10,000 people, the median of the positives was 1.6 ^g/L (ppb), with
an occurrence rate of 57%. The nonrandomly chosen systems had a median
concentration for the positives of 1.9 /ig/L (ppb) and an occurrence rate of
53% (Westrick et-al. 1983).
The National Screening Program for Organics in Drinking Water (NSP).
sponsored by the EPA, was conducted from June 1977 to March 1981. The survey
sampled 169 systems nationwide. Samples were collected at the treatment faci-
lities. For chloroform, the mean and median concentrations for 132 positives
IV-6
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were 45 ng/L (ppb) and 29 jtg/L (ppb) , respectively, with a maximum concentra-
tion of 204 ftg/L (ppb) (Boland 1981).
Thirty- five water utilities nationwide, of which 10 were located in
California, were sampled for chloroform in clearwell effluent (after final
disinfection, but before distribution). Source water used by the 35 utilities
included groundwater (7), lake/reservoir (17), and flowing stream (11).
Samples were taken for four quarters (spring, summer, and fall in 1988 and
winter in 1989). The median for all four quarters was 14 /ig/L (ppb), with the
medians of the individual quarters reported as 15, 15, 13 and 9.6 jtg/L (ppb),
respectively. The maximum value was .130 pg/L (ppb). For all four quarters,
75% of the data were below 33 jig/L (ppb) . The detection limit was not
reported (Krasne.r et al. 1989; U.S. EPA 1989a, 1989b) .
The Technical Support Division (TSD) of the Office of Ground Water and
Drinking Water (OGWDW) maintains a ground water contaminant database. For
chloroform, the database contains 5,806 measurements taken at treatment
facilities from 19 states between 1984 and 1991. The mean and median
chloroform concentrations were determined to be 17 ^g/L (ppb) and 5 jjg/L
(ppb), respectively (U.S. EPA 1991).
The EPA's Technical Support Division (TSD) has compiled a database
from its disinfection by-products field studies. The field studies included a
chlorination by-products survey, conducted from October 1987 to March 1989..
In this survey, chloroform in finished water from the treatment plant and in
the distribution system was sampled. For surface water systems, the mean
concentrations, in finished water for systems serving greater than and less
than 10,000 people were 38.9 jjg/L (ppb) (90th percentile based on 42 samples,
IV- 7
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74.4 jtg/L (ppb)) and 42.8 ^g/L (ppb) (90th percentile based on 20 samples.
63.5 /ig/L (ppb)), respectively. In the distribution system, the means were
58.7 Mg/L (ppb) (90th percentile, 141 Mg/L(ppb)) for 39 samples from plants
serving greater than 10,000 people and 77.2 jig/L (ppb) (90th percentile,
110 jig/L(ppb)) for 11 samples from plants serving fewer than 10,000 people.
The ground water systems serving less than 10,000 people had mean chloroform
concentrations in seven finished water samples and five distribution system
samples of 2.8 ^g/L (ppb) (90th percentile, 10.3 ^g/L (ppb)) and 3.6 Mg/L
(ppb) (90th percentile, 9.4 ^g/L (ppb)), respectively. Only one ground water
system serving greater than 10,000 people was sampled; the concentrations at
the plant and in the distribution system were 0.6 and 0.8 /ig/L (ppb),
respectively (U.S. EPA 1992).
The Rhode Island Private Well Study was conducted in 1986 by the Rhode
Island Department of Environmental Management (RIDEM). Private wells that
were located in land use areas that posed a threat to groundwater (e.g.
industrial areas, unsewered areas, junkyards, and agricultural areas) were
sampled for contamination. A total of 485 samples were taken from 463 private
wells. Chloroform concentrations ranged from below the detection limit of
5 Mg/L (ppb) to 8 Mg/L (ppb) (mean. 3.9 Mg/L (ppb)) (RIDEM 1990).
Fair et al. (1988) analyzed drinking water from three community water
supplies for chlorination by-products. Chloroform concentrations reported for
each of the plants ranged from 11 to 100 jig/L (ppb) in finished water at the
plants and from 21 to 160 ^g/L (ppb) in the distribution systems.
The EPA's five-year Total Exposure Assessment Methodology (TEAM) study
measured the personal exposures of urban populations to a number of organic
IV-8
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chemicals in the air and drinking water of several U.S. cities between 1981
and 1987. As part of the study, running tap water samples, collected from
residences of nearly 850 study participants during the icrning and the
evening, were analyzed for chloroform content. Table IV-2 shows chloroform
concentrations found in drinking water from the six cities surveyed.
Uden and Miller (1983) sampled drinking water from two treatment
plants in Amherst, Massachusetts. Two sets of tap water samples were
collected at each plant;, the first set of samples was analyzed immediately
following collection, and the second set was allowed to sit for 24 hours to
mimic distribution levels. Chloroform concentrations for the two treatment
plants were 39.6 jig/L (ppb) and 87.4 ng/L (ppb) immediately after disinfection
and 139 /ig/L (ppb) and 190 /jg/L (ppb) after standing for 24 hours.
Howard (1990) reported results from several additional surveys in a
literature review. In a Federal survey of finished water supplies, chloroform
was found to occur in 70% of ground water supplies (Dyksen and Hess 1982).
Coleman et al. (1976) reported that a survey of drinking water in five cities
found concentrations ranging from 1 to 301 ^g/L (ppb) (mean, 85 ^g/L (ppb)).
In a nine-city survey, chloroform was found in eight of nine water supplies,
with concentrations ranging from below the detection limit to 58 jig/L (ppb);
the mean of the positive samples was 19.2 /ig/L (ppb) (Heikes 1987). Furlong
and D'itri (1986) reported that a survey of chlorinated drinking water from
40 plants in Michigan had a chloroform detection rate of 80%, with
concentrations ranging from below the detection limit to 201.4 jjg/L (ppb).
The mean of the positive samples was 41.8 ^tg/L (ppb), and the median was
16.2 Mg/L (ppb).
IV-9
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Table IV-2 Chloroform Concentrations in Drinking Water from the EPA TEAM study
Locat ion
El izabeth/Bayonne
New Jersey
Los Angeles,
California
Ant loch/Pittsburgh
California
Devils Lake,
North Dakota
Greensboro,
North Carolina
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
355
157
49
117
52
9
7
71
24
24
10
Concentration «g/L (ppb)
Mean
70
61
17
14
29
6.8
11
42
0.46
43
24
Median
67
55
16
14
33
7.5
9.6
49
0.38
44
24
Maximum
170
130
33
60 •'
52
13
18
99
1.4
91
35
Percent Measured
25% 75% 95%
83 102
77 104
24 30
5.6 20 32
9.2 42 49
......
13 65 91
- - . - - -
56
. .
Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al. 1987; and Wallace 1992
-------
As part of a study to determine the exposure of university students to
volatile organics during normal daily activities, tap water at two
universities was analyzed for chloroform. The University of North Carolina
(Chapel Hill, NC) was chosen to represent a non-industrial site, and Lamar
University (Beaumont, TX) was chosen to represent a petrochemical industry
area. Chloroform concentrations in 14 samples of tap water at the University
of North Carolina ranged from 180 to 260 /xg/L (ppb) (mean, 220 Mg/L (ppb)).
At Lamar University, chloroform concentrations in 25 tap water samples ranged
from 99 to 550 /xg/L (ppb) (mean, 150 /ig/L (ppb)). The levels of detection at
The University of North Carolina and Lamar University were 1.0 and 0.05 /ig/L
(ppb), respectively (Wallace et al. 1982).
A 1987 analysis of drinking water in Nassau County, New York, which
takes its drinking water from underground aquifers, found chloroform to be
present in approximately 3% of the drinking water samples (detection limit
1 ppb); however, the chloroform concentration exceeded 10 ppb in only 1% of
the samples (Moon et al. 1990).
The exposure to chloroform in drinking water from ground water sources
has been estimated from the median levels found in the GWSS. Based on the
range of median levels, 1.4-1.9 /ig/L (ppb), and a consumption rate of two
liters per day for adults, the median exposure to chloroform was determined to
be 2.8-3.8 /ig/day. The data from NOMS, which sampled mainly surface water
systems, has been used to estimate exposure to chloroform in drinking water
from surface water supplies. Based on the range of medians, 22-54.5 ng/L
(ppb), the exposure is estimated to range from 44 to 109 jjg/day. The NOMS
data, however, were collected prior to promulgation of the total
trihalomethane rule; therefore, the exposures based on this data may be
IV-11
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overestimated. Exposure to chloroform in drinking water also has been
estimated from mean levels found at the tap in the TEAM studies. The mean
concentrations ranged from 24 to 49 ^g/L (ppb) for the four TEAM study cities
using surface water, and the mean concentration for Devils Lake, which used
ground water exclusively, was 0.46 /jg/L (ppb). The mean concentration in Los
Angelos, where both surface and ground water were used, was 15 /ig/L (ppb).
The exposure to chloroform from the tap is estimated to be from 0.48 to
98 jig/day for surface water users, but only 1 ^g/day for ground water users in
the TEAM study. The exposure estimates assume a consumption rate of two
liters per day. The data used in estimating chloroform exposure is limited
and contains considerable uncertainty. There may be sampling errors that
arise from uncertainties relating to the representativeness of actual samples
being measured, .and there may be measurement errors, that arise from random
and systematic error in a given measurement technique.
3. Brominated Trihalomethanes
The occurrence of brominated THMs, including bromodichloromethane and
bromoform, in U.S. drinking water was described in eight national surveys, and
dibromochloromethane occurence has been described in nine national surveys.
In 1975, the National Organics Reconnaissance Survey (NORS), conducted by the
EPA, collected drinking water samples from 80 cities nationwide. The survey
sampled for several organics, including brominated trihalomethanes, at the
water treatment facilities. The sampling method employed was refrigeration
without quenching; therefore, brominated THM concentrations may have increased
following collection. Dibromochloromethane was found in 90X of the systems
sampled at a median concentration of 2 ^S/L (ppb)• Bromodichloromethane was
found in 98% of the systems sampled. The median concentration was 8 /ig/L
IV-12
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(ppb), and the maximum level was 116 ^g/L (ppb). The detection limit for
dibromochloromethane and bromodichloromethane was 0.1 ^g/L (ppb). The median
concentration for bromoform was below the detection limit of approximately
5 jzg/L (ppb), and the maximum level found was 92 /xg/L (ppb) (Symons et al.
1975) . NORS was performed prior to the promulgation of the total trihalo-
methane regulation; therefore, these results may be higher than current
levels.
The National Organics Monitoring Survey (MOMS) was conducted by the
EPA from March 1976 to January 1977. 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
all three sampling methods. During Phase 1, all samples were refrigerated.
In Phase 2, the samples were allowed to stand at 20-25°C for 2-3 weeks to
maximize THM formation. In Phase 3T, one set of samples was allowed to stand
an additional 2-3 weeks. In Phase 3Q, a quenching agent, sodium thiosulfate,
was added. As expected, the highest THM values occurred in Phases 2 and 3T.
Bromodichloromethane was detected in over 90% of the systems sampled. The
median concentration under the various sampling conditions ranged from 5.9 to
14 /jg/L (ppb) , and the maximum concentration was 183 ng/'L (ppb) . Dibromo-
chloromethane was detected in 73% of the systems sampled. The median
concentration ranged from below the detection limit to 3 jig/L (ppb), and the
maximum value was 280 ^g/L (ppb). The median bromoform concentration under
all sampling conditions was below the detection limit of 0.3 jig/L (ppb); the
maximum value was 280 /ig/L (ppb). NOMS was conducted before the promulgation
of the total trihalomethane regulation; therefore, these results may be higher
than current levels.
IV-13
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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. 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 ng/L (ppb), respectively. The mean of the positives for ground
water supplies was 5.8 ng/L (ppb), and the overall median was below the
minimum reporting limit of 0.5 jig/L (ppb). For dibromochloromethane, 97% of
the surface water supplies and 34% of the ground water supplies were positive.
For surface water supplies, the mean of the positives and the overall median
were 5.0 and 1.5 ^g/L (ppb), respectively. The mean of the positives for
ground water supplies was 6.6 ^tg/L (ppb), and the overall median was below the
minimum reporting limit of 0.5 ptg/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 fig/L (ppb),
and the overall median was less than 1.0 jig/L (ppb). The mean of the
positives for ground water supplies was 11 /jg/L (ppb), and the overall median
was below the minimum reporting limit of 0.5 /xg/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 bromodichloromethane, 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
IV-14
-------
supplies, the mean of the positives and the overall median concentrations were
17 ^g/L (ppb) and 11 ng/L (ppb) for bromodichloromethane, 8.5 ng/L (ppb) and
0.3 /ig/L (ppb) for dibromochloromethane, and 8.7 ng/L (ppb) and <0.5 ^g/L
(ppb) for bromoform. For the ground water supplies, the mean of the positives
was 7.7 fig/L (ppb) for bromodichloromethane, 9.9 /xg/L (ppb) for dibromochloro-
methane, and 12 /ig/L (ppb) for bromoform. The overall median was below the
minimum reporting limit of 0.5 /xg/L (ppb) for all three brominated trihalo-
methanes (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 THM 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 ^g/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 of 3.9 jig/L (ppb) and an
occurrence rate of 46%. For bromoform, the median positive concentration was
IV-15
-------
2.4 MS/L (ppb) for the randomly chosen systems serving greater than and
3.8 jjg/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
(ppb) and an occurrence rate of 31% (Westrick et al. 1983).
The Technical Support Division (TSD) 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 ng/L
(ppb), and the median was 3 /xg/L (ppb). For dibromochloromethane, the mean
concentration was 3.0 /ig/L (ppb), and the median was 1.7 jig/L (ppb). For
bromoform, the database contains 1,409 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 /ig/L (ppb), respectively (U.S. EPA
1991).
Thirty-five water utilities nationwide, of which 10 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 bromo-
dichloromethane concentration for all four quarters was 6.6 jig/L (ppb), with
the medians of the individual quarters reported as 6.9, 10, 5.5 and 4.1 /xg/L
(ppb), respectively, and with a maximum value of 82 fjg/L (ppb). For all four
quarters, 75% of the data were less than 14 jig/L (ppb). The median dibromo-
chloromethane 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
IV-16
-------
(ppb) , respectively, and with a. maximum value of 63 /ig/L (ppb) . For all four
quarters, 75% of the data were below 9.1 yg/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 data were below 2.8 /ig/L (ppb) (Krasner et al. 1989;
U.S. EPA 1989a 1989b).
The EPA's Technical Support Division (TSD) has 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.
The mean concentration of bromodichloromethane was 12.7 /ig/L (ppb) in
42 samples from systems serving more than 10,000 people (90th percentile,
25.0 /tg/L (ppb)) and 17.0 /ig/L (ppb) for 20 samples from the smaller systems
(90th percentile, 29.5 /ig/L (ppb)). The mean dibromochloromethane concentra-
tions was 4.7 /ig/L (ppb) for 42 samples from the larger systems (90th percen-
tile, 13.8 /ig/L (ppb)) and 6.9 /ig/L (ppb) for 20 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
IV-17
-------
(ppb) (90th percentile, 4.9 /ig/L (ppb)) in 42 samples from the larger systems
and 20 samples from the smaller systems, respectively (U.S. EPA 1992).
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 greater
than 10,000 people, and 11 samples were from systems serving less 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 ng/L (ppb)) and 24.8 Ag/L (ppb) (90th percentile, 51.0 Mg/L (ppb)),
respectively. The mean dibromochloromethane concentrations were 6.3 /jg/L
(ppb) (90th percentile, 17.3 /jg/L (ppb)) and 10.4 jig/L (ppb) (90th percentile,
35.0 ^tg/L (ppb))., respectively. Mean bromoform concentrations were 0.8 fig/L
(ppb) (90th percentile, 3.1 /ig/L (ppb)) and 1.4 j*g/L (ppb) (90th percentile,
5.1 Mg/L (ppb)), respectively (U.S. EPA 1992).
Ground water systems serving less than 10,000 people were analyzed for
bromodichloromethane, dibromochloromethane, and bromoform in 7 finished water
samples and in 5 distribution system samples.. Mean bromodichloromethane
concentrations were in the finished water samples and in the distribution
system samples were 1.1 /ig/L (ppb) (90th percentile, 2.6 fig/L (ppb)) and
2.2 ng/L (ppb) (90th percentile, 3.4 jig/L (ppb)), respectively. Mean
dibromochloromethane concentrations were 0.6 /ig/L (ppb) (90th percentile,
1.0 ng/L (ppb)) and 1.8 Mg/L (ppb) (90th percentile, 3.6 Mg/L (ppb)),
respectively. Mean bromoform concentrations were 0.6 /ig/L (ppb) (90th
percentile, 2.6 jjg/L (ppb)) and 2.3 /ig/L (ppb) (90th percentile, 10 /ig/L
(ppb)), respectively.
IV-18
-------
For ground water systems serving greater than 10,000 people, dibromo-
chloromethane and bromoform were not detected in single samples taken at the
plant and from the distribution system, based on a detection limit of 0.2 ^tg/L
(ppb). Bromodichloromethane concentrations in the plant and distribution
system samples were 0.2 and 0.4 jig/L (ppb), respectively (U.S. EPA 1992).
Several less comprehensive surveys have analyzed drinking water for
bromodichloromethane and Jsromoform content. The EPA's 5-year Total Exposure
Assessment Methodology (TEAM) study measured the personal exposures of urban
populations to various organic chemicals in air and drinking water in several
U.S. cities 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 bromodichloromethane and dibromo-
chloromethane concentrations. Tables IV-3, IV-4, and IV-5 show bromodichloro-
methane, dibromochloromethane, and bromoform concentrations found in drinking
water from the six cities surveyed.
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 ng/L (ppb); the
mean of the positive samples was 7.4 /ig/L (ppb). Dibromochloromethane also
was detected in 30 plants at a median concentration of 2.2 ng/L (ppb) and a
maximum of 39.6 pg/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 ng/l-- 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 1 fig/L (ppb) and a maximum level of 7 jtg/L (ppb). A total of 14% of the
IV-19
-------
Table IV-3 Bromodichloromet.hane Concentrations in Drinking Water from the EPA
TEAM Study
Ni
o
Local ion
El i zabe th/Bayonne ,
New Jersey
Los Angeles,
California
Antioch/Pittsburgh,
California
Devils Lake,
North Dakota
Greensboro ,
North Carolina
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
355
157
49
117
52
9
7
71
24
24
10
Concentration
Mean
13.6
13.6
5.4
11
20
19
26
21
0.21
7.1
10
Median
13
12
5.8
12
24
24
27
17
0.18
7.8
10
uR/L (ppb) Percent Measured
Maximum 25% 757. 95X
23 -- 15 18
54 --15 20
16 , -- 7.1 8.3
23 5.1 16 20
38 7.7 31 37
31
36
47 2.4 36 47
1.0
11 -- 9.2 --
13
Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al. 1987; and Wallace 1992.
-------
Table IV-4 Dibromochloromethane Concentrations in Drinking Water from the EPA
TEAM study
Location
El izabeth/Bayonne
New Jersey
Los Angeles,
California
Aritioch/Pittsburgh
Cal ifornia
Devils Lake,
North Dakota
Greensboro,
North Carolina
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
355
157
49
117
52
9
7
71
24
24
10
Concentration u£/L (ppb) Percent Measured
Mean
2.4
2.1
1.4
9.4
28
10
24.7
8
0.09
1.2
2.7
Median
2.4
1.9
1.6
11
32
12
18
6.4
0.06
1.2
2.6
Maximum 25%
8.4
7.2
3.0 , --
19 2.4
55 15
17
70
21 0.98
0.45
1.9
3.5
75%
2.7
2.4
1.8
15
42
15
0.06
1.5
95%
3.4
3.8
2.1
18
48
19
Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al. 1987; and Wallace 1992.
-------
Table IV-5 Bromoforin Concentrations in Drinking Water from the EPA TEAM Study
Locat ion
Los Angeles,
Cal i fornia
Ant ioch/Pittsburgh ,
California
Date
Sampled
Winter 1984
Summer 1984
Winter 1987
Summer 1987
Spring 1984
Sample
Size
117
52
9
7
71
Concentration «R/L (ppb)
Mean
0.78
8.08
3.2
25.5
0.78
Median
0.54
3.0
3.2
9.6
0.58
Maximum
12
78
4.7
113
2.0
Percent Measured
25% 75% 95%
0.34 0.92 1.5
2.00 5.9 53
. .
0.19 1.2 1.9
Adapted from Wallace, 1992.
-------
locations sampled contained detectable levels of bromoform (U.S. EPA 1980b).
Kelley (1985) surveyed 18 drinking water plants in Iowa for trihalomethanes,
detecting bromoform in five water supplies at concentrations ranging from 1.0
to 10 /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 Chan 0.5 to 19 jig/L (ppb) in finished water at the plant and from
less than 0.5 to 23 fig/L (ppb) .in the distribution systems. Bromoform
concentrations ranged from less than 0.5 to 2.5 j*g/L (ppb) in finished water
and from less than 0.5 to 3.1 jxg/L (ppb) in the distribution systems.
The National Screening Program for Organics in Drinking Water (NSP),
sponsored by the EPA, was conducted from June 1977 to March 1981. The survey
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 ^g/L (ppb) (Boland 1981).
As part of a study to determine individual exposure to volatile
organics during normal daily activities of students at the University of North
Carolina, Chapel Hill, tap water was analyzed for bromodichloromethane.
Bromodichloromethane concentrations in tap water ranged from 15 to 20 /ig/L
(ppb), with a mean of 17 /ig/L (ppb). The level of detection was 0.1 /ig/L
(ppb) (Wallace et al. 1982).
IV-23
-------
Chang and Singer (1984) analyzed the bromoform concentration in
drinking water samples prepared by the desalination of seawater. After
pretireatment using either activated carbon or ultrafiltration, but prior to
the reverse osmosis treatment, bromoform concentrations were 13+14 and
110+59 ^g/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 /jg/L (ppb)) when activated carbon was used as a
pretreatment and 127 jig/L (ppb) when ultrafiltration was used. In the reverse
osmosis treatment, three reverse osmosis membranes were evaluated. The
cellulose triacetate filter resulted in concentrations of 51 /ig/L (ppb),
making it less efficient in removing bromoform, compared to the polyether/urea
thin film spiral wound membrane and the polysulfone membrane filters whcih
resulted in concentrations of 5.0 jig/L (ppb) and 2.25 /ig/L (ppb),
respectively.
Broraodichloromethane, dibromochloromethane, and bromoform were
detected in 9.5-12.8% 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 concen-
tration profiles, with approximately 10% and 8.5% of the samples containing
less than 4.9 ppb of the respective chemicals. The detection limit was 1 ppb
for each chemical. Bromoform was detected at less than 4.9 ppb in 8% of the
samples, at 5-9.9 ppb in 2.5% of the samples, and 10-49.9 ppb in less than 1%
of the samples. 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).
IV-24
-------
Exposure co 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 ^g/L (ppb), and for
bromoform, median exposure may range from 4.8 to 8.4 .>ig/day. Exposure to
bromodichloromethane from, ground water can be estimated based on the range of
medians observed under different conditions in NOMS, which mainly sampled
surface water systems. Based on a range of 5.9-14 ^g/L (ppb), exposure to
bromodichloromethane from surface water is estimated, to be between 12 and
28 /jg/day. Similarly, based on the range of medians reported for
dibromochloromethane concentrations, the median exposure is estimated to be up
to 6 /jg/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 jig/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 ^g/L (ppb), the exposure to
bromoform is estimated to range from 1 to 2 /ig/day for surface water supplies.
Exposure to bromodichloromethane, dibromochloromethane and bromoform in
drinking water has also been estimated from mean or median levels found at the
tap in the TEAM studies. For the six cities in the TEAM study, the mean
concentrations for bromodichloromethane and dibromochloromethane ranged from
0.21 to 22.5 jig/L (ppb) and from 0.09 to 17.3 ^g/L (ppb), respectively. The
exposure to bromodichloromethane and dibromochloromethane from the tap is
estimated to be 0.42-45 /ig/day and 0.18-34.6 ^g/day, respectively, assuming a
consumption rate of two liters per day. If samples were taken over more than
one season, the medians of the seasons were averaged to reflect temporal
IV-25
-------
changes. To estimate exposure to bromoform at the tap, the median
concentrations were used instead of the mean to negate the effect of one
sample with a very high level of bromoform. Over 90% of all samples had
bromoform levels below the detection limit; therefore, only data from two of
the six cities are available to estimate exposure. The median concentrations
ranged from 0.58 to 6.4 ^g/L (ppb), and based on a tap water consumption rate
of two liters per day, the exposure to bromoform from the tap is estimated to
be 1.16-12.8 fig/day. The data used in estimating bromodichloromethane,
dibromochloromethane, and bromoform exposure is limited and contains consider-
able uncertainty. There may be sampling errors that arise from uncertainties
relating to the representativeness of actual samples being measured, and there
may be measurement errors, that arise from random and systematic error in a
given measurement technique.
B. Exposure to Sources Other Than Drinking Water
1. Dietary Intake
a. Chloroform
No exposure estimates were found for chloroform in food. Several
studies, however, analyzed foods for chloroform content. Daft (1987, 1988,
1989) analyzed foods in three studies. The most recent and comprehensive of
these surveys analyzed 549 food items. Foods were chosen from the FDA's
market basket and were made table-ready (i.e cooked, peeled, etc.) prior to
analysis. Chloroform was detected in 302 of the foods at an average
concnetration of 71 ng/g (ppb), with concentrations ranging from 2 to 830 ng/g
(ppb). In an earlier study, 231 food items obtained from the FDA's market -
IV-26
-------
basket collection were analyzed. Chloroform was detected in 94 samples at an
average concentration of 52 ng/g (ppb) with concentrations ranging from 4 to
312 ng/g (ppb). Ir- -he third study, 16 unprepared, uncooked, or off-the-shelf
food items were analyzed. Chloroform was detected in three items: instant hot
cereal at 30 ng/g (ppb), golden cake mix at 10 ng/g (ppb), and pancake mix at
70 ng/g (ppb).
A Total Exposure Assessment Methodology (TEAM) study measured
chloroform in five composite food samples. In accordance with the FDA's Total
Diet Market Basket Study, 39 different food items were purchased at retail
markets in three geographical areas (Elizabeth, NJ; Chapel Hill, NC; and
Washington, D.C.) (Entz et al. 1982) . Twenty food composites,' comprising
four different fpod groups (dairy, meats, oils-and-fats, and beverages), were
analyzed. Chloroform was detected in one dairy composite at 17 ng/g (ppb),
one oils-and-fats composite at trace levels (less than 12 ng/g (ppb)) and
three beverage composites at concentrations of less than 12, 6, and 12 ng/g
(ppb). Subsequent analysis of individual food items detected chloroform in
four cola soft drink samples (178, 22, 9, and 36 ng/g (ppb)), two non-cola
soft drink samples (32 and 14.5 ng/g (ppb)), milk (17 ng/g (ppb)), ice cream
(23 ng/g (ppb)), processed American cheese (17 ng/g (ppb)), natural cheese
(15 ng/g (ppb)), butter (56 ng/g (ppb)), and mayonnaise (34 ng/g (ppb)).
Heikes (1987) analyzed 18 table-ready food items, representative of
the 234 items in the FDA Total Diet Study. A total of 56% of the foods tested
were positive for chloroform-at the following levels: chocolate chip cookies,
22 ng/g (ppb); plain granola, 57 ng/g (ppb); cheddar cheese, 80 ng/g (ppb);
peanut butter, 29 ng/g (ppb); butter, 670 ng/g (ppb); fried breaded shrimp,
24 ng/g (ppb); scalloped potatoes, 7.1 ng/g (ppb); cream styled corn, 6.1 ng/g
IV-27
-------
(ppb) ; frozen fried chicken dinner, 29 ng/g (ppb); and high meat baby food
dinner, 17 ng/g (ppb). Those items shown to be high in volatile halocarbons
were further studied. Samples cf individual food items, representative of
2 or 3 regions of the country, were analyzed and found to have the following
mean concentrations: 14 butter and margarine samples, 364 ng/g (ppb);
8 samples of 4 types of cheeses, 182 ng/g (ppb); 11 samples of ready-to-eat
cereal products, 60.1 ng/g (ppb); 7 samples of peanut butter, 51.3 ng/g (ppb);
and 12 samples of highly processed foods, 122 ng/g (ppb).
Uhler and Diachenko (1987) sampled food products from 15 food
processing plants located in nine states. Plants were chosen from areas where
contamination of water used in processing would be most probable. Results
of analysis showed that out of 37 food items tested, chloroform was detected
in 13 samples at the foloowing levels: 2 samples of clear sodas, 2.3 and
15.6 ng/g (ppb); 1 sample of dark cola, 12.3 ng/g (ppb); 4 samples of cheese,
2.4 to 10.9 ng/g (ppb); and 6 samples of ice cream, 4.6 to 31.2 ng/g (ppb).
Abdel-Rahman (1982) analyzed various soft drinks for chloroform and found
average levels ranging from 9 to 61 ^g/L (ppb) for colas and 2.7 to 10.9 jig/L
(ppb) for clear soft drinks.
Wallace (1992) reported che results of sampling margarine products for
volatile organic carbons in 18 grocery stores and 19 manufacturing plants.
The FDA study detected chloroform in 5 of 18 samples in grocery stores and in
13 of 19 finished samples at the manufacturing plants. Levels in the samples
at the manufacturing plants were higher than at the stores. Two samples had
concentrations of 100 and 150 ppb; however, the remaining samples from the
manufacturing plants ranged from 15-50 ppb.
IV-28
-------
Kroneld and Reunanen (1990) sampled human milk and pasteurized and
unpasteurized cow's milk from a suburban area of Turku, Finland. The
chloroform concentration in pasteurized milk ranged from unde.tectable to
3.1 ng/L (ppb) (mean, 2.2 M§/L (PPM). Chloroform was not detected in either
the human or unpasteurized cow's milk. The detection limit was'not reported.
Toyoda et al. (1990) analyzed the dietary chloroform intake of
30 Japanese housewives in Nagoya and Yokohama, Japan. Duplicate portions of
3 meals (the type of food was not reported) were sampled for chloroform. The
detection limit was 0.5 ppb. Chloroform concentrations ranged from undetect-
able to 106.8 ppb (mean, 19.6+18.1 ppb); the mean dietary intake was
40.0+25.4 Mg/day.
No exposure estimates concerning the use of chloroform as a food
additive are available. Chloroform has been approved for use as an indirect
food additive. Chloroform may be used as a component in adhesives for
packaging foods (FDA 1977 (21 CFR 175.105 4-1-93 Edition)), and it may be used
in the production of polycarbonate resins. Polycarbonate resins are articles
or components of articles intended for use in producing, manufacturing,
packing, processing, preparing, treating, packaging, transporting, or holding
food (FDA 1988).
b. Brominated Trihalomethanes
No information is available concerning the occurrence of dibromochloro-
methane or bromoform in food in the United States. The FDA does not analyze
for dibromochloromethane or bromoform in foods. However, chlorine is used in
food production for applications such as the disinfection of chicken in
IV-29
-------
poultry plants and the superchlorination of water at soda and beer bottling
plants (Borum 1991). Therefore, the possibility exists for dietary exposure
from the by-products of chlorination in foods.
Entz et al. (1982) analyzed food samples from Elizabeth, NJ, Chapel
Hill, NC, and Washington, D.C. 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. From 20 food composites
covering four food groups, 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 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 7 ppb. In a second study, analysis of various soft drinks
found average levels ranging from 0.2 to 6.6 /ig/L (ppb) for'colas and from
0.1 to 0.2 ng/L (ppb) for clear soft drinks (Abdel-Rahman 1982). Uhler and
Diachenko (1987) sampled food products from 15 food processing plants in nine
states. Plants were chosen from areas where contaminated water would most
likely be used in processing. Bromodichloromethane was detected in six out of
37 food items tested at the following levels:, two samples of clear sodas at
1.2 and 2.3 ng/g (ppb); one sample of dark cola at 1.2 ng/g (ppb); and three
samples of ice cream at 0.6 to 2.3 ng/g (ppb). Bromodichloromethane also was
identified in bacon, but no concentrations were given (U.S. EPA 1985b).
Toyoda et al. (1990) analyzed the dietary intake of bromodichloro.-
methane, dibromochloromethane, and bromoform for 30 Japanese housewives in
Nagoya and Yokohama, Japan. Duplicate portions of 3 meals (the type of food
was not reported) were sampled for all three chemicals. The detection limits
were 0.1, 0.2, and 0.5 ppb, for bromodichloromethane, dibromochloromethane,
IV-30
-------
and bromoform, respectively. The concentration of bromodichloromethane ranged
from undetectable to 1.7 ppb (average, 0.3+0.3 ppb). The detection limit was
0.1 ppb. The mean daily intake of broroodichlorcice thane was 0.6+0.5 ^g/dav.
The concentration of dibromochloromethane ranged from undetectable to 0.6 ppb
(average, 0.1+0.2 ppb), and the mean dietary intake was 0.3+0.3 ^g/day. The
detection limit was 0.2 ppb. The concentration of bromoform ranged from
undetectable to 8.1 ppb (average, 0.5+1.3 ppb). The mean dietary intake of
bromoform was 0.9±1.3 fig/day. The detection limit was 0.5 ppb.
In a study that determined volatile organic compounds in human milk
and pasteurized and unpasteurized cow's milk in Turku, Finland, the average
concentration of bromodichloromethane measured in pasteurized milk was
0.008 ^g/L (ppb). (range, undetectable to 0.03 ^ig/L (ppb)). Dibromochloro-
methane was detected in only one sample of pasteurized milk at 5 /ig/L (ppb).
Traces of bromoform also were detected but were not quantified. Bromo-
dichloromethane and dibromochloromethane were not detected in human or
unpasteurized milk. The presence of the brominated trihalomethanes in
pasteurized milk was most likely due to the use of' chlorinated water during
processing (Kroneld and Reunanen 1990).
2. Air Intake
a. Chloroform
Inhalation exposure to chloroform in indoor and outdoor air has been
surveyed in several studies. The major source of these data is the EPA's
5-year Total Exposure Assessment Methodology (TEAM) study measuring the
personal exposures of urban populations to a number of organic chemicals in
IV-31
-------
the air and drinking water of several U.S. cities between 1981 and 1987. As
part of the study, personal air samples and outdoor air samples were analyzed
for chloroform. Personal air samples were collected for each individual for
two 12-hour periods, excluding showers, and outdoor samples also were
collected in two 12-hour periods. Table IV-6 shows chloroform concentrations
in personal air samples, and Table IV-7 shows concentrations found in outdoor
air samples. Based on personal air results, the median exposure to chloroform
from air intake would range from 0.6 to 64 /xg/day, using an inhalation rate of
20 m3/day.
Additional studies have measured exposure levels similar to those
found in the TEAM studies. Wallace (1992) reported results from short-term
measurements of ambient air in 10 cities between 1979 and 1981. For a total
of 2,577 measurements, the 25th percentile, median, and 75th percentile air
concentrations were 0.14, 0.20, and 0.78 /ig/m3, corresponding to exposures of
2.8, 4.0, and 16 /jg/day, respectively. Ambient air samples at four urban/in-
dustrial sites in the California South Coast air basin were surveyed from
November 1982 to December 1983 for the presence of halogenated hydrocarbons.
Chloroform was detected above the quantitation limit of 0.02 ppb in 96% of the
samples. Concentrations generally increased by a similar degree at all sites
during the summer months. The highest concentration, 3.0 ppb, was recorded
for downtown Los Angeles, as was the highest monthly mean (0.78 ppb) and the
highest composite mean (0.13 ppb) (Shikiya et al. 1984). Using an inhalation
rate of 20 m3/day, potential human exposure could be as high as 14 jig/day,
based on the highest observed composite mean of 0.13 ppb.
Singh et al. (1981) reported results from field studies conducted in
three cities: Los Angeles, California; Oakland, California; and Phoenix,
IV-32
-------
Table IV-6 Chloroform in Personal Air Samples from the EPA TEAM Study'
Concentration ita/m3
Location
Elizabeth/Bayonne ,
New Jersey
Los Angeles,
California
Antioch/Pittsburgh,
Cal ifornia
Devils Lake,
North Dakota
Greensboro,
North Carolina
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer
Winter
Winter
Summer
Winter
Summer
Spring
1982b
1983
1984
1984
1987
1987 .
1984
Fall 1982
Fall 1982
Spring
1987
Sample
Size
340
150
49
117
52
88
78
68
23
24
116
Mean
8
4
4
1
1
1
2
0
2
2
5
.0
.3
.0
.9
.1
.6
.4
.64
.02
.2
.8
Median
3.
0.
2.
1.
0.
0.
0.
0.
0
1
3.
2
8
2
25
49
71
48
03
.38
.7
05
Maximum
Percent
Measured
(Night/Day) 25% 75%
210/89
35/140
16/40
9.7/92
20/12
42/4.2
7.6/76
6.3/4.2
2.8/50
5.5/7.5
38/51
7
6
' -- 5
0.69 2
0.16 1
0.23 1
0.15 0
0.03 0
0
3
0
.8
.0
.3
.3
.2
.7
.99
.72
.77
.2
.75
95%
26
14.5
16.5
4.8
4.4
6.5
47.5
2.65
18
Adapted from Wallace 1986; Wallace et al. 1986a; and Wallace et al. 1986b.
• Average of night and day measurements
6 These values were affected by contamination of sampling cartridges.
-------
Table IV-7 Chloroform in Outdoor Air Samples From the EPA TEAM Study *
Concentration ug/m3
Location
Elizabeth/Bayonne ,
New Jersey
Los Angeles,
California
Antioch/Pittsburgh,
California
Devils Lake,
North Dakota
Greensboro,
North Carolina
Date
Sampled
Fall 1981
Summer 1982b
Winter 1983
Winter 1984
Summer 1984
Spring 1984
Fall 1982
Fall 1982
Sample
Size
86
60
9
24
23
10
5
6
Maximum Percent Measured
Mean Median
1.4 0.63
13 0.11
0.3 0.07
0.7 0.3
0.3 0.03
0.3 0.11
0.05
0.14
Night/Day 25%
22/8.8
130/230
1.2/1.2
1
5.9/1.5
2.4/2.4
1.5/0.23
0.78
11.0
75% 95%
1.7 5.9
9.8 78
0.63 --
0.45 1.2
0.03 1.1
0.33 0.6
. .
. .
v
Adapted from Wallace 1986; Wallace et al. 1986a; and Wallace et al. 1986b.
• Average of night and day measurements
bThese values were affected by contamination of sampling cartridges.
-------
Arizona. Ambient air samples were collected at each site over 2-week periods
in 1979 and analyzed for chloroform. Results of the analyses indicated that
for iihe Los Angeles area, Che mean concentration was 0.088 ppb and the range
was 0.024-223 ppb, while Oakland had a mean of 0.032 ppb and a range of
0.013-0.060 ppb. In Phoenix, a mean of 111 ppb and range of 0.027-514 ppb was
reported. Based on the range of means, the average exposure to chloroform due
to inhalation would be from 3.2-10.8 /jg/day, using an inhalation rate of
20 m3/day.
Although TEAM studies do not collect personal air samples during
showers, attempts have been made to measure the contribution to airborne
chloroform levels. Wallace (1992) reported results from a 1987 EPA study
which found chloroform levels in the air of a shower to increase from 2 to
100 ppb during a 10-minute shower with chlorinated water.
Jo et al. (1990a, 1990b) reported separate inhalation and dermal
exposure concentrations for chloroform exposure in the shower. Volunteers
took showers with and without wetsuits to estimate the fraction of chloroform
absorbed dermally. Chloroform concentrations in the tap water prior to the
shower ranged from 5.3 to 35.9 /ig/L (ppb) (mean, 23.5 ;ig/L (ppb)). The total
mean absorbed chloroform dose for a 10-minute shower was 0.47 ^g/kg/day. A
chloroform absorption efficiency rate of .0.77 was used for inhalation. Body
burden from dermal exposure was estimated to be 93% of the body burden from
inhalation exposure. The inhalation and dermal exposure components were •
0.24 jjg/kg/day and 0.23 M§Ag/day. respectively. Chloroform concentrations
were slightly higher in air in the shower stall in the presence of a showering
individual than in an empty shower with the water running. In the absence of
a showering individual, chloroform concentrations ranged 58.1 to 326.9
IV-35
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(11.9 to 66.9 ppb) (mean, 157 /jg/m» (32.2 ppb)), and in the presence of a
showering individual, chloroform concentrations ranged from 119 to 313.4
(24.4 to 64.18 ppb) (mean, 186 H&/K* (38.1 ppb)) (Jo et al., 1990b).
Tancrede et al. (1992) reported that volatilization of chloroform
during a shower increased by 21% when the water temperature was increased from
25 to 42°C. Additionally, the fraction of chloroform in the shower water that
volatilized (42 to 64%) increased by 15% when the water flow rate was
increased from 9.7 to 13.5 L/min. Wallace (1992) reported that 55% of the
chloroform in tap water volatilized during an 11 minute shower with a 5 L/min
rate of flow. The initial chloroform concentration in the water was 580 ^ig/L
(ppb), and the shower water effluent contained 260 jig/L (ppb) chloroform.
Furthermore, the. longer the shower was on, the greater the volatilization
rate. The low-volatility compounds rapidly reached a steady state, while the
high-volatility compounds reached a steady state more slowly. Therefore, the
concentration of chloroform in the air increases over time. An example of a
high-exposure scenario is a shower facility in a locker room where showers are
on for long periods of time (Little 1992).
Heating and boiling water reduced chloroform levels; this reduction
increased with the temperature and duration of boiling. Chloroform was
reduced by SOX after one minute ac 80°C, by approximately 70% immediately
after the water began to boil, approximately 80% after the water had boiled
for about one minute, and by 90% when the water had boiled for five minutes.
Dibromochloromethane, bromodichloromethane, and bromoform decreased by 86, 90,
and 81%, respectively, when the water had been boiled for five minutes (Lahl
et al. 1982).'
IV-36
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Wallace (1992) analyzed indoor air concentrations of chloroform
resulting from the use of hot water. Seven volunteers in four homes took long
showers, boiled water, and washed clothes while ir.dcor air concentrations were
being monitored. Washing clothes and dishes increased levels of chloroform in
the house from undetectable (<4.5 /ig/m3 (0.92 ppb)) to 44 jig/m3 (9.00 ppb)
over a period of 5-11 hours. Limited data indicate that taking long showers
and boiling water did not increase chloroform levels in the rest of the house.
Further measurements are required to determine whether long showers or boiling
water increases chloroform concentrations outside of the bathroom or kitchen.
The TEAM program analyzed personal air concentrations of chloroform
for lifeguards at three swimming pools (2 outdoor and 1 indoor). The personal
air levels of ch.loroform at the indoor pool were 95, 46, and 68 jig/m3 (19.5,
9.4, and 13.9 ppb) for the three lifeguards. Chloroform concentrations
measured at the homes of the lifeguards were 2.2, 5.2 and 2.0 ^g/m3, (0.45,
1.00, and 0.41 ppb). The chloroform levels in the air at outdoor pools did
not differ from the concentrations measured at the lifeguards' homes. Levels
of chloroform in the pool water ranged from 48 to 153 jig/L (ppb) for all pools
(Wallace 1992).
Armstrong and Golden (1986) measured chloroform 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
above the water. Chloroform concentrations in the water- in the outdoor pools
ranged from 4 to 402 jig/L (ppb) (mean, 128 ^ig/L (ppb)), while levels in the
indoor pools ranged from 3 to 580 /ig/L (ppb) (mean, 133 ^g/L (ppb)). The
levels of chloroform in the hot tubs ranged from <0.1 to 530 /xg/L (ppb) (mean,
115 jig/L (ppb)). Means and ranges of concentrations (in parentheses) of
IV-37
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chloroform measured two meters above the water surface for outdoor pools.
indoor pools, and hot tubs, respectively, were: <0.1 /ig/m3 (0.02 ppb)
(<0.1-1 /ig/m3 (0.02-0.2 ppb)), 90 Mg/m3 (13.4 ppb) (<0.1-260 Mg/m3
(0.02-53.2 ppb)), and 12 /jg/m3 (2.5 ppb) (<0.1-47 Mg/m3 (0.02-9.6 ppb)).
b. Brominated Trihalomethanes
Brominated trihalomethanes usually are found in air at low concentra-
tions. Brodzinsky and Singh (1983) reported ambient outside air concentra-
tions for several urban locations across the United States. Ambient air
samples also were analyzed for bromodichloromethane and dibromochloromethane
at Magnolia, AZ, El Dorado, TX, Chapel Hill, NC, and Beaumont, TX. Bromo-
dichloromethane was detected at mean concentrations of 0.76 ppt, 1.40 ppt,
120 ppt, and 180 ppt, respectively. Based on an inhalation rate of 20 m3/day,
daily intake of bromodichloromethane was estimated to range from 0.12 to
26 ^tg/day. Dibromochloromethane was detected in the air samples from
Magnolia, AZ, El Dorado, TX, Chapel Hill, NC, Beaumont TX, and Lake Charles,
LA at concentrations of 0 ppt, 0.48 ppt, 14 ppt, 14 ppt, and 19 ppt,
respectively. From 1976 to 1977 at El Dorado., Texas, bromoform was detected
in 76% of 46 samples at a mean concentration of 0.81 jjg/m3 (0.08 ppb), with
concentrations ranging from below the detection limit to 2.7 /ig/m3 (0.3 ppb).
In air samples collected in 1978 at Lake Charles, Louisiana, bromoform was
detected in all of four samples, with a mean of 50 ng/m3 (4.8 ppt) and a range
of 6.6-7.1 ng/m3 (0.66-0.71 ppt). In 1977, bromoform was detected in 89.3% of
28 ambient air samples from Magnolia, AZ. Concentrations ranged from not
detected to 8.3 ng/m3 (0.83 ppt) , with a mean of 1.5 ng/m3 (0.15 ppt).
Overall, the average bromoform concentration was 37 ng/m3 (0.36 ppt)
(Brodzinsky and Singh 1983).
IV-38
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From November 1982 Co December 1983, ambient air samples at four
urban/industrial sites in the California South Coast air basin were surveyed
for the presence of halogenated hydrocarbons. Thirty-five percent of the
samples had bromodichloromethane levels above the quantitation limit of
0.01 ppb, 17% percent had dibromochloromethane levels above the quantitation
limit of 10 ppt, and 31% had bromoform levels above.the quantitation limit of
10 ppt. 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, 30 ppt. The maximum reported value was
40 ppt, while the composite means ranged from 20 to 100 ppt. The composite
sample was a collection of several individual samples mixed together before
analysis, and the concentration of any chemical in a composite sample is equal
to or greater than the concentration in individual samples. For dibromo-
chloromethane, the highest reported concentration, monthly mean, and mean
composite, respectively, were 290 ppt, 280 ppt, and 50 ppt; all were recorded
in-downtown Los Angeles in July. Mean concentrations ranged from 10 to
50 ppt. 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) and the highest monthly mean (310 ppt) in June
1983 (Shikiya et al. 1984). Based on the range of mean composites, daily
intake was estimated to range from 2.8 to 15 ^g/day (Shikiya et al. 1984).
Replicate air samples were collected 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 (Atlas and Schauffler 1991).
IV-39
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Wallace et al. (1982) conducted a pilot study designed to field test
personal air-quality monitoring methods. .Personal air samples were collected
frorr. students at two universities: Laraar University, Tor.as, located near a
petrochemical manufacturing area, and the University of North Carolina (UNC),
located in a nonindustrialized area. Bromodichloromethane was detected in 64%
of personal air samples from 11 Lamar students,' with a mean of 1.23 /ig/m3
(0.18 ppb), a median of 1 ng/m2 (0.15 ppb), and a range of 0.12-3.72 ^g/m3
(0.017-0.56 ppb). The limit of detection was 0.24 jig/m3 (0.04 ppb). At UNC,
17% of the samples from 6 students had detectable levels of bromodichloro-
methane. Concentrations ranged from 0.12-4.36 /jg/m3 (0.017-0.65 ppb) (mean,
0.83 /ig/m3 (0.12 ppb); median, 0.12 jig/m3 (0.017 ppb)). Based on the above
information, the average daily intake of bromodichloromethane from air using
an inhalation rate of 20 nr/day was estimated to be 25 ^g/day for Lamar
students and 17 /ig/day for UNC students. Dibromochloromethane was not
detected above 0.12 /ig/m3 at either site.
Armstrong and Golden (1986) measured bromodichloromethane, dibrorao-
chloromethane, 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. The
bromodichloromethane concentrations of water in the outdoor pools ranged from
1 to 72 fig/L (mean, 33 /ig/m3) . Levels in the indoor pools ranged from 1 to
90 jig/L (mean, 16 /ig/L) . The levels of bromodichloromethane in the hot tubs
ranged from S0.1 to 105 /ig/L (mean, 17 jtg/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 /xg/nr (0.02 ppb)
(mean only), 1.7 ng/m* (0.25 ppb) (<0.1-10 Mg/m3 (0.25-1.49 ppb)), and
1.4 jig/m3 (0.21 ppb) (<0.1-10 /ig/m3 (0.25-1.49 ppb)). The dibromochloro-
IV-40
-------
methane concentration of water in the outdoor pools ranged from <0.1 to 8 jjg/L
(ppb) (mean, 4.2 jig/m3, (ppb)). Levels in the indoor pools ranged from 0.3 to
30 fig/L (ppb) (mean, 9.5 /ig/L (ppb)). The level of dibromochloromethane ir.
the hot tubs ranged from sO.l 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 jtg/m3 (0.01 ppb) (mean only), 0.9 /ig/m3 (0.1 ppb) (<0.1 to 5 ng/n?
(0.011-0.59 ppb)), and 0.3. ;ig/m3 (0.08 ppb) (<0.1 to 5 /ig/m3 (0.01-0.59 ppb)).
The mean bromoform concentration in the outdoor pools was less than 0.1 jig/L
(ppb). Levels in the indoor pools ranged from less than 0.1 to 20 /ig/L (ppb)
(mean, 6 ^ig/L (ppb)). The levels of bromoform in the hot tubs ranged from
less than 0.1 to 62 ^g/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 jig/m3 (9.6 ppt) (mean
only), 9 Mg/m3 (87° PPC) (<0.1-14 ng/m3 (9.6-1354 ppt)), and 8 ^g/m3 (773 ppt)
(<0.1-14 Mg/m3 (9.6-1354 ppb)).
Based on the above surveys, exposure to bromodichloromethane, dibromo-
chlorome thane and bromoform due to inhalation can be estimated based on an
inhalation rate of 20 m3/day. Based on an ambient air concentration of
0.76-180 ppt, daily intake of bromodichloromethane was estimated to range from
0.12 to 26 fig/day. For dibromochloromethane, assuming an ambient air concen-
tration of 1-50 ppt, exposure may be as low as 0.18 /ig/day or as high as
9.4 /ig/day. Using an ambient air concentration of 0.021-3.5 /jg/m,
(0.002-0.34 ppb), mean exposure may be as low as 0.42 ^g/day or as high as
70 ^g/day for bromoform. The use of ambient air concentrations, due to the
lack of data on indoor air concentrations, will underestimate the exposure.
IV-41
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Brominaced THM levels are higher in indoor air compared to oucdoor air due co
confined space and additional indoor air sources of THMs.
C. Overall Exposure
1. Chloroform
The EPA has set the maximum contaminant level goal (MCLG) for
chloroform at zero because it has been determined that it is a probable human
carcinogen. Therefore, a relative source contribution (RSC) analysis is not
relevant because it is the Agency's policy to perform RSC analyses for only
non-carcinogens.
2. Brominated Trihalomethanes
The EPA has set the maximum contaminant level goal (MCLG) for dibromo-
chloromethane and bromoform at zero because it has been determined that these
chemicals are probable human carcinogens. Therefore, a relative source
contribution (RSC) analysis is not relevant for these chemicals because it is
the Agency's policy to perform RSC analyses for only non-carcinogens.
A relative source contribution (RSC) analysis for bromodichloromethane
cannot be performed due to the limited occurrence and exposure data. Based on
the available water and air data, it appears that, on average, drinking water
may be the predominant source of bromodichloromethane exposure, but that air
may contribute a significant amount. Based on these anecdotal data, an RSC
value of 80% for drinking water is believed to be on the high side.
IV-42
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D. Body Burden
1. Chloroform
a. Breath
The largest existing data set on chloroform concentrations in breath
is from the TEAM study which reports measurements on 800 people taken from
fall 1981 to summer 1987; the study provides more than 1,200 breath samples.
Mean concentrations ranged from 0.5 to 3 /ig/m3 (0.1 to 0.6 ppb) , with the
lowest levels in California. Wallace et al. (1982) analyzed chloroform
concentrations in breath for two groups of university students. Students at
Lamar University., Texas, resided in the vicinity of oil wells and oil storage
tanks, and students at the University of North Carolina (UNC) resided in a
nonindustrial area. Chloroform was detected in the breath of all students at
Lamar University, with concentrations ranging from 0.22 to 2.48 jig/m3
(0.05-0.51 ppb) (mean, 0.42 ng/m3 (0.09 ppb)). At UNC, 100% of the students
had detectable levels of chloroform in their breath with concentrations
ranging from 1.70 to 5.06 ng/v? (0.35-1.04 ppb) (mean, 2.86 ng/mz (0.56 ppb)).
The detection limit was 0.11 ^g/m3 (0.02 ppb)
Jo et al. (1990a, 1990b) measured breath concentrations of chloroform
pre- and post-exposure in the shower and for inhalation -only exposure in the
shower. The post-exposure breath concentrations after a normal (10-minute)
shower ranged from 6.0 to 21 /ig/m3 and from 2.4 to 10 jig/m3 for inhalation-
only exposure showers. The chloroform body burden after showering was 14 to
49 times higher than background chloroform body burden. The pre -shower breath
concentrations of chloroform were all below the detection limit of 0.86 jig/m3 .
IV-43
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b. Blood
Wallace (1992) reported that chloroform concentrations in blood were
measured (in triplicate) in 25 female volunteers using a purge and trap
method. Five subjects had blood concentrations below 10 ^ig/L (ppb), 6
subjects ranged from 10 to 25 /ig/L (ppb), with a small standard deviation
(<10 Mg/L (ppb)), and another 5 subjects were in the same range with a large
standard deviation (>20 fig/L (ppb)). Seven subjects exceeded 25 jig/L (ppb).
All values above 20 ppb were confirmed with gas chromatography/mass
spectrography. The author noted that the analytical method used thermally
decomposes trichloroacetic acid (TCA) in the blood to chloroform. Subse-
quently, the measured chloroform may include original chloroform in the blood
and "derived" chloroform from the decarboxylation of TCA. Therefore, the
measured values are upper limits with respect to chloroform exposure and may
be due to chlorinated VOC's as well as chloroform. Hajimiragha et al. (1986)
analyzed blood chloroform concentrations in 39 subjects with no known
occupational exposure. Chloroform was detected in 60-95% of the samples
analyzed, with concentrations ranging from <0.1 to 7 ^ig/L (ppb) (mean,
0.2 jig/m3 (ppb)). The analytical method used .was headspace gas
chromatography.
Antoine et al. (1986) analyzed the blood of 250 environmentally
sensitive patients for 18 volatile organic compounds. Chloroform
concentrations ranged from undetectable to 7.0 /zg/L (ppb) (mean, 1.5 pg/L
(ppb)). Hajimiragha et al. (1986) measured chloroform concentrations in the
blood of dry cleaners. Concentrations ranged from 0.2 to 51.2 jjg/L (ppb)
(mean, 12.2 ^g/L (ppb)). Wallace (1992) reported that plasma chloroform
IV-44
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levels in 127 swimmers using indoor pools in Italy ranged from 0.1 to 3.1
(ppb) shortly after swimming.
c. Mother's Milk
Pellizzari et al. (1982) analyzed mother's milk for chloroform by
using gas chromatography/mass spectrometry . 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. Seven of the 49 samples contained detectable
levels of chloroform. Actual concentrations and detection limits were not
reported.
d. Adipose Tissue
The EPA's National Human Adipose Tissue Survey (NHATS) , in 1982 I
quantified the prevalence of toxic compounds detected in the general public of
the United States. Several hundred samples of adipose tissue were collected,
pooled into 46 composite samples by age and geographic re-gion, and analyzed.
Chloroform was detected at 5-580 ng/g (ppb) in 29 of the samples (Phillips
1992; Wallace 1992). The results were divided into nine geographical regions,
and chloroform exposure was ranked highest in the East North Central Region,
which consisted of Ohio, Indiana, Illinois, Michigan and Wisconsin (Phillips
and Birchard 1991) .
IV-45
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2. Brominaced Trihalomethanes
a. Blood
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 jjg/L (ppb), with a mean of
0.6 Mg/L (ppb).
b. Mother's Milk
Pellizzari et al. (1982) analyzed mother's milk for dibromochloro-
methane using gas chromatography/mass spectrometry. 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. One of the 49 samples contained
detectable levels of dibromochloromethane. Actual concentrations and
detection limits were not reported.
E. Summary
Trihalomethanes are found in virtually all treated drinking water;
however, concentrations of individual forms vary widely depending on the type
of water treatment, locale, time of year, and source of the drinking water.
Chloroform is the most commonly detected THM and usually occurs at the
greatest concentrations. Chloroform concentrations in drinking water have
ranged from less than 0.5 to 550 ^ig/L (ppb). Bromodichloromethane concen-
trations in drinking water ranged from 0.2 to 183 jzg/L (ppb), while dibromo-
IV-46
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chloromethane and bromoforra concentrations ranged from less than 0.5 to
280 ^g/L (ppb). Concentrations of all trihalomethanes in drinking water were
generally lower when the raw water is derived from Around water sources rat-her
than surface water sources. In any study analyzing THMs in water, the type of
sampling method used should be considered because some methods restrict THM
formation by refrigeration or the use of quenching agents, whereas others
maximize THM formation.
Chloroform has been detected in food although data on the brominated
forms is not available in the U.S.. Chloroform was detected in food at
concentrations ranging from nondetectable to 830 ng/g (ppb), and concentra-
tions of bromodichloromethane detected in food ranged from nondetectable to
70 ng/L (0.07 ppb). No studies were located that analyzed dibromochloro-
methane and bromoform in food in the United States. Bromodichloromethane,
dibromochloromethane, and bromoform concentrations in various foods in studies
conducted in Japan and Finland ranged from undetectable to 1.7 ppb, undetect-
able to 0.6 ppb, and undetectable to 8.1 ppb, respectively. Chloroform is
approved by FDA for use in food packaging.
THMs are ubiquitous in air, although the concentrations are highly
variable depending on the ambient environment. Chloroform concentrations tend
to be higher in indoor air compared to outdoor air because of the confined
space and release of chloroform from various indoor sources. Chloroform
concentrations in personal air and outdoor air ranged from 0.06 to 215 jig/m3
(0.01-44 ppb) , and from 0.04 to 21.5 ^ig/m3 (0.008-44 ppb), respectively. One
major source of chloroform in indoor air appears be tap water that releases
chloroform when it is used for showers or washing. One study indicated that
concentrations of chloroform in shower stall air samples during a 10-minute
IV-47
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shower ranged from 10 to 500 Mg/m3 (2.05-112 ppb). The absorbed inhalation
and dermal doses were 0.24 and 0.23 jig/kg/day, respectively for a combined
absorbed chloroform dose for the 10-rcinute shower of 0.47 /ig/kg/day.
Concentrations of other THMs, in indoor or outdoor air are generally lower
than chloroform. Bromodichloromethane concentrations in personal air ranged
from 0.12 to 4.36 /jg/m3 (0.017-0.65 ppb), whereas concentrations in outdoor
air ranged from 0.006 to 1.3 ^g/m3 (0.9-194 ppt). Bromoform and
dibromochloromethane concentrations in outdoor air ranged from below the
detection limit to 3.5 Mg/m3 (0.34 ppb), and from 0.00 to 0.47 jig/m3
(0-0.06 ppb), respectively.
The use of chlorine to disinfect swimming pools and hot tubs also
results in the release of THMs to the overlying air. One study indicated that
chloroform, bromodichloromethane, dibromochloromethane, and bromoform
concentrations in swimming pool and hot tub water ranged from less than 1 to
530 /ig/L (ppb), from 1 to 105 jig/L (ppb), from 0.1 to 48 ^g/L (ppb), and from
less than 0.1 to 62 ^g/L (ppb), respectively. Concentrations of these same
THMs in the air two meters above the pool water ranged from 0.1 to 260 ng/m3
(0.2-53 (ppb)), less than 0.1 to 14 /ig/m3 (0.15-2.00 (ppb)), from less than
0.1-10 ng/nf (0.01 to 1.17 (ppb)), and from less than 0.1 to 5.0 /^g/m3 (0.014
33-0.48 ppb), respectively.
Chloroform exhaled in breath is related to body burden of chloroform
and recent exposure to chloroform in air or water. Background chloroform
concentrations measured in breath have ranged from 0.22 to 5.06 ^ig/m3
(0.05-1.04 ppb), and reported breath concentrations after a 10-minute shower
ranged from 6 to 21 ^g/m3 (1.23-4.3 ppb). THMs also have been detected in the
blood, milk and adipose tissue of humans. Chloroform concentrations in blood
IV-48
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have ranged from less chan 0.1 to greater than 25 ^g/L (ppb), and bromoform
has been detected in blood at levels up to 3.4 jig/L (ppb). Chloroform has
been detected in the milk of 7 of 49 lactacing women living in industrial
areas; however, actual concentrations were not reported.
IV-49
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V. 'HEALTH EFFECTS IN ANIMALS
A. Short-Term Exposure
1. Lethality
Table V-l summarizes reported oral LDSO values in rats and mice for
the trihalomethanes. Reported values for chloroform range from 119 to
2,000 mg/kg, with most of the values falling in the range of 908 to
2,000 mg/kg. Values for the brominated trihalomethanes range from 450 to
1.-550 mg/kg. Bowman et al. (1978) concluded that male mice were more sensi-
tive to the lethal effects of trihalomethanes than females (see Table V-l).
Hill (1978) repo.rted single-dose LD50 values for chloroform ranging from 119
to 490 mg/kg in three strains of mice. Intraperitoneal LD50 values for
chloroform in mice range from 1,780 to 3,280 mg/kg (Kutob and Plaa 1962;
Klaassen and Plaa 1967b), and an intraperitoneal LD50 of 1,490 mg/kg has been
reported in dogs (Klaassen and Plaa 1967a).
2. Other Effects •
a. Chloroform
A number of studies have indicated that the organs most affected by
acute exposure to chloroform are the liver and kidneys. Jones et al. (1958)
administered single oral doses of chloroform (in olive oil) by gavage to male
and female Swiss mice at levels from 7 to 1,100 mg/kg (10 animals/dose).
Animals were sacrificed after 72 hours and livers were examined histolo-
gically. The authors observed minimal midzonal fatty infiltration at 35 mg/kg
V-l
-------
TABLE V-l Summary of Oral Lethality of Trihalomethanes
Compound
CHC13
CHBr3
CHBr2Cl
CHBrCl2
^50
Mouse3
Male Female
1,120 1,400
119-490C
1,400 1,550
800 1,200
450 900
(mg/ke)
Ratb
Male Female
908 1,117
2,000d
1,388 1,147
1,186 848
916 969
aBowman et al. (1978) .
bChu et al. (1980).
cHill (1978).
dTorkelson et al . (1976).
V-2
-------
and minimal cencral fatty changes at 70 mg/kg. These fatty changes, in the
absence of other histopathological signs, are not considered to be adverse,
and the NOAEL for liver damage was 35 to 70 mg/kg. However, the 3-day period
between dosing and observation may have been too long to detect transient
effects at the lower doses. The authors observed moderate to severe hepatic
injury (massive fatty infiltration with necrosis) at doses of 140 to
350 mg/kg. Based on this, 140 mg/kg was identified as the LOAEL.
Hill (1978) investigated strain and sex differences in chloroform-
induced toxicity in mice. Male mice of three strains (DBA/2J, B6D2F1/J, and
C57BL/6J) were given single oral doses of chloroform in oil. No clear
difference in hepatotoxicity between strains was observed; centrilobular
necrosis occurre.d at doses greater than 250 mg/kg in all three strains. In
contrast, there were strain-specific differences in renal toxicity. Doses of
89 mg/kg caused glucosuria and/or proteinuria in half of the DBA/2J animals,
while doses of 119 and 163 mg/kg were required to produce these effects in
half the B6D2F1/J and C57BL/6J animals, respectively. Plasma levels of
testosterone in resistant strains tended to be lower than levels in
susceptible strains. The author conjectured that testosterone may act by
sensitizing the renal proximal convoluted tubules to chloroform through a
testosterone receptor mechanism. This study identified a LOAEL of 89 mg/kg,
based on renal injury in male mice of sensitive strains.
Reitz et al. (1980) administered single oral doses of 15, 60, or
240 mg CHCl3/kg to male B6C3F1 mice (two/group) and examined liver and kidney
tissues 48 hours later. Severe diffuse renal necrosis occurred after a. single
dose of 240 mg/kg, and focal tubular regeneration occurred after single doses
of 60 or 240 mg/kg. .These effects were not observed at 15 mg/kg. Liver
V-3
-------
damage (hepatocellular necrosis and swelling with inflammatory cell infiltra-
tion) was observed only at the highest dose. This study identified a NOAEL of
15 nig/kg and a LOAEL of 60 mg/kg for renal injury in male mice, but too few
animals (two/dose) were used to allow a firm conclusion.
Larson et al. (1993) administered a single oral gavage dose of
chloroform in corn oil to male F344 rats (three to five/group) and to female
B6C3F1 mice (three to five/group). Doses of 0, 34, 180, or 477 mg/kg were
administered to the rats, and 0, 34, 238, or 477 mg/kg was administered to the
mice. The kidney and liver were examined histologically 1 day posttreatment.
As an indirect quantitative measure of necrosis, cell proliferation was
determined in the mid-dose mice and the mid- and high-dose rats by determining
the labeling index, a measure of the percentage of nuclei in S-phase. Mild to
severe proximal tubular necrosis was observed in the kidney of the rats at all
treatment levels, in a dose-related manner. There was no effect on BUN or
urinary protein or glucose at any dose. The labeling index was increased
20-fold at 180 mg/kg. Centrilobular necrosis was observed in the livers of
rats in all treatment groups with a dose-related severity; the severity in the
mid- and high-dose groups was characterized as slight to moderate. The
labeling index was increased 10-fold in the livers of high-dose rats but was
unaffected in the mid-dose group. In mice, renal lesions were not observed,
but centrilobular hepatic lesions were observed with a dose-related severity
in mice treated with at least 238 mg/kg. In a separate experiment, female
mice received an oral gavage dose of 350 mg/kg and were necropsied at varying
times after treatment. The renal labeling index in these mice was increased
only 2-fold over controls, but the hepatic labeling index was increased
38-fold. The study authors noted that the organ- and species-specific pattern
of cytotoxicity and resulting cell proliferation was the same as the pattern
V-4
-------
of tumorigenicity at similar doses. Because the kidney and liver effects
observed in the low-dose rats were limited to scattered necrotic tubules and
soatt-ered necrotic foci iu the liver, the NOAEL for tats in this study was
identified as 34 mg/kg; the LOAEL was 180 mg/kg, based on liver and kidney
histopathology. In mice, the NOAEL was 34 mg/kg and the LOAEL was 238 mg/kg,
based on liver histopathology.
Larson et al. (19.94) measured cell proliferation in the liver
following administration of chloroform by gavage in corn oil or in drinking
water. Female B6C3F1 mice were administered doses of 0, 3, 10, 34, 90, 238,
or 477 mg/kg for 4 days or 5 days/week for 3 weeks, or provided drinking water
containing 0, 60, 200, 400, 900, or 1,800 ppm for 4 days or 3 weeks. The
average daily doses in the 4-day drinking water study were 0, 16, 26, 54, 81,
or 105 mg/kg/day and the average doses in the 3-week study were 0, 16, 43, 83,
184, or 329 mg/kg/day. Mice administered 34 or 90 mg/kg/day by oil gavage for
4 days exhibited mild degenerative changes in centrilobular hepatocytes; more
severe changes were observed at 3 weeks. Centrilobular necrosis was observed
at the higher doses (238 or 477 mg/kg/day in oil), with increased severity
following the longer treatment period. The hepatic labeling index was
significantly (p < 0.05) increased at both dosing durations following gavage
dosing with 238 or 477 mg/kg/day, and following 3 weeks of dosing with
90 mg/kg/day. No histopathological changes and no increase in the labeling
index was observed in any mice receiving chloroform in drinking water for
either duration. Thus, cytolethality and cell proliferation were observed-
following gavage dosing in oil, but not following drinking water administra-
tion. The study authors noted that sustained cell proliferation was observed
only under the conditions that induce tumors (oil gavage dosing; see Section
V.E.I.). Using the pharmacokinetic model of Corley et al. (1990), they
V-5
-------
predicted that the bolus dosing in oil results in a higher rate of chloroform
metabolism in the liver than would be seen following drinking water
adminis tration.
Culliford and Hewitt (1957) (reviewed in U.S. EPA 1985a) reported sex-
and strain-dependent differences in mice exposed to chloroform by inhalation.
Adult male mice of two strains (CBA and WH) developed extensive necrosis of
the renal tubules after exposure to low concentrations of chloroform vapor
(7 to 10 mg/L for 2 hours), while adult females showed no renal damage after
equivalent exposure. Assuming an inhalation absorption fraction of 0.63
(Lehmann and Hasegawa 1910), a body weight of 0.03 kg, and an inhalation rate
of 0.052 m3/day (U.S. EPA 1986), this concentration corresponds to an absorbed
dose range of 63.7 to 910 mg/kg. Adult females became fully susceptible to
renal necrosis after treatment with androgens, and the susceptibility of males
was greatly reduced by treatment with estrogens. Castration removed the
susceptibility of the males of one strain, but did not completely remove it in
another. The residual susceptibility of castrates was abolished by
adrenalectomy. Male mice under 11 days old were not susceptible to renal
necrosis, even after massive doses of androgen. Between 11 and 30 days, they
were susceptible if given androgen. Thereafter, they became spontaneously
susceptible. In contrast to thest- sex hormone-dependent effects on the
kidney, liver damage occurred in nearly all exposed mice and was not
correlated with sex hormone status.
Klaassen and Plaa (1967a) investigated the effects of ethanol pre-
treatment on chloroform-induced liver damage in dogs. Groups of four to six
mongrel dogs (7 to 14 kg) were administered either a-single dose of 4 g/kg of
ethanol by gavage or an equicaloric dose of dextrose solution. Twenty-four
V-6
-------
hours later, the animals received an intraperitoneal injection of chloroform
(300 mg/kg) or corn oil; serum glutamic pyruvic transaminase (SGPT) activity
was measured 24 hours after that. In control animals, the average SGPT level
was 29 units. The values increased slightly following exposure to either
ethanol alone (42 units) or chloroform alone (44 units). When chloroform was
given to animals pretreated with ethanol, the values increased dramatically
(1,760 units). The authors concluded that ethanol pretreatment markedly
increased the potential fo.r chloroform hepatotoxicity. These authors obtained
similar results in mice (Klaassen and Plaa 1966). The mechanism of this
potentiation is not known, but may be related to induction of enzymes by
ethanol that result in increased metabolism of chloroform.
Munson e.t al. (1982) administered chloroform (aqueous) by gavage to
male and female CD-I mice. Dosage levels of 0, 50, 125, or 250 mg/kg/day were
administered to groups of 14 to 24 animals for either 14 or 90 days.
Parameters measured included body weights, organ weights, hematology, clinical
chemistry, hepatic microsomal enzymes, and cell-mediated and humoral immunity.
In the 14-day study, dose-related increases in liver weight (both absolute and
relative to total body weight) were observed in mid- and high-dose males.
Increased relative liver weights (not dose related) were observed in all
female groups. Increases of 2- to 36-fold (mean 8.4-fold) were reported in
SGPT values in high-dose males and females and in serum glutamic oxaloacetic
transferase (SCOT) levels in high-dose females. The authors observed a
decrease in spleen antibody-forming cells at all doses in males and females
(p < 0.05), but no effects on hemagglutination titers or alterations in cell-
mediated immunity. This study identified a NOAEL of 125 mg/kg/day and a LOAEL
of 250 mg/kg/day, based on elevated serum enzyme levels. In the 90-day study,
the authors viewed differences or improvements in a number of clinical
V-7
-------
chemistry parameters compared to the 14-day study as indicative of recovery
from or induction of tolerance to chloroform during the longer experiment.
Condie et al. (1983) reported dose-dependent increases in the
incidence and severity of centrilobular cytoplasmic pallor, mitotic figures,
and focal inflammation of the liver in mice given doses of 0, 37, 74, or
148 mg/kg/day chloroform in oil for 14 days. Body weights were decreased in
animals at the highest dojse. Decreased renal uptake of PAH, accompanied by an
increased incidence of microscopic changes, were observed in the two highest
dose groups. Blood urea nitrogen and SGPT were increased in the high-dose
group.
Chu et a.l. (1982a) exposed male Sprague-Dawley rats to 0, 5, 50, or
500 ppm chloroform in drinking water for 28 days (ten animals/group). Based
on measured water intake, these doses corresponded to 0, 0.74, 7.4, or
62.9 mg/kg/day. At the end of the experiment, animals were sacrificed and the
authors measured serum biochemical and hematological parameters and hepatic
microsomal and soluble enzyme activities and performed gross histopathological
examinations. No changes were observed in any of these parameters, except a
decreased number of neutrophils in the high-dose rats (1.0±0.27 versus
0.52±0.3xl03 cells/jiL). The authors stated that they observed no treatment-
related histopathological changes, but they provided no data or details. This
study identified a NOAEL of 7.4 mg/kg/day. The significance of the decrease
in neutrophil count in the high-dose rats is not certain.
Plummer et al. (1990) compared the liver effects of exposing male
Wistar rats (12/group) for 4 weeks to 50 ppm in air for 24 hours/day,
7 days/week (continuous profile) or 275 ppm for 6 hours/day, 5 days/week
V-8
-------
(fluctuating profile). The total exposure was about 31,600 ppm-hour in each
group. Minor liver pathology was observed in both exposed groups, but quali-
tative and quantitative assessment revealed more injury in the continuously
exposed group. The continuous exposure group had microvesicular fatty change,
largely in zone 3 hepatocytes (cells furthest from the terminal" afferent
vessel), while only scattered hepatocytes containing small fat droplets were
observed in the fluctuating profile group. Necrosis scores were also higher
in the continuous profile group. The difference in toxicity was attributed to
saturation of chloroform metabolism in the fluctuating profile group. This
hypothesis was supported by data regarding the kinetics of chloroform uptake
(two rats/dose) that suggested saturation of chloroform metabolism at levels
above 100 ppm.
Table V-2 summarizes the short-term ef-fects of chlotoform.
b. Brominated Trihalomethanes
Bowman et al. (1978) studied the acute oral toxicity of bromoform,
bromodichloromethane, and dibromochloromethane in mice. Groups of ten male
(30 to 35 g) and ten female (25 to 30 g) ICR Swiss mice were treated with
doses ranging from 500 to 4,000 mg/kg. Compounds were solubilized in
emulphor: alcohol:saline (1:1:8) and administered by gavage. Ataxia,
sedation, and anesthesia occurred with bromodichloromethane or dibromochloro-
methane at 500 mg/kg, but a 1,000-mg/kg dose of bromoform was required to
produce these effects. These endpoints are insufficiently sensitive to
identify useful acute NOAEL or LOAEL values.
V-9
-------
TABLE V-2 Summary of Short-Term Health Effects Data on Chloroform
Reference Species
Jones et al. House
(1958)
Hill (1978) Mouse
Reitz et al. Mouse
(1980)
Larson et al. Rat
(1993)
Mouse
Route
Gavage
(oil)
Gavage
(oil)
Gavage
Gavage
(oil)
Gavage
(oil)
dose
Sex Duration (mg/kg/day)
M,F One dose 35-70
140-350
M One dose 89
250
M One dose 15
60
240
M One dose 34
180
F One dose 34
238
Results
Fatty liver (NOAEL)
Fatty liver, hepatic
necrosis
Renal damage (LOAEL)
Liver damage
NOAEL
Focal renal
necrosis (LOAEL)
Diffuse renal
necrosis, liver
damage
Trace liver and kidney
necrosis (NOAEL)
Liver and kidney necrosis
(LOAEL)
NOAEL
Liver necrosis (LOAEL)
Klaassen and Dog
Plaa (1967a)
Intraperi- M,F One dose
toneal
injection
Munson et al. Mouse Gavage M,F 14 days
(1982) (aqueous)
Condie et al. Mouse
(1983)
Gavage
(oil)
14 days
Chu et al.
(1982a)
Rat Drinking M,F 28 days
water
300 Slight increase in
SCOT levels;
potential ion
by ethanol
pretreatment
125 Increased liver
weight (NOAEL)
250 Increased serum
enzymes (LOAEL)
37 Decreased PAH uptake,
moderate renal and
hepatic histo-
pathotogy (LOAEL)
0.74-7.4 NOAEL (histologic,
hematologic,
biochemical
evaluation)
63 Decreased
neutrophils
V-10
-------
NTP (1987) administered single oral doses of bromodichloromechane
(150, 300, 600, 1,250, or 2,500 mg/kg) by gavage in corn oil to F344/N rats
and S6C3F1 mice (five/sex/'dose). 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
included lethargy and labored breathing. 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. This study is not
appropriate for the identification of a reliable NOAEL or LOAEL because no
histopathological or biochemical endpoints were monitored.
In a preliminary range-finding study by NTP (1985), F344/N rats and
B6C3F1 mice (five/sex/dose) were administered single doses of 160, 310, 630,
1,250, or 2,500 mg/kg dibromochloromethane in corn oil by gavage. All rats
that received 2,500 mg/kg were dead by day 3. At 1,250 mg/kg, only one of
five male rats survived, while four of five females survived. A dose of
310 mg/kg caused no deaths but produced lethargy in all animals. All mice
receiving 2,500 mg/kg, and all male mice and four of five female mice
receiving 1,250 mg/kg, died betwem days 2 and 8. No females died at
630 mg/kg or lower, while three of five males died at 630 mg/kg and one of
five males died at 310 mg/kg. Livers with discolored foci and kidneys with
dark red or pale medullae were seen more frequently in dosed animals than in
control animals. Single oral doses of 310 mg/kg of dibromochloromethane were
hazardous in this study, but mortality is an insufficiently sensitive endpoint
for the identification of a useful NOAEL or LOAEL.
V-ll
-------
NTP (1989a) investigated the acute oral toxicity of bromoform in
F344/N rats and B6C3F1 mice. Groups of five males and five females of each
species were administered a single oral dose of bromoforra (by gavage, in corn
oil) at dose levels of 125, 250, 500, 1,000, or 2,000 mg/kg. There were no
controls. In rats, 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. In mice, 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
main clinical sign observed was shallow breathing. This study is also not
appropriate for identification of a reliable NOAEL or LOAEL because no
histopathological or biochemical endpoints were monitored.
Munson et al. (1982) administered bromodichloromethane, dibromochloro-
methane, and bromoform (aqueous) by gavage to CD-I male and female mice (14 to
25/group) for 14 days at levels of 0, 50, 125, or 250 mg/kg/day. Parameters
observed included body and organ weights, hematology, serum enzyme levels
(SCOT, SGPT), and humoral and cell-mediated immune system functions. At the
high dose (250 mg/kg/day) of all three compounds, body weights were generally
decreased and SCOT and SGPT levels were clearly increased (p < 0.05). These
effects were not significant at the mid or low doses. Bromodichloromethane
and dibromochloromethane appeared to affect the humoral immune system, as
judged by decreased antibody-forming (ABF) cells in serum and by decreased
hemagglutination titers. 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. The authors judged that the
humoral immune system was not significantly affected by bromoform (although a
decrease in ABF cells was reported for high-dose males). On the basis of
decreased immune function in females, this study identified a NOAEL of
50 mg/kg/day and a LOAEL of 125 mg/kg/day for bromodichloromethane and
V-12
-------
dibromochloromethane. For bromoform, this study identified a NOAEL of
125 mg/kg/day and a LOAEL of 250 mg/kg/day, based on elevated serum enzymes.
Condie et al. (1983) investigated the renal and hepatic toxicity of
the brominated trihalomethanes. Groups of 8 to 16 male CD-I mice were admin-
istered the chemicals by gavage, using corn oil as the vehicle, for 14 days.
Doses tested were 0, 37, 74 or 148 mg/kg/day for bromodichloromethane; 0, 37,
74, or 147 mg/kg/day for _dibromochloromethane; and 0, 72, 145, or 289 mg/kg/
day for bromoform. Biochemical evidence of liver and kidney damage (decreased
PAH uptake by kidney slices and elevated SGPT release from liver) was consis-
tently observed at the high dose but not at the mid or low doses (Table V-3).
Similarly, histological examinations revealed no consistent or important
changes at the low or mid doses, with slight to moderate liver and kidney
injury at the high dose (Table V-4). On this basis, this study identified
NOAEL values of 74, 74, and 145 mg/kg/day and LOAEL values of 148, 147 and
289 mg/kg/day for bromodichloromethane, dibromochloromethane, and bromoform,
respectively.
NTP (1987) administered doses of 0, 38, 75, 150, 300, or 600 mg/kg/day
of bromodichloromethane in corn oil by gavage to F344/N rats (five/sex/dose)
for 14 days. A necropsy was performed on all animals. One of five female
rats that received 38 mg/kg and one of five female rats that received
600 mg/kg died before the end of the study. All rats receiving 600 mg/kg 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. In the 300-mg/kg groups, body weights in males
and females decreased by 21% and 7%, respectively. At 600 mg/kg, body weights
decreased by 44% and 22%, respectively. Renal medullae were reddened in five
V-13
-------
TABLE V-3 Effects of Brominated Trihalomethanes in
Mice Dosed by Gavage for 14 Days
Chemical
CHBrCl2
CHBr2Cl
CHBr3
Dose
(mg/kg)
0
37
74
148
0
37
74
147
0
72
145
289
Mean
Body Weight
Gain (g)
2
0
1
-0
-0
0
0
-1
0
0
1
2
.0
.7
.2
.3
.1
.1
.2
.3
.8
.8
.1
.7
PAH Uptake
(slice/ BUN
medium ratio) (mg%)
18.
18.
13.
8.
14.
12.
14.
10.
19.
18.
18.
13.
3
1
4a
8a
3
5
0
4a
1
5
1
3a
24
20
20
24
20
24
26
25
22
22
23
25
.6
.7a
.5a
.0
.1
.6
.2
.2
.9
.4
.9
.0
Creatinine SGPT
(mg%) (Units/L)
0
0
0
0
0
0
0
0
0
0
0
0
.44
.41
.47
.51
.49
.49
.55
.48
.42
.46
.38
.42
20
26
21
51a
16
16
21
35a
21
21
17
30a
ap < 0.05 compared to control.
Adapted from Condie et al. (1983).
V-14
-------
Table V-4 Incidence and Severity of Microscopic Changes in Kidney and Liver
of Mice Dosed with Brominated Trihalomethanes for 14 Days
Kidney
Dose
Compound (mg/kg/day)
Number
of
Animals
Examined
Intra-
tubular
Mineral-
ization
Epithe-
lial
Hyper-
plasia
Cyto-'
megaly
Mesan-
gial
Hyper-
trophy
Mesan-
gial
Neph-
rosis
Centri-
lobular
Pallor
Liver
Mitotic
Figures
Focal
Inflam-
mation
Cyto-
plasmic
Vacuo-
lation
Severity of Chanae3
Corn oil
(vehicle)
CHBrCl2
CHBr2Cl
CHBr3
0
37
74
148
37
74
147
72
145
289
16
9
10
10
5
10
10
5
10
10
ABCD
0000
0000
0100
2111
0000
0000
0000
0000
0000
1000
ABCD
2000
1000
0100
1521
0000
0000
2110
0000
1000
3200
ABCD
0000
0000
1000
5100
0000
0000
0000
0000
0000
0000
ABCD
0000
0000
0000
0000
4000
7000
4300
0000
2000
1000
ABCD
0000
0000
0000
0000
0000
0000-
0000
0000
4100
6100
ABCD
0000
2000
6210
5220
0000
0000
0000
0000
6000
5111
ABCD
0000
0000
1320
0100
0000
1010
3100
0000
1000
1010
ABCD
1000
2000
4000
5110
0000
1000
1000
0000
1000
3300
ABCD
1000
0000
0000
1000
2100
4000
3500
2000
0000
0100
Categories of severity ratings:
A ~ minimal
B = slight
C = moderate
D = moderately severe/severe.
Numbers of animals in each category are listed in columns.
Adapted from Condie et al. (1983).
-------
of five male rats in the 600-mg/kg group, one of five female vehicle controls.
one of five females in the 38-mg/kg group, and one of five females in the
600-mg/kg group. Based on body weight; gain, this study identified a NOAEL of
150 mg/kg/day and a LOAEL of 300 mg/kg/day in rats.
In a similar experiment, NTP (1987) administered doses of 0, 19, 38,
75, 150, or 300 mg/kg/day broraodichloromethane in corn oil by gavage to
B6C3F1 mice (five/sex/doae) for 14 days. All male mice that received 150 or
300 ntg/kg bromodichloromethane died before the end of the study. 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 of five males
receiving 150 mg/kg, five of five males receiving 300 mg/kg, and one of five
females receiving 150 mg/kg. Based on behavior, appearance, gross necropsy,
and mortality, this study identified a NOAEL of 75 mg/kg/day in mice.
In a 14-day study by NTP (1985), groups of F344/N rats (five/sex/dose)
were administered 0, 60, 125, 250, 500, or 1,000 mg/kg/day dibromochlorome-
thane in corn oil by gavage. Animals were observed twice daily for mortality
and were weighed once per week. Necropsies were performed on all animals.
All of the rats of each sex that rt-ceived 1,000 mg/kg dibromochloromethane and
all of the female rats that received 500 mg/kg were dead by day 6. No deaths
occurred at 250 mg/kg/day. Lethargy, ataxia, and labored breathing were
observed in rats of each sex that received 500 or 1,000 'mg/kg. Mottled livers
and reddened, darkened renal medullae were seen at gross necropsy in male and
female rats administered 500 or 1,000 mg/kg, but no gross lesions were obser-
ved in animals dosed with 250 mg/kg/day or less. Based on behavior, gross
pathology, and mortality, this study identified a NOAEL of 250 mg/kg/day.
V-16
-------
In a parallel study in B6C3F1 mice (NTP 1985), groups of five animals/
sex/dose were administered doses of 0, 30, 60, 125, 250, or 500 mg/kg/day of
dibromochloromethane in corn oil by gavage for 14 dzys. Deaths of four of
five males and three of five females that received 500 mg/kg were considered
to be compound related. Clinical signs at this dose included lethargy,
ataxia, and labored breathing. Mottled livers and reddened renal medullae
were seen in male and female mice administered 500 mg/kg; these changes were
considered to be compound related. Stomach lesions (white papillomatous
nodules) were seen in male mice that received 125, 250, or 500 mg/kg and in
female mice that received 250 or 500 mg/kg. Based on gross lesions, this
study identified a NOAEL of 60 mg/kg/day and a LOAEL of 125 mg/kg/day in mice.
NTP (1989a) investigated the subacute oral toxicity of bromoform in
F344/N rats and B6C3F1 mice. Groups of male and female rats (five/sex/dose)
and female mice (five/dose) 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. All rats that received
600 or 800 mg/kg/day and one male rat that received 400 mg/kg/day died before
the end of the study. These rats exhibited lethargy, labored breathing, and
ataxia. Rats receiving 400 mg/kg/day had final body weights 14% less than
controls. In mice, no effects were detected at 200 mg/kg/day, but ataxia and
lethargy were noted at 600 mg/kg/day. One male died at 600 mg/kg/day, and one
female died at 800 mg/kg/day. Raised stomach nodules were observed in males
and females at doses of 400 mg/kg/day or higher. Based on body weight and
mortality in rats and stomach nodules in mice, this study identified a NOAEL
of 200 mg/kg/day and a LOAEL of 400 mg/kg/day.
V-17
-------
Chu et al. (1982a) administered bromodichloromethane, dibromochloro-
methane, or bromoform to male Sprague-Dawley rats (150 to 200 g) in drinking
water for 28 days at dose levels of 0, 5, 50, or 500 ppm (10 animals per
group). Based on recorded fluid intake, these levels corresponded to doses of
0.8, 8.6, or 68 mg/kg/day, as calculated by the authors. The authors observed
no effects on growth rate or food consumption from any of the compounds and no
signs of toxicity throughout the exposure. No dose-related biochemical or
histological changes were, detected (no data provided). This study identified
a NOAEL of 68 mg/kg/day for each of the brominated trichloromethanes, but the
reported data were too limited to allow a. firm conclusion.
Aida et al. (1992a) investigated the effects of administering
bromoform, dibrojnochloromethane, or bromodichloromethane to groups of seven
male and seven female Slc:Wistar rats for 1 month. The test material was
microencapsulated and mixed with powdered feed; placebo granules were used for
the control groups. Clinical effects, body weight, food consumption, hemato-
logical parameters, serum chemistry, and histopathology of all major organs
were determined.
Bromodichloromethane was administered at dietary levels of 0%, 0.024%,
0.072%, or 0.215% of diet for males, and 0%, 0.024%, 0.076%, or 0.227% of diet
for females. Based on the mean food intakes, the study authors reported the
mean compound intakes for the 1-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. Body weight
gain was significantly (p < 0.01) decreased in the high-dose groups. 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 in the low-dose groups were limited to decreased
V-18
-------
Laccate dehydrogenase (LDH) levels, but the biological significance of this
effect is unclear. As shown in Table V-5, other significant, dose-related
changes included 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
cholesterol levels were within normal ranges at all doses. Chemical-related
lesions were limited to the liver and were rated as very slight or slight.
Histopathology was mostly.-..confined to the high-dose groups, and the incidence
was lower than for the other two brominated trihalomethanes (Table V-6). The
vacuolization observed in mid-dose females, and in single low-dose males was
not considered an adverse effect. Other observed effects included swelling of
hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct
proliferation. JExcept for very slight to slight changes in individual low-
dose males, these lesions were observed only in high-dose males and females.
There was no effect on any hematological parameter. Based on the histopath-
ology observed in high-dose males and females, the LOAEL identified in this
study for bromodichloromethane in rats was 189 mg/kg/day in males and
204 mg/kg/day in females; the NOAEL was 62 (males) and 66 (females) mg/kg/day.
Dibromochloromethane was administered at dietary levels of 0%, 0.020%.
0.062%, or 0.185% of diet for males, and 0%, 0.038%, 0.113%, or 0.338% of diet
for females. Based on.the mean food intakes, the study authors reported the
doses as 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. Body weight gain was significantly (p < 0.01)
reduced in high-dose females. High-dose females also exhibited slight
piloerection and emaciation. Dose-related increases in relative liver weight
were observed in males at the high dose and females of all dosing groups, and
relative kidney weight increased in the high-dose females. Significant
V-19
-------
decreases in alkaline phosphatase (AP) and LDH were observed, but the biolo-
gical significance of these changes is unclear (Table V-5). Significant.
dcss-related changes in serum biochemistry included reduced nonesterified
fatty acids (FAA) in high-dose males, reduced total serum triglycerides
(T-GLY) in high-dose males and females, and increased cholesterol in mid- and
high-dose males and in females at all dosing levels. However, the cholesterol
levels were within normal ranges at all dose levels. Serum cholinesterase
activity was also significantly decreased in high-dose males and mid- and
high-dose females. Liver cell vacuolization was 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
severity of the effects at the low dose were similar to those observed in the
control groups, and were not considered adverse. Swelling and single cell
necrosis were also observed, largely in the high-dose groups (Table V-6).
There was no effect on any hematological parameter. Based on the
histopathology and serum biochemistry results, NOAELs of 56 (males) and 101
(females) mg/kg/day and LOAELs of 173 (males) and 332 (females) mg/kg/day were
identified for dibromochloromethane in rats.
Bromoform was administered at dietary levels of 0%, 0.068%, 0.204%, or
0.612% of diet for males, and 0%, 0.072%, 0.217%, or 0.651% of diet for
females. 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. Body weight gain was significantly.
(p < 0.01) reduced in high-dose males, and high-dose animals of both sexes
exhibited slight piloerection and emaciation. Relative liver weight was
significantly (p < 0.05) increased in mid- and high-dose males and females.
Serum glucose, T-GLY, blood urea nitrogen (BUN), and cholinesterase levels
V-20
-------
Table V-5 Serum Biochemical Levels8 in Rats Fed Brominated
Trihalomethanes for One Month
Dietary
Chemical Sex Level (X)
CHBrCl2 M 0
0.024
0.072
0.215
F 0
0.024
0.076
0.227
CHBr2Cl. M 0
0.020
0.062
0.185
F 0
0.038
0.113
0.338
CHBr3 M 0
0.068
0.204
0.612
F 0
0.072
0.217
0.651
Blood
Urea
Nitrogen
16±2
15±1
14±2
14±1
15±1
15±2
15±3
15±2
18±2
17±1
18±2
17±2
19±1
19±1
18±1
20±1
17±2
19±3
18±2
17±2
18±3
15±3
13±2*
12±3**
Tri-
glycerides
170±51
142±13
129±20
49 ±18**
92±25
89±24
60±12
38±6**
129±24
124±16
112±16
65±20**
63±14
62±10
60±10
58±6*
99±26
84±25
74±16
31±4"
75±14
62 ±14
45±4"
36±4**
Cholesterol
52±5
58±3
65±3**
60±7*
92±60
67±8
69±2
62±5
58±2
59±4
68±4**
74±6**
59±5
67±7*
74 ±5**
74±6**
46±4
52±5
57±6**
58±7**
62±8
67±3
76±6**
71±6**
Nonesterif led
Fatty
Acids
0.37±0.12
0.37±0.06
0.46±0.15
0.25±0.09
0.25±0'.04
0.29±0.07
0.27±0.05
0,22±0.07
0.52 ±0.14
0.50±0.04
0.40±0.09
0.31±0.06*
0.54±0.17
0.50±0.13
0.56±0.11
0.39±0.15
0.34±0.07
0.30±0.05
0.33±0.09
0.29±0.07
0.43±0.07
0.40±0.10
0.32±0.08
0.36±0.10
Serum
Glucose Cholinesterase
165±17
159±15
157±26
138±9*
114±36
126±10
130±6
119±5
119±9
113±7
116±6
117±6
85±12
82±12
76±9
87±12
151±14
136±10*
139±10
117±6**
128±25
114±13
106±14*
105±9*
547±208
463±42
489±79
328±131*
1449±257
1300±172
939±lll"
498±118**
600±108
624±73
542±55
399±76"
1459±67
1320±73
1004±31**
748±124"
537±97
466±87
378±50
301±22**
1306±161
1029±105**
723±60**
624±83**
aResults from seven animals/sex/dose. Cholinesterase activity expressed as mU/mL; other parameters as mg/clL.
*p less than 0.05 compared to control
**p less than 0.01 compared to control
Adapted from Aida et al. (1992a).
-------
Table V-6 Incidence and Severity of Microscopic Changes in the Liver
of Rats Dosed with Brominated Trihaloinethanes for One Month
ro
to
Hepatocyte
Dietary Vacuoli- Hepatocyte Focal
Compound Sex Level (%) zation Swelling Necrosis
Single
Cell
Necrosis
Hepatic Bile Duct
Cord Prolifer.1-
Irregularity tion
Severity of Change3
CHBrCl2 M 0.024
0.072
0.215
F 0.024
0.076
0.227
CHBr2Cl M 0
0.020
0.062
0.185
F 0
0.038
0.113
0.338
CHBr3 M 0.068
0.204
0.612
F 0
0.072
0.217
0.651
ABCD
0100
0000
2400
0000
3200
2300
3000
4100
4300
0061
6000
4100
1600
0052
1000
0430
0025
2000
2410
0016
0016
ABCD
0100
0000
2300
0000
0000
1100
0000
0000
0000
0006
0000
0000
2000
0700
0000
2000
2210
0000
2100
4300
0700
ABCD
0000
0000
0001
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
ABCD
pooo
0000
4100
.0000
' 0000
0000
0000
1000
0000
3300
0000
0000
0000
4000
0000
2000
1000
0000
0000
0000
0000
ABCD
0000
0000
3000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
4100
6100
0000
0000
0000
0000
ABCD
0100
0000
1100
0000
0000
1100
0000
0000
0000
0000
0000
0000
0000
0000
0000
6000
511.1
0000
0000
0000
0000
aCat.egories of severity ratings:
A = very slight change B = slight change C = moderate change D = remarkable change
Numbers of animals in each category are listed in cplumns; seven animals were tested per dose.
Adapted from Aida et al. (1992a).
-------
were decreased in males and females in a dose-related manner, while
phospholipids (males only) and cholesterol were increased in the mid- and
high-dose groups (Table V-5). The only change of clear biological signifi-
cance at the low dose was a decrease in cholinesterase activity in females.
LDH and AP activity and creatinine levels were decreased at the low dose in
females, but the physiological significance of these effects is unclear.
There was no effect on any hematological parameter. .Chemical-related lesions
were limited to the liver^ (Table V-6). 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. Females were more
sensitive, but the histopathology observed in all low-dose females was not
considered an adverse effect. Based on the histopathology and serum chemistry
changes in the mid-dose animals, NOAELs of 62 mg/kg/day for males and
56 mg/kg/day for females, and LOAELs of 187 and 208 mg/kg/day for males and
females, respectively, were identified for bromoform in rats.
Balster and Borzelleca (1982) investigated the effects of trihalo-
methanes on behavior in mice. Groups of six to eight adult male ICR mice (19
to 24 g) were dosed (aqueous) by gavage for 14 or 90 days with bromodichloro-
methane (1.2 or 11.6 mg/kg/day), dibromochloromethane (1.0 or 10.0 mg/kg/day),
or bromoform (0.9 or 9.2 mg/kg/day). .These treatments caused no significant
effects in a. number of behavioral tests (swimming endurance, screen test,
hole-board). Dosing with these compounds for 30 days at 100 mg/kg/day also
produced no effect on passive avoidance learning. Animals dosed for 60 days
with 100 mg/kg/day of bromodichloromethane or bromoform exhibited decreased
response rates (p < 0.05) in an operant behavior test; all three brominated
trihalomethanes produced this decrease after dosing for 60 days at
400 mg/kg/day. The greatest effects were observed early in the regimen, with
V-23
-------
no evidence of progressive deterioration. In fact, partial tolerance was
observed in all cases. Based on behavioral effects, this study identified a
subacute (30-day) NOAEL of 100 mg/kg/day for all three brominated
trihalomethanes.
Tables V-7, V-8, and V-9 summarize the short-term effects of the
brominated trihalomethanes.
B. Longer-Term Exposure
1. Chloroform
Chu et aJL. (1982b) administered chloroform in drinking water
to weanling Sprague-Dawley rats (94 to 100 g) for 90 days. Animals
(20/sex/group) received 0, 5, 50, 500, or 2,500 ppm chloroform, corresponding
to approximately 0, 0.7, 6, 50, or 180 mg/kg/day. After 90 days, one-half of
each group was sacrificed; the remaining rats were observed for an additional
90 days. At the high dose, increased mortality, decreased growth rate, and
decreased food intake were reported. Mild to moderate liver and thyroid
lesions were observed in the prerecovery groups, but these were not signifi-
cantly different from controls except the thyroid lesions at the high dose in
males. After the 90-day recovery period, these changes became very mild and
did not differ significantly from controls. No significant changes were
observed in serum biochemical profiles or hematological parameters. This
study identified a NOAEL of 50 mg/kg/day.
Bull et al. (1986) studied the effect of vehicle on the hepatotoxicity
of chloroform in mice. Chloroform was administered by gavage to 80 male
V-24
-------
TABLE V-7 Summary of Short-term Health Effects Data
on Bromodichloromethane
Reference
Bowman et a I.
(1978)
NTP
(1987)
Munson et al.
(1982)
Condie et al.
(1983)
NTP
(1987)
Chu et al.
Aida et al.
(1992a)
Aida et al.
(1992a)
Balster and
Borzelleca
(1982)
Species
Mouse
Rat
Mouse
Mouse
Mouse
Rat
•
Mouse
Rat
Rat
Rat
Mouse
Route Sex
Gavage M,F
(aqueous)
Gavage M.F
(oil)
Gavage M,F
(aqueous)
---.
Gavage M
(oil)
Gavage M,F
(oil)
Gavage M,F
Drinking M
Diet M
Diet F
Gavage M
(aqueous)
Duration
One dose
One dose
. ". days
U days
14 days
14 days
28 days
1 month
1 month
30 days
60 days
90 days
Dose
(mg/kg/day)
500
150-300
600
1,250-2,500
50
125
250
37-74
148
38-150
300
600
19-75
150-300
0.8-68
21-62
189
21-66
204
100
100-400
12
Results
Ataxia, sedation
No deaths
Some deaths
Complete mortality
NOAEL
Decreased immune
function
Increased serum
enzymes
NOAEL
Decreased PAH
uptake, moderate
liver and kidney
histopathology
(LOAEL)
NOAEL
Decreased body
weight gain (LOAEL)
Hyperactivity,
renal pathology
NOAEL
Mortality, renal
histopathology
NOAEL
NOAEL
Liver
histopathology
(LOAEL)
NOAEL
Liver
histopathology
(LOAEL)
NOAEL
Decreased operant
response
NOAEL
V-25
-------
TABLE V-8 Sununary of Short-term Health Effects Data
on Dibromochloromethane
Reference
Bowman et al.
(1978)
NTP (1985)
NTP (1985)
Munson
et al.
(198Z)
Condi e
et. al.
(1983)
NTP (1985)
Chu et al.
(1982a)
A Ida et al.
Aida et al.
(1992a)
Balster and
Borzelleca
(1982)
Species Route
Mouse Gavage
(aqueous)
Rat Gavage
(oil)
Mouse Gavage
(oil)
Mouse Gavage
(aqueous)
Mouse Gavage
(oil)
Rat Gavage
(oil)
Mouse Gavage
(oil)
Rat Drinking
water
Rat Diet
Rat Diet
Mouse Gavage
(aqueous)
Sex Duration
M,F One dose
M,F One dose
M,F One dose
' M,F U days
M 14 days
M,F 14 days
M,F 14 days
M 28 days
M 1 month
F 1 month
M 30-60 days
60 days
90 days
Dose
(mg/kg/day)
500
160
310
630-2.500
160
310
50
125
250
37-74
147
60-250
500-1,000
30-60
125
500
0.8-68
18-56
173
34-101
332
100
400
10
Results
Ataxia, sedation
No clear effects
Lethargy
Mortality
No clear effects
20% mortality
NOAEL
Decreased immune
f unct i on
Increased serum
. enzymes
NOAEL
Decreased PAH
uptake, moderate
liver and kidney
histopathology
(LOAEL)
NOAEL
Mortality; liver
and renal gross
pathology
NOAEL
Stomach lesions
(LOAEL)
Mortality, liver
and renal gross
pathology
NOAEL .
NOAEL
Liver histo-
pathology, serum
enzymes (LOAEL)
Relative liver
weight increased
(NOAEL)
Liver histo-
pathology, serum
enzymes (LOAEL)
NOAEL
Decreased operant
response
NOAEL
V-26
-------
TABLE V-9 Summary of Short-term Health Effects Data
on Bromoform
Reference
Bowman et a I .
(1978)
NTP (1989a)
NTP <1989a)
Munson et a I .
(1982)
Condi e
et al.
(1983)
Species Route Sex
Mouse Gavage M,F
(aqueous)
Rat Gavage M,F
(oil)
House Gavage M,F
(oil)
House Gavage M,F
(aqueous)
House Gavage M
(oil)
Duration
One dose
One dose
One dose
14 days
14 days
Dose
(mg/kg/day)
500
1.000
'500
1,000
250
500
50-125
250
72-145
289
Results
NOAEL
Ataxia, sedation
No deaths
60% mortality
No deaths
10% mortality
NOAEL
Increased serum
enzymes
NOAEL
Decreased PAH
uptake, moderate
histopathological
changes (LOAEL)
NTP (1989a)
NTP (1989a)
Chu et al.
Aida et al.
(1992a)
Balster and
Borzelleca
(1982)
Rat Gavage
(oil)
House Gavage
(oil)
Rat Drinking
Rat Diet
House Gavage
(aqueous)
H,F 14 days
M,F 14 days
H 28 days
M,F 1 month
H 30 days
60 days
90 days
200
400
600
200
400
600
0.8-68
62 (M)
56 (F)
187 (M)
208 (F)
100
100-400
9
NOAEL
Decreased body
weight gain, 1/5 died
100% mortality
NOAEL
Stomach nodules
Ataxia, lethargy.
1/5 died
NOAEL
NOAEL
•
Hepatic vacuol-
ization, serum
chemistry (LOAEL)
NOAEL
Decreased operant
response
NOAEL
V-27
-------
(8 weeks old) and 80 female (6 weeks old) B6C3F1 mice at doses of 60, 130, or
270 mg/kg/day for 90 days, using either corn oil or 2% emulphor as the
vehicle. At sacrifice, body and organ weights were measured (Table V-10), and
blood was recovered to perform several serum chemistry measurements. The
liver was sectioned for histopathological examination. In the group given
chloroform in corn oil, mice administered 270 mg/kg/day displayed significant-
ly elevated SCOT levels (p < 0.05, see Table V-10), a significant degree of
diffuse parenchymal degeneration (5 of 10 males and 1 of 10 females), and mild
to moderate early cirrhosis (5 of 10 males and 7 of 10 females). Serum tri-
glyceride levels were significantly decreased (p a 0.05) for males adminis-
tered 130 or 270 mg/kg/day and for females administered 270 mg/kg/day
(Table V-ll). Total liver lipid was elevated in males (not significantly) and
females (p & 0.0.1) at 60 mg/kg/day (Table V-12) . Significant pathological
lesions were not observed in the animals administered corn oil without chloro-
form or in mice receiving chloroform in 2% emulphor. These data suggested to
the authors that administration of chloroform by corn oil gavage results in
more marked hepatotoxic effects than when it is provided in an aqueous suspen-
sion. The authors could not determine if this was due to differences in
absorption kinetics or if the effect resulted from an interaction between
chloroform and the corn oil vehicle. These data identified 270 mg/kg/day
chloroform as a LOAEL when given in oil but as a NOAEL when given in aqueous
vehicle.
Jorgenson and Rushbrook (1980) exposed 6-week-old male Osborne-Mendel
rats weighing 190 g to chloroform in drinking water for 30, 60, or 90 days.
Animals received chloroform at levels of 200, 400, 600, 900, or 1,800 ppm
(30 animals/dose). There were two control groups of 40 animals each; one
V-28
-------
TABLE V-10 Effects of Vehicle on the Subchronic Toxicity
of Chloroform in B6C3F1 Mice
Chloroform Dose Male Female
(mg/kg/day) 2% Emulphor Corn Oil 2% Emulphor Corn Oil
Final Body Weight (g + SEM)
0 33.6 + 0.5 35.5 + 0.4b-c 26.5 ± 0.6 25.4 ± 0.4
60 31.0 + 0.8 31.7 ± 0.4 25.4 ± 0.6 25.5 ± 0.7
130 31.6 + 0.8 30.6 + 0.5 26.4 ± 0.6 26.0 ± 0.3
270 29.3 +~0.6C • 26.6 ± 0.3b-c 25.4 ± 0.3 24.0+0.7
Liver Weight (g + SEM)
0 1.29 ± 0.03 1.17 + 0.03b 0.98+0.03 1.00 ± 0.04
60 1.18 ± 0.03 1.32 + 0.03b-c 1.09 ± 0.04C 1.19 + 0.05C
130 1.33 + 0.05 1.36 ± 0.03C 1.13 ± 0.04C 1.22 + 0.03C
270 1.36 + 0.03 1.50 ± 0.02b-c 1.22 + 0.03C 1.39 ± 0.03b-c
Liver/Body Weight Ratiod (g/lOOg + SEM)
0 4.02 ± 0.49 3.58 ± 0.10b- 4.32 ± 0.10 4.38 + 0.15 .
60 4.28 + 0.06 4.67 ± 0.06b-c 4.84 + 0.10C 5.25 + 0.10b-c
130 4.84+0.11° 5.09+0.09C 4.94 + 0.12C 5.47 + 0.08b-c
270 5.34 + 0.07C 6.64 + 0.09b'c 5.58 + 0.12C 6.86 + 0.16b-c
Liver/Brain Weisht Ratio (g/g + SEM)
0
60
130
270
2.31 + 0.30
2.47 + 0.04
2.72 + 0.10
2.87 + 0.06C
2.33 + 0.05b
2.72 + 0.07b-c
2.74 + 0.05b
3.10 + 0.07c-e
1.90 + 0.05
2.14 + 0.08C
2.15 + 0.08C
2.42 + 0.05C
1.96 + 0.07
2.31 + O.llc
2.39 + 0.05c-e
2.83 + 0.03b-c
aFor each sex and dose level, significant differences between emulphor and
corn oil groups of p & 0.05 or p * 0.01, based on Student's t-test.
Number of animals per group = 9 or 10.
bp s. 0.01, as in footnote a.
cSignificantly different from corresponding control at p s 0.05 by one-way
analysis of variance and pairwise Student's t-test.
dLiver/body weight ratios were determined using the fasted body weights rather
than the final body weights.
ep s. 0.05, as in footnote a.
Adapted from Bull et al. (1986).
V-29
-------
TABLE V-ll Comparison of Clinical Chemistry Parameters of Mice Treated
with Chloroform in Corn Oil Versus Emulphor for 90 Days
Chloroform Dose Male
(mg/kg/day) 2% Emulphor
Corn
Oil
Female
2% Emulphor
Corn Oil
SCOT (mU/mL)
Control
60
130
270
284 ± 53a
142 -±- 32b
176 ± 43
167 + 31
181 ±
158 ±
116 ±
298 ±
36
31
14
31b,d
151 ± 27
113 ± 26
169 ± 25
117 ± 17
193 ± 22
126 ± 16
127 ± 14C
335 ± 60b-d
Triglvcerides (rag, %)
Control
60
130
270
58 ± 8
75 ± 12
54 ± 6
60 ± 4
80 ±
75 ±
60 ±
43 ±
10
4
5b
3b,d
63 ± 10
62 ± 9
50 ± 4
55 ± 3
64 ± 5
65 ± 5
55 ± 5
41 ± 3b-d
aMean ± Standard error of the mean (SEM). Four to 10 animals per group.
bSignificantly different from corresponding control at p s. 0.05.
cFor each sex and dose level, significant differences of p s 0.05 between
emulphor and corn oil groups were based on Student's t-test.
dSignificant differences at p < 0.01, as in footnote c.
Adapted from Bull et al. (1986).
V-30
-------
TABLE V-12 Effect of Vehicle on Chloroform-Induced Accumulation of
Lipid in the Liver of B6C3F1 Mice
Chloroform Dose
(mg/kg/day)
0
60
130
270
Male Female
2% Emulphor Corn Oil 2% Emulphor Corn Oil
6.6 ± 1.1 7.9 ± 0.7 6.8 ± 0.4 7.5 ± 0.8
6.8+0.6 9.4+0.8 7.5+0.3 13.1 + l.lb
7.6_.+ 0.7 7.8 + 0.4 6.9 + 0.3 9.2 + 0.8
5.3 + 0.6 8.0 + 0.3_ 6.0 + 0.4 8.4 + 0.4
aAverage lipid content in percent net-weight ± S.D. Nine or 10 animals per
group.
bSignificantly different from corresponding control at p s 0.01.
Adapted from BuJ.1 et al. (1986).
V-31
-------
group was an ad libitum control, and the second group matched the water con-
sumption of the high-dose group. Based on reported water intakes, the levels
of chloroform administered corresponded to doses of 0, 20, 38, 57, 81, or
160 mg/kg/day. The authors then examined body weight, kidney-fat-to-kidney-
weight ratios, serum biochemical parameters, and gross and microscopic
pathology findings. Treatment decreased the body weight gain of rats receiv-
ing the high concentration by 25% compared to the ad libitum control, but
there was no clear decrease compared to the matched control. Therefore, the
decreased body weight was probably secondary to decreased water consumption,
probably due to poor palatability. No effect was observed on the percentage
of kidney fat. Most serum biochemical parameters were unchanged, but some
showed changes at doses of 400 to 1,800 ppm. The authors judged these changes
to also be secondary to reduced water intake. Gross and microscopic pathology
findings generally were slight or mild in severity, not dose related, and
either appeared adaptive (occurred in rats sacrificed after 30 or 60 days, but
not in those sacrificed after 90 days) or were sporadic (by nature and/or
incidence) and not considered related to treatment. These data indicate that
dose's up to 160 mg/kg/day are without significant adverse effect, identifying
160 mg/kg/day as the NOAEL.-
Jorgenson and Rushbrook (1980) performed a similar experiment using
6-week-old B6C3F1 female mice (19 g). Groups of 30 animals were given water
containing 200, 400, 600, 900, 1,800, or 2,700 ppm chloroform for 30 to
90 days. Body weight and water consumption were monitored throughout. Using
reported water intakes, calculated dose levels were approximately 0, 32, 64.
97, 145, 290, or 436 mg/kg/day. Groups of 10 animals were sacrificed at 30.
60, or 90 days, organ fat/organ weight ratios were measured, and gross and
microscopic pathologic examinations were performed. Mice receiving 900,
V-32
-------
1,800, or 2,700 ppm sustained body weight losses during the first week, but
all body weights thereafter were comparable to those of controls. Because
there was considerable variability in water consumption, dose-dependency was
not evident. Liver fat increased significantly at the 2,700-ppm level
throughout the study, ranging from 160% to 250% of the control value. Gross
pathological examinations revealed occasional, Very slight hemorrhaging in the
lungs of mice from all dose levels. Histologically,.mild centrilobular fatty
changes in mouse livers appeared in the two highest dose groups. Reversible
fatty changes also appeared at doses as low as 64 mg/kg/day at the 30-day
sacrifice. Values for SCOT and LDH (lactate dehydrogenase) in plasma were
highly variable but tended to decrease at the high-dose levels. The authors
also observed extramedullary hematopoiesis in the liver, but did not consider
this effect to be related to treatment because it was sporadic and not dose-
related. Lymphoid atrophy of the spleen at the high dose was considered
treatment-related. These data indicate that the dose of chloroform of
290 mg/kg/day produced mild adverse effects on liver and other tissues
(LOAEL), while doses of 145 mg/kg/day or lower were without significant effect
(NOAEL).
Heywood et al. (1979) administered chloroform to beagle dogs orally in
a toothpaste base in gelatin capsules 6 days/week for 7.5 years, followed by a
20- to 24-week recovery period. Groups of 16 males and females received
0.5 mL/kg/day of the vehicle (toothpaste without chloroform), and eight dogs
of each sex.remained untreated. Treated groups, comprising eight dogs of each
sex, received doses equivalent to 15 or 30 mg chloroform/kg/day in the tooth-
paste vehicle; another group of the same size received an alternative
nonchloroform toothpaste (0.5 mL/kg/day). Eleven of the 96 dogs died during
the study, one from each chloroform-treated group. SGPT levels rose moder-
V-33
-------
ately in che high-dose group during the treatment period. The effect was
detectable after 6 weeks of exposure (34 versus 29 mU/mL, p < 0.05) and
reached a peak in the sixth year of the study (134 versus 49 mU/mL,
p < 0.001). Slight, but statistically significant, increases in SGPT were
also detected after 130 weeks in the low-dose group. The authors concluded
that this effect probably corresponded to minimal liver damage. The most
prominent histopathological effect was the presence of hepatic "fatty cysts"
which, although observed .in the control groups, were larger and more numerous
in the treated animals (Table V-13). Nodules of altered hepatocytes were also
observed in all groups, but the frequency of nodules was not obviously dose
related (Table V-13). Based on SGPT levels and increased frequency of fatty
cysts, this study identified a LOAEL of 15 mg/kg/day.
Palmer et al. (1979) dosed groups of Sprague-Dawley rats (50/sex/dose)
by gavage (toothpaste vehicle) with 0 or 60 mg/kg/day chloroform, 6 days/week,
for 80 weeks. Toxic effects attributable to treatment were absent except for
a marginal, though consistent and progressive, retardation of weight gain
(=10%) in both sexes. A decrease in plasma, but not in erythrocyte, cholines-
terase activity, was observed in the treated female rats with a maximum at
week 52. There were no significant differences in the timing or numbers of
deaths during the study between treated and control groups. The only notewor-
thy finding in organ-weight analyses was a significant (p < 0.01) decrease in
relative liver weight for treated female rats. The authors observed minor
histological changes in the liver but no severe fatty infiltration, fibrosis,
or marked bile duct abnormalities. There was no evidence of any treatment-
related toxic effect in the liver or any macroscopic or microscopic treatment-
related changes in the brain. Slight increases in the incidence of moderately
V-34
-------
TABLE V-13 Hepatic Changes Observed in Dogs Administered
Chloroform Orally for 7.5 Years
Chloroform
Treatment
(mg/kg/day)
Control
Vehicle
control
Nonchloroform
toothpaste
15
30
Sex
M
F
M
F
M
F
M
F
M
F
Number of Animals
Examined
Histopathologically
7
5
15
12
8
7
7
8
7
8
Number of
Animals with
Nodules
1
1
0
3
0
1
1
1
0
4
Number of
with Fattv
Minimal
2
1
7
3
2
0
0
2
1
0
Animals
Cvsts
Moderate
0
0
1
0
0
0
6
3
6
7
Adapted from Heywood et al. (1979).
V-35
-------
severe glomerulonephritis were of uncertain significance. This study identi-
fied a LOAEL of 60 mg/kg/day for decreases observed in body weight gain,
relative liver weight, and plasma cholinesterase activity.
Jorgenson et al. (1982) administered chloroform to male Osborne-Mendel
rats at concentrations of 0, 200, 400, 900, or 1,800 ppm in drinking water for
23 months. Corresponding dose groups contained 330, 330, 150, 50, and
50 animals, respectively^.. Based on measured water consumption and body
weights, the time-weighted 23-month average doses were 0, 19, 38, 81, or
160 mg/kg/day (Jorgenson et al. 1985). Water consumption was lower in
chloroform-exposed animals than in controls, especially at the two highest
doses. In order to compensate for the effect of decreased water consumption,
therefore, the authors included a matched control group of 50 animals, for
which water consumption was restricted to that of the high-dose group. Body
weight gain was inversely proportional to chloroform dose, probably as a
result of reduced water consumption, since there was no consistent difference
between the high-dose and matched control groups. Survival was proportional
to dose (lowest in controls, highest in high-dose animals). The authors
indicated that this result may have been due to the beneficial effect of
reduced body weight. Groups of 10 rats/dose were sacrificed at 3 or 6 months,
and liver triglyceride levels were- measured. No increase in mean percent
liver fat was observed at any dose level, except for a small increase (from
4.5% to 5.1%) at the 1,800-ppm dose at 6 months (p < 0.05). Groups of 20 rats
were sacrificed at 6, 12, or 18 months of exposure, and a number of blood
parameters were evaluated. White blood cell values were lower in the
1,800-ppm group and in the matched controls at 6 and 12 months. Differences
in erythrocyte and hemoglobin parameters at 12 months suggested hemoconcen-
tration in the treated groups, but no significant differences were apparent at
V-36
-------
18 months. Various blood chemistry parameters differed significantly from the
control values, according to a number of apparent trends. Chloride, potas-
sium, phosphorus, bilirubin, alkaline phosphatase (A?), total iron, albumin,
and the albumin/globulin ratio tended to be higher in treated groups than in
the controls. Cholesterol, triglycerides, LDH, and globulin tended to be
lower in treated groups than in the controls. With the exception of serum
triglyceride levels, changes in hematologic and blood chemistry parameters
observed in the treated .groups were also evident in the matched control group;
the authors, therefore, 'concluded that the changes observed were secondary to
reduced water and food consumption. However, decreased water intake alone
produced a 40% decline in serum triglyceride levels, compared to an 89%
decrease observed in the high-dose group. The significance of these observa-
tions is uncertain.
Jorgenson et al. (1982) also exposed female B6C3F1 mice to chloroform
in drinking water for 23 months. Drinking water concentrations were 0, 200.
400, 900, or 1,800 ppm. Based on measured water consumption and body weight,
these doses corresponded to time-weighted average doses of 0, 34, 65, 130, and
263 mg/kg/day (Jorgenson et al. 1985). Dose groups contained 430, 430, 150.
50, and 50 animals, respectively. A matched control group of 50 animals was
included, in which water consumption was restricted to that of the high-dose
group. Decreased survival at 3 weeks (99%, 94%, 74%, and 76% for the 200-,
400-, 900-, and 1,800-ppm groups, respectively) and behavioral effects (e.g..
lassitude, lack of vigor) in the 900- and 1,800-ppm dose groups were
attributed to markedly decreased fluid consumption. The percent fat in the
liver of the mice was significantly increased (p & 0.01) in the 400- to
1,800-ppm dose groups at 3 months and in the 900- and 1,800-ppm dose groups
(p s 0.05) at 6 months. These data indicate that doses of 65 to 263 mg/kg/dav
V-37
-------
may produce adverse effects in mice, possibly secondary to decreased water
consumption. However, because no sensitive index of liver toxicity was
monitored, the data were not adequate to identify a useful NOAEL or LOAEL
value.
Klaunig et al. (1986) examined the effect of chronic chloroform
exposure in mice. Two groups of 35 male B6C3F1 mice (4 weeks old) were
supplied with drinking water containing 600 or 1,800 ppm of chloroform
(equivalent to 86 or 258 mg/kg/day). Animals were sacrificed after 24 weeks
(10 mice) or 52 weeks (25 mice), and body weight and water consumption were
monitored throughout the study. Both groups displayed a statistically signi-
ficant (p < 0.05) decrease in drinking water intake. The 1,800-ppm group
exhibited statistically significant (p < 0.05) decreases in mean body weight,
but this effect may have been due to decreased water intake; no matched
control was included. No deaths occurred in the control group during the
52-week study period, but 2/35 mice in the low-dose group and 3/35 in the
high-dose group died between weeks 40 and 52. Focal areas of cellular
necrosis were found in the kidneys and liver of chloroform-treated mice at 24
and 52 weeks (no data reported). Focal areas of hepatic•lipid accumulation
were also seen in the 1,800-ppm mice. This study suggested that 86 mg/kg/day
may be a LOAEL in mice, but the data were not adequate to provide a firm
conclusion.
Table V-14 summarizes the longer-term health effects data on
chloroform.
V-38
-------
TABLE V-14 Summary of Longer-term Studies of Chloroform
Reference
Species Route
Dose
Sex Duration (mg/kg/day)
Results
Chu et al.
(1982b)
Bull et al.
(1986)
Rat
Jorgenson and
Rushbrook
(1980)
Heywood
et al.
(1979)
Palmer
et al.
(1979)
Jorgenson
et al.
(1982)
Rat
House
Rat
Rat
Drinking
water
Mouse Gavage
(oil)
Drinking
water
M,F 90 days 0.7-50
180
M,F 90 days -60-130
270
M,F 90 days 60-270
M 30-90 days 20-160
30-90 days 32-145
290
M,F 7.5 years 15
Gavage
(aqueous)
Drinking
water
Drinking
water
Oral
(toothpaste
base
gelatin
capsule)
Gavage M,F 80 weeks
(toothpaste
base)
60
23 months 19-160
NOAEL
Increased mortality;
mild thyroid lesions
Decreased serum
triglycerides,
elevated liver fat
Elevated SCOT,
diffuse liver
degeneration
NOAEL
Serum chemistry
changes (judged
secondary to
reduced water
intake) (NOAEL)
NOAEL
Mild hepatic
fatty change
(LOAEL)
Rise in SGPT and
fatty cysts (LOAEL)
Decreased body weight
liver weight and
plasma cholinesterase
activity. Minor
liver changes.
(LOAEL)
Minimal changes
in various blood
chemistry
parameters and
mean percent liver
fat, probably
secondary to
decreased water
consumption
V-39
-------
Table V-14 (Continued)
Dose
Reference Species Route Sex Duration (mg/kg/day) Results
Jorgenson Mouse Drinking F 23 months 65 Increased mean
et at. (1982) water percent liver fat
(continued) (3 months)
130-263 Rejection of
chloroform
solutions leading
to high initial
mortality;
Increased mean
percent liver
fat (3 and 6
months).
Klaunig Mouse Drinking M 52 weeks 86-258 Kidney and liver
et al. water histological
(1986) changes; LOAEL (?)
V-40
-------
2. Brominated Trihalomethanes
Chu et al. (19S2b) administered bromodichloromethane, dibromochloro-
methane or bromoform to weanling Sprague-Dawley rats for 90 days. The
compounds were added to drinking water at levels of 0, 5, 50, 500, or
2,500 ppm (20 rats/sex/dose). 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.-... Using the authors' calculations of trihalomethane
ingested per rat per day and reported average body weights, these levels
corresponded to doses of approximately 0, 0.6, 7, 52, or 250 mg/kg/day. Food
consumption was depressed in all of the 2,500-ppm groups, although growth
suppression occurred only in the 2,500-ppm bromodichloromethane group. Mild
histological changes occurred in the liver and thyroid. Although neither
incidence nor severity were clearly dose related, these parameters did tend to
increase with dose (Table V-15). Observed lesions included increased
cytoplasmic volume and vacuolation due to fatty infiltration. Statistically
significant (p < 0.05) increases in the severity of hepatic lesions were
observed at the highest dose for all three chemicals. The authors indicated
that in both sexes the hepatotoxicity of the brominated trihalomethanes
followed the order: bromoform > bromodichloromethane > dibromochloromethane.
The only serum biochemical parameter affected was LDH, which was significantly
reduced in rats given 2,500 ppm bromoform (both sexes). Ninety days after
cessation of treatment, histological changes were reversed, although LDH
levels in plasma remained reduced. These data identified a NOAEL of
52 mg/kg/day and a LOAEL of 250 mg/kg/day for all three brominated
trihalomethanes.
V-41
-------
Table V-15 Incidence and Severity of Liver and Thyroid Lesions in Rats
Fed Brominated Trihalomethanes for 90 Days
Liver
Male
Group
Control (H20)
Control (Vehicle)
(1% Emulphor)
Bromodichlorome thane
5 ppm
50 ppm
500 ppm
2,500 ppm
Dibromochl or ome thane
5 ppm
50 ppm
500 ppm
2,500 ppm
Bromoform
5 ppm
50 ppm
500 ppm
2,500 ppm
Female
Incidence Severity3 Incidence
0/10
2/9
1/10
8/10
8/10
9/10
3/10
4/10
5/10
6/9
5/10
4/10
7/10
9/9
1.0
1.2
1.1
2.2
2.0
2.3
1.3
1.4
1.7
2.0
1.6
1.4
1.8
2.7
± 0.4
±0.3
± 0.8b
± 0.7b
± 0.7b
±0.5
±0.5
± 0.8
± 0.9b
±0.7
±0.5
± 0.6
± 0.7b
0/10
0/10
3/10
5/10
3/10
4/10
3/10
5/10
3/10
4/10
3/10
0/10
4/10
6/10
Male
Soever ity Incidence
1.0
1.0
1.3
1.6
1.3
1.5
1.3
1.6
1.3
1.6
1.3
1.0
1.5
1.7
±0.5
±0.7b
±0.5
± 0.7b
±0.5
± 0.7b
±0.5
± 0.8b
±0.5
± 0.7b
± 0.7b
3/10
3/9
1
2/10 .
5/10
5/10
4/10
2/10
3/10
6/10
3/10
3/10
5/10
4/10
5/10
Thyroid
Female
Severity Incidence
1.3
1.3
1.2
1.7
1.8
1.5
1.3
1.3
1.9
1.4
1.3
1.6
1.5
1.7
±0.5
±0.5
± 0.4
± 0.8
± 0.9
±0.7
±0.7
±0.5
± 0.9
±0.7
±0.5
±0.7
±0.7
±0.7
2/10
0/10
0/10
1/10
3/10
2/10
3/10
1/10
0/10
3/10
I/ 10
0/10
3/10
5/10
Severity
1.2
1.0
1.0
1.1
1.3
1.2
1.3
1 .1
1.0
1.3
1.1
1.0
1.4
1.7
±0.4
±0.3
±0.5
±0.4
±0.5
±0.3
±0.5
± 0.3
±0.7
±0.8
aSeverity rating for lesions range from 1 (no effect) to 10 (malignant/complete necrosis).
Significantly different from vehicle control (p < 0.05).
Adapted from Chu et al. (1982b).
-------
NTP (1987) administered doses of 0, 19, 38, 75, 150, or 300 mg/kg/day
of bromodichloromethane to F344/N rats (10/sex/dose) by gavage in corn oil,
5 days/week, for 13 weeks. (The IS-mg/kg/day group was administered
1.9 mg/kg/day for the first 3 weeks of the study.) A necropsy was performed
on all animals. Five of ten male rats and two of ten female rats that
received 300 mg/kg died before the end of the study. Final mean body weights
of male rats that received 150 or 300 mg/kg and of f.emale rats that received
300 mg/kg were 30% to 55%.. or more lower than those of the vehicle controls;
the final mean body weight of female rats at 150 mg/kg was 12% lower than that
of the vehicle controls. No other compound-related clinical signs were
reported. Compound-related lesions were observed at 300 mg/kg but not at
150 mg/kg. In high-dose males, centrilobular degeneration of the liver and
occasional necro.tic cells were observed in four of nine animals. Mild bile
duct hyperplasia was also observed in these animals. Degeneration of renal
proximal tubular epithelial cells was observed in four of nine males, and two
of nine males had definite foci of coagulative necrosis of the tubular
epithelium. Lymphoid atrophy of the thymus, spleen, and lymph nodes and mild
to moderate atrophy of the seminal vesicles and/or prostate were present in
four of nine high-dose males. In high-dose females, enlarged hepatocytes were
observed in two of nine animals. Atrophy of the thymus, lymph nodes, and
spleen occurred in high-dose female rats but was much less than that observed
in males. Rats in the high-dose groups were emaciated and appeared to consume
less feed on dosing days (although actual feed consumption was not measured).
Based on reduced body weight gain, this study identified a NOAEL of
75 mg/kg/day and a LOAEL of 150 mg/kg/day.
In a similar experiment, NTP (1987) administered bromodichloromethane
in corn oil by gavage to B6C3F1 mice (10/sex/dose), 5 days/week, for 13 weeks.
V-43
-------
Doses were 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 mean body weight of males that received 100 mg/kg was 9%
lower than that of the vehicle controls. The final mean body weight of
females that received 200 or 400 mg/kg was 4% to 5% lower than that of the
vehicle controls. Focal necrosis of the proximal renal tubular epithelium
occurred in 6/10 males at 100 mg/kg, and nephrosis of minimal severity
occurred in 2/10 males at this level. No compound-related lesions were seen
in the 50-mg/kg groups. . Hepatocytes in the centrilobular area of the liver of
8/10 female mice at 400 mg/kg and 7/10 female mice at 200 mg/kg were greatly
enlarged with vacuolated or foamy cytoplasm characteristic of lipid accumu-
lation. Microgranulomas were present in the liver of 7/10 female mice that
received 200 mg/kg, and no compound-related lesions were noted in females at
100 mg/kg. No compound-related clinical signs-were noted. Based on renal
histopathology, this study identified a NOAEL of 50 mg/kg/day and a LOAEL of
100 mg/kg/day in male mice.
NTP (1985) administered dibromochloromethane by gavage (in corn oil)
to 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 and a thorough
histological examination was performed on all animals, either at the time of
death or at the end of the study. Only one male and one female in the high-
dose group survived, with most deaths occurring in weeks 8 to 10. Histo-
logical examination revealed severe lesions and necrosis in kidney, liver, and
salivary glands. Body weight gain of males receiving 125 mg/kg/day was 85% of
the corresponding control value. A dose-dependent increase in the frequency
of clear cytoplasmic vacuoles indicative of fatty metamorphosis was observed
in males; this effect was statistically significant at doses of 60 mg/kg/day
V-44
-------
or higher. On this basis, this study identified a. NOAEL of 30 mg/kg/day and a
LOAEL of 60 mg/kg/day in rats for dibrompchloromethane.
NTP (1985) performed a similar 13-week gavage study with dibromochloro-
methane in male and female B6C3F1 mice (10/sex/dose). The doses and dosing
schedule were the same as for the study with rats. No effects on body weight
or histopathology were observed at doses of 125 mg/kg/day or lower. Fatty
liver and toxic nephropathy were observed in males, but not females, receiving
250 mg/kg/day. Both males and females gained less weight at this dose level.
This study identified a NOAEL of 125 mg/kg/day and a LOAEL of 250 mg/kg/day in
mice.
NTP (1989a) exposed F344/N rats to bromoform by gavage, 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. Animals receiving 100 or
200 mg/kg/day were lethargic. At sacrifice, tissues were examined for gross
and histological changes. A dose-dependent increase in the frequency of
hepatocellular vacuolation was observed in male rats, which became statis-
tically significant (p < 0.05) at 50 mg/kg/day (IRIS 1988). These hepatic
effects were not observed in females. On the basis of the hepatic vacuolation
seen in male rats, this study identified a NOAEL of 25 mg/kg/day and a LOAEL
of 50 mg/kg/day.
In a parallel study, NTP (1989a) exposed B6C3F1 mice to bromoform by
gavage, 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
there were no other deaths, even at the higher doses. At sacrifice, tissues
V-45
-------
were examined for gross and histological changes. Body weights were not
significantly affected, although males receiving 400 mg/kg/day had body
weights about 8% less than controls. An increase in the number of hepato-
cellular vacuoles was seen in male mice (5/10 at 200 mg/kg and 8/10 at
400 mg/kg) but not in females. Based on hepatocellular vacuolation, this
study identified a NOAEL of 100 mg/kg/day and a LOAEL of 200 mg/kg/day in male
mice.
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. Ophthalmo-
scopic examinations were performed prior to treatment and during the last week
of the study, and hematological analysis, serum clinical chemistry, and a
thorough histopathological examination were conducted. There were no deaths,
clinical signs of toxicity, or treatment-related changes in the ophthalmo-
scopic examinations or hematology. Body weight gain was significantly reduced
in the high-dose groups, to less than 50% of the controls (males) and less
than 70% of the controls (females). Clinical chemistry values indicative of
hepatotoxicity and suggestive of nephrotoxicity included increased levels of
AP (high-dose males and females), alanine aminotransferase (also known as
SGPT) (mid- and high-dose males) and creatinine (mid- and high-dose males and
high-dose females), and decreased potassium levels (high-dose males). Centri-
lobular 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. Centrolobular necrosis was also observed in
V-46
-------
high-dose males and females. Slight-to-moderate degeneration within the
proximal kidney tubular cells occurred in all high-dose males and females, and
to a lesser extent in mid-dose males and low- and inid-do3e 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.
Tobe et al. (1982) evaluated the chronic effects of bromodichloro-
methane, dibromochloromethane, and bromoform administered in the diet to
groups of 40 male and 40 female Slc:Wistar SPF rats for 24 months. The
histopathology data from this study for the bromodichloromethane-exposed
animals were reported by Aida et al. (1992b). No histopathology data has been
published for the other two chemicals. The animals were 5 weeks old at the
start of the tes.t and weighed approximately 100 g. Each trihalomethane was
microencapsulated, and an appropriate amount was mixed with powdered feed.
Control groups (70 rats/sex) received microcapsules without trihalomethanes.
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. Interim sacrifices of at least 9 animals/sex/
control group and 5 animals/sex/dose group were conducted for bromodichloro-
methane at 6, 12, and 18 months; all surviving animals were sacrificed at
2 years. For bromoform and dibromochloromethane, data were reported from the
sacrifices of 9 animals/sex in the control group and 5/sex/dose group at
18 months; all surviving animals were sacrificed at 24 months. At each time
of sacrifice, necropsies, hematological studies, and serum biochemical studies
were conducted.
Bromodichloromethane was administered in the diet at levels of 0.0%.
0.014%, 0.055%, or 0.22%. Based on mean food intakes, the reported average
V-47
-------
doses were about 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 all dose groups, and
relative kidney weight was significantly increased (p < 0.01) in males and
females in the high-dose group. Dose-dependent reductions in T-GLY levels and
increases in vglucaniyl transpeptidase (y-GTP) activity (indicative of bile
duct proliferation) were observed at all sacrifice times in both sexes
(Table V-16). Serum cholinesterase activity was also decreased in a dose-
related manner in both sexes. The most sensitive markers at 24 months were
T-GLY and serum cholinesterase, with significant (p < 0.05) changes seen in
low-dose males. .Gross necropsy revealed dose-related yellowing and roughening
of the surface of the liver. Treatment-related.lesions were limited to the
liver and included fatty degeneration and granuloma in all dosage groups, and
cholangiofibrosis in the high-dose groups (Table V-17). Bile duct prolifera-
tion was observed in most high-dose animals at 6 months, but by 24 months it
was prevalent in the controls and all dosage groups. Histopathology was
observed in all dose groups, except for low-dose females, as early as
6 months. At 24 months, fatty degeneration and granuloma 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).
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 and the LOAEL was identified as 26 (males), to
32 (females) mg/kg/day, based on serum enzyme changes and altered liver
appearance. However, taking into consideration the histopathology data
reported for this study by Aida et al. (1992b), the entire study identified a
V-48
-------
Table V-16 Serum Biochemical Levels8 in Rats Fed Brominated
Trihalomethanes for 18 to 24 Months
<
I
vD
SCOT
Dietary
Chemical Sex Level (X)
CHBrCl2 M 0
0
0
0,
F 0
0
0
0.
CHBr2Cl M 0
0
" 0
0
F 0
o
o
0
CHBr3 M 0
o
0
0
F 0
0
0
0
.on
.055
.220
.014
.055
220
.022
.088
.350
.022
.068
.350
.04
.160
.650
.04
. 160
.650
18
106
77'
122
85
92
100
103
96
106
92
110
175
92
121'
148"
124"
106
115
202
229'
92
112
112
21B'
24
123
95
95
119
95
156
88
102
123
110
107
187
95
94
123
no
123
83
96
198
95
121
127
182*
SGPT
18
42
39
59
47
37
56
53
43
42
45
57'
92"
41
52
81*
42
42
47
106
100"
41
53
48
100"
24
35
45
34
44
46
47
42
39
35
36
45
58
46
42
54
42
35
31
40
92"
46
44
69
75"
yGTP
18
0.90
0.35
3.38
6.08"
0.27
0.50
1.85"
9.14"
0.90
0.78
3.13"
21.59"
0.27
1.48*
5.03'
11.87"
0.90
0.34
2.84
4.94"
0.26
1.11"
1.07"
7.66"
24
2.25
2.45
3.50
9.51"
1.83
3.92
2.72
9.49"
2.25
3.73
5.46*
17.26"
1.83
2.11
4.09*
8.57"
2.25
0.19
0.53
7.50"
1.83
2.18
4.06"
6.61"
LDH
18
2610
1900
1925
1220"
1770
1250
1400
1440
2610
1670
1370"
mo"
1770
1880
2120
1940
2610
2850
3160
2830
1770
2210
2130
2210
24
1890
1910
2260
2480"
1300
1440
1300
1340
1890
2160
2640
2000
1300
1250
1600
1430
1890
1560
2050
1350"
1300
1420
1460
1570
TGLY
18
153
168
112"
54"
117
96
68*
43"
153
160
94"
69"
117
164"
79"
37"
153
263
168
52"
117
173"
120
34"
24
321
164"
106"
77"
334
194
172"
97"
321
209"
195
50"
235
178
106"
50"
321
232
231
46"
234
238
179
47"
CHL
18
954
877
786
556"
1962
1338"
1133"
758"
954
831
839
568"
1962
1456"
1202"
737"
954
1067
798
467"
1962
1729
1436"
674"
24
1538
994"
772"
627"
1739
1469
1293"
865"
153B
1150
1044*
582"
1739
1465
1235"
866"
1538
1313
1179
393"
1739
1720
1443
837"
FAA
18
0.59
0.44
0.43
0.36"
0.74
0.52"
0.48
0.46"
0.59
0.62
0.50
0.40"
0.74
0.87
0.59
0.49
0.59
0.66
0.39"
0.39"
0.74
0.91
0.54
0.37"
0
0
0
0
0
0
0
0
0.
0.
0
0
0
0
0
0
0
0
0.
0.
0.
0.
0.
0.
24
.60
.56
.53
.52
.83
.81
. 79
57"
60
63
.55
.30"
.83
.84
.64
.47"
.60
.46"
40"
34"
83
99
52"
39"
'Results from nine animals/sex in the control group and five/sex/dose In the treatment groups at the 18-month sacrifice, and 12 animals/sex in the
control group and seven/sex/dose in the treatment groups at 24 months. Enzyme activities expressed as mU/mL, TGLY in mg/dl.. and NEFA in mEq/L
"p less than 0.05 compared to control
"p less than 0.01 compared to control
Adapted from Tobe et al. (1982).
-------
Table V-17 Liver Lesions In Rats Fed Bromodichloromethane for 2 Years
Ul
o
Dose
Control
0.014
0.055
0.22
Control
0.014
0.055
0.22
Control
0.014
0.055
0.22
Control
0.014
0.055
0.22
Number of
Animals/Sex
Examined
Histologically
10
6
6
6
9
5
5
5
9
5
5
5
24a (32)b
14 (19)
13 (18)
19 (18)
: Fatty
Bile
Duct Altered Cholangio-
Degeneration Granuloma Proliferation Cell Foci fibrosis
M
- .
6
6
6
.. .
5
5
5
1
3
5
5
5
12
19
F
--
5
6
--
5
4
1
5
5
2
8
18
18
M
6 Months
--
1
--
12 Months
--
1
18 Months
--
2
4
24 Months
4
9
19
F
_ .
3
6
--
5
5
--
5
5
--
17
18
M
--
5
1
1
--
5
9
5
5
5
24
13
13
19
F M F M
- - - - - - _.
~~l ~"
6 .... 6
* .„ __
3
5 5
6 2
2
4
5 -- --3
28 12 15
16 69--
17 7 6 --
18 734
F
_ _
6
5
--
4
12
aNuinber of males
bNumber of females
Adapted from Aida et al.
(1992b).
-------
LOAEL of 6 mg/kg/day for bromodichloromethane in male rats and 8 mg/kg/day in
female rats, based on histopathology and serum biochemistry.
Dibromochloromethane was administered at dietary levels of 0.0%,
0.022%, 0.088%, or 0.35%. Based on reported body weights (150 to 475 g) and
food consumption (15 to 20 g/day), these levels correspond to doses of
approximately 0, 10, 39, or 210 mg/kg/day for males and 0, 17, 66, or
350 mg/kg/day for females. Marked suppression of body weight gain was seen in
males and females of the high-dose group, and mild (about 10%) suppression of
body weight gain was seen in males and females of the mid-dose group.
D.ecreased T-GLY and serum cholinesterase activity and increased y-GTP were
seen in the mid-dose males and females (Table V-16). Yellowing and roughening
(high-dose males_ only) of the surface of the liver were noted in the mid- and
high-dose groups. Based on the serum biochemistry data, decreased body
weight, and gross necropsy results, this study suggests a NOAEL of
10 mg/kg/day in males and 17 mg/kg/day in females, and a LOAEL of 39 (males)
and 66 (females) mg/kg/day. However, no histopathology results were
presented.
Bromoform was administered at dietary levels of 0.0%, 0.04%, 0.16%,
or 0.65%. Based on reported bodv weights (150 to 475 g) and food consumption
(15 to 20 g), these levels correspond to doses of about 0, 18, 71, or
480 mg/kg/day for males and 0, 30, 120, or 870 mg/kg/day for females. Marked
suppression of body weight gain was seen in males and females of the high-dose
group, and mild (about 15%) suppression of body weight gain was seen in males
and females of the mid-dose group. Dose-related decreases in FAA (non-esteri-
fied fatty acids) were observed in all treated males, and mid- and high-dose
females had increased levels of y-GTP. Other serum biochemistry changes in
V-51
-------
the high-dose groups included decreased T-GLY and increased SCOT and SGPT
activity (Table V-16). Elevations in SCOT and SGPT are indicative of hepato-
cellular necrosis. Yellowing, small white spots, and roughening of the
surface (high-dose only) were seen in the livers of the mid- and high-dose
animals. Based on the necropsy findings and the serum biochemistry data, this
study indicates a NOAEL of 18 mg/kg/day in males and 30 mg/kg/day in females,
and a LOAEL of 71 (males) and 120 (females) mg/kg/day. However, no histo-
pathology results were presented.
The authors concluded that serum biochemical changes and the necropsy
findings in the liver among the groups administered high doses of the three
brominated trihalomethanes were primarily a result of abnormal lipid
metabolism.
NTP (1987) administered doses of 0, 50, or 100 mg/kg/day of bromo-
dichloromethane in corn oil by gavage to F34A/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 histopathologic examinations were 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 histolo-
gically. Survival of dosed rats was comparable to that of vehicle controls.
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 were 86% and 70% of the corresponding vehicle-control values. Body
V-52
-------
weight gains of low-dose male and female rats were comparable to those of the
vehicle control group. No compound-related clinical signs were observed.
Compound-related nonneoplastic lesion?; arp shown in Table V-18. In males,
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. Based on these
histological findings, this study identified a LOAEL of 50 mg/kg/day in rats.
NTP (1987) administered bromodichloromethane in corn oil by gavage to
B6C3F1 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 o.f dosed 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; 15/50), 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% 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 weights of high-dose female mice were 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. Compound-
related nonneoplastic lesions are shown in Table V-18. In males, changes
included fatty metamorphosis of the liver, renal cytomegaly, and folliculap
cell hyperplasia of the thyroid gland. In females, hyperplasia of the thyroid
gland was observed. Based on these histological findings, this study
identified a LOAEL of 25 mg/kg/day in male mice.
V-53
-------
TABLE V-18 Nonneoplastic Lesions in Rats and Mice
Exposed to Bromodichloromethane for 2 Years
Frequency
Animal
Male rat
Female rat
Male mouse
Female mouse
Tissue
Kidney
Liver
Liver
Kidney
Liver
Kidney
Thyroid
Thyroid
Lesion
Cytomegaly
Tubular hyperplasia
Necrosis
" Fatty -metamorphosis
Fatty metamorphosis
Focal cellular change
Clear cell change
Eosinophilic cytoplasm
Tubular hyperplasia
Fatty metamorphosis
Cytomegaly
Follicular cell hyperplasia
Follicular cell hyperplasia
Control
0/50
0/50
1/50
36/50
7/50
4/50
4/50
0/50
0/50
4/49
0/49
0/48
6/50
Low
Dose
18/50
0/50
4/50
48/50
22/50
. 4/50
6/50
1/50
1/50
8/50
41/50
3/44
18/45
High
Dose
44/50
3/50
6/50
47/50
13/50
11/50
39/50
11/50
4/50
19/50
47/50
5/49
21/48
Adapted from NTP (1987).
V-54
-------
NTP (1985) investigated the chronic oral toxicity of dibromochloro-
methane in 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), 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 gain was 88% of the control value.
Females in both dose groups gained more weight than did the controls.
Histologic lesions in liver were observed in both males and females at both
dose levels (p < 0.05). _Qhanges included fat accumulation, "ground glass"
appearance of the cytoplasm, and altered basop^hilic staining. This study
identified a LOAEL of 40 mg/kg/day for dibromochloromethane in rats.
NTP (1985) performed a similar chronic oral study with dibromochloro-
methane using male and female B6C3F1 mice. Groups of 50 animals/sex/dose were
administered doses of 0, 50, or 100 mg/kg/day by gavage (in corn oil),
5 days/week for 105 weeks. Survival in females was not different from
controls, while survival in high-dose males was decreased (p < 0.05). 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 weights were decreased
in high-dose males and females but not in low-dose females. Treatment-related
hepatocytomegaly and focal necrosis were observed (p < 0.05) in livers of
males (high dose). Females showed liver calcification (high dose) and fatty
metamorphosis (both low and high doses) . This study identified a LOAEL of
50 mg/kg/day for dibromochloromethane in mice.
NTP. (1989a) exposed groups of 50 male and 50 female F344/N rats to
bromoform by gavage 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 was performed on all animals, as was
V-55
-------
a thorough histological examination of tissues. Body weight gain was
decreased by 37% in high-dose females and by 29% in high-dose males, compared
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 B6C3F1 mice by gavage 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. An increased incidence of minimal to
mild fatty changes was 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, and a NOAEL of
100 mg/kg/day for male mice.
Tables V-19, V-20, and V-21 summarize the longer-term studies of
brominated trichloromethane toxicity.
V-56
-------
TABLE V-19 Summary of Longer-term Studies of Bromodichloromethane
Reference
Species Route
Pose
Sex Duration (mg/kg/day)
Results
Chu et al.
(1982b)
NTP
(1987)
Rat
Rat
Mouse
Rat
Drinking
water
Gavage
(oil)
Mouse Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Mouse Gavage
(oil)
M,F 90 days 0.6-52
250
M,F 13 weeks 19-75
150
300
M 13 weeks 6.25-50
100
13 weeks
M,F 2 years
25-100
200-400
50
M.F 2 years 25
Mild and reversible
changes (NOAEL)
Hepatic lesions
(LOAEL)
NOAEL
Decreased weight
gain (M)UOAEL)
Hepatic and renal
pathology
NOAEL
Decreased body
weight, renal and
hepatic pathology
(LOAEL)
NOAEL
Hepatic pathology
(LOAEL)
Renal and hepatic
histopathology
(LOAEL)
Renal and hepatic
histopathology
(LOAEL)
Aida et al. Rat Diet
(1992b); Tobe
et al. (1982)
M,F 2 years 6 (M)
8 (F)
Hepatic vacuol-
ization, serum
chemistry (LOAEL)
V-57
-------
TABLE V-20 Summary of Longer-term Studies of Dibromochloromethane
Reference
Species
Route
Sex Duration (nig/kg/day)
Results
Chu et. al.
(1982b)
NTP
(1985)
Rat
Rat
Drinking
water
Gavage
(oil)
Daniel et al.
(1990)
NTP
(1985)
House
Rat
Rat
Mouse
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Gavage
(oil)
Tobe et al.
(1982)
Rat
Diet
M,F 90 days 0.6-52
250
M,F 13 weeks 30
60
125
250
•M.F 13 weeks 15-125
250
M,F 90 days
M,F 2 years
M,F 2 years
50
100
200
40
50
100
M,F 2 years 10 (M)
17 (F)
39 (M)
66 (F)
Mild and reversible
changes (NOAEL)
Hepatic lesions
(LOAEL)
NOAEl
Hepatic vacuolation
(LOAEL)
Decreased body
weight (males)
Severe hepatic and
renal lesions,
mortality
NOAEL
Fatty liver, toxic
nephropathy in
males (LOAEL)
Hepatic vacuolization
(M), renal lesions (F)
(LOAEL)
Hepatic and renal
lesions
Decreased body weight
Histologic changes
in liver (LOAEL)
Liver calcification,
fatty metamorphosis
(F) (LOAEL)
Decreased body
weight (M,F),
decreased survival
(M)
NOAEL
Enzyme changes and
altered liver
appearance at
necropsy
(LOAEL)
V-58
-------
TABLE V-21 Summary of Longer-term Studies of Bromoform
Reference
Chu et al.
(1982b)
Species Route
Rat Drinking
water
Dose
Sex Duration (mg/kg/day)
M,F 90 days 0.6-52
250
Results
Mild and reversible
changes (NOAEL)
Hepatic lesions
. UOAEL)
NTP (1989a)
Tobe et al.
(1982)
NTP (1989a)
Rat Gavage
Mouse Gavage
Rat Diet
Rat Gavage
Mouse Gavage
M,F 13 weeks 25
(5 day/wk) 50
M,F
13 weeks 100
(5 day/wk) 200
M,F 2 years 18 (M)
30 (F)
71 CM)
120 (F)
M,F 103 weeks 100
(5 day/wk)
103 weeks 100
(5 day/wk)
103 weeks 100
(5 day/wk)
NOAEL
Hepatic vacuolation
in males UOAEL)
NOAEL
Hepatic vacuolation
in males (LOAEL)
NOAEL
Enzyme changes and
altered liver appear-
ance at necropsy
(LOAEL)
Decreased body weight,
lethargy, mild
hepatotoxicity (LOAEL)
NOAEL
Decreased body weight,
mild hepatotoxicity
(LOAEL)
V-59
-------
C. Reproductive and Developmental Effaces
1. Chloroform
Thompson et al. (1974) studied the effects of chloroform on the
embryonic and fetal development of Sprague-Dawley rats. In a preliminary
range-finding study, groups of six pregnant females given chloroform in corn
oil by intubation at doses of 79, 126, 300, 316, or 516 mg/kg/day on days 6 to
15 of gestation developed alopecia, rough hair, and eczema. At levels of
126 mg/kg/day and greater, food consumption and body weight gain were signifi-
cantly suppressed. Doses of 316 mg/kg/day caused acute toxic nephrosis,
hepatitis, and maternal death, as well as fetotoxicity. In the main study,
groups of 25 pregnant rats (181 to 224 g) were administered chloroform in corn
oil at doses of 0, 20, 50, or 126 mg/kg/day by oral intubation on days 6 to 15
of gestation. Dams receiving 50 or 126 mg/kg/day displayed signs of maternal
toxicity (decreased weight gain, mild fatty changes in the liver). Fetuses
were removed by Caesarean section 1 or 2 days prior to expected parturition
and examined for external, skeletal, and/or soft tissue abnormalities. There
were no fetal malformations, but the incidence of bilateral extra lumbar ribs
was significantly (p < 0.05) increased at the high dose. Fetal weight was
also reduced at the high dose (p < 0.05). This study identified a NOAEL of
20 mg/kg/day and a LOAEL of 50 mg/kg/day in rats.
In the same study, Thompson et al. (1974) administered chloroform (in
corn oil) to Dutch-Belted rabbits. In a preliminary range-finding study,
doses of 0, 25, 63, 100, 159, 251, or 398 mg/kg/day were administered to
pregnant rabbits on days 6 to 18 of gestation. High levels of maternal death
(60% to 100%) were observed at doses of 100 mg/kg/day and above. Adverse
V-60
-------
effects at 63 mg/kg/day included anorexia, weight loss, diarrhea, abortion.
and one maternal death. No overt signs of toxicity other than mild diarrhea
and intermittent anorexia were observed in dams dosed with 25 mg/kg/day. In
the main study, groups of 15 dams (1.7 to 2.2 kg) were dosed by oral
intubation with chloroform at 0, 20, 35, or 50 mg/kg/day on days 6 to 18 of
gestation. Decreased maternal weight gain was observed in dams given
50 mg/kg/day. Four high-dose dams died from hepatotoxicity. Four high-dose
dams aborted, but this was not considered to be a treatment-related effect
because three control animals aborted. No evidence of maternal toxicity was
noted at 35 mg/kg/day. Small reductions in body weights were observed in
fetuses from dams administered 20 or 50 mg/kg/day (p < 0.05), and an increased
incidence of incompletely ossified skull bones (usually parietals) was
observed at 20 ajid 35 mg/kg/day (p < 0.05). The authors did not consider
these effects to be evidence of teratogenicity or fetotoxicity. These data
indicate doses resulting in maternal toxicity are lower than those resulting
in fetotoxicity. On this basis, this study identified a NOAEL of 35 mg/kg/day
and a LOAEL of 50 mg/kg/day.
Ruddick et a-1. (1983) investigated the potential developmental
toxicity of chloroform in groups of 15 mated rats. Pregnant dams (8 to
14 animals per dose group) were given 0, 100, 200, or 400 mg/kg chloroform in
corn oil on days 6 to 15 of gestation. Maternal weight gain was depressed by
at least 20% at all dose levels. In addition, all dose levels of chloroform
produced maternal liver enlargement, decreased hemoglobin, and decreased
hematocrit. Levels of serum inorganic phosphorus and cholesterol were ele-
vated in the dams at the highest exposure level. Fetal weight was decreased
by about 19% at the highest dose level. There were no fetal malformations,
but sternebra aberrations were observed with a dose-dependent incidence at
V-61
-------
200 mg/kg/day and 400 mg/kg/day. Interparietal deviations also occurred ac
the high dose. Although there was a clear increase in the incidence of these
variations, no statistical analysis v;as performed.
In a study using inhalation exposure, Schwetz et al. (1974) exposed
pregnant Sprague-Dawley rats (approximately 250 g) to chloroform at 30, 100,
or 300 ppm (147, 488, or 1,466 mg/m3) for 7 hours daily on days 6 to 15 of
gestation. The number of animals exposed in the three dose groups was 31, 28,
and 20, respectively. At the highest dose level, fertility was decreased (15%
compared with 88% to 100% in controls, p < 0.05). At 100 ppm, there was an
increase in acaudia (short tail) and imperforate anus (p < 0.05). At 30 ppm.
there was an increased incidence of delayed skull ossification and wavy ribs
(p < 0.05), but no other adverse developmental effects occurred when compared
to controls. The authors concluded that chloroform was not highly teratogenic
but was highly embryotoxic.
Murray et al. (1979) observed teratogenic effects in mice exposed to
chloroform in air. Groups of 34 to 40 pregnant females were exposed to 0 or
100 ppm for 7 hours/day on days 8 to 15 of gestation. Exposure to chloroform
resulted in a significant increase (p < 0.05) in the incidence of cleft
palate. Exposure on gestational days 1 to 7 or 6 to 15 caused reduced litter
size but no malformations, suggesting the possibility that lethality to the
early embryo obscured other effects. In this study, the cleft palates were
seen predominately in fetuses with retarded growth, suggesting to the authors
that chloroform might have induced an indirect, rather than a direct,
teratogenic effect.
V-62
-------
2. Brominated Trihalomethanes
Borzelleca and Carchman (1982) conducted a two-generation reproductive
study in ICR Swiss mice. Nine-week-old mice (10 males and 30 females per dose
group) were continuously maintained on drinking water containing 0, 0.1, 1.0,
or 4.0 mg/mL dibromochloromethane (0, 17, 171, 'or 685 mg/kg/day). After
35 days on the test solutions, the F/0 mice were randomly mated to produce the
F/la litters. Rematings to produce F/lb and F/lc generations occurred 2 weeks
after weaning the previous generation. Thus, the F/0 mice were exposed for a
total of 27 weeks prior to sacrifice and necropsy. Body weight gain was
significantly reduced in both males and females at the high dose (685 mg/kg/
day) and in females at the mid dose (171 mg/kg/day). Animals in both of these
groups exhibited^ enlarged livers with gross morphological changes interpreted
by the authors to indicate hepatotoxicity. Fertility (mating index) was
reduced in the high-dose group for the F/lc generation but not the F/la or
F/lb generations. Gestational index was significantly reduced for all three
F/l generations at the high dose but not at the lower doses.
The F/lb generation was treated with test solutions for 11 weeks prior
to mating for the F/2a generation. Remating to produce the F/2b generation
occurred 2 weeks after weaning. The F/lb generation was sacrificed after
weaning of the F/2b generation, resulting in a total exposure period for the
F/lb generation of 24 weeks. Effects on body weight and liver morphology were
very similar to those seen in the F/0 group. Fertility was reduced at the.
highest dose between the F/lb and F/2a generation, but not in the F/2b
generation. Gestational index was not affected at any dose.
V-63
-------
A 21-day litter survival study was performed on all matings. In
addition, the final matings of both generations (F/lc and F/2b) were utilized
for dominant lethal and teratology studies. Parental ingestion of dibromo-
chloromethane at a dosage level of 685 mg/kg/day caused decreased litter size
in all generations (F/la, F/lb, F/lc, F/2a, and F/2b), a decreased viability
index in four of the five generations (F/la, F/lb, F/lc and F/2a), a decreased
lactation index in the F/2b generation, and a decrease in the postnatal body
weight of the F/2b generation. Ingestion of 171 mg/kg/day decreased the
litter size of the F/lc generation, decreased the viability index of the F/lb
generation, decreased the lactation index of the F/lb and F/2b generations,
and decreased the postnatal body weight of the F/2b generation. The only
effect observed at 17 mg/kg/day was decreased postnatal body weight in the
F/2b generation.. No dominant lethal or teratogenic effects were seen in the
F/lc or F/2b generations.
Based on maternal toxicity (weight loss, liver pathology) and possible
fetotoxicity (decreased pup weight and viability in some generations), this
study identified a NOAEL of 17 mg/kg/day and a LOAEL of 171 mg/kg/day for
dibromochloromethane.
Ruddick et al. (1983) investigated the potential developmental toxi-
city of bromoform, bromodichlorotnr thane, and dibromochloromethane in groups of
15 mated rats. Pregnant dams (9 to 15 animals per dose group) were admini-
stered 0, 50, 100, or 200 mg/kg/day on days 6 to 15 of gestation. Maternal
weight gain was depressed by over 25% in the high-dose groups for bromodi-
chloromethane and dibromochloromethane. Relative maternal liver weight was
increased in 'all bromodichloromethane-exposed groups, and the kidney weight
was increased at the highest dose. There were no fetal malformations, but
V-64
-------
scernebra aberrations were observed with a dose-dependent incidence in all
bromodichloromethane and bromoform groups. Interparietal deviations also
occurred at the mid and high doses of bromoform. Although there was a clear
increase in the incidence of these variations, no statistical analysis was
performed.
NTP (1989b) investigated the effect of bromoform on fertility and
reproduction in Swiss CD-I mice. Twenty male-female pairs were dosed by
gavage with 0, 50, 100, or 200 mg/kg/day for 105 days. There was no detect-
able effect of treatment on fertility, litters per pair, live pups per litter,
proportion of pups born alive, sex of live pups, or pup body weights. This
study identified a reproductive NOAEL of 200 mg/kg/day.
D. Mutagenicitv and Genotoxicity
1. Chloroform
The overall evidence regarding chloroform genotoxicity is
inconclusive. In vitro. chloroform has yielded mixed but mainly negative
results in a number of assays of mutagenic activity. Some of these results,
however, are inconclusive because of inadequacies in experimental protocols,
especially in the failure to use an appropriate (reconstituted) activation
system or to take precautions to prevent the escape of volatilized chloroform
(U.S. EPA 1985a; Rosenthal 1987). In vitro assays for early evidence of DNA
damage (sister chromatid exchanges or DNA damage in yeast) and in vivo assays
for chromosomal damage tend to give positive results. Specific studies are
summarized below. Unless specified otherwise, S9 fractions were prepared from
the liver of Aroclor 1254-induced male rats.
V-65
-------
a. In Vitro Studies
Chloroform has been reported to be nonmutagenic in both Salmonella
typhimurium and Escherichia goli reverse mutation assays under conditions
designed to reduce chloroform loss through volatilization. The inclusion of
exogenous metabolic activation derived from the livers or kidneys of untreated
or Aroclor-pretreated mice or rats did not alter these findings.
Gocke et al. (1981) assessed the mutagenicity of chloroform in £.
typhimurium strains TA1535, TAlOO, TA1538, TA98 and TA1537 in the presence and
absence of S9. Chloroform at levels up to 30 jimol/plate (3,600 /ig/plate) did
not increase the mutation frequency in any tested strain.
Van Abbe et al. (1982) also tested chloroform mutagenicity in these
five tester strains of S^. typhimurium. In a standard mutation assay, there
was no evidence of mutagenicity at 84 ^mol/plate (10,000 ^g/plate) in the
presence or absence of microsomal preparations from the liver or kidney of
rats and mice. There was also no evidence of mutagenicity in strains TA1535
or TA1538 exposed to a stream of chloroform vapor at 32 mL/hour in the
presence or absence of rat liver S9.
Van Abbe et al. (1982) also tested chloroform mutagenicity in these
five tester strains of S.. typhimurium. using a closed system to reduce
volatilization of the test material. There was no evidence of mutagenicity at
84 ^mol/plate in the presence or absence of microsomal preparations from the
liver or kidney of rats and mice.
V-66
-------
Uehleke et al. (1977) found that 5 mM chloroform was not mutagenic in
£. tvphimurium strains TA1535 or TA1538 in the presence or absence of
exogenous metabolic activation. Liver micrcscmes prepared from male rats and
mice were used as the activation system. Similar negative results were also
reported by Simmon et al. (1977).
Kirkland et al. (1981) investigated the mutagenicity of chloroform in
E. coli over a wide range of concentrations (8.4 x 10"^ to 84 /jmol/plate; 0.1
to 10,000 jig/plate), using a closed system to prevent chloroform
volatilization. Negative results were reported in a standard assay and in a
preincubation assay, both in the presence and in the absence of S9 microsomes.
In contr.ast to the sizable number of negative results, Varma et al.
(1988) reported that 0.2 or 0.4 /unol/plate chloroform induced a mutagenic
response in four strains of S_. tvphimurium and the response was lower with
activation than without. Normal background levels were observed at higher
doses (0.8 to 4.2 /xmol/plate). This spike in mutation frequency at the low
dose is very unusual, especially since the number of revertants was almost
identical in strains that detect frameshifts and those that detect base
substitutions. It is possible that the reported data may have resulted from
cytotoxicity, although the number of revertants at the norunutagenic doses was
comparable Co background levels. A methanol/water mixture was used as a
solvent and the authors did not specify whether the assay was conducted in a
closed system.
Sturrock (1977) investigated the mutagenic potential of chloroform in
Chinese hamster lung fibroblasts at the 8-azaguanine locus in the absence of
exogenous metabolic activation. Chloroform was tested as a vapor at
V-67
-------
concentrations of 10,000 to 25,000 ppm. These concentrations did not produce
any significant increase in the incidence of mutation.
Kirkland et al. (1981) did not observe an increase in the frequency of
chromosomal aberrations or sister chromatid exchanges in human lymphocytes
exposed to 0.21 to 3.35 mM of chloroform. Incubations were performed in a
closed system to prevent chloroform volatilization. However, the positive
control was not run concurrently.
Morimoto and Koizumi (1983) also used human lymphocytes to test
chloroform for its potential to induce sister chromatid exchanges. No
activation system was used. Significant increases were observed at chloroform
concentrations o.f 10 mM or higher.
Sobti (1984) assessed the potential of S9-activated chloroform to
induce sister chromatid exchanges in human lymphocytes. Microsomes prepared
form the liver of phenobarbital-induced rats were used as the activating
system. Significant increases (p < 0.01) in sister chromatid exchanges were
observed at concentrations of 0.1 mM or higher.
White et al. (1979) investigated the ability of chloroform to induce
sister chromatid exchanges in Chinese hamster ovary (CHO) cells in the
presence of an activation system. Chloroform was tested as a vapor at only
one concentration (7,100 ppm). This concentration did not significantly
increase the incidence of sister chromatid exchanges.
Callen et al. (1980) used the D7 strain of Saccharomyces cerevisiae to
study the genotoxicity of chloroform in the absence of S9 activation.
V-68
-------
Incubations were carried out in screw-capped glass tubes to prevent
volatilization of the chloroform. Positive results were obtained for gene
conversion, gene reversion and mitotic crossing over at the highest dose
(54 mM) . At lower concentrations (21 mM and 41 mM), small dose-related
increases in gene convertants were observed.
Crebelli et al. (1988) used the fungus Aspergillus nidulans to
investigate the ability of chloroform to cause chromosome malsegregation
(nondisjunctional diploid and haploid segregants). The incidence of these
events was increased at a very high concentration (20 mM; 0.16% by volume).
Table V-22 summarizes data on the genotoxic potential of chloroform
from these in vitro studies.
b. In Vivo Studies
Reitz et al. (1980) studied the potential of chloroform to cause DNA
alkylation in vivo in mice and rats. Animals were given a single oral dose of
240 mg/kg 14C-chlorofonn and were sacrificed four hours later. DNA was
isolated from the livers and kidnevs and evaluated for label binding. No
significant increase in DNA alkylation was observed.
Colacci et al. (1991) administered chloroform to rats and mice
intraperitoneally, and observed covalent binding of radiolabeled chloroform to
DNA isolated from the liver, kidney, lung, and stomach. Chloroform binding to
calf thymus DNA was also observed in vitro in the presence of mouse liver
microsomes, but only at very low levels in the absence of microsomes or
cytosol.
V-69
-------
TABLE V-22 Summary of In Vitro Genotoxicity Data on Chloroform
Endpoint
Assay System
Results (w/wo
Activation)
References
Gene mutation
Salmonella
tvphimurium
TA1535, TA100,
TA1538, TA98,
TAl537b
TA98, TA100,
TA1535, TA1537
TAl538b
TA1535;' TA153-83
TA1535, TA15383
TA1535, TA1537,
TA1538, TA98,
TA1003
TAlOOb
TA1537, TA1535,
TA98, TA100b
Gocke et al . 1981
Van Abbe et al. 1982
Van Abbe et al. 1982
Uehleke et al. 1977
Simmon et al. 1977
Rapson et al. 1980
Varma et al. 1988
Chromosome
aberration
Sister
chromatid
exchange
DNA damage
Chromosome
malsegregatio-
Ischerichia colia
Chinese hamster
lung fibroblasts3
Human
lymphocytes3
Chinese hamster
ovary cells3
Human
lymphocytes3
Human
lymphocytes'5
Human
lymphocytes'*
Saccharomvces
cerevisiae3
Aspergillus
nidulans3
V-
NTC/-
( vapor)
-/NT
-/NT
(vapor)
-/NT
NT/+
+/NT
NT/+
NT/+
Kirkland et al
Sturrock 1977
Kirkland et al
. 1981
. 1981
White et al . 1979
Kirkland et al
Morimoto and
Koizumi 1982
Sobti 1984
Callen et al .
Crebelli et al
. 1981
1980
. 1988
3Assay was conducted in a closed system.
^he authors did not specify whether or not the assay was conducted in a
closed system.
°Not tested.
V-70
-------
Gocke et al. (1981) conducted studies on the potential genotoxicity of
chloroform in two in vivo assay systems. Ir. the first assay, Drosophila were
exposed to one chloroform concentration (25 mM), and chromosomes were
evaluated for sex-linked recessive lethal mutations. No increased incidence
of this type of mutation was detected. In the clastogenicity assay, mice were
given two doses of 238 to 952 mg/kg chloroform, and polychromatic erythrocytes
from the bone marrow were scored for micronucleus formation. No significant
increase was detected. However, the protocol (two treatments separated by 24
hours with sampling at 6 hours after the second treatment) was incomplete.
The sample should have been collected at least 48 hours after the first
treatment (Mavournin et al. 1990).
Agustin and Lim-Sylianco (1978) investigated the mutagenicity of
chloroform in a host-mediated assay. Salmonella strains TA1535 and TA1537
were used as indicator organisms and injected into male and female mice. The
authors reported that male, but not female, mice metabolized chloroform to a
form that was mutagenic in strain TA1537. Although the results suggest a
positive response in males, incomplete reporting of the data and the
procedures used prohibit a definitive conclusion (U.S. EPA 1985a).
Fujie et al. (1990) analyzed chromosome aberrations in bone marrow
cells from Long-Evans rats (three/sex/dose) following oral (males only) or
intraperitoneal (males and females) exposure to chloroform. Oral administra-
tion was by gavage in saline for five consecutive days, and the animals were
sacrificed 18 hours after the last dose. Dose-related increases in the inci-
dence of aberrant cells were observed at all levels (1.2 to 119.4 mg/kg/day),
but the effect was significant (p < 0.01) only at the high dose. More pro-
V-71
-------
nounced increases in clastogenic activity were noted when comparable doses
were administered once by intraperitoneal injection. Regardless of the route,
the predominant types of induced aberrations were chromatid and chromosome
breaks.
Liang et al. (1983) investigated the ability of chloroform to produce
chromosome aberrations in grasshopper embryos exposed in vivo. The incidence
of chromosome aberrations was increased in embryos exposed to 620 to
1,200 ^mol/jar (0.05 to. 0.1 mL/jar).
Land et al. (1981) exposed mice to 400 or 800 ppm chloroform in air
for 4 hours/day for 5 days, and observed an increase in the frequency of sperm
head abnormalities when compared to the negative controls (p < 0.01).
Although the results suggested a positive response, the appropriateness of the
statistical analysis has been questioned (U.S. EPA 1985a). Topham (1980) did
not observe any sperm-head abnormalities in mice given 0.037 to 0.37 mg/kg/day
by intraperitoneal injection.
Morimoto and Koizumi (1983) investigated the potential of chloroform
to cause sister chromatid exchanges in mice. The mice were given 25 to
200 mg/kg/day chloroform by gavage (in olive oil) for four days. Significant
increases (p < 0.05) were detected at doses of 50 mg/kg/day or more.
Vogel and Nivard (1993) found that chloroform at concentrations
ranging from 2,000 ppm to a lethal dose of 16,000 ppm did not increase the
frequency of interchromosomal mitotic recombination at the white locus in
Drosophila melanogaster.
V-72
-------
Table V-23 summarizes data on the genotoxic potential of chloroform
from these in vivo studies.
2. Brominated Trihalomethanes
A number of in vitro and in vivo studies have been performed to inves-
tigate the genotoxicity of brominated trihalomethanes. In general, these
studies have yielded mixed results, and in some cases are subject to uncer-
tainty due to the tendency of these compounds to volatilize from the test
systems. However, a number of positive findings have been reported, both in
vitro and in vivo. for all three compounds. These data generally indicate
that bromodichloromethane and bromoform are genotoxic. The data on dibromo-
chloromethane are less conclusive, but are also suggestive of genotoxicity.
Data are summarized in Tables V-24 (bromodichloromethane), V-25 (dibromo-
chloromethane), and V-26 (bromoform). More detailed descriptions of these
studies are provided below.
a. In Vitro Studies
Simmon and Tardiff (1978) reported that nonactivated bromodichloro-
methane, dibromochloromethane, and bromoform were mutagenic in S^. tvphimurium
strain TA100 when assayed in a desiccator containing an atmosphere with the
test compound. The minimal amount added to the dessicator resulting in a
mutagenic response was 600, 57, and 570 jjmol (50, 5, and 50 /tL) for
bromodichloromethane, dibromochloromethane, and bromoform, respectively.
Zeiger (1990) found that bromoform vapor was mutagenic in S_.
tvphimurium strain TA98 when tested as a vapor in a closed system, but not
V-73
-------
TABLE V-23 Summary of In Vivo Genotoxicicy Data on Chloroform
Endpoint
Assay System
Results
Reference
DNA alkylation
DNA binding
Gene mutation
Micronuclei
Chromosome
aberrations
Sperm
abnormalities
Sister
chromatid
exchange
Mitotic
recombination
Mouse and rat
Mouse and rat
Drosophila
Host (mouse) mediated
assay (£. tvptiimurium
TA 1537)
Host (mouse) mediated
assay (S.. typhimurium
TA1535)
Mouse
Rat
Rat
(intraperitoneal)
Grasshopper
embryo
Mouse
(vapor)
Mouse
(intraperitoneal)
Mouse
Drosophila melanogaster
(vapor)
Reitz et al. 1982
Colacci et al. 1991
Gocke et al. 1981
Agus t in and
Lim-Sylianco
1978
Agustin and
Lim-Sylianco
1978
Gocke et al. 1981
Fujie et al. 1990
Fujie et al. 1990
Liang et al. 1983
Land et al. 1981
Topham 1980
Morimoto and
Koizumi 1983
Vogel and Nivard 1993
aPositive in male mice but not in female mice.
V-74
-------
TABLE V-24 Summary of Genotoxicity Data on Bromodichloromethane
Endpoint
Assay System
Results (w/wo
Activation)
References
In Vitro Studies
Gene mutation
Chromosome
aberration
DNA damage
Sister
chromatid
exchange
Salmonella
typhimurium
TAIOO3
TA98, TAIOO, -
TA1535, TA1537C
TA100C
TA15370
TA1535, TA98,
TA100C
Mouse lymphoma
cellsc
Chinese hamster
fibroblastsc
Chinese hamster
ovary cells0
Saecharomyces
cerevisiae3
Human
lymphocytes3
Human
lymphocytes3
Rat liver cells9
Chinese hamster
ovary cellsc
NTb/+
V-
V-
NT/+
+/NT
+/NT
Simmon and Tardiff
1978
NTP 1987
Ishidate et al. 1982
Varma et al. 1988
Varma et al. 1988
NTP 1987
Ishidate et al. 1982
NTP 1987; Anderson
et al. 1987
Nestmann and Lee
1985
Morimoto and Koizumi
1983
Sobti 1984
Sobti 1984
NTP 1987; Anderson
et al. 1987
V-75
-------
Table V-24 (Continued)
Endpoint
In Vivo Studies
Micronuclei
Chromosome
aberrations
Sister
chromatid
exchange
Assay System Results
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow +
cells
Rat bone marrow +
cells (i.p.)
Mouse bone +
marrow cells
References
Ishidate
Hayashi
Fuj ie et
Fuj ie et
Morimoto
1983
et al. 1982
et al. 1988
al. 1990
al. 1990
and Koizumi
aAssay was conducted in a closed system.
kNot tested.
cAuthors did not specify whether or not the assay was conducted in a closed
system.
d£quivocal
V-76
-------
TABLE V-25 Summary of Genotoxicity Data on Dibromochloromethane
Endpoint
Assay System
Results (w/wo
Activation)
References
In Vitro Studies
Gene mutation
Salmonella
typhimurium
TA1003
TA98, TA100, "
TA1535, TA1537C
TA1537, TA1535C
TA98, TA100C
TA100C
NTb/+
Simmon and Tardiff
1978
NTP 1985
Varma et al. 1988
Varma et al. 1988
Ishidate et al. 1982
Chromosome
aberration
DNA damage
Sister
chromatid
exchange
Mouse lymphoma
cells3
Chinese hamster
fibroblastsc
Chinese hamster
ovary cellsc
Saccharomyces
cerevisiae3
Human
lymphocytes3
Human
lymphocytes3
Rat liver cellsc
Chinese hamster
ovary cells0
NT/+
NT/+
+/NT
+/NT
McGregor et al. 1991
Ishidate et al. 1982
Loveday et al. 1990
Nestmann and Lee
1985
Morimoto and Koizumi
1983
Sobti 1984
Sobti 1984
Loveday et al. 1990
V-77
-------
Table V-25 (Continued)
Endpoint
In Vivo Studies
Micronuclei
Chromosome
aberrations
Sister
chromatid
exchange
Assay System Results
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow +
cells
Rat bone marrow +
cells (i.p.)
Mouse bone +
marrow cells
References
Ishidate
Hayashi
Fuj ie et
Fuj ie et
Morimoto
1983
ec-al. 1982
et al. 1988
al. 1990
al. 1990
and Koizumi
aAssay was conducted in a closed system.
''Not tested.
cAuthors did not specify whether or not the assay was conducted in a. closed
system.
dNot specified.
V.-78
-------
TABLE V-26 Summary of Genotoxicity Data on Bromoform
Endpoint
Assay System
Results (w/wo
Activation)
References
In Vitro Studies
Gene Mutation
Chromosome
aberration
Sister
chromatid
exchange
Salmonella
typhimurium
TA1535, TA1003
TAl535riAl537c
TA100.
TA98, TA98
TA100C
TA98a
TA100, TA15383
Mouse lymphoma
cells0
Chinese hamster
fibroblastsc
Chinese hamster
ovary cellsc
Toadfish
leukocytes3
Human
lymphocytes0
Chinese hamster
ovary cells0
NTb/+
?"/-
-/+
+/+
-/-
+/-
NT/-
NT/+
-/mixed
Simmon and Tardiff
1978
NTP 1989a
NTP 1989a
NTP 1989a
Ishidate et al .
1982
Zeiger 1990
Zeiger 1990
NTP 1989a
Ishidate et al . 1982
NTP 1989a
Maddock and Kelly
1980
Morimoto and Koizumi
1983
NTP 1989a
V-79
-------
Table V-26 (Continued)
Endpoint
In Vivo Studies
Micronuclei
Chromosome
aberration
Sister
chromatid
exchange
Sex -linked
recessive
lethal
mutations
Assay System
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow
cells
Rat bone marrow
cells (i.p.)
Mouse bone
marrow cells
Mouse bone
marrow cells (i.p.)
Drosophila
Results References
Ishidate et al. 1982
Hayashi et al. 1988
NTP 1989a
+ Fuj ie et al . 1990
+ Fujie et al . 1990
+ Morimoto and Koizumi
1983
+ NTP 1989a
+ NTP 1989a
aAssay was conducted in a closed system.
''Not tested.
cAuthors did not
system.
dEquivocal
specify whether or not
the assay was conducted in a closed
V-80
-------
when tested in an open system using a preincubation protocol. Positive
results were observed at levels of at least 114 ^mol/desiccator (0.01 mL/
dessicator), in the presence and absence of S9 prepared from rat or hamster
liver. Bromoform was negative in the closed system with strains TAlOO and
TA1538 +/- rat or hamster liver S9.
Ishidate et al. (1982) conducted studies on the mutagenicity of
bromodichloromethane, dibromochloromethane, and bromoform with £. typhimurium
strain TAlOO. Increased mutation frequencies were observed with all three
nonactivated compounds, but not with the compounds in the presence of S9. All
three compounds also induced chromosomal aberrations in Chinese hamster
fibroblasts, in the presence, but not the absence, of rat liver S9. The
concentrations tested in these two assays were not reported.
Varma et al. (1988) tested bromodichloromethane and dibromochloro-
methane for mutagenicity in J5. typhimurium strains TA1535, TA1537, TA98, and
TAlOO. Bromodichloromethane at nonactivated concentrations of 2.4 to
3.2 /xmol/plate induced mutations in strain TA1537. There was no effect in the
other strains. Dibromochloromethane produced a significantly increased
mutation frequency at the lowest S9-activated dose (0.12 ^mol/plate) in all
four strains. Dosing with nonactivated dibromochloromethane at this level
also resulted in increased mutation frequencies in strains TA1535 and TA1537.
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-81
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NTP (1987) reported that bromodichloromethane was not mutagenic when
tested using a preincubation protocol in S.. typhimurium strains TA1535,
TA153"?, TA98, or TA100 at concentrations reaching cytotoxic levels
(20 /jmol/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
it did induce dose-related increases in forward mutations at S9-activated
concentrations greater than or equal to 2,000 jiM (300 ^g/mL). Cytogenetic
tests with CHO cells were reported here and by Anderson et al. (1990). There
was no evidence of induction of chromosomal aberrations following treatment
with up to 30,00.0 /iM (5,000 jjg/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 /xM (4,000 ^g/mL). However, these results are
difficult to interpret because cytotoxicity was observed at similar levels in
the other trials.
NTP (1985) reported that dibromochloromethane (0.5 to 50 ^mol/plate;
100 to 10,000 /ig/plate) was not mutagenic in strains TA1535, TA1537, TA98, or
TAlOO when tested in the presence or absence of Aroclor-induced Sprague-Dawley
rat or Syrian hamster liver S9 fractions. The volatilization of the test
compound was again considered to be a possible explanation for the negative
results.
V-82
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NTP (L989a) studied the genotoxic potential of bromoform in several
test systems. Concentrations of 0.04 to 13 ^mol/plate (10 to 3,333 fig/plate)
produced no evidence of mutager.icity in j5. typhiguriun: strains TA1535 or
TA1537, with or without exogenous metabolic activation derived from rat or
hamster liver. Equivocal evidence of mutagenicity was noted in strain TAlOO
(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 nonactivated bromoform concentrations greater than or equal to
2,300 /iM (200 nL/mL) or S9-activated concentrations of at least 300 ^M
(25 nL/mL) resulted in forward mutations at the thymidine kinase (tk) locus.
One of two laboratories reported increased sister chromatid exchanges in CHO
cells exposed to 3,800 fM (290 jjg/mL) bromoform in the absence of exogenous
activation; neither laboratory observed increases in the presence of S9. S9-
activated bromoform did not induce chromosome aberrations in CHO cells;
results in the absence of exogenous activation were equivocal.
Nestmann and Lee (1985) investigated the mutagenicity of bromodichloro-
methane at 12 to 1,200 /JU (0.001 to 0.1 /*L/mL) and dibromochloromethane at 11
to 5,700 jiM (0.001 to 0.5 /zL/mL) in S. cerevisiae strains .07 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 with bromodichloromethane +/-
S9 or S9-activated dibromochloromethane. Nonactivated dibromochloromethane
produced an increased incidence of gene convertants in strain D7 at
concentrations greater than 1,140 /^M (0.1 jxL/mL), but there was no effect on
revertants. The high dose was cytotoxic.
Dibromochloromethane induced mutations at the tk locus of L5178Y mouse
lymphoma cells when tested at concentrations greater than or equal to 480 jiM
V-83
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(100 ^g/rnL) 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 at S9-activated levels that caused cell-
cycle delay (12,200 /iM; 2,540 jig/mL) or at nonactivated levels that were cyto-
toxic with a standard harvest time (6,000 /iM; 1,240 /ig/mL) . Sister chromatid
exchange was induced in CHO cells by S9-activated 3,600 jiM (740 jjg/mL) with a
delayed cell harvest, while the nonactivated test material had no effect up to
cytotoxic levels (1,200 /jM; 247 jig/mL) .
Morimoto and Koizumi (1983) investigated the ability of the brominated
trihalomethanes .to induce sister chromatid exchanges in human lymphocytes in
vitro in the absence of S9 activation. All three compounds (bromodichloro-
methane, dibromochloromethane, and bromoform) caused a dose-dependent increase
in sister chromatid exchanges. Bromoform was more potent than bromodichloro-
methane or dibromochloromethane. The increases were significant (p < 0.05) at
concentrations greater than or equal to 400 /iM, 400 /jM, and 80 /jM for bromo-
dichloromethane, dibromochloromethane, and bromoform, respectively.
The potential of S9-activared bromodichloromethane and dibromochloro-
methane to induce sister chromatid exchanges in vitro was also investigated by
Sobti (1984). A dose of 100 /iM of either trihalomethane produced an increased
frequency of sister chromatid exchange in rat liver cells. Bromodichloro-
methane produced the same effect in human lymphocytes at a concentration of
100 jiM, as did dibromochloromethane at 1 jiM. Maddock and Kelly (1980)
reported that bromoform did not induce an increase in sister chromatid
exchanges in toadfish leukocytes exposed to concentrations of 0.4 to 400 /iM.
V-84
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b. In Vivo Studies
Fujie et al. (1990) analyzed chromosome aberrations in bone marrow
from Long-Evans rats (3/sex/dose) following oral (males only) or intraperi-
toneal (males and females) exposure to bromoform, dibromochloromethane, or
bromodichloromethane. 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. The other two
chemicals induced smaller, dose-related increases in breaks. More pronounced
increases in clastogenic activity were observed following a single intraperi-
toneal dose, with significant (p < 0.05) effects at 16.4, 20.8, and 25.3 mg/kg
for bromodichlorpmethane, dibromochloromethane, and bromoform respectively.
Regardless of the route, the predominant types of induced aberrations were
chromatid and chromosome breaks.
Hayashi et al. (1988) measured induction of micronucleated polychro-
matic erythrocytes in ddY mice by intraperitoneal administration of bromodi-
chlorome thane, dibromochloromethane, or bromoform at single doses up to 500,
1,000 and 1,400 mg/kg in corn oil, respectively. No evidence of clasto-
genicity was observed with any of the chemicals. However, the sampling time
was not long enough for dibromochloromethane and bromoform, and there was no
clear evidence of toxicity or cytotoxicity in the target tissue.
Ishidate et al. (1982) investigated the in vivo clastogenicity of
bromodichloromethane, dibromochloromethane, and bromoform using ddY and MS
mice and Wistar rats. Animals were administered 125 to 500 mg/kg/day trihalo-
methane in olive oil by intraperitoneal injection, and were sacrificed at 18,
V-85
-------
24, 30, 48, and 72 hours after dosing. No significant induction of micro-
nucleus formation was reported in mice or rats given any of the brominated
trihalomethanes.
Morimoto and Koizumi (1983) used male ICR/SJ mice to investigate the
potential of trihalomethanes to produce sister chromatid exchanges i.n vivo.
Animals were given 0, 25, 50, 100, or 200 mg/kg/day of bromodichloromethane,
dibromochloromethane, or bromoform for four days by olive oil gavage. All
three brominated trihalomethanes were similarly potent and produced a roughly
linear dose - dependent increase in sister chromatid exchange frequency. These
increases were statistically significant (p < 0.05) at 50, 25, and
25 mg/kg/day for bromodichloromethane, dibromochloromethane, and bromoform,
respectively. 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, although only short-term exposure periods were investigated.
NTP (1989a) studied the genotoxic potential of bromoform in several
test systems. Feeding adul-t male Drosophila with a 1,000-ppm solution of
bromoform increased the frequency of sex-linked recessive lethal mutations but
had no significant effects on reciprocal translocations. 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.
NTP concluded that the genotoxicity of bromoform has been demonstrated in a
variety of test systems, both in vivo and in vitro (see above), and indicated
that this property may be involved in the carcinogenicity of bromoform. This
is consistent with the general observation that sister chromatid exchanges are
usually observed at lower doses than are chromosome aberrations.
V-86
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E. Carcinogenicitv
1. Chloroform
Eschenbrenner and Miller (1945) induced hepatomas in mice with
chloroform. Three-month-old Strain A mice (historical spontaneous hepatoma
rate of <1% at 16 months of age) were administered chloroform at dose levels
of 150, 300, 600, 1,200, or 2,400 mg/kg in olive oil by gavage (five/sex/
group). "Chemically pure" chloroform was used, but chemical analysis was not
indicated. Controls received olive oil only. The animals were dosed every
4 days for a total of 30 doses (120 days) and were examined for hepatomas
30 days after the last dose. Twenty-four hours before necropsy the animals
were given an additional dose of chloroform. Tissues and organs were examined
histopathologically. Five other groups of mice (one male and two females per
group) were given single doses of chloroform (same dose levels as used
previously) 24 hours before liver removal. No males administered doses of at
least 600 mg/kg and no females in the high-dose group survived the study. All
deaths occurred 24 to 48 hours after the first or second chloroform dose. All
surviving females dosed with chloroform at 600 or 1,200 mg/kg developed
hepatomas. Liver necrosis was observed in both sexes in the three highest-
dose groups. Males in all treatment groups developed kidney necrosis, whereas
kidney necrosis was not apparent in any females (Table V-27).
Necrosis was not observed in hepatoma cells. Hepatomas contained
cords of enlarged liver-like cells that formed disorganized anastomosed
columns. The hepatomas did not appear invasive and no metastasis was found.
Renal necrosis in males was localized in the areas of the proximal and distal
tubules; glomeruli and collecting tubules appeared normal. The severity of
V-87
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TABLE V-27 Liver and Kidney Necrosis and Hepatomas in Strain A Mice
Following Repeated Oral Administration of Chloroform in Olive Oil
Chloroform Dose (mg/kg)
Observation
Liver necrosis3
Kidney necrosis
Deaths
Hepatomasb
Sex
F
M
F
M
F
M
F
M
150
0
0
0
+ '
0/5
0/5
0/5C
0/5
300
0
0
0
+
0/5
2/5
0/5
0/3
600 1,200 2,400 Control
+ + + 0
+ + + 0
000 0
+ + + 0
2/5 1/5 5/5 0/5
5/5 5/5 5/5 0/5
3/3 4/4 --d 0/5
0/5
a+ = necrosis.; 0 = no indication of necrosis.
bln animals surviving the dosing regimen.
cPositive occurrences/animals observed.
"^No animals survived.
Adapted from Eschenbrenner and Miller (1945)
V-88
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renal necrosis was dose related. The authors speculated that the different
renal responses by males and females to chloroform treatment may have been due
to the unique lining of the Bowman's capsules with flat or cuboidal epithelium
in females and males, respectively (an anatomic sexual dimorphism in mice).
In the single-dose experiment, there was a sharp distinction between
normal and necrotic cells in the liver. Doses of 1,200 and 2,400 mg/kg
produced extensive necrosis in all liver lobules, and the 600 mg/kg dose
produced necrosis in some lobes. U.S. EPA (1985a) stated that the Eschen-
brenner and Miller study (1945) indicated that hepatomas in female mice were
induced at chloroform doses that also produced liver necrosis. Early
mortality precluded the development of hepatomas in all animals administered
doses that produced liver necrosis. The observation of kidney necrosis in
males without tumor formation and the lack of necrosis in hepatomas suggested
to the authors that livers in Strain A mice were uniquely sensitive to tumor
induction at necrotizing doses of chloroform, or that there might be other
factors affecting liver tumor formation, in addition to necrosis. Also,
because a dose of chloroform was given 1 day before sacrifice (a factor which
in itself could have been responsible for producing necrosis), the extent of
necrosis during the last month of observation while the animals were untreated
is not clear.
The National Cancer Institute (NCI 1976) performed a carcinogenic
bioassay of chloroform on Osborne-Mendel rats. Chloroform was administered by
oral gavage (in corn oil) to 50 animals/sex five times per week for 78 weeks.
Male rats (52 days old) were administered doses of 90 or 180 mg/kg/day.
Female rats ('52 days old) were initially administered doses of 125 or
250 mg/kg/day, but the doses were reduced to 90 or 180 mg/kg/day after
V-89
-------
22 weeks (average doses of 100 or 200 mg/kg/day). All animals were sacrificed
after 111 weeks. Decreases in survival rates and weight gain were evident for
all treated groups. The incidence of renal epithelial tumors in male rats was
24% in the high-dose groups (p = 0.0016). An increase in thyroid tumors was
observed in treated female rats, but this finding was not considered
biologically significant. Table V-28 summarizes tumor frequencies for all
groups in the study.
NCI (1976) performed a similar carcinogenic bioassay of chloroform in
B6C3F1 mice. Animals (35 days old) were administered initial dose levels of
100 or 200 mg/kg/day for males and 200 or 400 mg/kg/day for females. These
doses were increased after 18 weeks to 150 or 300 mg/kg/day for males and 250
or 500 mg/kg/day for females (average doses of 138 or 277 mg/kg/day for males,
238 or 477 mg/kg/day for females). The animals were sacrificed after 92 to
93 weeks. Survival rates and weight gains were comparable for all groups
except high-dose females. Significant increases (p < 0.001) in hepatocellular
carcinomas were observed in all treated groups. Table V-29 summarizes tumor
frequencies for all groups in the study. Nodular hyperplasia of the liver was
observed in many low-dose male mice that had not developed hepatocellular
carcinomas.
Roe et al. (1979) studied the carcinogenicity of chloroform in
toothpaste in four strains of mice (C57BL, CBA, CF/1, and ICI) . In three
different studies, 10-week-old mice were administered chloroform by gavage
6 days per week for 80 weeks, followed by a 13- to 24-week observation period.
In the first study, ICI mice (52/sex/dose) were given chloroform (in
toothpaste) at 17 or 60 mg/kg/day. One control group per sex of ICI mice was
given toothpaste without chloroform. The second study included 260 ICI mice
V-90
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TABLE V-28 Summary of Tumor Frequencies in Rats
Administered Chloroform for 78 Weeks
Hepatocellular
Carcinomas/Number
of Animals
Kidney Epithelial
Tumors/Number
of Animals
Thyroid Tumors/
Number of Animals
Treatment
Control -colony
Control -matched
Low dose
High dose
Sex
M
F
M
F
M
F
M
F
Examined (%)
1/99
0/98
0/19.
0/20
0/50
0/49
1/50
0/48
(1)
(0)
(0)
(0)
(0)
(0)
(2)
(0)
Examined (%)
0/99
0/98
0/19
0/20
4/50
0/49
12/50
2/48
(0)
(0)
(0)
(0)
(8)
(0)
(24)
(4)
Examined
6/99
1/98
4/19
1/19
3/49
8/49
4/48
10/46
(%)
(8)
(1)
(21)
(5)
(6)
(16)
(8)
(22)
Adapted from NCI (1976).
V-91
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TABLE V-29 Summary of Tumor Frequencies in Mice
Administered Chloroform for 78 Weeks
Hepatocellular
Carcinomas/Number
of Animals
Kidney Epithelial
Tumors/Number
of Animals
Thyroid Tumors/
Number of Animals
Treatment
Control -colony
Control -matched
Low dose
High dose
Sex
M
F
M
F
M
F
M
F
Examined (%)
5/77
1/80
•"• 1/18
0/20
18/50
36/45
44/45
39/41
(6)
(1)
(6)
(0)
(36)
(80)
(98)
(95)
Examined (%)
1/77
0/80
1/18
0/20
1/50
0/45
2/45
0/40
(1)
(0)
(6)
(0)
(2)
(0)
(4)
(0)
Examined (%)
0/77
0/80
0/17
0/20
0/48
0/41
0/43
0/36
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
Adapted from NCI (1976)
V-92
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given toothpaste without chloroform and a group of 52 mice administered
chloroform at 60 mg/kg/day in toothpaste;.a second control group consisted of
52 untreated mice. In the third study, four groups oT 32 male mice (one
group/strain) were administered chloroform in toothpaste at a dose of
60 mg/kg/day. A fifth group (52 male ICI mice) was given the same dose of
chloroform in arachis oil. This study contained three control groups: an
untreated control (100 ICI mice), a group given toothpaste without chloroform
(52 mice), and a group given arachis oil only (52 ICI mice). Body weights
were recorded in each study, and food consumption was estimated in the second
and third studies. In each study the animals were necropsied, and tumors,
lesions, and selected tissues and organs were examined histopathologically.
Adrenals, kidneys, livers, lungs, and spleens were weighed.
Differences in survival, body weight, and food consumption between
control and treatment groups were not statistically significant (p > 0.05).
Median survival was approximately 73 weeks in the first and second studies.
In the third study, 52% to 79% of the C57BL and CBA mice and 12% to 31% of the
CF/1 and ICI mice were alive at the end of the observation period. Liver and
kidney weights were slightly lower (data not reported) in male ICI mice given
chloroform in toothpaste.
The incidence of tumors did not differ significantly between control
and chloroform-treated groups of male or female C57BL, CBA or CF/1 mice, or
between control and treated female ICI mice. Increased frequency of kidney
tumors was observed in treated male ICI mice (Table V-30). Malignant tumors
were identified as hypernephromas, and benign kidney tumors were characterized
as cortical adenomas. The authors observed a significantly higher incidence
of moderate to severe kidney "changes" in treated CBA and CF/1 males
V-93
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TABLE V-30 Kidney Tumor Incidence in Male ICI Mice
Treated with Chloroform
Dose Group
Numbers of Mice
Examined
Histologically
Number of Mice with Kidnev Tumors
Benign Malignant Total
First study:
Vehicle-control3
17 mg/kg/dayb
60 mg/kg/day*
Second study:
Untreated control
Vehicle-control3
60 mg/kg/dayf
Third study:
72
37
38
45
237
49
0
0
5C
1
6
7C
0
0
3d
0
0
2d
0
0
8e
1
6
9e
Untreated control
Vehicle -control9
60 mg/kg/dayh
Vehicle -control1
60 mg/kg/dayj
83
49
47
50
48
0
1
2
1
3
0
0
3
0
9C
0
1
5
0
12e
toothpaste-base vehicle without chloroform, eucalyptol or peppermint oil.
bChloroform given in toothpaste base with eucalyptol and peppermint oil.
cStatistically significant versus vehicle-control (p < 0.05).
dStatistically significant versus vehicle-control (p < 0.01).
Statistically significant versus vehicle-control (p < 0.001).
*Chloroform given in toothpaste base without eucalyptol or peppermint oil.
9Toothpaste-base vehicle without chloroform.
hChloroform given in toothpaste base.
'Arachis oil.
^Chloroform given in arachis oil.
Adapted from Roe et al. (1979)
V-94
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(p < 0.001, chi-square test) and moderate to severe kidney disease (p < 0.05.
chi-square test) in treated ICI males compared to controls. (These results
"ere described by the authors without presentation cf data.)
Jorgenson et al. (1985) (also detailed in SRI 1985) studied the
carcinogenic activity of chloroform administered at 0, 200, 400, 900, or
1,800 mg/L in drinking water to male Osborne-Mendel rats and female B6C3F1
mice for 104 weeks. Based on measured water intake and body weights, these
exposures corresponded to time-weighted average doses of 0, 19, 38, 81, or
160 mg/kg/day in rats and 0, 34, 65, 130, or 263 mg/kg/day in mice. The water
consumption of a second control group included in the study was restricted to
that of the high-dose group. Group sizes at low doses were adjusted so that a
detectable tumor, response would result at the lowest dose if there were a
linear relationship with dose. (Group sizes were 330, 150, 50, and 50 for the
low- to high-dose rat groups, and 430, 150, 50, and 50 for the low- to high-
dose mice groups.) Chloroform increased the incidence of renal tumors
(metastatic carcinomas, transitional cell carcinomas, tubular cell adenomas,
and a'denocarcinomas and nephroblastomas) in male rats in a dose-related manner
(Table V-31). The incidences for all kidney tumors were 5/301 (2%), 1/50
(2%), 6/313 (2%), 7/148 (5%), 3/48 (6%), and 7/50 (14%) for the control,
matched control, 200-, 400-, 900-, and 1,800-mg/L groups, respectively.
Chloroform in the drinking water did not increase the incidence of hepato-
cellular carcinomas in female B6C3F1 mice. The combined incidence of hepato-
cellular adenomas and carcinomas was 2% in the high-dose group compared to 6%
in the control groups. The authors speculated that the differences observed
between this study and the NCI (1976) bioassay may be related to differences
in the mode of administration (in drinking water versus in corn oil by
gavage).
V-95
-------
Table V-31 Compound-Re la ted Increased Incidences of Neoplasms in Rats and Mice
Exposed to Chloroform in the Drinking Water for 2 Years
<
o>
Animal
Male rat
Female mice
Tissue/Tumor
Kidney
Tubular cell adenoma
Tubular cell adenocarclnoma
Nephroblastoma
Metastatic carcinoma
Transitional cell carcinoma
Combined
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Control
4/301
0/301
0/301
0/301
1/301
5/301
19/415
2/415
21/415
Matched
Control
0/50
1/50
0/50
0/50
0/50
1/50
0/47
0/47
0/47
Tumor
200
mg/L
2/313
,2/313
5/313
0/313
0/313
1 6/313
8/410
7/410
15/410
Frequency
400
mg/L
3/148
1/148
3/148
1/148
0/148
7/148
8/142
1/142
9/142
900
mg/L
2/48
1/48
0/48
0/48
0/48
3/48
0/47
0/47
0/47
1 , 800
mg/L
5/50
2/50
0/50
0/50
0/50
7/50
0/44
1/44
1/44
Adapted from Jorgenson et al. (1985) and SRI (1985).
-------
Tumasonis et al. (1987) exposed groups of 32 male and 45 female Wistar
rats to chloroform in drinking water until all of the animals died (up to
185 weeks). The exposure level was 2,900 mg/L for 72 weeks and was reduced to
1,450 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
200 mg/kg/day for females and about 150 mg/kg/day for males. Exposed animals
of both sexes gained significantly less weight than did control animals.
There was a statistically significant (p < 0.01) increase in incidence of
hepatic neoplastic nodules in exposed females compared to control females (25%
versus 0%), but not in males (18% versus 23%). Hepatic adenofibrosis occurred
in 61% of the males and 85% of the females, but not in control animals.
However, neoplas.tic nodules and adenofibrosis are not specific pathology
terms, and it is unclear whether these lesions were actually tumors.
Increases in tumor frequency were not noted in other tissues, although
statistically significant decreases occurred in several tumor types (mammary
tumors and pituitary tumors in females, and lymphosarcomas in males). This
study is also limited because the study ran until all of the animals died,
rather than having a scheduled sacrifice. SLnce the control animals had
shorter lifespans than did the exposed animals, age-related tumors could not
be distinguished from treatment-related ones.
Voronin et al. (1987) investigated the carcinogenicity of chloroform
in mice when administered in oil or water. Chloroform produced an increased
incidence of tumors (tissue not specified) in mice given 250 mg/kg/day chloro-
form in oil but not in those given 15 mg/kg/day. No such increase was
reported in mice given 0.0042 to 42 mg/kg/day chloroform in drinking water.
V-97
-------
Interpretation of this study is limited since the only information available
was obtained from an abstract.
A number of studies have investigated the carcinogenic potential of
chloroform using initiation-promotion protocols. Pereira et al. (1985) and
Herren-Freund and Pereira (1987) administered 0, 5, or 20 mg/kg
ethylnitrosourea (EMU) to groups of male and female 15-day-old CD-I Swiss
mice. At weaning, groups.,of 23 to 45 per dose level were supplied with
drinking water containing 0 or 1,800 ppm chloroform, equivalent to about
0 or 270 mg/kg/day, assuming consumption of 150 mL/kg/day (Arrington 1972).
After 46 weeks of exposure (at 51 weeks of age), animals were sacrificed and a
complete histopathological examination was performed on liver, lung, kidney,
and all gross le.sions. In males, ENU treatment alone resulted in a dose-
dependent production of liver adenomas and hepatocellular carcinomas
(Table V-32). Subsequent administration of chloroform inhibited the
occurrence of tumors both in animals that did not receive ENU and in those
that did (Table V-31). No significant effect of chloroform exposure was
observed in female mice. The authors concluded that, in Swiss mice,
chloroform in drinking water acts as an inhibitor of hepatocarcinogenesis. In
considering why the results of this study differed from those reported by NCI
(1976), the authors speculated that the difference may be due either to the
toxicokinetic difference between administration of chloroform as a bolus by
gavage in corn oil and continuous dosing in water or, alternatively, to a
possibly synergistic interaction between chloroform and corn oil.
Klaunig et al. (1986) examined the effects of chronic oral chloroform
exposure on liver and lung tumor incidence in mice. Thirty-five male B6C3F1
mice (4 weeks old) were initiated by treatment with diethylnitrosamine (DENA)
V-98
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TABLE V-32 Effects of Chloroform Exposure on Liver Tumors Initiated by
Ethylnitrosourea Exposure in Male CD-I Mice
Exposure
ENU CHC13
Sex (mg/kg) (1,800 ppm)
Males 0
0 +
5
5 + ""
20
20 +
Females 0
0 +
5
5 . +
20
20 +
Animals Animals with
with Adenomas Adenomas Carcinomas Carcinomas
(%) per Animal (%) per Animal
5
0
21
5
73
41
0
0
0
4
0
4
0.19
0
0.51
0.04
3.13
1.00
0
0
0
0.24
• 0
0.11
5
0
5
0
33
17
0
0
0
0
0
0
0.
0
0.
0
0.
0.
0
0
0
0
0
0
08
10
83 .
21
Adapted from Pereira et al. (1985) and Herren-Freund and Pereira (1987).
V-99
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(10 mg/L) in drinking water for 4 weeks, while a second group of 35 received
deionized drinking water. Each group was subsequently supplied with drinking
water containing 0, 600, or 1,800 mg chloroform/L, corresponding to doses of
about 0, 0.7, or 1.8 mg/kg/day. Animals were sacrificed after 24 weeks
(10/dose) or 52 weeks (25/dose) and were examined grossly and histologically
for liver and lung tumors. In mice not initiated with DENA, exposure to
chloroform did not cause an increase in the incidence of these tumors. In
mice initiated with DENA.,. chloroform inhibited lung and liver tumorigenesis.
These results are shown-in Table V-33. The authors noted that these results
were different from earlier studies that had reported carcinogenic effects of
chloroform. Like Pereira et al. (1985), they speculated that the reason
chloroform did not appear to be carcinogenic in'this study was that water (as
opposed to corn .oil) was used as the vehicle.
Herren-Freund and Pereira (1986) studied the cancer initiating and
promoting activity of chloroform in a short-term assay in rats. The assay
used an increase in the number of gamma-glutamyltranspeptidase (GGT) foci in
regenerating liver as an indicator of carcinogenicity. This system has been
found to detect the activity of both hepatic and nonhepatic carcinogens. Rats
underwent a two-thirds partial hepatectomy, followed 18 to 24 hours later by
administration of the initiator. Exposure to promoter was begun 7 days after
the hepatectomy and was continued for at.least 10 weeks. After sacrifice,
liver slices were prepared and examined for the presence of GGT-positive foci
and any other lesions. Chloroform was negative in both'the initiation assay
(a single [presumably oral] dose of 130 or 260 mg/kg [1.1 or 2.2 mmol/kg] of
chloroform in an unspecified vehicle, followed by 500 mg/L phenobarbital in
drinking water as the promoter) and the promotion assay (a single oral dose of
0.3 mmol/kg of DENA as initiator followed by 1,800 mg/L chloroform in drinking
V-100
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TABLE V-33 Effect of Chloroform Exposure on Liver
and Lung Tumors in Male B6C3F1 Mice
Exposure
CHC13
(mg/L)
0
0
600
600
1,800
1,800
DENA Liver Tumor
(10 mg/L) 24 weeks
0/10
+ 7/10
0/10
+ 4/10
0/10
+ 3/10
Incidence
52 weeks
5/25
25/25
3/25
25/25
4/25
20/25
Lung Tumor
24 weeks
0/10
1/10
0/10
0/10
0/10
1/10
Incidence
52 weeks
2/25
18/25
0/25
13/25
0/25
6/25
Adapted from Klaunig et al. (1986).
V-101
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water). When chloroform (1,800 mg/L) was given concurrently with weekly doses
of DENA (8.2 mg/kg), there was a slight increase in the incidence of liver
tumors and a slight decrease in the mean number cf GGT-foci, but neither of
these effects was statistically significant.
Demi and Oesterle (1987) administered one oral dose of DENA (8 mg/kg)
to female Sprague-Dawley rats. One week later, chloroform was administered by
gavage in oil to groups of 4 to 6 rats at doses of 0, 25, 100, 200 or
400 mg/kg, twice per week for 11 weeks. After this time, animals were
sacrificed and the livers were examined histochemically for GGT-foci. No
significant effect was observed in low-dose animals,.but increases in
preneoplastic islands were observed in animals dosed with 100 mg/kg or above.
Chloroform alone, at these doses did not result in increases in preneoplastic
islands. The observation of a promoting effect in this study, where
chloroform was administered in oil, but not in studies where chloroform was
administered in drinking water, supports the hypothesis that the tumor-
promoting activity of chloroform is vehicle-dependent.
2. Brominated Trihalomethanes
NTP (1987) administered doses of 0, 50, or 100 mg/kg of bromodichloro-
methane in corn oil by gavage to F344/N rats (50/sex/dose), 5 days/week for
102 weeks. In a similar experiment, B6C3F1 mice (50/sex/dose) were adminis-
tered doses of 0, 25, or 50 mg/kg/day (males) or 0, 75, or 150 mg/kg/day
(females). All animals were examined grossly and microscopically for
neoplastic lesions. As shown in Table V-34, bromodichloromethane caused
compound-related increases in the incidences of neoplasms of the large
intestine and kidney in male and female rats, the kidney in male mice, and the
V-102
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TABLE V-34 Tumor Frequencies in Rats and Mice Exposed to
Bromodichloromethane in Corn Oil for 2 Years
Animal
Tissue/Tumor
Tumor Frequency
Male rat
Large intestine3
Adenomatous polyp
Adenocar c inoma
Combined
Kidney3
Tubular cell adenoma
Tubular cell adehocar cinema
Combined
Large intestine and/or kidney combined3
Female rat
Large intestineb
Adenomatous polyp
Adenocarc inoma
Combined
Kidney
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Large intestine and/or kidney combined0
Male mouse
Kidneyd
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female mouse
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Control
0/50
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
0/46
Control
1/46
0/46
1/46
Control
1/50
2/50
3/50
50 mg/kg
3/49
11/49
13/49
1/49
0/49
1/49
13/49
50 mg/kg
0/50
0/50
0/50
1/50
0/50
1/50
1/50
25 mg/kg
2/49
0/49
2/49
75 mg/kg
13/48
5/48
18/48
100 mg/kg
33/50
38/50
45/50
3/50
10/50
13/50
46/50
100 mg/kg
7/47
6/47
12/47
6/50
9/50
15/50
24/48
50 mg/kg
6/50
4/50
9/50
150 mg/kg
23/50
10/50
29/50
aOne rat died at week 33 in the low-dose group and was eliminated from the cancer
risk calculation.
blntestine not examined in four rats from control group and three rats from high-dose
group.
C0ne rat in high-dose group not examined for intestinal tumors had kidney tumors.
dln 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.
Adapted from NTP (1987).
V-103
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liver in female mice. The authors noted that neoplasms of the large intestine
and kidney are uncommon tumors in F344/N rats and B6C3F1 mice, and concluded
that under the conditions of these 2-year gavage studies, there was clear
evidence of carcinogenic activity for male and female rats and mice.
Tumasonis et al. (1987) exposed groups of 58 male and female Wistar
rats to bromodichloromethane in drinking water until all of the animals died
(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 than control animals. There
was a statistically significant (p < 0.01) increase in incidence of hepatic
neoplastic nodules in exposed females compared to control females (32% versus
0%), but not in males (13% versus 23%). Increases in tumor frequency were not
noted in other tissues, although statistically significant decreases occurred
in several tumor types (mammary tumors and pituitary tumors in females, and
lymphosarcomas in males). This study is limited because the study ran until
all of the animals died, rather than having a scheduled sacrifice. Since the
control animals had shorter lifespans than did the exposed animals, age-
related tumors could not be distinguished from treatment-related ones.
Aida et al. (1992b) administered bromodichloromethane to Sic:Wistar
rats (40/sex/dose for the treatment groups and 70/sex/dose for the 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
(males) and 0, 8.0, 31.7, or 168.4 mg/kg/day (females). The observed liver
V-104
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tumors were three cholangiocarcinomas and two hepatocellular adenomas in the
high-dose females, one hepatocellular adenoma in a control female, one
cholar.giocarcinoma 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 in feed was carcinogenic in Wistar rats. The study
authors further noted that the first indication of liver changes in this study
was fatty degeneration, although bile duct proliferation and cholangiofibrosis
were also observed. This was in contrast with the usual progression leading
to cholangiocarcinomas of bile duct cell proliferation, cholangiofibrosis,
benign cystic cholangiomas, cholangiofibromas, and eventually
cholangiocarcinomas.
Theiss et al. (1977) examined the carcinogenic activity of bromoform
and bromodichloromethane in Strain A mice. Male animals, 6 to 8 weeks old,
were injected intraperitoneally up to three times weekly over a period of
8 weeks. Three dose levels (20 mice/group) were used (4, 48, or 100 mg/kg
bromoform and 20, 40, or 100 mg/kg bromodichloromethane). A positive and a
negative control group each contained 20 animals. 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. Bromoform produced a
significant increase (p •= 0.041) in tumor frequency only at the intermediate
dose. Bromodichloromethane produced a marginally significant (p =• 0.062)
increase at the high dose. U.S. EPA (1980b) concluded that these results were
suggestive of carcinogenic activity but were not an adequate basis for the
development of a risk assessment.
V-105
-------
In a carcinogenesis study reported by NTP (1985), dibromochloromethane
was administered by gavage in corn oil five times per week for 104 weeks to
groups of 50 male and 50 female F344/N rats at 0, 40, or 80 mg/kg/day and to
groups of 50 male and 50 female B6C3F1 mice for 105 weeks at doses of 0, 50,
or 100 mg/kg/day. Survival of dosed male and female rats and 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. High-dose male rats and
male and female mice had lower body weights compared with the vehicle
controls. 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 low-dose male mouse' group was therefore
considered to be. inadequate for analysis of neoplasms. Compound-related
nonneoplastic lesions were found primarily in -the livers of male and female
rats (fatty metamorphosis and ground-glass cytoplasmic changes), male mice
(hepatocytomegaly, necrosis, fatty metamorphosis) and female mice (calcifi-
cation and fatty metamorphosis). Nephrosis was observed in male mice and
female rats. A summary of tumor frequencies in the mice is presented in
Table V-35. Administration of dibromochloromethane significantly increased
the incidence of hepatocellular adenomas and the combined incidences of
hepatocellular adenomas or carcinomas in high-dose female mice. The incidence
of hepatocellular carcinomas was significantly increased in high-dose male
mice. Significant increases in the combined incidence of hepatocellular
adenomas or carcinomas was detected by the life table test, but not by the
incidental tumor test. Negative trends in several common tumors were found in
dosed animals in the 2-year study. These neoplasms included fibroadenomas of
the mammary gland and endometrial stromal polyps of the uterus in female rats
V-106
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TABLE V-35 Frequencies of Liver Tumors in Mice Administered
Dibromochloromethane in Corn Oil for 104 Weeks
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
27/50=
19/50d
aMale low-dose group was inadequate for statistical analysis.
bp < 0.05 relative to controls.
cp < 0.01 (life table analysis); p = 0.065 (incidental tumor test) relative
to controls.
^ < 0.01 relative to controls.
Adapted from NTP (1985).
V-107
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and malignant lymphomas in male mice. There was no evidence of carcino-
genicity in rats administered dibromochloromethane. Fatty metamorphosis and
ground-glass cytoplasmic changes observed in the rat livers were, however,
related to administration of dibromochloromethane.
The authors concluded that this study provided equivocal evidence of
dibromochloromethane carcinogenicity in male B6C3F1 mice, some evidence of
carcinogenicity in female. B6C3F1 mice and no evidence of carcinogenicity in
male or female F344/N rats.
NTP (1989a) exposed groups of 50 male B6C3F1 mice by gavage (corn oil)
to doses of 0, 50, or 100 mg/kg/day of bromoform for 103 weeks (5 days/week).
Groups of 50 females received doses of 0, 100, or 200 mg/kg/day. At
termination, all animals underwent gross necropsy and thorough histological
examinations of tissues. An increased incidence of follicular cell hyper -
plasia was.noted in high-dose females, but no increase in tumors was reported
in any tissue in any group. A decreased incidence of lung tumors was noted in
males. The NTP concluded there was no evidence of carcinogenic activity in
male or female mice.
NTP (1989a) exposed groups of 50 male and 50 female F344/N rats to
bromoform by gavage for 103 weeks (5 days/week) at doses of 0, 100, or
200 mg/kg/day. 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 V-36).
Despite the small number of tumors found, the increase was considered to be
significant because these tumors are very rare in the rat. The NTP concluded
V-108
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Animal
TABLE V-36 Tumor Frequencies in Rats Exposed to
Bromoform in Corn Oil for 2 Years
Tumor Frequency
Tissue/Tumor
Control 100 mg/kg 200 mg/kg
Male rat
Female rat
Large intestine
Adenocar c inoma
Polyp (adenomatous)
Large intestine
Adenocarc inoma
Polyp (adenomatous)
0/50
0/50
0/48
0/48
0/50
0/50
0/50
1/50
1/50
2/50
2/50
6/50
Adapted from NTP (1989a).
V-109
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that there was some evidence for carcinogenic activity in male rats and clear
evidence in female rats.
Herren-Freund and Pereira (1986) used the rat liver GGT-foci assay to
study the initiating activity of oral exposure to bromoform. The authors
reported that bromoform at 250 mg/kg (1 mmol/kg) in an unspecified vehicle did
not initiate GGT-foci in this test.
F. Summary
1. Health Effects of Acute and Short-Term Exposure of Animals
Large oral doses of trihalomethanes are lethal to laboratory animals.
Reported acute LD50 values range from 119 to 2,000 mg/kg for chloroform, 450
to 969 mg/kg for bromodichloromethane, 800 to 1,200 mg/kg for dibromochloro-
methane, and 1,388 to 1,550 mg/kg for bromoform. Death from acute high-dose
trihalomethane exposure was usually found to be due to central nervous system
depression and cardiac effects, and .was usually accompanied by histopatho-
logical changes in the liver and kidney.
Acute oral exposure to sublethal doses of trihalomethanes can also
produce effects on the liver, kidney and central nervous system. In mice,
single oral doses of 60 to 89 mg/kg chloroform produced kidney damage, with
doses of 140 to 250 mg/kg producing liver damage. Organ damage was character-
ized by fatty infiltration, cellular necrosis, vacuolization, enzyme level
changes, and/or organ weight changes. Ataxia and sedation were noted in mice
receiving 500 mg/kg chloroform, 500 mg/kg bromodichloromethane, 500 mg/kg
dibromochloromethane, or 1,000 mg/kg bromoform.
V-110
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Short-term exposures of laboratory animals to trihalomethanes has been
observed to cause effects on the liver, kidney, central nervous system, and
immune system. Hepatic effects, including organ weight changes, elevated
serum enzyme levels, and histopathological changes, were reported in mice
and/or rats administered 37 to 290 mg/kg/day chloroform, 148 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-arainohippurate uptake, histopathological changes, and organ weight
changes, were reported in mice and/or rats administered 37 to 148 mg/kg/day
chloroform, 148 to 600 mg/kg/day bromodichloromethane, 147 to 500 mg/kg/day
dibromochloromethane, or 289 mg/kg/day bromoform for 14 days. Hyperactivity
and/or a decreased operant response were observed in mice and/or rats after
ingesting 100 to. 600 mg/kg/day bromodichloromethane, 400 mg/kg/day dibromo-
chloromethane, or 100 mg/kg/day bromoform for 14 to 60 days.
2. Health Effects of Longer-Term Exposure of Animals
The predominant effects of longer-term oral exposure to trihalo-
methanes occur in the liver and kidney. The .effects produced on these two
organs are similar in nature to those described for short-term exposures, with
liver appearing to be the most sensitive target organ. Hepatic effects were
reported in mice and/or rats administered 15 to 180 mg/kg/day chloroform, 6 to
300 mg/kg/day bromodichloromethane, 39 to 250 mg/kg/day dibromochloromethane,
or 187 to 250 mg/kg/day bromoform. In general, these dose ranges are slightly
lower than those reported to'cause effects following short-term exposures.
Renal effects were reported in mice and/or rats administered 25 to
'300 mg/kg/day bromodichloromethane or 250 mg/kg/day dibromochloromethane.
V-lll
-------
3. Reproductive/Developmental Effects in Animals
Data concerning the developmental effects of trihalomethanes indicate
that these chemicals are toxic to the mother and fetus at high doses and
suggest that reproductive and developmental effects may occur as well. Signs
of maternal toxicity (decreased body weight and changes in organ weight) were
reported in rats, rabbits and/or mice administered 50 to 100 mg/kg/day chloro-
form, 200 mg/kg/day bromodichloromethane, or 171 to 200 mg/kg/day dibromo-
chloromethane. Fetotoxicity, as indicated by decreased fetal body weights,
was evident in the offspring of rats administered 121 to 400 mg/kg/day
chloroform or in mice administered 685 mg/kg/day dibromochloromethane. Oral
exposure of animals to trihalomethane has caused variations, mainly in the
skeletal system.. Delayed ossification and sternebral aberrations have been
reported in rats and/or rabbits administered 20 to 200 mg/kg/day chloroform,
50 to 200 mg/kg/day bromodichloromethane or 50 to 200 mg/kg/day bromoform.
The study authors generally considered these effects to be secondary to
maternal toxicity. Malformations (cleft plate, imperforate anus, acaudia,
delayed ossification) have been observed in inhalation studies in which mice
and/or rats were exposed to 30 to 100 ppm chloroform, suggesting that
chloroform may be weakly teratogenic.
Two studies were located which investigated the effects of trihalo-
methanes on reproduction. In one study, oral doses of 685 mg/kg/day of
dibromochloromethane administered to mice for two generations led to decreased
fertility and gestational indices. In the second study, doses of
200 mg/kg/day bromoform had no effe.ct on the fertility of mice.
V-112
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4. Mutagenicicy and Genotoxicity Studies
In vitro and in vivo studies or> the mutagenic a.nd genotoxic potential
of the trichloromethanes have yielded mixed results. Interpretation 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 positive results for
bromoform and bromodichloromethane, and evidence of mutagenicity is considered
adequate for these chemicals. Studies on the mutagenicity of dibromochloro-
methane and chloroform were mixed, and the overall evidence for mutagenicity
of these two chemicals is judged to be inconclusive.
5. Carcinogenicity Studies in Animals
The carcinogenic potential of each of the four trihalomethanes has
been investigated in chronic oral exposure studies in animals. Ingestion of
chloroform in oil has been found to cause liver tumors in male and female
mice, but these tumors were not detected in mice exposed to chloroform in
drinking water. Renal tumors were detected in male rats exposed to chloroform
in either oil or. water, and renal tumors have been reported in male mice
exposed to chloroform in a toothpaste base.
Ingestion of bromodichloromethane in oil has been found to cause 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 in oil has been found to cause liver tumors in male and
V-113
-------
female mice, and ingestion of bromoform in oil has been found to cause
intestinal tumors in male and female rats.
V-114
-------
VI. HEALTH EFFECTS IN HUMANS
A. Clinical Case Studies
The mean lethal oral dose of chloroform in humans is estimated to be
about 630 mg/kg (Gosselin et al. 1976). Fatalities may occur at doses as low
as 211 mg/kg, with death attributable to respiratory or cardiac arrest (U.S.
EPA 1985a).
Schroeder (1965) reported a case history of a 27-year-old white male
who drank 4 fluid ounces of chloroform. Assuming a body weight of 70 kg, the
approximate dose ingested was 2,500 mg/kg. He was deeply unconscious and
cyanosed, and relaxation of his jaw had obstructed his upper respiratory
tract. His pupils were dilated and did not react to light. The patient
responded to medical treatment which included lavage. Blood urea reached high
levels in the first few days and then returned to normal. Urinary output was
scanty during the first 2 days, and urinalysis indicated albuminuria,
glucosuria, ketonuria, and the presence of bilirubin, red cells, and granular
casts. Excessive excretion of leucine, alanine, glutamine, glutamic acid,
serine, and tyrosine occurred during the acute phase. Liver function tests
indicated elevated levels of bilirubin, AP, and SCOT, indicating liver damage.
Clinical sequelae included slight jaundice and an enlarged liver. A liver
biopsy was performed 26 days later; the histological appearance of the
specimen was reported as consistent with recovering from toxic liver damage
(no other data were provided in this report).
In the past, oral dosing with bromoform was used as a sedative for
children with whooping cough. Typical doses were usually around one drop
VI-1
-------
(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 probably about 300 mg/kg, and the LOAEL for mild sedation is about
54 mg/kg/day.
No clinical case studies relevant to the ingestion of bromodichloro-
methane or dibromochloromethane were located.
B. Epidemiological Studies
Challen et al. (1958) studied the inhalation exposure of industrial
workers to chloroform in a confectionary firm in England. In 1950, the
wqrkers exposed to chloroform vapor given off during the production of
lozenges were placed on a reduced work week to alleviate complaints of
lassitude, flatulence, dry mouth, thirst, depression, irritability, and
"scalding" micturition. This action was not successful and the employees
refused to work on that particular process. In 1954, a new team of operators
was engaged, and in 1955, the firm installed an exhaust ventilation system,
after which manufacturing proceeded without interruption.
Within this study, one group of eight employees, "long service
operators," refused to continue in the lozenge department after experiencing
the previously described symptoms. This group of workers had been observed
staggering about the work area when exposed to chloroform vapor in concen-
trations ranging from 376 to 1,158 mg/m3. After terminating work in the
VI-2
-------
lozenge department, this group reported experiencing nausea after even short
exposures to chloroform.
A second group of nine employees, the "short service operators,"
replaced the "long service operators" and worked in locations where the
chloroform concentrations ranged from 112 to 347 mg/m3. Two of these nine
employees did not report unpleasant experiences from chloroform exposure.
Among the other seven, fiwe reported dryness of the mouth and throat at work;
two were subject to lassitude in the evening; one complained of lassitude and
flatulence at work; and the two others experienced similar symptoms to those
of the "long service operators."
A third group of five employees who worked in other departments of the
firm served as controls and exhibited no symptoms. Tests of the liver
function (thymol flocculation, direct van den Bergh, and indirect serum
bilirubin), clinical examinations and urinary urobilinogen measurements failed
to show significant differences among the three groups of workers.
Bomski et al. (1967) reported liver injury from inhalation exposure to
chloroform among workers in a pharmaceutical factory in Poland. The
concentration of chloroform ranged from 9.8 to 1,002 mg/m3. Sixty-eight
workers were exposed to chloroform for 1 to 4 years and were still in contact
with chloroform; 39 had been exposed to chloroform previously, but were no
longer exposed; 23 had viral hepatitis with jaundice 2 to 3 years earlier and
were designated as posticteric controls and were working in a germ-free area:
and 165 worked in a germ-free area and had no history of viral hepatitis
(controls). Blood pressure, blood morphology, urinalysis, blood albumin,
serum protein, thymol turbidity, zinc sulfate turbidity, urobilinogen, SCOT
VI-3
-------
and SGPT were measured in all workers; Che "Takata-Ara" sulfate (colorimetric)
test was performed, and a complete medical history was taken. Sixty workers
were hospitalized for determination of bromosulfophthalein (BSP) clearance and
urinary urobilinogen.
The frequency of viral hepatitis and jaundice among the 68 pharmaceu-
tical workers currently exposed to chloroform was compared with that of a
group of city inhabitants, 18 years of age and older. In 3 successive years,
the incidence of viral hepatitis among the pharmaceutical workers was
significantly higher than the incidence of hepatitis within the city group.
The authors suspected that the toxic liver changes that occurred as a result
of chloroform exposure promoted a viral infection; however, the incidence of
viral hepatitis among the other groups of plant workers was not reported. The
majority of the workers exposed to chloroform during this study complained of.
headaches, nausea, belching and loss of appetite. Among the 68 workers
working with chloroform, 19 cases of splenomegaly were reported; none were
observed in the controls. The frequency of enlarged livers (25%) among the
chloroform-exposed workers exceeded that of the other two groups (13% and 9%).
In 3 of the 17 chloroform workers with enlarged livers, toxic hepatitis was
diagnosed on the basis of elevated serum enzyme activities and elevated serum
gamma globulin. In the remaining 14 cases of liver enlargement, fatty liver
was diagnosed.
Kramer et al. (1992) conducted a population-based case-control
analysis to determine if exposure to trihalomethanes in drinking water is
associated with low birthweight, prematurity, or intrauterine growth
retardation (lower than the 5th percentile of weight for gestational age). A
separate analysis was conducted for each endpoint, using five randomly
VI-4
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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. A significant association was
observed between exposure to water chloroform levels of at least 10 jig/L and
intrauterine growth retardation (odds ratio = 1.8, 95% confidence interval =
1.1-2.9). Associations were also observed between exposure to at least
10 fig/L chloroform and low birthweight (odds ratio = 1.3), exposure to
intermediate levels of chloroform (1 to 9 ^g/L) and intrauterine growth
retardation, and exposure to at least 10 ^g/L bromodichloromethane and
intrauterine growth retardation (odds ratio = 1.7). However, the confidence
interval in these cases included one, indicating that the increases were not
statistically significant. The elevated risk of intrauterine growth retar-
dation associated with high chloroform levels remained when only chlorinated
water sources were included. The study authors could not control directly for
the presence of other chemicals. However, the effect remained when the only
water source was deep wells. Since deep wells are separated from groundwater,
they are less likely to be contaminated by pesticides and other chemicals,
aside from disinfectant byproducts. The study authors noted that the
association with chloroform may result at least partially from chloroform
acting as a marker for other organic halides. They also noted that ambient
conditions may- have led to higher trihalomethane levels in the water from 1989
VI-5
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to 1990 than in 1987, so the relative chloroform concentrations should be
interpreted qualitatively rather than quantitatively.
Shaw et al. (1991) conducted a case-control study of 141 mothers of
children born with a cardiac anomaly and 176 controls. No association was
found between a first trimester residence that received chlorinated water, or
drinking of water potentially containing trihalomethanes, and babies born with
cardiac anomaly. This study was reported in an abstract.
A number of epidemiological studies suggest there may be an associ-
ation between water chlorination and cancer mortality rates (see Table VI-1).
These studies are discussed in more detail in the Drinking Water Criteria
Document on Chlorine (EPA 1994). It cannot be concluded from these studies,
however, that trihalomethanes are carcinogenic-in humans, since chlorinated
water contains many chemical by-products in addition to the trihalomethanes.
Morris et al. (1992) conducted a meta-analysis of nine case-control studies
and one cohort study investigating an association between chlorinated water or
trihalomethane exposure and cancer. The overall relative risk estimate for
bladder cancer was 1.21 (95% CI: 1.09, 1.34), and the risk estimate for
rectal cancer was 1.38 (95% CI: 1.01, 1.87). Although objective criteria
were used to assess the quality of the studies and a pre-determined protocol
was used for the analysis, it is unclear whether meta-analysis can be
productively applied to observational epidemiological studies.
C. High-Risk Populations
No data were located that indicate any human subpopulation may be at
greater risk from exposure to trihalomethanes than the general population,
VI-6
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TABLE VI-1 Epidemiological Studies Investigating an Association
Between Cancer and Chlorinated Water
Reference
Study Description
Observation
Alavanja et al.
(1978)
Case control study in seven
New York State counties.
Cantor et al.
(1978)
Hogan et al.
(1979)
Ecological study using age-
standardized cancer mortality
rates, 1968-71; and halomethane
levels from U.S. EPA surveys.
Ecological study using NCI
cancer mortality data 1950-
1969. Chloroform levels in
finished drinking water
from U.S. EPA surveys.
Struba (1979)
Brenniman et
al. (1980)
Case-control study of mortality
in North Carolina, 1975-1978.
Case-control study in 70
Illinois communities, 1973-76.
Questionnaires sent to water
treatment plants to verify
1963 inventory data on chlorine
levels.
Greater risk of
gastrointestinal and
urinary tract cancer
mortality, both sexes,
in chlorinated water
areas of the counties.
Strongest correlation
between bromine -
containing trihalo-
methanes and bladder
cancer.
Significant positive
correlations between
chloroform levels and
cancer mortality in
white females for
bladder, rectum and
large intestine; in
white males for stomach
cancer.
Small but significant
odds ratios for rectum,
colon and bladder
cancers in rural areas
but not in urban areas.
Statistically signifi-
cant relative risks of
cancer of gall bladder,
large intestine, and
total gastrointestinal
and urinary tract in
females served by
systems with chlorinated
versus.nonchlorinated
- continued
VI-7
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Table VI-1 (Continued)
Reference
Study Description
Observation
Brenniman - continued
Gottlieb et
al. (1981)
Young et
al. (1981)
Cragle et
al. (1985)
Young et
al. (1987)
Morris et
al. (1992)
Case-control study using
mortality data in Louisiana
and estimations of exposure.
Case-control, State of
Wisconsin, 1972-1977.
Questionnaires sent to
waterworks superintendents on
chlorine content.
Case-control study using
colon cancer cases from
seven hospitals in North
Carolina.
Case-Control study of colon
cancer cases in Wisconsin.
Water consumption was deter-
mined by interview, and
chloroform levels by
historical records and
measurement
Meta-analysis of nine
case-control studies and one
cohort study analyzing cancer
and consumption of chlorinated
water or water containing high
significant increase in
chloroform levels.
ground water. Due to
many uncontrolled
confounding factors,
authors concluded that
chlorination was not a
major factor in the
etiology of gastro-
intestinal and urinary
tract cancers.
Rectal cancer
significantly
elevated with
respect to surface
or Mississippi River
water consumption.
Colon cancer showed
significant (p < 0.05)
association with
chlorine intake in all
three dosage categories
Consumption of
chlorinated water
strongly associated
with colon cancer,
above age 60.
No association found
between trihalomethane
exposure and colon
cancer incidence.
Statistically signifi-
cant relative risk of
rectal cancer and
bladder cancer in
exposed groups. No
colon cancer.
VI-8
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although studies in animals suggest that adult males may be at a greater risk
from chloroform exposure than young males or females (Culliford and Hewitt
1957; Eschenbrenner and Miller 1945). Also, studies in animals suggest that
ethanol may potentiate the hepatotoxic effects of chloroform (Klaassen and
Plaa 1966, 1967a), so alcoholics may be at increased risk. As described in
Chapter VII, chloroform-induced liver hepatotoxicity was potentiated in a rat
model of diabetes (Hanasono et al. 1975). This suggests that diabetics may
also be at increased risk.
D. Summary
In a case study of a young man who ingested 4 ounces of chloroform (a
dose of about 2,.500 mg/kg), prominent clinical findings included jaundice, an
enlarged liver, increased serum levels of bilirubin, alkaline phosphatase and
SCOT along with albuminuria, glucosuria, ketonuria and the presence of red
cells and granular casts in the urine. These observations indicated that in
humans, as in animals, the liver and kidneys are the organs most affected by
chloroform ingestion.
In the past, bromoform was given orally as a sedative to children
suffering from whooping cough. Doses of 50 to 100 mg/kg/day usually produced
sedation without any apparent adverse effects. Some cases of severe toxicity
or death were reported, but these were generally attributed to accidental
overdoses. No data were located on human exposure to either bromodichloro-
methane or dibromochloromethane.
Workers exposed to chloroform by inhalation at levels of 112 to
1,158 mg/m3 for 1 or more years complained of nausea, lassitude, dry mouth,
VI-9
-------
flatulence, thirst, depression, irritability and "scalding" micturition, but
clinical examination and tests of liver function failed to detect any
abnormalities. Inhalation exposure of workers to chloroform at levels of
about 10 to 1,000 mg/m3 for 1 to 4 years was reported to be associated with an
increased incidence of viral hepatitis and enlarged liver.
Some epidemiological studies suggest there may be an association
between water chlorination and increased cancer mortality rates. An
association has also been reported between exposure to water chloroform levels
of at least 10 ^g/L and intrauterine growth retardation. However, since
chlorinated water contains many by-products, it cannot be directly concluded
from such studies that trihalomethanes are human carcinogens or developmental
toxicants.
No data were located regarding whether any human subpopulation may be
at. greater risk to trihalomethane exposure than the general population. Data
from animal studies suggest that males may be more sensitive than females to
kidney effects, that alcohol consumption may increase toxicity, and that
diabetics may be at increased risk.
VI-10
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VII. MECHANISM OF TOXICITY
A. Role of Metabolism
It is generally believed Chat the toxicity of the trihalomethanes is
related to their metabolism. This conclusion is based mainly on the
observation that the chief target tissues for these compounds (liver, kidney)
are also the primary sites of their metabolism. Moreover, treatments which
increase or decrease metabolism also tend to increase or decrease trihalo-
methane-induced toxicity in parallel. Representative studies that demonstrate
correlations between metabolism and toxicity are presented below.
Brown et. al. (1974b) compared the effects of inhalation exposure to
chloroform (0.5% or 1%) in rats with and without pretreatment with phenobar-
bital (an inducer of cytochrome P-450). The authors reported that pretreat-
ment led to increased chloroform metabolism (as measured by formation of
covalent adducts), and this was paralleled by increased hepatotoxicity (as
measured by triglyceride levels, destruction of cellular microsomal enzymes,
and histopathological examination). Thorton-Manning et al. (1993) found that
pretreatment with acetone, a cytochrome P4502E1 inducer, potentiated the
hepatotoxicity of bromodichloromerhane in male rats.
Gopinath and Ford (1975) reported that various inducers of microsomal
hydroxylases (phenobarbitone, phenylbutazone, and chlorpromazine) potentiated
the hepatotoxicity of chloroform in male rats. Conversely, inhibitors of
microsomal oxidase (SKF-525A, sodium diethyl-dithiocarbamate, and carbon
disulfide) acted to protect against the hepatotoxic effect of chloroform. The
VII-1
-------
cycochrome P450 inhibitor' 1-aminobenzotriazole prevented bromodichloromethane-
induced hepatotoxicity in rats (Thorton-Manning et al. 1993).
Smith and Hook (1983) reported that 2H-chloroform (which is less
readily metabolized than 1H-chloroform) was less effective in inducing
nephrotoxicity in male mouse renal cortical slices than was an equimolar
concentration of chloroform. Nephrotoxicity was assessed iu vitro by
measuring decreased PAH and tetraethylammonium accumulation by kidney slices.
Ruch et al. (1986) reported that chloroform-induced release of LDH
from cultured B6C3F1 mouse hepatocytes into the culture medium (an index of
cell injury or death) was decreased by adding SKF-525A to the cultures, and
could also be decreased by adding antioxidants such as N-N'-diphenyl-
p-phenylenediamine, a-tocopherol (Vitamin E) or superoxide dismutase.
Addition of diethylmaleate, which depletes intracellular glutathione, led Co
increased chloroform toxicity.
Gao et al. (1993) evaluated the effects of glutathione on bromo-
dichloromethane toxicity in vivo and in vitro. Depletion of glutathione by
pretreatment of male rats with the glutathione synthesis inhibitor buthionine
sulfoximine led to increased hepatotoxicity in vivo. The addition of
glutathione to a reaction mixture of rat hepatic microsomal fraction and
radiolabeled bromodichloromethane resulted in a 90% reduction in protein
binding by bromodichloromethane.
Sex and species differences in the metabolism of trihalomethanes also
appear to correlate with sex and species differences in toxicity. Taylor
et al. (1974) reported that the kidneys from male mice have a higher capacity
VII-2
-------
to metabolize chloroform than kidneys from female mice, and male mice are also
more sensitive than female mice to the renal toxic effects of chloroform
(Culliford and Hewitt 1957; Eschenbrenner and Miller 1945). Similarly, mice
appear to have a higher capacity to metabolize trihalomethanes than rats , both
via the oxidative pathway (Mink et al. 1986) and the reductive pathway (Testai
and Vittozzi 1986; Testai et al . 1987), and mice also appear to be more
sensitive than rats to chloroform, at least for renal toxicity (Klaunig et al.
1986; Munson et al. 1982).
*
B. Biochemical Basis of Toxicity
/
I
The precise biochemical mechanisms which link metabolism to toxicity
are not certain,, but many researchers have proposed that toxicity results from
the production of reactive intermediates, either from the oxidative pathway
(dihalocarbonyls) or the reductive pathway (free radicals). These reactive
intermediates are known to form covalent adducts with various cellular
molecules (see Section III.C), which could presumably impair the function of
those molecules and cause cell injury. However, direct evidence showing a
relation between the degree of covalent binding and the extent of toxicity is
limited.
Ilett et al. (1973) reported that in mice administered 10 /iCi of
14C- chloroform, the amount of label bound to proteins in the liver and kidneys
paralleled the extent of hepatic and renal necrosis, both in normal animals
and in animals pretreated with an inducer (phenobarbital) or inhibitor
(piperonyl butoxide) of cytochrome P-450. Furthermore, autoradiograms
revealed that incorporation of radioactivity occurred primarily at the sites
of necrotic lesions.
VII-3
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Another mechanism chat has been proposed for trihalomethane toxicicv
is lipid peroxidation induced by free radical metabolites. Although evidence
is limited that lipid peroxidation actually accounts for observed cellular
toxicity, several studies have established that lipid peroxidation does occur
in conjunction with trihalomethane metabolism.
Brown et al. (1974b) exposed rats to chloroform in air at levels of
0.5% or 1% for 2 hours. Livers were removed and levels of conjugated dienes
were measured in hepatic microsomes. This endpoint was taken to reflect the
level of hepatic lipid peroxidation. Chloroform exposure resulted in a
significant increase in diene conjugation in phenobarbital-treated rats, but
not in untreated rats. The authors speculated that chloroform metabolism
leads to free radicals which induce destructive lipid peroxidation.
More recently, de Groot and Noll (1989) reported that all four
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 02) .
The authors interpreted these data to support the concept that lipid
peroxidation is caused by free radical metabolites that are generated by the
reductive metabolism of trihalomethanes.
Cohen and Chance (1990) measured hepatic chemiluminescence as an in
vivo measure of lipid peroxidation in two male phenobarbital-induced Wistar
rats. Increased luminescence was observed within about 15 minutes after the
initiation of inhalation exposure to an unspecified concentration of
chloroform.
VII-4
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C. Mechanism of Carcinogenesis
The mechanism by which trihalomethanes induce i-umors in laboratory
animals is not known. However, two possible mechanism have been proposed.
Metabolism of trihalomethanes to reactive intermediates could lead to
formation of covalent DNA-trihalomethane adduct's. As discussed in
Section V.D., there is some direct evidence that chloroform can bind to DNA
Colacci et al. (1991). This is consistent with positive findings in
genotoxicity tests, although the overall evidence regarding chloroform
genotoxicity is inconclusive.
Alternatively, the induction of tumors by trihalomethanes could
involve an epigenetic mechanism. Induction of tumors in animal studies has
been noted to occur primarily at sites where significant cytotoxicity was
observed (i.e., liver and kidney), and there is a correlation between
hepatotoxic.ity and liver tumorigenicity of trihalomethanes in mice (bromo-
dichloromethane > chloroform = dibromochloromethane > bromoform). This raises
the possibility that regenerative hyperplasia caused by the cytotoxic effects
of the trihalomethanes may be important in the tumorigenic potential of these
chemicals. However, because of the apparent genotoxicity of the trihalo-
methanes (evidence is considered adequate for bromoform and bromodichloro-
methane and marginal for dibromochloromethane and chloroform), EPA does not
believe it is appropriate at present to assume that an epigenetic pathway is
operating.
VII-5
-------
D. Interactions
A variety cf chemicals have been shown to potentiate the toxicity of
chloroform. The potentiating activity of inducers of microsoraal hydroxylases
was described earlier in this chapter. Nontoxic levels of several ketones and
compounds that are metabolized to ketones also increase the hepatotoxicity of
chloroform, but this effect cannot be attributed solely to induction of
hepatic enzymes. Although pretreatment with the insecticide mirex induced
hepatic mixed function oxidases to a greater extent than did pretreatment with
its ketone analog, chlordecone, pretreatment with chlordecone, but not mirex,
ma-rkedly increased chloroform binding to hepatic constituents (Cianflone et
al. 1980). Furthermore, chlordecone pretreatment resulted in a different
histological pat.tern of lesions than that observed following dosing with
chloroform alone at a level that produce a similar level of total abnormal
hepatocytes (Hewitt et al. 1979). Potentiation has also been observed with
various alcohols, which can be metabolized to ketones, and with other ketones
(Hewitt et al. 1980, 1986; Ray and Mehendale 1990). Rats with metabolic
ketosis due to the induction of diabetes by alloxan are also more sensitive to
the hepatotoxic effects of chloroform (Hanasono et al. 1975). Possible
mechanisms for potentiation by kerones include an effect on calcium pump
activity (Moore and Ray 1983) and increased susceptibility of organelles
resulting from ketone exposure (Hewitt et al. 1990).
Davis (1992) found that dichloroacetic acid administered at nontoxic
levels (three doses of 2.45 mmol/kg/dose [315.8 mg/kg] in 24 hours) increased
chloroform hepatotoxicity (measured as increased plasma alanine amino-
transferase) and nephrotoxicity (measured as increased BUN) in female Sprague-
Dawley rats. Trichloroacetic acid at the same molar level increased chloro-
VII-6
-------
form nephrotoxicity. Histopathology was not conducted, and no mechanism of
interaction was proposed. Borzelleca et al. (1990) used a statistical
technique called response surface methodology to predict the synergistic
interactions between chloroform and carbon tetrachloride at varying
concentrations of each chemical, based on measurements of plasma enzyme levels
following oral administration of these chemicals singly and in combination.
Lilly et al. (1992) found that concurrent oral administration of
chloroform at doses up to 1.5 mg/kg (1 mL/kg) and trichloroethylene at 1 mL/kg
to adult male F344 rats resulted in decreased toxicity relative to that seen
following chloroform administration alone. Hepatic and renal toxicity were
observed at this dose of chloroform alone, while trichloroethylene alone was
not overtly toxic to either organ. Trichloroethylene antagonism of chloroform
toxicity appeared to be independent of dosing vehicle, and was observed
following administration in oil or an aqueous vehicle.
The severity of trihalomethane toxicity is markedly affected by the
vehicle of administration. Several s-tudies showing that chloroform was more
toxic following administration in oil than in an aqueous vehicle were
discussed in Chapter V (e.g., Bull et al. 1986; Jorgenson et al. 1985). Lilly
et al. (1992) found that chloroform administration in corn oil caused
substantially greater hepatic and renal toxicity in adult male F344 rats than
did administration in an aqueous vehicle. 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).
VII-7
-------
E. Summary
Three lines of evidence indicate that trihalomethane metabolism is
essential for toxicity: (1) the tissues that most actively metabolize the
trihalomethanes (liver, kidney) are also the chief target tissues;
(2) chemical treatments that increase or decrease metabolism also tend to
increase or decrease toxicity in parallel; and (3) species- and sex-related
differences in metabolism are paralleled by similar differences in toxicity.
The detailed biochemical mechanisms by which trihalomethane metabolism leads
to toxicity are not certain, but covalent binding of reactive metabolites to
key cellular molecules is one likely mechanism. Such metabolites are produced
both by oxidative metabolism to dihalocarbonyls and reductive metabolism to
free radicals. Free radical production may also lead to cell injury by
inducing lipid peroxidation in cellular membranes.
Formation of DNA adducts might also account for the genotoxic and
carcinogenic potential of the trihalomethanes. Alternatively, carcinogenesis
may be related, at least in part, to increased cell proliferation following
direct tissue injury. However, neither of these potential mechanisms have
been definitively linked to trihalomethane carcinogenesis.
Chloroform toxicity is potentiated by various chemicals. At least
some of the potentiation by ketones appears to occur by a mechanism other than
induction of microsomal enzymes. Some quantitative data are available
regarding the interactions between chloroform and dichloroacetic acid, carbon
tetrachloride, or trichloroethylene. The vehicle (corn oil versus aqueous)
used for oral dosing also affects toxicity, with toxicity generally being more
severe following administration in oil.
VII-8
-------
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
The quantificaticn of Lexicological effects of a chemical consists of
separate assessments of noncarcinogenic and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarcinogenic health effects occur,
while carcinogens are assumed to act without a threshold.
Quantification of NoncarcinoEenic Effects
In the quantification of noncarcinogenic effects, a Reference Dose
(RfD), (formerly called the Acceptable Daily Intake (ADI)), is calculated.
The RfD is an es.timate of a daily exposure to the human population that is
likely to be without appreciable risk of deleterious health effects, even if
exposure occurs over a lifetime. The RfD is derived from a NOAEL, or LOAEL,
identified from a subchronic or chronic study, and divided by an uncertainty
factor(s). The RfD is calculated as follows:
D_ (NOAEL or LOAEL) .. , ..
RfD = rr-4 : "T—r = mg/kg bw/day
Uncertainty Factor(s) &/ e> / J
Selection of the uncertainty factor to be employed in the calculation
of the RfD is based on professional judgment and consideration of the entire
data base of toxicological effects for the chemical. To ensure that
uncertainty factors are selected and applied in a consistent manner, the
Office of Water (OW) employs a modification of the guidelines proposed by the
National Academy of Sciences (NAS 1977, 1980), as follows:
VIII-1
-------
• An uncertainty factor of 10 is generally used when good chronic or
subchronic human exposure data identifying a NOAEL are available
and are supported by good chror.ic or subchronic tcxicity data in
other species.
• An uncertainty factor of 100 is generally used when good chronic
toxicity data identifying a NOAEL are available for one or more
animal species., (and human data are not available), or when good
chronic or subchronic toxicity data identifying a LOAEL in humans
are available.
• An uncertainty factor of 1,000 is generally used when limited or
incomplete chronic or subchronic toxicity data are available, or
when good chronic or subchronic toxicity data that identify a LOAEL
but not a NOAEL for one or more animal species are available.
The uncertainty factor used for a specific risk assessment is based
principally on scientific judgment rather than scientific fact and accounts
for possible intra- and interspecies differences. Additional considerations
not incorporated in the NAS/OW guidelines for selection of an uncertainty
factor include the use of a less-rhan-lifetime study for deriving a RfD, the
significance of the adverse health effect and the counterbalancing of
beneficial effects.
From the RfD, a Drinking Water Equivalent Level (DWEL) can be
calculated. The DWEL represents a medium specific (i.e., drinking water)
lifetime exposure at which adverse, noncarcinogenic health effects are not
anticipated to occur. The DWEL assumes 100% exposure from drinking water and
VIII-2
-------
provides the noncarcinogenic health effects basis for establishing a drinking
water standard. For ingestion data, the DWEL is derived as follows:
ntTPT (RfD) x (Body Weight in ks) ,,
DWEL = TT—7—r~r 77 ' , . n :——-~~— = mg/L
Drinking Water Volume in L/day °
where:
Body weight is as'sumed to be 70 kg for an adult.
Drinking water volume is assumed to be 2 L per day for an adult.
In addition to the RfD and the DWEL, Health Advisories (HAs) for
exposures of shorter duration (One-day, Ten-day and Longer-term) are
determined. The HA values are used as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
The HAs are calculated using a similar equation to the RfD and DWEL; however.
the NOAELs or LOAELs are identified from acute or subchronic studies. The HAs
are derived as follows:
(NOAEL or LOAEL) x (bw) /T
(UF) x ( L/day) = mg/L
Using the above equation, the following drinking water HAs are
developed for noncarcinogenic effects:
1. One-day HA for a 10-kg child ingesting 1 L water per day.
2. Ten-day HA for a 10-kg child ingesting 1 L water per day.
3. Longer-term HA for a 10-kg child ingesting 1 L water per day.
4. Longer-term HA for a 70-kg adult ingesting 2 L water per day.
VIII-3
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The One-day HA calculated for a 10-kg child assumes a single acute
exposure to the chemical and is generally derived from a study of less than
7 days' duration. The Ten-day HA assumes s. limited exposure period of 1 to
2 weeks and is generally derived from a study of less than 30-days' duration.
A Longer-term HA is derived for both the 10-kg child and a 70-kg adult and
assumes an exposure period of approximately 7 years (or 10% of an individual's
lifetime). A Longer-term HA is generally derived from a study of subchronic
duration (exposure for 10% of an animal's lifetime).
Quantification of Carcinogenic Effects
The EPA categorizes the carcinogenic potential of a chemical based on
the overall weight-of-evidence, according to the following scheme:
• Group A: Known Human Carcinoeen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
• Group B: Probable Human Carcinogen. Sufficient evidence of
/
carcinogenicity in animals with limited (Group Bl) or inadequate
(Group B2) evidence in humans.
• Group C: Possible Human Carcinogen. Limited evidence of
carcinogenicity in animals in the absence of human data.
• Group D: Not Classified as to Human Carcinogenicity. Inadequate
human and animal evidence of carcinogenicity or for which no data
are available.
VIII-4
-------
• Group E: Evidence of Noncarcinogenicitv for Humans. No evidence
of carcinogenicity in at least two adequate animal tests in
different species or in both adequate epidemiologic and animal
studies.
If toxicological evidence leads to the classification of the
contaminant as a known, probable or possible human carcinogen, mathematical
models are used to calculate the estimated excess cancer risk associated with
the ingestion of the contaminant in drinking water. The data used in these
estimates usually come from lifetime exposure studies in animals. In order to
predict the risk for humans from animal data, animal doses must be converted
to equivalent human doses. This conversion includes correction for
noncontinuous exposure, less-than-lifetime studies and differences in size.
The factor that compensates for the size difference is the cube root of the
ratio of the animal and human body weights. It is assumed that the average
adult human body weight is 70 kg and that the average water consumption of an
adult human is 2 L of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure via
ingestion of water. The cancer unit risk is usually derived from a linearized
multistage model with a 95% upper confidence limit providing a low-dose
estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper limit estimate and, in fact, may be lower. Excess
cancer risk estimates may also be calculated using other models such as the
one-hit, Weibull, logit and probit. There is little basis in the current
understanding of the biological mechanisms involved in cancer to suggest that
VIII-5
-------
any one of these models is able to predict risk more accurately than others.
Because each model is based upon differing assumptions, the estimates derived
for each model can differ by several orders of magr.itv.de.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an inherent uncertainty due to the systematic and
random errors in scientific measurement. In most cases, only studies using
experimental animals have been performed. Thus, there is uncertainty when the
data are extrapolated to humans. When developing cancer risk rate levels,
several other areas of uncertainty exist, such as incomplete knowledge con-
cerning the health effects of contaminants in drinking water; the impact of
the experimental animal's age, sex and species; the nature of the target organ
system(s) examined; and the actual rate of exposure of the internal targets in
experimental animals or humans. Dose-response data usually are available only
for high levels of exposure, not for the lower levels of exposure closer to
where a standard may be set. In exposures to more than one contaminant,
additional uncertainty results from a lack of information about possible
synergistic or antagonistic effects.
A. Noncarcinogenic Effects
1. Chloroform
a. One-day Health Advisory
Table VIII-1 summarizes studies that were considered for calculating
the One-day HA for chloroform. All four studies (Jones et al. 1958; Hill
1978; Reitz et al. 1980; Larson et al. 1993) indicate moderate to severe
VIII-6
-------
Table VIII-1 Summary of Candidate Studies for Derivation
of the One-day Health Advisory for Chloroform
Reference
Jones et al.
(1958)
Hill (1978)
Rei r.2 et al .
(1980)
Larson et al.
(1993a)
Species Route
Mouse Gavage
(oil)
Mouse Gavage
' (oil)
Mouse Gavage8
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure
Duration
Single
dose
Single
dose
Single
dose
Single
dose
Single
dose
Endpoints
Histology
(liver)
Histology
(liver),
urinalysis
Histology
(liver,
kidney)
Histology
(liver,
kidney)
Histology
(liver,
kidney)
NOAEL
(mg/kg/day)
35-70
(Mild fatty
infiltration
of liver)
~ ~
15
34
34
LOAEL
(mg/kg/day)
140
(Marked fatty
infiltration,
necrosis)
89
(Glucosuria,
proteinuria)
60
(Renal tubular
regeneration)
180
(Liver and kidney
necrosis)
238
(Mild liver
necrosis)
' •
BThe vehicle was not reported by Reitz et al. (1980) but is presumed to be oil.
-------
liver and/or kidney damage following single oral doses of 140 to 250 mg/kg
chloroform in oil. The study by Jones et al. (1958) indicates that only mild
hepatic, fat accumulation, without overt liver injury, occurs following doses
of 35 mg/kg. The data of Hill (1978) indicate that renal effects (evidenced
by urinary loss of glucose or protein) occur at doses of 89 to 163 mg/kg, and
liver histopathology occurs at 250 mg/kg. Reit'z et al. (1980) reported
regenerative changes in renal tubules of mice given 60 mg/kg (although only
two animals per dose were used). Larson et al. (1993) reported scattered
necrotic tubules in rats at 34 mg/kg, with no consistent effects on BUN or
urinary protein or glucose. Taken together, these studies indicate that renal
and hepatic injury begins to become manifest following doses of about 60 mg/kg
and becomes severe following doses of 140 to 250 mg/kg. However, confidence
in these values ;Ls limited because of the use of an oil vehicle,3 which may
have contributed to the observed hepatotoxicity. Nevertheless, the NOAEL of
35 mg/kg identified by Jones et al. (1958) is selected as the most appropriate
basis for calculation of the One-day HA value.
Using this value, the One-day HA for the 10-kg child is calculated as
follows:
^ j u. (35 mg/kg/day) (10 kg) _ c .. . , . . , ,T,
One-day HA - (100) (1 L/day) mg/
where:
35 mg/kg/day = NOAEL, based on absence of hepatotoxicity in mice given
single oral doses of chloroform via gavage
'The vehicle was not reported by Reitz et al. (1980) but is presumed to be oil.
VIII-8
-------
10 kg = assumed weight of a child
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
No existing guidelines or standards were located for acute oral
exposure to chloroform. A concentration of 10 ppm in air has been proposed as
a Time-Weighted Average-Threshold Limit Value (TWA-TLV) by the American
Conference of Governmental Industrial Hygienists (ACGIH 1991) . A concentra-
tion of 2 ppm in air has been proposed as a Short-Term Exposure Limit (STEL)
(NIOSH 1990). The Occupational Safety and Health Administration (OSHA)
established an 8-hour time-weighted average (TWA) permissible exposure limit
of 2 ppm (54FR2332, January 19, 1989).
b. Ten-day Health Advisory for Chloroform
Table VIII-2 summarizes studies that were considered for calculation
of the Ten-day HA for chloroform. The studies by Munson et al. (1982) and by
Thompson et al. (1974) identify hepatotoxicity as the most appropriate
endpoint of short-term exposure to chloroform. Munson et al. (1982) detected
no significant changes in serum enzymes in mice given up to 125 mg/kg/day
chloroform in water for 14 days, and Chu et al. (1982a) reported no change in
serum enzymes or gross pathology in the liver of rats given 63 mg/kg/day in
the drinking water for 28 days. However, Thompson et al. (1974) noted
maternal toxicity (decreased weight gain and fatty liver) in pregnant rats and
VIII-9
-------
Table VIII-2 Summary of Candidate Studies for Derivation
of the Ten-day Health Advisory for Chloroform
Reference
Munson et al.
(1982)
Chu et al.
(1982a)
Thompson
et al. (1974)
Thompson
et al. (1974)
Species Route
Mouse Gavage
(aqueous)
Rat Drinking
water
Rat Gavage
(oil)
Rabbit Gavage
(oil)
Exposure .
Duration
14 days
28 days
Days
6-15 of
gestation
Days
6-15 of
gestation
NOAEL
Endpoints (mg/kg/day)
Liver weight, 125
serum enzymes,
immune function. ,
Serum 7.4
biochemistry ,
hematology ,
gross pathology
Maternal 20
toxicity ,
fetotoxicity ,
teratogenicity
Maternal 35
toxicity,
fetotoxicity,
teratogenicity
LOAEL
(rag/kg/day)
250
(Elevated
serum
enzymes)
63
(Decreased
neutrophil
count; no liver
toxicity)
50
(Decreased
maternal
weight gain;
fatty liver)
50
(Decreased
maternal
weight gain;
fatty liver)
-------
rabbits given 50 mg/kg/day chloroform in oil on days 6 to 15 of gestation;
this dose was identified as the LOAEL. Doses of 20 mg/kg/day in rats and
35 mg/kg/day in rabbits were without significant maternal toxicity,
fetotoxicity, or teratogenicity. The dose of 35 mg/kg/day from the rabbit
study was selected as the NOAEL. Even though this study employed the use of a
corn oil vehicle that may have contributed to maternal hepatotoxicity, these
NOAELs are supported by the drinking water study by Chu et al. (1982a).
Using this value, the Ten-day HA for the 10-kg child is calculated as
follows:
_ , „. (35 mg/kg/dav) (10 kg) , ,
Ten-day HA = (100) (1 L/day) '5 mg/L (rounded to 4 mS/L)
where:
35 mg/kg/day = NOAEL, based on absence of maternal or fetal toxicity in
rabbits exposed to chloroform via gavage during days 6 to
15 of gestation
10 kg = assumed weight of a child
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
VIII-11
-------
No existing guidelines or standards were located for short-term oral
exposure to chloroform. OSHA currently limits occupational exposure to
chloroform Co a ceiling level of 50 ppm (29 GFR 1910.1000).
c. Longer-term Health Advisory for Chloroform
Table VIII-3 summarizes studies that were considered for calculating
the Longer-term HA values for chloroform. These studies indicate that
exposure of rats and mice to chloroform at doses of 50 to 270 mg/kg/day in
water or 130 mg/kg/day in oil does not result in significant liver injury or
other evidence of toxicity, while doses of 270 to 290 mg/kg/day produce mild
changes that may reflect incipient injury. Decreased growth rate was observed
at a lower dose .(180 mg/kg/day). This conclusion is not consistent with
short-term studies (see Tables VIII-1 and VIII-2), which indicate that doses
of 50 mg/kg/day or higher produce hepatic or renal injury. The reasons for
this discrepancy are not certain, but the difference may be due in part to
metabolic adaptation during continued exposure or to differences in exposure
route (drinking water versus gavage) or vehicle (aqueous versus corn oil).
Since the data identify a longer-term NOAEL that is higher than short-term
LOAEL values, it is recommended that the DWEL for chloroform (0.4 mg/L,
calculated below) be taken as an appropriate estimate of the Longer-term HA
value for adults. For children, che Longer-term HA value may be estimated by
calculation of an adjusted DWEL value that accounts for the higher water
intake of children, as follows:
UA . , .. .. (0.01 mg/kE/dav)(10 kg)
Longer-term HA (child) = : ~ T'
i. Li/ o.ay
=0.1 mg/L (rounded to 0.1 mg/L)
VIII-12
-------
Table VIII-3 Summary of Candidate Studies for Derivation
of the Longer-term Health Advisory for Chloroform -
Reference Species
Chu et al . Rat
(1982b)
Jorgenson Rat
and Rushbrook
(1980)
Jorgenson Mouse
and Rushbrook
(1980)
Bull et al. Mouse
(1986)
Bull et al. Mouse
(1986)
Exposure
Route Duration
Drinking 90 days
water
Drinking 90 days
water
Drinking 90 days
water
Gavage 90 days
(oil)
Gavage • 90 days
(aqueous)
Endpoints
Serum
biochemistry,
hematology ,
histology,
growth
Growth, serum
biochemistry,
histopathology,
organ fat
Growth, serum
biochemistry,
histopathology ,
organ fat
Serum
biochemistry,
. liver histology
Serum
biochemistry,
NOAEL
(mg/kg/day)
50
160
145
130
(Increased
liver fat,
decreased
serum tri-
glycerides)
270
LOAEL
(mg/kg/day)
180
(Thyroid
lesions ,
decreased
growth)
290
(Increased
liver fat)
270
(Elevated
serum
enzymes ,
diffuse
liver
pathology)
liver histology
-------
where:
0.01 mg/kg/day = RfD (see below)
10 kg = assumed weight of a child
1 L/day = assumed water consumption by a 10-kg child
The Longer-term HA for a 70-kg adult consuming 2 L/day of water is
calculated as follows:
T „ UA (0.01 mg/kg/day) (70 kg) _ ,, • , . , . .
Longer-term HA = J ?2 L/d ( ^~ = °-35 m§/L (rounded to 0.4 mg/L)
d. Reference Dose and Drinking Water Equivalent Level for Chloroform
Table VIII-4 summarizes studies that were considered for deriving the
RfD and DWEL for chloroform. The studies by Palmer et al. (1979) and
Jorgenson et al. (1982) indicate that chronic exposure of rats to chloroform
at doses ranging from 60 mg/kg/day in toothpaste to 160 mg/kg/day in drinking
water did not result in any clear hepatotoxicity, although decreased weight
gain (probably secondary to reduced water consumption) was noted in both
studies. The study by Heywood et al. (1979) indicated that chronic exposure
of dogs (eight/sex) to doses of 15 mg/kg/day (6 days/week) resulted in minimal
liver injury (evidenced by slightly elevated SCOT levels and an increased
number of fatty cysts in the liver). This study is selected as the most .
appropriate for derivation of the DWEL because exposure was chronic
(7.5 years), there were 16 animals per dose group, and sensitive indices of
hepatotoxicity (serum enzyme levels, liver histology) were monitored.
VIII-14
-------
Table VIII-4 Summary of Candidate Studies for Derivation
of.the DWEL for Chloroform
Reference
Heywood
et al .
(1979)
Palmer
et al.
(1979)
Jorgenson
et al.
(1982)
Exposure
Species Route Duration
Dog Oral 7.5 years
(toothpaste
base in
capsules)
Rat Cayage 80 weeks
(toothpaste
base)
Rat Drinking 23 months
water
Endpolnts
Serum enzymes ,
hlstopathology
Weight gain,
organ weight,
histology
Weight gain,
liver fat,
hematology ,
serum
chemistry
NOAEL
(mg/kg/day)
1
60
(Decreased
body
weight)
160
(Decreased
.body weight
secondary to
decreased water
LOAEL
(mg/kg/d,ay)
15
(Elevated
SCOT, fatty
cysts in
liver)
consumption;
hemoconcentration)
-------
Using this study, the DWEL is derived as follows:
Step 1: Determination of Reference Dose (RfD)
T,.rrv (15 mg/kg/davH6/7) -. _.n _ .. .. . , , „ «, „
RfD = J ?1 000) — " 0.013 mg/kg/day (rounded to 0.01 mg/kg/day)
where:
15 mg/kg/day - LOAEL, based on mild hepatotoxicity in dogs exposed to
chloroform via gelatin capsules for 7.5 years
1,000 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a LOAEL from a study in animals is
employed
Step 2: Determination of DWEL
DWEL = - (70 kg.) _ ^ mg/L (rounded to Q 4 mg/L)
where:
0.01 mgAg/day = RfD
70 kg = assumed weight of an adult
2 L/day = assumed water consumption by a 70-kg adult
VII1-16
-------
An interim Maximum Contaminant Level (MCL) for total trihalomethanes
of 100 jig/L was established by the U.S. EPA in November 1979 (44FR68624).
Although some estimates of cancer risk were performed, this limit was set
primarily on the basis of technological and economic feasibility. The basis
and purpose of this regulation are discussed in a report prepared by OW
(U.S. EPA 1979). Based on its carcinogenic potential, the U.S. EPA (1980a)
proposed an ambient water quality criterion of zero for chloroform. On the
basis of reports of carcinogenicity and embryotoxicity, the ACGIH classified
chloroform as a suspected human carcinogen (ACGIH 1991) and recommended a
threshold limit value (TLV) of 10 ppm. Based on positive carcinogenic
findings (NCI 1976), NIOSH (1977) recommended that exposure to chloroform be
limited to 2 ppm (the lowest detectable level using the recommended sampling
and analysis techniques).
2. Bromodichloromethane
a. One-day Health Advisory for Bromodichloromethane
Studies by Bowman et al. (1978) and NTP (1987) indicate that single
oral doses of 500 mg/kg or higher of bromodichloromethane produce profound
central nervous system (CNS) depression and may lead to death. However, no
studies were located that identified a NOAEL or LOAEL based on more sensitive
endpoints suitable for derivation of the One-day HA value. In the absence of
adequate data, it is recommended that the Ten-day HA of 6 mg/L be taken as a
conservative estimate of the One-day HA value.
No existing guidelines or standards for acute oral or inhalation
exposure to bromodichloromethane were located.
VIII-17
-------
b. Ten-day Health Advisory for Bromodichloromethane
Table VIII -5 summarizes studies that were considered for calculating
the Ten-day HA for bromodichloromethane . The 30-day study by Aida et al .
(1992a) was selected for the calculation of the Ten-day HA since it resulted
in a lower LOAEL than the only other study of appropriate duration that
included a histological examination (Condie et al. 1983). This study
identified a NOAEL (in males) of 62 mg/kg/day and a LOAEL of 189 mg/kg/day
when microencapsulated bromodichloromethane was administered in feed to rats,
based on histopathology of the liver, along with sensitive biochemical tests
of liver and kidney injury. This NOAEL value is strongly supported by
drinking water studies (Munson et al. 1982; NTP 1987; Chu et al. 1982a) and a
study using oil gavage administration (Condie et al. 1983), all of which
identified NOAEL values from 50 to 150 mg/kg/day.
Using the NOAEL identified by Aida et al. (1992a) , the Ten-day HA for
the 10-kg child is calculated as follows:
Ten-day HA = <62 ffffi **> = 6.2 mg/L (rounded to 6 mg/L)
where:
62 mg/kg/day = NOAEL, based on absence of reduced body weight and
histopathological effects on the liver of rats fed
bromodichloromethane for 30 days
10 kg = assumed weight of a child
VIII-18
-------
Table V1II-5 Summary of Candidate Studies for Derivation
of the Ten-day Health Advisory for Bromodichloromethane
Reference
Munson et al.
(1982)
Condie et al.
(1983)
NTP
(1987)
NTP
(1987)
Chu et al.
(1982a)
Aida et al.
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)
Mouse Gavage 14 days
(oil)
Rat Gavage 14 days
(oil)
Mouse Gavage 14 days
(oil)
Rat Drinking 28 days
water
Rat Feed 1 month
NOAEL
Endpoints (mg/kg/day)
"Body and organ 50
weight, serum
chemistry ,
hematology ,
immune function '.
Serum enzymes, 74
histopathology ,
PAH uptake in
vitro
Body weight, 150
clinical signs,
gross necropsy
Body weight, 75
clinical signs,
gross necropsy
Clinical signs, 68
serum chemistry,
histology
Clinical signs, 62
body weight, serum
LOAEL
(mg/kg/day)
125
(Depressed
humoral
immunity)
148
(Elevated SCOT, •
decreased PAH,
histopathology in
liver, kidney)
300
(Decreased
weight gain)
150
(Mortality,
lethargy,
gross renal
pathology)
189
(Hi stopathology
biochemistry,
histology,
hematology
in liver)
-------
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
No existing guidelines or standards for short-term oral or inhalation
exposure to bromodichloromethane were located.
c. Longer-term Health Advisory for Bromodichloromethane
Table VIII-6 summarizes studies that were considered for calculation
of the Longer-term HA values for bromodichloromethane. The studies by Chu
et al. (1982b) and NTP (1987) are in good agreement, each identifying NOAEL
values of 50 to 75 mg/kg/day in oil or water. The study by NTP (1987) is
selected as the most appropriate for deriving the Longer-term HA values,
because clear dose-response trends were observed and the doses given
(separated by factors of 2.0) defined the threshold more sharply than in the
Chu study (where doses were separated by factors of 10.0). The NOAEL of
50 mg/kg/day in male mice is selected as the most appropriate value because
the LOAEL in this group (100 mg/kg/day) was lower than that for female mice
(LOAEL =• 200 mg/kg/day) and that for rats (LOAEL - 150 mg/kg/day).
Using the NTP (1987) study, the Longer-term HA for the 10-kg child is
calculated as follows:
Longer-term HA - <5° " 3'6 mS/L <™»ded to
VIII-20
-------
Table VIII-6 Summary of Candidate Studies for Derivation of
the Longer-term Health Advisory for Bromodichloromethane
Reference
Chu et al .
(1982b)
NTP
(1987)
NTP
(1987)
Species Route
Rat Drinking
water
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure
Duration
90 days
13 weeks
(5 days/
week)
13 weeks
(5 days/
week)
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
Body weight, 75
clinical signs,
histology
Body weight, 50
clinical signs,
histology
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
150
(Decreased
weight gain)
300
(Hepatic,
renal lesions)
100
(Renal
lesions)
-------
where:
50 mg/kg/day = NOAEL, based en absence of clinical signs or histological
changes in male mice exposed to bromodichloromethane via
gavage for 13 weeks
10 kg = assumed weight of a child
5/7 = correction factor to account for exposure 5 days/week
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
The Longer-term HA for a 70-kg adult consuming 2 L of water per day is
calculated as follows:
Longer-term HA = (5° = 12'5 ^/L
-------
d. Reference Dose and Drinking Water Equivalent Level for
Bromodichloromethane
Table VIII-7 summarizes studies that were considered for derivation of
the RfD and DWEL for bromodichlorome thane . The dietary study by Tobe et al.
(1982) identified a NOAEL of 6 mg/kg/day (males) based on gross pathology and
serum biochemistry. Histological examination of tissues in the Tobe et al.
(1982) study was recently reported in Aida et al. (1992b) , and a LOAEL of
6 mg/kg/day was identififed, based on fatty degeneration and granuloma of the
liver. The oil gavage study by NTP (1987) included a thorough histological
examination of control and exposed animals. The authors observed histological
lesions in the liver, kidney, and thyroid of rats exposed to 50 mg/kg/day and
of mice exposed to 25 mg/kg/day chloroform in oil. Before the publication of
the histology data in Aida et al . (1992b), the NTP (1987) study was considered
most appropriate for the derivation of the RfD and DWEL. Since a NOAEL value
was not observed in either species, the lowest LOAEL value (25 mg/kg/day in
mice) is selected as the most appropriate basis for deriving the RfD and the
DWEL. This choice is currently under review in light of Aida et al. (1992b) .
Using the NTP (1987) value, the DWEL is derived as follows:
Step 1: Determination of Reference Dose (RfD)
RfD = (25 "ft (5/7) = 0.0179 mg/kg/day (rounded to 0.02 mg/kg/day).
VIII-23
-------
Table VI11-7 Summary of Candidate Studies for Derivation
of the DWEL for Bromodichloromethane
Reference
NTP
(1987)
NTP
(1987)
Aida et al.
(1992b)
Species Route
Rat Gavage
(oil)
Mouse Gavage
(oil)
Rat Diet
Exposure . NOAEL
Duration Endpoints (mg/kg/day)
102 weeks Clinical signs,
body weight,
gross necropsy, .
histology
102 weeks Clinical signs,
body weight,
gross necropsy,
histology
24 months Clinical signs,
body weight,
LOAEL
(mg/kg/day)
50
(Renal and
hepatic
lesions)
25
(Lesions of
liver, kidney,
thyroid)
6.1
(Liver fatty
serum biochemistry,
gross necropsy,
histology
degeneration
and granuloma)
-------
where:
25 mg/kg/day - LOAEL, based on histological lesions in liver, kidney and
thyroid in mice exposed to bromodichloromethane via gavage
for 102 weeks
5/7 = correction for exposure 5 days/week
1,000 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a LOAEL from a study in animals is
employed
Step 2: Determination of DWEL
DWEL = (0.02 ms/ka/day) <70 - 0.7 mg/L
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
No existing guidelines or standards were located for chronic oral
exposure to bromodichloromethane. The interim MCL for total trihalomethanes
is 100 /xg/L (44FR68624). Although some estimates of cancer risk were
VIII-25
-------
performed, this limit was set primarily on the basis of technological and
economic feasibility. The basis and purpose of this regulation are discussed
in a report that was prepared by the OW (U.S. EPA 1979").
3. Dibromochloromethane
a. One-day Health Advisory for Dibromochloromethane
Studies by Bowman et al. (1978) indicate that single oral doses of
500 mg/kg or higher of dibromochloromethane produce profound CNS depression,
and NTP (1985) data show that doses from 310 to 2,500 mg/kg/day may lead to
death in rats and mice. However, no studies were located that identified a
NOAEL or LOAEL based on more sensitive endpoints suitable for derivation of
the One-day HA value. In the absence of adequate data, it is recommended that
the Ten-day HA of 6 mg/L be considered as a conservative estimate of the One-
day HA value.
No existing guidelines or standards for acute oral or inhalation
exposure to dibromochloromethane were located.
b. Ten-day Health Advisory for Dibromochloromethane
Table VIII-8 summarizes studies that were considered for calculating
the Ten-day HA for dibromochloromethane. The study by Aida et al. (1992a) was
selected as the basis for the calculation of the Ten-day HA. In this study,
microencapsulated dibromochloromethane was administered in feed to rats for
one month. Based on liver histopathology and increased cholesterol levels in
male rats, this study identified a NOAEL of 56 mg/kg/day and a LOAEL of
VIII-26
-------
Table VIII-8 Summary of Candidate Studies for Derivation
of the Ten-day Health Advisory for Dibromochloromethane
Reference
Munson et al .
(1982)
Condie et al.
(1983)
NTP (1985)
NTP (1985)
Chu et al.
(1982a)
Aida et al .
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)
Mouse Gavage 14 days
(oil)
Rat Gavage 14 days
(oil)
Mouse Gavage 14 days
(oil)
Rat Drinking 28 days
water
Rat Feed 1 month
NOAEL
Endpoints (mg/kg/day)
"Body and organ 50
weight, serum
chemistry,
hematology ,
immune function '•
Serum enzymes, 74
histopathology ,
PAH uptake in
vitro
Body weight, 250
clinical signs,
gross necropsy
Body weight, 60
clinical signs,
gross necropsy
Clinical signs, 68
serum chemistry,
histology
Clinical signs, 56
body weight, serum
LOAEL
(mg/kg/day)
125
(Decreased
humoral
immunity)
147
(Elevated SCOT,
decreased PAH,
histopathology
in liver,
kidney)
500
(Lethargy,
gross pathology,
mortality)
125
(Stomach
lesions)
173
(Histopathology
biochemistry,
histology,
hematology
in liver,
increased
cholesterol |M|)
-------
173 mg/kg/day. Histopathology was also assessed in the oil gavage study of
Condie et al. (1983), which identified a NOAEL of 74 rag/kg/day. These data
are strongly supported by the other drinking water and corn oil vehicle
studies (Munson et al. 1982; NTP 1985; Chu et al. 1982a), all of which
identified NOAEL values from 50 to 250 mg/kg/day.
Using the NOAEL identified by Aida et al. (1992a), the Ten-day HA for
the 10-kg child is calculated as follows:
(rounded to 6 ms/L)
where:
56 mg/kg/day = NOAEL, based on absence of hepatic effects in rats exposed
to microencapsulated dibromochloromethane in feed for one
month
10 kg = assumed weight of a child
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
No existing guidelines or standards for short-term oral or inhalation
exposure to dibromochloromethane were located.
VIII-28
-------
c. Longer-term Health Advisory for Dibromochloromethane
Table VIII-9 summarizes studies that were considered for calculation
of the Longer-term HA values for dibromochloromethane. The 90-day drinking
water study by Chu et al. (1982b) and the corn oil study by NTP (1985) are in
good agreement, each identifying NOAEL values of about 30 to 50 mg/kg/day and
LOAEL values of 60 to 250 mg/kg/day. The drinking water study by Borzelleca
and Carchman (1982) is also consistent with these values, identifying a NOAEL
of 17 mg/kg/day and a LOAEL of 171 mg/kg/day after somewhat longer exposure
(24 to 27 weeks). The LOAEL of 50 mg/kg/day identified by Daniel et al.
(1990) in a corn oil study is consistent with the threshold suggested by NTP
1985). The NOAEL value of 30 mg/kg/day chloroform in oil identified in rats
by NTP (1985) is. selected as the most appropriate value for derivation of the
Longer-term HA values and is supported by two drinking water studies.
Using this study, the Longer-term HA for the 10-kg child is calculated
as follows:
T - UA (30 mg/kg/dav) (10 kg) (5/7) ... „ • .
Longer-term HA = J (100) (1 L/dav) — =2.1 mg/L (rounded to 2 mg/L)
where:
30 mg/kg/day = NOAEL, based on absence of clinical signs or histological
changes in rats exposed to dibromochloromethane via gavage
for 13 weeks
10 kg = assumed weight of a child
VIII-29
-------
Table VIII-9 Summary of Candidate Studies for Derivation of
the Longer-term Health Advisory for Dibromochloromethane
Reference
Chu et al.
(1982b)
NTP (1985)
NTP (1985)
Daniel et al.
(1990)
Borzelleca
and Carchman
(1982)
Exposure
Species Route Duration
Rat Drinking 90 days
water
Rat Gavage 13 weeks
(oil)
Mouse Gavage 13 weeks
(oil)
Rat Gavage 90 days
(oil)
Mouse Drinking 27 weeks
water
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
t
Body weight, 30
clinical signs,
histology
Body weight, 125
clinical signs,
histology
Body weight, ' --
clinical signs,
histology, serum
biochemistry, gross
necropsy
Maternal body 17
weight, gross
pathology, fetal
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
60
(Hepatic
vacuolation)
250
(Renal and
hepatic
lesions)
50
(Hepatic
vacuolizat ion,
serum
biochemistry)
171
(Maternal
toxic. ity ,
weight, survival,
teratogeriicity
possible
fetotoxicity)
-------
5/7 = correction factor to account for exposure 5 days/week
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
The Longer-term HA for a 70-kg adult consuming 2 L/day of water is
calculated as follows:
T UA (30 mg/kg/dav) (70 kg) (5/7) ... ...
Longer-term HA = J (100) (2 L/dav) — =7.5 mg/L (rounded to 8 mg/L)
No existing guidelines or standards were located for longer-term
(subchronic) oral or inhalation exposure to dibromochloromethane.
d. Reference Dose and Drinking Water Equivalent Level for
Dibromochloromethane
Table VIII-10 summarizes studies that were considered for derivation
of the RfD and DWEL for dibromochloromethane. The dietary study by Tobe
et al. (1982) identified a NOAEL of 10 mg/kg/day based on gross pathology and
serum biochemistry, but mild weight suppression did occur, and histological
examination of tissues was not performed. The chronic corn oil vehicle study
by NTP (1985) indicated that doses of 40 and 50 mg/kg/day produce histological
lesions in the liver of rats and mice, respectively. However, the chronic
studies do not identify a reliable NOAEL. The subchronic (13-week) study by
NTP (1985) identified a LOAEL and NOAEL of 60 and 30 mg/kg/day, respectively.
VIII-31
-------
Table VIII-10 Summary of Candidate Studies for Derivation
of the DWEL for Dibromochloromethane
Reference
Tobe et al.
(1982)
NTP (1985)
NTP (1985)
Species Route
Rat Diet
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure NOAEL
Duration Eridpoints (mg/kg/day)
24 months Body weight, 10
serum
biochemistry, ,
gross
pathology
102 weeks Clinical signs,
body weight,
gross necropsy,
histology
102 weeks Clinical signs,
body weight,
gross necropsy, •
LOAEL
(mg/kg/day)
39
(Serum enzyme
changes and
altered liver
appearance)
40
(Hepatic
lesions)
50
(Hepatic
lesions)
histology
-------
Use of the chronic LOAEL of 40 mg/kg/day would result in a slightly less
protective DWEL of 1 mg/L. Therefore, greater confidence is placed in the
NOAF.L of 30 mg/kg/day identified in rats in the subchronic (13-week) study by
NTP (1985).
Using this value, the DWEL is derived as follows:
Step 1: Determination of Reference Dose (RfD)
RfD = (30 ^As/day) (5/7) _ Q 02U mg/kg/day (rounded to 0.02 mg/kg/day)
(1,000)
where:
30 mg/kg/day = NOAEL, based on absence of histological lesions in liver
of rats exposed to dibromochloromethane via gavage for
13 weeks
5/7 = correction factor to account for exposure 5 days/week
1,000 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a less-than-lifetime
study in animals is employed to derive an RfD
Step 2: Determination of DWEL
DWEL =
VIII-33
-------
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
No existing guidelines or standards were located for chronic oral
exposure to dibromochloromethane. The interim MCL for total trihalomethanes
is 100 /ig/L (44FR68624). Although some estimates of cancer risk were
performed, this limit was set primarily on the basis of technological and
economic feasibility. The basis and purpose of this regulation are discussed
in a report that was prepared by the OW (U.S. EPA 1979).
4. Bromoform
a. One-day Health Advisory for Bromoform
Although the data are limited, information from reports on the use of
bromoform as a sedative in children is adequate to establish that the normal
therapeutic dosing regimen (about one drop given three to four times per day,
equivalent to a total daily dose of about 540 mg) produced mild sedation, with
more severe reactions typically being associated with accidental overdoses
(Burton-Fanning 1901; Dwelle.1903; Benson 1907). On this basis, a dose of
54 mg/kg/day (540 mg/day given to a 10-kg child) is identified as a LOAEL in
humans, and the One-day HA for the 10-kg child is calculated as follows:
VIII-34
-------
n A* UA (54 mg/kg/dav) (10 kg) = . ... , .
One-day HA = (1 L/day) = 5'4 mS/L (rounded to 5 mg/L)
where:
54 mg/kg/day =• LOAEL, based on sedation in children given bromoform by
mouth
10 kg — assumed weight of a child
100 = uncertainty factor; selected in accordance with NAS/OW
guidelines in which a LOAEL from a study in humans is
employed
1/L day •= assumed water consumption by a 10-kg child
No existing guidelines or standards for acute oral exposure to
bromoform were located. A concentration of 0.5 ppm in air has been proposed
as a Time-Weighted Average-Threshold Limit Value (TWA-TLV) by the American
Conference of Governmental Industrial Hygienists (ACGIH 1991). The OSHA
permissible exposure limit (PEL) for bromoform is 0.5 ppm as the TWA exposure
that is not to be exceeded in any up 8-hour work shift of a 40-hour work week
(54FR2332, January 19, 1989).
b. Ten-day Health Advisory for Bromoform
Table VIII-11 summarizes studies that were considered for calculating
the Ten-day HA for bromoform. The study by Condie et al. (1983) identified a
VIII-35
-------
Table V11I-11 Summary of Candidate Studies for Derivation
of the Ten-day Health Advisory for Bromoform
Reference
Munson et al.
(1982)
Condie et al.
(1983)
Chu et al.
(1982a)
NTP (1989a)
Aida et al.
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)
Mouse Gavage 14 days
(oil)
Rat Drinking 28 days
water
Rat Gavage 14 days
(oil)
Rat Feed 1 month
NOAEL
Endpoints (mg/kg/day)
Body and organ 125
weight, serum
chemistry,
hematology,
immune function
Serum enzymes, 145
histopathology ,
PAH uptake in
vitro
Clinical signs,' 68
serum chemistry,
histology
Body weight, 200
clinical signs
Clinical signs, 62
body weight, serum
LOAEL
(mg/kg/day)
250
(Elevated
serum enzymes)
289
(Elevated SCOT,
decreased PAH,
histopathology
in li.ver,
kidney)
• -
400
(Decreased
body weight)
187
(Histopathology
biochemistry,
histology,
hematology
in liver,
decreased
cholinesternse)
-------
NOAEL of 145 mg/kg/day and a LOAEL of 289 mg/kg/day chloroform in oil, based
on histological examination of the liver and kidney along with sensitive
biochemical tests of liver and kidney injury. This NOAEL value is supported
by the drinking water and corn oil vehicle studies of Munson et al. (1982),
Chu et al. (1982a) and NTP (1989a) , which identified NOAEL values of 125, 68,
and 200 mg/kg/day, respectively. A NOAEL of 62 mg/kg/day and a LOAEL of
187 mg/kg/day was determined in a recent feed study based on liver
histopathology and decreased serum cholinesterase (Aida et al. 1992a).
However, based on a NOAEL of 62 mg/kg/day in animals, the resulting
Ten-day HA for a 10-kg child would be 6 mg/L. Since this is larger than the
One-day HA calculated from human data (above), it is recommended that the
Longer-term HA value of 2 mg/L (calculated below) be taken as a conservative
estimate of the Ten-day HA value.
No existing guidelines or standards for short-term oral or inhalation
exposure to bromoform were located.
c. Longer-term Health Advisory for Bromoform
Three studies were located chat are candidates for calculating the
Longer-term HA values for bromoform (Table VIII-12). Chu et al. (1982b)
identified a NOAEL of 52 mg/kg/day and a LOAEL of 250 mg/kg/day chloroform in
the drinking water, based on mild histologic lesions in liver. The study by
NTP (1989a) identified a NOAEL of 25 mg/kg/day and a LOAEL of 50 mg/kg/day
chloroform administered by corn oil.gavage, based on histological signs of
hepatotoxicity in rats, while mice appeared to be somewhat less sensitive.
VIII-37
-------
Table VIII-12 Summary of Candidate Studies for Derivation of
the Longer-term Health Advisory for Broraoform
00
Reference
Chu et al .
(1982b)
NTP (1989a)
NTP (1989a)
Species Route
Rat Drinking
water
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure •
Duration
90 days
13 weeks
(5 days/
week)
13 weeks
(5 days/
week)
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
i
Clinical signs, 25
body weight,
necropsy,
histopathology
Clinical signs, 100
body weight,
necropsy,
histopathology
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
50 (Hepato-
cellular
vacuolation)
200 (Hepato-
cellular
vacuolation)
-------
The NOAEL of 25 mg/kg/day in rats is selected as the most appropriate basis
for derivation of the Longer-term HA, which is calculated as follows:
UA (25 mg/kg/dav) (10 kg) (5/7) . 0 .. , ' _,
Longer-term HA - (100) (1 L/day) = 1 • 8 mg/L (rounded to 2 mg/L)
where:
25 mg/kg/day = NOAEL, based on absence of clinical or histological
effects in rats exposed to bromoform by gavage for
13 weeks
10 kg = assumed weight of a child
5/7 = correction for exposure 5 days/week
100 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a study in animals is
employed
1 L/day = assumed water consumption by a 10-kg child
The Longer-term HA for a 70-kg adult consuming 2 L/day of water is
calculated as follows:
T UA C25 mg/kg/dav) (70 kg) (5/7) ^ A r- f. m n ^
Longer-term HA = J (100) (2 L/day) =6.2 mg/L (rounded to 6 mg/L)
VIII-39
-------
No existing guidelines or standards were located for longer-term
(subchronic) oral exposure to bromoform. The OSHA has established an 8-hour
TWA permissible exposure limit of 0.5 ppm (54FR2322, January 19, 1989).
d. Reference Dose and Drinking Water Equivalent Level for Bromoform
Three studies were located that are candidates for deriving the RfD
and DWEL for bromoform (Table VIII-13). Tobe et al. (1982) identified a NOAEL
of 18 mg/kg/day, based on gross pathology and serum biochemistry in rats
exposed to bromoform in the diet for 2 years. However, this study did not
include any histological examination of tissues following bromoform exposure,
and the data have not been published or formally peer reviewed. On this
basis, this study is judged as not adequate for derivation of an RfD and a
DWEL for bromoform. A LOAEL of 100 mg/kg/day was identified by the chronic
(2-year) corn oil gavage study by NTP (1989a) for decreased body weight and
liver effects in rats and mice. A reliable NOAEL was not identified by this
study since only high doses (100 or 200 mg/kg/day) were tested. The subchro
nic (13-week) study by NTP (1989a) tested lower doses and identified NOAEL and
LOAEL values of 25 and 50 mg/kg/day, respectively. Use of the chronic LOAEL
of 100 mg/kg/day would result in a less protective DWEL of 2.5 mg/L.
Therefore, the subchronic NOAEL of 23 mg/kg/day is selected as the most
appropriate basis for the derivation of an RfD and DWEL.
Based on this value, the DWEL is calculated as follows:
VIII-40
-------
Table VIII-13
Summary of Candidate Studies for Derivation of
DWEL for Bromoform
Reference
Tobe et al.
(1982)
Exposure
Species Route Duration
Rat Diet 2k months
NOAEL
Endpoints (mg/kg/day)
Body weight, 18 (M)
serum chemistry, 30 (F)
gross pathology ,
LOAEL
(mg/kg/day)
71 (M)
120 (F)
(Decreased
NTP (1989a)
Rat
NTP (1989a)
Mouse
Gavage 103 weeks Clinical signs,
(oil) . (5 days/ body weight,
week) survival, gross
necropsy, histo-
pathology
Gavage 103 weeks Clinical signs, 100 (M)
(oil) (5 days/ body weight,
week) survival, gross
necropsy, histo-
pathology
body weight,
elevated serum
enzymes)
100 (M)(F)
(Decreased
body weight,
lethargy, mild
liver histo-
pathology)
100 (F)
(Decreased
body weight,
mild liver
histopathology)
-------
Seep 1: Determination of RfD
RfD = (25 mR/kfi/dav) (5/7) _ Q Qlg mg/kg/day (rounded to 0.02 mg/kg/day)
(1,000)
where:
25 mg/kg/day = NOAEL, based on absence of clinical or histological
effec-ts in rats exposed to bromoform in the diet for
13 weeks
5/7 = correction factor to account for exposure 5 days/week
1,000 = uncertainty factor; chosen in accordance with NAS/OW
guidelines in which a NOAEL from a less-than-lifetime
study in animals is employed to derive an RfD
Step 2: Determination of DWEL
DWEL = (0-02 ,R/kK/d.y) (70 = Q ? mg/L
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
VIII-42
-------
B. Carcinogenic Effects
1. Categorization of Carcinogenic Potential
a. Chloroform
The International Agency for Research on Cancer (IARC) has performed
an assessment of the degree of evidence for the carcinogenicity of chloroform
to humans and experimental animals (IARC 1982, 1987). This assessment
concluded that chloroform is a Group 2B chemical (sufficient evidence of
carcinogenicity in animals but inadequate evidence of carcinogenicity in
humans).
The U.S. EPA (1985a) has reviewed the evidence on the carcinogenicity
of chloroform and has ranked it as a Group B2 chemical (probable human
carcinogen) (IRIS 1990).
b. Brominated Trihalomethanes
The Carcinogenic Risk Assessment Verification Endeavor (CRAVE) group
of the U.S. EPA has recently reviewed the available evidence on the carcino-
genicity of the brominated trihalomethanes. Based on this review, CRAVE has
assigned bromoform and bromodichloromethane to Group B2 and dibromochloro-
methane to Group C (IRIS 1991). IARC has evaluated the carcinogenic
potentials of brominated trihalomethanes (IARC 1991a, 1991b, 1991c). IARC
concluded that there is sufficient .evidence of carcinogenicity for
bromodichloromethane in experimental animals, but inadequate evidence in
humans. For dibromochloromethane and bromoform, IARC concluded that there is
VIII-43
-------
limited-evidence of carcinogenicity in experimental animals and inadequate
evidence in humans. IARC suggested that bromodichloromethane is a Group 2B
carcinogen (possibly carcinogenic to humans) and r.h^t dibromochloromethane and
bromoform are Group 3 carcinogens (not classifiable as to their carcinogen-
icity to humans).
2. Quantitative Carcinogenic Risk Estimates
a. Chloroform
Five data sets were used by the U.S. EPA (1985a) to estimate the
carcinogenic potency of chloroform, as shown in Table VIII-14. The unit risks
at 1 mg/kg/day, calculated by the linearized multistage model, were judged to
be comparable for each of these data sets. The values of q^ derived from
these five data sets were 2.0xlCT1, 3.3xlO'2, 2.35xlO"2, l.OxlO"1 and
4.41xlO"3 (mg/kg/day)"1, respectively. The geometric mean of the slope
estimates calculated from liver tumors in male and female mice (S.lxlO"2
[mg/kg/day]"1) was taken to represent the carcinogenic potency. Based on
this, the upper-bound estimate of the cancer risk due to 1 ^g/L in water was
2.3xlO"6. This estimate was judged to be consistent with the limited
epidemiologic data available for humans (U.S. EPA 1985a). Since risk is
linear in this range, risk factors of 10"4, 10"5, and 10"6 correspond to
concentrations of 40, 4, and 0.4 /ig/L, respectively.
More recently, the U.S. EPA (1987) reviewed these studies and
concluded that the quantitative risk estimate for chloroform should be based
only on the incidence of renal tumors in rats reported in the study by
Jorgenson et al. (1985) and should not consider the incidence of liver tumors
VIII-44
-------
TABLE VIII-14 Upper-Bound Estimates of Cancer Risk of 1 mg/kg/day
of Chloroform, Calculated by Four Models on the
Basis of Various Data Sets
Data Base Multistage Probit
Liver tumors in female 1.8 x 10"1 2.1 x 10"1
mice (NCI, 1976)
Liver tumors in male 3.3 x 10"2 6.7 x 10"2
mice (NCI, 1976)
Kidney tumors in male 2.4 x 10"2 3.9 x 10"5
rats (NCI, 1976)
Kidney tumors in male 1.0 x 10"1 NAb
mice (Roe et al. , 1979)
Kidney tumors in male 4.4 x 10~3 9.0 x 10"5
rats (Jorgenson et al.,
1985)
Weibull One-Hit
4.8 x 10'1 1.8 x 10'1
3.2 x 10-3 1.6 x lO'1
3.0 x 10'3 2.5 x ID'2
NA 1.0 x 10'1
4.8 x ID'4 5.4 x 10'3
aUpper-bound estimates are calculated by the one-sided 95% confidence limit.
''NA = not applicable; models are not applicable because there is only one
dosed group
Adapted from U.S. EPA (1985a).
VIII-45
-------
reported in mice by NCI (1976). This decision was based on the observation
that liver tumors were not increased in mice exposed to chloroform via
drinking water, suggesting that the vehicle may have played a role ir. the
hepatic tumors following exposure in oil. This concept is supported by the
observation that corn oil appears to potentiate the hepatotoxic effects of
chloroform in B6C3F1 mice (Bull et al. 1986). The slope factor based on renal
tumors in rats was recalculated employing the incidence of tubular cell
adenomas, tubular cell adenocarcinomas, and nephroblastomas, but excluding
metastatic and transitional tumors (see Table V-31). The resulting slope
factor is 6.IxlO'3 (mg/kg/day)'1 (IRIS 1992). Based on this, the unit risk
for chloroform is 1.7xlO~7 (jig/L)'1, and the drinking water concentration
corresponding to the 10'6 risk level is 6 ^g/L (IRIS 1992) .
The NAS (1987) has also reviewed available data on the carcinogenicity
of chloroform and calculated several different risk estimates using the
linearized multistage model. The results of their calculations are shown in
Table VIII-15. Because of the same concern discussed above regarding the
apparent oil vehicle effect on liver tumors in mice, the NAS Subcommittee on
Health Effects of Disinfectants and Disinfectant By-Products also recommended
that the cancer slope factor for chloroform be based on the kidney tumor data
in rats reported by Jorgenson et al (1985).
b. Brominated Trihalomethanes
The NAS (1987) utilized the data reported by NTP (1985) on the
frequency of liver tumors in female B6C3F1 mice exposed to dibromochloro-
methane to calculate an excess lifetime cancer unit risk of 8.3xlO"7, using
the linearized multistage model and assuming consumption of 1 L of water per
VIII-46
-------
TABLE VIII-15 Carcinogenic Risk Estimates for
Chloroform Calculated bv NAS
Reference
Jorgenson et al .
(1985)
NCI (1976)
NCI (1976)
Roe et al .
(1979)
Concentration
Equivalent to
Unit Cancer Riskb Risk of 10'6
Species Sex Tumor (Upper 95% Limit) (/ig/L)c
Osborne -Mendel M Kidney 1.1 x 10"7 4.5
rat
B6C3F1* F Liver 1.9 x 10'6 0.3
mouse
Osborne -Mendel M Kidney 4.7 x 10'8 10.6
rat
ICI M Kidney 3.7 x 10'7 1.4
mouse
aAdapted from NAS (1987).
bAssuming consumption of 1 L/day of water.
°Assuming consumption of 2 L/day of water.
VIII-47
-------
day containing 1 pig/L of dibromochloromethane. Based on this calculation, the
concentration associated with a risk of 10"6 is 0.6 ng/L, assuming consumption
of 2 L of water per day.
More recently, the U.S. EPA has calculated quantitative cancer risk
estimates for bromodichloromethane, dibromochloromethane, and bromoform, based
on the data from the three 2-year oral exposure studies performed by NTP
(1987, 1985, 1989a) (see Tables V-31, V-32, V-33). The resulting slope
factors, unit risks, and risk-specific concentration values are summarized in
Table VIII-16, along with a summary of the tumor data used to derive the
values.
Evaluation of the quantitative cancer risk estimates for the
brominated trihalomethanes based on the three NTP rodent studies (1985, 1987,
1989a) is complicated by the use of a corn oil vehicle in these studies.
Although a vehicle effect has not been investigated for brominated
trihalomethanes, it can be inferred from studies of chloroform carcinogenicity
(see above) 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). Therefore, the slope factor based on renal
tumors in male mice (6.2xlO~2 per mg/kg/day) is the recommended value.
In the case of dibromochloromethane, the only tumor data available are
for liver tumors in mice. Therefore, the slope factor (8.4xlO'2
[mg/kg/day]"1) is based on these data, but some uncertainty exists regarding
VIII-48
-------
TABLE VIII-16 Carcinogenic Risk Estimates for Brominated Trihalomethanes
Trihalomethane Tumor Site
Sromodichloro- Liver
methane
Kidney
Large
intestine
Large intestine
and kidney
(combined)
Spec i es
House
Rat
Mouse
Rat
Rat
Sex
Female
Male
Female
Male
Male
Female
Male
Female
Slope Factorl
(mg/kg-day)"
1.3 x 10'1
8.7 x 10^
9.5 x 10
6.2 x 10"2
2.5 x 10"*
4.9 x 10"3
2.4 x 10'*
7.9 x 10 •
Unit
Risk
((ig/L)"1
3.7 x 10"6
2.5 x 10"'
2.7 x 10
1.8 x 10"6
7.1 x 10"'
1.4 x 10
6.9 x 10''
2.3 x 10"7
10"5 Risk
Concen-
tration
Ug/D
3
40
37
6
14
72
15
44
Oibromochloro- Liver
methane
Bromoform
Large intestine
Mouse
Rat
Female
Female
8.4 x 10"
7.9 x 10"
2.4 x 10"
2.3 x 10"
40
Adapted from IRIS (1992).
VIII-49
-------
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 ro clarify this issue.
In the case of bromoform, the cancer risk estimate (7.9xlO~3 [tng/kg/
day]"1) is based on the incidence of intestinal"tumors in rats. There are no
data to suggest that tumor incidence in this tissue is influenced by the use
of an oil vehicle, so thi.s risk estimate is believed to be applicable to
drinking water exposures.
C. Summary
Table VIII-17 summarizes HA and DWEL values (calculated on the basis
of noncarcinogenic endpoints) and the 10~6 excess cancer risk levels
(calculated using the linearized multistage model) for chloroform,
bromodichloromethane, dibromochloromethane, and bromoform.
VIII-30
-------
TABLE VIII-17
Summary of Quantification of Toxicological
Effects for Trihalomethanes
Value
Chloroform
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
OWEL (70-kg adult)
Excess cancer risk (10" )
--•.
Bromodi ch 1 oromethane
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
DUEL (70-kg adult)
Excess cancer risk (10~6)
Dibromoch I oromethane •
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
DWEL (70-kg adult)
Excess cancer risk (10~6)
Drinking Uater
Concentration
4 mg/L
4 mg/L
0.1 mg/L
0.4 mg/L
0.4 mg/L
6 »ig/L
4.5 A9/L
6 mg/L*
6 mg/L
4 mg/L
13 mg/L
0.7 mg/L
0.6 jig/L
6 mg/L*
6 mg/L '
2 mg/L
8 mg/L
0.7 mg/L
0.4
-------
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