United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Criteria and Standards Division
Washington DC 20460
EPA 440/5-80-033
October 1980
c/EPA
Ambient
Water Quality
Criteria for
Chloroform
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AMBIENT WATER QUALITY CRITERIA FOR
CHLOROFORM
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, 1NTIS), Springfield, Virginia 22161.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. ,','owever, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects:
Leland J. McCabe, HERL (author)
U.S. Environmental Protection Agency
Debdas J. Mukerjee (doc. mgr.)
ECAO-Cin
U.S. Environmental Protection Agency
Jerry F. Stara (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Patrick Durkin
Syracuse Research Corporation
Roy E. Albert*
Carcinogen Assessment Group
U.S. Environmental Protection Agency
David J. Hansen, ERL-Gulf Breeze
U.S. Environmental Protection Agency
Julian Andelman
University of Pittsburgh
Herbert Cornish
University of Michigan
Joseph Borzelleca
Medical College of Virginia
Steven D. Lutkenhoff, ECAO-Cin
U.S. Environmental Protection Agency
Si Duk Lee, ECAO-RTP
U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, C. Russom, B. Gardiner.
*CAG Participating Members:
Elizabeth L. Anderson, Larry Anderson, Dolph Arnicar, Steven Bayard,
David L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman,
Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosen-
blatt, Dharm V. Singh, and Todd W. Thorslund.
IV
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TABLE OF CONTENTS
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Chronic Toxicity B-l
Miscellaneous B-2
Summary B-2
Criteria B-3
References B-7
Mammalian Toxicology and Human Health Effects C-l
Exposure C-l
Ingestion from Water C-l
Ingestion from Food C-3
Inhalation C-4
Dermal C-5
Pharmacokinetics C-5
Absorption C-5
Distribution C-6
Metabolism C-6
Excretion C-9
Effects C-9
Acute, Subacute and Chronic Toxicity C-9
Synergism and/or Antagonism C-l7
Teratogenicity C-18
Mutagenicity C-19
Carcinogeniclty C-20
Criterion Formulation C-29
Existing Guidelines and Standards C-29
Current Levels of Exposure C-31
Basis and Derivation of Criteria C-35
References C-41
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CRITERIA DOCUMENT
CHLOROFORM
CRITERIA
Aquatic Life
The available data for chloroform indicate that acute toxicity to fresh-
water aquatic life occurs at concentrations as low as 28,900 yg/1, and would
occur at lower concentrations among species that are more sensitive than the
three tested species. Twenty-seven-day LCgn values indicate that chronic
toxicity occurs at concentrations as low as 1,240 yg/1, and could occur at
lower concentration among species or other life stages that are sensitive
than the earliest life cycle stage of the rainbow trout.
The data base for saltwater species is limited to one test and no state-
ment can be made concerning acute or chronic toxicity.
Human Health
For the maximum protection of human health from the potential
carcinogenic effects due to exposure of chloroform through ingestion of
contaminated water and contaminated aquatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10~5, 10 , and 10 . The
corresponding recommended criteria are 1.90 yg/1, 0.19 ug/1, and 0.019 yg/1,
respectively. If the above estimates are made for consumption of aquatic
organisms only, excluding consumption of water, the levels are 157 ug/1,
15.7 ug/1, and 1.57 ug/1, respectively.
VI
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INTRODUCTION
Chloroform (CHC1-) was first employed as an anesthetic agent in 1847.
Only a small amount was necessary to induce narcosis, and its action was
more complete than ether. Today, it has been replaced by other anesthetics
with more desirable properties; but it is used widely as a chemical solvent
and as an intermediate in the production of refrigerants, plastics, and
Pharmaceuticals (U.S. EPA, 1975). Current annual production of chloroform
approaches 120,000 metric tons (U.S. EPA, 1977).
Chloroform (CHCl^; molecular weight 119.39), at ordinary temperatures
and pressures, is a clear, colorless, volatile liquid with a pleasant,
etheric, nonirritating odor and sweet taste (Hardie, 1964; Windholz, 1976).
It has a boiling point range of 61-62'C, a melting point of -63.5"C, and is
nonflammable. There is no flash point (Hardie, 1964; Windholz, 1976).
Chloroform is slightly soluble in water (7.42 x 106 pg/1 of water at
25°C). It is miscible with alcohol, benzene, ether, petroleum ether, carbon
tetrachloride, carbon disulfide, and oils (Windholz, 1976). Chloroform is
highly refractive and has a vapor pressure of 200 mm Hg at 25°C (Irish,
1962; Windholz, 1976). Because of its volatile nature, chloroform has the
potential for evaporation to the air from pollution sources or from the
water column.
At ambient environmental temperatures, chloroform is thermostable and
resists decomposition (Hardie, 1964). However, slow decomposition occurs
following prolonged exposure to sunlight and in darkness when air is present
(Hardie, 1964). Chloroform has the potential to react with, and thereby de-
plete, the ozone layer; studies have shown that phosgene is a decomposition
A-l
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product of ozone and chloroform (Hardie, 1964). There is no appreciable de-
composition of chloroform at ambient temperatures in water, even in the pre-
sence of sunlight (Hardie, 1964), Aqueous degradation of chloroform is ac-
celerated in the presence of aerated waters and metals, such as iron, with
hydrogen peroxide representing a reaction product (Hardie, 1964).
Chloroform appears to be ubiquitous in the environment in trace amounts,
and discharges into the environment result largely from chlorination of
water and wastewater (U.S. EPA, 1975).
A-2
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REFERENCES
Hardie, D.W.F. 1964. Chlorocarbons and Chlorohydrocarbons: Chloroform.
ln_: D.F. Kirk and D.E. Othmer (eds.). Encyclopedia of Chemical Technology.
2nd ed. John Wiley and Sons, Inc., New York.
Irish, D.D. 1962. Aliphatic Halogenated Hydrocarbons. j_n: Industrial Hy-
giene and Toxicology. 2nd ed. John Wiley and Sons, Inc., New York.
U.S. EPA. 1975. Development document for interim final effluent limita-
tions guidelines and new source performance standards for the significant
organic products segment of the organic chemical manufacturing point source
category. EPA 440/1-75-045. U.S. Environ. Prot. Agency, Washington, D.C.
U.S. EPA. 1977. Determination of sources of selected chemicals in water
and amounts from these sources. Area 1. Task 2. Draft final report. Con-
tract No. 68-01-3852. U.S. Environ. Prot. Agency, Washington, D.C.
VJindholz, M. (ed.) 1976. The Merck Index. 9th ed. Merck and Co., Inc.,
Rahway, New Jersey.
A-3
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Aquatic Life Toxicology*
INTRODUCTION
Chloroform has been most commonly tested under static conditions with no
measurement of the concentrations of chloroform to which the organisms are
exposed. Consequently, the acute toxicity data base will probably under-
estimate the toxicity because concentrations in static tests are likely to
diminish during the progress of the exposure as a result of loss from water
to air.
EFFECTS
Acute Toxicity
A 48-hour static test with Daphnia magna resulted in an LCcn of 28,900
yg/1 (Table 1). Bentley, et al. (1975) compared the toxicity of chloroform
to rainbow trout and to bluegill and found (Table 1) that the trout was more
sensitive. All 96-hour LC5Q values for freshwater fish, using static
methods and unmeasured concentrations, were between 43,800 and 115,000 ug/1.
Only one appropriate acute test has been reported on the toxicity of
chloroform to saltwater aquatic life. Bentley, et al. (1975) conducted a
static test with pink shrimp and determined a 96-hour IC™ value of 81,500
ug/1 (Table 1).
Chronic ^oxicity
No chronic effects of chloroform on freshwater or saltwater species are
available other than those in Table 3.
*The reade^ is referred to the Guidelines for Deriving Water Quality Cri-
teria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
conta-'n the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various measures of tox-
icity as described in the Guidelines.
B-l
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Residues
After a 14-day exposure (U.S. EPA, 1978) to radiolabeled chloroform, the
bluegill bioconcentrated chloroform by a factor of 6 times (Table 2) and the
tissue half-life was less than 1 day. This degree of bioconcentration and
short biological half-life suggest that residues of chloroform would not be
an environmental hazard to consumers of aquatic life.
Miscellaneous
Most of these data are compiled from short exposures of minutes to a few
hours in duration (Table 3). With stickleback, goldfish, and orangespotted
sunfish, anesthetization or death occurred at concentrations between 97,000
and 296,640 ug/1. Birge, et al. (1979) conducted flow-through tests with
measured chloroform concentrations in closed systems. Exposures of rainbow
trout began within 20 minutes after fertilization and ended eight days after
hatching. There was no additional mortality between the fourth and eighth
days after hatching. The 27-day IC™ values for soft and hard water were
2,030 and 1,240 ug/1, respectively. There was a 40 percent incidence of
teratogenesis in the embryos at hatching.
Summary
Two freshwater fish and one invertebrate species have been acutely test-
ed under standard conditions and 50 percent effect concentrations were be-
tween 28,900 and 115,000 ug/1. Embryo-larval tests with rainbow trout at
two levels of hardness provided 27-day LCro values of 2,030 and 1,240
ug/1. There was a 40 percent occurrence of teratogenesis after a 23-day ex-
posure of rainbow trout embryos. The equilibrium bioconcentration factor
for the bluegill was 6, which indicates that residues should not be a pro-
blem in the aquatic ecosystem.
B-2
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Only one test has been conducted with chloroform and saltwater organ-
isms. The 96-hour LC5Q for the pink shrimp was 81,500 ug/1.
CRITERIA
The available data for chloroform indicate that acute toxicity to fresh-
water aquatic life occurs at concentrations as low as 28,900 ng/1, and would
occur at lower concentrations among species that are more sensitive than the
three tested species. Twenty-seven-day LC^g values indicate that chronic
toxicity occurs at concentrations as low as 1,240 yg/1, and could occur at
lower concentrations among species or other life stages that are more
sensitive than the earliest life cycle stage of the rainbow trout.
The data base for saltwater species is limited to one test and no state-
ment can be made concerning acute and chronic toxicity.
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TabUi 1. Acuttt value* for chloroform
Spocles
LC3iO/EC!!iO
Method* (Vi
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TabI« 2. Residues for chloroform (U.S. EPA, 1978)
Species
Blueglll,
Lepomls nacrochlrus
Bloconcantratlon
Tlssut Factor
FRESHWATER SPECIES
whole body
Duration
(day*)
14
B-5
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TobU 3. Oth«r data for chloroform
Species
Rainbow trout
(embryo-larva I),
Salmo galrdnorI
Rainbow trout
(ombryo- larva I ),
SaImo ijalrdnerI
Rainbow trout (ombryo),
Sa I mo ga Irciner I
Orangespotted sunflsh,
Lepomls humlI Is
Goldfish,
Uarass I us juratus
Threesplne stickleback,
Gasterosteus aculeatus
Nlnesplne stickleback,
Pungltlus pungitlus
Duration Effect
FRESHWATER SPECIES
Result
27 days LC50 at 50 mg/l
hardness
21 days LC50 at 200 mg/l
hardness
1 hr
30-60 mln
90 mln
2,030
blrge, et al. 1979
1,240 Blrge, et al. 1979
23 days 40J teratogenes I s 10,600 Ulrge, el al. 1979
Death
50J anesthetized
Anesthesia with
recovery
Avoldance
106,890-
152,700
97,000-
167,000
146,320-
296,640*
Cloyberg, 1917
Gherkin i Catchpool,
1964
207,648» Jones, I947a
Jones, 1947b
* Corrected from vol/vol to
B-6
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REFERENCES
Bentley, R.E., et al. 1975. Acute toxicity of chloroform to bluegill
(Lepomis macrochirus), rainbow trout, (Salmo gairdneri), and pink shrimp
(Penaeus durprarum). Contract No. WA-6-99-1414-B. U.S. Environ. Prot.
Agency.
Birge, W.J., et al. 1979. Toxicity of organic chemicals to embryo-larval
stages of fish. EPA-560/11-79-007. U.S. Environ. Prot. Agency.
Gherkin, A. and J.F. Catchpool. 1964. Temperature dependence of anesthesia
in goldfish. Science. 144: 1460.
Clayberg, H.D. 1917. Effect of ether and chloroform on certain fishes.
Biol. Bull. 32: 234.
Jones, J.R.E. 1947a. The oxygen consumption of Gasterosteus aculeatus L.
to toxic solutions. Exp. Biol. 23: 298.
Jones, J.R.E. 1947b. The reactions of Pygosteus pungitius L. to toxic
solutions. Jour. Exp. Biol. 24: 110.
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. U.S. Environ. Prot. Agency, Contract No. 68-01-
4646.
B-7
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Ingestion from Water
In an 80-city study, chloroform was found in all finished
drinking water supplies produced from raw water which had been
chlorinated (Symons, et al. 1975). Chloroform usually was found in
the highest concentration among the four trihalomethanes usually
detected. In finished drinking water supplies, the respective
levels of chloroform, bromodichloromethane, dibromochloromethane,
and bromoform ranged from less than 0.1 jug/1 to 311 ug/1, undetect-
ed up to 116 iig/1, undetected up to 100 Jug/1, and undetected up to
92 ug/1. The highest concentrations of total trihalomethanes were
found in finished drinking water supplies for which surface water
was used as the source; the source water was chlorinated and the
free chlorine residual from this chlorination was greater than 0.4
mg/1. Total trihalomethane concentrations were generally related
to the organic content of the raw water when sufficient chlorine
was added to create a chlorine residual. Analysis of the raw
source waters showed only minor contributions to the chloroform
levels of the finished drinking waters, thereby inferring the pro-
duction of chloroform in the chlorination process.
In its Statement of Basis and Purpose for an Amendment to the
National Interim Primary Drinking Water Regulations for Trihalo-
methanes, 1978, the U.S. EPA (1978b) reviewed the latest data on
chloroform exposure from drinking water. Data derived from the
National Organics Monitoring Study (NOMS) (U.S. EPA, 1977) noted
C-l
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that with an average per capita consumption figure of 2 liters per
day and 100 percent body absorption of chloroform, a total chloro-
form uptake from water was estimated to be a mean value of 61
mg/year and maximum value of 343 mg/year. The corresponding NOMS
mean and maximum chloroform concentrations for drinking water were
0.083 mg/1 and 0.47 rag/1.
Additional evidence of chloroform production as a result of
chlorination practices in water renovation was provided by Bellar,
et al. (1974). Chloroform concentrations in the influent and ef-
fluent of the Cincinnati, Ohio sewage treatment plant where chlori-
nation was practiced were 9.3 ug/i and 12.1 ug/'l, respectively.
Much higher levels of chloroform have been found in wastewater
effluents and also as the result of accidental industrial spills.
Wastewater effluents from rubber and chemical companies in the
Louisville, Kentucky area have had chloroform levels as high as
22,000 ug/1 (National Academy of Sciences (NAS), 1978a). An acci-
dental spill into the Mississippi River was studied in detail by
Neely, et al. (1976); the damage involved the rupturing of two
barge tanks and the release of 1.75 million pounds (0.79 x 10 kg)
of chloroform. Numerous spills have been detected in the upper
Ohio River (Thomas, 1979), and levels of 50 ug/1 persisted for five
days in March 1978. Both of these rivers serve as raw water sources
for finished drinking water supplies, and it is obvious that these
incidences contributed abnormally high exposure of chloroform to
the human population.
C-2
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Ingestion from Food
McConnell, et al. (1975) reviewed the incidence, significance,
and movement of chlorinated hydrocarbons in the food chain. They
concluded that chloroform is widely distributed in the environment
and is present in fish, water birds, marine mammals, and various
foods.
A bioconcentration factor (BCF) relates the concentration of a
chemical in aquatic animals to the concentration in the water in
which they live. The steady-state BCFs for a lipid-soluble com-
pound in the tissues of various aquatic animals seems to be propor-
tional to the percent lipid in the tissue. Thus, the per capita
ingestion of a lipid-soluble chemical can be estimated from the per
capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and shellfish, and a steady-state BCF
for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States was analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the per capita con-
sumption of freshwater and estuarine fish and shellfish in the
United States is 6.5 g/day (Stephan, 1980). In addition, these
data were used with data on the fat content of the edible portion of
the same species to estimate that the weighted average percent
lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
A measured steady-state bioconcentration factor of 6 was ob-
tained for chloroform using bluegills (U.S. EPA, 1978a). Similar
bluegills contained an average of 4.8 percent lipids (Johnson,
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1980). An adjustment factor of 3.0/4.8 = 0.625 can be used to ad-
just the measured BCF from the 4.8 percent lipids of the bluegill
to the 3.0 percent lipids that is the weighted average for consumed
fish and shellfish. Thus, the weighted average bioconcentration
factor for chloroform and the edible portions of all freshwater and
estuarine aquatic organisms consumed by Americans is calculated to
be 6 x 0.625 = 3.75.
In food, the typical range of chloroform was 1 to 30 wg/kg.
The highest concentration noted was in Cheshire cheese, at 33
ug/kg. It was concluded that chloroform levels in food would not
be acutely toxic to humans. Pearson and McConnell (1975) also
reviewed the incidence of chlorinated hydrocarbons in various mar-
ine organisms and water birds and found that the concentrations of
chloroform in edible fish and marine organisms ranged from 3 to 180
ug/kg.
Potrepka (1976) estimated that the consumption of products
such as bread derived from chloroform-treated (as a fumigant)
grains would contribute 0.56 ug of chloroform per day to the adult
human diet. This number was derived assuming: (1) consumption of
140 g of bread per day, (2) a chloroform level of 0.4 ^ig/g in the
bread where chloroform was used as the grain fumigant, and (3)
chloroform comprises only one percent of total fumigant use in the
United States.
Inhalation
The National Academy of Sciences (NAS, 1978a) provided data on
the occurrence of six halomethanes in the air. The general back-
ground tropospheric concentration of chloroform ranged from
C-4
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9.8 x 10 to 19.6 x 10 mg/m , with higher concentrations in mar-
ine air; lower levels were normally found in continental air sam-
ples. Over urban areas, there can be higher concentrations of car-
bon tetrachloride, chloroform, and methylene chloride. Bayonne,
N.J. had the highest measured ambient air concentration of chloro-
form at 0.073 mg/m . Automobile exhausts have been implicated in
high urban area chloroform concentrations. Typically, automobile
exhausts have chloroform levels of 0.027 mg/m . The concentration
of chloroform in indoor air rarely exceeds 4.9 x 10~ mg/m .
Dermal
At one time, chloroform was administered as an anesthetic by
absorption through the skin. The American Conference of Govern-
mental Industrial Hygienists (ACGIH, 1977) has stated the potential
danger of percutaneous chloroform poisoning. Today, dermal expo-
sure is rare and is applicable to the small segment of the popula-
tion engaged in the manufacture and use of chloroform and its prod-
ucts.
PHARMACOKINETICS
Absorption
Chloroform is well absorbed via the respiratory system (49 to
77 percent). In an early study by Lehman and Hasegawa (1910),
chloroform required 80 to 100 minutes to reach equilibrium between
blood concentration and inhaled air concentration. Chloroform ab-
sorption from the gastrointestinal tract approximates 100 percent
(Fry, et al. 1972).
Inhalation studies of CHC1, in experimental animals have been
summarized by von Oettingen (1955). At an exposure level of 8,000
C-5
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ppm of CHC1.,, mice were dead within three hours, and at 12,500 ppm,
animals died within two hours. At high levels of exposure, anes-
thesia occurred within a few minutes, indicating rapid absorption
and distribution via the respiratory system. Gastrointestinal ab-
sorption was slower, but lethal tissue levels could be attained
within minutes to a few hours, depending on the dose. Fry, et al.
(1972) reported that gastrointestinal absorption approximates 100
percent.
Distr ibution
Being a lipid soluble compound, CHC1, passes readily through
cell membranes (primarily by simple diffusion) and easily reaches
the central nervous system to produce narcosis, an effect common to
most of the halogenated hydrocarbon solvents (Cornish, 1975).
Cohen and Hood (1969) demonstrated the long-term retention of CHC13
in body fat, with increased levels occurring in liver during the
post-exposure period. Thus, there is redistribution of CHC1., in
body tissues as it slowly builds up in fatty tissues during the
post-exposure period.
Metabolism
As early as 1964, Van Dyke, et al. (1964) demonstrated that
labeled CO- appeared in expired air less than an hour after an in-
14
jection of C-labeled chloroform. This amounted to 4 to 5 percent
of the total dose being exhaled as C02 over the subsequent 12 hours
and about 2 percent exhaled as other labeled metabolites. This
represents considerable metabolism of a relatively inert chemical
solvent. Other unidentified metabolites also were reported in the
urine during this early study. The chloride ion (JOC1) also has
C-6
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been found in the urine of rats after the intraperitoneal dose of
labeled CHC13.
It has been suggested that the formation of CO- results from
the degradation of CHC1., to methylene chloride (CH-Clj) and thence
to formaldehyde, formic acid, and CO- (Rubenstein and Kanics,
1964). However, the formation of CH2C12 has not been well estab-
lished.
Scholler (1970) reported that the hepatotoxicity of chloroform
was markedly enhanced by phenobarbital, a known inducer of the
mixed function oxidase (MFO) system. Conversely a decrease in
hepatotoxicity of CHCl^ occurred in animals pretreated with SKF
525-A, an inhibitor of the MFO enzyme system (Gopinath and Ford,
1975). Chloroform metabolism depleted liver glutathione, and this
depletion was stimulated by liver microsomal enzyme inducers, such
as phenobarbital (Ilett, et al. 1973). These authors also reported
that after phenobarbital treatment there was an increased biliary
excretion of labeled metabolites of CHC1, in the bile of rats.
The formation of a chemically reactive CHC1-, metabolite, which
may bind covalently to tissue macromolecules, has been reported by
several investigators (Ilett, et al. 1973; Uehleke and Werner,
1975). Covalent binding in both liver and kidney was increased
following microsomal enzyme induction. In vitro studies (Ilett, et
al. 1973) indicated that the formation of CHC1- metabolites capable
of covalent binding is NADPH-dependent and inhibited by carbon mon-
oxide. There is a suggestion that a different or additional path-
way of metabolism also may operate in the kidney, since there is a
C-7
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minimal requirement for NADPH and no requirement for 0- in the in
£ ——
vitro microsomal system.
Recent reports have shown the in vitro formation of a systemic
metabolite of chloroform (2-oxothiazolidine-4-carboxylic acid)
during incubation with liver microsomes (Pohl, et al. 1977). This
compound is readily formed by the reaction of cysteine and phos-
gene, raising again the suggestion of phosgene as an intermediate
in the metabolism of chloroform. Pohl (1979) suggested the initial
formation of unstable trichloromethanol via the cytochrome P450
system, spontaneous elimination of HC1 to yield the reactive phos-
gene which binds with cysteine and other tissue macromolecules.
The author also reported data indicating that deuterium (D)-labeled
chloroform (CDC1-,) was less toxic and less readily metabolized than
CHC1.,, suggesting that the cleavage of the C-H bond is the rate-
limiting step in the process resulting in the hepatotoxicity of
chloroform. Free radical formation also has been proposed as a
metabolic pathway of CHC1, which would lead to reactive intermedi-
ates (Smuckler, 1976; Reynolds, 1977; Royer, et al. 1978).
As a result of these studies, it is quite apparent that the
microsomal enzyme system plays an important role in the metabolism
and toxicity of chloroform. However, several pathways and interme-
diates have been proposed as the relevant ones. Additional clari-
fication at the molecular level is still necessary to determine the
operative in vivo pathways involved in the metabolism of chloroform
in animals and in man.
C-8
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Excretion
Fry, et al. (1972) studied a group of adult humans who ingest-
ed capsules containing 500 mg of C-labeled chloroform. More than
96 percent of the administered isotope was exhaled within 8 hours.
Unchanged chloroform was excreted by this route with an efficiency
of 18 to 67 percent. Less than one percent of the isotope appeared
in the urine. Those people with a higher fat content exhaled less
unchanged chloroform in the 8-hour period and presumably more C02-
A kinetic analysis of Fry's data on two people by Chiou (1975)
showed that, extrapolated to infinite time, the fraction metab-
olized to CO- is 46 percent for a male and 58 percent for a female,
and the rest is exhaled as chloroform. The half-life of chloroform
in the blood and in expired air is approximately 1.5 hours.
EFFECTS
Acute, Subacute, and Chronic Toxicity
Human exposure to chloroform may be via inhalation, ingestion,
or by cutaneous contact (Gonzales, et al. 1954; Schroeder, 1965).
The first reported case of death as a result of chloroform anes-
thesia-induced liver damage occurred in 1894 (Guthrie, 1894).
Toxic effects include local irritation (hyperemia, erythemia, mois-
ture loss) at the site of skin absorption (Malten, et al. 1968),
central nervous system depression, gastrointestinal irritation
(Challen, et al. 1958), hepatic and renal damage, and possible car-
diac sensitization to adrenalin (Fuhner, 1923; Althausen and
Thoenes, 1932; Cullen, et al. 1940).
Chloroform is considered to be moderately toxic. It is sever-
al times more potent than carbon tetrachloride as a depressant of
C-9
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the central nervous system when inhaled, but clinical experience
suggests that it is less toxic than carbon tetrachloride when taken
orally. The ingestion of 263 g has been possible, although inges-
tion of much smaller amounts has produced serious illness. The
mean lethal dose is approximately 44 g (Gosselin, et al. 1976).
The National Institute for Occupational Safety and Health
(NIOSH) Criteria Document (1974) contains a tabulation of the ef-
fects of chronic chloroform exposure on humans. One 33-year-old
male, who habitually had inhaled chloroform for 12 years, was noted
to have the psychiatric and neurologic symptoms of depression, loss
of appetite, hallucination, ataxia, and dysarthria. Other symptoms
from habitual use are moodiness, mental and physical sluggishness,
nausea, rheumatic pain, and delirium.
Most human toxicological data have resulted from the use of
chloroform as a general anesthetic in operations. Delayed chloro-
form poisoning has often occurred after delivery in obstetrical
cases. The delayed toxic effects were usually preceded by a latent
period ranging from a few hours to one day. Initially drowsiness,
restlessness, jaundice, and vomiting occurred, followed by fever,
elevated pulse rate, liver enlargement, abdominal tenderness, de-
lirium, coma, and abnormal findings in liver and kidney function
tests were also reported. Death often ensued, three to ten days
post partum. Autopsy reports generally described the liver as hav-
ing a bright yellowish color, fatty infiltration with necrosis was
found. Other hepatotoxic effects have been reviewed (NIOSH, 1974).
Numerous animal studies have shown that chloroform causes fatty
infiltration and necrosis of the liver. None of these studies
C-10
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involved long-term exposures to low concentrations. However, these
studies show that hepatotoxic effects of chloroform can occur as
the result of ingestion, inhalation, or intravenous administration
(NIOSH, 1974). Although the causes of death in most cases of
chloroform poisoning have been attributed to necrosis of the liver,
there also has been evidence at autopsy of renal damage, including
albumin and red blood cells in urine, elevated blood urea, an 18
percent decrease in prothrombin after surgery, and fatty degenera-
tion.
A case of pulmonary toxicity resulting from an intentional
intravenous injection of chloroform has been reported (Timms and
Moser, 1975). Chloroform poisoning has resulted in symptoms simi-
lar to those of marked hemolytic anemia. Chloroform has induced
hemolysis of human erythrocytes in vitro (Belifore and Zimmerman,
1970).
Malten, et al. (1968) reported that chloroform exposure ulti-
mately results in an injury to only the horny layer of skin in hu-
mans, and that the skin often responds with the formation of a
temporary protective barrier.
There have been few studies of industrial worker exposure.
Challen, et al. (1958) reported a study of workers in a confection-
ary firm in England that manufactured medicinal lozenges. In 1950,
the workers began to complain of chloroform vapor given off during
the production of the lozenges. These workers were placed on a
reduced work week to alleviate their complaints of lassitude, flat-
ulence, water brash (British term indicative of symptoms of dyspep-
sia), dry mouth, thirst, depression, irritability, and frequent and
C-ll
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"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, a system of exhaust
ventilation was installed, after which manufacturing proceeded.
without interruption.
Clinical investigations of three different groups of workers
in this manufacturing plant were performed by Challen, et al.
(1958). One group of eight employees was termed the "long service
operators." These were people who refused to continue in the loz-
enge department after they experienced the previously described
symptoms. This group of workers, when exposed to chloroform vapor
in concentrations ranging from 376 to L,158 nig/m , had been observ-
ed staggering about the work area. After terminating work in the
lozenge department, the "long service operators" reported experi-
encing nausea after even short exposures to chloroform.
A second group of nine employees, termed the "short service
operators," were the replacements for the "long service operators."
Two of these nine employees did not report unpleasant experiences
from chloroform exposure. Among the other seven, five reported
dryness of the mouth and throat at work; two were subject to lassi-
tude in the evening; one complained of lassitude and flatulence at
work; and the experiences of two others were similar to those of
the "long service operators." The "short service operators" worked
in locations where the chloroform concentrations ranged from 112 to
347 mg/m3.
A third group of five employees who worked in other depart-
ments of the firm served as controls and exhibited no symptoms.
C-12
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Tests of liver function (thymol turbidity, thymol flocculation,
direct van den Bergh, and indirect serum bilirubin), clinical exam-
inations, and urinary urobilinogen failed to show significant
differences among the three groups of workers.
Bomski, et al. (1967) reported on liver injury from chloroform
exposure among workers in a pharmaceutical factory in Poland. The
study included the entire group of 295 workers who were exposed to
chloroform in the course of production. Of these, 68 were exposed
to chloroform for 1 to 4 years and still were in contact with chlor-
oform, 39 had chloroform contact at one time, but were no longer
exposed, 23 had viral hepatitis with icterus two to three years
earlier and were designated as posticterus controls and were work-
ing in a germ-free area, and 165 worked in a germ-free area with no
history of viral hepatitis. Blood pressure, blood morphology,
urinalysis, blood albumin, serum protein, thymol turbidity, zinc
sulfate turbidity, urobilinogen, SCOT, and SGPT were measured in
all; the "Takata-Ara" sulfate (colorimetric) test was also per-
formed. A complete medical history was taken. Sixty of the people
were hospitalized for determination of BSP clearance and urinary
urobilinogen.
The air in the production room was sampled, and chloroform
concentrations were determined with the Grabowicz method. The con-
centration of chloroform ranged from 9.8 to 1,002 mg/m . No other
concentration measurements were reported, nor was there any mention
of the frequency of sampling.
The authors compared the frequency of viral hepatitis and
jaundice among a group of inhabitants of the city, 18 years of age
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and older, with that of the same 68 pharmaceutical workers who were
exposed to chloroform. The results showed that in I960, 0.35 per-
cent of city inhabitants had viral hepatitis, while 16.67 percent
of the chloroform-exposed workers had viral hepatitis. In 1961,
the frequency of viral hepatitis for city inhabitants was 0.22 per-
cent, and the frequency among the chloroform workers was 7.50 per-
cent. In 1962, the frequency of viral hepatitis was 0.38 percent
for city inhabitants and 4.4 percent for workers exposed to chloro-
form. The authors suspected that the toxic liver changes occurring
as a result of exposure to chloroform promoted a viral infection in
such cases, but they did not give information on the incidence of
viral hepatitis among the other groups of plant workers. This in-
formation might have helped resolve questions about sanitary prac-
tices and facilities in the plant.
The majority of the workers who were in contact with chloro-
form during the investigation period covered in this study com-
plained of headache, nausea, belching, and loss of appetite. Among
the 68 workers using chloroform, 19 cases of splenomegaly were
found; none was found in the controls.
The frequency of enlarged livers (17 of 68) among workers
exposed to chloroform exceeded the frequency of enlarged livers
in two of the other groups (5 of 39 and 2 of 23). Livers were
judged to be enlarged if they extended at least 1 cm beyond the rib
arch in the midclavicular line. The upper margin was apparently
not measured. In 3 of the 17 chloroform workers with enlargement
of the liver, toxic hepatitis was diagnosed on the basis of ele-
vated serum enzyme activities and elevated serum gairjna globulin,
C-14
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but the measured amounts of these serum constituents in these 3
workers were not reported. In the remaining 14 cases of liver en-
largement, fatty liver was diagnosed.
Intraperitoneally-injected chloroform can be nephrotoxic in
mice (Klaassen and Plaa, 1967a). The acute LDcn value for male
mice was 1,800 mg/kg body weight; for females, 1,900 mg/kg body
weight. Male mice demonstrated renal dysfunction at 116 mg/kg body
weight, but females did not exhibit renal dysfunction at any time
during or after exposure to even a lethal dose of chloroform.
Intragastric administration of 250 mg of chloroform/kg body
weight to rats showed gross pathological changes in both renal and
hepatic tissues (Torkelson, et al. 1976). The intragastric LD50
for chloroform was 2,000 mg/kg body weight, with most deaths occur-
ring from 2 to 4 hours.
Rats, guinea pigs, and rabbits received repeated exposure to
chloroform vapor at 85 ppm, 50 ppm, and 25 ppm (415, 244, and 122
mg/m , respectively) for 7 hours per day, 5 days per week for up to
203 days. Dogs were similarly exposed to a chloroform concentra-
tion of 122 mg/m . The results of these studies are reported in
Table 1.
The effects of chloroform on kidney and liver function in mon-
grel dogs have been reported (Klaassen and Plaa, 1967b). Male and
female dogs received intraperitoneal injections of chloroform in
corn oil. The 24-hour LD5Q was estimated to be 1,483 mg/kg body
weight using the "up and down" method of Browning (1937).
Toxic effects by dermal administration have been demonstrated
in both humans and other mammals. Torkelson, et al. (1976) reported
C-15
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TABLE 1
Effects of Chloroform Inhalation on
Four Laboratory Animal Species*
Animal
Rats :
Guinea Pigs:
Rabbits :
Rats:
Guinea Pigs:
Rabbits :
Dogs:
Concentration
06 X ^
(mg/m )
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
415
415
415
415
415
415
244
244
244
244
244
244
122
122
Effects
Pneumonia, renal
symptoms, hepatic
degradation
Renal & hepatic
pathology
No effects
Pneumonitis
Pneumoni tis ,
hepatic necrosis
Hepatic, renal
pathology
Symptoms less
severe than re-
ported at 415 mg/m
Less affected than
males
Normal
Normal
Normal
Normal
Normal
Microscopic
changes in kidney
*Source: Torkelson, et al. 1976
C-16
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on adverse effects in the rabbit. One to two 24-hour applica-
tions, by a cotton pad bandaged on the shaven belly of rabbits,
produced a slight hyperemia with moderate necrosis and a resulting
eschar formation. Healing appeared to be delayed on the site and
on abraded areas treated in the same way. Single applications as
low as 1,000 mg/kg of body weight for 24 hours under an impermeable
plastic cuff resulted in degenerative changes in the kidney tu-
bules. Chloroform dropped into the eyes of rabbits produced slight
injury which required one week to heal.
Synergism and/or Antagonism
In male rats, chloroform has been demonstrated to be hepato-
toxic. Experiments have elucidated the role of microsomal amidopy-
rine N-demethylase activity in the hepatotoxic response (Gopinath
and Ford, 1975). Pretreatment with phenobarbitone sodium or
phenylbutazone, from 1 hour to 14 days prior to single oral doses
of chloroform, induced this enzyme and potentiated the hepatotoxic
effects of chloroform, i.e., necrotized cells, up to 1,000 percent
over control values. On histological examination, degenerative
cells were also apparent. Pretreatment with sodium diethyl-
dithiocarbamate and carbon disulfide has been shown to protect
against liver damage, with no necrotized regions apparent histo-
logically.
Pretreatment with alcohols, barbiturates, and other chemicals
such as DDT increased the toxic effects of chloroform, apparently
by lowering the threshold for its necrotic action. Studies by
Ilett, et al. (1973) indicate that this synergistic effect may be
related to enhanced tissue binding.
C-17
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Kutob and Kutob (1961) found that ethanol pretreatment of mice
increased the toxic effects of chloroform on the liver. McLean
(1970) demonstrated the potentiating effects of other agents.
Phenobarbital and DDT increased the liver hydroxylating enzyme ac-
tivity, and the toxicity of chloroform was more than doubled by the
pretreatment with these chemicals as measured by the LD .
Animals on high fat or protein-poor diets are more susceptible
to hepatotoxicity from chloroform, whereas diets high in carbohy-
drates and proteins have a protective effect (von Oettingen, 1964).
Teratogenicity
In 1974, Schwetz, et al. demonstrated the effects of repeated
exposures to chloroform on rat embryos and fetal development.
Pregnant Sprague-Dawley rats were exposed to airborne chloroform at
147, 489, and 1,466 mg/m for 7 hours per day on days 6 to 15 of
gestation. Pregnant rats exposed to 489 mg/m showed a significant
increased incidence of fetal abnormalities compared with controls.
There were significantly increased incidences of acaudia (no
tails}, imperforate anus, subcutaneous edema, missing ribs, and de-
layed sternebrae ossification. Rats exposed to 147 mg/m showed
significantly increased incidences of delayed skull ossification
and wavy ribs, but exhibited no other deleterious effects compared
with controls.
Thompson, et al. (1974) reports in a range-finding study on
rats, oral doses of chloroform (126 mg/kg/day and greater) pro-
duced dose-related maternal toxicity. Doses of 316 mg/kg/day and
greater caused acute toxic nephrosis and hepatitis and death of
dams, as well as fetotox icity. Results of the Thompson, et al.
C-18
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(1974) study in the rabbit suggest this species to be more sensi-
tive to the effects of chloroform, in that oral doses of 100
mg/kg/day or higher were toxic to both the dam and fetus.
In these teratology studies, the occurrence of adverse clini-
cal effects in the females of both species and of hepatotoxicity in
the rabbit indicates that maximum tolerated doses of chloroform
were used. At levels toxic to the mother, only mild fetal toxicity
in the form of reduced birth weights was observed. Dose levels as
high as 126 mg/kg/day in the rat and 50 mg/kg/day in the rabbit were
neither embryocidal nor teratogenic.
The occurence of fetal anomalies (Schwetz, et al. 1974) following
exposure of pregnant rats to chloroform by inhalation, and the
absence of effects following oral exposure (Thompson, et al. 1974)
may be attributed to the difference in routes of administration.
Blood concentrations and tissue distribution of chloroform in ma-
ternal and fetal compartments would undoubtedly be affected by the
route and duration of maternal exposure which differed in the two
studies, i.e., continuous exposure seven hours daily in the inhala-
tion study compared with one or two short periods of exposure per
day in the oral study.
Mutagenicity
Chloroform, tested by the histidine-revertant mutation system
employing Salmonella typhimur imr» tester strains, was found to be
negative. The other trihalomethanes formed by chlorination of
drinking water were positive in such tests (Simmon, et al. 1977).
C-19
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Carcinogen!city
Eschenbrenner and Miller (1945) studied the effect of repeated
oral doses of chloroform on the induction of hepatomas in mice. A
graded series of necrotizing and nonnecrotizing doses of chloroform
was administered. Three-month-old strain A mice which had an in-
cidence of spontaneous hepatomas of less than one percent at 16
months were given intragastric doses of chloroform in olive oil
solutions at 5 ml/kg body weight. The chloroform content of the
solutions varied so that the chloroform doses were 1.6, 0.8, 0.4,
0.2, or 0.1 ml/kg, respectively (2,373, 1,187, 593, 297, 148
mg/k'g).
The presence or absence of liver necrosis was determined by
microscopic examination of liver sections taken 24 hours after
administration of a single dose of chloroform. The livers of ani-
mals receiving doses of 297 and 148 mg/kg of chloroform showed no
necrosis. However, with these doses, necrotic areas were observed
in the kidneys of males, but not of females. This sex difference of
renal necrotic lesions was observed at all concentrations. No sex
difference was observed for liver necrosis. Twenty-four hours
after a single dose of 593 mg/kg or more of chloroform, there was
extensive necrosis of liver cells around the central veins. Thirty
doses were given at four-day intervals to test for any carcinogenic
effect. (This was the schedule under which a hepatoma incidence of
100 percent was obtained when carbon tetrachloride was used).
Hepatomas were found only in animals that received necrotizing
doses of chloroform (at least 593 mg/kg) and which were killed one
month after the last dose. These were seen only in female mice,
C-20
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which could reflect the lower tolerance to chloroform for males,
i.e., the males might have died earlier of renal necrosis, before
onset of malignant changes. The authors suggested that necrosis
was a prerequisite to tumor induction.
In 1976, the National Cancer Institute (NCI) released its Re-
port on Carcinogenesis Bioassay of Chloroform. This study followed
the protocol that had been developed for testing a series of chemi-
cals by the Carcinogenesis Bioassay Program of the Division of Can-
cer Cause and Prevention. The work was carried out under contract
with the Hazelton Laboratories of America, Inc.
A Carcinogenesis bioassay of USP grade chloroform was conduct-
ed using male Osborne-Mendel rats and both male and female B6C3F,
mice. Chloroform was administered orally (by gavage) in corn oil
to 50 animals of each sex and at two dose levels 5 times per week
for 78 weeks. Rats were started on the test at 52 days of age and
killed after 111 weeks. The dose levels for males were 90 and 180
mg/kg body weight. Female rats were started at 125 and 250 mg/kg,
reduced to 90 and 180 mg/kg after 22 weeks, with an average level of
100 and 200 mg/kg for the study. A decrease in survival rate and
weight gain was evident for all treated groups. The most signifi-
cant observation (p = .0016) was kidney epithelial tumors in male
rats with incidences of: 0 percent in controls, 8 percent in the
low-dose groups, and 24 percent in the high-dose groups (Table 2).
An increase in thyroid tumors was also observed in treated female
rats, but this finding was not considered statistically signifi-
cant.
C-21
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TABLE 2
Statistically Significant Tumor Incidence in Rats*
Males
Controls
Colony Matched Low Dose High Dose
Kidney epithelial tumors/animals 0/99 0/19 90 mg/kg 4/50 180 mg/kg 12/50
(8%) (24%)
p value 0.0000 0.0016
*Source: NCI, 1976
C-22
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The epithelial tumors varied from circumscribed, well-differ-
entiated tubular-cell adenomas to highly pleomorphic, poorly dif-
ferentiated carcinomas which had invaded and metastasized. The
cells in adenomas were relatively uniform and polygonal, with abun-
dant eosinophilic cytoplasm. Nuclei were central or basal in loca-
tion, with minimal atypia and little increase in mitotic index.
Most carcinomas were very large and replaced a considerable portion
of the renal parenchyma. They were infiltrated surrounding normal
tissues and were poorly circumscribed. These cells assumed the
form of irregular sheets, nests, and tubular arrangements with
varying degrees of anaplasia and increased nuclear/cytoplasmic
ratio. The nests of cells were often surrounded by a delicate
fibrovascular stroma, and central necrosis was sometimes present in
the more anaplastic neoplasms. A papillary glandular pattern was
rarely observed.
Mice were started on test at 35 days and killed after 92 to 93
weeks. Initial dose levels were 100 and 200 mg/kg for males and 200
and 400 mg/kg for female mice. These levels were increased after
18 weeks to 150/300 and 250/500 mg/kg, respectively, so that the
average levels were 138 and 277 mg/kg for male and 238 and 477 mg/kg
for female mice. Survival rates and weight gains were comparable
for all groups except high, dose females which had a decreased sur-
vival. Highly significant increases (p = ,001) in hepatocellular
carcinoma were observed in both sexes of mice with incidences of 98
percent and 95 percent for males and females at the high dose, and
36 and 80 percent for males and females at the low dose (Table 3).
This compares with six percent in both matched and colony control
C-23
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TABLE 3
Hepatocellular Carcinoma Incidence in Mice*
Controls
Colony Matched Low Dose High Dose
Male 5/77 1/18 138 mg/kg 18/50 277 rag/kg 44/45
(6%) (6%) (36%) (98%)
Female l/80a 0/20 238 mg/kg 36/45a 477 mg/kg 39/41
(1%) (0%) (80%) (95%)
*Source: NCI, 1976
aData used for calculation of cancer risk in Criteria Formulation section of this
document.
C-24
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males, zero percent in matched control females, and one percent in
colony control females. Nodular hyperplasia of the liver was ob-
served in many low dose male mice that had not developed hepato-
cellular carcinoma.
The incidence of hepatocellular carcinoma is significantly
elevated in both sexes of mice. A high incidence of these tumors
was observed in all treated groups, and the difference was deter=
mined to be statistically significant at the p< 0.001 level. The
lesions were observed in animals which died as early as 54 weeks
following initial exposure. The increase in lesion development ob-
served is due to the occurrence of a specific type of tumor, hepa-
tocellular carcinoma. The tumors varied from those composed of
well-differentiated hepatocytes with a relatively uniform arrange-
ment, to those which were very anaplastic and poorly differentiated
with numerous mitotic figures. Various types of hepatocellular
carcinomas described in the literature were seen, including those
with an orderly cord-like arrangement of neoplastic cells, those
with a pseudoglandular pattern resembling adenocarcinoma, and those
composed of sheets of highly anaplastic cells with little tendency
to form a cord or gland-like arrangement. The diagnosis of hepato-
cellular carcinoma was based primarily on histologic character-
istics of the neoplasm. Hepatocellular carcinomas were found to
have metastasized to the lung in two low-dose males a.id two high-
dose females, and to the kidney in a high-dose male.
A search of the literature has not revealed long-term followup
studies on industrially-exposed populations. It is expected that
there would be a long latency period. A survey of plant workers who
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had been exposed for only a few years would not be expected to show
a significant increase in cancer.
When data on chloroform concentrations became available from
the U.S. EPA's surveys of drinking water, a correlation was noted
with cancer death rates for all sites in the survey (McCabe, 1975).
Fifty cities, where at least 70 percent of the population was
served by the water sampled, had chloroform concentrations measured
in 1975 that could be compared with cancer mortality in 1969-71. A
statistically significant correlation was reported between the age,
sex, and race-adjusted death rate for total cancer and chloroform
levels.
Epidemiology studies of cancer frequency for trihalomethanes,
of which chloroform is a primary chemical species, began to appear
in 1974. EPA asked a number of research groups to evaluate whether
there was a relationship between cancer rates and chloroform and
other trihalomethanes (THM) in water supplies. Most of the EPA re-
quested studies used indirect evidence of the presence of THM in
water supplies while two others used direct measurements of chloro-
form and other THM.
This type of study has been extended by Cantor, et al. (1977)
who looked at the association between each of 16 cancer rates in
whites, by sex and levels of trihalomethanes separated into Chloro-
form and nonchloroform components. Exposure information came from
the National Organics Reconnaissance Survey and the U.S. EPA Region
V Survey of 1975. Seventy-six counties, in which more than 50 per-
cent of the population was served by the sampled and assayed water
supply, were included in the study. The most consistent finding
C-26
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was an association between bladder cancer mortality rates and tri-
halomethane levels. The association was observed in both sexes and
showed a gradient of increasing degree of correlation when counties
were grouped by percentage of the county population served by the wa-
ter supply. The correlations noted were stronger with the bromi-
nated trihalomethanes than with chloroform.
Hogan, et al. (1977) used approximately the same data base and
applied various statistical procedures to the data in order to de-
termine the appropriateness of the statistical model. The results
were similar to previous studies showing positive correlation be-
tween rectal-intestinal and bladder cancer mortality rates and
chloroform levels in drinking water, when a weighted regression
analysis was applied.
Given the number of existing epidemiology studies, the EPA
asked the National Research Council to review the studies. The
National Academy of Sciences (NAS, 1978b) provided such a review of
10 epidemiology studies, including the ones previously mentioned.
It is useful to quote from their summary and conclusions:
The studies that the subcommittee reviewed were divided
into two groups: those in which nonspecific measures of
exposures to putative carcinogens in water (e.g., the use
of surface water vs. ground water) were examined and
those in which water quality was characterized by mea-
surements of trihalomethane (THM) concentrations. The
subcommittee gave greater weight to the conclusions of
the latter group of studies because crude measures of
exposure, which lead to the comparisons of cancer between
surface water users and ground water users, must be of
limited value. They do not permit the quantitation of
exposure to contaminants in water consumed, which is
needed to determine dose-response relationships between
THM concentrations and cancer frequencies and to estimate
the effects of reducing THM concentrations.
C-27
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The conclusions drawn in the second group of studies, in
which many cancer sites were examined, suggest that high-
er concentrations of THMs in drinking water may be
associated with an increased frequency of cancer of the
bladder. The results do not establish causality, and the
quantitative estimates of. increased or decreased risk are
extremely crude. The effects of certain potentially
important confounding factors, such as cigarette smok-
ing, have not been determined.
C-28
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CRITERION FORMULATION
Existing Guidelines and Standards
The Occupational Safety and Health Administration (OSHA) limit
for chloroform in work place air is 50 ppm, or 244 mg/m . This is a
"ceiling value" for a maximum 10-minute exposure that at no time
should be exceeded. NIOSH recommended a criterion of 10 ppm (48.9
mg/m ) in 1974. This criterion was applied to a time-weighted ex-
posure for as high as 10 hours per day and a 40-hour work week.
Following the National Cancer Institute (NCI) study of chloroform,
NIOSH on June 9, 1976, reduced this allowable time-weighted average
exposure criterion to 2 ppm (9.8 mg/m ).
Based on available health information, a safe level of air-
borne exposure to halogenated agents could not be defined. Since a
safe level of occupational exposure to halogenated anesthetic
agents could not be established by either animal or human invest-
igations, NIOSH recommended that airborne exposure be limited to
levels no greater than the lowest level detectable using the recom-
mended sampling and analysis techniques (NIOSH, 1977). At the pre-
sent, chloroform is not usually used as an anesthetic; this use is
included in the criterion and is limited to 2 ppm or 9.8 mg/m .
If a procedure that converts this air limit to a water limit
is employed, the equivalent exposure in water would be 34.9 mg/1
(Stokinger and Woodward, 1958). In this method, complete absorp-
tion from inhalation and ingestion is assumed. The inhalation
absorption may be closer to 50 percent and the equivalent water
exposure would then be 17.4 mg/1. The occupational limits apply to
the healthy working-age population, and even then if exposure level
C-29
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is half the limit, comprehensive medical surveillance is required.
To use the occupational limit as a guide for the general popula-
tion, an application factor of 100 can be used. Thus, an equi-
valent level in water would be 174 jug/1. It must be remembered that
the occupational limit for chloroform is based on the lowest level
detectable in air using NIOSH recommended analytical techniques and
does not necessarily represent a level adequate to protect man.
In general, the use of inhalation data assumes an 8-hour day,
time-weighted average, occupational exposure in the working place
with workers inhaling the toxic substance throughout such a period.
Exposures for the general population should be considerably less.
Such worker-exposure inhalation standards are inappropriate for the
general population since they presume an exposure limited to an
eight-hour day, an age bracket of the population that excludes the
very young and the very old, and a healthy worker prior to expo-
sure. Ingestion data are far superior to inhalation data when the
risks associated with the food and water of the aquatic environment
are being considered.
Following the NCI study of chloroform,- the Food and Drug Ad-
ministration took action to halt the use of chloroform in drug pro-
ducts, cosmetic products, and food contact materials (41 FR 15026,
15029). The EPA has issued a notice of "rebuttable presumption"
against continued registration of chloroform-containi .g pesticides
(41 FR 14588).
The EPA has also proposed an amendment which would add to the
National Interim Primary Drinking Water Regulations a section
on the control of organic halogenated chemical contaminants in
C-30
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drinking water (43 FR 5756). The proposed limit for total trihalo-
methanes, which includes chloroform, is 100 pg/1. This limit was
set largely on the basis of technological and economic feasibility.
Originally the limit will apply only to water supplies serving
greater than 75,000 consumers; this is intended to provide an or-
derly upgrading of drinking water treatment in the country. The
basis and purpose of the regulation are discussed in a paper by the
Office of Drinking Water issued in January, 1978 (U.S. EPA, 1978b).
This document contains a number of estimates of cancer risk attri-
butable to the presence of chloroform in drinking water. One of
these, performed by NAS, using a linear non-threshold extrapolation
from animal data, estimated that the lifetime risk would fall be-
tween 1.5 X 10"7 and 17 X 10~7 ug/CHC!3/l of water consumed daily
depending upon the data set employed. The upper 95 percent confi-
dence estimates would range between 3 X 10~ and 22 X 10~
iig/1/day.
Current Levels of Exposure
The National Academy of Science (NAS, 1978a) assembled data
based on human exposure to chloroform. Their calculations of human
uptake are based on fluid intake, respiratory volume, and food con-
sumption data for "reference man" as compiled by the International
Commission for Radiological Protection. Table 4 from the NAS re-
port is reproduced to show their estimates of chloroform uptake
from fluids. Table 5 presents the data on relative human uptake
from the three sources.
According to the NAS report the uptake of chloroform from the
atmosphere at minimum levels of exposure is about 10 times greater
C-31
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TABLE 4
Chloroform Uptake From Fluids (mg/yr) Assuming 100 Percent Absorption8'
Exposure
Minimum
Concentration
Exposure
(0.0001 mg/1)
Median
Concentration
Exposure
(0.021 mg/1)
Max imuro
Concentration
Exposure
(0.366 mg/1)
Fluid
Tap
Water
Other1
Total
Fluid
Tap
Water
Other11
Total
Fluid
Tap
water
Other1
Total
Fluid
Adult
Mln.
Fluid
Intake
0.016
0.012
0.037
3.44
2.45
7.57
60.0
42.7
134
Man
Max.
Fluid
Intake
0.027
0.053
0.088
5.59
11.1
18.4
97.5
194
321
Refer .
Man
Intake
0.005
0.055
0.071
1.15
11.5
14.9
20.1
200
261
Adult
Min.
Fluid
intake
0.016
0.012
0.037
3.44
2.45
7.67
60.0
42.7
134
Woman
Max.
Fluid
Intake
0.027
0.053
0.086
5.59
11.1
18.4
97.5
194
321
Refer.
Man
Intake
0.004
0.040
0.051
0.77
8.43
10.7
13.4
147
187
Child
(5-14 Yr) (10 Yr)
Min. Max. Refer.
Fluid Fluid Man
Intake Intake Intake
0.007
0.020 0.029
0.027
0.036 0.061 0.051
1.53
4.14 6.06
5.75
7.67 12.8 10.7
26.7
72.1 106 100
134 223 187
aCalculated by multiplying the exposure concentration (ug/1) x fluid intake (1/yr) for minimum and
maximum Intakes, and dividing by 1000 ng/mg - mg/yr.
Includes water based drinks, such as tea, coffee, soft drinks, beer, cider, wine.
*Source: NAS, 1978a
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TABLE 5
Relative Human Uptake of Carbon Tetrachloride (CC1.) and
Chloroform (CHC1-J from Environmental Sources (rag/year)*
At Minimum Exposure Levels3
Source
Fluid Intake
Atmosphere
Food Supply
Total
Adult
cci4
0.73
3.60
0.21
4.54
Man
CHC13
0.037
0.41
0.21
0.66
Adult
cci4
0.73
3.30
0.21
4.24
Woman
CHC13
0.037
0.37
0.21
0.62
cci4
0.73
2.40
0.21
3.34
Child
CHC13
0.036
0.27
0.21
0.52
At Typical Exposure Levels
Source
Fluid Intake
Atmosphere
Food Supply
Adult
cci4
1.78
4.80
1.12
Man
CHC13
14.90
5.20
2.17
Adult
CC1.
4
1.28
4.40
1.12
Woman
CHC13
10.70
4.70
2.17
cci4
1.28
3.20
1.12
Child
CHC13
10.70
3.40
2.17
Total
7.70
22.27
6.80
17.57
5.60
16.27
C-33
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TABL3 5 (Continued)
Source
Adult Man
CC1, CHC1.
At Maximum Exposure Levels
Adult Woman
Fluid Intake
Atmosphere
Food Supply
Total
629
811
CC1.
CHC1
578
771
Child
CC1
CHC1.
4.05
618
7.33
321
474
16.4
4.05
567
7.33
321
434
16.4
1.83
405
7. 33
223
310
16.4
414
549
*Source: NAS , 1978a
(a)
(b)
(c)
Minimum conditions of all variables assumed: Minimum exposure-minimum in-
take for fluids; minimum exposure-minimum absorption for atmosphere; and
minimum exposure-minimum intake for food supplies.
Typical conditions of all variables assumed. For CC1.: 0.0025 mg/1-refer-
ence man intake for fluids; average of typical minimum and maximum absorp-
tion for atmosphere; and average exposure and intake for food supplies. For
median exposure-reference man intake for fluids; average of typical
and maximum absorption for atmosphere; and average exposure and in-
take for food supplies.
minimum
Maximum conditions of all variables assumed: maximum exposure intake for
fluids; maximum exposure-maximum absorption for atmosphere; and maximum ex-
posure-maximum intake for food supplies.
C-34
-------
than from fluids. At maximum exposure levels, the chloroform up-
take from fluids is slightly less than that from the atmosphere.
At typical exposure levels, however, the human uptake from fluids
is 2 to 3 times greater than from the atmosphere, with slight vari-
ation by sex and age noted.
In its Statement of Basis and Purpose for an Amendment to the
National Interim Primary Drinking Water Regulations on Trihalo-
methanes, the U.S. EPA (1978b) estimated the total human exposure
to chloroform (Table 6). The estimates have basic assumptions that
are comparable to those used by the NAS (1978a), are based on newer
values for chloroform content in drinking water from NOMS data, and
provide estimates for human adults only.
The two exposure estimates, NAS (1978a) and the U.S. EPA
(1978b) demonstrate that chloroform intake from ingesting water is
likely to range from a modest to predominant percentage of total
exposure with a simple minimum, mean and maximum exposure scenario.
% total exposure % total exposure
from water from water
(U.S. EPA) (NAS)
Minimum Exposure 23% 6%
Mean Exposure 69% 67%
Maximum Exposure 61% 40%
Basis and Derivation of Criteria
Chloroform has several adverse effects on the human body.
Safe levels of chloroform in water necessary to avoid some of these
effects would be difficult to establish because adequate studies
have not been conducted. The most serious effect to consider is
C-35
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TABLE 6
Uptake of Chloroform for the Adult Human
from Air, Water, and Food*
Source
Atmosphere
Water
Food Supply
Total
Atmosphere
Water
Food Supply
Total
Atmosphere
Water
Food Supply
Total
Adult
mg/yr
Maximum Conditions
204
343
16
563
Minimum Conditions
0.41
0.73
2.00
3.14
Mean Conditions
20.0
64.0
9.00
93
Percent
uptake
36
61
3
100.00
13
23
64
100.00
22
69
10
101.00
*Source: U.S. EPA, 1978b
C-36
-------
the cancer-causing potential of the chemical. Current knowledge
leads to the conclusion that carcinogenesis is a non-threshold,
nonreversible process. The non-threshold concept implies that many
tumors will be produced at high doses, but any dose, no matter how
small, will have the probability of causing cancer. Even small
carcinogenic risks have a serious impact on society when the ex-
posed population is large, because it is likely that some cancers
will be caused by chloroform. The nonreversible concept implies
that once the tumor growth process has started, growth will con-
tinue and may metastasize and involve other organs until death
ensues.
Chloroform has been shown to induce cancer in two species of
experimental animals. This conclusion is neither confirmed nor
denied by the results of numerous epidemiology studies now avail-
able, although from a public health point of view, a suspicion of a
qualitative weight of evidence for confirmation probably exists.
The available information on total human exposure to chloro-
form from air, water, and food sources suggests that drinking water
contributes from 6 to 69 percent of the total exposure. Studies in
which water quality was characterized by measurements of THM con-
centrations suggest that higher trihalomethanes (TKM) concentra-
tions in drinking water may be associated with an increased fre-
quency of cancer of the bladder. The results do not establish
causality, and the estimates of increased or decreased risk are
extremely crude.
It is therefore proposed that the total risk for carcinogenic
response be allocated to the ambient water exposure conditions of
C-37
-------
ingesting 2 liters/day of water and consuming 6.5 grams of poten-
tially contaminated fish products.
Under the Consent Decree in NRDC v. Train, criteria are to
state "recommended maximum permissible concentrations (including
where appropriate, zero) consistent with the protection of aquatic
organisms, human health, and recreational activities." Chloroform
is suspected of being a human carcinogen. Because there is no
recognized safe concentration for a human carcinogen, the recom-
mended concentration of chloroform in water for maximum protection
of human health is zero.
Because attaining a zero concentration level may be infeasible
in some cases, and in order to assist the Agency and states in the
possible future development of water quality regulations, the con-
centrations of chloroform corresponding to several incremental
lifetime cancer risk levels have been estimated. A cancer risk
level provides an estimate of the additional incidence of cancer
that may be expected in nn exposed population. A risk of 10 , for
example, indicates a probability of one additional case of cancer
for every 100,000 people exposed; a risk of 10~ indicates one addi-
tional case of cancer for every million people exposed, and so
forth.
In the Federal Register notice of availability of draft am-
bient water quality criteria, EPA stated that it is considering
— 5 — fi
setting criteria at an interim target risk level of 10 ,10 , or
10~ , as shown in the following table.
C-38
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Exposure Assumptions Risk Levels
(per day) and Corresponding Criteria (1)
1£"7 _10"6 1£~5
2 liters of drinking 0.019 ug/1 0.19 ug/1 1.90 pg/l
water and consumption
of 6.5 grams of
fish and shellfish (2)
Consumption of fish 1.57 ug/i 15.7 ug/1 157 ug/i
and shellfish only.
(1) Calculated by applying a linearized multistage model, as dis-
cussed in the Human Health Methodology Appendices to the
October 1980 Federal Register notice which announced the
availability of this document, to the animal bioassay data
presented in Appendix I and in Table 3. Since the extrapola-
tion model is linear at low doses, the additional lifetime
risk is directly proportional to the water concentration.
Therefore, water concentrations corresponding to other risk
levels can be derived by multiplying or dividing one of the
risk levels and corresponding water concentrations shown in
the table by factors such as 10, 100, 1,000, and so forth.
(2) Approximately 1 percent of the chloroform exposure results
from the consumption of aquatic organisms which exhibit an
average bioconcentration potential of 3.75-fold. The remain-
ing 99 percent of chloroform exposure results from drinking
water.
Concentration levels were derived by assuming a lifetime expo-
sure to various amounts of chloroform: (1) occurring from the con-
sumption of both drinking water and aquatic life grown in waters
containing the corresponding chloroform concentrations; and (2) oc-
curring solely from consumption of aquatic life grown in the waters
C-39
-------
containing the corresponding chloroform concentrations. Although
total exposure information for chloroform is discussed and an esti-
mate of the contributions from other sources of exposure can be
made, these data will not be factored into ambient water quality
criteria formulation until additional analysis can be made. The
criteria presented, therefore, assume an incremental risk from amb-
ient water exposure only.
C-40
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C-45
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APPENDIX I
Derivation of Criterion for Chloroform
The NCI (1976) bioassay with female mice given a time-weighted
average dose of chloroform at 238 or 477 mg/kg by stomach tube 5
times per week for 78 weeks is used to derive the water quality cri-
terion. The treatment induced hepatocellular carcinomas in the
tested animals and controls as outlined in the table below. Assum-
ing that the fish bioaccumulation factor is 3.75, the parameters of
the extrapolation model are:
Dose Incidence
(mg/kg/day) (no. responding/no, tested)
0 0/20
238 x 5/7 = 170 36/45
477 x 5/7 = 341 39/41
le = 546 days w = 0.030 kg
Le = 644 days R = 3.75 I/kg
L = 644 days
With these parameters the carcinogenic potency for humans,
q *, is 0.18272 (mg/kg/day ). The result is that the water con-
centration should be less than 1.90 ug/1 in order to keep the indi-
vidual lifetime risk below 10~ .
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