CHLOROFORM
Interim 3: 03/2009
CHLOROFORM
(CAS Reg. No. 67-66-3)
PROPOSED ACUTE EXPOSURE GUIDELINE LEVELS (AEGLs)
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PREFACE
Under the authority of the Federal Advisory Committee Act (FACA) P. L. 92-463 of
1972, the National Advisory Committee for Acute Exposure Guideline Levels for Hazardous
Substances (NAC/AEGL Committee) has been established to identify, review and interpret
relevant toxicologic and other scientific data and develop AEGLs for high priority, acutely toxic
chemicals.
AEGLs represent threshold exposure limits for the general public and are applicable to
emergency exposure periods ranging from 10 minutes to 8 hours. Three levels — AEGL-1,
AEGL-2 and AEGL-3 — are developed for each of five exposure periods (10 and 30 minutes, 1
hour, 4 hours, and 8 hours) and are distinguished by varying degrees of severity of toxic effects.
The three AEGLs are defined as follows:
AEGL-1 is the airborne concentration (expressed as parts per million or milligrams per
cubic meter [ppm or mg/m3]) of a substance above which it is predicted that the general
population, including susceptible individuals, could experience notable discomfort, irritation, or
certain asymptomatic, non-sensory effects. However, the effects are not disabling and are
transient and reversible upon cessation of exposure.
AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a substance above
which it is predicted that the general population, including susceptible individuals, could
experience irreversible or other serious, long-lasting adverse health effects or an impaired ability
to escape.
AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a substance above
which it is predicted that the general population, including susceptible individuals, could
experience life-threatening health effects or death.
Airborne concentrations below the AEGL-1 represent exposure levels that could produce
mild and progressively increasing but transient and nondisabling odor, taste, and sensory
irritation or certain asymptomatic, non-sensory effects. With increasing airborne concentrations
above each AEGL, there is a progressive increase in the likelihood of occurrence and the
severity of effects described for each corresponding AEGL. Although the AEGL values
represent threshold levels for the general public, including susceptible subpopulations, such as
infants, children, the elderly, persons with asthma, and those with other illnesses, it is recognized
that individuals, subject to unique or idiosyncratic responses, could experience the effects
described at concentrations below the corresponding AEGL.
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TABLE OF CONTENTS
PREFACE 1
EXECUTIVE SUMMARY 5
LIST OF TABLES 3
LIST OF APPENDICES 3
1. INTRODUCTION 8
2. HUMAN TOXICITY DATA 9
2.1 Acute Lethality 9
2.2 Nonlethal Toxicity 9
2.2.1 Epidemiologic Studies 10
2.3 Reproductive/Developmental Toxicity 11
2.4 Genotoxicity 12
2.5 Carcinogenicity 12
2.6 Summary 12
3. ANIMAL TOXICITY DATA 14
3.1 Acute Lethality 14
3.1.1 Rats 14
3.1.2 Mice 17
3.1.3 Dogs 17
3.1.4 Summary of Lethal Toxicity in Animals 17
3.2 Nonlethal Toxicity 18
3.2.1 Rats 18
3.2.2 Mice 20
3.2.3 Dogs 22
3.2.4 Cats 22
3.2.5 Summary of Nonlethal Toxicity in Animals 23
3.3 Developmental/Reproductive Toxicity 25
3.3.1 Rats 25
3.3.2 Mice 28
3.4 Genotoxicity 29
3.5 Carcinogenicity 29
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4. SPECIAL CONSIDERATIONS 32
4.1 Metabolism and Disposition 32
4.2 Mechanism of Toxicity 33
4.3 Structure-Activity Relationships 35
4.4 Other Relevant Information 35
4.4.1 Species Variability 35
4.4.2 Concurrent Exposure Issues 35
5. DATA ANALYSIS I OR AEGL-1 35
5.1 Summary of Human Data Relevant to AEGL-1 35
5.2 Summary of Animal Data Relevant to AEGL-1 36
5.3 Derivation of AEGL-1 36
6. DATA ANALYSIS FOR AEGL-2 37
6.1 Summary of Human Data Relevant to AEGL-2 37
6.2 Summary of Animal Data Relevant to AEGL-2 37
6.3 Derivation of AEGL-2 37
7. DATA ANALYSIS FOR AEGL-3 38
7.1 Summary of Human Data Relevant to AEGL-3 38
7.2 Summary of Animal Data Relevant to AEGL-3 39
7.3 Derivation of AEGL-3 39
8. SUMMARY OF AEGLS 40
8.1 AEGL Values and Toxicity Endpoints 40
8.2 Comparison with Other Standards and Guidelines 41
8.3 Data Quality and Research Needs 43
9. REFERENCES CITED 4
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LIST OF TABLES
TABLE 1. Physicochemical Data for Chloroform 8
TABLE 2. Nonlethal Toxicity of Chloroform in Humans Following Acute
Inhalation Exposure 13
TABLE 3. Lethal Toxicity of Chloroform in Laboratory Species Following
Acute Inhalation Exposure 18
TABLE 4. Nonlethal Toxic Effects of Chloroform in Laboratory Species Following
Acute Inhalation Exposure 24
TABLE 5. Embryotoxicity and Fetotoxicity of Chloroform in Rats Following
Gestational Exposure 25
TABLE 6. Litter data from Wistar rats exposed to chloroform on gestation days 7-16
(Baeder and Hoffman, 1988 27
TABLE 7. Litter data from Wistar rats exposed to chloroform on gestation days 7-16
(Baeder and Hoffman, 1991) 27
TABLE 8. Skeletal/ossification variations in Wistar rats exposed to chloroform on
gestation days 7-16 (Baeder and Hoffman, 1991) 28
TABLE 9. Developmental Toxicity of Chloroform in Mice Exposed During
Gestation (Baeder and Hoffman, 1991) 29
TABLE 10. AEGL-1 Values for Chloroform 36
TABLE 11 AEGL-2 Values for Chloroform 38
TABLE 12. AEGL-3 Values for Chloroform 40
TABLE 13 Proposed AEGL Values for Chloroform 40
TABLE 14 Extant Standards and Guidelines for Chloroform 42
APPENDIX A (Derivation of AEGL Values) 51
APPENDIX B (Derivation Summaries for Chloroform) 57
APPENDIX C (Carcinogenicity Assessment for Chloroform) 63
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SUMMARY
Chloroform is a volatile liquid with a pleasant, nonirritating odor. The chemical is miscible with
organic solvents but only slightly soluble in water. Although previously used as an anesthetic
and in pharmaceutical preparations, these uses are no longer allowed in the U.S. The chemical is,
however, produced and imported in large quantities for use in chemical syntheses, as a solvent,
and in the manufacture of some plastics.
Human data regarding acute inhalation exposure to chloroform are limited to older studies
involving the exposure of human subjects to various exposure regimens (3-30 minutes and 680-
7200 ppm) and resulting in effects ranging from detection of strong odor, headaches and
dizziness, to vertigo. Analyses of published reports of surgical patients anesthetized with
chloroform, although lacking precise exposure terms, suggested that such exposures (generally
in excess of 13,000 ppm) may produce cardiac arrhythmias and transient hepatic and renal
toxicity. Quantitative data regarding human fatalities following acute inhalation exposure to
chloroform are not currently available.
Only limited data are available pertaining to lethality in animals following acute exposure to
chloroform. Definitive quantitative data are limited to a 4- hr LC50 of 9780 ppm in rats and a 7-
hr LC50 of 5687 ppm in mice. Remaining data indicate notable lethality following exposures
ranging from 5 minutes ("saturated" concentration probably equivalent to approximately 25,000
ppm) to 12-hour exposure to 726 ppm. Data regarding the nonlethal toxicity in animals focus on
biochemical (elevated serum enzyme activity indicative of hepatic damage) and histopathologic
indices of hepatic toxicity in laboratory species. Data regarding reproductive/developmental
toxicity in animals are equivocal. One study provided evidence of fetotoxicity in rats following
gestational exposure to chloroform at 30 ppm although another study found no evidence of such
toxicity following gestational exposures to 2232 ppm.
Although chloroform has been shown to be tumorigenic in rats (kidney tumors in male but not
female rats) and mice (hepatocarcinomas in male and female mice) following oral exposure,
there are no inhalation exposure studies demonstrating carcinogenic responses to chloroform.
Currently available data on the mechanism of chloroform toxicity and tumorigenicity imply that
the tumorigenic response occurs following chloroform exposures great enough to cause cell
death and proliferative cellular regeneration. As such a linear low-dose extrapolation for cancer
risk may not be appropriate. For this reason and because the inhalation slope factor for
chloroform is based upon effects following oral administration, the AEGL values for chloroform
are based upon noncarcinogenic endpoints.
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Metabolism and disposition studies have affirmed that metabolism of chloroform to phosgene is
mediated by P-450IIE1 and that phosgene along with depletion of reduced glutathione and
formation of trichlorocarbon-radical intermediates are responsible for the toxicity of chloroform.
Data from several studies indicate that the metabolism and, therefore, the rate of production of
reactive metabolites is greater in rodents than in humans.
AEGL-1 values were not recommended. Based upon the available data, attempts to identify a
critical effect consistent with the AEGL-1 definition were considered tenuous and uncertain.
Exposures of humans to concentrations approaching those inducing narcosis or possibly causing
hepatic and renal effects are not accompanied by overt signs or symptoms. Furthermore, the
odor of chloroform is not unpleasant or irritating.
The AEGL-2 values for chloroform were based upon fetotoxicity and embryolethality in rats
(Schwetz et al., 1974) resulting from exposure of dams to 100 ppm, 7 hours/day on gestation
days 6-15. For AEGL-2 development, an assumption was made that the effects could be caused
by only a single 7-hr exposure. Because available data on metabolism and kinetics indicate that
rodents are more sensitive than humans to the toxic effects of chloroform, an interspecies
variability uncertainty factor was not applied. An intraspecies uncertainty factor of 3 was
applied to account for variability in metabolism and disposition among individuals and to protect
more susceptible individuals (e.g., P-450 induction by alcohol use or exposure to other inducers
of P-450 monooxygenase). No additional reduction in the AEGL-2 values was warranted
because the critical effect and the assumption of a single-exposure scenario provided a
conservative point of departure. For AEGL development, data were unavailable for empirically
deriving a chemical-specific time scaling relationship (Cn x t = k). The concentration-exposure
time relationship for many irritant and systemically acting vapors and gases may be described by
C" xt = k, where the exponent n ranges from 0.8 to 3.5 (ten Berge et al., 1986). In the absence
of data with which to empirically derive a chemical-specific scaling exponent (//), temporal
scaling was performed using n = 3 when extrapolating to shorter exposure durations or n = 1
when extrapolating to longer exposure durations.
The AEGL-3 values for chloroform were based upon a 560-minute mouse LCtso of 4500 ppm.
Because data were unavailable for quantitatively estimating a lethality threshold, the LC50 was
reduced 3-fold to 1500 ppm, an exposure level unlikely to cause lethality based upon
comparisons to other available human and animal exposure data. Uncertainty factor application
was limited to 3 for protection of sensitive individuals. As in AEGL-2 derivations, the
intraspecies uncertainty factor of 3 was selected because it is unlikely that induction of
metabolism would increase toxic effects by an order of magnitude. Available data indicate that
rodents metabolize chloroform at a greater rate than do humans resulting in production of
reactive, toxic intermediates at a greater rate. Results of PBPK model studies have shown that
rodents, especially mice, are considerably more susceptible to the lethal effects of chloroform
than are human. Therefore, the AEGL-3 values were increased three-fold by a weight-of-
evidence adjustment factor of 1/3. This adjustment is further justified by surgical anesthesia data
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showing cumulative exposures of >675,000 ppm-minute and exposures to 22,500 ppm for up to
120 minutes resulted in surgical anesthesia and cardiac irregularities but not death. Time scaling
was performed using an n of 3 to extrapolate from the 560-minute exposure duration of the
point-of-departure to the shorter AEGL- time periods. To minimize uncertainties of
extrapolating from the 560-minute experimental exposure period to the 10-minute AEGL-3
period, the 30-minute AEGL-3 value of 4000 ppm was adopted for the 10-minute period as well.
Assessments of carcinogenic potential following single, acute exposure to chloroform indicated
that AEGL-2 values based upon noncancer toxicity endpoints were slightly greater than those
based on cancer risk. However, the carcinogenic response to chloroform appears to be a
function of necrosis and subsequent regenerative cellular proliferation that are not relevant to a
single acute exposure.
TABLE 13. Pro
posed AEGL Values for Chloroform (ppm [mg/m3])
Classific
ation
10-min
30-min
1-hour
4-hour
8-hour
Endpoint (Reference)
AEGL-1
NR
NR
NR
NR
NR
Not recommended; data
insufficient to develop
AEGL-1 values; AEGL-1
effects unlikely to occur in
the absence of notable
toxicity.
AEGL-2
120 ppm
580
mg/m3
80 ppm
390
mg/m3
64 ppm
312 mg/m3
40 ppm
195 mg/m3
29 ppm
141 mg/m3
Fetotoxicity/embryo-lethality
in rats exposed for 7 hrs/day
on gestation days 6-15
(Schwetz et al., 1974); single
exposure assumed
AEGL-3
4000 ppm
[19,000
mg/m3]
4000 ppm
[19,000
mg/m3]
3200 ppm
[16,000
mg/m3]
2000 ppm
[9,700
mg/m3]
1600 ppm
[7,800
mg/m3]
Estimated lethality threshold
for mice; 3-fold reduction
560-min LC50 of 4500 ppm to
1500 ppm (Gehring, 1968)
References
Gehring, P.J. 1968. Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to
their narcotic and lethal potencies in mice. Toxicol. Appl. Pharmacol. 13: 287-298.
Schwetz, B.A., Leong, B.K.J., Gehring, P.J. 1974. Embryo- and fetotoxicity of inhaled
chloroform in rats. Toxicol. Appl. Pharmacol. 28: 442-451.
ten Berge, W.F., Zwart, A., Appelman, L.M. 1986. Concentration-time mortality response
relationship of irritant and systemically acting vapours and gases. J. Hazard. Materials. 13: 301-
309.
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1. INTRODUCTION
Chloroform is a volatile liquid with a pleasant, nonirritating odor. The chemical is miscible
with organic solvents but only slightly soluble in water. Although previously used as an
anesthetic and in pharmaceutical preparations, these uses are no longer allowed in the U.S.
Chloroform is produced and imported in large quantities (-93-350 million pounds/year) and
used in chemical syntheses, for refrigeration, as a solvent, and in the manufacture of
polytetrafuoroethylene plastics (DeShon, 1978; Li et al., 1993). Chloroform is also a byproduct
of wood pulp chlorination for production of paper products. Physicochemical data for
chloroform are shown in Table 1.
The AIHA (AIHA, 1989) reported an odor threshold of 192 ppm based upon a geometric
mean of acceptable values (133-276 ppm). An odor detection of 6.1 ppm was reported by the
U.S. EPA(USEPA, 1992a).
TABLE 1. Physicochemical Data for Chloroform
Parameter
Value
Reference
Synonyms
trichloromethane, methenyl
chloride, methyl trichloride
DeShon, 1978
CAS Registry No.
67-66-3
Budavari etal., 1996
Chemical formula
CHC13
Budavari etal., 1996
Molecular weight
119.39
Budavari etal., 1996
Physical state
liquid
Budavari etal., 1996
Vapor pressure
159.6 mmHg@20°C
DeShon, 1978
Density
1.484 @20°C
Budavari etal., 1996
Boiling/melting point
61-62°C/-63.5°C
Budavari etal., 1996
Solubility
1 ml/200 ml water @ 20 °C
Budavari etal., 1996
Conversion factors in air
1 ppm =4.87 mg/m3
1 mg/m3 = 0.21 ppm
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2. HUMAN TOXICITY DATA
2.1 Acute Lethality
Quantitative data regarding acute inhalation exposures to chloroform resulting in death were
not available.
2.2 Nonlethal Toxicity
Several reports are available regarding the effects of acute inhalation exposure of humans to
chloroform and serve to qualitatively characterize the health effects of chloroform inhalation.
Hutchens and Kiing (1985) reported nausea, appetite loss, transitory jaundice, cardiac
arrhythmias, arterial hypotension, mild intravascular hemolysis, and unconsciousness in an
individual following intentional, nonsuicidal inhalation of chloroform.
Lehmann and Hasegawa (1910) conducted controlled exposure studies on human subjects.
The results of this study showed that a 3-minute exposure to 920 ppm induced vertigo and
dizziness and a 30-minute exposure to 680 ppm produced moderately strong odor. A 30-minute
exposure to 1400 ppm produced lightheadedness, giddiness, lassitude, and headache while
exposure to 3000 ppm resulted in gagging and pounding of the heart. Twenty-minute exposure
at 4300 to 5100 ppm or 15 minute exposure at 7200 ppm produced light intoxication and
dizziness. These data appeared to be derived from exposure of only three subjects and methods
of exposure generation and measurements are unavailable. The signs and symptoms of exposure
described in this report appear to be consistent with early stages of narcosis.
Lehmann and Flury (1943) reported that exposure of humans to 389 ppm for 30 minutes is
tolerated without complaint but that exposure to 1030 ppm resulted in dizziness, intracranial
pressure, and nausea within 7 minutes and headache that persisted for several hours.
Whitaker and Jones (1965) analyzed the clinical effects of chloroform anesthesia from 1502
surgery patients. Although the duration of anesthesia varied from <30 minutes to over two
hours, the chloroform concentration never exceeded 2.25% (22,500 ppm). For most (1164 of
1502) of the cases, anesthesia was for less than 30 minutes. Clinical observations regarding the
chloroform anesthesia included tachypnea, bradycardia, cardiac arrhythmias, hypotension, one
case of transient jaundice, and one death (this was complicated by renal insufficiency and could
not necessarily be attributed to the chloroform anesthesia). Although the maximum chloroform
concentration was provided, the exposure time required to attain anesthesia was not stated. It
could be assumed; however, that onset of anesthesia likely occurred within a few minutes. These
observations do, however, demonstrate that a short exposure to 22,500 ppm will induce a
surgical plane of anesthesia concurrent with various physiologic responses.
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The clinical effects associated with chloroform-induced anesthesia were also studied by
Smith et al. (1973) in an attempt to justify the resurrection of chloroform as an accepted
anesthetic agent. However, the utility of data from this study for AEGL development are
compromised by confounders including premedication with diazepam and pentobarbital or with
hydroxyzine and pentobarbital. The inspired chloroform concentration appeared to vary between
0.85 (8,500 ppm) and 1.3% (13,000 ppm) and the average duration of anesthesia was
112.0+60.38 minutes among the 58 surgical patients. Upon recovery, 46% of the patients
receiving chloroform experienced nausea and vomiting. Clinical assessment of liver function
and toxicity indicated transient alterations. One ventricular tachycardia occurred that
necessitated pharmacologic correction. Data from a single patient indicated that chloroform at
8500 ppm would induce anesthesia.
McDonald and Vire (1992) examined the possible health hazards associated with chloroform
use in endodontic procedures. Two industrial hygiene monitors sampled air in the treatment
operatory and additional sampling devices were attached to the dentist and the dental assistant.
The operatory area samples measured <0.57 ppm for a 5.5-hour period and the individual
breathing air samples (dentist and assistant) measured <0.88 ppm over a 150-minute period.
Health screening tests for the dentist and assistant revealed no signs of liver, kidney or lung
damage five hours post exposure or at one year after the study.
Although specific data were not presented, Snyder and Andrews (1996) report that humans
may tolerate up to 400 ppm chloroform for 30 minutes without complaint but may experience
dizziness and gastrointestinal upset at 1000 ppm for seven minutes, and narcosis following
exposure to 14,000 ppm (no duration specified).
2.2.1 Epidemiologic Studies
Several epidemiologic studies have been conducted regarding occupational exposure to
chloroform. These studies involve worker populations exposed to the chemical for periods of
time in excess of what would be considered acute exposure, and are not directly applicable to
developing AEGL values. They do, however, provide some insight regarding the relationship
between proposed AEGL values and the health effects that may be associated with long-term
exposures.
Challen et al. (1958) evaluated workers in a pharmaceutical manufacturing process that
involved exposure to chloroform vapor. Data regarding exposure terms are limited to eight
"long- service operators" (3 to 10-year exposures) exposed to 77 to 237 ppm, nine employees
termed "short-service operators" (10 to 24-month exposures) who were replacements for the long
service operators and were exposed to 23 to 71 ppm, and a group of five controls who were not
exposed to processes involving chloroform. All workers in these groups were women whose
ages ranged from 34 to 60 years. Some "long-service operators" had been observed staggering
about the work area. All "long-service" workers experienced alimentary effects (e.g., nausea,
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flatulence, thirst), increased micturition and urinary discomfort, and behavioral effects
(depression, irritability, poor concentration ability, motor deficiencies) during employment. All
experienced nausea and stomach upset upon smelling chloroform after leaving their
employment. Two of nine "short service operators" reported no effects from chloroform
exposure, five reported dryness of the mouth and throat while at work, two had similar
experiences as the "long service operators", and several reported lassitude.
Bomski et al. (1967) reported the results of a study on workers in a Polish pharmaceutical
factory with a special emphasis on examining chloroform-induced susceptibility to viral
infection. Chloroform exposures were determined to be 2 to 205 ppm although frequency of
sampling was not provided. The authors found that the incidence of viral hepatitis was greater in
chloroform-exposed workers than in non-exposed inhabitants of the city and postulated that
chloroform-induced hepatic damage may have predisposed the workers to the viral infection.
Increased incidences of spleen and liver enlargement were also found in the chloroform-exposed
workers.
Li et al., (1993) conducted surveys of chloroform-producing facilities in Shanghai, China.
Most of the workers exposed to chloroform were associated with production of perspex
(polymethylmethacrylate) and chemical synthesis. In the three facilities sampled (where no
effective preventive equipment or measures were in place), chloroform concentrations ranged
from 4.27 to 147.91 mg/m3 (0.88 to 31.06 ppm) with a geometric mean of 21.38 mg/m3 (4.49
ppm) for 119 samples. Chloroform concentrations were <20 mg/m3 (4.20 ppm) in 45.5% of the
119 samples. Exposure groups were classified as Exposure I (13.49 mg/m3 [2.83 ppm]; 1-15
years exposure) and Exposure 2 (29.51 mg/m3 [6.20 ppm]; 1-15 years exposure). The exposure
groups and control group (no obvious chloroform or other hazardous exposures) included males
and females as well as smokers and nonsmokers; all groups had an average age of approximately
36 years. The investigators concluded that long-term exposure to chloroform at 29.51 mg/m3
(6.20 ppm) resulted in functional liver damage as determined by changes in various serum
enzymes (ALT [alanine aminotransferase], gamaglutamyltransferase, and adenosine deaminase),
prealbumin levels, serum transferrin, and blood urea nitrogen.
2.3 Reproductive/Developmental Toxicity
Wennborg et al. (2000) conducted a study in a cohort of Swedish women who had worked in
laboratory or non-laboratory jobs for one or more years during 1990-1994. The investigators
obtained data from questionnaires to 763 women (66 were excluded for various reasons) that
assessed reproductive history, health status, time-to-pregnancy, personal habits, specific work,
and exposure to various agents and specific time at which these exposures occurred. The data
from these women were compared to respective birth information from the Swedish Medical
Register. Parameters examined included spontaneous abortion (SAB), birth weight, preterm
delivery, small-for-gestation age (SGA), large-for-gestation age, and congenital deformities. A
number of confounding variables were considered (high blood pressure, smoking, gynecological
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and chronic disease, sexually transmitted infectious diseases, father's work and potential
exposures during time of conception, previous abortions, etc.). Information regarding
consumption of alcohol, teas, and coffee, and stress levels was not included. The analysis
included 869 pregnancies but did not involve specific exposure concentrations, and did not
account for exposures to other chemicals. There was no association between laboratory work
and SABs. A weak association was found between women who had worked with chloroform
prior to conception and SABs but there was no significant association between chloroform
exposure and SGA or body weight.
2.4 Genotoxicity
No studies were located in the available literature regarding the genotoxicity of chloroform
in humans.
2.5 Carcinogenicity
Although epidemiology studies have been conducted to assess the carcinogenic potential of
chloroform in drinking water, no inhalation studies are available regarding the carcinogenic
potential of chloroform in humans following inhalation exposure. The U.S. EPA (1992b) has
developed an inhalation slope factor of 6.1 x 10"3 (mg/kg/day)"1 based upon an increased
incidence of renal tumors in male rats following long-term exposure to chloroform in drinking
water (Jorgenson et al., 1985). Route-to-route extrapolation was required for its derivation as
inhalation exposure data were not available.
2.6 Summary
Quantitative data regarding human lethality following acute exposure to chloroform are
unavailable. Although lacking quantitative exposure terms and often pertaining to oral
exposures, clinical reports affirm the hepatotoxicity and renal toxicity of chloroform as well as
the neurological effects. The available data on nonlethal responses indicate that acute inhalation
of chloroform may result in narcosis and may be preceded by signs and symptoms characteristic
of early stages of anesthesia. Early reports in which the effects of chloroform inhalation were
observed in human subjects are limited by uncertainties in the measurements of exposure
concentrations but do provide information regarding the human experience that does not appear
to be inconsistent with other data. A summary of data relevant to acute nonlethal exposure of
humans to chloroform is presented in Table 2.
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TABLE 2. Nonlethal Effects of Chloroform in Humans Following Acute Inhalation Exposure
No.of subjects
Exposure
concentration
(ppm)
Exposure
duration (min)
Effect
Reference
3
920
3
vertigo
Lehmann and
Hasegawa,1910
3
680
30
strong odor
Lehmann and
Hasegawa,1910
3
1400
30
light headedness, lassitude,
headache
Lehmann and
Hasegawa,1910
3
3000
30
pounding heart, gagging
Lehmann and
Hasegawa,1910
NA
4300-5100
20
intoxication, dizziness
Lehmann and
Hasegawa,1910
NA
7200
15
intoxication, dizziness
Lehmann and
Hasegawa,1910
NA
389
30
no complaints
Lehmann and Flury,
1943
NA
1030
7
dizziness, intracranial
pressure, nausea, persistent
headache
Lehmann and Flury,
1943
1502
22,500
<30 - >120
(most <30)
surgical plane anesthesia,
cardiac irregularities
Whitaker and Jones,
1965
58
8500-13,000
113
(mean duration)
surgical plane anesthesia
Smith et al., 1973
2
<0.5
330
no effects*
McDonald and Vire,
1992
2
<0.88
150
no effects*
McDonald and Vire,
1992
* Health screening conducted at 5 hours postexposure and at one year after exposure
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3. ANIMAL TOXICITY DATA
3.1 Lethal Toxicity
3.1.1 Rats
Results of preliminary range-finding experiments for a large number of chemicals were
reported by Smyth et al. (1962). Concentrated chloroform vapor (presumably a saturated
concentration [-25,000 ppm] but no quantitative data provided) killed all six of the albino rats
(strain not specified) exposed for five minutes. A four-hour exposure to 8000 ppm (nominal
concentration; no analytical determination) killed five or six albino rats.
The results of 4-hour inhalation study in rats were briefly described in report to E. I. du Pont
de Nemours and Co. (Haskell Laboratory, 1964). The study, designed to assess the toxicity of
Freon TC® and Freon-113®, also included experiments with chloroform (a component of Freon
TC®). For the experiments with chloroform, four rats (gender and strain not specified) were
exposed to chloroform at concentrations of 5000, 3700, or 3000 ppm for 4 hours. Four rats
exposed to clean air served as controls. For the 5000, 3700, and 3000-ppm exposures, mortality
was 3 of 4, 3 of 4, and 0 of 4. Deaths occurred at 2 to 3 days postexposure; the four rats in the
3000-ppm group were terminated at 14 days postexposure. No information was provided
regarding the methods for measurement of chloroform concentrations (atmosphere produced by
heating chloroform and injection into the chamber via a nebulizer); only nominal exposure
concentrations were reported. There were no histopathology data provided for the chloroform-
treated rats.
In experiments to assess the effect of chloroform inhalation on barbiturate metabolism and
narcosis, Puri et al. (1971) exposed male Sprague-Dawley rats to 726 ppm chloroform for up to
48 hours (continuous exposure). Although the study focused on the effect of chloroform on
barbiturate activity and metabolism, one group of rats was exposed to chloroform alone. Based
upon the graphic presentation of the data, continuous 12-hour exposure resulted in at least 10
deaths. It is unclear if any deaths occurred prior to the 12-hour data point.
Lundberg et al. (1986) reported a 4-hour LC50 of 47,702 mg/m3 (9780 ppm) for groups of ten
female Sprague-Dawley rats exposed to a geometric series of chloroform concentrations
(specific exposure concentrations for the series were not provided but stated as being equivalent
to equivalent to 1/2, 1/4, 1/8, 1/16, or 1/32 of the LC50 or the saturation concentration. Mortality
was determined at 24 hours after exposure. The exposure concentrations were measured by
infrared detection in a suitably designed apparatus.
3.1.2 Mice
The results of studies with mice exposed to chloroform were reported by Fiihner (1923).
Groups of mice (sex and strain not reported; 30 mice total) were exposed to various
concentrations of chloroform (12 to 38 mg/L or -2458 to 7782 ppm). The mice were exposed
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individually in 10-liter bottles in which chloroform was vaporized to achieve the desired
concentration. Concentrations were not determined analytically. Five mice exposed to 2458 to
5120 ppm exhibited reflex loss at 48 to 215 minutes of exposure but there were no deaths.
Exposure to 4710 to 5529 ppm resulted in reflex loss at 30 to 90 minutes with recovery of 12 of
18 animals and the death of six. Deaths occurred at 71 to 175 minutes of exposure. Out of seven
mice exposed to 6758 to 7782 ppm, six mice exhibited reflex loss at 13 to 46 minutes of
exposure and one mouse died after a 35-minute exposure (reflex loss occurred at 8 minutes).
The absence of validated exposure concentrations limits the quantitative validity of these data.
Four additional mice were exposed to 5585 ppm for 120 or 135 minutes. For the three mice
exposed for 120 minutes, death occurred at 105, 130, and 140 minutes after the start of exposure,
and the one mouse exposed for 135 minutes died 95 minutes after exposure. Under the
conditions of these experiments, the findings suggest that exposure concentrations in the vicinity
of 4710 ppm may represent a lethal threshold for mice following 1 to 2-hour exposure.
A 7-hour LC50 of 5687 ppm for mice was reported by von Oettingen et al. (1949). These
experiments used 20 adult white mice (strain and gender not specified) exposed to chloroform in
a bell jar. The chloroform concentrations were calculated based upon the amount of test material
volatilized over time and the volume of air passed through the chamber. The concentrations
were also determined by chemical analysis. Analysis of the graphic representation of the
experimental results indicated an LC30 of 5529 ppm and an LC90 of 6963 ppm. At the
concentrations tested (4915 ppm to 7372 ppm), the mice exhibited progressive central nervous
system depression followed by rapidly occurring narcosis. Death of the mice started occurring at
3 to 5 hours of exposure.
In a study by Deringer et al. (1953), the nephrotoxic and lethal effects of inhaled chloroform
were examined using male and female C3H mice. In this study, three groups of six male and six
female mice (2 months old) were exposed for 1, 2, or 3 hours to chloroform at concentrations of
3.38 to 5.4 mg/L (693 to 1,106 ppm). Additionally, three groups (six males and six females per
group) of 8-month old mice were also exposed similarly. Twenty two male and 20 female mice
served as untreated controls. Mice were observed daily for deaths or morbidity, and were
examined weekly for tumors or other abnormal conditions. Necropsies were performed on all
moribund or dead mice and any female mice exhibiting mammary tumors. Regardless of the
exposure duration or specific concentration (693 to 1106 ppm), all of the male mice (except one)
exposed to chloroform exhibited evidence of kidney damage. Within 11 das following the
exposure, 15 of 18 eight-month old males and 7 of 18 two-month old males had died. The
remainder of the 8-month old males survived 5 to 7 months and the remainder of the 2-month old
male mice survived 14 to 18 months. Generally, deaths occurred earlier in the mice exposed for
2 or 3 hours than in those exposed for only 1 hour; specific data, however, were not provided.
Histologic findings in mice that died included necrosis and calcification of the proximal and
distal convoluted tubules. Necrosis appeared to be more severe with earlier deaths.
Additionally, hepatic necrosis was also observed in mice exposed to the higher end of the
concentration range (i.e., 942 to 1106 ppm) that died within six days. For male mice surviving
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longer and in all female mice, hepatic damage was not notable. Based upon the various
chloroform concentrations and exposure durations reported, the results of this study show that 3-
hour exposure of male C3H male mice to chloroform at a concentration as low as 692 ppm or 1-
hour exposure to a concentration as low as 921 ppm resulted in severe renal damage and death.
The influence of sex hormone status on gender-specific chloroform-induced nephrotoxicity
in mice was studied by Culliford and Hewitt (1957). Although the primary objective of the study
was to verify the influence of androgens on chloroform-induced nephrotoxicity, the initial results
of the study provided evidence of nearly complete tubular necrosis in two strains of male mice
following 2-hour inhalation exposures. Male WH (Westminster Hospital in-house, uniform
heterozygous) mice exposed to 3.3 to 7.0 mg/L (676 to 1434 ppm) and male CBA mice exposed
to 1.2 to 5 mg/L (246 to 1024 ppm) all exhibited complete tubular necrosis 24 hours following
the exposure. Female mice of these strains did not exhibit any evidence of renal damage. The
study went on to show that administration of estrogen to male mice abolished the susceptibility
to the nephrotoxic response, and that the administration of testosterone to female mice increased
susceptibility. The chloroform concentrations were calculated based upon the amount of
chloroform added to the 6-L exposure chamber, and the assumption of complete vaporization at
80°F and uniform dispersal. No analytical measurements were made, thereby imparting some
uncertainty regarding the actual chamber concentrations.
In studying the hepatotoxicity of chlorinated hydrocarbons, Gehring (1968) calculated a
4500-ppm LCtso of 560 minutes (540 -585 minutes, 95% C.I.) for female Swiss-Webster mice as
well a 4500-ppm ECt50 of 35 minutes (31.0 - 39.6 minutes, 95% C.I.) for narcosis, and a 4500-
ppm ECtso of 2.3 minutes (1.9 - 2.8 minutes, 95% C.I.) for elevated serum glutamic pyruvic
transaminase (SGPT) activity. Groups of mice (10/group for narcosis determination and
20/group for lethality determination) were exposed to 4500 ppm chloroform and the number of
responders for the endpoint of concern noted relative to exposure duration. The control group
consisted of 254 mice representing a composite group of controls for all of the chlorinated
hydrocarbons tested. Chloroform concentrations were attained by metering the chloroform into a
heated tube for vaporization. Actual concentrations were measured by continuous flow of the
atmosphere through an infrared spectrophotometric cell. If the chloroform concentration varied
by more than 7% the experiment was repeated. Mortality responses to 4500-ppm chloroform
ranged from approximately 5% at 400 minutes exposure duration to 80% at 700 minutes
exposure duration. An exposure-response for narcosis was also determined and was shown to
exhibit the same slope. These data suggest that, at an exposure of 4500 ppm, there is
approximately a 16-fold difference between the time-to-narcosis (35 minutes) and the time-to-
death (560 minutes) for mice exposed under the conditions of this study. Elevation of SGPT was
also reported and exhibited a notably different exposure-response relationship (see Section
3.2.2).
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3.1.3 Dogs
The effect of chloroform-induced anesthesia in dogs was studied by Whipple and Sperry
(1909). Details regarding the exposure concentrations are limited to notation of the amount of
chloroform (in ounces) used on each dog. Anesthesia duration varied from 1.5 to 2.5 hours and
chloroform amounts varied from <1 to 3 ounces. Some of the dogs died although it was not
possible to ascertain a definitive dose response relationship from the data.
In addition to studies with mice, von Oettingen et al. (1949) also studied the effects of
chloroform (exposure to 15,000 ppm nominal; 14,376 ppm determined) on dogs (10 beagles,
gender not specified) that had been surgically prepared with a tracheal cannula, and carotid and
femoral artery cannulae to which pressure transducers were attached. Following recovery from
the pentothal-induced surgical anesthesia (beginning of voluntary movement and "lively" reflex)
, the dogs were exposed continuously to the chloroform. The average survival time was 202
minutes with extremes of 60 and 285 minutes.
3.1.4 Summary of Lethal Toxicity In Animals
The lethality of inhaled chloroform in laboratory species is summarized in Table 3. With the
exception of the rat 4-hour LC50 value (9780 ppm) reported by Lundberg et al. (1986) and the
mouse LCtso (4500 ppm; 560 minutes) reported by Gehring (1968), the data tend to be of a more
qualitative nature. Data from older studies lack details regarding generation and measurement of
exposure atmospheres. The available data do not present a clear delineation of the lethality of
acute inhalation exposure to chloroform.
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TABLE 3. Lethal Toxicity of Chloroform in Laboratory Species Following Acute Inhalation Exposure
Species
Exposure
concentration
(ppm)
Exposure
duration (min)
Effect
Reference
Rat
9780
240
4-hr LCsot
Lundberg et al., 1986
Rat
3000
240
100% mortality
Haskell Laboratory, 1964
Rat
3700
240
75% mortality*
Haskell Laboratory, 1964
Rat
5000
240
75% mortality*
Haskell Laboratory, 1964
Rat
8000
240
80% mortality
Smyth etal., 1962
Rat
"saturated conc."
5
100% mortality
Smyth etal., 1962
Rat
726
720
lethality (no
specifics provided)
Purietal., 1971
Mouse
5529
420
7-hr LC30
von Oettingen et al., 1949
Mouse
5687
420
7-hrLCso
von Oettingen et al., 1949
Mouse
6963
420
7-hr LC90
von Oettingen et al., 1949
Mouse
4,710-5,529
71-175
66% mortality
Fiihner, 1923
Mouse
6,758-7,782
35
14% mortality
Fiihner, 1923
Mouse
2,458-5,120
48-215
no deaths
Fiihner, 1923
Mouse
5,585
120
75% mortality}:
Fiihner, 1923
Mouse
4500
560 min
50% lethality
(LCtso)
Gehring (1968)
t Mortality at 24 hours postexposure
* Deaths determined at 2-3 days postexposure
t Deaths occurred at 105-140 minutes after exposure
3.2 Nonlethal Toxicity
3.2.1 Rats
In experiments reported by Brown et al. (1974b), groups of 3-9 male Sprague-Dawley rats
were used to assess the effect of P-450 induction by phenobarbital on chloroform-induced effects
on reduced glutathione (GSH). Both induced and control rats were exposed for two hours to
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chloroform at concentrations of 0.5% (5000 ppm) or 1.0% (10,000 ppm). Although control rats
(non-induced) exhibited no decrease in GSH levels, rats with induced P-450 exhibited an
approximately 70% and 83% decrease in GSH for the 0.5% and 1.0% chloroform treatment
groups, respectively. The absence of GSH decrease in the control rats exposed to chloroform
suggests that at these exposures GSH levels are more than sufficient for scavenging reactive
intermediates of chloroform metabolism.
Brondeau et al. (1983) examined the effect of a 4-hour inhalation exposure of rats on serum
enzyme activities (GLDH- glutamate dehydrogenase; GOT - glutamic oxaloacetic transaminase;
GPT - glutamic pyruvic transaminase; SDH- sorbitol dehydrogenase). In this study, groups of
eight male Sprague-Dawley rats were exposed by whole-body inhalation to chloroform at
concentrations of 137, 292, 400, 618, 942, or 1,075 ppm. A control group consisted of eight rats
exposed to clean air. Chamber atmospheres were analyzed by gas chromatography (sample loop
compared to a known concentration standard) and by analysis of a solid absorbent (activated
charcoal or silica gel subjected to appropriate solvent extraction and gas-liquid chromatography).
Exposure to the lowest concentration failed to significantly alter the activity levels of any the
tested enzymes. The 4-hour exposure to chloroform, even at the highest concentration, resulted
only in <2-fold to 7-fold increase in serum enzyme activities. Statistically significant elevations
in GLDH and SDH were observed in rats exposed to 292 ppm for four hours. GLDH and SDH
appeared to be most affected, although none of the changes in activity levels demonstrated a
definitive exposure-response relationship. Although some of the increases (especially for GLDH
and SDH) were statistically significant p<0.05 and 0.02), the toxicological relevance of these
changes is uncertain above and beyond being biological indicators of exposure. A second phase
of the study exposed rats to 301 ppm (the concentration selected by the investigators as a
threshold for alteration of serum enzyme activity based upon the single 4-hour experiments) for
two 6-hour or four 6-hour exposures. GLDH and SDH activities exhibited somewhat greater
increases following the four 6-hour exposures than the single 4-hour or two 4-hour exposures.
Statistically significant increases in serum SDH activity were also reported by Lundberg et
al. (1986) for female Sprague-Dawley rats exposed for four hours to chloroform concentrations
as low as 153 ppm (1/64 of the LC50 for chloroform as determined by Lundberg et al.).
Although useful as biomarkers of exposure, the elevation of serum enzyme activity in the
absence of clinical correlates would be of limited use as an endpoint for AEGL derivation.
In experiments to study the interaction of carbon tetrachloride and chloroform in ethanol-
treated rats, Ikatsu and Nakajima (1992) presented data for groups of four rats exposed in a
dynamic airflow chamber to chloroform-only controls (0, 50, or 100 ppm for 8 hours).
Chloroform concentrations were monitored every 15 to 30 minutes by gas chromatography.
Hepatotoxicity was determined by assessing changes in serum glutamic oxaloacetic transaminase
(SGOT), serum glutamic pyruvate transaminase (SGPT), liver malondialdehyde (MDA) and
plasma MDA. Only marginal and statistically insignificant changes were detected for these
indices following 8-hour exposure to 50 or 100 ppm chloroform, thereby indicating that the 8-
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hour exposure of rats to 50 or 100 ppm chloroform was without appreciable effect.
Histopathologic examination revealed only negligible fat deposits in the liver of rats treated with
100 ppm chloroform. These findings are consistent with those of Brondeau et al. (1983) in the
previously described study. In rats pretreated with ethanol (2 g ethanol/80 ml liquid diet fed
daily for six weeks), only SGOT levels were increased significantly (3-fold; p<0.05) following
exposure to 50 ppm chloroform. Exposure of ethanol-treated rats to 100 ppm chloroform,
however, resulted in significant (p<0.05) increases in SGOT (7.5-fold) and SGPT (14-fold).
There was no effect on liver MDA or plasma MDA. In ethanol-treated rats, ballooned
hepatocytes in midzonal areas were observed but only in the high-dose (100 ppm) chloroform
group. The results indicate that 8-hour exposure of rats to 50 or 100 ppm chloroform produce
only minor effects that are indicative of indices of exposure rather than outright toxicity.
Ethanol pretreatment followed by 8-hour exposure to 100 ppm chloroform produced notable
signs of toxicity as determined by serum enzyme and histopathologic evaluations.
The hepatotoxicity and renal toxicity of inhaled chloroform was studied in male F-344 rats
(five animals per group) following a 7-day exposure to 1, 3, 10, 30, 100, or 300 ppm chloroform
for 6 hrs/day (Larson et al., 1994). The effects on nasopharyngeal tissue were also examined
after the 7-day exposure (Mery et al., 1994). Cage-side observations indicated no observable
signs of toxicity during the exposure period although there was a significant dose-dependent
decrease in body weight gain at 10 ppm and above. Mild centrilobular vacuolation was observed
only in the 300 ppm group and histopathologic changes (hyperplasia) were observed in the 10-
ppm and above groups at the end of the 7-day exposure period. Two-treatment-related lesions
were observed in the nasal region of the chloroform-exposed rats. An increase in the size of
goblet cells of the nasopharyngeal meatus was observed in rats exposed to 100 or 300 ppm.
Also, region-specific changes were observed in the olfactory mucosa and bone of the ethmoid
turbinates of rat exposed to chloroform at or above 10 ppm. Although not providing data
appropriate for derivation of AEGL values, the results of this study may be used to evaluate the
protectiveness of proposed AEGL values.
In studies to assess the impact of ethanol on the metabolism and toxicity of chloroform by
various routes of administration, Wang et al. (1994) provided nonlethal effects data for male
Wistar rats exposed by inhalation to chloroform alone (50, 100, or 500 ppm for six hours).
Indices of hepatotoxicity (GOT, GPT, and GSH) were evaluated in groups of five rats. Rats in
the 50- and 100-ppm chloroform-only groups exhibited no significant changes in any serum
enzyme activities. Both GOT and GPT were significantly (p<0.05) elevated following the 6-
hour exposure to 500 ppm (about 1.6-fold and 1.2-fold, respectively). Such increases, although
numerically significant, are not indicative of severe hepatotoxicity. Ethanol pretreatment
increased these enzyme activities approximately two-fold above that of chloroform alone, and
failed to alter the GSH levels.
3.2.2 Mice
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In addition to providing limited qualitative data on the lethality of mice exposed to
chloroform, Fiihner (1923) provided similar data regarding nonlethal responses of mice exposed
to chloroform. Mice exposed to 2,458 to 5,120 ppm exhibited reflex loss at 48 to 215 minutes of
exposure but there were no deaths. Exposure to 4,710 to 5,529 ppm resulted in reflex loss at 30
to 90 minutes with recovery of 12 of 18 animals and the death of six. Deaths occurred at 71 to
175 minutes of exposure. For mice exposed to 6,758 to 7,782 ppm, six mice exhibited reflex
loss at 13 to 46 minutes of exposure and one mouse died after a 35-minute exposure (reflex loss
occurred at 8 minutes). The absence of analytical measurement of exposure concentrations
limits the quantitative validity of these data.
Kylin et al. (1963) reported on the hepatotoxicity of a single inhalation exposure of mice to
chloroform. A pilot study to determine the time to maximum elevation of serum ornithine
carbamyl transferase (OCT) was conducted using groups of five female albino mice exposed to
chloroform (3000 ppm) for four hours with a group being terminated at 1, 2, 4, 8, or 16 days
after the exposure. In the main study, groups of 10 female albino mice were exposed for four
hours to 100, 200, 400, or 800 ppm chloroform. Controls were exposed similarly but without
chloroform in the chamber. Histopathologic exam of the liver and measurement of serum OCT
were used as indices of effect at 24 and 72 hours after the single exposure. The chloroform was
vaporized prior to injection into the constant-flow chamber. No information was provided
regarding the measurement of test material concentration in the chamber. In the pilot study,
maximum serum OCT elevations were observed at four days postexposure. In the main study,
fatty infiltration of the liver was observed at one day following a single exposure to 100 ppm
chloroform. At higher exposure concentrations, the extent and severity of the fatty degeneration
was increased. The authors concluded that the minimal chloroform concentration to produce
fatty infiltration of the liver of mice after a 4-hour inhalation exposure was <100 ppm.
Histologic changes (fatty infiltration and necrosis) also appeared to be greater at 24 hours after
exposure than at 72 hours after exposure.
Gehring (1968), in addition to examining indices of lethality, determined 4500-ppm ECt
values for narcosis and for significant elevation of SGPT in female Swiss-Webster mice. Groups
of mice (10/group for narcosis determination and 8-10/group for SGPT determination) were
exposed to 4500 ppm chloroform and the response rate noted relative to exposure duration. The
control group consisted of 254 mice representing a composite group of controls for all of the
chlorinated hydrocarbons tested. SGPT elevations greater than 54 Reitman-Frankel units were
considered as statistically significant (control values were 24.4+14.7 R-F units). Chloroform
concentrations were attained by metering the chloroform into a heated tube for vaporization.
Actual concentrations were measured by continuous flow of the atmosphere through an infrared
spectrophotometric cell. If the chloroform concentration varied by more than 7% the experiment
was repeated. The 4500-ppm ECt50 for narcosis was 35 minutes (31.0- 39.6 minutes, 95% C.I.)
with 10% response occurring at 15 minutes and 80% response occurring at approximately 40
minutes of exposure. The 4500-ppm ECtso for significant elevation of SGPT was 13.5 min (10.1
-18.1 minutes, 95% C.I.). A 20% response was observed at about 6 minutes duration and a 90%
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response as early as 20 minutes of exposure. The exposure-response relationship for SGPT
elevation was notably different than that observed for narcosis and lethality. The authors noted
that elevation of SGPT activity occurred much earlier than narcosis or lethality and, therefore,
that chloroform was inducing liver damage prior to the onset of narcosis.
The hepatotoxicity and renal toxicity of inhaled chloroform was studied in female B6C3Fi
mice (five animals per group) following a 7-day exposure to 1, 3, 10, 30, 100, or 300 ppm
chloroform for 6 hrs/day (Larson et al., 1994). The effects on nasopharyngeal tissue were also
examined after the 7-day exposure (Mery et al., 1994). Centrilobular hepatocyte necrosis and
severe vacuolation in centrilobular hepatocytes were observed in mice of the 100-ppm and 300-
ppm groups. Mild to moderate vacuolar changes were observed in the 10-ppm and 30-ppm
groups. Notable renal toxicity was observed only in the 300-ppm group. Histologic changes in
the nasal region of the female mice were limited to increased cell proliferation at 10 ppm and
above and a slight indication of new bone growth in the endoturbinate of one mouse in the 300-
ppm group. In a later report (Larson et al., 1996), however, it was noted that the nasal lesions
induced in female mice following 6 hr/day exposures to chloroform (10, 30, or 90 ppm) were
transient and not sustained in mice similarly exposed for up to 13 weeks.
3.2.3 Dogs
Renal toxicity in dogs following inhalation exposure to chloroform was reported by Whipple
and Sperry (1909). Details of experimental protocol and were limited and lacked definitive
exposure terms. The report provided only qualitative information regarding the clinical signs
(vomiting, diarrhea, lassitude) of animals subjected to the chloroform treatment. Additionally,
gross pathology and histopathology evidence of hepatotoxicity and renal toxicity was reported
for dogs on successive days after inhaling 1-2 oz of chloroform over 1-2 hours.
von Oettingen et al. (1949) described alterations in various physiologic functions in dogs
surgically prepared for monitoring of respiration and blood pressure (see Section 3.1.3).
Although the continuous exposure to 15,000 ppm ultimately resulted in the deaths of all ten dogs
(6-285 minutes), it was reported that the dogs exhibited notable cardiovascular responses
(decreased arterial blood pressure), decreased respiratory rate and body temperature, and
depression of voluntary and involuntary reflexes within 35 minutes. Although it is uncertain if
discontinuation of the exposure at or below 35 minutes would have prevented a fatal response,
the data serve to provide a qualitative description of the response of this species to very high
concentrations.
3.2.4 Cats
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Nonlethal effects of acute exposure to chloroform in cats was reported by Lehmann and
Schmidt-Kehl (1936). In this study, adult cats were exposed to chloroform concentrations of
7200 to 22,000 ppm. The chloroform concentrations were determined by chemical reaction
(hydrolysis with alkali in alcohol). At 7500 ppm the cats exhibited light narcosis at 78 minutes
and deep narcosis after 93 minutes. Light and deep narcosis were induced after 10 minutes and
13 minutes, respectively for exposures to 22,000 ppm. This exposure also reportedly produced
mucous membrane irritation in the eyes, mouth and nose.
3.2.5 Summary of Nonlethal Toxicity In Animals
The nonlethal toxicity of chloroform in laboratory species (rats, mice, and cats) following
acute inhalation exposure is summarized in Table 4. As would be expected of a known
hepatotoxicant, many of the nonlethal effects reported for inhalation exposures of laboratory
species focused on indices of liver damage. Acute exposures (1 to 4 hours) to chloroform
concentrations of 100 to 292 ppm have resulted in some degree of hepatic injury as determined
by elevated serum enzyme activities and histopathologic examination. Without histopathologic
correlates, however, marginal elevations (although statistically significant) in serum enzyme
activities may not be indicative of a serious toxic response. Renal toxicity has also been
demonstrated in mice at exposures that are relatively low (e.g., 246-665 ppm for 2 hours or 693
ppm for 1 hour) compared to those inducing narcosis (e.g., 4500 ppm for 35 minutes). Data in
cats are limited to high, narcosis-inducing exposures.
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TABLE 4. Nonlethal Effects of Chloroform in Laboratory Species Following Acute Inhalation Exposure
Species
Exposure
concentration
(ppm)
Exposure
duration
Effect
Reference
Rat
500
6 hrs
statistically significant elevation
in serum enzyme activity
Wang et al.,1994
Rat
10
6 hrs/day for 7
days
histopathologic changes in the
liver
Larson etal. 1994
Rat
50
8 hrs
no increase in liver weight
Ikatsu and Nakajima, 1992
Rat
100
8 hrs
marginal, biologically
insignificant increase in serum
enzyme activity
Ikatsu and Nakajima, 1992
Rat
153
4 hrs
elevated serum enzyme activity
Lundberg et al., 1986
Rat
292
4 hrs
elevated serum enzyme activity
Brondeau et al. (1983)
Rat
10,000
2 hrs
no effect on hepatic GSHa
Brown etal., 1974b
Mouse
2,458-5,120
48 min
reflex loss
Fiihner, 1923
Mouse
100
4 hrs
fatty infiltration of the liver
Kylinetal., 1963
Mouse
693
1 hr
renal toxicity
Deringer et al. (1953)
Mouse
246
2 hrs
renal tubular necrosis
Culliford and Hewitt
(1957)
Mouse
665
2 hrs
renal necrosis in males
Culliford and Hewitt
(1957)
Mouse
4500
35 min
50% narcosis (ECt50)
Gehring (1968)
Mouse
4500
13.5 min
50% significantly13 elevated
SGPT (ECt50)
Gehring (1968)
Cat
7500
78 min
light narcosis
Lehmann and Schmidt-
Kehl, 1936
Cat
22,000
10 min
narcosis, eye, mouth and nose
irritation
Lehmann and Schmidt-
Kehl, 1936
a Narcosis and significant reduction in GSH was found in phenobarbital-induced rats exposed for 2 hrs to 5,000
ppm chloroform.
b Approximately 2.2-fold increase relative to unexposed controls; considered by investigators to be statistically
significant.
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3.3 Developmental/Reproductive Toxicity
3.3.1 Rats
The embryotoxicity and fetotoxicity of inhaled chloroform in Sprague-Dawley rats was
studied by Schwetz et al. (1974). Pregnant rats were exposed to 30 ppm (22 dams), 100 ppm (23
dams), or 300 ppm (3 dams) chloroform for 7 hours/day on gestation days 6 through 15; control
rats (68) were exposed to filtered air (Table 5). The exposure concentrations were subanesthetic
and varied <5% from the target concentrations. The chloroform concentrations were monitored
three times per day using an infrared spectrophotometer. The 300-ppm exposure produced a
marked anorexia at the end of the treatment period although comparison with a pair-fed control
group (8 dams) later showed that inanition was not a contributor to the observed embryotoxicity
and fetotoxicity. Chloroform at 30 ppm induced some evidence of embryotoxicity and
fetotoxicity while the 100- and 300-ppm exposures caused significant toxicity (Table 5).
TABLE 5. Embryotoxicity and Fetotoxicity of Chloroform in Rats Following Gestational Exposure
Parameter
Control
Pair-fed
control
30 ppm
100 ppm
300 ppm
% pregnancy (pregnant/bred)
88 (68/77)
100 (8/8)
71 (22/31)
82 (23/28)
15 (3/20)b
corpora lutea/dam
14±2
14±2
16±3b
14±2
14± 1
live fetuses/litter
10±4
10±4
12±2
11±2
4±7b
% reabsorptions/implantations
8(63/769)
7(6/87)
8(24/291)
6(16/278)
61(20/3 3)b
fetal body weight (g)
5.69±0.36
5.19±0.29b
5.51±0.20
5.59±0.24
3.42±0.02b
fetal crown-rump length (mm)
43.5± 1.1
42.1± l.lb
42.5±0.6b
43.6±0.7
36.9±0.2b
total gross anomalies3
1/68
0/8
0/30
13/23b
0/3
total skeletal anomalies3
46/68
3/8
20/22b
17/23
2/3
total soft tissue anomalies3
33/68
3/8
10/22
15/23
1/3
a litters affected/litters examined.
b Significantly different from control; p<0.05.
The investigators concluded that exposure to 30 ppm chloroform produced minor effects on
the embryo and fetus, exposure to 100 ppm was highly embryotoxic and fetotoxic, and that
exposure to 300 ppm was embryocidal as well as highly embryotoxic and fetotoxic. The
observed effects could not be correlated with maternal toxicity or inanition.
Newell and Dilley (1978) conducted experiments in which Sprague-Dawley rats were
exposed to 942, 2232, or 4117 ppm chloroform 1 hour/day on gestation days 7-14. Controls
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were exposed to clean air. There was an increase in the number of resorptions (45% relative to
unexposed controls) and decrease in average fetal body weight in the high-exposure group and
no notable effects in the low- or mid-exposure group. There was no evidence of teratologic
effects.
A series of experiments (two preliminary and one main study) were reported by Baeder and
Hoffman (1988) to assess developmental toxicity of chloroform in Wistar rats. In one
preliminary study, time-mated Wistar rats (4-6/group) were exposed to chloroform for 6
hours/day at concentrations of 0, 10, 30, or 100 ppm on gestation days 7-11 and 14-16. At 10
ppm, two dams had no fetuses and a single implantation site. At 30 ppm, one dam had only one
fetus and three empty implantation sites. No such effects were reported for the 100-ppm group.
In the second preliminary experiment, Wistar rats exposed to 100 and 300 ppm (6 hours/day) on
gestation days 7-16 exhibited decreased feed consumption and body weight loss. Fetal weights
in two litters in the 100-ppm group were lightly decreased while in the 300-ppm group three
dams had normally developed fetuses, one dam had totally resorbed fetuses, and one dam had
only empty implantation sites. In the main study, groups of 20-23 time-mated Wistar rats were
exposed to chloroform (7 hours/day, gestation days 7-16) at concentrations of 0, 30, 100, or 300
ppm. During exposure, chloroform-exposed rats exhibited decreased feed consumption and
body weight gain (p<0.05 for all exposure groups except body weight gain for 30-ppm group on
gestation day 21) relative to unexposed controls. Litter data for the main study are summarized
in Table 6. Although fetal weight is significantly decreased for the 300-ppm group and crown-
rump length is significantly decreased in all chloroform-exposed groups, these effects may be a
function of the maternal feed consumption/body weight effects. Incidences of external and
TABLE 6. Litter data from Wistar rats exposed to chloroform on gestation days 7-16.
(Baeder and Hoffman, 1988)
Parameter
Exposure concentration (ppm)
0
30
100
300
no. pregnant/no. sperm plugs
20/20
20/20
20/21
20/23
no. lost litters
0
2
3
8
no. live litters
20
18
17
12
resorptions/live litters (mean)
0.75
0.22
0.53
0.92
live fetuses/litter (mean)
12.4
12.8
12.8
13.4
fetal wt. (g)
3.19±0.30
3.16±0.19
3.13±0.21
3.00±0.19*
fetal crown-rump length (cm)
3.52±0.17
3.38±0.12*
3.39±0.10*
3.39±0.12*
significantly different from control group; p<0.
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A follow-up study was conducted by Baeder and Hoffman (1991) in which groups of 20
time-mated Wistar rats were exposed to chloroform (0, 3, 10, or 30 ppm, 7 hrs/day) on gestation
days 7-16. Feed consumption during the first week of exposure was significantly (p<0.05)
decreased and remained so for the 30-ppm group to the end of the study. Body weight of the 3-
ppm group was unaffected but an exposure-dependent decrease was detected by gestation day
17. Body weights remained lower than controls on gestation day 21 for the 10-ppm and 30-ppm
groups. Analysis of litter data by the investigators revealed a significant decrease in fetal weight
and crown-rump length in the 30-ppm group (Table 7). Significantly increased incidences of
some ossification variations were observed, especially for the 30-ppm group (Table 8).
TABLE 7. Litter data from Wistar rats exposed to chloroform on gestation days 7-16.
Baeder and Hoffman, 1991)
Parameter
Exposure concentration (ppm)
0
3
10
30
no. pregnant
20
20
20
20
no. lost litters
0
0
30
0
no. live litters
20
20
20
19
resorptions/live litters (mean)
0.55±0.89
0.40±0.60
0.75± 1.02
0.84± 1.42
live fetuses/litter (mean)
12.4±2.4
12.4±3.5
12.9±3.0
12.5± 1.9
fetal wt. (g)
3.4±0.3
3.2±0.3
3.2±0.3
3.2±0.3*
fetal crown-rump length (mm)
35.8±2.0
35.5±2.1
34.4±2.6
34.0± 1.9*
* significantly different from control group; p<0.05
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TABLE 8. Skeletal/ossification variations in Wistar rats exposed to chloroform on
gestation days 7-16 (Baeder and Hoffman, 1991)
Parameter
Exposure concentration (ppm)
0
3
10
30
no. pregnant
20
20
20
20
no. lost litters
0
0
30
0
no. live litters
20
20
20
19
resorptions/live litters (mean)
0.55±0.89
0.40±0.60
0.75± 1.02
0.84± 1.42
live fetuses/litter (mean)
12.4±2.4
12.4±3.5
12.9±3.0
12.5± 1.9
fetal wt. (g)
3.4±0.3
3.2±0.3
3.2±0.3
3.2±0.3*
fetal crown-rump length (mm)
35.8±2.0
35.5±2.1
34.4±2.6
34.0± 1.9*
* significantly different from control group; p<0.05
3.3.2 Mice
Murray et al. (1979) examined the developmental toxicity of inhaled chloroform in CF-1
mice following gestational exposure. Groups of 34-40 pregnant mice were exposed to
chloroform (100 ppm) for 7 hours/day on gestation days 6-15, days 1-7, or days 8-15. Controls
were exposed to filtered room air. Chloroform concentrations were monitored by infrared
spectrophotometry and found to vary <3% from the target concentration. Maintenance of
pregnancy was significantly (p<0.05) decreased in the dams exposed on gestation days 1-7 (44%
pregnant vs 74% in controls) and 6-15 (43% pregnant vs 91% in controls) but not for those
exposed on days 8-15 (decreased, but not significantly so). The significant developmental
toxicity findings are shown in Table 9. The incidences of delayed ossification of skull bones and
sternebrae (not for days 6-15) were significantly increased in the chloroform-treated groups
compared to the respective control groups. However, these data were not presented in the report
tables. There was also evidence of hepatotoxicity in the chloroform-exposed dams as
demonstrated by significantly increased absolute and relative liver weights, and by elevated
SGPT activity. The study authors concluded that exposure of pregnant mice to 100 ppm
chloroform (7 hrs/day) on gestation days 1-7 or 6-15 decreased the ability to maintain pregnancy
but was not teratogenic. Exposure on gestation days 8-15 did not affect pregnancy maintenance
but resulted in an increased incidence of cleft palate.
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TABLE 9. Developmental Toxicity of Chloroform in Mice Exposed During Gestation
Parameter
Days 1-7
Days 6-15
Days 8-15
Control
100 ppm
Control
100 ppm
Control
100 ppm
Litters examined
22
11
29
12
24
18
Resorptions/litter
2±2
4±5a
2±2
1±1
2±2
2±2
Fetal body weight (g)
1.02±0.10
0.92±0.07a
0.99±0.11
0.95±0.13
1.00±0.12
0.85±0.17a
Fetal crown-rump
length
24.7± 1.0
23.6± 1.2a
23.7± 1.3
23.2± 1.1
24.1± 1.1
22.9±2.2a
Cleft palate
fetuses (litters) affected
3(1)
0
0
0
1(1)
10(4)a
a Significantly different from control (p<0.05)
Land et al. (1981) studied the morphologic changes in spermatozoa of C57B1/C3H mice.
The mice were observed 28 days after exposure to chloroform (4 hrs/day for 5 days) at
concentrations of 0.1 or 0.05 of the MAC (Minimal Alveolar Concentration). The chloroform
was delivered via calibrated vaporizers and the concentration was monitored by gas
chromatography. The mice were terminated 28 days after the last exposure and the spermatozoa
(1,000 /slide) examined independently by two pathologists. Based upon data from groups of five
mice, the percent of abnormal spermatozoa was 1.42+0.08, 2.76+0.31, and 3.48+0.66 for the
control (clean air), 0.5 and 1.0 ppm chloroform groups respectively. Both treatment groups were
significantly different (p<0.01) than the controls. The abnormalities identified included flattened
spermatozoa, amorphous spermatozoa and spermatozoa with abnormal hook formation.
3.4 Genotoxicity
Numerous genotoxicity assays have been performed with chloroform (ATSDR, 1997).
Generally, the results of these bioassays indicate chloroform to be a weak mutagen with low
potential for interaction with DNA.
3.5 Carcinogenicity
Renal and hepatic tumors have been reported for rodents following chronic oral
administration of chloroform (reviewed in ATSDR, 1997). The results of cancer bioassays
appear to be substantially influenced by the method of administration (gavage vs drinking water)
and by the vehicle (corn oil vs water). Currently, inhalation exposure studies addressing the
tumorigenic potential of chloroform are limited to a 90-day study in F-344 rats by Templin et al.
(1996a), a short-term exposure study by Templin et al. (1996b), and a report on a long-term
inhalation study by Yamamoto et al. (1994).
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In the study by Templin et al. (1996a), male and female F-344 rats were exposed to
chloroform (0, 2, 10, 20, 30, 90, or 300 ppm), 6 hours/day, 7 days/week. Groups of rats (15-60
per group) were subjected to different exposure protocols: male rats were exposed for 4 days or
3, 6, or 13 weeks, and female rats were exposed for 3 or 13 weeks. The exposure atmospheres
were monitored by infrared gas analysis. Average analytically determined concentrations were
always within 4.5% of the target concentration. Results of the study revealed the liver, kidneys,
and nasal passages as primary targets of toxicity. Cytolethality and regenerative cell
proliferation were significant at the 300 ppm exposure. Although long-term exposure to 300
ppm would likely induce a tumorigenic response, this exposure was considered by the
investigators to be highly cytotoxic (in excess of the MTD) with no relevance for extrapolating
to low dose responses. Statistically significant body weight loss was observed in the male rats
exposed for four days but kidney lesions were seen only in rats exposed to 30 (1 of 5 rats), 90 (3
of 5 rats), or 300 ppm (5 of 5 rats).
Templin et al. (1996b) conducted studies in BDFi mice to affirm the role of cytotoxicity and
regenerative cell proliferation in the tumorigenic response to chloroform. Groups of male and
female mice were exposed to 0, 0.3, 5, 30, or 90 ppm chloroform 6 hours/day for 4 days.
Bromodeoxyuridine (BrdU) was administered by osmotic pumps implanted 3.5 days prior to
necropsy and served to provide a labeling index (LI) for S-phase cells. Additional groups of
mice were exposed to chloroform at 30 or 90 ppm for 5 days/week for 2 weeks. Degenerative
lesions and a 7- to 10-fold increase in the LI were observed in the kidneys of male but not female
mice treated exposed to 30 or 90 ppm. Liver lesions and an increased hepatocyte LI were
observed in male mice exposed to 30 and 90 ppm and in female mice exposed to 90 ppm. A
40% and 80% lethality, respectively, were observed in the 30- and 90-ppm groups exposed for
two weeks; severe kidney damage was evident in these animals. These findings show that in the
2-year assays, these exposures actually exceeded the MTD and were tolerated only because of
the step-wise exposure protocol allowing the animals to metabolically accommodate to the high
exposures. Templin et al. questioned the validity of low-dose extrapolation from tumor data of
this type (e.g., non-genotoxic-cytotoxic mechanism that is secondary to organ-specific toxicity).
In a preliminary report of a 2-year cancer bioassay, Yamamoto et al. (1994) observed no
increase in tumor incidences in male and female F-344 rats exposed to chloroform (10, 30, or 90
ppm), 5 days/week. No further details are available on this study.
Several issues, however, are relevant to the carcinogenic potential of chloroform. These are
especially relevant regarding the estimation of carcinogenic risk following a single acute
exposure. As reviewed by Conolly (1995) and Golden et al. (1997), the tumorigenic dose-
response of mice and rats to chloroform appears to be nonlinear and is secondary to cytotoxicity
(i.e., cell necrosis and subsequent cellular regeneration) following exposures that induce frank
toxicity in tissues that are tumor sites and exposure that often exceed the maximum tolerated
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dose (MTD). Additionally, both in vivo and in vitro genotoxicity data indicate the absence of a
genotoxic mechanism for chloroform.
The significance of regenerative cell proliferation in chloroform-induced cancer was also
examined by Butterworth et al. (1995) and Wolf and Butterworth (1997). Generally, an analysis
of the available data indicates that chloroform acts through a nongenotoxic, cytotoxic
mechanism. In rodent studies, toxicity is not observed when chloroform is not metabolized to
reactive metabolites at a rate sufficient to cause cytolethality. As such, a linearized extrapolation
from high doses that produce tumors to very low doses is considered inappropriate.
Additionally, the current inhalation cancer risk is 2.3 x 10"5 (/ig/in3)"' (USEPA, 1992b) and is
based upon a tumorigenic response (hepatocellular carcinomas) in B6C3Fi mice administered
chloroform by gavage (NCI, 1976) and, therefore, involves the uncertainties associated with
route-to-route extrapolation.
Butterworth et al. (1995) and Wolf and Butterworth (1997) have compared the application of
the linearized multistage model for low dose extrapolation in cancer risk assessment to an
assessment based upon the use of a threshold response (i.e., cytolethality and cellular
regeneration). The resulting outcomes are remarkably different. Application of the linear
multistage model to tumor incidence data from a gavage study with mice (NCI, 1976) results in a
virtually safe dose (VSD; relative to a 10"6 cancer risk) of 0.000008 ppm. However, a VSD of
0.01 ppm is obtained using a safety factor approach (10 x 10 x 10 for interspecies, intraspecies,
and use of subchronic study) and inhalation data from rodents showing that exposure to 10 ppm
does not produce cytolethality or a regenerative cell proliferation. The investigators justify the
approach because of the apparent need for cytolethality and cellular regeneration in the
tumorigenic response.
Melnick et al. (1998), however, have provided data and alternate interpretations regarding
the relevance of cytolethality and proliferative cellular regeneration to the tumorigenic response
observed in rodents following oral administration of chloroform in corn oil. Following gavage
dosing of female B6C3Fi mice (10/group) with chloroform (5 times/week for 3 weeks at doses
of 55,110, 238, or 477 mg/kg)), biochemical indices of toxicity (ALT, SDH), labeling index
(bromodeoxyuridine [BrdU]) for S-phase hepatocytes, and histopathologic examination were
conducted to ascertain the relationship between regenerative hyperplasia and tumor induction.
As expected, a dose-related response was observed for liver-to-body weight ratio, increase in
ALT and SDH activity, severity and incidence of hepatocyte hydropic degeneration, and labeling
index. The investigators compared the dose-response curves for tumor incidence (using data
from previous cancer bioassays) and hepatocyte labeling index and reported that the processes
are not causally related. In other words, an elevated labeling index resulting from cellular
proliferation is not required for a tumorigenic response.
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4. SPECIAL CONSIDERATIONS
4.1 Metabolism and Disposition
The metabolism of chloroform has been thoroughly studied (reviewed in ATSDR, 1997).
Although metabolism via cytochrome P-450 IIE1 is well established, a minor anaerobic pathway
also exists resulting in a dichloromethyl radical intermediate. Phosgene, formed by P-450-
mediated dehydrochlorination, may react with cellular proteins or be converted to hydrochloric
acid and carbon dioxide (Pohl et al., 1981). Phosgene may also react with glutathione to form
diglutathionyl dithiocarbonate which is then metabolized to 2-oxothiazolidine-4-carboxylic acid
(Pohl et al., 1977; Mansuy et al., 1977; Branchflower et al., 1984).
Brown et al. (1974a) studied the metabolism of orally administered [14C]-chloroform (60
mg/kg) in male Sprague-Dawley rats, male CBA, CF/LP, and C57 mice, and squirrel monkeys.
In all test species, 14CC>2 was a major excretory product but species-dependent variability was
observed in its elimination. For all three strains of mice, 14CC>2 represented approximately 80%
of the administered dose while for rats only about 60% of the dose was eliminated as 14CC>2 and
for squirrel monkeys only 20% of the dose was excreted as carbon dioxide.
Fry et al. (1973) reported that 17.8-66.6%) of an oral dose of radiolabeled chloroform (500
mg) was expired unchanged by eight human volunteers over an 8-hour period. Maximum
excretion of chloroform occurred at 40 minutes to two hours after administration. Carbon
dioxide excretion was measured in one male and one female volunteer. Over a 450-minute
period, 48%> (woman) and 50%> (man) of the dose was expired as carbon dioxide. The study
authors also reported decreased excretion of chloroform by obese subjects and suggested this
was due to uptake of chloroform by greater amounts of adipose tissue. Peak blood
concentrations 1 Mg/ml) occurred at about 45 minutes after dosing. Elimination from the
blood appeared to be biphasic: an initial rapid clearance within an hour and a slower clearance
over the next six hours. As chloroform concentration in the blood increased, pulmonary
excretion increased.
Corley et al. (1990) developed a physiologically based pharmacokinetic model for
chloroform based upon a kinetic constant from in vivo studies on rats and mice, in vitro
enzymatic studies with human tissue samples, and physiologically-based estimates for
absorption, distribution, metabolism, and excretion processes. Macromolecular binding was
considered as a measure of internal dose. The model was validated by comparison of predicted
values with experimental data from mice, rats, and humans. Human metabolic and
macromolecular binding constants for VmaxC (15.7 mg/hr/kg) and Km (0.448 mg/L) were
derived. It was also shown that metabolic activation of chloroform to reactive intermediates
such as phosgene was greatest in mice. Metabolic activation was less in rats and lowest in
humans. Therefore, it was estimated that exposure to equivalent concentrations of chloroform
would result in a lower delivered dose in humans than in laboratory species. Species variability
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was also reported by Brown et al. (1974a), who reported that conversion of chloroform to carbon
dioxide was highest in mice (80%) and lowest in squirrel monkeys (18%). In rats and mice,
[14C]-urea was detected in the urine along with two unidentified metabolites and parent
compound was found in the bile of the squirrel monkeys. In mice, radioactivity in the blood
peaked at 1 hour after dosing and decreased gradually over the next 24 hours.
The chloroform PBPK model developed by Corely et al. (1990) was used by Delic et al.
(2000) to develop models for humans and rats in an effort to compare rates of metabolism in the
context of assessing the validity of uncertainty factors used in developing occupational exposure
limits. The study also utilized Monte Carlo analysis to determine the extent of variability within
human and animal model populations. The results demonstrated that even at the most extreme
ranges within the human population, levels of toxic metabolites necessary for induction of a
toxic response would not be generated at rates comparable to that in rats. Specifically, the
model showed that the mean peak rate of metabolism of inhaled chloroform (at the mouse
NOAEL of 10 ppm) is approximately 78-fold lower in humans and that the chloroform exposure
required toachieve peak metabolism rate in humans would be 65-fold higher than that in mice.
Monte Carlo analysis of population variability also indicated that chloroform metabolism rates
between mice and humans varied by 25 to 50-fold. Overall, the work clearly demonstrated that
human require considerably higher exposure concentrations than do mice to induce a toxic
response.
Data regarding the distribution of chloroform among brain, lung and liver tissue of humans
was attained by Gettler and Blume (1931) from suicide victims or deaths during chloroform
anesthesia. The brain and lungs consistently contained the highest levels of chloroform (60-480
mg/g brain tissue; 24-485 mg/g lung tissue). Liver tissue tended to contain lower levels (24-238
mg/g liver tissue) than did brain and lung tissue. Due to the nature of the subjects examined,
these values reflect tissue burdens following high exposures.
The distribution of [14C]-chloroform in pregnant C57BL mice following a single 10-minute
inhalation exposure (approximately 16 mmoles based upon specific activity) was studied by
Danielsson et al. (1986). Assessments were conducted at 0.5, 4, and 24-hour time points. At all
time points, radioactivity was greatest in the lungs, liver, and kidneys. Pulmonary radioactivity
decreased with time while radioactivity in the liver and kidneys peaked at 0.5 hours followed by
a successive decrease. Radioactivity in the respiratory tract was associated with epithelial tissue
(nasal mucosa, trachea, and bronchi). Radioactivity was also found in the fetus and placenta at
all time points, peaking at 0.5 hours and gradually decreasing over the 24-hour time frame. In
addition to total radioactivity, the investigators also determined bound radioactivity in various
tissues and found that the respiratory tract and centrilobular portion of the liver contained bound
radioactivity possibly indicative of on-site production of reactive metabolites.
Wang et al. (1997) reported on the effects of ethanol pretreatment (2 g/rat/day for 3 weeks)
on the metabolism and hepatotoxicity of chloroform in rats following administration of
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chloroform by various routes (ip, po, and inhalation). The ethanol pretreatment increased
cytochrome P-450 from 0.74 nmol/mg protein to 1.10 nmol/mg protein and increased the
metabolism of inhaled chloroform 7-fold in rats exposed to 500 ppm for 6 hours but did not
increase the metabolism of chloroform in rats exposed to 50 ppm for 6 hours. Hepatotoxicity, as
determined by GPT, GOT, and GSH activity, was unaffected in the 50-ppm group and increased
approximately 6-fold in the 500-ppm group.
4.2 Mechanism of Toxicity
The noncarcinogenic and carcinogenic mechanisms of chloroform have been previously
reviewed (Butterworth et al.,1995; Conolly, et al., 1995; Templin et al., 1996a, b; ATSDR, 1997;
Golden et al., 1997; Wolf and Butterworth, 1997). Chloroform toxicity may be generally
categorized as effects on the central nervous system, hepatic and renal effects, and cardiac
effects (primarily the result of myocardial sensitization to epinephrine).
The precise mechanism of chloroform on neural activity is unknown. It is generally assumed
that general anesthetics act by influencing synaptic transmission (e.g., potentiating transmitter
release at inhibitory synapses and/or inhibiting release at excitatory synapses). These actions
may be the result of interaction with protein-lipid interfaces (Kennedy and Longnecker, 1996).
The underlying mechanism of chloroform hepatic and renal toxicity is the binding of reactive
intermediates, such as phosgene (Pohl et al., 1977), to cellular macromolecules, the depletion of
these macromolcules and subsequent cell death.
Brown et al. (1974b) exposed phenobarbital-treated rats for 2 hours to 0.5% (5,000 ppm) or
1.0% (10,000) chloroform and found, respectively, a 70% and 83% reduction in hepatic
glutathione in rats (p<0.001). At these exposure levels, however, normal (non-induced) rats
exhibited no significant change in GSH levels.
The importance of GSH depletion was also demonstrated by Docks and Krishna (1976), who
showed that administration of chloroform (80 mg/kg, i.p.) to phenobarbital-treated rats decreased
glutathione and that this depletion resulted in massive liver necrosis. Because of the greater
depletion of glutathione by chloroform than by halomethanes known to be metabolized to the
trichlorocarbon radical (CCI3 •)> Docks and Krishna postulated that chloroform-mediated
decrease in glutathione was not due to the trichlorocarbon radical.
The mechanism of chloroform toxicity in isolated rat hepatocytes was studied by El-shenawy
and Abdel-Rahman (1993). The results of this study supported the contentions of Docks and
Krishna regarding the depletion of glutathione as a causative precursor for cytotoxicity. In this
study, isolated rat hepatocytes exposed to chloroform at concentrations of 1, 10, 100, or 1000
ppm exhibited a concentration-dependent decrease in viability (statistically significant at p<0.05
at all concentrations). Leakage of AST occurred with all concentrations although was significant
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only after 60 and 30 minutes for the 1-ppm and 10-ppm tests, respectively. Leakage of ALT was
significant at 60 minutes and 30 minutes in the 100 and 1000-ppm tests. Glutathione was
significantly decreased at all time points from 15 to 120 minutes following incubation of
hepatocytes with 1000 ppm chloroform. At 100 ppm and 10 ppm, glutathione depletion became
significant at 30 minutes and 120 minutes, respectively.
4.3 Structure-Activity Relationships
Assessment of structure-activity relationships was not instrumental is deriving AEGL values
for chloroform.
4.4 Other Relevant Information
4.4.1 Species Variability
Strain, species, and gender variability in the metabolism and toxicity of chloroform has been
demonstrated. As previously noted, male mice exhibit both renal and hepatotoxicity following
exposure to chloroform whereas female mice exhibit only hepatotoxicity. This has been shown
to be due to hormone-specific cytochrome P-450 in the kidneys of male mice. By examining
differences in the biotransformation of chloroform to phosgene, Pohl et al. (1984) clearly
demonstrated strain and sex differences in chloroform-induced renal toxicity. The differences
could be attributed to strain and gender-dependent differences in the rate of phosgene production
by microsomal and mitochondrial fractions form the kidneys. A notable difference was observed
between sensitive male DBA/2J mice and less sensitive C57BL/6J mice. Male mice exhibited
nearly an order of magnitude more rapid formation of phosgene than did female mice.
Additionally, based upon results of PBPK model studies using metabolism and disposition data,
humans appear to be less sensitive than rodent species, and the mouse appears to be the most
sensitive.
4.4.2 Concurrent Exposure Issues
Because the biotransformation of chloroform to reactive intermediates is mediated by
cytochrome P-450IIE1, exposures to chemicals that induce P-450 may increase the toxic
response of chloroform. From a practical standpoint, special concern would be directed to
alcohol consumption.
5. DATA ANALYSIS FOR AEGL-1
5.1 Summary of Human Data Relevant to AEGL-1
Human exposure data consistent with AEGL-1 effects are limited to the Lehmann and
Hasegawa (1910) study using human volunteers, and a study of dental workers (McDonald and
Vire (1992). Lehmann and Hasegawa reported that 2-3 minute exposures to 920-1100 ppm
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resulted in vertigo and 15-30 minute exposures to concentrations as high as 1400 ppm produced
a condition of lassitude, vertigo, and headache. Some individuals exposed for 30 minutes to 620
ppm reported only the sensation of a not unpleasant odor and no neurological symptoms.
Because vertigo may affect escape from a potentially hazardous condition, those exposures
inducing this condition may be inappropriate for development of AEGL-1 values. The data of
Lehmann and Hasegawa lack details on exposure methods and validity of exposure
measurements. The McDonald and Vire (1992) report is limited to very low exposures
encountered during endodontic procedures (<0.57 ppm for 5.5 hrs and <0.88 ppm for over 150
minutes). These exposures did not result in any signs or symptoms even following clinical
screening at five hours and one year after exposure. No additional human data consistent with
AEGL-1 level effects were available.
5.2 Summary of Animal Data Relevant to AEGL-1
Animal data consistent with AEGL-1 level effects were limited to alterations in clinical
chemistry determinations (specifically serum ALT, AST, GLDH and SDH activity) and minor
histopathologic findings in the liver and kidneys of rats and mice. Elevated serum enzyme
activities were observed in rats exposed for four hours to 153 ppm (Lundberg et al., 1986) or 292
ppm (Brondeau et al., 1983). Six-hour exposure of rats to 500 ppm produced statistically
significant elevations in serum enzyme activity (Wang et al., 1994). Eight-hour exposures of
rats to 50 ppm produced no increase in liver weight while exposure to 100 ppm resulted in a
slight increase in serum enzyme activity (Ikatsu and Nakajima, 1992). Although statistically
significant increases in serum enzyme levels were reported in several studies, the increases were
not necessarily indicative of biologically relevant hepatic damage (some of the enzyme activities
were increased only 2-fold and histologic correlates were negligible) and, therefore, would not
be appropriate as AEGL-1 endpoints.
5.3 Derivation of AEGL-1
Human data sets for AEGL-1 determination are limited by poorly defined exposure values
with poorly described methodology. Animal data consistent with the AEGL-1, although having
more definitive exposure data, are limited to clinical chemistry findings that are more indicative
of biological indices of exposure than overt toxicity. Based upon currently available data, it is
difficult to identify exposures producing effects consistent with AEGL-1. Exposures that do not
produce overt signs of toxicity in humans are neither irritating nor with unpleasant odor. As a
result, it was the consensus of the NAC/AEGL that AEGL-1 values not be recommended due to
the properties of the chemical (Table 10). Specifically, it would be difficult to identify
exposures that would produce notable discomfort or mild sensory irritation without approaching
levels that may be near a threshold for narcosis.
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TABLE 10. AEGL-1 Values for Chloroform (ppm [mg/m3])
AEGL Level
10-min
30-min
1-hr
4-hr
8-hr
AEGL-1
NR
NR
NR
NR
NR
vfR: Not recommended due to properties of the chemical.
6. DATA ANALYSIS FOR AEGL-2
6.1 Summary of Human Data Relevant to AEGL-2
In an assessment of 1502 surgical patients anesthetized with chloroform (never exceeding
22,500 ppm) for <30 minutes to >120 minutes, Whitaker and Jones (1965) reported cardiac
irregularities (bradycardia 8.1%; arrhythmias 1.3%) in some patients. Protection against
narcosis even in the absence of toxic effects would appear to be at least one focus of the AEGL-
2, thereby rendering the Whitaker and Jones data inappropriate for AEGL-2 derivation.
Lehmann and Hasegawa (1910) reported "intoxication and dizziness" following exposure of a
human subject(s) to 4300-5100 ppm for 20 minutes or 7200 ppm for 15 minutes. In this same
study, three human volunteers reported pounding heart and experienced gagging during a 30-
minute exposure to 3000 ppm and "light-headedness" and lassitude following 30-minute
exposure to 1400 ppm. Smith et al. (1973) evaluated surgical patients anesthetized with
chloroform (8,500-13,000 ppm; concentration never exceeded 2% [20,000 ppm]) for a mean
duration of 112.96 minutes. Cardiac arrhythmias of various types were detected in 1-17 of the
patients. With the exception of a slight elevation of LDH, serum enzyme values (SGPT, SGOT,
alkaline phosphatase) were not altered by the chloroform anesthesia. Nausea and vomiting
occurred in 46% of the patients.
6.2 Summary of Animal Data Relevant to AEGL-2
Several studies in rats indicate that signs of hepatotoxicity (fatty infiltration) and renal
damage (tubular necrosis) may occur at cumulative exposures of 400-1330 ppm • hr that
encompass exposure durations of 1-4 hours and concentrations of 100-693 ppm (Deringer et al.,
1953; Culliford and Hewitt, 1957; Kylin et al., 1963). Exposure of pregnant rats during
gestation (7 hrs/day on gestation days 6-15) to 30 ppm chloroform produced minor effects on the
embryo and fetus and exposure to 100 ppm was significantly embryotoxic and fetotoxic
(Schwetz et al., 1974). Newell and Dilley (1978), however, found that gestational exposure of
rats to chloroform at concentrations as high as 2232 ppm (1 hr/day on gestation days 7-14) did
not cause developmental effects although exposure to 4117 ppm increased resorptions 45% and
decreased fetal body weight.
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6.3 Derivation of AEGL-2
Protection against severe hepato- or renal toxicity, or narcosis initially appear to be critical
effects for the development of AEGL-2 values. Human data suggest that exposures to 8500 ppm
will induce anesthesia; although the duration of this exposure is unknown, it is assumed that the
exposure duration would be on the order of minutes. The human data reported by Lehmann and
Hasegawa (1910) suggest that exposure to 7500 ppm for 15 minutes or 4300-5100 ppm for 20
minutes were approaching narcosis-inducing effects as determined by signs and symptoms of
dizziness, and "intoxication". These data and the anesthesia data reported by Whitaker and Jones
(1965) are, however, compromised by the uncertainties regarding determination of exposure
concentrations and specific concentration-duration relationships. Alternately, the increased
fetotoxicity and embryolethality reported by Schwetz et al. (1974) for rats exposed to 100 ppm
(7 hrs/day) on gestation days 6-15 was considered a sensitive critical effect and point-of-
departure for developing AEGL-2 values. The assumption was made that the reported effects
(increased fetotoxicity and embryolethality) occurring following the 10-day gestational exposure
could result from a single 7-hour exposure. This contention is not without precedent as has been
shown by analyses of developmental toxicity data for other chemicals (van Raaij et al., 2003).
An intraspecies uncertainty factor of 3 was applied to account for individual variability in
metabolism and disposition of chloroform. No adjustment was made for interspecies variability
because available metabolism/kinetics data and PB-PK models (Corley et al., 1990) indicate that
humans are less sensitive than laboratory species to the toxic effects of chloroform. The
attenuated uncertainty factors were justified by the sensitive point of departure selected for
AEGL-2 development and the results of another study (Newell and, 1978) that showed
gestational exposure of rats to chloroform at concentrations as high as 2232 ppm (1 hr/day on
gestation days 7-14) was without effect.
Data were unavailable for calculating an exponent (n) for use in temporal extrapolation (Cn x
t = k). The concentration-exposure time relationship for many irritant and systemically acting
vapors and gases may be described by Cn x t = k, where the exponent n ranges from 0.8 to 3.5
(ten Berge et al., 1986). In the absence of chemical-specific data, an exponent (n) of 1 or 3 was
used in the equation, C" xt = k. The exponent of 1 applied for extrapolation to exposure
durations longer than that of the experimental exposure while the exponent of 3 was applied for
time scaling to shorter exposure durations. The resulting AEGL-2 values are presented in Table
11 and Appendix A.
TABLE 11. AEGL-2 Values for Chloroform
AEGL Level
10-min
30-min
1-hr
4-hr
8-hr
AEGL-2
120 ppm
580 mg/m3
80 ppm
390 mg/m3
64 ppm
312 mg/m3
40 ppm
195 mg/m3
29 ppm
141mg/m3
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7. DATA ANALYSIS FOR AEGL-3
7.1 Summary of Human Data Relevant to AEGL-3
Definitive lethality data for humans are unavailable. Although the weight of evidence
indicates that acute exposure to high concentrations of chloroform may result in narcosis and
subsequent death, the precise exposure concentrations and durations for such exposures are
unavailable. The available human data generally suggest that concentrations in excess of 10,000
ppm are required for an unspecified, although short, exposure duration for surgical plane
anesthesia. In the analysis of surgical patients anesthetized with chloroform, Whitaker and Jones
(1965) reported that the 22,500 ppm exposure for surgical anesthesia also produced evidence of
potentially serious cardiovascular effects. While these data are superficially compelling for
development of AEGL values, specific exposure duration terms are lacking (i.e., it is not
possible to associate a specific exposure concentration with a specific exposure duration).
Additionally, the concentrations were likely variable over the duration of anesthesia. This is not
unexpected; chloroform anesthesia utilizes very high initial exposures (25,000 to 30,000 ppm) of
very short duration (2 to 3 minutes) for the purpose of induction but lower exposure
concentrations are utilized for maintenance of surgical plane anesthesia (ATSDR, 1997; NRC,
2000). Therefore, it is unlikely that these patients were exposed to the highest concentrations
(e.g., 22,500 ppm) for AEGL-specific durations. Arrhythmias were also reported by Smith et al.
(1973) in some patients anesthetized for 113 minutes with chloroform concentrations (at least
initially) of 8,500 to 13,000 ppm. The available data suggest that surgical plane narcosis would
occur at or above 8,500 ppm following short duration exposure. Based upon the available
human data, it is not feasible to extrapolate to an exposure duration that would result in death.
7.2 Summary of Animal Data Relevant to AEGL-3
Data regarding the acute lethality of animals following acute inhalation exposure to
chloroform are limited to rats and mice. Four-hour exposures to concentrations of 3000 to 8000
ppm resulted in 75-100% mortality in rats (lethality determined 2-3 days post exposure) (Haskell
Laboratory, 1964; Smyth et al., 1962), and a 4-hour LC50 of 9780 ppm was reported by
Lundberg et al. (1986). For mice, 75% mortality was observed following 120-minute exposure
to 5585 ppm, a 66% mortality was reported for exposures to 4710 - 5529 ppm for durations of
71-175 minutes, and 14% mortality for 35-minute exposure to 6758 - 7782 ppm (Fiihner, 1923).
However, Fiihner (1923) observed no deaths at exposures of 2458 - 5120 ppm for 48-215
minutes. If the aforementioned responses are converted to cumulative exposures, the
inconsistency among the data becomes apparent. For example, no deaths were observed at 2458
- 5120 ppm for 48- 215 minute exposures (i.e., a maximum of 1,100,800 ppm • min), yet 66%
mortality was observed following exposures of 71 - 175 minute duration to 4710 - 5529 ppm (a
minimum of 334,410 ppm • min). A well-conducted study by Gehring (1968) reported a 4500-
ppm LCtso of 560 minutes (540 -585 minutes, 95% C.I.) for female Swiss-Webster mice.
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7.3 Derivation of AEGL-3
The available data do not allow for the identification of a definitive lethality threshold in
humans for acute exposure to chloroform. Data regarding chloroform as an anesthetic for
humans suggest that very high concentrations (in excess of 8,500 ppm) are tolerated for brief
durations, although quantitative concentration-time data are lacking in this respect. These
limitations preclude the use of the human data in the estimation of a lethality threshold for
humans.
Animal data are inconsistent regarding the lethality of acute inhalation exposure to
chloroform. Data for mice is highly variable although this species appears to be the most
sensitive, which is also affirmed by PBPK models. Four-hour exposure to concentrations of
3000 to 8000 ppm reportedly produced 75-100% mortality in rats (Smyth et al., 1962; Haskell
Laboratory, 1964). Assuming the mouse to be the most sensitive species, the 560-minute LC50
of 4500 ppm reported by Gehring (1968) appears to be a valid basis for development of the
AEGL-3 values. A 3-fold reduction in this value results in a point-of departure of 1500 ppm as
an estimate of the lethality threshold for mice. Consistent with the AEGL Standing Operating
Procedures for development of AEGLs (NRC, 2001) an exponent of 3 was applied for time
scaling (Cn xt = k) because data were insufficient for empirically deriving a time-scaling
exponent. Because the point-of-departure was based upon a 560-minute exposure duration, the
10-minute AEGL-3 was set equivalent to the 30-minute AEGL-3 to avoid the uncertainties
inherent in a 560-minute to 10-minute extrapolation (NRC 2001). Consistent with the AEGL
Standing Operating Procedures, an uncertainty factor of 3 was applied to account for responses
of potentially sensitive individuals such as those exposed to inducers of cytochrome P-450 IIE1
(e. g., ethanol consumption). No interspecies uncertainty factor was applied because currently
available data indicate that laboratory species metabolize chloroform more rapidly than do
humans and, therefore, are more susceptible to the toxic effects of the more rapidly formed toxic
intermediates. PBPK models (Corley et al., 1990) justify this contention. Further, human
anesthesia data show that cumulative exposures considerably greater than those associated with
the AEGL-3 values are not lethal. A more recent study using the PBPK model to compare the
metabolism of chloroform in mice and humans demonstrated quantitatively the overwhelmingly
greater sensitivity of mice (due primarily to a 25 to 50-fold difference in the rate of metabolism
of chloroform) and the overly protective nature of typically applied uncertainty factors. These
findings and the overall weight-of-evidence indicating the greater sensitivity of rodents to
chloroform-induced toxicity justified further adjustment in the AEGL-3 values. This adjustment,
applied as a weight-of evidence factor of 1/3, effectively increases the AEGL-3. The resulting
AEGL-3 values are shown in Table 12 and Appendix A.
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TABLE 12. AEGL-3 Values for Chloroform
AEGL
Level
10-min
30-min
1-hr
4-hr
8-hr
AEGL-3
4000 ppm
[19,000 mg/m3]
4000 ppm
[19,000 mg/m3]
3200 ppm
[16,000 mg/m3]
2000 ppm
[9,700 mg/m3]
1600 ppm
[7,800 mg/m3]
8. SUMMARY OF PROPOSED AEGLS
8.1 AEGL Values and Toxicity Endpoints
The proposed AEGL values for chloroform are summarized in Table 13.
TABLE 13. Pro
posed AEGL Values for Chloroform (ppm [mg/m3])
Classific
ation
10-min
30-min
1-hour
4-hour
8-hour
Endpoint (Reference)
AEGL-1
NR
NR
NR
NR
NR
Not recommended; AEGL-1
effects unlikely to occur in
the absence of notable
toxicity.
AEGL-2
120 ppm
580
mg/m3
80 ppm
390
mg/m3
64 ppm
312 mg/m3
40 ppm
195 mg/m3
29 ppm
141 mg/m3
Fetotoxicity/embryo-lethality
in rats exposed for 7 hrs/day
on gestation days 6-15
(Schwetz et al., 1974); single
exposure assumed
AEGL-3
4000 ppm
[19,000
mg/m3]
4000 ppm
[19,000
mg/m3]
3200 ppm
[16,000
mg/m3]
2000 ppm
[9,700
mg/m3]
1600 ppm
[7,800
mg/m3]
Estimated lethality threshold
for mice; 3-fold reduction in
560-min LC50 of 4500 ppm to
1500 ppm (Gehring, 1968)
The AEGL-1 values were not recommended because of the inability to determine an
exposure that would be consistent with the AEGL definition. The properties of chloroform are
such that the odor is not unpleasant and is not irritating even at exposures that approach levels
inducing narcosis.
The AEGL-2 values were developed using embryolethality/fetotoxicity in rats as the critical
effect. This was considered to be a very sensitive endpoint especially with the assumption of a
single-exposure response (i.e., fetotoxic effects resulting from 7-hour exposure on gestation days
6-15 were assumed possible following only one 7-hr exposure).
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The AEGL-3 values were developed based upon a rat 4-hour LC50 value (9780 ppm) and a 3-
fold reduction of this as an estimate of the lethality threshold. A 3-fold reduction of the LC50
value resulted an exposure of 3260 ppm which, when compared to available human and animal
data, appeared to represent an exposure that would not likely result in life-threatening exposures.
The AEGL values were developed using an uncertainty factor of 3 for protection of sensitive
individuals. Because chloroform is metabolized to toxic intermediates (i.e., phosgene) by
cytochrome P-450 IIE1, induction of this enzyme by inducers such as ethanol potentially
increase susceptibility to chloroform-induced toxicity although they do not appear to do so by an
order of magnitude (e.g., Brown et al., [1974b] reported a 2.6-fold increase in P-450 levels
following induction by phenobarbitol, a more effective P-450 inducer than ethanol).
Furthermore, dose rate appears to be a relevant factor in toxicity outcomes following exposure to
halogenated hydrocarbons such as chloroform, a fact that may justify the application of an
intraspecies uncertainty factor of less than an order of magnitude. Due to effects on P-450 and
GSH levels, single exposures result in toxic outcomes that are different from those following
repeated exposures. Available data and application of pharmacokinetic modeling indicate that
rodents metabolize chloroform more rapidly than do humans. Therefore the application of an
interspecies uncertainty factor was minimized. Furthermore, human data reveal surgical
anesthesia at cumulative exposures of >675,000 ppm-minute and that exposures to 22,500 ppm
for up to 120 minutes resulted in surgical anesthesia and cardiac irregularities but not death.
These data suggest that the AEGL-3 values represent NOAELs for lethality.
When compared to occupational exposure data reported by Challen et al. (1958) for
pharmaceutical workers, the AEGL values appear to be sufficiently protective. In this study, it
was found that workers exposed to <71 ppm (4 hrs/day for 10-24 months) experienced mild
symptoms (dryness of mouth and throat) while workers exposed to 77-232 ppm over a period of
3-10 years exhibited notable signs of exposure (staggering). It should be noted than the Challen
et al. findings are the result of repeated exposures and that it was not specified if any of the
workers represented a sensitive population.
8.2 Comparison with Other Standards and Guidelines
Standards and guidance values for workplace and community exposures are summarized in
Table 14. The cancer notation provided for some of the criteria was not considered to be
appropriate for the AEGL values.
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TABLE 14. Extant Standards and Guidelines for Chloroform
Guideline
Exposure Duration
10 minutes
30 minutes
1 hour
4 hours
8 hours
AEGL-1
(Nondisabling)
NR
NR
NR
NR
NR
AEGL-2
(Disabling)
120 ppm
80 ppm
64 ppm
40 ppm
29 ppm
AEGL-3
(Lethal)
4000 ppm
4000 ppm
3200 ppm
2000 ppm
1600 ppm
ERPG-1a
NA
ERPG-2
50 ppm
ERPG-3
5000 ppm
NRC SPEGLb
NRC SMAC°
NRC F.F.I.''
200 ppm
(30 ppm 24-
hr)
NIOSH IDLHe
NIOSH RELf
500 ppm
2 ppm
OSHA
STEL/Ceil.8
50 ppm
ACGIH
TLV-TWAh
10 ppm
MAK (Germany)1
2.5 mg/m3
MAC (the
Netherlands)"
5 ppm
(15-min)
1 ppm
aERPG (Emergency Response Planning Guidelines, American Industrial Hygiene Association (AIHA 2002)
The ERPG-1 is the maximum airborne concentration below which it is believed nearly all individuals could be
exposed for up to one hour without experiencing other than mild, transient adverse health effects or without
perceiving a clearly defined objectionable odor.
The ERPG-2 is the maximum airborne concentration below which it is believed nearly all individuals could be
exposed for up to one hour without experiencing or developing irreversible or other serious health effects or
symptoms that could impair an individual's ability to take protection action.
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The ERPG-3 is the maximum airborne concentration below which it is believed nearly all individuals could be
exposed for up to one hour without experiencing or developing life-threatening health effects.
bNRC SPEGL (Short-term Public Emergency Guidance Level).
CNRC SMAC (Spacecraft Maximum Allowable Concentration).
dNRC EEL (Emergency Exposure Guideline) (NRC, 1984).
eIDLH (Immediately Dangerous to Life and Health, National Institute of Occupational Safety and Health)
(NIOSH 2003) represents the maximum concentration from which one could escape within 30 minutes without
any escape-impairing symptoms, or any irreversible health effects. IDLH carries a cancer notation.
fNIOSH REL-TWA (National Institute of Occupational Safety and Health, Recommended Exposure
Limits - Time Weighted Average) is defined analogous to the-TLV-TWA, with cancer notation (ACGIH,
2003).
gOSHA PEL-TWA (Occupational Health and Safety Administration, Permissible Exposure Limits
Time Weighted Average) is defined analogous to the ACGIH-TLV-TWA, but is for exposures of no more than
10 hours/day, 40 hours/week (OSHA, 1993).
hACGIH TLV-TWA (American Conference of Governmental Industrial Hygienists, Threshold Limit
Value - Time Weighted Average) is the time-weighted average concentration for a normal 8-hour workday and
a 40-hour workweek, to which nearly all workers may be repeatedly exposed, day after day, without adverse
effect. ACGIH, 2002.
'MAC (Maximaal Aanvaaarde Concentratie [Maximal Accepted Concentration]) (SDU Uitgevers [under the
auspices of the Ministry of Social Affairs and Employment], The Hague, The Netherlands 2002)
is defined analogous to the ACGIH-TLV-TWA.
jMAK (Maximale Argeitsplatzkonzentration [Maximum Workplace Concentration]) (DFG 2002, Deutsche
Forschungsgemeinschaft [German Research Association]) is defined analogous to the ACGIH-TLV-TWA.
Cancer category 4 noted.
8.3 Data Quality and Research Needs
Much of the human experience data are from older studies that lacked information regarding
analytical techniques used to determine exposure concentrations. The human anesthesia data
focus on initial concentration and duration of anesthesia and were not sufficient for developing
AEGL values.
The most obvious data deficiency regarding development of AEGL values for chloroform is
the lack of data with which to determine a lethality threshold. There is also a paucity of reliable
data demonstrating definitive concentration-response relationships. The human experience data
are deficient in exposure-time relationships or are unreliable and difficult to validate. The
animal data are variable. Acute exposure studies providing exposure-response data for specific
toxicity endpoints (e.g., hepatotoxicity, renal toxicity, narcosis threshold, lethality) in two or
more species would be desirable.
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McDonald, M.N., Vire, D.E. 1992. Chloroform in the endodontic operatory. J. Endodontics 18:
301-303.
Melnick, R.L., Kohn, M.C., Dunnick, J.K., Leininger, J.R. 1998. Regenerative hyperplasia is not
required for liver tumor induction in female B6C3Fi mice exposed to trihalomethanes.
Toxicol. Appl. Pharmacol. 148: 137-147.
Mery, S., Larson, J.L., Butterworth, B.E., Wolf, D.C., Harden, R., Morgan, K.Y. 1994. Nasal
toxicity of chloroform in male F-344 rats and female B6C3Fi mice following a 1-week
inhalation exposure. Toxicol. Appl. Pharmacol. 125: 214-227.
Murray, F.J., Schwetz, B.A., McBride, J.G., Staples, R.E. 1979. Toxicity of inhaled chloroform
in pregnant mice and their offspring. Toxicol. Appl. Pharmacol. 50: 515-522.
NCI (national Cancer Institute). 1976. Carcinogenesis Bioassay of Chloroform. National Tech.
Inform. Service No. PB264011/AS, National Cancer Institute, Bethesda,MD.
Newell, G.W., Dilley, J.V. 1978. Teratology and acute toxicology of selected chemical
pesticides administered by inhalation. Report by Stanford Research Institute, Menlo Park,
CA. Health Effects Research Laboratory, Office of Research and Development, U.S.
Environmental Protection Agency, Research Triangle Park, NC. (Cited in ATSDR, 1997).
NIOSH (National Institute for Occupational Safety and Health). 2003. NIOSH Pocket Guide to
Chemical Hazards. Publication 97-140, U.S. Department of Health and Human Services;
U.S. Government Printing Office, Washington, DC.
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NIOSH (National Institute for Occupational Safety). 1974. Occupational exposure to chloroform.
Criteria for a recommended standard. U.S. Dept. Health, Education, and Welfare, NIOSH.
NRC (National Research Council), 1984. Emergency and Continuous Exposure Guidance Levels
for Selected Airborne Contaminants. Committee on Toxicology, Board on Toxicology and
Environmental Health, Commission on Life Sciences. National Academy Press, Wash., D.C.,
Vol. 5, pp. 57-76.
NRC (National Research Council), 2001. Standing Operating Procedures for Developing Acute
Exposure Guideline Levels for Hazardous Chemicals. Committee on Toxicology, Board on
Toxicology and Environmental Health, Commission on Life Sciences. National Academy
Press, Wash., D C.
OSHA (Occupational Safety and Health Administration). 1993. Air Contaminants Rule. 29 CFR
Part 1910. Fed. Reg. 58: 35345.
Pohl, L.R., Bhooshan, B., Whitaker, N.F., Krishna, G. 1977. Phosgene: A metabolite of
chloroform. Biochem. Biophys. Res. Comm. 79: 684-691.
Pohl, L. R., Branchflower, R.V., Highet, R.J., Martin, J.L., Nunn, D.S., Monks, T.J., et al. 1981.
The formation of diglutathionyl dithiocarbonate as a metabolite of chloroform,
bromotrichloromethane, and carbon tetrachloride. Drug Metab. Dispos. 9: 334-339.
Pohl, L.R., George, J.W., Satoh, H. 1984. Strain differences in chloroform-induced
nephrotoxicity: Different rates of metabolism of chloroform to phosgene by the mouse
kidney. Drug. Metab. Dispos. 12: 304-308.
Puri, S.K., Fuller, G.C., Lai, H. 1971. Effect of chloroform inhalation on barbiturate narcosis and
metabolism in normal and phenobarbital pretreated rats. Pharmacol. Res. Comm. 3: 247-
254.
Schwetz, B.A., Leong, B.K.J., Gehring, P.J. 1974. Embryo- and fetotoxicity of inhaled
chloroform in rats. Toxicol. Appl. Pharmacol. 28: 442-451.
SDU Uitgevers [under the auspices of the Ministry of Social Affairs and Employment], 2000.
The Hague, The Netherlands.
Smith, A.A., Volpitto, P.P., Gramling, Z.W., DeVore, M.B., Glassman, A.B. 1973. Chloroform,
halothane, and regional anesthesia: a comparative study. Anesth. Analg. 52: 1-11.
Smyth, H.F., Carpenter, C.P., Weil, C.S., Pozzani, U.C., Striegel, J.A. 1962. Range-finding
toxicity data: list VI. Am. Ind. Hyg. Assoc. J. 23: 95-107.
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Snyder, R., Andrews, L.S. 1996. Toxic Effects of Solvents and Vapors. In: Klaassen, C.D.,
Amdur, M.O., Doull, J., Eds. Casarett and Doull's Toxicology: The Basic Science of
Poisons, 5th ed. McGraw Hill, New York. p. 748.
Templin, M.V., Larson, J.L., Butterworth, B.E., Jamison, K.C., Leininger, J.R., Mery, S., et al.
1996a. A 90-day chloroform inhalation study in F-344 rats: Profile of toxicity and relevance
to cancer studies. Fundam. Appl. Toxicol. 32: 109-125.
Templin, M.V., Jamison, K.C., Sprankle, C.S., Wolf, D.C., Wong, B.A., Butterworth, B.E.
1996b. Chloroform induced cytotoxicity and regenerative cell proliferation in the kidneys
and liver ofBDFi mice. Cancer Letters 108: 225-231.
ten Berge, W.F., Zwart, A., Appelman, L.M. 1986. Concentration-time mortality response
relationship of irritant and systemically acting vapours and gases. J. Hazard. Materials: 13:
301-309.
U.S. EPA (United States Environmental Protection Agency), 1992a. Reference Guide to Odor
Thresholds for Hazardous Air Pollutants Listed in the Clean Air Act Amendments of 1990.
Office of Research and Development, Washington, D.C. EPA/600/R-92/047, March 1992.
U.S. EPA (United States Environmental Protection Agency). 1992b. Chloroform
Carcinogenicity Assessment. Integrated Risk Information System.
U.S. EPA (United States Environmental Protection Agency). 2005. Chloroform:
Carcinogenicity Assessment. Integrated Risk Information System. On-line retrieval
http://www.epa.gov/iris/subst/0025.htm.
van Raaij, M.T.M., Janssen, P.A.H., Piersma, A.H. 2003. The relevance of developmental
toxicity endpoints for acute limit setting. RIVM Report 601900004/2003. Rijksinstituut voor
Volksgezondheid en Milieu.
Von Oettingen, W.F., Powell, C.C., Sharpless, N.E., Alford, W.C., Pecora, L.J. 1949. Relation
between the toxic action of chlorinated methanes and their chemical and physicochemical
properties. Experimental Biology and Medicine Institute, National Institutes of Health Bull.
No 191., pp. 5-35.
Wang, P-Y., Kaneko, T., Tsukada, H., Nakano, M., Sato, A. 1997. Dose- and route-dependent
alterations in metabolism and toxicity of chemical compounds in ethanol-treated rats:
Difference between highly (chloroform) and poorly (carbon tetrachloride) metabolized
hepatotoxic compounds. Toxicol. Appl. Pharmacol. 142: 13-21.
50
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Wang, P-Y., Kaneko, T., Tsukada, H. Sato, A. 1994. Dose and route dependency of metabolism
and toxicity of chloroform in ethanol-treated rats. Arch.Toxicol. 69: 18-23.
Wennborg, H., Bodin, L., Vainio, H., Axelsson, G. 2000. Pregnancy outcome of personnel in
Swedish biomedical research laboratories. J. Occup. Envrion. Med. 42: 438-446.
Whitaker, A.M., Jones, C.S. 1965. Report of 1500 chloroform anesthetics administered with a
precision vaporizer. Anesth. Analg. 44: 60-65.
Whipple, G.H., Sperry, J. A. 1909. Chloroform poisoning - liver necrosis and repair. Bull. Johns
Hopkins Hosp. 20: 278-289. (as cited in NIOSH, 1974).
Wolf, D.C., Butterworth, B.E. 1997. Risk assessment of inhaled chloroform based on its mode of
action. Toxicol. Pathol. 25: 49-52.
Yamamoto, S., Aiso, S., Ikawa, N., Matsushima, T. 1994. Carcinogenesis studies of chloroform
in F344 rats and BDFi mice (abstract). Proc. 53rd Annual Meeting, Japanese Cancer
Association, (cited in Templin et al., 1996)
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APPENDIX A
DERIVATION OF AEGL VALUES
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CHLOROFORM
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DERIVATION OF AEGL-1 VALUES
AEGL-1 values were not recommended by the NAC/AEGL due to properties of the chemical.
Based upon the available data it was not possible to identify a definitive effect consistent with
the AEGL-1 definition. Exposures to concentrations approaching those inducing narcosis or
hepatic and renal effects are not accompanied by overt signs or symptoms. Furthermore, the
odor of chloroform is not unpleasant or irritating.
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DERIVATION OF AEGL-2
Key study: Schwetz et al., 1974
Toxicity endpoint: absence of developmental effects in rats
Scaling: Cn x t = k , where n= 1 or 3. Data were unavailable for empirically
determining the exponent "n". The concentration-exposure time
relationship for many irritant and systemically acting vapors and gases
may be described by C1 x t = k, where the exponent n ranges from 1 to 3.5
(ten Berge et al., 1986). In the absence of chemical-specific data,
temporal scaling was performed using n = 3 when extrapolating to shorter
time points and n= 1 when extrapolating to longer time points using the
C" xt = k equation:
(100 ppm)1 x 7 hrs = 700 ppm-hr
(100 ppm)3 x 7 hrs = 7,000 ppm-hr
Uncertainty factors: No interspecies uncertainty factor was applied because the available
metabolism/kinetics data and PB-PK models (Corley et al., 1990) indicate
that humans may be less sensitive than laboratory animals to the toxic
effects of chloroform. Additional adjustments were considered
unnecessary because a single 7-hour exposure was utilized for AEGL-2
development rather than the full exposure period specified in the study
protocol (7 hrs/day on gestation days 6-15).
An intraspecies uncertainty factor of 3 was applied to account for
individual variability in metabolism and disposition of chloroform.
Additional adjustment was not made because the point of departure
(embryolethality) and assumption of a single-exposure effect was
considered conservative.
Total uncertainty factor application of 3 was applied.
10-min AEGL-2
C3 x 0.1667 hr = 7,000,000 ppm-hr
C = 348 ppm
10-min AEGL-2 =348 ppm/3 =116 ppm (rounded to 120 ppm)
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30-min AEGL-2
1-hr AEGL-2
4-hr AEGL-2
8-hr AEGL-2
C3 x 0.5 hr = 7,000,00 ppm-hr
C =241 ppm
30-min AEGL-2 = 241 ppm/3 = 80.3 ppm (rounded to 80 ppm)
C3 x 1 hr = 7,000,000 ppm-hr
C = 191 ppm
1-hr AEGL-2 =191 ppm/3 =63.7 ppm (rounded to 64 ppm)
C3 x 4 hrs = 7,000,000 ppm-hr
C = 121 ppm
4-hr AEGL-2 =121 ppm/3 =40.3 ppm (rounded to 40 ppm)
C1 x 8 hrs = 700 ppm-hr
C = 87.5 ppm
8-hr AEGL-2 = 87.5 ppm/3 = 29.2 ppm (rounded to 29 ppm)
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DERIVATION OF AEGL-3
Key study: Based upon a 3-fold reduction in a 560-minute LC50 (4500 ppm) in mice
(Gehring, 1968); 4500 ppm/3 = 1500 ppm.
Toxicity endpoint: Lethality
Scaling: Cn x t = k , where n = 3. Data were unavailable for empirically determining
the exponent "n". The concentration-exposure time relationship for many
irritant and systemically acting vapors and gases may be described by C1 x t =
k, where the exponent n ranges from 1 to 3.5 (ten Berge et al., 1986). In the
absence of chemical-specific data, temporal scaling was performed using n =
3 when for extrapolating to shorter time points using the C" x t = k equation:
(1500 ppm)3 x 9.3 hrs = 3.1 x 1010 ppm3-hr
Due to uncertainties in extrapolating from a 560-minute exposure duration to
a 10-minute duration, the 10-minute AEGL-3 was set equivalent to the 30-
minute AEGL-3.
Uncertainty factors: No adjustment was made for interspecies variability (animal-to-human
adjustment) regarding the lethal response to chloroform.
Metabolism/kinetics data and PB-PK models (Corley et al., 1990; Delic et
al., 2001) indicate that humans may be less sensitive than laboratory
animals to the toxic effects of chloroform.
An intraspecies uncertainty factor of 3 was applied to account for
individual variability in metabolism and disposition of chloroform (e.g.,
induction of P-450 enzymes and subsequent enhancement of toxicity).
Comparison with available anesthesia data in humans precluded
incorporation of additional uncertainty factor adjustment.
Due to the results of PBPK models (Corley et al., 1990; Delic et al., 2001)
showing that mice are considerably more sensitive (25 to 50-fold
difference in rate of metabolism of chloroform) to the toxic effects of
inhaled chloroform than are humans, an additional adjustment factor of
1/3 has been applied resulting in overall net adjustment of 1.
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10-min AEGL-3
The 10-min value is set equivalent to the 30-minute value (4000 ppm) to
minimize uncertainty associated with extrapolation from the 560-minute
exposure duration for the point-of-departure.
30-min AEGL-3
C3 x 0.5 hr = 3.1 x 1010 ppm3-hr
C = 3,979 ppm
30-min AEGL-3 =3,979 ppm/1 =3,979 ppm (rounded to 4000 ppm)
1-hr AEGL-3
C3x 1 hr = 3.1 x 1010 ppm3-hr
C =3158 ppm
1-hr AEGL-3 =3158 ppm/1 =3158 ppm (rounded to 3200 ppm)
4-hr AEGL-3
C3 x 4 hrs = 3.1 x 1010 ppm3-hr
C = 1989 ppm
4-hr AEGL-3 = 1989 ppm/1 = 1989 ppm (rounded to 2000 ppm)
8-hr AEGL-3
C3 x 8 hrs = 3.1 x 1010 ppm3-hr
C = 1579
8-hr AEGL-3 = 1579 ppm/1 = 1579 ppm (rounded to 1600 ppm)
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CHLOROFORM Interim 3: 03/2009
APPENDIX B
DERIVATION SUMMARIES FOR CHLOROFORM
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CHLOROFORM Interim 3: 03/2009
ACUTE EXPOSURE GUIDELINES FOR CHLOROFORM
(CAS NO. 67-66-3)
AEGL-1 VALUES
10 minutes
30 minutes
1 hour
4 hours
8 hours
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Reference: not applicable
Test Species/Strain/Number: not applicable
Exposure Route/Concentrations/Durations: not applicable
Toxicity Endpoint: not applicable
Time Scaling: not applicable
Concentration/Time Selection/Rationale: not applicable
Uncertainty Factors/Rationale
Total Uncertainty Factor: not applicable
Modifying Factor: not applicable
Animal-to-Human Dosimetric Adjustments: not applicable
Data Adequacy: AEGL-1 values were not recommended by the NAC/AEGL due to properties of
the chemical. Based upon the available data it was not possible to identify a definitive effect
consistent with the AEGL-1 definition. Exposures to concentrations approaching those inducing
narcosis or hepatic and renal effects are not accompanied by overt signs or symptoms.
Furthermore, the odor of chloroform is not unpleasant or irritating.
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ACUTE EXPOSURE GUIDELINES FOR CHLOROFORM
(CAS NO. 67-66-3)
AEGL-2 VALUES
10 minutes
30 minutes
1 hour
4 hours
8 hours
120 ppm
80 ppm
64 ppm
40 ppm
29 ppm
Reference: Schwetz, B.A. et al., 1974.
Test Species/Strain/Number: Sprague Dawley rats; 68, 8, 22, 23, and 3 dams for the control,
pair-fed control, low-, mid-, and high-dose groups, respectively
Exposure Route/Concentrations/Durations: inhalation (whole body); 0, 30,100, or 300 ppm, 7
hrs/day on gestation days 6-15.
Toxicity Endpoint: fetotoxicity (total gross anomalies) expressed as litters affected/litters
examined
Effect
Control
Pair-fed
30 DPm
100 DDm*
300 DDm
Total gross
1/68
0/8
0/22
3/23a
0/3
anomalies
Total skeletal
46/68
3/8
20/22a
17/23
2/3
anomalies
Total soft tissue
33/68
3/8
10/22
15/23
3/3
anomalies
Reduced fetal
bw(g)
5.69
5.19
5.51
5.59
3.42a
Fetal crown/rump
length (mm)
43.5
42.1
42.5a
43.6
36.9a
><00.05
* Determinant for AEGL-2 (100 ppm); although the effects reported in the study were the
result of 7-hr exposures on gestation days 6-15, for AEGL-2, it was assumed that the effects
were the result of a single 7-hr exposure.
Time Scaling: The concentration-exposure time relationship for many irritant and systemically
acting vapors and gases may be described by C x t = k, where the exponent n ranges from 0.8 to
3.5 (ten Berge et al., 1986). In the absence of chemical-specific data, temporal scaling was
performed using n = 3 when extrapolating to shorter time points and n = 1 when extrapolating
to longer exposure durations.
Concentration/Time Selection/Rationale: a 7-hr exposure to 100 ppm was selected based upon
total anomalies occurring in rat fetuses from dams exposed on gestation days 6-15. The mid
dose was chosen in conjunction with the assumption of a single 7-hr exposure. The fetotoxicity
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endpoint is considered to represent a sensitive indicator of potential serious and irreversible
effects in a susceptible population.
Uncertainty Factors/Rationale:
Total Uncertainty Factor: 3
Interspecies: none; available metabolism/kinetics data and PB-PK models (Corley et al.,
1990) indicate that humans are less sensitive than rats to the toxic effects of
chloroform.
Intraspecies: 3; to account for individual variability in metabolism and disposition of
chloroform and protection of individuals with altered
metabolism/disposition(e.g.,users of alcohol); the fetus is considered a
sensitive population and, therefore, no additional reduction is warranted.
Modifying Factor: none
Animal-to-Human Dosimetric Adjustments: insufficient data
Data Adequacy: AEGL-2 development used a conservative approach to select the point of
departure (assumption of a single 7-hr exposure). The values are considered to be protective of
human health consistent with the AEGL-2 definition.
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ACUTE EXPOSURE GUIDELINES FOR CHLOROFORM
(CAS NO. 67-66-3)
AEGL-3 VALUES
10 minutes
30 minutes
1 hour
4 hours
8 hours
4000 ppm
4000 ppm
3200 ppm
2000 ppm
1600 ppm
Reference: Gehring, 1968
Test Species/Strain/Number: 4500-ppm LCtso of 560 minutes (540 -585 minutes, 95%
C.I.) for female Swiss-Webster mice (20/group)
Exposure Route/Concentrations/Durations: inhalation/various time frames and
exposures utilized
Toxicity Endpoint: lethality threshold estimated as 3-fold reduction of the 560-minute
LC50 of 4500 ppm
Time Scaling: The concentration-exposure time relationship for many irritant and
systemically acting vapors and gases may be described by C xt = k (ten Berge et al.,
1986), where the exponent n ranges from 0.8 to 3.5. In the absence of chemical-specific
data, temporal scaling was performed using n = 3 when extrapolating to shorter time points.
Concentration/Time Selection/Rationale: estimated lethality threshold for 4-hour
exposure (3-fold reduction in the 4-hr LC50 of 9780 ppm)
Uncertainty Factors/Rationale:
Total Uncertainty Factor: 1
Interspecies: No adjustment; currently available data indicate that laboratory
species metabolize chloroform more rapidly than do humans and,
therefore, are likely to be more susceptible to the toxic effects of the
more rapidly formed toxic intermediates. PB-PK models (Corley et
al., 1990) justify the adequacy of the uncertainty factor.
Intraspecies: 3 to account for individual variability in the sensitivity to chloroform-
induced toxicity (e.g., alcohol-potentiated hepatotoxicity)
An additional adjustment (weight-of-evidence factor) of 1/3 was applied to account for
the PBPK findings indicating that the mouse is notably more susceptible to chloroform
toxicity due metabolism factors
Modifying Factor: none applied
Animal-to-Human Dosimetric Adjustments: insufficient data
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Data Adequacy: Human lethality data are lacking and lethality data in laboratory
species are limited. However, when compared to human anesthesia data, the AEGL-3
values appear to be sufficient. PBPK models affirm that rodents, especially mice, are a
considerably more sensitive species than are humans to the toxic effects of chloroform
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CHLOROFORM Interim 3: 03/2009
APPENDIX C
CATEGORY PLOT FOR CHLOROFORM
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CHLOROFORM
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100000
10000
1000 •£*[
I. 100
10
n
60
Chemical Toxicity - TSD All Data
Chloroform
-HJ-
~
120
180
240 300
Minutes
360
420
~
Human - No Effect
~
Human - Discomfort
Human - Disabling
o
Animal - No Effect
o
Animal - Some Lethality
Animal - Lethal
480
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CHLOROFORM Interim 3: 03/2009
APPENDIX D
CARCINOGENICITY ASSESSMENT FOR CHLOROFORM
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CANCER ASSESSMENT OF CHLOROFORM
The currently available cancer slope factor for chloroform is 2.3 x 10"5 (/ig/m3)"1 (USEPA, 1992b;
2005) and is based upon a tumorigenic response (hepatocellular carcinomas) in B6C3Fi mice
administered chloroform by gavage (NCI, 1976). Based upon this slope factor, the upper-bound unit
4 7 3 6 3 3
risks of 10" to 10" are 4x10" to 4x10" mg/m assuming an inhalation rate of 20 m /day for a 70 kg
individual. At the 10"4 risk level, the virtually safe dose (d) is 4 //g/m3.
To convert a 70-year exposure to a 24-hour exposure:
24-hr exposure = d x 25,600; where d = 4 Mg/m3
= (4 Mg/m3) x 25,600 days
= 102,400 Mg/m3 (102.4 mg/m3)
To account for uncertainty regarding the variability in the stage of the cancer process at which
carbon tetrachloride or its metabolites may act, a multistage factor of 6 is applied (Crump and Howe,
1984):
(102.4 mg/m3)/6 = 17.07 mg/m3
Therefore, based upon the potential carcinogenicity of carbon tetrachloride, an acceptable 24-hr
exposure would be 17.07 mg/m3 (3.58 ppm).
If the exposure is limited to a fraction (f) of a 24-hr period, the fractional exposure becomes 1/f x 24
hrs (NRC, 1984).
24-hr exposure = 17.07 mg/m3 (3.58 ppm)
8-hr = 51.21 mg/m3 (11 ppm)
4-hr = 102.42 mg/m3 (22 ppm)
1-hr = 409.68 mg/m3 (86 ppm)
0.5 hr = 819.36 mg/m3 (172 ppm)
The AEGL-2 values based upon acute toxicity were somewhat greater than the values derived based
on potential carcinogenicity. However, the data are compelling regarding the carcinogenic response
to chloroform being a threshold response necessitating the need for repeated exposures that result in
tissue necrosis and regeneration.
Note: A virtually safe dose of 0.01 ppm (48.7 Mg/m3) was derived by Butterworth et al. (1995) and
Wolf and Butterworth (1997) based upon a 10 ppm NOAEL in mice and the contention that the
tumorigenic response observed in mice is secondary to necrosis and regenerative cell proliferation
(i.e., a threshold response). Cancer risk based upon this approach is 12-fold less than those derived
from the 10"4 unit risk number.
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