United States Office of Wate. EPA 440/5-80-026
Environmental Protection Regulations and Standards October 1980
Agency Criteria and Standards Division
Washington DC 20460 n _ /
SEPA Ambient
Water Quality
Criteria for
Carbon Tetrachloride
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AMBIENT WATER QUALITY CRITERIA FOR
CARBON TETRACHLORIDE
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.O.C. 1976), modified, 12 £RC 1833 (D.D.C. 1979):
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
John H. Gentile, ERL-Narragansett
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects:
Cynthia Sonich (author), HERL
U.S. Environmental Protection Agency
Debdas Mukerjee (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Bonnie Smith (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Herbert Cornish
University of Michigan
Si Duk Lee, ECAO-Cin
U.S. Environmental Protection Agency
Robert McGaughy, CAG
U.S. Environmental Protection Agency
Ester Rinde, ODW
U.S. Environmental Protection Agency
Roy E. Albert, CAG*
U.S. Environmental Protection Agency
Julian Andelman
University of Pittsburgh
Joseph Borzelleca
Medical College of Virginia
Patrick Durkin
Syracuse Research Corporation
Steven D. Lutkenhoff, ECAO-Cin
U.S. Environmental Protection Agency
Mike Norvell
Mobil Oil Company
Alan Rubin
U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell,. P. Gray, B. Gardiner.
*CAG Participating Members: Elizabeth L. Anderson, Larry Anderson, Ralph Arnicar,
Steven Bayard, David L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman,
Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosenblatt,
Dharm V. Singh, and Todd W. Thorslund.
IV
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TABLE OF CONTENTS
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Chronic Toxicity B-l
Plant Effects B-2
Residues B-2
Summary B-2
Criteria B-2
References B-8
Mammalian Toxicology and Human Health Effects C-l
Exposure C-l
Ingestion from Water C-l
Ingestion from Food C-2
Inhalation C-9
Dermal C-l2
Pharmacokinetics C-12
Absorption C-12
Distribution C-17
Metabolism C-19
Excretion C-23
Effects C-24
Acute, Subacute and Chronic Toxicity C-24
Synergism and/or Antagonism C-33
Teratogenicity C-35
Mutagenicity C-36
Carcinogenicity C-36
Criterion Formulation C-77
Existing Guidelines and Standards C-77
Current Levels of Exposure C-83
Special Groups at Risk C-86
Basis and Derivation of Criteria C-87
References C-91
Appendix I C-lll
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CRITERIA DOCUMENT
CARBON TETRACHLORIDE
CRITERIA
Aquatic Life
The available data for carbon tetrachloride indicate that acute toxicity
to freshwater aauatic life occurs at concentrations as low as 35,200 yg/1
and would occur at lower concentrations among species that are more sensi-
tive than those tested. No data are available concerning the chronic toxic-
ity of carbon tetrachloride to sensitive freshwater aauatic life.
The available data for carbon tetrachloride indicate that acute toxicity
to saltwater aauatic life occurs at concentrations as low as 50,000 yg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
carbon tetrachloride to sensitive saltwater aauatic life.
Human Health
For the maximum protection of human health from the potential
carcinogenic effects due to exposure of carbon tetrachloride through
ingestion of contaminated water and contaminated aauatic organisms, the
ambient water concentrations should be zero based on the non-threshold
assumption for this chemical. However, zero level may not be attainable at
the present time. Therefore, the levels which may result in incremental
increase of cancer risk over the lifetime are estimated at 10~5, 10~6,
and 10~ . The corresponding recommended criteria are 4.0 yg/1, 0.40ug/l,
and 0.04 yg/1, respectively. If the above estimates are made for
consumption of aauatic organisms only, excluding consumption of water, the
levels are 69.4 yg/1, 6.94 yg/1, and 0.69 yg/1 respectively.
VI
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INTRODUCTION
Carbon tetrachloride (CC1J is a haloalkane with a wiae range of in-
dustrial and chemical applications. The chemical is also known as tetra-
chloromethane and perchloromethane. Approximately 423,000 metric tons
(932.7 million pounds) are produced each year at 11 plant sites in the U.S.
(U.S. EPA, 1977; John, 1976). Most of this chemical is used in the manufac-
ture of fluorocarbons (95 percent in 1973), which were once used primarily
as aerosol propellants. Carbon tetrachloride is used as a component of fire
extinguisher solutions and as an industrial and chemical solvent. Its use
in grain fumigation is being largely replaced by other registered pesticide
products. Its use as a degreaser in the dry-cleaning industry has been
largely replaced by perchloroethylene (Johns, 1976).
Carbon tetrachloride has a molecular weight of 153.82, a melting point
of -22.99°C, and a boiling point of 76.54°C (Weast, 1972). It is a heavy
(density of 1.594 g/ml), colorless liquid at room temperature (Hardie,
1964). The compound is relatively nonpolar and miscible with alcohol, ace-
tone, and most other organic solvents. Its solubility in water at 25°C is
800,000 yg/1, and its vapor pressure at 10°C is 55.65 mm Hg (Hardie, 1964).
It has an octanol/water partition coefficient of 2.73 (U.S. EPA, 1978).
Carbon tetrachloride may be quite stable under certain environmental
conditions. An estimated 70,000 years are required for half of a given
quantity of CCl* to decompose in water (Johns, 1976). This decomposition
rate is considerably accelerated in the presence of metals such as iron
(Pearson and McConnell, 1975). Hydrolytic decomposition as a means of re-
moval from water appears to be insignificant as compared to evaporation.
Dill ing, et al. (1975) determined that CC14 has an evaporative half-life
of 29 minutes in water at ambient temperatures.
A-l
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Volatilization is the major transport process for removal of tetra-
chloromethane from aquatic systems. Once in the troposphere, tetrachloro-
methane remains stable; it exhibits an extremely slow rate of reaction with
hydroxyl radicals present in the troposphere. Tetrach1oromethane eventually
diffuses into the stratosphere or is carried back to the earth during the
precipitation process. Once in the stratosphere, tetrachloromethane is
degraded on exposure to shorter wavelength, higher energy ultraviolet light
to eventually form phosgene as the principal initial product (U.S. EPA,
1979).
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REFERENCES
Dilling, W.L., et al. 1975. Evaporation rates and reactivities of methyl-
ene chloride, chloroform, 1,1,1-trichloroethane, trichloroethylene, tetra-
chloroethylene, and other chlorinated compounds in dilute aqueous solutions.
Environ. Sci. Techno!. 9: 833.
Hardie, D.W.F. (ed.) 1964. Kirk-Othmer Encyclopedia of Chemical Technolo-
gy. 2nd ed. John Wiley and Sons, Inc., New York.
Johns, R. 1976. Air pollution assessment of carbon tetrachloride. Pre-
pared under contract for U.S. Environ. Prot. Agency. Mitre, Corp., McLean,
Virginia.
Pearson, C.R. and G. McConnell. 1975. Chlorinated C^ and C2 hydrocar-
bons in the marine environment. Proc. Royal Soc. London B. 189: 305.
U.S. EPA. 1977. Determination of sources of selected chemicals in waters
and amounts from these sources. Area 1. Task 2. Final Rep. Contract No.
68-01-3852. U.S. Environ. Prot. Agency, Washington, O.C.
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. EPA 68-01-4646. U.S. Environ. Prot. Agency,
Washington, D.C.
U.S. EPA. 1979. Water-related environmental fate of 129 priority pollut-
ants. EPA 68-01-3852. U.S. Environ. Prot. Agency, Washington, D.C.
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Weast, R.C. (ed.) 1972. Handbook of Chemistry and Physics. CRC Press,
Cleveland, Ohio.
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Aquatic Life Toxicology*
INTRODUCTION
The majority of the acute toxicity data for carbon tetrachloride and
aquatic organisms has been determined using static procedures with unmea-
sured test concentrations. Results of these tests may underestimate the
acute toxicity of carbon tetrachloride due to its volatility. No acute or
chronic effects were observed at a concentration lower than 3,400 yg/1.
EFFECTS
Acute Toxicity
The 48-hour EC™ is 35,200 yg/1 for Daphnia magna (Table 1). The
bluegill has been tested (Oawson, et al. 1977, U.S. EPA, 1978) and the 96-
hour LC5Q values are 125,000 and 27,300 yg/1, respectively (Table 1). The
reason for this large difference is not clear, but may have been caused by
the volatility of this compound. There appears to be no great difference in
sensitivity between the two tested species. A flow-through test result for
the fathead minnow is 43,100 yg/1. However, no comment can be made concern-
ing the effect of test conditions on test results.
Only two saltwater fish and no invertebrate species have been tested and
the 96-hour LC5Q for the tidewater silversides is 150,000 yg/1 (Table 1).
The other datum is an estimated 96-hour LC5Q for the dab of about 50,000
yg/1 (Table 4).
Chronic Toxicity
No chronic test has been conducted with a freshwater invertebrate spe-
cies or any saltwater species. An embryo-larval test with the fathead min-
*The reader is referred to the Guidelines for Deriving Water Quality Crite-
ria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various measures of tox-
icity as described in the Guidelines.
B-l
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now (U.S. EPA, 1978) was conducted, and no adverse effect was observed at
carbon tetrachloride concentrations up to 3,400 wg/1 (Table 2).
Plant Effects
There are no data describing the effects of carbon tetrachloride on any
alga or aquatic plant.
Residues
The bluegill bioconcentrated carbon tetrachloride at equilibrium to a
factor of 30 times within 21 days (Table 3). The biological half-life in
these tissues was less than 1 day. In addition, Neely, et al. (1974) ex-
posed the rainbow trout to carbon tetrachloride and estimated a steady-state
bioconcentration factor of 17 (Table 4). These results indicate that tissue
residues of carbon tetrachloride should not pose a potential environmental
hazard to aquatic life.
Summary
Only two freshwater fish and one invertebrate species have been acutely
tested and a 96-hour LC5Q has been determined as low as 27,300 yg/1. No
definitive chronic data are available. Tissue residues of carbon tetra-
chloride do not appear to be a problem since available data suggest a bio-
concentration factor of less than 30.
CRITERIA
The available data for carbon tetrachloride indicate that acute toxicity
to freshwater aquatic life occurs at concentrations as low as 35,200 wg/l
and would occur at lower concentrations among species that are more
sensitive than those tested. No data are available concerning the chronic
toxicity of carbon tetrachloride to sensitive freshwater aquatic life.
The available data for carbon tetrachloride indicate that acute toxicity
to saltwater aquatic life occurs at concentrations as low as 50,000 yg/1 and
R-?
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would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
carbon tetrachloride to sensitive saltwater aquatic life.
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Table 1. Acute values for carbon tetrad)IorIda
Species Method*
ta
i
Cladoceran,
Daphnla magna
LC50/EC50 Species Acute
lvo/1) Value (uq/l)
FRESHWATER SPECIES
S, U 35,200 35.200
Fathead minnow, FT, M
Plmephales prone Ias
Blueglll, s, U
Lepcxnls macrochlrus
Blueglll, S, U
Lepomls macrochlrus
Tidewater sllversldes, S, U
Men IdI a bery111na
43,100 43,100
125,000
27,300 58,000
SALTWATER SPECIES
150,000 150,000
Reference
U.S. EPA, 1978
Klmball, manuscript
Dawson, et al. 1977
U.S. EPA, 1978
Dawson, et a I. 1977
* S = static, FT = flow-through, U = unmeasured, M = measured
No Final Acute Values are calculable since the minimum data base requirements are not met.
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tt)
Oi
Table 2. Chronic values for carbon tetrachlorlde (U.S. EPA, 1978)
Chronic
Limits Value
Species Method* (i»q/l) tug/I)
FRESHWATER SPECIES
Fathead minnow, E-L >3,400 >3,400
Plmephales promelas
* E-L = embryo-larva I
No acute-chronic ratio Is calculable.
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Table 3. Residues for carbon tetrachlorlde (U.S. EPA, 1978)
BloconoentratIon Duration
sPecles Tissue Factor (days)
FRESHWATER SPECIES
Blueglll, whole body 30 21
Lepomls macrochlrus
B
I
TV
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Table 4* Other data for carbon tetrachlorlde
Result
Species Duration Effect (ug/l) Reference
FRESHWATER SPECIES
01
I
Rainbow trout, - Estimated steady- - Neely, et al. 1974
Salmo galrdnerl state bl oconcentra-
tlon factor = 17
SALTWATER SPECIES
Dab, 96 hrs LC50 ca. 50,000 Pearson &
Llmanda 11manda McConnefl, 1975
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REFERENCES
Dawson, G.W., et al. 1977. The acute toxicity of 47 industrial chemicals
to fresh and saltwater fishes. Jour. Hazard. Mater. 1: 303.
Kimball, G. The effects of lesser known metals and one organic to fathead
minnows (Pimephales promelas) and Daphnia magna. (Manuscript).
Neely, W.B., et al. 1974. Partition coefficient to measure bioconcentra-
tion potential of organic chemicals in fish. Environ. Science and Tech.
8: 1113.
Pearson, C.R. and G. McConnell. 1975. Chlorinated C, and C2 hydrocar-
bons in the marine environment. Proc. Royal Soc. London, Ser. B. 189: 305.
U.S. EPA. 1978. In-depth studies of health and environmental impacts of
selected water pollutants. Contract No. 68-01-4646, U.S. Environ. Prot.
Agency, Cincinnati, Ohio.
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Mammalian Toxicology and Human Health Effects
EXPOSURE
Humans are exposed to carbon tetrachloride (CC14) and other
nonfluorinated haloraethanes by three primary routes: intake of
water and other fluids, inhalation, and ingestion of foodstuffs
(National Research Council (NRG), 1978). Of all of the nonfluor-
inated halomethanes, only carbon tetrachloride and chloroform have
been observed or studied extensively in all three of these primary
routes. It is not known if humans absorb CC14 more efficiently
when exposed via one of these routes versus another. However, it
appears as though inhalation is generally the most important route
of entry (NRG, 1978). Absorption through the skin is a common
route in occupational exposures. The National Institute for Occupa-
tional Safety and Health (NIOSH) estimates that 160,000 people are
potentially exposed to carbon tetrachloride in their working envi-
ronment (NIOSH, 1975).
In the early 1920's, CC14 was used as an effective oral an-
thelmintic (NIOSH, 1975; Hall, 1921a,b). This is the only known
intentional oral exposure aside from suicidal attempts.
Ingestion from Water
Carbon tetrachloride has been found in many sampled waters
(including rain, surface, potable, and sea) in the sub-part per
billion (sub-ppb) range (McConnell, et al. 1975). Low levels have
even been detected in snow. In the National Organic Reconnaissance
Survey (NORS) of 80 cities CC1, was found in 10 percent of the U.S.
drinking water supplies at levels less than 2 to 3 ug/1 (Kopfler,
C-l
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e_t al. 1976). In New Orleans, CC14 was found in drinking water.
However, CC14 was found in only four samples of raw water with a
maximum concentration of 4 jug/1. Confirmatory analyses performed
by an alternate analytical technique indicated a level of 2 jug/1.
A more recent drinking water study, the National Organics Mon-
itoring Survey (NOMS), sampled 113 public water systems and found
carbon tetrachloride at very low concentrations relative to levels
of chloroform and other organics (U.S. EPA, 1977). Positive re-
sults were noted in about 10 percent of the samples with mean val-
ues ranging from 2.4 to 6.4 pg/1 depending on sampling and analyti-
cal procedures.
Carbon tetrachloride is a chlorinated hydrocarbon. However,
unlike other members of this, group of organic chemicals, CC14 is
not produced in finished drinking water as a result of the chlori-
nation process (NRC, 1977, 1978).
Ingestion from Food
Carbon tetrachloride has been detected in a variety of food-
stuffs other than fish and shellfish in levels ranging from 1 to 20
ug/kg. A rough summation of the different food categories that
have been shown to be particularly susceptible to CC14 contamina-
tion follows (McConnell, et al. 1975).
C-2
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dairy 0.2-14.0 ug/kg
meat 7.0- 9.0 yg/kg
oils and fats 0.7-18.0 ug/kg
beverages 0.2- 6.0 ug/kg
fruits and vegetables 3.0- 8.0 ug/kg
black grapes (imported) 19.7 jug/kg
fresh bread 5.0 jug/kg
Although the highest amount of CC14 detected in food has been 19.7
ug/kg in imported black grapes, the concentration permitted by the
FAO/WHO expert committee is 50 Jig/kg for cooked cereal products
(NRC, 1978). McConnell, et al. (1975) note that there is no evi-
dence of significant bioaccumulation of CC14 via the food chain to
higher trophic levels.
Carbon tetrachloride is used as a food fumigant either alone
or in admixture with ethylene dichloride (EDC), ethylene dibromide
(EDB), methyl bromide (MB), and/or other solvents. Some common
commercial mixtures cited by a National Research Council report
(1978) are given in Table 1.
Residues of CC1. have been found in commercially fumigated
wheat, corn, and milo in amounts ranging from 2.9 to 20.4 mg/kg
after 1 to 3 hours of storage (McMahon, 1971). Residues have also
been detected in food products containing grain. From 1964 to
1966, Wit (1972) analyzed a number of samples of cereals imported
into the Netherlands. Carbon tetrachloride residues ranged from
0.1 to 1.0 mg/kg in 20 percent of the samples, 0.5 to 1.0 mg/kg in 5
percent of the samples, 1.0 to 5.0 mg/kg in 8 percent of the
C-3
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TABLE 1
Commercial Mixtures of Fumigants Containing
Carbon Tetrachloride*
Mixture Number Chemical Contents Amounts (%)
1 ethylene dichloride (EDC) 75
carbon tetrachloride (CC1.) 25
2 carbon tetrachloride (CC1.) 80
carbon disulfide (CS,) 20
3 carbon tetrachloride (CC1.) 60
ethylene dichloride (EDC) 35
ethylene dibromide (EDB) 5
4 trichloroethylene (CHC1=CC1,) 64
carbon disulfide (CS~) 26
carbon tetrachloride (CC1.) 10
5 chloroform (CHC1-) 37
trichloroethylene (CHC1=CC1,) 32
carbon disulfide (CS2) 26
carbon tetrachloride (CC14) 5
*Source: Bielorai and Alumot, 1966
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samples, and greater than 5.0 mg/kg in 3 percent of the samples.
The maximum amount of CC14 residue found was 53 mg/kg (NRC, 1978.)
Commercially fumigated samples of flour contained CC14 resi-
dues from 0.2 to 0.3 mg/kg (Bondi and Alumot, 1972). Bread and bis-
cuits made from this same flour were free of any detectable amounts
of CC14 (i.e., less than 0.005 mg/kg).
Through laboratory experiments, researchers have managed to
simulate commercial fumigation conditions. Results similar to
those obtained from tests performed on commercially fumigated com-
modities have been obtained. A report by the National Research
Council (1978) contains a discussion of such laboratory studies
that have been performed to date. The summary of this report is
included in the following discussion.
Wit, et al. (1972) analyzed 75 kg sacks of wheat that were
fumigated with a mixture of CC14-EDC-EDB (10.2:8:1 by weight) and
then were aerated for several weeks. Carbon tetrachloride residues
within the sacks ranged from 20 to 62 mg/kg. White flour processed
from this wheat had residues ranging from 2 to 10 mg/kg, and bread
from this wheat had residues up to 0.007 mg/kg.
Scudamore and Heuser (1973) analyzed wheat and corn samples
following 3 to 6 days of application by vaporization of a weighed
amount of CC14 sufficient to give an average vapor concentration of
80 mg/1 air. The initial residues ranged from 200 to 400 mg/kg.
After six months of aeration the residues ranged from 1 to 10 mg/kg.
Residues up to 4.7 mg/kg were found in whole kernel wheat after 12
months aeration, indicating that carbon tetrachloride residues can
be very persistent.
C-5
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Bielorai and Alumot (1966) analyzed wheat and barley following
CC14 treatment at 40 gm of commercial mixture No. 5 (Table 1) per m3
of air for 72 hours. The initial residues of 1.53 and 2.2 mg/kg in
wheat and barley, respectively, decreased to 0.7 and 0.6 mg/kg by
day 17. In 1969, Alumot and Bielorai (1969) analyzed several
grains fumigated with commercial mixture No. 5 (Table 1) and aired
at different temperatures. These data were further analyzed in
1975 (Bielorai and Alumot, 1975) along with new data on carbon
tetrachlorlde and other fumigants from both laboratory and field
studies. They found more rapid desorption of fumigant residues
from whole cereal grain aired at low (15°C) rather than high (30°C)
temperatures, although the absorption rate of CC14 was less temper-
ature dependent than those of the other fumigants. However, for
ground grains the temperature effect was annulled, indicating that
grain structure is the principal factor involved. Bielorai and
Alumot (1975) suggest that unchanged fumigant residues are present
in two forms, loosely and firmly bound, and that the loosely bound
desorbs rapidly while the firmly bound desorbs more slowly and is
the temperature dependent component.
Lynn and Vorches (1957) reported carbon tetrachlorlde residues
in fumigated wheat and wheat fractions that were analyzed before
and after treatment with commercial fumigant mixtures No. 2 and No.
3 (Table 1) at dosages recommended by the U.S. Dept. of Agricul-
ture. The normal dosage for each mixture is 2 gallons/1,000 bush-
els. The residues found are given in Table 2 (mixture numbers re-
ferred to in the table are described in Table 1).
C-6
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TABLE 2
CC1. Residues, parts per million (mg/kg)
Normal Dosage Normal Dosage
Before #2 Commercial #3 Commercial
Product Fumigation Mixture .Mixture
(CC14-CS2) (CC14-EDC-EDB)
Wheat (soft) 0.5 115 (76 ppm org. Cl)
Flour 0.5 21 10
Shorts 39 28
Bran 88 43
Triple Dosage Triple Dosage
, Before #2 Commercial #3 Commercial
product Fumigation Mixture Mixture
(CC14-CS2) (CC14-EDC-EDB)
Wheat (soft) 0.5 270 140
Flour 0.5 74 34
Shorts 79 72
Bran 67 204
*
Source: Lynn and Vorches, 1957
C-7
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Berck (1974) analyzed wheat, wheat fractions, and bread frac-
tions for carbon tetrachloride residue following treatment with
Dowfume EB-5® (CC14, EDC, EDB; 63:30:7 percent respectively, by
weight) at a dosage of four imperial gallons/1,000 bushels. Sam-
ples were analyzed after different periods of aeration. The high-
est residues were found in the fumigated wheat; they ranged from
72.6 mg/kg (1-week aeration) to 3.2 mg/kg (7-weeks aeration). The
wheat fractions had residues ranging from 0.20 to 0.93 mg/kg for
flour, 0.43 to 3.53 mg/kg for bran, and 0.20 to 1.65 mg/kg for
middlings. Bread made from wheat aerated for three days had res-
idues of about 0.04 mg/kg in the upper and lower crusts and 0.13
mg/kg in the crumbs. In bread made from wheat aerated for seven
weeks, the upper crusts had no residue, the lower crust had 0.2
mg/kg, and the crumbs had 0.01 mg/kg.
Results of these studies summarized by the National Research
Council (1978) indicate that the amount of carbon tetrachloride
residue depends on the fumigant dosage, storage conditions, length
of aeration, and extent of processing. Usually, proper storage and
aeration reduce CC14 residues to trace amounts. However, several
studies indicate that, despite prolonged aeration and proper stor-
age conditions, the residues may persist at low levels for as long
as a year.
A bioconcentration factor (BCF) relates the concentration of a
chemical in aquatic animals to the concentration in the water in
which they live. The steady-state BCFs for a lipid-soluble com-
pound in the tissues of various aquatic animals seem to be propor-
tional to the percent lipid in the tissue. Thus, the per capita
ingestion of a lipid-soluble chemical can be estimated from the per
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capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and shellfish, and a steady-state BCF
for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States were analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the per capita con-
sumption of freshwater and estuarine fish and shellfish in the
United States is 6.5 g/day (Stephan, 1980). In addition, these
data were used with data on the fat content of the edible portion of
the same species to estimate that the weighted average percent
lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
A measured steady-state bioconcentration factor of 30 was ob-
tained for carbon tetrachloride using bluegills (U.S. EPA, 1978).
Similar bluegills contained an average of 4.8 percent lipids
(Johnson, 1980). An adjustment factor of 3.0/4.8 = 0.625 can be
used to adjust the measured BCF from the 4.8 percent lipids of the
bluegill to the 3.0 percent lipids that is the weighted average
bioconcentration factor for carbon tetrachloride and the edible
portion of all freshwater and estuarine aquatic organisms consumed
by Americans is calculated to be 30 x 0.625 = 18.75.
Inhalation
Carbon tetrachloride has been measured extensively in the
atmosphere; its distribution is well understood. Of all the non-
fluorinated halomethanes, CC14 has been studied most extensively.
Historically, CC1. was used as an inhalation anesthetic
(NIOSH, 1975). Smith (1867) reported the results of 52 cases in
-------
which carbon tetrachloride was the anesthetic agent. He concluded
that it was useful in removing pain while producing no nausea or
sickness (NIOSH, 1975).
The occurrence of CC14 in the atmosphere is due largely to its
volatility. A number of researchers have measured the amounts of
CC14 in the atmosphere. Their results are given in Table 3. It is
evident that there are no major gradients in the atmospheric dis-
tribution of CC14 between the continental and marine air masses.
Comparison of the amounts of CC14 in the Southern Hemisphere re-
veals a slightly lower concentration than that found in the North-
ern Hemisphere (NEC, 1978); however, the gradient is decreasing as
emissions in the more industrialized Northern Hemisphere have
stabilized with the new era of environmental awareness. Thus, the
global atmospheric distribution is approaching homogeneity. Some
extremely high CC14 concentrations have been reported in urban air.
An average annual amount of 0.0091 mg/m3 was found in Tokyo between
April 1974 and April 1975 (Ohta, et al. 1976). This was the high-
est level ever measured over an extended period of time; the data
have not been confirmed (NEC, 1978). The maximum quantity measured
in the atmosphere was detected by Lillian, et al. (1975); they
found 0.117 mg/m in Bayonne, New Jersey. Su and Goldberg (1976)
detected a high CC14 concentration in Grenoble, France: 0.0098
mg/m .
CC14 is primarily of anthropogenic orgin (Altshuller, 1976;
Lovelock, et al. 1974; Wilkniss, et al. 1973; Singh, et al. 1976).
Of all of the halocarbons, it is the most widely distributed (NRG,
1978).
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O
I
TABLE 3
Summary of Atmospheric Concentrations
of Carbon Tetrachloride (mg/m )
Compound Continental
Carbon .00013 ±
Tetrachloride .00079 +
(CC14) .00086 +
.00075 +
.00078 +
a. Grimsrud and Rasmussen,
b. Cronn, et al. 1976
c. Pierotti and Rasmussen,
d. Singh, et al. 1976
e. Pierotti, et al. 1976
f. Cox, et al. 1976
g. Lillian, et al. 1975
h. Ohta, et al. 1976
i. Su and Goldberg, 1976
Background Marine Background Urban Range
.000065a
.000085b .000083 + .0000266 .00087 + .00013d
.000065° .00083 + .00010d .00078 + .1179
.000052d .00072 + .00007f .0091h
.0000983 .00078 + .009751
1975
1976
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Dermal
It has long been known that chlorinated aliphatic hydrocarbon
solvents can penetrate animal or human skin (Stewart and Dodd,
1964). Absorption of these solvents through animal skin has been
investigated in the laboratory, but there have been few controlled
studies of human exposure.
Historically, CC14 was used as a waterless shampoo (NIOSH,
1975). In 1909 recommendations were made to label the compound as
a poison after its use in this capacity resulted in death. It con-
tinued to be used in some European countries as a hair shampoo de-
spite further reports of serious illness and death (NIOSH, 1975).
Early in the 20th century, health hazards were being reported
from industrial uses of CC14. In Germany, Lehmann confirmed evi-
dence of "unwelcome effects" when CC14, used as a cleaning agent,
was brushed on by hand (NIOSH, 1975). In 1915, Hamilton reported
that men working with CC14 without the protection of gloves devel-
oped dermatitis on their hands and arms (NIOSH, 1975).
Information was not found on levels or frequencies of dermal
exposures. Carbon tetrachloride is no longer on the market as a
hair shampoo, and through the practice of strict safety regula-
tions, dermal exposure in the work environment has been minimal.
PHARMACOKINETICS
Much of this section is extracted from reviews compiled by
NIOSH (1975) and von Oettingen (1964).
Absorption
Carbon tetrachloride is readily absorbed through the lungs and
more slowly through the gastrointestinal tract (Nielsen and Larson,
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1965). It can also enter the body by penetration through the skin.
The rate and amount of absorption are enhanced with the ingestion
of fat (Nielsen and Larson, 1965) and alcohol (Nielsen and Larson,
1965; Folland, et al. 1976; Moon, 1950). Nielsen and Larson (1965)
found high CC1, concentrations in animal testicular fatty tissues,
liver, brain, bone marrow, and kidneys.
Lehmann and Hasegawa (1910) showed that the rate of absorption
through the lungs decreases with the duration of exposure. Von
Oettingen, et al. (1949, 1950) followed the absorption of CC14
through the lungs by determining its level in the blood at differ-
ent intervals. The results are given in Table 4.
These findings confirm the observation of Lehmann and Hasegawa
(1910) that the rate of absorption decreases gradually and finally
reaches an equilibrium. Furthermore, this equilibrium is estab-
lished earlier with high concentrations because the vascular col-
lapse and depression of respiration will later interfere with the
absorption. Lazarew (1929) suggested that the absorption of CC14
is limited because of its moderate solubility in water. Powell
(1945) determined that the distribution coefficient (concentration
in the liquid phase divided by that in the gaseous phase) between
CC14 dissolved in water and that present in air is 1.04 at 20 C and
0.46 at 37°C. The concentration in air varies between 0.33 and
3.44 mg/100 ml, corresponding to 500 and 5,000 ppm (mg/kg). He
showed that the distribution coefficient between CC1, dissolved in
blood and that present in air is constant for a given specimen
throughout the above range of concentrations and varies with dif-
ferent specimens between 3.6 and 5.2 at 20°C and between 1.8 and
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TABLE 4
Concentration of Carbon Tetrachloride in the Blood of Dogs (mg/1)
During Exposure to Concentrations of 15,000 and 20,000 mg/1 CC1. in Air
(Averages of 5 Experiments)*
TIME (in minutes)
10 30 60 90 120 150 180 210 240 300 360 420 480 Later
A. CONCENTRATION = 15,000 mg/1
£ 13.33 19.22 23.08 27.05 29.53 27.57 27.23 28.78 29.92 34.17 32.09 36.23 35.64 35.26
B. CONCENTRATION = 20,000 mg/1
17.73 18.94 29.55 33.17 33.39 35.40 36.49 - 36.20 38.10 - - - 33.5
*Source: von Oettingen, et al. 1949
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2.5 at 37°C. These findings support the observation that changes
in the CC14 level in the blood are due to physiological functional
changes.
An investigation of the absorption of CC14 from the gastro-
intestinal tract of dogs was performed by Robbins (1929). He found
that considerable quantities are absorbed from the small intestine,
lower quantities from the colon, and still lower quantities from
the stomach. From an observation by Lamson, et al. (1923) it ap-
pears that the absorption from the gastrointestinal tract may vary
with different species because it occurs more rapidly in rabbits
than in dogs.
The amount of CC1. absorbed through the skin does not appear
to be significant when compared to the other routes of entry.
Lapidus (1929) studied CC14 in the blood, liver, and brain of the
rabbit. Four animals were used in this investigation, which en-
tailed immersion of one ear of each rabbit into CC14- Ear immersion
times were 5, 6, 8, and 9 hours; precautions were taken to avoid
inhalation of CC14 vapor. Following exposure, analysis for CC14
showed that the blood contained 0.12 to 0.13 mg/g; the liver, 0 to
90 mg/g; and fat, 0 to 300 mg/g. A trace of CC14 was found in the
brain of the rabbit whose ear was immersed for 9 hours, but none was
detected in the brains of the remaining three rabbits. The lowest
detectable limit of the analytical technique used for the brain
analysis was 5 mg.
Contrary to this finding, Kionka (1931) claimed that CC14 is
not absorbed through intact or scarified skin but is absorbed quite
readily through burnt (denuded) skin. However, McCord (1932)
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administered CC14 under a leak-proof bandage placed on the clipped
abdominal skin of three animals (species not specified). He in-
jected either 7.5, 1.6, or 1.2 mg/kg CC14 under the bandages three
times per day for 7 or 8 days. The animal receiving 7.5 mg/kg CC1.
died on the seventh day. The animal receiving the 1.6 mg/kg injec-
tion died on the eighth day. The remaining animal, receiving 1.2
mg/kg CC14, was killed on the seventh day. Autopsies were per-
formed but were limited to macroscopic observations. Subcutaneous
necrosis was evident where the CC14 had been applied; the livers
showed a dark purple mottling, and inflammation was seen throughout
the body of each animal. This study suggests that CC14 is absorbed
through animal skin.
Lande and Dervillee (1936) studied Kionka's hypothesis in rats
and guinea pigs. They applied CC14 to normal, scarified, and burnt
skin and also to skin with open lesions. No evidence was found that
it was absorbed through intact or scarified skin even when applica-
tion continued for 6 to 8 hours, when applied to open lesions or
burnt areas, sufficient quantities were absorbed so as to result in
liver and kidney injury. Tabusso (1941) produced chronic CC14 poi-
soning in rabbits by daily application of a 5 percent solution in
ether to the shaved skin. Beamer, et al. (1950) studied in monkeys
the cutaneous absorption of CC14 vapors containing radioactive car-
bon. They found that after a 270-minute exposure to concentrations
of 1,150 ppm (mg/1), the radioactivity of the blood was equivalent
to 0.30 mg CC14/1 and that the expired air contained 0.003 mg/1.
These experiments show that carbon tetrachloride vapors will pene-
trate through the intact skin of animals in very small amounts.
C-16
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Larger amounts may be absorbed through the human skin (von
Oettingen, 1964).
Stewart and Dodd (1964) investigated the absorption of CC14
through human skin. Three subjects each immersed one thumb in CC14
for 30 minutes. The concentrations of CC1. in the exhaled air were
measured at 10, 20, and 30 minutes of immersion. Ranges of CC14
concentrations in the exhaled air measured at these time periods
were 0.0250 to 0.819 ug/1, 0.25 to 3.27 ug/1, and 0.69 to 5.23 ug/1,
respectively. Five hours later, CC1. was still measurable in the
exhaled air of these subjects. The investigators concluded that
the amount of CC1. that could penetrate the skin depends on the
type of skin, the area exposed, and the duration of exposure.
Using the data from the experimental exposure of one thumb, they
estimated that the amount of CC14 absorbed during topical exposure
of both hands for 30 minutes would be equivalent to a vapor expo-
sure of about 10 mg/1 for 3 hours.
Distribution
Robbins (1929) studied the distribution of CC14 in dogs after
oral administration. He found the highest concentration in the
bone marrow. The amount found in the liver, pancreas, and spleen
was one-fifth of the amount found in the bone marrow. Von
Oettingen, et al. (1949, 1950) determined the concentration of CC14
in various organs of dogs exposed to 97,500 and 130,000 mg/m CC14
in air. The results are summarized in Table 5. These findings
indicate that with inhalation, the concentration in the brain is
higher than that in the heart, liver, and blood. Findings further
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TABLE 5
Concentration of Carbon Tetrachloride in Various
Organs of Dogs Exposed to CC1. in Air*
Dog
Number
20
21
22
23
24
AVERAGE
29
27
25
28
26
AVERAGE
AVERAGE
DOGS 28
AND 26
Exposure
Time
(Minutes)
640
485
505
495
250
475
15
30
45
335
345
154
OF
340
Concentration (mg/100 ml)
Heart
A.
56.80
73.30a
40.10
48.05
49.62
53.57
(48.64)°
B.
25.53
33.00
19.30
69.80
85.09
46.54
77.44
Liver
97,500 mg/m3.
43.35
31.63
31.63
46.46
25.06
35.63
130,000 mg/m3.
9.85
25.83
17.50
60.66
57.63
34.29
59.14
Brain
71.40
62.03
48.05
74.20
67.90
64.72
29.55
44.62
32.56
84.80
82.60
54.83
83.70
Blood
44.15
31.16
32.40
29.75
30.58
33.61
19.19
21.50
10.45
43.20
33.80
25.63
38.50
*Source: von Oettingen, et al. 1949
aHeart showed fatty degeneration.
Average value excluding No. 21.
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indicate that the concentration in the brain increases with time of
exposure and the concentration of CC1. in the air.
McCollister, et al. (1950) studied the organ distribution in
monkeys after inhalation of radioactive CC1. in concentrations of
299 mg/ra for 139 and 334 minutes. They reported the following
ratios:
Blood 1.00
Depot fat 7.86
Liver 3.00
Bone marrow 2.97
Bone, lung, muscle,
spleen, heart,
kidney & brain 0.14-0.96
McConnell, et al. (1975) found CC14 in human tissues as fol-
lows (jug/kg, wet tissue):
Kidney 1-3
Liver 1-5
Body fat 1-13.6
Recknagel and Litteria (1960) worked with rats and demon-
strated that after oral administration, the liver concentration of
CC14 increases for 1.5 hours and then continuously decreases. It
appears that the organ distribution of CC14 varies with the route
of administration, its concentration, and the duration of exposure.
On the cellular level, McLean, et al. (1965) found CC14 in all cell
fractions with higher concentrations in ribosomes.
Metabolism
When CC14 is administered to mammals, it is metabolized to a
small extent (most is excreted through the lungs). The metabolites
include chloroform, hexachloroethane, and carbon dioxide (C02).
Research efforts have revealed that these metabolites play an im-
portant role in the overall toxicity of CC14. Some of the CC1.
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metabolic products are incorporated into fatty acids by the liver
and into liver microsomal proteins and lipids (Gordis, 1969).
The chemical pathology of liver injury induced by CC1. is gen-
erally viewed as an example of lethal cleavage (Recknagel and
Glende, 1973). The initial event is thought to involve the homo-
lytic cleavage of a C-C1 bond of CC14, thus liberating trichloro-
methyl and chlorine free radicals. There are two major views on
the consequences of this cleavage; both views consider the high
reactivity of the free radical products of the cleavage.
The first possibility is direct attack (via alkylation) by
toxic free radical metabolites of CC1. metabolism on cellular con-
stituents, especially protein sulfhydryl groups (Butler, 1961). In
homolytic fission, the two odd-electron fragments formed would be
trichloromethyl and monatomic chlorine free radicals (e.g.,
CC14—^CC^ +C1'). Verifying this hypothesis, Fowler (1969) de-
tected hexachloroethane (CC1-CC13) in tissues of rabbits following
CC1. intoxication.
The alternative view has emphasized peroxidative decomposi-
tion of lipids of the endoplasmic reticulum as a key link between
the initial bond cleavage and pathological phenomena characteristic
of CC14 liver injury (Tracey and Sherlock, 1968). Thus, CC14 binds
to cytochrome P-450 apoprotein and is cleaved at the locus to yield
extremely short-lived free radicals which initiate peroxidative
decomposition of polyenoic lipids (Fishbein, 1976). The auto-
catalytic decomposition of the lipid spreads from the initial locus
and lipid peroxides, and hydroperoxides probably also move to more
distant sites where they decompose to yield new free radicals
C-20
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(Fishbein, 1976). Rapid breakdown of structure and function of the
endoplasmic reticulura is due to decomposition of the lipid and to
attack on protein functional groups, especially sulfhydryl groups,
by lipid peroxides (Recknagel and Glende, 1973).
This intracellular process of lipid peroxidation has been
linked to adverse effects. Recknagel, et al. (1973) explain that
peroxidative decomposition of the membrane structural lipids dis-
rupts normal structure and function, usually catastrophically. For
example, peroxidative decomposition of red cell lipids has been
correlated with an increase in red cell permeability and hemolysis
in vitamin E deficiency.
It has been hypothesized that CC14 is the initiator of some
process which proceeds autocatalytically. A study in which rats
were treated with a variety of drugs has supplied further support
to this hypothesis. For example, administration of phenobarbital
for several days increases the available activity of the liver
microsomal drug-metabolizing enzyme system. Rats treated with such
a drug show an enhanced susceptibility to a fixed dose of CC14, and
the double bond shift in the microsomal lipids of such rats in re-
sponse to a fixed dose of CC14 is markedly enhanced. When a drug
which inhibits microsomal electron flow at the cytochrome P-450
level is administered, the toxic effect of CC14 is ameliorated
(Recknagel, et al. 1973). Studies with rats reveal that pretreat-
ment with phenobarbital to stimulate cytochrome P-450 increased
metabolism of orally administered CC14 to C02, markedly increased
fat in the liver, and increased plasma concentrations of bilirubin
(McLean and McLean, 1966; Garner and McLean, 1969).
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Pre-exposure to DDT has also been shown to increase both the
P-450 content of rat liver cells and subsequently the toxicity of
CC14 (McLean and McLean, 1966; McLean, 1971). Ethanol administra-
tion increased the activity of the liver hydroxylating enzyme sys-
tem. Ethanol pretreatment increased the necrotic liver injury due
to CC14 but had less effect on the liver fat accumulation caused by
CC14 (Wei, et al. 1971).
Litterst, et al. (1973) found that dogs chronically treated
with phenobarbital for 12 months and then given a single dose of
CC14 were affected to a greater extent than dogs given only CC14/
as indicated by increased liver triglyceride content, increased
diene conjugates, and increased serum glutamic-oxalacetic trans-
am inase (SCOT).
Klaassen and Plaa (1969) found that CC14 promotes lipid per-
oxidation in the liver of rats at oral doses of 0.3 to 1.0 ml/kg,
but not at doses of 0.1 ml/kg. Hashimoto, et al. (1968) compared
lipid peroxidation in the liver of a woman who died with massive
liver necrosis after drinking CC14 with that of a victim who died
from a traffic accident. They concluded, on the basis of liver
lipid conjugated dienes, that extensive peroxidative degeneration
occurred as a result of the CC14 poisoning.
Investigations have been made of the mechanism of action of
CC14. Nakata and Higaki (1969) summarized the action of CC14 on an
isolated perfused rat liver:
1. Temperature increase in hepatic blood flow in the
early stages and marked decrease in the advanced
stages following the toxic injury of the liver cell.
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2. Impairment of bile production.
3. Extremely low oxygen saturation of the hepatic vein
blood flow, apparently the result of deterioration
of oxygen uptake by the liver cells injured by CC1,.
4. Histological lesions comprising:
a. Eosinophilic degeneration and decrease in
amount of glycogen in the liver cell at the
inital stage of intoxication.
b. Marked swelling of the liver cell accompanied
by marked stenosis of the sinusoid at the mid-
dle zone of the liver lobule.
c. Necrosis of the liver cell at the central zone
of the liver lobule.
d. Proliferation of macrophages which obliterates
the sinusoidal lumen in the central zone of the
liver lobule at the advanced stage of CC1.
intoxication.
Excretion
Lehmann and Hasegawa (1910) stated that 78.7 percent of the
amount of inhaled CC14 is excreted through the lungs within six hours
after exposure. McCollister, et al. (1950) found that approximately
50 percent of absorbed radioactive CC1, is eliminated through the
lungs. The remaining 50 percent is eliminated in some form in the
urine and feces. According to Robbins (1929), it is not excreted,
as such, in the urine by dogs. This finding was confirmed by
Barrett, et al. (1939). All investigators agree that the largest
portion of the absorbed CC14 is rapidly excreted. Beamer, et al.
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(1950) reported that following inhalation of labeled CC1., no
radioactive CC14 was detectable in the blood after 48 hours, nor
was it detectable in the expired air after 6 days.
A study on "Drinking Water and Health" (NEC, 1977) concurs
that CC14 is primarily excreted through the lungs in both animals
and humans. A summary of this study's findings reveals that the
excretion products are 85 percent as the parent compound, 10 per-
cent carbon dioxide, and smaller quantities of other products in-
cluding chloroform.
EFFECTS
Acute, Subacute, and Chronic Toxicity
Norwood, et al. (1950) reported the occurrence of 2 fatal-
ities, 1 near fatality, 4 poisonings requiring hospitalization, and
51 mild industrial poisonings in two communities over a period of 1
year. Smyth (1935) noted 28 fatalities, 14 of which resulted from
the ingestion of CC1.; 120 acute and subacute poisonings; and 7
cases of chronic poisoning. Subsequently, 28 poisonings resulting
from CC14 ingestion (including 10 fatalities) and 202 cases from
inhalation (including 29 fatalities) have been reported. The actu-
al incidence of such poisonings is doubtless much greater because
many poisonings are not attributed to CC14 and others are not pub-
lished in the medical literature (von Oettingen, 1964).
Many poisonings have resulted from the accidental or suicidal
ingestion of CC14 or from its medicinal use as an anthelmintic.
The vast majority, however, have resulted from the inhalation of
its vapors when used as a solvent or dry cleaning agent (von
Oettingen, 1964). Still other poisonings have been the result of
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dermal exposures through the use of CC14 in shampoos (NIOSH, 1975).
Finally, some have resulted from its use in fire extinguishers
(Dudley, 1935).
Direct application of CC14 onto the human skin causes a burn-
ing and stinging sensation within 5 minutes (Oettel, 1936). The
maximum pain is reached 6 minutes later and is associated with
erythema, hyperemia, and wheal formation, later followed by vesica-
tion {Oettel, 1936).
Hall (1921a,b) demonstrated the effectiveness of CC14 as a
vermicidal agent in treatment of hookworm infestations. The usage
of CC14 in such capacity stimulated considerable research efforts
to investigate the pharmacologic and physiologic effects of CC1. on
humans (NIOSH, 1975). The effects of oral doses of CC14 as a human
anthelmintic, administered to condemned prisoners in Ceylon has
been reported (Docherty and Burgess, 1922; Docherty and Nicholls,
1923). Three of the prisoners received 4 ml CC14, two received 5 ml
CC14, and one received 5 ml plus an additional 3 ml two weeks after
the first dose. Execution of the prisoners occurred 3 to 15 days
after the CC14 administration. Autopsies were performed and the
findings varied. The livers of some showed no major microscopic or
macroscopic changes whereas the livers of others showed marked fat-
ty degeneration. From such data, a dose-response relationship
would be difficult to determine (NIOSH, 1975).
The therapeutic dose recommended for adults was 2 to 3 ml in
capsule form and 0.13 ml/year for infants and children up to 15
years of age (von Oettingen, 1964). As emphasized by von Oettingen
(1964), such doses, which are followed by doses of Epsom salts,
C-25
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have caused toxic effects only exceptionally. Horrocks (1934)
reported one fatality from its medicinal use.
However, oral poisonings have occurred to a great extent, as
reported by a number of authors (Docherty and Burgess, 1922;
Beattie, et al. 1944; NIOSH, 1975; Kirkpatrick and Sutherland,
1956; Dawborn, et al. 1961). A paraphrased summary of the symptoms
of such oral poisoning is given below (von Oettingen, 1964). Fol-
lowing ingestion of CC14, the patient experiences a burning sensa-
tion in the mouth, esophagus, and stomach. Depending upon the
dose, this is sooner or later complicated by abdominal pain, nau-
sea, and vomiting. Some patients develop hiccoughs. The tongue is
coated. These symptoms are soon followed by diarrhea, which later
may be followed by constipation and occasionally by gastric and
intestinal hemorrhages which, in rare cases, may also be seen in
the mouth and pharynx. Again, depending upon the dose along with
other factors, the patient becomes jaundiced, the liver becomes
enlarged and tender, and this may be associated with ascites and
generalized edema. Soon after the ingestion, the patient feels
dizzy, may suffer from headache and become confused, semiconscious,
and delirious. The patient may become restless and develop chorea-
tic movements. Finally, consciousness is lost and the patient pas-
ses into coma. Some patients complain of visual disturbances and
edema of the eyelids and develop hemorrhages of the sclerae. In
severe cases, circulatory disturbances may develop, characterized
by lowered or increased blood pressure, thin and rapid pulse, and
signs of congestive heart failure with cyanosis. Nakata and Higaki
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(JL969) demonstrated these types of changes in vitro, through exper-
iments with the rat liver.
Conway and Hoven (1946) point out that certain electrocardio-
graphic changes may be observed indicating degenerative processes
in the heart muscle, such as sinus bradycardia, followed by auric-
ulo-ventricular rhythm, auricular fibrillation, and sinus arrhyth-
mias. The respiration varies with the condition of the patient.
If he is in collapse, it will be rapid and shallow; if he is coma-
tose, it may be labored and dyspneic, and pulmonary edema and hem-
orrhages may develop. Eventually, disturbances develop character-
ized by polyuria and followed by oliguria which may pass into anu-
ria. The urine of such patients is rich in albumin and may contain
blood and casts. If the liver is damaged, the urine will contain
urobilinogen, urobilin, and bile pigments. The nonprotein nitrogen
level in the blood will be increased and the patient may suffer
from hypoprothrombinemia, hypochloremia, and signs of acidosis.
Death may ensue after 8 hours, or 3, 5, or 10 days, and sometimes
later.
Postmortem reports on pathological changes in patients after
the ingestion of CC14 are not numerous. McMahon and Weiss (1929)
examined a 34-year-old male alcoholic who died five days after
drinking one ounce of CC14. They discovered some reddish-brown
fluid in the abdominal cavity, early atheromatous lesions in the
heart, congested and edematous lungs with scattered petechial hem-
orrhages, enlarged and congested kidneys, marked erosion of the eso-
phagus, and a congested and enlarged fatty liver.
Acute toxicity of CC14 by inhalation for humans has been re-
ported by different investigators (Davis, 1934; Stewart, et al.
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1961; Smith, 1950; NIOSH, 1975; von Oettingen, 1964). Inhalation
studies have also been performed on animals (Adams, et al. 1952;
Prendergast, et al. 1967; Wong and DiStefano, 1966). The human
studies indicate that with single exposure to low concentrations,
there is considerable variation in symptoms among different persons
and that the acute toxicity is relatively low in contrast to that
with repeated exposure. Cases in which exposure is light may be
restricted to such symptoms as moderate irritation of the eyes,
moderate dizziness, and headache, which disappear promptly upon
discontinuation of the exposure.
The immediate effects from acute inhalation exposure to higher
concentrations of CC1. consist of the same symptoms as described
above, but in addition the patient may become nauseated and suffer
from loss of appetite, mental confusion, agitation, and the feeling
of suffocation. In severe cases, the patient may lose conscious-
ness and develop fever and chills. The tongue may be furred and the
patient may suffer from vomiting with bloody or bile-stained vomi-
tus which may last for days, colicky pain, and diarrhea with liquid
brown-black or bloody stools (von Oettingen, 1964). This tendency
for hemorrhages may also result in bleeding from the gums and nose,
hemorrhages under the skin, and macular papular rashes. The colic-
ky pain may be associated with a marked abdominal resistance simu-
lating the "acute abdomen" and thus has been mistaken for append-
icitis and peptic ulcer. Following such an acute episode, the
patient feels tired and weak and frequently suffers from headache.
The patient may develop muscular twitchings and epileptic
C-28
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convulsions. In a few instances, paralysis (hemiplegia) , and poly-
neuritis have been reported (von Oettingen, 1964).
In more severe inhalation poisoning blood pressure may be
lowered, but as renal complications develop, the blood pressure is
usually elevated and the cardiac output decreases because of in-
creased peripheral resistance. The pulse may be accelerated. In
the case of severe inhalation poisoning, the patient may collapse.
Electrocardiograms have shown changes characteristic of myocardial
injury characterized by sinus bradycardia and followed by auriculo-
ventricular rhythm, auricular fibrillation, and sinus arrhythmias
(von Oettingen, 1964).
Depending upon the condition of the patient, respiration may
be normal, rapid and shallow, or slow and labored. The latter is
evident especially if circulatory failure is imminent and pulmonary
edema develops. Thompson (1946) found that early roentgenograms of
the lungs may show pulmonary involvement.
In most instances after the severe inhalation exposure, the pa-
tient develops signs of liver injury within a few days. The pa-
tient becomes jaundiced and the liver becomes enlarged and tender.
This is toxic hepatitis, which may pass into yellow atrophy and, in
more protracted cases, eventually into cirrhosis of the liver. In
the early stages of liver injury, even before a marked enlargement
occurs and while liver function tests such as the cephalin-floccu-
lation test are still normal, the level of serum glutamic-oxala-
cetic transaminase (SCOT) may be markedly elevated (von Oettingen,
1964).
C-29
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As signs of liver injury develop, and sometimes in their ab-
sence, injury of the kidneys may dominate the clinical picture and
be responsible for early death (von Oettingen, 1964). Kittleson
and Borden (1956) characterized renal failure by three phases. The
first phase is characterized by polyuria and nocturia, which may
result in severe dehydration, followed by oliguria and finally by
diuresis. The renal injury may result in acute nephritis with
albumin, red and white cells, and casts in the urine. In some pa-
tients, the presence of acetone and sugar in the urine has been re-
ported. The oliguria may be associated with increased blood levels
of potassium, indican, phenol, cresol, creatinine, and urea; the
latter may result in uremia. In other instances, the injury may
consist in necrotizing nephrosis with comparatively little changes
in the urinary composition. The renal blood flow and glomerular
filtration rate are decreased, and the former seems to be mainly
responsible for the maintenance of oliguria, being the sequela
rather than the cause of renal failure (von Oettingen, 1964).
During the early stage of oliguria, abnormal tubular back diffusion
of the filtrate may play an important role. Oliguria may develop
as early as 24 hours or 3 to 4 days after onset of the poisoning and
may persist for 12 to 14 days and even longer (von Oettingen,
1964).
In the early stages after severe inhalation poisoning and dur-
ing the period of polyuria, the blood may show some polycythemia,
but later this may be followed by anemia and lowering of the hema-
tocrit levels because of hemodilution. The most important changes
in the blood are, however, related to the biochemical composition
C-30
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of the blood which reflects the renal and hepatic injury. As soon
as the renal injury develops, the nonprotein nitrogen and urea-
nitrogen levels in the blood are increased and may reach extremely
high values. The creatinine, indican, phenol, and cresol levels
may also be increased. In the case of liver injury, as related to
the blood, the icteric index is usually increased, and the levels
of sugar and phospholipids, along with the ratio of cholesterol
esters over cholesterol, are reduced. The prothrombin time and the
fibrinogen content may be reduced, resulting in an increased clot-
ting time. The chloride level is frequently lowered by hemodilu-
tion or severe vomiting, and the potassium level may be elevated.
This increase in potassium may contribute to ventricular fibrilla-
tion or cardiac arrest (von Oettingen, 1964).
Carbon tetrachloride poisoning can also result in blurred and
double vision. Constriction of the visual field and toxic amblyo-
pia have been reported (NIOSH, 1975; von Oettingen, 1964). Conjunc
tival hemorrhages are common. Retinal hemorrhages and exceptional
cases of the degeneration of the optic nerve have been reported
(von Oettingen, 1964).
Two cases involving the pancreas following inhalation exposure
to CC14 were reported by Jahnke (1953). Both patients became list-
less and developed hepatic and circulatory disturbances and sensi-
tivity of the pancreas to pressure. Such disturbances were long-
term and had not completely subsided after 10 months.
Chronic inhalation poisoning is the result of continued low
exposures. Cases of such occurrences have been reported by Butsch
(1932), Wirtschafter (1933), Strauss (1954), von Oettingen (1964),
C-31
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and others. The clinical picture of chronic CC1. poisoning is much
less characteristic than that of acute poisoning. Von Oettingen
(1964) has reviewed the symptoms. Patients suffering from this
condition may complain of fatigue, lassitude, giddiness, anxiety,
and headache. They suffer from paresthesias and muscular twitch-
ings and show increased reflex excitability. They may be moderate-
ly jaundiced, have a tendency to hypoglycemia, and biopsy specimens
of the liver may show fatty infiltration. Patients may complain of
lack of appetite, nausea, and occasionally of diarrhea. In some
instances, the blood pressure is lowered which is accompanied by
pain in the cardiac region and mild anemia. Other patients develop
pain in the kidney region, dysuria, and slight nocturia and have
urine containing small amounts of albumin and a few red blood
cells. Burning of the eyes and, in a few instances, blurred vision
are frequent complaints of those exposed. If these symptoms are
not pronounced or of long standing, recovery usually takes place
upon discontinuation of the exposure if the proper treatment is
received (von Oettingen, 1964).
Postmortem reports on pathological changes in patients after
inhalation of CC14 are generally limited to findings in the liver
and kidneys^ The liver may show nutmeg appearance and fatty degen-
eration even in the absence of clinical signs and symptoms of liver
injury. In other instances, centrilobular necrosis and hemorrhages
with infiltration of leukocytes and histiocytes and collapse of the
lobules with condensation of the reticular framework within these
areas are seen. After chronic exposure, there may be evidence of
regeneration of the liver cells (von Oettingen, 1964).
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Postmortem changes in the kidney are characterized by nephro-
sis, by a distention of Bowman's capsule with albuminous precipi-
tates, and by swelling of the lining cells. The cells of the con-
voluted tubules may be swollen and vacuolated; later, degenerative
changes may be seen in Henle's loops, associated with granular,
hyaline, and cellular casts in the tubules. After chronic expo-
sure, regenerative changes may be visible in these regions. In
other cases, the kidneys may offer the picture of acute hemorrhagic
nephritis (von Oettingen, 1964).
Other postmortem organ changes are less characteristic for
CC14 poisoning and vary considerably with the clinical picture.
Some changes may occur that are a direct result of the changes
occurring in the primary target organs of CC14. Stasis of various
organs is the most outstanding feature of cardiac failure. The
brain and lungs may be edematous. The intestines may be hyperemic
and covered with numerous petechial hemorrhages, and the spleen may
be enlarged and hyperemic. Occasionally the adrenal glands may
show degenerative changes of the cortex, and the heart may undergo
toxic myocarditis (von Oettingen, 1964).
Synergism and/or Antagonism
A description of the entire clinical picture of the toxicity
of CC14 should consider the role played by alcohol in the genesis
of severe CC14 poisoning (von Oettingen, 1964). A number of re-
searchers have reported on this phenomenon (Stevens and Forster,
1953; Kirkpatrick and Sutherland, 1956; Joron, et al. 1957; New, et
al. 1962; Traiger and Plaa, 1971). It has been established that
habitual ingestion of alcoholic beverages and also occasional use
C-33
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may increase the dangers from comparatively moderate exposure.
This fact is illustrated by reports on simultaneous exposure of
abstinent persons and consumers of alcohol to the same concentra-
tion with only the latter becoming seriously ill (von Oettingen,
1964).
Traiger and Plaa (1971) investigated the differences in the
potentiation of CC1, by pretreatment with methanol, ethanol, and
isopropanol in rats. The activity of serum glutamic-pyruvic trans-
aminase (SGPT) was monitored to assess the effects of hepato-
toxicity. Methanol, ethanol, and isopropanol displayed potentiat-
ing ability and produced elevated activity of SGPT. The most
marked potentiation was produced by isopropanol. The administra-
tion of the alcohols or CC1. alone did not change the levels of
SGPT.
Wei, et al. (1971) investigated the potentiation of CC1. hepa-
totoxicity by ethanol and cold. This was accomplished by pretreat-
ing rats with ethanol and exposing rats to a cold temperature (18
hours at 4°C). Indices of hepatotoxicity were SGPT levels and
liver triglyceride levels. In both male and female rats, the SGPT
levels increased after both ethanol and cold exposures in response
to the CC14. The authors postulate that the ethanol releases
norepinephrine, which increases the susceptibility of the liver to
CC1.. According to Davis (1934), very obese or undernourished per-
sons suffering from pulmonary diseases or gastric ulcers or having
a tendency to vomiting, liver or kidney diseases, diabetes, or
glandular disturbances are especially sensitive to the toxic effect
of CC1. (von Oettingen, 1964).
C-34
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As to the antagonistic compounds associated with CC1., Hafeman
and Hoekstra (1977) report a protective effect of dietary vitamin
E, selenium, and methionine against lipid peroxidation induced by
CC14. They monitored lipid peroxidation by the evolution of eth-
ane, an autoxidation product of cj-3-unsaturated fatty acids. The
authors concluded that the toxicity of CC14 decreased in correla-
tion with ethane evolution. Thus, methionine and selenium protect-
ed against CCl4~induced lipid peroxidation, probably by maintaining
intracellular glutathione and glutathione peroxidase. Vitamin E
also exhibited this protection. The authors also found that sub-
stituting cod liver oil (which is rich in (J-3-unsaturated fat) for
lard in the basal diet increased CCl4-induced ethane evolution by a
factor of six.
Teratogenicity
Data concerning the teratogenicity of CC14 are scarce.
Schwetz, et al. (1974) administered CC14 to Sprague-Dawley rats at
300 or 1,000 mg/1 for 7 hours per day on days 6 to 15 of gestation.
Results indicated that CC14 was not highly embryotoxic but that it
does cause some degree of retarded fetal development such as de-
layed ossification of sternebra. Maternal toxicity was found.
Bhattacharyya (1965) studied fetal and neonatal responses to
hepatotoxic agents. He found that subcutaneous injection of CC1.
into pregnant rats and subcutaneous and intra-amniotic injection
into fetuses only occasionally give rise to changes in fetal liver.
When changes do occur, they vary from sinusoidal dilation and con-
gestion or well-marked variability of staining of liver lobules to
occasional centrilobular or (rarely) massive necrosis.
C-35
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Mutagenicity
Very little information was found on the mutagenicity of CC1,.
Kraemer, et al. (1974) found that CC14 is not mutagenic in the Sal-
monella typhimurium or Escherichia coli reversion tests.
Carcinogenicity
In a number of studies, CC1. has been shown to be carcinogenic
in animals, the target organ being the liver. Some of these stud-
ies will be reviewed in this section.
Rueber and Glover (1967) studied cholangiofibrosis of the
liver in male and female Buffalo strain rats of varying ages. Some
of the rats were given CC1. subcutaneously, while others were fed
3-methylcholanthrene (MCA) in the diet. Cholangiofibrosis is a
lesion made up of ducts lined by irregular epithelial cells and
surrounded by connective tissue. The lesion is a precursor of
cholangiocarcinomas of the liver (Rueber and Glover, 1967).
Of the female rats injected with CC1., 4/11 52-week-old rats
(36 percent) had cholangiofibrosis. The lesion was present in
11/12 8-week-old male rats (92.5 percent), 6/11 12-week-old male
rats (55 percent), all of the 24- and 52-week-old male rats (100
percent), and 13/14 76-week-old male rats (93 percent). Cholan-
giofibrosis was less developed in the younger rats and most ad-
vanced in the oldest rats. There were atypical cells in the 24-
week-old male and 52-week-old female rats. The lesions were larger
in the 52-week-old rats.
Cholangiofibrosis was increased in 5-week-old male rats
and 24-week-old females (50 percent) given both CC14 and MCA.
C-36
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Cholangiofibrosis was decreased in male rats 8 to 76 weeks of age
receiving both chemicals.
The cholangiofibrosis in male rats given only CC1. was often
found in livers with severe cirrhosis. Male rats receiving CC1,
and MCA with severe cirrhosis developed less cholangiofibrosis. In
female rats given CC1., cholangiofibrosis was not related to the
severity of the cirrhosis. (Results are given in Table 6.)
In summary, cholangiofibrosis of the liver developed in male
and female rats receiving injections of carbon tetrachloride. The
lesion was present in male rats of all ages, except those four
weeks of age. The lesion was increased in male rats five weeks of
age given both CC1. and MCA, whereas it was decreased in rats of all
other ages. Most female rats given both chemicals also had cholan-
giof ibrosis.
Rueber and Glover (1967) also investigated hyperplastic and
neoplastic lesions of the liver. Inbred Buffalo male and female
rats 4, 12, 24, 52, and 76 weeks old were given subcutaneous in-
jections of carbon tetrachloride (CC1,) twice a week for 12 weeks.
There were 10 to 14 rats of each sex and age. Rats were given 1.3
ml/kg of body weight of a 50 percent solution of CC1. and corn oil.
Control rats, six per group, were injected with the same amount of
corn oil.
Rats survived for the 12 weeks of the study. During this per-
iod, the 52-week-old rats lost an average of 15 to 30 g, the 24-
week-old rats maintained their weight, and the 12-week-old rats
each gained from 20 to 30 g. The 4-week-old females weighed three
C-37
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TABLE 6
Cholangiofibrosis in Male and Female Rats Given Subcutaneous
Carbon Tetrachlo^ide and Methylcholanthrene*
Age
(Weeks
4-5
8
12
24
52
76
Males
) CC14
0/14
11/12
6/11
11/11
14/14
13/14
(0%)
(92%)
(55%)
(100%)
(100%)
(93%)
cci4
8/17
1/15
1/16
2/16
3/13
3/14
and MCA
(47%)
(7%)
(6%)
(13%)
(23%)
(21%)
Females
cci4
0/11
1/12
1/11
0/10
4/11
3/15
(0%)
(8%)
(9%)
(0%)
(36%)
(20%)
cci4
0/18
1/16
0/14
8/16
2/12
1/13
and MCA
(0%)
(6%)
(0%)
(50%)
(17%)
(8%)
*Source: Rueber and Glover, 1967
C-38
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times their starting weights and males weighed four times their
starting weights.
At sacrifice complete necropsies were done. All organs were
examined histologically, including such tissues as diaphragm,
tongue, and skeletal muscle. Special staining was done for gly-
cogen, mucin, connective tissue, ceroid, canaliculi, hemosiderin,
and lipid.
The males given injections at 52 weeks of age had more hyper-
plastic lesions than the other males. Six of 14 rats (43 percent)
had hyperplastic nodules with one having a small hepatic carcinoma.
The only other males with nodules, 2/11 (18 percent) were the 24-
week-old rats. The remaining 52-week-old rats, and all except for
one of the 24-week-old rats, had hyperplasia of the liver. Hyper-
plasia developed in less than half of the 12-week-old rats, whereas
most of the 76-week-old rats had hyperplasia. Hyperplastic lesions
and hyperplasia were not observed in control male rats.
The 24- and 52-week-old females had more hyperplastic nodules
than did the younger females. The most striking lesions were in
the 24-week-old rats. In this group, 8/10 rats (80 percent) had
hyperplastic nodules and one rat had a small carcinoma of the
liver. There were more hyperplastic nodules per liver and larger
lesions in the females than in the males. The lesions in the 76-
week old female rats were similar to those in the male rats. Le-
sions were not present in control female rats.
There were two kinds of hyperplastic lesions in the liver, one
located in the periportal region and the other around central
veins. Cirrhosis varied from mild to severe, but was unrelated to
C-39
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the hyperplastic lesions in individual rats. The severity and the
histologic pattern of the cirrhosis were related to age and sex.
Hyperplastic nodules are accepted by many investigators as being
preneoplastic. If the study had been continued for a longer period
of time, the hyperplastic nodules could thus have become overt tu-
mors. Results of this study are given in Table 7.
In summary, 24- and 52-week-old rats of both sexes given sub-
cutaneous carbon tetrachloride developed more hyperplastic hepatic
nodules, as well as an occasional early carcinoma of the liver,
than did rats of other ages. The number of hyperplastic lesions
per liver and the size of lesions were larger in females than in
males. Four-day-old rats died with necrosis of the liver and kid-
ney.
Rueber and Glover (1970) gave subcutaneous injections to
Japanese, Osborne-Mendel, Wistar, Black Rat, and Sprague-Dawley
stocks of male rats 12-weeks-old. The injections were of a 50 per-
cent solution of CC1. and corn oil, two times per week. The dosage
was 1.3 ml/kg of body weight. There were 12 to 17 rats in each
treatment group and 12 of each stock in the control groups. Con-
trol rats were given corn oil.
Rats were killed when they became moribund. Surviving con-
trols for each strain were killed when the last experimental rat
was killed. Complete necropsies were done. Special staining was
done for glycogen, mucin, connective tissue, ceroid, hemosiderin,
bilirubin, lipid, and fibrin.
Japanese rats survived an average of 47 weeks and Osborne-Men-
del rats for 44 weeks. Black Rats and Sprague-Dawley rats were
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TABLE 7
Lesions of the Liver in Male Rats Given
Subcutaneous Carbon Tetrachloride*
Age
(Weeks
4
12
24
52
76
. Hyperplasia
6/14
4/11
8/11
7/14
10/12
(43%)
(36%)
(73%)
(50%)
(83%)
Hyperplastic
Nodules
0/14
0/11
2/11
6/14
0/12
(0%)
(0%)
(18%)
(43%)
(0%)
Carcinoma
0/14
0/11
0/11
1/14
0/12
(0%)
(0%)
(0%)
(7%)
(0%)
Nodules Plus
Carcinoma
0/14
0/11
2/11
7/14
0/12
(0%)
(0%)
(18%)
(50%)
(0%)
Lesions of the Liver in Female Rats Given
Subcutaneous Carbon Tetrachloride*
Age
(Weeks
4
12
24
52
76
. Hyperplasia
4/11
5/11
1/10
4/11
10/13
(36%)
(45%)
(10%)
(36%)
(77%)
Hyperplastic
Nodules
0/11
3/11
8/10
6/11
2/13
(0%)
(27%)
(80%)
(54%)
(15%)
Carcinoma
0/11
0/11
1/10
1/11
0/13
(0%)
(0%)
(10%)
(9%)
(0%)
Total
Nodules Plus
Carcinoma
0/11
3/11
9/10
7/11
2/13
(0%)
(27%)
(90%)
(64%)
(15%)
*Source: Rueber and Glover, 1967
C-41
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dead in an average of 11 and 13 weeks. The average survival of
Wistar strain rats was between these two extremes, at 33 weeks.
Rats with severe cirrhosis usually died with recent hemorrhage
into the stomach and/or small intestine, as well as ascites.
The strains of rats could be divided into three distinct
groups by survival time, severity of cirrhosis, and the development
of carcinomas of the liver. Carcinomas of the liver were present
in 12/15 Japanese male rats (80 percent), 8/13 Osborne-Mendel male
rats (62 percent), and 4/12 Wistar rats (33 percent), whereas Black
Rat and Sprague-Dawley rats did not develop carcinomas, possibly
due to their short survival. Some rats which did not have carcin-
omas had hyperplastic, hepatic nodules.
The cirrhosis was most severe in rats surviving for a short
time, i.e., Black Rat and Sprague-Dawley. Japanese and Osborne-
Mendel strains tended to have mild or moderate cirrhosis. The
degree of cirrhosis in Wistar rats again was somewhere between that
of the other two groups. One-half of the animals developed severe
cirrhosis; the remaining had moderate cirrhosis. Carcinomas of the
liver developed with mild or moderate, rather than severe, cirrho-
sis.
Small carcinomas were less than 5 mm in diameter. Large car-
cinomas measured between 1.2 and 3.1 cm. The first large carcinoma
was observed after 68 weeks.
The carcinomas were usually well-differentiated, hepatocellu-
lar carcinomas in which the cells retained characteristics of nor-
mal parenchymal cells. Poorly differentiated, hepatocellular car-
cinomas had smaller cells, with basophilic cytoplasm. The cells in
C-42
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the undifferentiated carcinomas were unlike hepatic parenchymal
cells. They varied in size and shape. The nuclei were large and
the cytoplasm was basophilic. The well-differentiated carcinomas
were observed in Japanese, Osborne-Mendel, and Wistar strains; how-
ever, the less-differentiated carcinomas were seen only in Japanese
rats. There were small metastases in the lung from a well-differ-
entiated carcinoma in one Japanese rat.
Hepatic vein thrombosis was noted in two Osborne-Mendel and
two Japanese rats. Cholangiofibrosis was seen in four Osborne-
Mendel rats and one of the Japanese strain.
Spleens were enlarged. Hemangiomas of the spleen were present
in two Japanese rats and in one of the Osborne-Mendel strain.
Atrophy of the testes, prostate, and seminal vesicles was propor-
tionate to the degree of cirrhosis of the liver. There were car-
cinomas of the thyroid gland in three Osborne-Mendel and three
Japanese rats, and one Japanese rat had a subcutaneous leiomyosar-
coma. Two Osborne-Mendel and three Japanese rats had chronic renal
disease.
In summary, the development of carcinomas of the liver in rats
given subcutaneous injections of CC14 was inversely related to the
severity of cirrhosis and survival time. It appeared that Sprague-
Dawley, Black Rat, and, to a lesser extent, Wistar male rats died
from moderate or severe cirrhosis before they could develop car-
cinomas of the liver. Japanese and Osborne-Mendel male rats, on
the other hand, were less susceptible to the development of cir-
rhosis; they survived for a long period and had hepatocellular car-
cinomas of the liver; one had metastases to the lung (Table 8).
C-43
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TABLE 8
Cirrhosis, Hyperplastic Nodules, and Hepatic Carcinomas
in Male Rats Given Subcutaneous Carbon Tetrachloride*
Hyperplastic
Strain Nodules Carcii
Japanese
Osborne-
Mendel
Wistar
Black Rat
Sprague-
D aw ley
3/15
4/13
7/12
7/17
2/16
12/15
8/13
4/12
0/17
0/16
Cirrhosis
lomas Mild Moderafce
(80%) 9 5
(62%) 2 7
(33%) 0 6
(0%) 0 4
(0%) 0 0
Severe
1
4
6
13
16
Total
15/15
13/13
12/12
17/17
16/16
*Source: Rueber and Glover, 1970
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Cameron and Karunaratne (1936) looked at CC14 cirrhosis in
relation to liver regeneration in the rat. Albino rats weighing
about 150 g each were injected subcutaneously with 0.1 to 0.25 ml
carbon tetrachloride twice a week.
After 6 to 10 doses, changes which may have developed in the
liver disappeared within 7 to 10 days after cessation of treatment.
With longer periods of exposure, the liver showed less and less
tendency to return to a normal appearance when the chemical was
discontinued. Cirrhosis of the liver developed after several doses
and was severe and irreversible after 40 doses.
The liver was pale, tough, and finely granular. There was ex-
tensive fibrosis radiating from the portal areas, thereby dividing
the liver into small irregular masses. Hyperplastic nodules were
seen in different parts of the liver.
In this study, rats given subcutaneous injections of carbon
tetrachloride readily developed cirrhosis of the liver. Also,
there were presumably preneoplastic and hyperplastic nodules of the
liver.
Rueber in 1970 performed a study similar to his previous one
on the accentuation of hyperplastic and neoplastic hepatic lesions
by methylcholathrene. Inbred Buffalo strain male and female rats
5, 8, 12, 24, 52, and 76 weeks old were used. Groups of rats of
each age and sex were treated with either: (1) only carbon tetra-
chloride (CC14); (2) only 3-methylcholanthrene (MCA); or (3) CC14
and MCA simultaneously. There were 10 to 17 rats in each group.
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Laboratory meal was ingested ad libitum. At a dosage of 1.3
ml/kg body weight, CC14 was injected subcutaneously twice weekly;
0.033 percent MCA was added to the meal.
Rats given CC14 and MCA simultaneously were killed when mori-
bund or at the end of 12 weeks. Rats given only CC14 were killed
between 6 and 12 weeks to correspond to the time of death for those
receiving CC14 and MCA. Rats receiving only MCA, as well as con-
trol rats, were killed at the end of 12 weeks.
Complete necropsies were done, and all tissues were examined
histologically. Special staining was carried out for glycogen,
mucin, connective tissue, ceroid, canaliculi, hemosiderin, and
lipid.
Rats given both CC14 and MCA lost weight, whereas those given
only CC14 or MCA gained weight. Terminally, the male rats receiv-
ing both chemicals weighed 45 to 100 g less than those given CC1.;
the females weighed 20 to 55 g less. The controls and the rats in-
gesting MCA gained more weight than those on CC14.
Male rats of all ages treated with CC14 and MCA survived an
average of 9.2 weeks (range, 5.7 to 12), and the females lived an
average of 11.0 weeks (range, 6.2 to 12). Survival times of rats
receiving CC14 were similar because of the experimental procedure.
Rats fed MCA and the controls lived for 12 weeks.
Hyperplastic nodules were observed in rats 8, 24, and 52 weeks
of age (those given only CC14). There was one small, hepatic car-
cinoma in an 8-week-old rat. Hyperplastic nodules were induced by
MCA and CC14 in rats of all ages. Carcinomas were found in rats 12
weeks of age and older. The incidence of nodules and carcinomas
increased with increasing age of the rats, with one exception. The
C-46
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incidence of nodules and carcinomas was lower in the 8-week-old
male rats given both chemicals than in those treated with CC14
alone. In rats of all other ages receiving both chemicals, there
were more nodules and more carcinomas per liver. In females, the
total number of nodules and carcinomas increased with the age of
the rats.
The incidence of hyperplastic nodules in female rats 12 to 52
weeks old that received only CC14 was greater than in male rats.
Females 24 and 52 weeks of age developed the highest incidence of
nodules. In comparison to the 5-, 8-, and 12-week-old rats, 76-
week-old females were less prone to develop nodules. Males 8 weeks
old were more susceptible to the growth of nodules and carcinomas
than were females of the same age. One 24-week-old and another 52-
week-old female had early hepatic carcinomas.
Nodules and early carcinomas increased notably in females 24,
52, and 76 weeks of age that were treated with CC14 and MCA simul-
taneously. Almost all 52- and 76-week-old rats had nodules or car-
cinomas of the liver. The increase in numbers of nodules and car-
cinomas per liver was even more striking in the females than in the
males.
Control rats or rats ingesting MCA did not have hepatic
lesions. Rats given injections of CC14 had only mild or moder-
ate cirrhosis. Those fed MCA in the diet simultaneously with
injections of CC14 developed severe cirrhosis. The incidence of
hepatic vein thrombosis was markedly increased in rats given
both chemicals, except for both 8-week-old males and females.
C-47
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Cholangiofibrosis was increased in 5-week-old males and 24-week-old
females treated with both chemicals.
There was a transitional cell carcinoma of the urinary bladder
in one 8-week-old female rat receiving both chemicals. All results
are given in Table 9.
In summary, methylcholathrene increases the incidence of
hyperplastic, hepatic nodules and early carcinomas in rats of all
ages. The difference was greater in rats 12 weeks of age and older.
Females were more susceptible to the development of hyperplastic
nodules and carcinomas than were males. Multiple nodules and car-
cinomas were observed in the livers of rats given both chemicals,
whereas rats receiving only carbon tetrachloride had fewer lesions
per liver. Cirrhosis of the liver was more advanced in rats given
methylcholanthrene and carbon tetrachloride simultaneously.
A number of studies were performed utilizing mice to test for
a relationship between CC14 exposure and carcinomas. Edwards, et
al. (1942) did such an investigation. The mice used in this study
were inbred strain L (their incidence of spontaneous hepatomas is
extremely low). Mice were 2.5 to 3.5 months or 3.5 to 7.5 months of
age at the start. The number of mice varied from 8 to 39 per group.
Carbon tetrachloride of a high degree of purity was admin-
istered in olive oil by stomach tube usually three, but occasion-
ally two, times weekly. Each treatment consisted of 0.1 cc of a 40
percent solution or 0.04 ml of CC14> Mice also ingested Purina dog
chow.
Mice were given 46 administrations of CC14 over a 4-month per-
iod and were killed and necropsied 3 to 3.5 months after the last
C-48
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TABLE 9
Lesions of the Liver in Male Rats Given Subcutaneous
Carbon Tetrachloride and Methylcholanthrene*
Age
(Weeks)
5
8
12
24
52
76
cci4
4/12
2/12
4/10
6/11
5/10
10/14
HY
(33%)
(17%)
(40%)
(54%)
(50%)
(71%)
0/12
7/12
0/10
1/11
3/10
0/14
HN
(0%)
(58%)
(0%)
(9%)
(30%)
(0%)
C
0/12
1/12
0/10
0/11
0/10
0/14
Total
(0%)
(8%)
(0%)
(0%)
(0%)
(0%)
0/12
8/12
0/10
1/11
3/10
0/14
(0%)
(66%)
(0%)
(9%)
(30%)
(0%)
HY
9/17
6/15
6/16
7/16
4/13
5/14
(53%)
(40%)
(38%)
(44%)
(31%)
(36%)
5/17
4/15
4/16
3/16
5/13
5/14
cci4
HN
(29%)
(27%)
(25%)
(19%)
(38%)
(36%)
& MCA
C
0/17
0/15
1/16
1/16
2/13
2/14
(0%)
(0%)
(6%)
(6%)
(15%)
(14%)
Total
5/17 (29%)
4/15 (27%)
5/16. (31%)
4/16 (25%)
7/13 (54%)
7/14 (50%)
o
£t
vo
Lesions of the Liver in Female Rats Given Subcutaneous
Carbon Tetrachloride and Methylcholanthrene*
Age
(Weeks)
5
8
2
24
52
76
5/12
7/12
5/11
3/11
5/11
11/14
cci4
HY
(42%)
(58%)
(45%)
(27%)
(45%)
(79%)
0/12
3/12
2/11
5/11
5/11
2/14
HN
(0%)
(25%)
(18%)
(45%)
(45%)
(14%)
C
0/12
0/12
0/11
1/11
1/11
0/11
Total
(0%)
(0%)
(0%)
(9%)
(9%)
(0%)
0/12
3/12
2/11
6/11
6/11
2/11
(0%)
(25%)
(18%)
(54%)
(54%)
(18%)
HY
8/18
6/16
4/16
4/16
1/13
2/13
(44%)
(38%)
(25%)
(25%)
(8%)
(15%)
6/18
5/16
8/16
7/16
8/13
6/13
cci4
HN
(34%)
(31%)
(50%)
(44%)
(62%)
(46%)
C, MCA
C
0/18
1/16
2/16
5/16
3/13
5/13
Total
(0%)
(6%)
(13%)
(31%)
(23%)
(38%)
6/18
6/16
10/16
12/16
11/13
11/13
(34%)
(38%)
(63%)
(75%)
(85%)
(85%)
*Source: Rueber, 1970
a HY = hyperplasia; HN = hyperplastic nodule; C = carcinoma; Total = nodules plus carcinoma.
-------
treatment. The mice varied from 8.5 to 14 months of age at necrop-
sy. The liver was examined histologically.
Thirty-four of 73 mice (47 percent) given CC14 developed hepa-
tomas. The incidence of tumors in the younger mice was essentially
similar to that for the older mice, with the exception of the older
females where the incidence was considerably lower. Tumors of the
liver were observed in 7/15 younger male mice (47 percent), 21/39
older male mice (54 percent), 3/8 younger females (38 percent), and
3/11 older females (27 percent). Cirrhosis of the liver was not
mentioned.
The tumors in mice given CC14 were usually multiple, appearing
as gray or grayish-yellow, bulging nodules ranging from 2 to 15 mm
in diameter. The tumors were not encapsulated and were not inva-
sive. The adjacent hepatic tissue was compressed. Tumor cells
closely resembled the hepatic parenchymal cells. The authors re-
ported that: "The tumor cells were arranged in cords which alter-
nated with endothelial-lined sinusoids...None of the tumors ob-
served appeared to invade blood vessels, and there were no metas-
tases" (Edwards, et al. 1942).
Hepatomas have been observed in 2/152 untreated strain L mice
(1 percent). One of 23 untreated virgin male mice (4 percent) and 0
of 28 females (0 percent), necropsied at 15 months of age, had
tumors of the liver. Tumors were not present in 22 males and 28
females 18 months of age or in 27 female breeders 12 to 23 months of
age. One of 24 male breeders (4 percent) had a tumor. The results
are summarized in Table 10.
C-50
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TABLE 10
Tumors of the Liver in Male and Female Mice Receiving
Carbon Tetrachloride by Stomach Tube*
Age (Months) Males Females
2.5 - 3.5
3.5 - 7.5
7/15 (47%)
21/39 (54%)
3/8 (38%)
3/11 (27%)
2.5 - 7.5a 28/54 (52%)b 6/19 (32%)b
*Source: Edwards, et al. 1942
aThese values represent total number of tumors observed in mice in
both age groups.
Old control mice of this strain exhibit a very low incidence, as
compared to CCl.-treated mice. Hepatomas were present in 2/71
untreated males (3%) and 0/81 untreated females (0%).
C-51
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In summary, strain L male and female mice were highly suscept-
ible to the induction of hepatomas by carbon tetrachloride, and
male mice were slightly more susceptible than female mice.
Eschenbrenner and Miller (1943) studied the effects of size
and spacing of multiple CC14 doses in the induction of hepatomas.
Strain A mice were used because of their normal low incidence of
tumors of the liver in untreated mice (1 percent or less). Male and
female mice were 2.5 to 3 months old at the beginning of the study.
They ingested Purina dog chow pellets and tap water ad libitum.
Carbon tetrachloride was of a chemically pure quality and was
diluted with U.S.P. olive oil. Solutions of CC1. in olive oil were
administered by stomach tube. All mice received 30 doses of the
solution or olive oil alone. Five dilutions of CC14 were used: 32,
16, 8, 4, and 2 percent solutions. Mice received 0.005 ml of solu-
tions per g body weight containing 16 x 10~4, 8 x 10~4, 4 x 10~4,
-4 -4
2 x 10 , or 1 x 10 ml, respectively, of CC14. Central necrosis
of the liver was produced by each of these doses. Control mice
received 0.005 ml of olive oil per g body weight.
The experimental and control groups were subdivided into 5
subgroups according to the interval between successive doses (1, 2,
3, 4, and 5 days) and the total period of treatment (29, 58, 87,
116, or 145 days). Equal numbers of male and female mice were used
in each of the experimental and the five control groups. All mice
were examined for the presence of hepatoma 150 days after the first
dose. Some of them were killed at that time; others were subjected
to laparotomy. It hepatomas were not present, laparotomies were
performed at monthly intervals thereafter to determine if hepatomas
C-52
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eventually did appear. The gross diagnoses of hepatoma were con-
firmed by histological examinations.
In the lower dosage and shorter interval groups, hepatomas
were few in number and small in size. With increases in dose and
increase in interval between successive doses, there was a progres-
sive increase in the number of small hepatomas and in the size of
hepatomas for a given mouse. There was no difference in the incid-
ence of tumors of the liver between males and females.
The authors distinguished between "spontaneous" and "gross"
hepatomas on the basis of gross and histological characteristics.
The tumors were suspected of being "spontaneous" by their yellow
color, as contrasted with the pale pink color of induced hepatomas,
and were distinguished from them histologically by their architec-
ture and by the structure of their mitochondria.
In this study, the incidence of hepatomas increased with an
increase in total time during which a given amount of carbon tetra-
chloride was administered. A given incidence of hepatomas was
obtained with progressively less total amount of carbon tetra-
chloride as the duration of administration was increased.
Andervont and Dunn (1955) did a study to compare the trans-
plantability of tumors of the liver and to compare the suscepti-
bility of male mice with female mice to CC1.. Carbon tetrachloride
was administered because, in a previous study, eight tumors of the
liver, induced by CC14, failed to grow when transplanted in new
hosts.
Strain C3H mice of both sexes, three and six months of age,
were used. Litter mates were divided equally between those receiving
C-53
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azo dye, o-aminoazotoluene (used as the control agent), and those
receiving CC14. Carbon tetrachloride was dissolved in olive oil
and administered by stomach tube. The first dose of 0.25 ml of a 4
percent solution of CC14 was toxic and killed a number of mice. One
week later, 0.15 ml of the same concentration also proved to be
toxic. In the following two weeks, the dose was lowered to 0.2 ml
of a 2 percent solution, which was tolerated by the mice. Each
mouse then was given, at weekly intervals, 17 treatments of 0.2 ml
of a 3 percent solution. Mice given o-aminoazotoluene each re-
ceived, at monthly intervals, four subcutaneous injections of 10 mg
dissolved in 0.5 ml of olive oil. The mice were killed when they
were 10 to 16 months old.
A portion of the hepatoma selected for transplantation was
prepared for histologic study, and pieces of the remainder were
implanted subcutaneously by means of the trocar technique. Thirty
hepatomas from CC14-treated mice were transplanted, of which two
failed to grow. Those that grew were carried through 4 to 6 trans-
plant generations. In addition, 9/10 "spontaneous" hepatomas and
5/6 o-animoazotoluene hepatomas grew.
Eschenbrenner and Miller (1946) performed another study inves-
tigating liver necrosis and the induction of CC14 hepatomas in
strain A mice. The mice ingested Purina dog chow pellets and water
ad libitum. Five male and five female mice were used in each of
seven different doses and two control groups. Treatment with CC1.
was started when the mice were three months of age and was termi-
nated when they were seven months old. Animals were necropsied at
C-54
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eight months. All mice were given one additional dose of the solu-
tion 24 hours prior to necropsy.
The CC14 was chemically pure and was diluted with U.S.P.
olive oil. Solutions of 1.0, 0.5, 0.25, or 0.125 percent were
administered by stomach tube based on body weight.
Mice in two groups at each dose level were administered the
same total amount of CC14 over the same period of time, but with a
variation in the number of doses into which the total amount was
divided, and therefore in the size of each dose. This first group
of mice per dose was given 30 doses of CC14 (0.02 ml solution/g body
weight) at intervals of four days, whereas those in group two re-
ceived 120 daily doses (0.005 ml solution/g body weight). The
doses for mice in group one were previously determined as being
"only necrotizing," and in group two as "necrotizing" and "non-
necrotizing." Two control groups of mice received 0.02 ml or 0.005
ml of olive oil per g body weight per dose. All results are summar-
ized in Tables 11 and 12.
Mice receiving the largest dose (1 percent solution) had
multiple hepatomas up to 1 cm in diameter; those given smaller
doses (0.5 or 0.25 percent solution) had two or more smaller tu-
mors. The hepatomas were larger and greater in number in mice that
received 120 doses than in those given 30 doses of a solution (in
both groups the total amount of CC14 was the same). Gross tumors
were not observed in mice given the 0.125 percent solution; how-
ever, there were early tumors on histological examination of the
liver in two mice. Mice given olive oil did not have tumors.
C-55
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TABLE 11
Hepatomas and Necrosis in Male Mice Given
Carbon Tetrachloride by Gastric Intubations'
Dose
(% Solution)
0
1/8
1/4
1/2
1
120 Doses
Necrosis
0/5
0/5
0/5
0/5
0/4
Tumor
0/5
0/5
5/5
5/5
4/4
Nee
0/5
0/5
3/5
4/4
30 Doses
rosis Tumor
0/5
2/5a
4/5
3/4
Source: Eschenbrenner and Miller, 1946
Microscopic Tumors.
C-56
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TABLE 12
Hepatomas in Female Mice Given
i
Carbon Tetrachloride by Gastric Intubations
Dose
(% Solution)
0
1/8
1/4
1/2
1
120
Doses
Necrosis Tumor
0/5
0/5
0/5
0/5
0/5
0/5
0/5
5/5
5/5
5/5
30 Doses
Necrosis
0/5
0/5
2/5
2/4
Tumor
0/5
0/5
3/5
2/4
___
*Source: Eschenbrenner and Miller, 1946
C-57
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The presence or absence of hepatomas and of hepatic necrosis
was determined. When necrosis of the liver was found in mice with
tumors, necrosis was not observed in the hepatomas. The locali-
zation of necrosis after chronic administration of CC1. did not ap-
pear to follow a definite pattern, in contrast to the regular pat-
tern of centrolobular necrosis seen after a single dose.
In summary, mice receiving "non-necrotizing" doses of CC1
developed as many, if not more, tumors of the liver than mice given
"necrotizing" doses despite the fact that equal amounts of CC14
were administered. Mice given the 0.125 percent solution did not
have gross tumors; most mice receiving either 0.25, 0.5 or 1 per-
cent solution did have tumors.
The National Cancer Institute (NCI, 1976) performed a study in
which B6C3F-L male and female mice, 35 days of age and 50 per group,
were used. Treatment by oral gavage 5 times per week occurred for
78 weeks. Surviving mice were s'acrificed at 92 weeks from the
start of the study. The doses of CC14 were 1250 or 2500 rag/kg of
body weight for mice of both sexes. There were 20 control mice of
each sex that were given corn oil only. Mice ingested Wayne Labor-
(O\
atory Blox ^ meal. A necropsy was performed on all mice. Complete
histological examinations were carried out.
Most male and female mice treated with CC14 were dead by 78
weeks (see Table 13). Hepatocellular carcinomas were found in
practically all mice receiving CC14, including those dying before
termination of the test (see Table 14). The first carcinomas were
observed in low dose female mice at 16 weeks, in high dose female
C-58
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TABLE 13
Survival of Male Mice Treated with
Carbon Tetrachloride*
Dose Initial
Control
Matched 20
Pooled 77
Low Dose 50
High Dose 50
78 Weeks 91-92 Weeks
13 (65%) 7 (35%)
53 (69%) 38 (49%)
11 (22%) 0 (0%)
2 (4%) 0 (0%)
Survival of Female Mice Treated with
Carbon Tetrachloride
Dose
Control
Matched
Pooled
Low Dose
High Dose
Initial
20
80
50
50
78 Weeks
'18 (90%)
71 (89%)
10 (20%)
4 (8%)
91-92 Weeks
17 (85%)
65 (81%)
0 (0%)
1 (2%)
*Source: NCI, 1976
C-59
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TABLE 14
Lesions of the Liver in Male Mice Treated
with Carbon Tetrachloride*
Dose
Hyperplastic
Nodules
Carcinomas
Total
Control
Matched
Pooled
Low Dose
High Dose
0/49 (0%)
a
2/19 (11%)
35/77 (6%)
*49/49 (100%)
47/48 (98%)
49/49 (100%)
Data used for calculation of cancer risk in Criterion Formulation
section of this document.
Lesions of the Liver in Female Mice Treated
with Carbon Tetrachloride
Dose
Hyperplastic
Nodules
Carcinomas
Total
Control
Matched
Pooled
Low Dose
High Dose
0/40 (0%)
1/20 (5%)
1/80 (1%)
40/40 (100%)
43/45 (96%)
40/40 (100%)
*Source: NCI, 1976
C-60
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mice at 19 weeks, in high dose males at 26 weeks, and in low dose
males at 48 weeks, compared to 72 weeks for pooled control males
and 90 weeks for pooled control females.
Cystic endometrial hyperplasia occurred in both control and
treated female mice. Thrombosis of the atrium of the heart was
seen in 9 or 41 high dose female mice (22 percent), all of which
died with carcinomas of the liver.
In summary, this study found carbon tetrachloride to be highly
carcinogenic for liver in mice.
Edwards (1941) studied hepatomas in mice induced with CC14.
Two-hundred and seven male C3H mice, aged 3 to 6 months, and 133
male and female strain Amice, aged 2 to 3h months, were used. They
were given 0.1 ml of a 40 percent olive oil solution of carbon
tetrachlor ide (0.04 cc CC14) by stomach tube two or three times
weekly for 8 to 16 weeks. Autopsy was performed up to 21 weeks
after the last treatment.
Olive oil was administered by stomach tube in doses of 0.1 ml
two or three times weekly to control male C3H and A mice from the
same stock as those mice used in treated groups. Twenty-three
strain C3H mice were given CC14 from 39 to 50 times and were killed
and examined from 9 to 11 months of age. A high percentage of the
treated animals developed hepatomas. Of 143 C3H mice, which varied
from 6 to 10 months of age at autopsy, 126, or 88.1 percent, showed
hepatomas (Table 15). Similar tumors were present in 54 of 54
strain A mice whose ages varied from 4.5 to 12 months (Table 16).
The incidence of spontaneous hepatomas in both the C3H and A
strains is markedly below that of the induced tumors in the treated
C-61
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TABLE 15
Incidence of Tumors in C3H Mice Ingesting
Carbon Tetrachloride
Group
Controls
Controls w/Olive Oil
Treated Animals
(Olive oil and
0.04 ml CC14)
Number of
Mice
Autopsied
17
23
143
Number of
Mice with
Hepatomas
0
1
126
Incidence
of Hepatomas
in Percent
0%
4.3%
88.1%
Source: Edwards, 1941
C-62
-------
TABLE 16
Incidence of Tumors in Strain A Mice Ingesting
*
Carbon Tetrachloride
Group
Number of
Mice
Autopsied
Number of
Mice with
Hepatomas
Incidence
of Hepatomas
in Percent
Controls
Controls w/Olive Oil
(0.1 ml 2 or 3X
weekly)
Treated Animals
(Olive oil and
0.04 ml CC14)
200
22
54
54
0.5%
0%
100.0%
Source: Edwards, 1941
C-63
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mice. Autopsies performed on 17 C3H male mice 8.5 to 9 months of
age and the same stock as that used in the study failed to show any
hepatic tumors.
The tumors were occasionally solitary, but generally several
tumors were found in the same liver. They were soft pink, pinkish
gray, grayish yellow, or yellow and varied in diameter from 2 to 20
mm. The majority protruded above the capsular surface, and a few
were pedunculated. No peritoneal implants or metastases were
found. There was striking microscopic similarity to the spon-
taneous hepatoma. The tumor cells closely resembled hepatic paren-
chymal cells, as in the spontaneous tumors. The pattern of the
tumor was that of epithelial cords, two cells thick, alternating
with endothelial-lined blood sinuses. The hepatomas were usually
sharply circumscribed, through not encapsulated, and there was no
invasion of intrahepatic blood vessels.
Confer and Stenger (1966) studied nodules in the livers of C3H
mice after long-term CC14 administration. Twenty-five male mice,
five weeks of age, received rectal installations of 0.1 ml of a 40
percent solution of carbon tetrachloride dissolved in olive oil two
times a week for 20 to 26 weeks. Ten control mice were given only
olive oil. Fourteen mice were killed nine days after the last
treatment, and the remaining mice were killed at periods of 3 to 37
weeks. The livers were examined by light and electron micropsy.
Five of the 14 mice (36 percent) killed after nine days, and 8
of 11 mice (73 percent) killed later, developed hyperplastic hepa-
tic nodules. Cirrhosis was not observed in the liver. The nodules
C-64
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were grossly pale pink or white, and measured 4 to 14 mm in diame-
ter. Histologically, the cells were uniform in size and shape.
In summary, mice given carbon tetrachloride by rectal instil-
lation had hyperplastic nodules that persisted after the discontin-
uation of the chemical, but did not develop cirrhosis of the liver.
Such hyperplastic nodules are precursors of carcinoma of the liver.
In 1942, using CC1., Edwards and Dalton (1942) studied the
induction of cirrhosis of the liver and hepatomas in mice. They
investigated the outcome of high dose, low dose, and limited treat-
ment.
For high dose administration, strain C3H male mice, male and
female strain A mice, and strain C female mice were used. The mice
ingested Purina dog chow. They were started on the study when 1 to
5 months of age.
The carbon tetrachloride contained no impurities. A dose of
0.1 ml of a 40 percent solution of CC14 in olive oil was admin-
istered by stomach tube two or three times per week. The number of
treatments varied from 23 to 58, but a number of mice were killed
after receiving 1 to 23 doses in order to study the early patho-
logic changes. In another study, male mice were given 0.1 ml of
olive oil two or three times a week for 39 to 62 doses.
Animals were killed at one year of age or younger by cervical
dislocation. Subcutaneous transplants of tumor tissue were made by
the trocar technique into mice of homologous strains. Special
histological techniques were used to examine a number of primary
and transplanted tumors. These include techniques for the presence
C-65
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of fat, glycogen, or alkaline phosphatase and those for studying
the mitochondria and Golgi apparatus.
Hepatomas were observed in 88 percent of C3H male mice treated
with CC14, whereas they occur in 4 percent of untreated mice of the
same age and strain. Tumors of the liver developed in 60 percent of
male and female Y strain mice, whereas only 2 percent were seen in
untreated mice of that strain. Liver tumors were seen in 98 per-
cent of strain A mice of both sexes, whereas only 2 percent of these
mice develop the tumor spontaneously. Hepatic tumors were found in
83 percent of C strain females, compared with 0 percent of untreat-
ed mice of the same age and strain. Results of both the treated and
controls are given in Tables 17 and 18.
The hepatic tumors observed in this study were usually multi-
ple - as many as 10 occurring in one liver. They varied from 0.1 to
2.0 cm in diameter, and there was some conciliation between the
size of the tumor and the duration. The smaller tumors were red-
dish gray and bulged above the hepatic capsule. The larger tumors
were soft, either gray or yellow, and a few of these were peduncu-
lated and hung suspended from the liver by a pedicle of shrunken
hepatic tissue; there also was cirrhosis of the liver.
Microscopically, the tumors were non-encapsulated, well-dif-
ferentiated hepatomas compressing the adjacent hepatic tissue. The
tumor was made up of cords, often one or two cells with endothelial
-lined sinusoids. The cells resembled hepatic parenchymal
cells from which they could usually be distinguished by their
faintly basophilic cytoplasm. The hepatoma cell varied
C-66
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TABLE 17
Hepatomas in Male and Female Mice Given
Carbon Tetrachloride by Stomach Tube
Strain
C3H
Y
C
A
Age (months) Males
6-10 126/143 (88%)
4-12
6-7
4-12
Females Both
-
9/15 (
34/41 (83%)
161/164 (
60%)
98%)
Hepatomas in Untreated Male and Female Mice
Strain
C3H
C3H
Y
C
A
A
Age (months) Males
8-11 2/50 (4%)
12-19 86/320 (27%)
10-16
13-24
4-8
12-16
Females Both
-
-
3/129 (
0/150 (0%)
0/400 (
8/400 (
2%)
0%)
2%)
Source: Edwards and Dalton, 1942
C-67
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TABLE 18
Hepatomas in Male Mice Given Olive Oil
*
by Stomach Tube
Strain
Age(months)
Incidence
C3H
C
A
10-11
12
5-12
4%
0%
0%
Source: Edwards and Dalton, 1942
C-68
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considerably in size. In many it was larger and in few it was smal-
ler than the normal hepatic cell.
Invasion of blood vessels by hepatomas was not seen/ even
though rather wide blood vessels were often in contact with the
periphery of the tumor nodules. Metastases were not observed.
Tumors did not appear to have been induced in any of the other
organs. The hepatoma that successfully grew on transplantation was
well differentiated; the subcutaneous transplants that resembled
the primary tumor were invasive.
Several special microscopic techniques were used to study a
number of primary and transplanted tumors. Much fat and glycogen
was present as large droplets in primary and transplanted tumor
cells. There was little or no alkaline phosphatase.
Low dose administration (0.1 ml of 5 percent CC14 in olive oil
0.005 ml) was administered three times weekly by stomach tube to
58 strain A female mice, 2.5 months of age, for 2 months. Mice were
necropsied 2 days to 4.5 months after the last treatment. Hepa-
tomas were present in 41 mice (71 percent), and some mice had cir-
rhosis of the liver.
The total dose (0.125 to 0.145 ml of CC14) is comparable to
the total dose of 0.120 ml of CC14 in the study in which mice were
given treatments of 0.04 ml each. The tumors of the liver were sim-
ilar in both studies.
Limited treatment involved strain A female mice, two months of
age. There were 21 to 62 mice in three treatment groups. The CC14
used was dissolved in olive oil, the volume of the mixture admin-
istered amounting to 0.1 ml. The mice were given 1 to 3 treatments.
C-69
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The doses, which were hepatotoxic, were 0.04, 0.01, or 0.005 ml of
CC1.. Eleven mice received olive oil only. The mice were necrop-
sied 2 to 12 months after the start of the study.
Tumors of the liver were not found in these mice. There was
pigment in Kupffer cells, foci of basophilic debris, and an in-
crease in connective tissue and reticulum.
In summary, carbon tetrachloride induced significant numbers
of tumors of the liver, as well as cirrhosis, in three strains of
mice. The neoplasms were similar to those induced in mice by an-
other hepatic carcinogen, o-aminoazotolene.
Since successful transplantation is frequently considered to
be a criterion of neoplasia, Leduc and Wilson (1959) attempted to
transplant CCl4-induced tumors of the liver in mice. At first
"numerous failures to establish a transplantable CCl4~induced hepa-
toma supported the idea that, if transplantability is a criterion,
the nodules might be hyperplastic but not neoplastic. Subsequent-
ly, however, several such hepatomas were successfully transplanted
from a host that was allowed to live for a long period after the
CC14 administration ceased" (Leduc and Wilson, 1959).
Male mice of the BOB strain were used. Spontaneous hepatomas
have not been found in this strain, which is now in its 40th genera-
tion. The mice ingested Purina Laboratory Chow.
Carbon tetrachloride was administered by stomach tube in doses
of 0.1 of a 40 percent solution in olive oil (0.04 ml of CC14) per
treatment. Carbon tetrachloride was given three times a week for a
total of 45 to 66 doses. About one third of the mice were given
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three daily intravenous injections of 0.2 ml of thorotrast before
CC14 administration was started.
The first-generation tumor transplants were made subcutan-
eously. Subsequently, both subcutaneous and intrasplenic trans-
plants were made. Under light ether anesthesia, implants of tumor
into the spleen were made by an incision through the dorsal body
wall. The spleens were examined periodically by laparotomy.
Hepatomas did not develop in 20 control mice given thorotrast
only. Hepatomas did occur in CCl4~treated mice that were free of
thoratrast.
The CC14 hepatomas (5 of 7) that were successfully transplant-
ed differed from those that did not grow in new hosts in previous
studies because a longer time period elapsed between CC1. admin-
istration and tumor transplantation. The five successful trans-
plants were obtained from a single host killed 8 months after the
last treatment, whereas those that did not grow were transplanted
11 weeks or so after the last treatment.
The authors note that:
Chronic CC1. injury to the liver induces the
development of both hyperplastic nodules and
hepatomas, and the livers of our CC1,-treated
mice were conspicuously cirrhotic with numer-
ous hyperplastic nodules. The nodules select-
ed as tumors differed from the rest principally
in size; histologically, there was little if
any difference, and it is possible that the
hepatomas which we transplanted 11 weeks or
less after CC1. treatment was (sic) not far
removed from the hyperplastic state. Thus,
this growth must have been dependent on the
particular conditions which were lacking in
the normal mice that were recipients of the
tumor implants.
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They concluded:
This suggests that there is a progressive in-
crease in the capacity for autonomous growth in
the primary hepatoma. This is based on the
observation that the primary hepatomas were
more readily transplanted when they had a long
sojourn in the host.
Delia Porta, et al. (1961) orally administered carbon tetra-
chloride to Syrian golden hamsters as a part of a larger investi-
gation of the response of this species to carcinogens that induced
neoplasms of the liver in other species. Ten female and 10 male
Syrian golden hamsters, 12 weeks old, were used. Males weighed an
average of 109 g and females 99 g. They were housed in plastic
cages on wood shavings in groups of five and were given Rockland
diet in pellets and tap water ad_ libitum. The treatment consisted
of weekly administration by stomach tube of a 5 percent solution of
CC14 in corn oil for 30 weeks. During the first seven weeks, 0.25
ml of the solution containing 12.5 jal of CC14 was given each week.
This dose was then reduced to 0.125 ml and contained 6.25 jul of
CC1.. After this treatment, the survivors were kept under observa-
4
tion- for 25 additional weeks and then killed.
Detailed histopathological examinations of all hamsters were
conducted, except for one female lost through cannabalism at the
28th week.
Weights of the hamsters varied irregularly during the period
following treatment. In general, the weights increased. Females
weighed an average of 114 g and males 118 g.
One female died at the 10th week of treatment; three females
and five males died or were killed between the 17th and the 28th
week. Three females died at weeks 41, 43, and 54. The surviving
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three females and five males were killed at the end of the 55th
week.
Hamsters dying during the treatment and at the 41st week had
cirrhosis, as well as hyperplastic nodules that were composed of
two to several layers thick; the cells showed irregularities in the
shape, size, and staining qualities of their cytoplasm and nucleus,
with an uneven distribution of glycogen.
All of the animals, five males and five females, dying or
killed 13 to 25 weeks after the end of the treatment, had one or
more liver-cell carcinomas (a total of 22 tumors: 12 in the 5 fe-
males and 10 in the males). These tumors varied in size from 4 to
30 mm and were located in all lobes: seven in the left, nine in the
right, and six in the posterior lobes. Grossly, their grayish pink
color, forms, and consistency differentiated them from the hyper-
plastic, regenerative nodules. Histologically, they were composed
of atypical cells, often in mitosis, either in solid masses without
any structure or arranged in small nests and in short trabeculae
surrounded by dilated vascular spaces with numerous endothelial
cells. Reticula on many occasions completely surrounded small
nests of tumor cells. This pattern was helpful in distinguishing
liver-cell carcinomas from adenomatous nodules. The tumors did not
have a capsule, but compressed and invaded the surrounding paren-
chyma. One of the larger liver-cell carcinomas metastasized to the
mesenteric and cervical lymph nodes. This tumor and six others
were transplanted unsuccessfully.
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The authors noted in the discussion that:
The diagnosis of malignancy in the liver tumors
observed was based on histological criteria
only, to which little was added by the solitary
case of metastasis. The negative result of the
transplantation study deserves further inves-
tigation. Many other tumors of hamsters have
been successfully transplanted to non-inbred
hamsters in this and other laboratories. Leduc
and Wilson (1959) suggested as an important
factor the length of the interval between the
last administration of carbon tetrachloride
and the transplantation (Delia Porta, et al.
1961).
In summary, carbon tetrachloride is a liver carcinogen in the
hamster. Hyperplastic nodules (adenomatous nodules) appeared dur-
ing treatment, and carcinomas appeared after CC14 administration
had been discontinued, which strongly suggests that the nodules or
benign tumors later became carcinomas. It should be noted that
this study is the only report of the induction of tumors in ham-
sters by CC14.
In concluding this section, it should be noted that some of
the research that has been reported suggests that hepatomas occur
only after liver necrosis and fibrosis have occurred (Edwards,
1941; Edwards and Dalton, 1942; Delia Porta, et al. 1961; Rueber
and Glover, 1967; Reuber and Glover, 1970). The results have been
interpreted to mean that "as far as the liver is concerned, hepa-
toma is an occasional consequence of the induction of post-necrotic
cirrhosis and that CC14 is not a direct liver carcinogen" (Louria,
1977). The results reported by Eschenbrenner and Miller (1946),
however, refute Louria1s statement. These authors decided that if
carbon tetrachloride is, in fact, a carcinogenic agent, tu-
mors should be obtained with non-necrotizing doses. A series of
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questions concerning the mechanisms of the toxicity and carcinogen-
icity of carbon tetrachloride led Eschenbrenner and Miller to a
series of experiments examining the issue. Their conclusions in-
cluded the following:
While it was found that a correlation exists
between the degree of liver necrosis and the
incidence of hepatomas in relation to dose, the
use of a graded series of necrotizing and non-
necrotizing doses indicated that repeated
liver necrosis and its associated chronic
regenerative state are probably not necessary
for the induction of tumors with carbon tetra-
chloride (Eschenbrenner and Miller, 1946).
A list of authors addressing the issue of liver necrosis in-
duced by carbon tetrachloride is provided in Table 19.
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TABLE 19
Studies in Which Liver Cancer was Induced Using
Carbon Tetrachloride
Author
Year
Species
1. McCord
2. Edwards
3. Edwards and Dalton
4. Eschenbrenner
and Miller
5. Delia Porta, et al.
6. Reuber and Glover
7. Hashimoto, et al.
8. Reuber and Glover
1932
1941
1942
1946
1961
1967
1968
1970
none identified —
subcutaneous injection
mice — gavage
mice — ingestion
mice -- ingestion
hamsters — ingestion
rats — subcutaneous
injection
human — ingestion
rats — subcutaneous
injection
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CRITERION FORMULATION
Existing Guidelines and Standards
At present, there is neither a water standard or an air stan-
dard for CC1,. However, a number of standards have been recommend-
ed for inhalation in the work environment. NIOSH (1975) has sum-
marized these standards. The following description is paraphrased
from this NIOSH report.
The Sub-Committee on Threshold Limits of the National Confer-
ence of Governmental Industrial Hygienists (NCGIH) published a list
in 1942 entitled, "Maximum Permissible Concentrations of Atmospher-
ic Contaminants as Recommended by Various State Industrial Hygiene
Units" (NCGIH, 1942). Thirteen states were listed as recommending
650 mg/m for carbon tetrachloride in air. The listing was pre-
sented without comment, other than that the tabulated values were
not to be construed as recommended safe concentrations.
Various standards for the inhalation of carbon tetrachloride
were the subject of discussion at the 7th Annual Meeting of NCGIH
(Bowditch, 1944). Manfred Bowditch, Director of the Massachusetts
Division of Occupational Hygiene, gave "temporary indisposition,"
indicated by nausea, as reason for a standard lower than 650 mg/m .
He reported that, as a consequence, the Division of Occupational
Hygiene of the Massachusetts Department of Labor and Industries
proposed lowering the standard for carbon tetrachloride to 260
mg/m . Other governmental agencies also considered 650 mg/m3 in-
effective and recommended a lower standard (Bowditch, 1944; Cook,
1945).
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A list of maximum allowable concentrations of atmospheric in-
dustrial contaminants compiled by Cook (1945) included the carbon
tetrachloride values of seven governmental agencies. These are
presented in Table 20.
These concentrations were all recommended as allowable for
prolonged exposures, usually assuming a 40-hour workweek (Cook,
1945).
In addition to tabulating these values, Cook (1945) reported
650 mg/m3 to be an accepted or tentative value based on the work
published by Smyth, et al. (1936). However, in his discussion of
carbon tetrachloride, Cook (1945) wrote that since Smyth's publi-
cation there was an increasing amount of evidence of injury to
health at lower concentrations, and he recommended that exposures
be at less than half the 650 mg/m then being used.
The American Conference of Governmental Industrial Hygienists
(ACGIH) (formerly NCGIH) adopted a list of "Maximum Allowable Con-
centrations of Air Contaminants for 1946," prepared by the Sub-
Committee on Threshold Limits (ACGIH, 1946) which, in accordance
with Cook's recommendation (Cook, 1945), selected a value of 325
mg/m for carbon tetrachloride.
The ACGIH Committee on Threshold Limits reported in 1949 that
it had received comments from outside the Conference that a value
of 325 mg/m3 for carbon tetrachloride was too low (ACGIH, 1949).
On the other hand, carbon tetrachloride was included in a list of
substances for which a reduction of the limit had been suggested by
members of the Conference.
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TABLE 20
1975 Carbon Tetrachloride Inhalation Standards
of Governmental Agencies*
Governmental Agency MAC, mg/m3
California Industrial Accident Commission 100
Connecticut Bureau of Industrial Hygiene 100
Massachusetts Department of Labor and Industries 50
New York State Department of Labor 75
Oregon State Board of Health 50
Utah Department of Health 100
United States Public Health Service 100
*Source: NIOSH, 1975
MAC = Maximum allowable concentration.
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The ACGIH recommended a threshold limit value (TLV) of 162.5
mg/m3 for carbon tetrachloride (ACGIH, 1953). A preface to future
tables of threshold limits was adopted and defined the values as
"maximum average atmospheric concentration of contaminants to which
workers may be exposed for an eight-hour working day without injury
to health" (ACGIH, 1953). The preface was modified in 1958 and
included the statement that "they (threshold limit values) repre-
sent conditions under which it is believed that nearly all workers
may be repeatedly exposed, day after day, without adverse effect"
(ACGIH, 1958).
The American Standard Maximum Acceptable Concentration of Car-
bon Tetrachloride (ASA Z37.17-1957), published in 1957, was 162.5
mg/m3 for exposures not exceeding 8 hours daily, with the under-
standing that variations should fluctuate around 65 mg/m (ASA,
1957). The 162.5 mg/m was understood to be a ceiling below which
all concentrations were to fall. It was based partly on the animal
experiments reported by Adams, et al. (1952) and partly on indus-
trial experiences of members of that Committee.
The Documentation of Threshold Limit Values (ACGIH, 1962)
referred to the reports of Adams, et al. (1952), Heimann and Ford
(1941), Kazantzis and Bomford (1960), and Elkins (1942), in its
support of the TLV for carbon tetrachloride of 162.5 mg/m . From
these data, it was considered that 162.5 mg/m was low enough to
prevent irreversible injury (ACGIH, 1962)-.
At the annual meeting of the ACGIH in 1962, the Threshold
Limit Committee recommended reducing the TLV for carbon tetra-
chloride to 65 mg/m3 because there were "increasing indications
C-80
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that exposure to carbon tetrachloride at 162.5 mg/m was exces-
sive" (ACGIH, 1962).
"Permissible Levels of Toxic Substances in the Working Envi-
ronment" for many countries was published by the International
Labour Office (1970). The reported carbon tetrachloride standards
are presented in Table 21. The USSR values (MAC) are absolute val-
ues never to be exceeded. They are set at a value which will not be
expected to produce, in any exposed person, any disease or other
detectable deviation from the normal. Some other countries tend to
follow this value in setting their standards, while still others
tend to follow the recommendations of the ACGIH. The intent is
indicated from some of the standards presented in Table 21.
The most recent documentation of the threshold limit values
was published by the ACGIH (1971). The reports of Heimann and Ford
(1941), Elkins (1942), Barnes and Jones (1967), Kazantzis and
Bomford (1960), Markham (1967), Adams, et al. (1952), and Stewart,
et al. (1961), were referred to in support of the TLV of 65 mg/m3,
which had been adopted in 1962, Information that some workmen
experienced nausea when average daily carbon tetrachloride expo-
sures approached 162.5 mg/m3, whereas no difficulties were exper-
ienced at 65 mg/m (based on a personal communication to the Com-
mittee), was used as additional support for the TLV. The TLV of 65
mg/m was recommended with the caution that peak exposures, even of
short duration, should not exceed 162.5 mg/m .
The Occupational Safety and Health Administration, U.S.
Department of Labor, adopted the American National Standards Insti-
tute (ANSI) standard Z37.17-1967 (ANSI, 1967) as the Federal
C-81
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TABLE 21
Carbon Tetrachloride Inhalation Standards of 10 Countries*
Country
Czechoslovakia
Finland
Hungary
Japan
Poland
Rumania
UAR and SAR
USSR
Yugoslavia
Standard
mg/m
50
250
160
20
100
10
20
50
625
20
65
Qualifications
Normal MAC
Single short exposure
8 hours continuous exposure
8-hour average
30 minutes
MAC
*Source: Adapted from NIOSH, 1975
MAC = Maximum Allowable Concentration.
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standard for carbon tetrachloride (29 CFR 1910.1000). This stan-
dard is 65 mg/m for an 8-hour TWA exposure, with an acceptable
ceiling exposure concentration of 162.5 mg/m , and an acceptable
maximum peak above the acceptable ceiling concentration for an
8-hour shift of 1,300 mg/m for 5 minutes in any four hours.
This ANSI standard was based on human experience and extensive
studies on animals. References cited to support it were Adams, et
al. (1952), Stewart, et al. (1961, 1965), Stewart and Dodd (1964),
von Oettingen (1964), and Irish (1963).
Finally, a standard decided upon by the FAO/WHO Expert Com-
mittee is 50 jug/kg for cooked cereal products.
Current Levels of Exposure
A brief review of some of the data presented in the exposure
section of this report will summarize the current levels of human
exposure. Carbon tetrachloride has been found in some waters. An
EPA survey of drinking water in the U.S. revealed that 10 percent
of the supplies surveyed had 2.4 to 6.4 jug/1 CC1..
Caroon tetrachloride (CC14) has been found in a variety of
food-stuffs ranging from 1 to 20 ug/kg. Residues have been found
in commercially fumigated wheat, corn, and milo in amounts ranging
from 2.9 to 20.4 mg/kg after storage for 1 to 3 hours. Carbon
tetrachloride residues ranging from 20 to 62 mg/kg in sacks of
wheat were found by Wit, et al. (1972), following fumigation with a
mixture of CC14-EDC-EDB (10.2:8:1 by weight) and then aerated for
several weeks. Residues as high as 72.6 mg/kg after 1 week of aera-
tion were detected in wheat by Berck (1974). After 7 weeks of aera-
tion, 3.2 mg/kg were found. Flour made from this wheat had
C-83
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residues of 0.20 to 0.93 mg/kg. Amounts of CC14 detected in bread
made from this wheat ranged from 0.04 to 0.13 mg/kg for wheat aer-
ated for 3 days and 0.01 to 0.2 mg/kg for wheat aerated for 7 weeks.
The most extensive measurements of CC14 have occurred in the
atmosphere. Virtually no variation has been found between land and
ocean, urban and rural, or Northern and Southern Hemispheres. The
maximum value detected was 0.117 mg/m in Bayonne, N. J.; however,
normal background levels range from 0.00078 to 0.00091 mg/m in the
continental and marine air masses.
The National Research Council (1978) in its assessment of
"Nonfluorinated Halomethanes in the Environment" estimated total
human exposure to CC14. Using drinking water concentrations of
less than 2.0 to 3.0 ug/1 and other conventional assumptions with
regard to human and environmental conditions, three ranges of expo-
sure were estimated (See Table 22).
Minimum, typical, and maximum exposure estimates of total CC14
uptake were 4.54 mg/year, 7.70 mg/year, and 629 mg/year, respec-
tively. The percentage from fluid sources (water) was 16 percent,
23 percent, and 0.6 percent, respectively. By far the highest up-
take of CC14 was estimated to come from atmospheric sources, 62 to
98 percent.
Although monitoring has provided more information on CC14
presence in the environment than most chemicals, there remain many
relative unknowns about absorption, synergism/antagonism, etc. The
estimated CC14 exposure from food sources is based upon only limit-
ed information which is compared to air and fluid uptakes. At face
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TABLE 22
Relative Human Uptake of Carbon Tetrachloride (CC1.)
£ ^
from Environmental Sources (mg/year)
At Minimum Exposure Levels3
Adult Man
Source
Fluid Intake
Atmosphere
Food Supply
Total
cci4
0.73
3.60
0.21
4.54
CHC13
0.037
0.41
0.21
0.66
At Typical Exposure Levels
Adult Man
Source
Fluid Intake
Atmosphere
Food Supply
Total
cci4
1.78
4.80
1.12
7.70
CHC13
14.90
5.20
2.17
22.27
At Maximum Exposure Levels0
Adult Man
Source
Fluid Intake
Atmosphere
Food Supply
Total
cci4
4.05
618
7.33
629.38
CHC13
494
474
16.4
984.4
Minimum conditions of all variables assumed: Minimum exposure-
minimum intake for fluids; minimum exposure-minimum absorption
for atmosphere; and minimum exposure-minimum intake for food sup-
bplies.
Typical conditions of all variables assumed.
For_CC14:_0.0025 mg/1-reference man intake for fluids; average of
typical minimum and maximum absorption for atmosphere; and average
exposure and intake for food supplies.
For CHCl^: median exposure-reference man intake for fluids; aver-
age of typical minimum and maximum absorption for atmosphere; and
caverage exposure and intake for food supplies.
Maximum conditions of all variables assumed: maximum exposure-
maximum intake for fluids; maximum exposure-maximum absorption for
atmosphere; and maximum exposure-maximum intake for food supplies.
*
Source: National Research Council, 1978
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value, the figures indicate that while none of the three routes of
exposure is negligible, inhalation is the most important for CC14-
Special Groups at Risk
As a result of the studies performed on animals it appears as
though older animals are more susceptible to the toxic effects of
CC1, than are younger animals (Rueber and Glover, 1967). Also,
male animals are more susceptible than females (Rueber and Glover,
1967). Chaturvedi (1969) examined age and sex as factors of CC14
toxicity. The findings revealed that female rats are less suscep-
tible to the ill-effects of different hepatotoxic agents and fare
better than males because of different hormonal and enzyme patterns
and the lack of certain proteins in contrast to the male liver. The
sex difference noticed in adult rats was not so apparent in young
rats.
The synergistic effects of alcohol must also be noted. Alco-
holics have a greater susceptibility to poisoning from CC14. As
described by Moon (1950), the frequent occurrence of a history of
alcoholism in cases of fatal CC14 poisoning indicates a synergistic
nephrotoxic, as well as hepatotoxic, effect between alcohol and
CC14.
Finally, as previously mentioned, very obese and under-
nourished persons suffering from pulmonary diseases, gastric ulcers
or a tendency to vomiting, liver or kidney diseases, diabetes or
glandular disturbances are especially sensitive to the toxic ef-
fects of CC14.
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Basis and Derivation of Criteria
Studies indicate that CC14 has a full spectrum of toxic ef-
fects. Industrial and accidental exposures to CC1, by ingestion,
inhalation, and dermal routes historically have produced acute, sub-
acute, and chronic poisoning, some of which were fatal. Acute tox-
icity for man and animals can be characterized generally as nodular
hyperplasia and cirrhosis of the liver and renal dysfunction.
Mutagenic effects have not been observed and teratogenic effects
have not been conclusively demonstrated.
The most significant effect to consider in terms of dose/re-
sponse is the cancer-causing potential of the chemical. Current
knowledge leads to the conclusion that carcinogenesis is a non-
threshold, nonreversible process. The nonthreshold concept implies
that many tumors will be produced at high doses, but any dose, no
matter how small, will have the probability of causing cancer.
Even small carcinogenic risks have a serious impact on society when
the exposed population is large, because it is likely that some
cancers will be caused by exposure to CC14. The nonreversible con-
cept implies that once the tumor growth process has started, growth
will continue and may metastasize and involve other organs until
death ensues.
There is sufficient evidence to conclude that CC1. is a car-
cinogen in laboratory animals and, with appropriate assumptions, is
interpreted to be a suspect human carcinogen.
Under the Consent Decree in NRDC vs. Train, criteria are to
state "recommended maximum permissible concentrations (including
where appropriate, zero) consistent with the protection of aquatic
C-87
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ojrganisms, human health, and recreational activities." Carbon
tetrachloride is suspected of being a human carcinogen. Because
there is no recognized safe concentration for a human carcinogen,
the appropriate concentration of carbon tetrachloride in water for
maximum protection of human health is zero.
Because attaining a zero concentration level may be infeasible
in some cases and in order to assist the Agency and states in the
possible future development of water quality regulations, the con-
centrations of carbon tetrachloride corresponding to several incre-
mental lifetime cancer risk levels have been estimated. A cancer
risk level provides an estimate of the additional incidence of can-
cer that may be expected in an exposed population. A risk of 10
for example, indicates a probability of one additional case of can-
cer for every 100,000 people exposed; a risk of 10 indicates one
additional case of cancer for every million people exposed, and so
forth.
In the Federal Register (44 FR 15930) notice of availability
of draft ambient water quality criteria, EPA stated that it is con-
sidering setting criteria at an interim target risk level of 10~ ,
10~6, or 10 ,as shown in the following table.
Exposure Assumptions Risk Levels
(per day) and Corresponding Criteria (1)
10"7 10"6 10"5
2 liters of drinking 0.04 ug/1 0.40 jug/1 4.0 jug/1
water and consumption
of 6.5 g fish and
shellfish. (2)
Consumption of fish 0.69 ug/1 6.94 jag/1 69.4 jug/1
and shellfish only.
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(1) Calculated by applying a linearized multistage model, as dis-
cussed in the Human Health Methodology Appendices to the
October, 1980 Federal Register notice which announced the
availability of this document, to the animal bioassay data
presented in Appendix I and in Table 14. Since the extrapola-
tion model is linear at low doses, the additional lifetime
risk is directly proportional to the water concentration.
Therefore, water concentrations corresponding to other risk
levels can be derived by multiplying or dividing one of the
risk levels and corresponding water concentrations shown in
the table by factors such as 10, 100, 1,000, and so forth.
(2) Approximately 6 percent of the carbon tetrachloride exposure
results from the consumption of aquatic organisms which exhib-
it an average bioconcentration potential of 18.75-fold. The
remaining 94 percent of carbon tetrachloride exposure results
from drinking water.
Concentration levels were derived by assuming a lifetime expo-
sure to various amounts of carbon tetrachloride: (1) occurring from
consumption of both drinking water and aquatic life grown in waters
containing the corresponding carbon tetrachloride concentrations;
and (2) occurring solely from consumption of aquatic life grown in
the waters containing the corresponding carbon tetrachloride con-
centrations. Although total exposure information for carbon tetra-
chloride is discussed and an estimate of the contributions from
other sources of exposure can be made, these data will not be
factored into ambient water quality criteria formulation until
C-89
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additional analyses can be made. The criteria presented, there-
fore, assume an incremental risk from ambient water exposure only.
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REFERENCES
Adams, E.M., et al. 1952. Vapor toxicity of carbon tetrachloride
determined by experiments on laboratory animals. Arch. Ind. Hyg.
Occup. Med. 6: 50.
Altshuller, A.P. 1976. Average tropospheric concentration of car-
bon tetrachloride based on industrial production, usage, and emis-
sions. Environ. Sci. Technol. 10: 596.
Alumot, E. and B. Bielorai. 1969. Residues of fumigant mixtures
in cereals fumigated and aired at two different temperatures.
Jour. Agric. Food Chem. 17: 869.
American Conference of Governmental Industrial Hygienists. 1946.
Report of the Sub-committee on Threshold Limits. in; Proc. 8th
Annu. Meet. Am. Conf. Gov. Ind. Hyg. Chicago, April 7-13.
American Conference of Governmental Industrial Hygienists. 1949.
Report of the Committee on Threshold Limits. In: Proc. llth Annu.
Meet. Am. Conf. Gov. Ind. Hyg. p. 63.
American Conference of Govenrmental Industrial Hygienists. 1953.
Report of the Committee on Threshold Limits. In; Proc. 15th Annu.
Meet. Am. Conf. Gov. Ind. Hyg. p. 45.
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American Conference of Governmental Industrial Hygienists. 1958.
Report of the Committee on Threshold Limits. In; Proc. 20th Annu.
Meet. Am. Conf. Gov. Ind. Hyg. p.138.
American Conference of Governmental Industrial Hygienists. 1962.
Carbon tetrachloride, in documentation of threshold limit values.
Cincinnati, Ohio.
American Conference of Governmental Industrial Hygienists. 1971.
Documentation of the Threshold Limit Values for Substances in Work-
room Air. 3rd ed. Cincinnati, Ohio.
American National Standards Institute. 1967. American standard
maximum acceptable concentration of carbon tetrachloride. Z37.17.
New York.
Andervont, H.B. and T.B. Dunn. 1955. Transplantation of hepatomas
in mice. Jour. Natl. Cancer Inst. 15: 1513.
Barnes, R. and R.C. Jones. 1967. Carbon tetrachloride poisoning.
Am. Ind. Hyg. Assoc. Jour. 28: 557.
Barrett, H.M., et al. 1939. Study of fate in organisms of some
chlorinated hydrocarbons. Jour. Ind. Hyg. Toxicol. 21: 479.
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APPENDIX I
Derivation of Criterion for Carbon Tetrachloride
Carbon tetrachloride has been studied extensively and admin-
istered orally in a number of studies in mice, rats, hamsters, and
dogs. However, in these studies, either the length of the study
was too short or the dose level was too high for a dose-response
estimation of lifetime exposure (NRC, 1978). The National Research
Council recognizes this problem and for this reason uses the NCI
(1976) bioassay for trichloroethylene in determining a carcinogenic
risk estimate for carbon tetrachloride.
This NCI study uses carbon tetrachloride as the positive con-
trol. Male mice receiving carbon tetrachloride by gavage at 1250
mg/kg 5 days per week for 78 weeks developed incidences of liver
tumors at 92 weeks, when the experiment was terminated, as shown
below. The parameters of the extrapolation model are:
Dose Incidence
(mg/kg/day) (no. responding/no, tested
0 5/77
1250 x 5/7 = 893 49/49
2500 x 5/7 = 1786 47/48
le = 78 weeks w = 0.028 kg
Le * 92 weeks R = 18.75 I/kg
L = 92 weeks
With these parameters the carcinogenic potency factor for hu-
* -l
mans, q1 , is 0.08275 (mg/kg/day) . The result is that the water
concentrations should be less than 4.0 ug/1 in order to keep the
individual lifetime risk below 10~5.
C— HI ft U. S GOVERNMENT PRINTING OFFICE . I960 720-016/4375
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