J Occup Health 2007; 49: 249–259
Journal of
Occupational Health
Thirteen-week Inhalation Toxicity of Carbon Tetrachloride in Rats
and Mice
Kasuke NAGANO, Yumi UMEDA, Misae SAITO, Tomoshi NISHIZAWA, Naoki IKAWA,
Heihachiro ARITO, Seigo YAMAMOTO and Shoji FUKUSHIMA
Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Japan
Abstract. Thirteen-week Inhalation Toxicity of
Carbon Tetrachloride in Rats and Mice: Kasuke
NAGANO, et al. Japan Bioassay Research Center,
Japan Industrial Safety and Health Association—
Subchronic toxicity of carbon tetrachloride (CCl4) was
examined by inhalation exposure of F344 rats and BDF1
mice of both sexes to 0, 10, 30, 90, 270 or 810 ppm (v/
v) CCl4 vapor for 13 wk (6 h/d and 5 d/wk). In the high
exposure levels at 270 and 810 ppm, altered cell foci
in the livers of both rats and mice, and fibrosis and
cirrhosis in the rat liver were observed. Hematoxylin
and eosin-stained altered cell foci of rats were
recognized as glutathione-S-transferase placental form
(GST-P) positive foci, which are preneoplastic lesions
of hepatocarcinogenesis. The most sensitive endpoint
of CCl4-induced toxicity was fatty change with large
droplets in rats of both sexes and male mice, and
cytoplasmic globules in male mice, as well as increased
relative liver weight in male rats. Those endpoints were
manifested at 10 ppm and the LOAEL was determined
as 10 ppm for the hepatic endpoints in rats and mice.
Enhanced cytolytic release of liver transaminases into
plasma in rats and mice and its close association with
hepatic collapse in mice were observed at medium and
high levels of inhalation exposure. Both CCl4-induced
hematotoxicity and nephrotoxicity were observed in
both rats and mice, but those toxicities were manifested
at higher exposure concentrations than hepatotoxicity.
The LOAEL for the hepatic endpoint and the GST-Pstained altered cell foci provide relevant animal data
for reconsidering the occupational exposure limit value
of 5 ppm for CCl4 and strengthen the evidence of CCl4induced hepatocarcinogenicity which is used in its
carcinogenicity classification.
(J Occup Health 2007; 49: 249–259)
Received Dec 6, 2006; Accepted Mar 8, 2007
Correspondence to: K. Nagano, Japan Bioassay Research Center,
2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan (e-mail: [email protected] )
Key words: Inhalation, Carbon tetrachloride, Mouse,
Rat, Liver, Kidney, Hepatotoxicity, Hematotoxicity,
Glutathione- S -transferase placental form (GST-P),
Preneoplastic lesion
The Montreal Protocol and its subsequent
amendments1) have established the phase-out by 1996 of
production and use of carbon tetrachloride (CCl4). United
States’ production of CCl4 peaked at 1,200 million pounds
in the early 1970s, then decreased steadily and production
of CCl4 was completely banned as of January 1, 20002).
The annual production volume of CCl4 in Japan remained
constant at about 50,000 tons from 1975 to 1993, and
then declined rapidly3). However, a sizable volume of
CCl 4 ranging from 60 to 300 tons/yr was still being
imported to Japan in the years following 2000 3). A
nationwide survey in Japan by Ukai et al.4) showed no
use of CCl4 as an organic solvent in small- and mediumscale industries in 1996, except for testing and research
purposes. Kauppinen et al.5) reported that CCl4 is one of
the most frequently reported exposures among laboratory
workers reported to the Finnish Register of Workers
Exposed to Carcinogens. Workers are at high risk of
exposure to high levels of CCl4 through inhalation and
dermal contact. On the other hand, the general population
may also be exposed to low levels of CCl 4 through
inhalation from the atmospheric environment 6) and
through ingestion of CCl4 contaminating public water7).
Occupational CCl4 poisoning has been characterized by
central nervous system depression, and disorders of the
liver and kidneys8, 9). In experimental toxicology, CCl4induced toxicity and its mechanisms have been extensively
investigated by oral administration of CCl4 to rats and mice
(see reviews9–11)), and some of these animal studies have
been used to assess health risks to humans from ingestion
of CCl4-contaminated water and food12–16). However, there
have been few inhalation studies17–19) on subchronic or
chronic CCl4 toxicity which have adequately delineated
dose-response relationships to determine the effective
250
endpoints for health risk assessment of workers exposed
to CCl4 vapor by inhalation. Data of animal toxicity from
inhalation exposure to CCl4 are more relevant for assessing
health risks to workers inhaling CCl4 than those from oral
administration, because inhalation is the principal route of
entry of CCl4 into the body, and because the hepatotoxicity
resulting from inhalation exposure is reported to be
different from that from oral administration of CCl420, 21).
The aim of the present study was to characterize
subchronic inhalation toxicity of CCl4, using two rodent
species of both sexes, with particular emphasis placed
on delineation of dose-response relationships to determine
the effective endpoints for health risk assessment of
workers inhaling CCl 4. For this purpose, the lowest
inhalation concentration of 10 ppm was selected in
consideration of the validity of the present occupational
exposure limit (OEL) value for CCl422–24). The OEL value
was established as 5 ppm by the American Conference
on Governmental Industrial Hygienists (ACGIH) in
198122) and by the Japan Society for Occupational Health
(JSOH) in 1991 23). However, the German Research
Foundation (Deutsche Forschungsgemeinschaft, DFG)
revised the maximum workplace concentration (MAK)
value24) from 10 ppm to 0.5 ppm in 2000. The high levels
of inhalation exposure to CCl4 used here were selected
to explore any precursor lesions that might develop to
tumors after long-term exposure to CCl4.
Materials and Methods
The present study was carried out with reference to
the Organisation for Economic Co-operation and
Development (OECD) Guideline for Testing of Chemicals
413 “Subchronic Inhalation Toxicity: 90-day Study”25).
The animals were cared for in accordance with the Guide
for the Care and Use of Laboratory Animals 26). The
present study was approved by the ethics committee of
the Japan Bioassay Research Center.
Chemicals
Analytical-grade CCl4 (greater than 99.8% pure) was
obtained from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). Each lot of CCl 4 was analyzed for
stability and purity by gas chromatography and infrared
spectrometry before and after use. Neither decomposition
products nor impurities were found in any lot of CCl4.
No gas chromatographic peak other than CCl 4 was
detected in the inhalation exposure chambers. Polyclonal
antibody of glutathione-S-transferase placental form
(GST-P) (Anti-GST-P) was obtained from Medical &
Biological Laboratories Co., Ltd. (Nagoya, Japan).
Animals
Four-wk-old F344/DuCrj rats and Crj:BDF1 mice of
both sexes were obtained from Charles River Japan, Inc.
(Kanagawa, Japan). The animals were quarantined and
J Occup Health, Vol. 49, 2007
acclimated for 2 wk, and then divided by stratified
randomization into 6 weight-matched groups, each
consisting of 10 rats and 10 mice of both sexes. Twelve
exposure chambers were installed in a barrier system
animal room in which a temperature of 24.5–25.1°C and
a relative humidity of 52.3–57.1% with 12–15 air
changes/h were maintained. The animals were housed
individually in stainless steel wire hanging cages (150
mm [W] × 216 mm [D] × 176 mm [H] for rats and 100
mm [W] × 116 mm [D] × 120 mm [H] for mice) in the
inhalation exposure chambers. The 12-h light/dark cycle
was automatically controlled. All rats and mice had free
access to sterilized commercial pellet diet (CRF-1,
Oriental Yeast Co., Ltd., Tokyo, Japan) and sterilized
drinking water supplied by an automatic watering system.
Exposure to CCl4
The animals were exposed to CCl 4 at a target
concentration of 10, 30, 90, 270 or 810 ppm for 6 h/d, 5
d/wk and 13 wk. Groups of 10 rats and 10 mice of both
sexes were exposed to clean air for 13 wk under the same
conditions, and served as respective controls.
The methods for generating airflow containing CCl4
vapor and supplying the airflow to the inhalation exposure
chamber were as described previously27). The vapor-air
mixture flow was generated by bubbling clean air through
CCl4 liquid in a solvent reservoir. The vapor-air mixture
was further diluted with clean air and supplied to the
inhalation exposure chambers. Air concentrations of CCl4
vapor in the exposure chambers were maintained constant
throughout the 6-h exposure period with the following
accuracy and precision: (mean ± standard deviation) 10.0
± 0.2 ppm, 30.0 ± 0.3 ppm, 90.4 ± 0.6 ppm, 272.0 ± 2.8
ppm and 811.0 ± 6.5 ppm for the rats; and 10.0 ± 0.1
ppm, 30.0 ± 0.3 ppm, 90.1 ± 1.0 ppm, 271.3 ± 3.5 ppm
and 810.3 ± 6.7 ppm for the mice throughout the 13-wk
exposure period.
Clinical observations and analysis, and pathological
examinations
The animals were observed daily for clinical signs and
mortality. Body weight and food consumption were
measured once a week. Urinary parameters were
measured in the last week of the 13-wk exposure period
with Ames Reagent Strips (Multistix for rats and UroLabstix for mice, Bayer Corporation, NY, USA). All
animals underwent complete necropsy. Blood was
collected for hematology and blood biochemistry under
etherization after overnight fasting at the end of the 13wk exposure period. The blood samples were analyzed
with an Automatic Blood Cell Analyzer (Coulter Counter
SP, Coulter Electronics Inc., FL, USA) for hematology,
and an automatic analyzer (Hitachi 705, Hitachi, Ltd.,
Ibaraki, Japan) and a flame analyzer (Hitachi 750, Hitachi,
Ltd., Ibaraki, Japan) for blood biochemistry. Organs were
Kasuke NAGANO, et al.: Subchronic Inhalation Toxicity of CCl4 in Rodents
251
Table 1. Terminal body weight, and relative liver and kidney weights of the rats exposed to CCl4 vapor by inhalation for 13 wk
Group (ppm)
No. of animals
examined
Body weight (g)
Organ weight
Liver (%)
Kidney (%)
Control
10
30
Male
90
270
810
Control
10
Female
30
90
270
810
10
10
10
10
10
10
10
10
10
10
10
10
301
296
304
302
289
244 **
176
178
185
184
175
169
2.44
0.57
2.59 **
0.61 **
2.89 **
0.59
3.27 **
0.63 **
3.91 **
0.70 **
4.23 **
0.83 **
2.33
0.65
2.47
0.65
3.19 **
0.65
4.41 ** 4.59 **
0.72 ** 0.83 **
Values indicate means. Significant difference; *: p≤0.05
**:
4.76 **
0.91 **
p≤0.01 by Dunnett’s tests.
removed, weighed, and examined for macroscopic lesions
at the necropsy. All the animals were examined for
histopathology. The tissues were fixed in 10% neutral
buffered formalin, and embedded in paraffin. Tissue
sections of 5 µm in thickness were prepared, and stained
with hematoxylin and eosin (H & E). Additionally, the
livers of 810 ppm-exposed and control groups of male
rats were sectioned for further examination of hepatic
altered cell foci, a preneoplastic lesion, by
immunohistochemical staining with anti-GST-P28–30) using
EnVision+ (EV+, Dako, Copenhagen, Denmark) of the
two-layer dextran polymer visualization system31).
Data analysis
Body weights, organ weights, and hematological and
blood biochemical parameters were analyzed by
Dunnett’s multiple comparison test or the same multiple
comparison test by rank. Two-sided analysis with a pvalue of 0.05 was performed. Pathological examinations
and urinary parameters were analyzed by Chi-square test
with a p-value of 0.05. Application of the statistics was
given in detail in the previous paper32).
Either a no-observed-adverse-effect-level (NOAEL) or
a lowest-observed-adverse-effect-level (LOAEL) was
determined according to the WHO definition33).
Results
Rat study
Survival, body and organ weights: No death occurred
in any of the CCl4-exposed rat groups of either sex. There
was no CCl4-related clinical sign except for soiling of
the perigenital and perinasal areas in male and female
rats exposed to 270 ppm and above. Terminal body
weight was significantly decreased only in the 810 ppmexposed male rats (Table 1). A significant increase in
relative organ weight was observed in the livers of male
rats exposed to 10 ppm and above and of female rats
exposed to 30 ppm and above. The relative kidney
weights of male rats exposed to 10 ppm and above, except
30 ppm, and of female rats exposed to 90 ppm and above
were significantly increased. A biologically meaningful
increase in the relative kidney weight was judged to have
occurred in both male and female rats exposed to 90 ppm
and above, because a dose-dependent increase was
observed in rats of both sexes exposed to 90 ppm and
above, and because the statistically increased relative
kidney weight of 10 ppm-exposed male rats was attributed
to a marginal decrease in the body weight.
Clinical chemistry: While red blood cell counts were
significantly decreased in the 810 ppm-exposed rats of
both sexes, hemoglobin and hematocrit were significantly
decreased in male and female rats exposed to 90 ppm
and above (Table 2). Aspartate aminotransferase (AST),
a l a n i n e a m i n o t r a n s f e r a s e ( A LT ) a n d l a c t a t e
dehydrogenase (LDH) tended to increase in male rats
exposed to 90 ppm and above and in female rats exposed
to 30 ppm and above, although the increases in AST and
LDH in the 90 ppm-exposed male rats were not
statistically significant. Alkaline phosphatase (ALP) was
significantly increased in male and female rats exposed
to 90 ppm and above. Total bilirubin was significantly
increased in male rats exposed to 810 ppm and in female
rats exposed to 270 and 810 ppm. Urinary protein was
significantly increased in male rats exposed to 270 and
810 ppm and in female rats exposed to 90 ppm and above.
Pathology: The liver surface was granular in 1 female
rat exposed to 90 ppm, in 5 male rats and 10 female rats
exposed to 270 ppm and all male and female rats exposed
to 810 ppm.
Incidences of selected microscopic hepatic lesions in
the CCl4-exposed rats as well as some of the averaged
severities are presented in Table 3. Fatty change (Fig.
1), as characterized by large droplets, which were greater
in diameter than the hepatocellular nuclei, was
significantly increased in both male and female rats
exposed to 30 ppm and above, and its averaged severity
was increased in a dose-related manner except for the
female groups exposed to 270 and 810 ppm. Notably,
the inhalation exposure to 10 ppm induced the fatty
change with such large droplets in 2 rats of both sexes.
252
J Occup Health, Vol. 49, 2007
Table 2. Hematological, blood biochemical and urinary parameters of the rats exposed to CCl4 vapor by inhalation for 13 wk
Male
Group (ppm)
Control
Hematology
No. of animals
10
examined
Red blood cell 10.16
(106/ml)
Hemoglobin
17.0
(g/dl)
Hematocrit (%) 48.1
Blood biochemistry
No. of animals
10
examined
AST (IU/l)
76
ALT (IU/l)
25
LDH (IU/l)
350
ALP (IU/l)
266
Total bilirubin 0.14
(mg/dl)
Urinalysis
No. of animals
10
examined
Protein a
0
10
30
90
270
810
Control
10
Female
30
90
10
10
10
10
10
10
10
9b
10.27
10.36
10.27
10.01
7.92 **
9.08
9.32
17.1
16.8
16.0 **
14.8 **
12.0 **
16.4
48.3
47.9
45.9 **
43.1 **
34.7 **
10
10
10
10
10
81
27
353
271
0.14
79
30
335
284
0.11
10
10
10
0
0
0
118
67 **
565
294 **
0.14
459 ** 1,465 **
303 **
465 **
*
739
642
497 ** 1,098 **
0.18
0.48 **
10
10
7 **
10 **
270
810
10
10
10
9.67 **
9.26
9.06
8.29 **
16.8
16.3
15.1 **
14.4 **
12.8 **
46.0
47.0
46.8
42.8 **
41.0 **
36.7 **
10
10
10
10
10
10
65
19
294
183
0.17
72
25
265
204
0.16
100 **
38 **
438 *
196
0.14
233 **
111 **
471 **
284 **
0.17
364 **
146 **
501 **
427 **
0.25 *
897 **
322 **
404
725 **
0.36 **
10
10
10
10
10
10
0
0
0
10 **
10 **
7 **
Values indicate means. Significant difference; *: p≤0.05 **: p≤0.01 by Dunnett’s test. AST: aspartate aminotransferase, ALT:
alanine aminotransferase, LDH: lactate dehydrogenase, ALP: alkaline phosphatase. a: Number of animals whose urinary protein
level exceeded 100 mg/dl was counted. b: Blood collection failed for one rat.
Table 3. Incidences and severities of selected histopathological lesions in the rats exposed to CCl4 vapor by inhalation for 13 wk
Male
Group (ppm)
10
30
90
270
810
Control
10
Female
30
90
10
10
10
10
10
10
10
10
10
0
0
0
0
0
0
8
(1.0)
8
(1.0)
0
0
2
(1.0)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Control
Number of animals
examined
Liver
Fatty change:
small droplet
Fatty change:
large droplet
Fibrosis
Cirrhosis
Altered cell foci
Acidophilic cell foci
Basophilic cell foci
Clear cell foci
Kidney
Glomerulosclerosis:
localized
10 **
10 **
(1.1)
(1.5)
270
810
10
10
10
0
0
0
10 **
(2.0)
10 **
(2.1)
0
2
(1.0)
10 **
10 **
10 **
10 *
(2.0)
(2.5)
(2.0)
(1.5)
0
0
0
0
0
0
10 **
2
3
2
1
0
10 **
10 **
10 **
7 **
6 **
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5 **
0
3
1
1
1
0
0
10 **
0
0
0
0
5 **
10 **
10 **
6 **
0
9 **
9 **
9 **
10 **
6 **
0
10 **
0
10 **
Values indicate number of animals bearing lesion. The values in parentheses indicate the average of severity grade index of the lesion.
The average of severity grade was calculated with the following equation. Σ (grade × number of animals with grade) / number of
affected animals. Grade: 1=slight, 2=moderate, 3=severe. Significant difference; *: p≤0.05 **: p≤0.01 by chi-square test.
Kasuke NAGANO, et al.: Subchronic Inhalation Toxicity of CCl4 in Rodents
Fig. 1. Fatty change (arrowheads) characterized by large
droplets in the centrilobular hepatocytes of a male rat
exposed to 10 ppm CCl4 for 13 weeks. CV: central
vein. Bar indicates 200 µm. H & E stain.
253
Fig. 2. Cirrhosis in the liver of a male rat exposed to 810 ppm
CCl4 for 13 wk. Bar indicates 200 µm. H & E stain.
Fig. 3. Serial sections of altered cell focus (acidophilic cell focus) in the liver of a male rat exposed to 810 ppm CCl4
for 13 wk. A: H & E stain. B: Immunohistochemical staining for GST-P. The altered cell focus was positively
stained with anti-GST-P antibody. Arrows indicate altered cell focus. Bars indicate 200 µm.
However, fatty change having small droplets was seen in
both control and 10 ppm-exposed female groups. The
incidence of fibrosis was significantly increased in male
rats exposed to 270 and 810 ppm and in female rats
exposed to 90 ppm and above. Cirrhosis (Fig. 2) occurred
in both male and female rats exposed to 270 and 810
ppm, although its incidence in the 270 ppm-exposed male
rats was not statistically significant. Either fibrosis or
cirrhosis was accompanied by increased mitosis and
nuclear enlargement of hepatocytes, proliferation of bile
duct and deposition of ceroid-like yellow pigment in the
connective tissue area. Altered cell foci (Fig. 3A), which
were characterized by expansile nodules composed of
the tinctorially distinguishable hepatocytes from the
adjacent parenchyma, were observed in male rats exposed
to 270 and 810 ppm, and in female rats exposed to 90
ppm and above, although the incidences in the 270 ppmexposed male rats and in the 90 ppm-exposed female rats
were not statistically significant. The altered cell foci
were further classified into acidophilic, basophilic or clear
cell foci, depending upon the tinctorial characteristics of
hepatocyte cytoplasm in the H & E-stained sections. It
was found that these H & E-stained altered cell foci seen
in the 810 ppm-exposed male rats were also stained
positively with the anti-GST-P antibody (Fig. 3B),
whereas there were no GST-P-positive foci in the livers
of male controls. The altered cell foci were
homogeneously composed of GST-P positive hepatocytes.
Localized glomerulosclerosis, which was characterized
by segmental deposition of hyaline material in several
glomeruli, was significantly increased in both male and
female rats exposed to 810 ppm (Table 3).
No histopathological change was observed in the
respiratory tracts, including the nasal cavity, larynx, trachea
or lungs, of any CCl4-exposed rat group of either sex.
Mouse study
Survival, body and organ weights: One male mouse
each of the control and 10 ppm-exposed groups died of
hydronephrosis, but their deaths were not related to the
exposure to CCl4. No CCl4-related clinical sign was found
in any of the CCl4-exposed mouse groups of either sex.
Terminal body weight was significantly decreased only
in male mice exposed to 30 ppm and above (Table 4). A
statistically significant increase in relative organ weights
occurred in the livers and kidneys of male mice exposed
254
J Occup Health, Vol. 49, 2007
Table 4. Terminal body weight, and relative liver and kidney weights of the mice exposed to CCl4 vapor by inhalation for 13 wk
Male
Group (ppm)
No. of animals
examined
Body weight (g)
Organ weight
Liver (%)
Kidney (%)
Control
10
30
90
270
810
Control
10
Female
30
90
9a
9a
10
10
10
10
10
10
10
31.1
31.2
28.6 *
27.4 **
27.0 **
26.3 **
22.5
21.8
3.49
1.38
3.64
1.36
3.80 *
2.19 **
4.24 **
1.63 **
4.42 **
1.59 **
4.76 **
1.62 **
3.97
1.34
4.13
1.36
Values indicate means. Significant difference; *: p≤0.05
**:
p≤0.01 by Dunnett’s test.
a:
270
810
10
10
10
22.4
21.4
21.8
21.5
4.11
1.38
4.25
1.39
4.79 **
1.49 **
4.74 **
1.49 **
One mouse died of hydronephrosis.
Table 5. Hematological and blood biochemical parameters of the mice exposed to CCl4 vapor by inhalation for 13 wk
Male
Group (ppm)
Control
Hematology
No. of animals
9a
examined
Red blood cell 11.22
(106/µl)
Hemoglobin
15.6
(g/dl)
Hematocrit (%) 48.7
Blood biochemistry
No. of animals
9a
examined
AST(IU/l)
47
ALT(IU/l)
14
ALP(IU/l)
145
10
9a
30
9b
90
9b
270
10
11.34
11.16
11.17
11.15
15.8
15.5
15.5
15.4
49.3
47.4
48.0
47.9
9a
43
11
153
9b
48
17
182 **
9b
66
43 **
209 **
10
65 *
55 **
218 **
810
8b
10.73
Control
9b
10
9b
Female
30
90
9b
10
270
8b
810
10
10.79 * 10.72 **
11.24
11.34
11.24
11.03
14.9 *
16.0
16.0
15.7
15.6
15.1 **
15.1 **
46.4
48.7
49.0
47.8
47.9
47.4
46.6 **
10
10
10
10
10
51
12
241
49
13
270
59
30 **
273
67
42 **
273
75
44 **
278
9b
100 **
71 **
229 **
9b
53
12
249
Values indicate means. Significant difference; *: p≤0.05 **: p≤0.01 by Dunnett’s test. AST: aspartate aminotransferase, ALT:
alanine aminotransferase, ALP: alkaline phosphatase. a: One mouse died of hydronephrosis. b: Blood collection failed for one or
two mice in each group.
to 30 ppm and above and of female mice exposed to 270
and 810 ppm. However, the statistically significant
increase in the relative kidney weight of the 30 ppmexposed male group was attributed to the markedly
increased kidney weight of 2 male mice suffering from
hydronephrosis. Therefore, a biologically meaningful
increase in relative kidney weight was judged to occur in
male mice exposed to 90 ppm and above and in female
mice exposed to 270 and 810 ppm.
Clinical chemistry: While only the 810 ppm-exposed
male mice exhibited significantly decreased hemoglobin,
the female mice exposed to 270 and 810 ppm showed a
significant decrease in red blood cell counts, hemoglobin
and hematocrit, except for the hematocrit value at 270 ppm
(Table 5). ALT was significantly increased in male and
female mice exposed to 90 ppm and above. A significant
increase in AST occurred only in male mice exposed to
270 and 810 ppm. ALP was significantly increased only
in male mice exposed to 30 ppm and above. No significant
change in LDH or in the urinary protein, occurred in any
CCl4-exposed male or female mouse.
Pathology: None of the mice in any of the CCl4exposed groups of either sex had livers with a granular
surface.
Incidences and averaged severities of selected
microscopic lesions in the livers of CCl4-exposed mice
are presented in Table 6. Cytoplasmic globules (Fig. 4) of
up to 20 µm in size, palely stained with eosin, were
observed in the centrilobular hepatocytes of the CCl4exposed mice. The incidence of cytoplasmic globules was
Kasuke NAGANO, et al.: Subchronic Inhalation Toxicity of CCl4 in Rodents
255
Table 6. Incidences and severities of selected histopathological lesions in the mice exposed to CCl4 vapor by inhalation for 13 wk
Male
Group (ppm)
Control
Number of animals
examined
Liver
Cytoplasmic
globules
Fatty change:
small droplet
Fatty change:
large droplet
Collapse
9
0
9
(1.0)
0
Nuclear enlargement
with atypia
Altered cell foci
Acidophilic cell foci
Basophilic cell foci
Clear cell foci
Mixed cell foci
0
0
0
0
0
0
0
a
10
9a
30
90
270
810
Control
10
30
Female
90
270
810
10
10
10
10
10
10
10
10
10
10
10 **
(2.0)
0
10 **
(2.0)
0
0
0
5
(1.0)
0
4
(1.0)
0
10 **
10 **
5 **
(1.0)
(1.0)
(2.0)
2
0
0
(1.0)
4*
7 **
8 **
(1.0)
(1.1)
(1.0)
0
9 **
10 **
(1.0)
(1.0)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10 **
(2.0)
9 **
10 **
(2.0)
10 **
0
0
0
0
5 **
3
0
3
0
7 **
0
3
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10 **
10 **
10 **
10 **
(1.0)
(2.0)
(2.0)
(2.0)
1
0
0
0
(1.0)
4*
3
3
1
(1.0)
(1.0)
(1.0)
(1.0)
10 **
9 **
10 **
10 **
(1.0)
(1.0)
(2.0)
(2.0)
0
0
10 **
10 **
0
0
0
0
0
0
0
0
0
0
2
0
2
1
0
0
0
0
0
0
Values indicate number of animals bearing lesion. The values in parentheses indicate the average of severity grade index of the
lesion. The average of severity grade was calculated with the following equation. Σ (grade × number of animals with grade) /
number of affected animals. Grade: 1=slight, 2=moderate, 3=severe. Significant difference; *: p≤0.05 **: p≤0.01 by Chi-square
test. a: One mouse died of hydronephrosis.
significantly increased in all CCl4-exposed male groups
and in female mice exposed to 30 ppm and above, and
their average severity increased in a dose-related manner.
Fatty change (Fig. 4) with large droplets was significantly
increased in male mice exposed to 10, 30 and 90 ppm and
in female mice exposed to 30 ppm, whereas no fatty change
with such large droplets occurred in either male or female
controls. However, fatty change having small droplets was
observed in both control and low-level-exposed mice of
both sexes, and neither its incidence nor severity was doserelated. A significantly increased incidence of collapse,
which was characterized by the shrunken parenchymal
tissue over the centrilobular area, presumably resulting
from the necrotic loss of hepatocytes, was observed in both
male and female mice exposed to 30 ppm and above, and
its averaged severity was increased in a dose-related
manner. Hepatic collapse was accompanied by both
proliferation of the bile ducts and oval cells and deposition
of ceroid-like yellow pigment. Nuclear enlargement of
hepatocytes with atypia, often having irregularly shaped
nuclei, was significantly increased in male and female mice
exposed to 270 and 810 ppm. Neither fibrosis nor cirrhosis
occurred in any of the CCl4-exposed mice of either sex.
Altered cell foci (Fig. 5) were significantly increased in
male mice exposed to 270 and 810 ppm. They were also
observed in female mice exposed to 270 ppm but their
incidence was not statistically significant. The altered cell
foci were characterized by a focal area composed of
hepatocytes tinctorially distinguishable from adjacent
parenchyma, and were further classified into acidophilic,
basophilic, clear or mixed cell foci, according to their
tinctorial characteristics.
No histopathological change was observed in the nasal
cavity, larynx, trachea or lungs of any of the CCl4-exposed
mouse groups of both sexes.
Discussion
The present study shows that repeated inhalation
exposure of rats and mice of both sexes to CCl4 vapor at
10 to 810 ppm for 13 wk induced mild to severe
hepatotoxicity including a preneoplastic lesion, as well
as renal and hematological toxicities to a less severe
extent. However, inhalation exposure of rats and mice
of both sexes to CCl 4 vapor did not produce any
histopathological change in the respiratory tracts,
including the nasal cavity, larynx, trachea and lungs. The
present study confirmed the pathological findings of
earlier inhalation studies 17–19), by demonstrating the
increased liver weight, enhanced release of transaminases
into plasma and fatty degeneration in rats and mice,
256
J Occup Health, Vol. 49, 2007
Fig. 4. Fatty change characterized by large droplets
(arrowheads) and cytoplasmic globules (arrows) in the
centrilobular hepatocytes of a male mouse exposed to
10 ppm CCl4 for 13 wk. CV: central vein. Bar indicates
100 µm. H & E stain.
Fig. 5. An altered cell focus (clear cell focus) in the liver of a
male mouse exposed to 810 ppm CCl4 for 13 wk. Bar
indicates 200 µm. H & E stain.
fibrosis and cirrhosis in rats, and cytoplasmic globules
and collapse in mice. Furthermore, the present study
extended these previously reported findings to the
histopathologically novel observations that inhalation
exposure to high levels of CCl4 for 13 wk induced the
hepatic altered cell foci mirrored by GST-P-positively
stained hepatocytes as a preneoplastic lesion28–30), while
the low levels of CCl4 designated 10 ppm for the most
sensitive sign as fatty change, cytoplasmic globules and
increased liver weight. Both the preneoplastic lesion and
the most sensitive sign would be available for health risk
assessment of workers exposed to CCl4 by inhalation.
It is especially noteworthy in the present study that
altered cell foci, which are known as a precursor lesion
of hepatocellular tumors 34), were found in male rats
exposed to 270 and 810 ppm, in female rats exposed to
90 ppm and above, in male mice exposed to 270 and 810
ppm and in female mice exposed to 270 ppm, and that
the altered cell foci of rats were stained positively with
anti-GST-P antibody. It has been reported that GST-P is
markedly induced in the liver foci and nodules in the
early stages of chemical hepatocarcinogenesis, while the
content of GST-P is very low in normal liver tissue35, 36).
GST-P has been reported to be an effective marker enzyme
for detection of preneoplastic lesions in chemical
hepatocarcinogenesis28–30). Ito et al.30, 37, 38) also reported
that almost all hepatocarcinogens induced GST-P positive
foci in the liver, which allowed prediction of
hepatocarcinogenicity with high probability. Therefore,
it can be concluded from the present result from positive
immunochemical staining of the altered cell foci with
GST-P that induction of the altered cell foci by 13-wk
inhalation exposure to CCl 4 might develop to
hepatocellular tumors after long-term inhalation exposure.
Well-known macro- and micro-scopic lesions of
fibrosis and cirrhosis were confirmed in rats of both sexes
exposed to high levels of CCl4 at 270 and 810 ppm as
well as in the 90 ppm-exposed female rats, whereas those
severe signs were not observed in any of the CCl4-exposed
mice. These results suggest that a difference in occurrence
of these severe hepatotoxic signs exists between rats and
mice, although any causative factor for the species
difference remains to be explored. Enhanced cytolytic
release of liver-associated transaminases into plasma,
reflecting a necrotic change in hepatocytes, occurred in
male rats and mice of both sexes exposed to 90 ppm and
above and in female rats exposed to 30 ppm, suggesting
that these biochemical alterations were induced at lower
exposure concentrations than the fibrotic and cirrhotic
changes. Hepatic collapse found in male and female mice
exposed to 30 ppm and above might have resulted from
hepatocellular necrosis acutely induced by CCl4 and thus
gave a good association with the enhanced cytolytic
release of ALT into plasma. The collapse was not
observed in any CCl4-exposed rat of either sex, and was
considered indicative of a necrotic lesion specifically
occurring in mice.
The most sensitive signs were found to be the increased
liver weight occurring in male rats exposed to 10 ppm,
the fatty change with large droplets occurring in male
and female rats and male mice exposed to 10 ppm and
the cytoplasmic globules occurring in male mice exposed
to 10 ppm. Both the fatty change and the cytoplasmic
globules were characteristic of excessive lipid
accumulation and the appearance of palely eosinophilic
materials in the hepatocellular cytoplasm, respectively.
Therefore, a LOAEL of 10 ppm can be determined as the
hepatic endpoint of the two rodent species on the
following basis. First, dose-response relationships were
found to hold for these hepatic endpoints except for the
fatty change with the large droplets in the exposed mice.
Second, both fatty change with large droplets and
cytoplasmic globules are thought to morphologically
reflect degenerative changes rather than any adaptive
change, aside from increased liver weight. The LOAEL
value of 10 ppm for CCl4 on the basis of 6 h/d found in
Kasuke NAGANO, et al.: Subchronic Inhalation Toxicity of CCl4 in Rodents
the present study is thought to be lower than the
previously reported NOAELs17, 18) determined by repeated
inhalation exposure of experimental animals to CCl 4
vapor, when daily exposure length of time was taken into
account. Adams et al.17) reported that while repeated
inhalation exposure of rats to 10 ppm CCl4 for 7 h/d and
192 d induced both increased liver weight and fatty
degeneration in the liver, 5 ppm did not produce any
change. Prendergast et al.18) reported that continuous
inhalation exposure of rats to 1 ppm CCl4 for 24 h/d and
90 d did not produce any specific pathologic change
attributable to the exposure, while 10 ppm induced fatty
change, fibroblastic proliferation and hepatic cell
degeneration and regeneration. Oral administration of
CCl 4 to rats and mice by gavage was also reported to
induce the same kinds of pathologic and biochemical
responses as those found in this inhalation study.
Bruckner et al.12) reported that oral administration of CCl4
to rats for 12 wk at a dose of 1 mg/kg elicited no apparent
adverse effects, whereas 10 mg/kg produced both
increased sorbitol dehydrogenase activity and hepatic
centrilobular vacuolization. Their data12) suggested 1 mg/
kg/d as a NOAEL. Condie et al.13) reported that an oral
dose of 12 mg CCl4/kg/d by gavage to CD-1 mice for 90
d induced hepatocellular vacuolar change, fatty
accumulation and increased serum activities of AST, ALT
and LDH, designating a lower dose of 1.2 mg/kg/d as a
NOAEL. Hayes et al.14) reported that oral administration
of CCl 4 to mice for 90 d induced fatty change and
increased LDH and ALT activities at a daily dose of 12
mg/kg. Their data14) suggested a LOAEL of 12 mg/kg/d.
Therefore, 6-h inhalation exposure of rats and mice to 10
ppm CCl4 vapor corresponds to a daily CCl4 uptake of 13
mg/kg and 29 mg/kg body weight, respectively, assuming
a minute volume of 561 ml/min/kg body weight for rats39)
and 1,239 ml/min/kg body weight for mice40) and a lung
absorption ratio of 100% for both rats and mice.
Therefore, the LOAEL value of 10 ppm for the hepatic
endpoint of rats and mice inhaling CCl4 appears to be
higher than the NOAEL and LOAEL values from the
oral administration of CCl4 to rats and mice by gavage.
This can be partly attributed to the first-pass effect of
CCl4 entering the liver directly through the
gastrointestinal tract by bolus gavage dose, as suggested
by Sanzgiri et al.20, 21).
CCl 4 -induced nephrotoxicity was manifested as
increased relative kidney weight in rats and mice, and a
significantly increased incidence of localized
glomerulosclerosis and increased urinary protein in rats.
CCl4-induced hematotoxicity occurred as decreases in
erythrocyte parameters in rats and mice. Both
nephrotoxicity and hematotoxicity were induced at higher
exposure concentrations than the CCl 4 -induced
hepatotoxicity. Consistently, medical case studies on
human CCl4 poisoning8, 41, 42) have reported that the liver
257
is a target organ of human CCl4 poisoning and the kidney
impairment and erythrocyte injury often accompany liver
damage.
It is interesting to note that the German MAK value of
0.5 ppm for CCl4 established in 200024) is one tenth lower
than the current OEL value of 5 ppm recommended by
the ACGIH in 1996 22) and the JSOH in 1991 23). The
lowered MAK value of 0.5 ppm which was based on the
result of marginal effects of exposure to CCl 4
concentrations of about 1 ml/m 3 in man (reduced
hematocrit) was not in conflict with the NOEL of 1 mg/
kg body weight 12) determined in studies with oral
administration to the rat. Exposure to CCl4 concentrations
of 0.5 ml/m3 for 8 h a day would correspond to a daily
uptake of 0.5 mg/kg body weight for man24).
Recommendations of the OEL value of 5 ppm for CCl4
by ACGIH and JSOH were based on both animal and
human studies22, 23). The hepatic endpoints of fatty change
in rats and mice and cytoplasmic globules in mice
employed for the determination of the LOAEL are not
adaptive but degenerative in nature. The route of
exposure and the daily and weekly exposure regimens
employed in this study closely simulate workers’
inhalation exposure in the workplace. Assuming an
uncertainty factor of 10 for extrapolation of rodent data
to humans43), one tenth of the present LOAEL value might
fall below the current OEL value of 5 ppm, suggesting
the need to reconsider the OEL for CCl4.
ACGIH classified carcinogenicity of CCl4 as A2, a
suspected human carcinogen, based on the increased liver
tumor incidence in mice and hamsters receiving oral
administration of CCl4, shedding light on a threshold
mechanism of carcinogenic action on the basis of both the
absent or very weak genotoxicity of CCl 4 and the
association with cytotoxicity and regenerative
hyperplasia22). DFG also assigned CCl4 to Carcinogen
category 424), indicating that genotoxicity plays no or at
most a minor part and no significant contribution to human
cancer risk is expected provided that the MAK value is
observed. Therefore, the present finding of occurrence of
the altered cell foci in the liver resulting from the
subchronic inhalation exposure of rats and mice to CCl4
provides cancer-related animal data strengthening the
evidence of CCl4-induced hepatocarcinogenicity which is
used to establish the carcinogenicity classifications by
ACGIH, DFG and JSOH, since the GST-P positive foci
are known as a preneoplastic lesion that allows prediction
of hepatocarcinogenicity with high probability30, 37, 38).
Further inhalation studies will be urgently needed to
confirm hepatocarcinogenicity by 2-yr inhalation exposure
of rats and mice of both sexes to CCl4 vapor.
As stated in the documentations by ACGIH 22) and
DFG24), involvement of genotoxicity in CCl 4-induced
carcinogenesis was negatively evaluated in the
carcinogenic risk assessment of CCl4. However, a recent
258
study from our laboratory 44) demonstrated that
mutagenicity of CCl4 was positive in E. coli WP2uvrA/
pKM101 and WP2/pKM101 with and without S9 mix
and in S. typhimurium TA98 without S9 mix. Thus, the
threshold mechanism based on a genotoxic mode-ofaction hypothesis will have to be re-examined for CCl4induced carcinogenesis.
In conclusion, the present study demonstrated that 13wk inhalation exposure of rats and mice of both sexes to
CCl4 induced mild to severe hepatotoxicity as well as
hemato- and nephro-toxicities to a lesser extent. Fibrosis
and cirrhosis were observed in the rats exposed to high
levels of CCl4, but not in any exposed mice. Altered cell
foci occurred in the livers of rats and mice exposed to
high levels of CCl4. Preneoplastic GST-P positive foci
of CCl4-exposed rats allowed prediction of CCl4-induced
hepatocarcinogenicity with high probability. The LOAEL
was estimated to be 10 ppm for the hepatic endpoint in
both rats and mice.
Acknowledgments: The present study was contracted
and supported by the Ministry of Labour of Japan (the
present Ministry of Health, Labour and Welfare). The
authors are deeply indebted to Dr. Taijiro Matsushima,
former Director of the Japan Bioassay Research Center
for his support and encouragement throughout this study.
References
1) United Nations Environment Programme (UNEP). The
1987 Montreal Protocol on Substances that Deplete
the Ozone Layer as adjusted and amended by the
second, fourth and seventh Meetings of the Parties. In:
Handbook for the international treaties for the
protection of the ozone layer, 4th ed. Nairobi: UNEP,
1996: 18–39.
2) Doherty RE: A history of the production and use of
carbon tetrachloride, tetrachloroethylene,
trichloroethylene and 1,1,1-trichloroethane in the
United States: Part 1—Historical background; carbon
tetrachloride and tetrachloroethylene. J Environ
Forensics 1, 69–81 (2000)
3) Chemical Daily. Carbon tetrachloride. In: Chemical
products handbooks issued from 1975 through 2006.
Tokyo: The Chemical Daily Co. Ltd. (in Japanese).
4) Ukai H, Inui S, Takada S, Dendo J, Ogawa J, Isobe K,
Ashida T, Tamura M, Tabuki K and Ikeda M: Types of
organic solvents used in small- to medium-scale
industries in Japan; a nationwide field survey. Int Arch
Occup Environ Health 70, 385–392 (1997)
5) Kauppinen T, Pukkala E, Saalo A and Sasco AJ:
Exposure to chemical carcinogens and risk of cancer
among Finnish laboratory workers. Am J Ind Med 44,
343–350 (2003)
6) Olansandan, Amagai T and Matsushita H: A passive
sampler-GC/ECD method for analyzing 18 volatile
organohalogen compounds in indoor and outdoor air
and its application to a survey on indoor pollution in
J Occup Health, Vol. 49, 2007
Shizuoka, Japan. Talanta 50, 851–863 (1999)
7) Japan Ministry of the Environment. Pollutant Release
and Transfer Register, Fiscal Year 2004. Environmental
Health and Safety Division, Environmental Health
Department, Japan Ministry of the Environment, 2006.
(online), available from <http://www.env.go.jp/en/
chemi/prtr.html>, (accessed 2006-11-22).
8) Reid JB. Carbon tetrachloride. In: Bingham E,
Cohrssen B, Powell CH, eds. Patty’s toxicology, Vol.
5. NY: John Wiley & Sons, Inc., 2001: 73–87.
9) International Programme on Chemical Safety.
Environmental health criteria 208. Carbon
tetrachloride. Geneva: WHO, 1999.
10) International Agency for Research on Cancer (IARC).
Carbon tetrachloride. In: IARC monographs on the
evaluation of the carcinogenic risk of chemicals to
humans, Vol. 20, Some halogenated hydrocarbons.
Lyon: IARC, 1979: 371–399.
11) International Agency for Research on Cancer (IARC).
Carbon tetrachloride. In: IARC monographs on the
evaluation of carcinogenic risks to humans, Vol. 71,
Re-evaluation of some organic chemicals, hydrazine
and hydrogen peroxide (Part Two). Lyon: IARC, 1999:
401–432.
12) Bruckner JV, MacKenzie WF, Muralidhara S, Luthra
R, Kyle GM and Acosta D: Oral toxicity of carbon
tetrachloride: Acute, subacute, and subchronic studies
in rats. Fundam Appl Toxicol 6, 16–34 (1986)
13) Condie LW, Laurie RD, Mills T, Robinson M and Bercz
JP: Effect of gavage vehicle on hepatotoxicity of carbon
tetrachloride in CD-1 mice: corn oil versus tween-60
aqueous emulsion. Fundam Appl Toxicol 7, 199–206
(1986)
14) Hayes JR, Condie LW Jr and Borzelleca JF: Acute,
14-day repeated dosing, and 90-day subchronic toxicity
studies of carbon tetrachloride in CD-1 mice. Fundam
Appl Toxicol 7, 454–463 (1986)
15) Weisburger EK: Carcinogenicity studies on
halogenated hydrocarbons. Environ Health Perspect 21,
7–16 (1977)
16) National Cancer Institute (NCI). Report on
carcinogenesis bioassay of chloroform. Bethesda: NCI,
1976.
17) Adams EM, Spencer HC, Rowe VK, McCollister DD
and Irish DD: Vapor toxicity of carbon tetrachloride
determined by experiments on laboratory animals.
AMA Arch Ind Hyg Occup Med 6, 50–66 (1952)
18) Prendergast JA, Jones RA, Jenkins LJ Jr and Siegel J:
Effects on experimental animals of long-term inhalation
of trichloroethylene, carbon tetrachloride, 1,1,1trichloroethane, dichlorodifluoromethane, and 1,1dichloroethylene. Toxicol Appl Pharmacol 10, 270–289
(1967)
19) Plummer JL, Hall P de la M, Ilsley AH, Jenner MA
and Cousins MJ: Influence of enzyme induction and
exposure profile on liver injury due to chlorinated
hydrocarbon inhalation. Pharmacol Toxicol 67, 329–
335 (1990)
20) Sanzgiri UY, Kim HJ, Muralidhara S, Dallas CE and
Bruckner JV: Effect of route and pattern of exposure
Kasuke NAGANO, et al.: Subchronic Inhalation Toxicity of CCl4 in Rodents
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
on the pharmacokinetics and acute hepatotoxicity of
carbon tetrachloride. Toxicol Appl Pharmacol 134,
148–154 (1995)
Sanzgiri UY, Srivatsan V, Muralidhara S, Dallas CE
and Bruckner JV: Uptake, distribution, and elimination
of carbon tetrachloride in rat tissues following
inhalation and ingestion exposures. Toxicol Appl
Pharmacol 143, 120–129 (1997)
American Conference on Governmental Industrial
Hygienists (ACGIH). Carbon tetrachloride. In:
Documentation of the threshold limit values (TLVs)
and biological exposure indices (BELs) [CD-ROM].
Cincinnati: ACGIH, 2001.
Japan Society for Occupational Health: Documentation
of the occupational exposure limit value for carbon
tetrachloride and chloroform. Jpn J Ind Health 33, 310–
312 (1991) (in Japanese)
Deutsche Forschungsgemeinschaft. Carbon
tetrachloride. In: Greim H, ed. Occupational toxicants.
Critical data evaluation for MAK values and
classification of carcinogens, Vol. 18. Weinheim:
Wiley-VCH Verlag GmbH, 2002: 81–106.
Organisation for Economic Co-operation and
Development (OECD). OECD Guideline for Testing
of Chemicals 413 “Subchronic Inhalation Toxicity: 90day Study”. Paris: OECD, 1981.
National Institutes of Health (NIH). Guide for the Care
and Use of Laboratory Animals. DHEW Publication
No. (NIH) 77–23. Bethesda: NIH, 1977.
Kano H, Goto K, Suzuki M, Yamazaki K, Nishizawa
T, Arito H, Yamamoto S and Matsushima T: An
exposure system for combined administration of an
organic solvent to rodents by inhalation and waterdrinking and its operational performance. J Occup
Health 44, 119–124 (2002)
Sato K, Kitahara A, Satoh K, Ishikawa T, Tatematsu
M and Ito N: The placental form of glutathione Stransferase as a new marker protein for preneoplasia
in rat chemical hepatocarcinogenesis. Gann 75, 199–
202 (1984)
Tatematsu M, Mera Y, Ito N, Satoh K and Sato K:
Relative merits of immunohistochemical
demonstrations of placental, A, B and C forms of
glutathione S-transferase and histochemical
demonstration of γ-glutamyl transferase as markers of
altered foci during liver carcinogenesis in rats.
Carcinogenesis 6, 1621–1626 (1985)
Ito N, Tsuda H, Tatematsu M, Inoue T, Tagawa Y, Aoki
T, Uwagawa S, Kagawa M, Ogiso T, Masui T, Imaida
K, Fukushima S and Asamoto M: Enhancing effect of
various hepatocarcinogens on induction of
preneoplastic glutathione S-transferase placental form
positive foci in rats—an approach for a new mediumterm bioassay system. Carcinogenesis 9, 387–394
(1988)
Vyberg M and Nielsen S: Dextran polymer conjugate
two-step visualization system for
32)
33)
34)
35)
36)
37)
38)
39)
40)
41)
42)
43)
44)
259
immunohistochemistry. A comparison of EnVision+
with two three-step avidin-biotin techniques. Appl
Immunohistochem 6, 3–10 (1998)
Aiso S, Arito H, Nishizawa T, Nagano K, Yamamoto S
and Matsushima T: Thirteen-week inhalation toxicity
of p-dichlorobenzene in mice and rats. J Occup Health
47, 249–260 (2005)
International Programme on Chemical Safety.
Environmental health criteria 170. Assessing human
health risks of chemicals: Derivation of guidance values
for health-based exposure limits. Geneva: WHO, 1994.
Eustis SL, Boorman GA, Harada T and Popp JA. Liver.
In: Boorman GA, Eustis SL, Elwell MR, et al., eds.
Pathology of the Fischer rats. Reference and atlas. San
Diego: Academic Press, Inc., 1990: 71–94.
Kitahara A, Satoh K, Nishimura K, Ishikawa T, Ruike
K, Sato K, Tsuda H and Ito N: Changes in molecular
forms of rat glutathione S-transferase during
hepatocarcinogenesis. Cancer Res 44, 2698–2703 (1984)
Satoh K, Kitahara A, Soma Y, Inaba Y, Hatayama I
and Sato K: Purification, induction, and distribution
of placental glutathione transferase: a new marker
enzyme for preneoplastic cells in the rat chemical
hepatocarcinogenesis. Proc Natl Acad Sci USA 82,
3964–3968 (1985)
Ito N, Hasegawa R, Imaida K, Hirose M, Shirai T,
Tamano S and Hagiwara A: Medium-term rat liver
bioassay for rapid detection of hepatocarcinogenic
substances. J Toxicol Pathol 10, 1–11 (1997)
Ito N, Imaida K, Asamoto M and Shirai T: Early
detection of carcinogenic substances and modifiers in
rats. Mutat Res 462, 209–217 (2000)
Mauderly JL, Tesarek JE, Sifford LJ and Sifford LJ:
Respiratory measurements of unsedated small
laboratory mammals using nonrebreathing valves. Lab
Anim Sci 29, 323–329 (1979)
Guyton AC: Measurement of the respiratory volumes
of laboratory animals. Am J Physiol 150, 70–77 (1947)
Deng J-F, Wang J-D, Shih T-S and Lan F-L: Outbreak
of carbon tetrachloride poisoning in a color printing
factory related to the use of isopropyl alcohol and an
air conditioning system in Taiwan. Am J Ind Med 12,
11–19 (1987)
Tomenson JA, Baron CE, O’Sullivan JJ, Edwards JC,
Stonard MD, Walker RJ and Fearnley DM: Hepatic
function in workers occupationally exposed to carbon
tetrachloride. Occup Environ Med 52, 508–514 (1995)
Barns DG and Dourson M: Reference dose (RfD):
Description and use in health risk assessments. Regul
Toxicol Pharmacol 8, 471–486 (1988)
Araki A, Kamigaito N, Sasaki T and Matsushima T:
Mutagenicity of carbon tetrachloride and chloroform
in Salmonella typhimurium TA98, TA100, TA1535, and
TA 1537, and Escherichia coli WP2uvrA/pKM101 and
WP2/pKM101, using a gas exposure method. Environ
Mol Mutagenesis 43, 128–133 (2004)