Evaluation of the carcinogenicity of carbon tetrachloride

Figures & data

Table 1. Incidences of selected histopathological lesions in the rats exposed to CCl₄ vapor by inhalation for 2 years (Nagano et al. 2007a).

Group	Male					Female
	Control	5 ppm	25 ppm	125 ppm	Peto	Control	5 ppm	25 ppm	125 ppm	Peto
No. of animals examined	50	50	50	50		50	50	50	50
Neoplastic lesions
Liver
Hepatocellular adenoma	0	1	1	21**	↑↑	0	0	0	40**	↑↑
Hepatocellular carcinoma	1	0	0	32**	↑↑	0	0	3	15**	↑↑
(Metastasis to lung)	(0)	–	–	(4)		–	–	(0)	(1)
Hepatocellular tumors^a	1	1	1	40**	↑↑	0	0	3	44**	↑↑
Pre-neoplastic lesions
Liver
Altered cell foci	14	12	13	16		3	4	24##	ND
Clear cell foci	10	9	7	3		2	2	14##	ND
Acidophilic cell foci	2	1	5	3		0	0	16##	ND
Basophilic cell foci	1	3	2	12##		0	1	6#	ND
Mixed cell foci	2	3	1	2		1	1	4	ND
Non-neoplastic lesions
Liver
Fatty change	4	7	39##	49##		6	7	49##	46##
Fibrosis	0	0	43##	2		0	0	45##	0
Cirrhosis	0	0	1	40##		0	0	2	50##
Kidney
Chronic progressive	49	49	50#	49##		44	45	49##	50##
nephropathy^b	(1.8)	(2.0)	(2.2)	(2.5)		(1.3)	(1.2)	(1.7)	(2.7)
Lung
Uremic pneumonia	1	5	2	19##		0	0	1	4#
Nasal cavity
Eosinophilic globules^b	43	47	50##	47##		39	49##	50##	50##
	(1.0)	(1.0)	(1.5)	(1.7)		(1.0)	(1.3)	(1.5)	(1.9)

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 is calculated with the following equation: ⅀ (grade x number of animals with grade)/number of affected animals. Grade: 1, slight; 2, moderate; 3, severe.

Significant difference indicated by *p ≤ .05, **p ≤ 0.01 by Fisher exact test; #p ≤ .05, ##p ≤ 0.01 by chi-square test; ↑ p ≤ .05, ↑↑

p ≤ .01 by Peto’s test. ND: Altered cell foci were not detected, since almost all area of the liver tissue was occupied by hepatocellular tumors.

^aHepatocellular tumors include hepatocellular adenoma and hepatocellular carcinoma.

^bNumber of animals with the lesion with average severity score in parenthesis. Statistical evaluation is for severity score since nearly all animals had the lesion.

Table 2. Incidences of selected histopathological lesions in the mice exposed to CCl₄ vapor by inhalation for 2 years (Nagano et al. 2007a).

Group	Male					Female
	Control	5 ppm	25 ppm	125 ppm	Peto	Control	5 ppm	25 ppm	125 ppm	Peto
No. of animals examined	50	50	50	50		50	49^a	50	49^a
Neoplastic lesions
Liver
Hepatocellular adenoma	9	10	27**	16	↑↑	2	8*	17**	5	↑↑
Hepatocellular carcinoma	17	12	44**	47**	↑↑	2	1	33**	48**	↑↑
(Metastasis to lung)	(3)	(3)	(10)	(14)		(1)	(0)	(4)	(8)
Hepatocellular tumors^b	24	20	49**	49**	↑↑	4	9	44**	48**	↑↑
Adrenal gland
Pheochromocytoma: benign	0	0	16**	31**	↑↑	0	0	0	22**	↑↑
Pre-neoplastic lesions
Liver
Altered cell foci	4	7	1	1		1	0	0	2
Clear cell foci	2	6	0	1		1	0	0	0
Acidophilic cell foci	1	1	1	0		0	0	0	1
Basophilic cell foci	1	1	0	0		0	0	0	1
Non-neoplastic lesions
Liver
Deposit of ceroid	2	1	36##	36##		0	0	28##	35##
Proliferation: bile duct	0	0	19##	22##		0	0	5##	9##
Hydropic change: centrilobular	1	0	8#	9##		1	0	13##	12##

Values indicate number of animals bearing lesion. Significant difference indicated by *p ≤ .05, **p ≤ 0.01 by Fisher exact test; #p ≤ .05, ##p ≤ 0.01 by chi-square test; ↑p ≤ .05, ↑↑p ≤ .01 by Peto’s test.

^aNumber of mice examined was 49 instead of 50, because one mouse accidentally died.

^bHepatocellular tumors include hepatocellular adenoma and hepatocellular carcinoma.

Table 3. Summary of principle chronic/carcinogenicity rodent studies of carbon tetrachloride.

Strain, species	Gender	Method of administration	Doses	Tumors	Reference
BDF1 mice	Male and female	Inhalation	0, 5, 25, 125 ppm	Hepatocellular tumors; adrenal pheochromocytoma	(Nagano et al. 2007a)
F344 rats	Male and female	Inhalation	0, 5, 25, 125 ppm	Hepatocellular tumors	(Nagano et al. 2007a)
C3H mice	Male	Gavage	0, 0.2, 0.4, 1.6 g/kg bw	Hepatocellular tumors	(Kiplinger and Kensler 1963)
B6C3F1 mice	Male and female	Gavage	1,250 and 2,500 mg/kg bw	Hepatocellular tumors	NCI 1976 (Weisburger 1977);

Table 4. Hematological, blood biochemical, and urinary parameters of rats exposed to CCl₄ vapor by inhalation for 13 weeks (Nagano et al. 2007b).

	Male						Female
Group (ppm)	Control	10	30	90	270	810	Control	10	30	90	270	810
Hematology
No. of animals examined	10	10	10	10	10	10	10	10	9^b	10	10	10
Red blood cell (10^6/ml)	10.16	10.27	10.36	10.27	10.01	7.92**	9.08	9.32	9.67**	9.26	9.06	8.29**
Hemoglobin (g/dl)	17.0	17.1	16.8	16.0**	14.8**	12.0**	16.4	16.8	16.3	15.1**	14.4**	12.8**
Hematocrit (%)	48.1	48.3	47.9	45.9**	43.1**	34.7**	46.0	47.0	46.8	42.8**	41.0**	36.7**
Blood biochemistry No. of animals examined	10	10	10	10	10	10	10	10	10	10	10	10
AST (IU/l)	76	81	79	118	459**	1,465**	65	72	100**	233**	364**	897*#
ALT (IU/l)	25	27	30	67**	303**	465**	19	25	38**	111**	146**	322**
LDH (IU/l)	350	353	335	565	739*	642	294	265	438*	471**	501**	404
ALP (IU/l)	266	271	284	294**	497**	1,098**	183	204	196	284**	427**	725**
Total bilirubin (mg/dl)	0.14	0.14	0.11	0.14	0.18	0.48**	0.17	0.16	0.14	0.17	0.25*	0.36**

Values indicate means. Significant difference; *:p ≤ .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 5. Hematological and blood biochemical parameters of mice exposed to CCl₄ vapor by inhalation for 13 weeks (Nagano et al. 2007b).

	Male						Female
Group (ppm)	Control	10	30	90	270	810	Control	10	30	90	270	810
Hematology
No. of animals examined	9^a	9^a	9^b	9^b	10	8^b	9^b	9^b	9^b	10	8^b	10
Red blood cell (10^6/μl)	11.22	11.34	11.16	11.17	11.15	10.73	11.24	11.34	11.24	11.03	10.79	10.72
Hemoglobin (g/dl)	15.6	15.8	15.5	15.5	15.4	14.9*	16.0	16.0	15.7	15.6	15.1**	15.1**
Hematocrit (%)	48.7	49.3	47.4	48.0	47.9	46.4	48.7	49.0	47.8	47.9	47.4	46.6**
Blood biochemistry
No. of animals examined	9^a	9^a	9^b	9^b	10	9^b	9^b	10	10	10	10	10
AST (IU/l)	47	43	48	66	65*	100*	53	51	49	59	67	75
ALT (IU/l)	14	11	17	43**	55**	71**	12	12	13	30**	42**	44**
ALP (IU/l)	145	153	182**	209**	218**	229**	249	241	270	273	273	278

Values indicate means. Significant difference; *:p ≤ .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.

Table 6. Incidences and severities of selected histopathological lesions in rats exposed to CCl₄ vapor by inhalation for 13 weeks (Nagano et al. 2007b).

	Male						Female
Group (ppm)	Control	10	30	90	270	810	Control	10	30	90	270	810
Number of animals examined	10	10	10	10	10	10	10	10	10	10	10	10
Liver
Fatty change:	0	0	0	0	0	0	8	8	0	0	0	0
Small droplet							(1.0)	(1.0)
Fatty change:	0	2	10**	10**	10**	10**	0	2	10**	10**	10**	10*
Large droplet		(1.0)	(1.1)	(1.5)	(2.0)	(2.1)		(1.0)	(2.0)	(2.5)	(2.0)	(1.5)
Fibrosis	0	0	0	0	10**	10**	0	0	0	5**	5**	9**
Cirrhosis	0	0	0	0	2	10**	0	0	0	0	10**	9**
Altered cell foci	0	0	0	0	3	10**	0	0	0	3	10**	10**
Acidophilic cell foci	0	0	0	0	2	7**	0	0	0	1	6**	6**
Basophilic cell foci	0	0	0	0	1	6**	0	0	0	1	0	0
Clear cell foci	0	0	0	0	0	0	0	0	0	1	9**	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. ⅀ (grade x number of animals with grade)/number of affected animals. Grade: 1=slight; 2=moderate; 3=severe. Significant difference; *:p ≤ .05, **:p ≤ 0.01 by chi-square test.

Table 7. Incidences and severities of selected histopathological lesions in mice exposed to CCl₄ vapor by inhalation for 13 weeks (Nagano et al. 2007b).

	Male						Female
Group (ppm)	Control	10	30	90	270	810	Control	10	30	90	270	810
Number of animals examined	9^a	9^a	10	10	10	10	10	10	10	10	10	10
Liver
Cytoplasmic	0	5**	10**	10**	10**	10**	0	0	10**	10**	10**	10**
Globules		(1.0)	(1.0)	(2.0)	(2.0)	(2.0)			(1.0)	(2.0)	(2.0)	(2.0)
Fatty change:	9	2	0	0	0	0	5	4	1	0	0	0
Small droplet	(1.0)	(1.0)					(1.0)	(1.0)	(1.0)
Fatty change:	0	4*	7**	8**	0	0	0	0	4*	3	3	1
Large droplet		(1.0)	(1.1)	(1.0)					(1.0)	(1.0)	(1.0)	(1.0)
Collapse	0	0	9**	10**	10**	10**	0	0	10**	9**	10**	10**
			(1.0)	(1.0)	(2.0)	(2.0)			(1.0)	(1.0)	(2.0)	(2.0)
Nuclear enlargement with Atypia	0	0	0	0	9**	10**	0	0	0	0	10**	10**
Altered cell foci	0	0	0	0	5**	7**	0	0	0	0	2	0
Acidophilic cell foci	0	0	0	0	3	0	0	0	0	0	0	0
Basophilic cell foci	0	0	0	0	0	3	0	0	0	0	2	0
Clear cell foci	0	0	0	0	3	2	0	0	0	0	1	0
Mixed cell foci	0	0	0	0	0	2	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. ⅀ (grade x number of animals with grade)/number of affected animals. Grade: 1=slight; 2=moderate; 3=severe. Significant difference; *:p ≤ .05, **:p ≤ 0.01 by chi-square test. ^a: One mouse died of hydronephrosis.

Table 8. Modes of action for hepatocellular carcinogenesis (modified from Cohen 2010).

i. DNA reactivity
ii. Non-DNA reactive (proliferation)
A. Receptor mediated
1. PPARα (peroxisome proliferation) activation
2. CAR activation
3. PXR activation
4. AHR activation
5. Estrogen activation
6. Cytotoxicity
a. Statins (HMG CoA reductase inhibition)
b. Porphyrias (porphyrin synthesis enzyme imitation)
c. Inherited disorders
B. Non-receptor mediated
1. Cytotoxicity
a. Infection
b. Metal overload (Cu, Fe)
c. Chemical cytotoxicity
d. Inherited disorders
e. Non-alcoholic steatohepatitis (NASH)

Table 9. Proposed mode of action for CCl₄-induced rodent tumors.

Key Events	References
Metabolism of CCl4 to the trichloromethyl radical by CYP2E1 and subsequent formation for the trichloromethyl peroxyl radical	(Albano et al. 1982; Ansari, Moslen, and Reynolds 1982; Fanelli and Castro 1995; Reinke and Janzen 1991); Pohl et al. 1984 (Wong, Chan, and Lee 1998);
Radical-induced hepatocellular cytotoxicity	(Dai and Cederbaum 1995; Reinke and Janzen 1991; Takahashi et al. 2002); Bruckner et al. 1990 (Diaz Gomez and Castro 1980; Packer, Slater, and Willson 1978)
Chronic and sustained necrosis (hepatotoxicity)	(Nagano et al. 2007b); Nakata et al. 1985 (Benson and Springer 1999; Doolittle, Muller, and Scribner 1987; Lee, Cameron, and Archer 1998);
Consequent regenerative increased cell proliferation	(Nagano et al. 2007b)
Formation of hepatocellular foci	(Nagano et al. 2007a)
Formation of hepatocellular neoplasms	(Nagano et al. 2007a)

Figure 1. Mode of action for carbon tetrachloride carcinogenesis.

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Data availability statement

This is a review manuscript, and all data referred to is available in the published literature.