Page 10: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Carbon Tetrachloride
Part II. Science and Technical Considerations - Continued
Acute exposure to carbon tetrachloride has been shown to cause central nervous system depression and gastrointestinal effects such as nausea and vomiting. The liver and kidneys have been shown to be the most sensitive target organs of carbon tetrachloride toxicity (ATSDR, 2005).
A number of historical cases of poisoning by oral exposure to carbon tetrachloride have been reported in the literature, with lethalities occurring over a wide range of doses (approximately 43-450 mg/kg bw) (Phelps and Hu, 1924; Umiker and Pearce, 1953; Guild et al., 1958). Acute exposure to carbon tetrachloride via inhalation has also been shown to cause various adverse effects, such as headaches and dizziness in workers exposed to 250 ppm for 4 h (Norwood et al., 1950), decreased serum iron in volunteers exposed to up to 50 ppm for 1-3 h (Stewart et al., 1961), and gastrointestinal irritation, nausea, proteinuria, and increased hepatic bilirubin in workers exposed to up to 200 ppm for 3 h (Barnes and Jones, 1967).
Alcohol consumption has been shown to increase the severity of acute carbon tetrachloride toxicity (ATSDR, 2005). Norwood et al. (1950) reported the death of one man with a history of alcoholism (after a 15-min exposure to 250 ppm carbon tetrachloride) due to liver, kidney, and lung failure. In addition, Folland et al. (1976) reported several cases of hepatic and renal injury among workers in an isopropyl alcohol packaging plant following accidental exposure to carbon tetrachloride. The most severe case exhibited acute renal failure and required dialysis.
Gastrointestinal effects (nausea, dyspepsia) at 20-50 ppm, central nervous system depression at 40 ppm, and narcosis at 80 ppm were found in humans occupationally exposed via inhalation for 2-3 months (Heimann and Ford, 1941; Elkins, 1942; Kazantzis and Bomford, 1960). Hepatic (fat accumulation) and renal (swelling) effects were also observed in workers after short-term (<3 h) exposure to 200 ppm carbon tetrachloride, similar to findings following acute exposure (Barnes and Jones, 1967).
Six male volunteers per group were exposed to carbon tetrachloride vapour (3 times, 4 weeks apart) at concentrations of 49 ppm for 70 min, 11 ppm for 180 min, and 10 ppm (64.1 mg/m³) for 180 min. At 49 ppm, all subjects were able to detect a sweetish odour. A decrease in serum iron concentration in two subjects was the only adverse effect observed at the highest dose. Carbon tetrachloride was detected in exhaled breath at all three exposure levels (Stewart et al., 1961). It is estimated that the threshold for central nervous system effects in humans is probably in the range of 20-50 ppm for an 8-h workday (ATSDR, 2005).
The effect of carbon tetrachloride on hepatic function following occupational exposure via inhalation was examined in a cross-sectional study of chemical plant workers. Historical personal monitoring data were used to categorize the exposed group into low, medium and high exposure groups. Alcohol consumption data were collected from control and exposed groups, and were found to be equivalent among groups. Multivariate analysis of serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and gamma-glutamyl transferase showed significant differences between exposed and control groups, however there were no significant differences between different exposure categories. Univariate analyses identified increases in only alkaline phosphatase and gamma-glutamyl transferase within the exposed group, and these did not show a significant dose-response. Although there was no evidence of significant clinical effects on the liver function of workers exposed to carbon tetrachloride, it is possible that some of the effects seen in liver function enzymes were due to carbon tetrachloride exposure. No evidence of any change in liver function was observed in a follow-up study conducted 3 years after the initial study (Tomenson et al., 1995).
No adverse effects on endocrine, cardiovascular, haematological, or musculoskeletal systems have been reported in the literature following dermal exposure to carbon tetrachloride in humans (ATSDR, 2005).
A case-control study was carried out in Montréal to estimate the association between 293 workplace substances (including carbon tetrachloride) and several types of cancer. Population subgroups were categorized according to ethnicity in an attempt to account for the effect of differing genetic or cultural characteristics that may have confounded the relationship between cancer and occupation. Approximately 4% of the population (firefighters, mechanics, and electricians) had been exposed to carbon tetrachloride mainly via inhalation. Elevated risks were observed for rectal cancer in all subjects (odds ratio [OR] = 2.0, 90% confidence interval [CI] = 1.2-3.3) and for bladder cancer in one ethnic subgroup (OR = 1.6, 90% CI = 0.9-2.8) (Siemiatycki, 1991).
Deaths due to cancer were analysed in 330 laundry and cleaning workers exposed dermally and via inhalation to carbon tetrachloride, trichloroethylene, and tetrachloroethylene. Eighty-seven deaths due to cancer were observed compared with the expected 67.9, indicating an increase in cancer risk. A significant increase in malignant neoplasms of the lung and cervix was observed, in addition to a slight increase in the incidence of leukaemia and liver cancer. Confounding factors such as exposure to multiple compounds and lack of adequate controls were noted in this study (Blair et al., 1979). Following investigation based on three occupational studies, associations between carbon tetrachloride and an increased risk of non-Hodgkin lymphoma and/or multiple myeloma were suggested, but were not statistically powerful and were observed in females only (Blair et al., 1990, 1998; IARC, 1999).
A nested case-control study within a cohort of workers from a large rubber and tire manufacturing plant was performed to examine the relationship between exposure to 24 solvents (including carbon tetrachloride) and the risk of cancer. The cohort study consisted of 6678 male plant workers (either active or retired), with a control group consisting of a 20% age-stratified random sample of the cohort (n = 1350) and cases composed of persons with fatal stomach cancer (n = 30), lung cancer (n = 101), prostate cancer (n = 33), lymphosarcoma (n = 9), or lymphatic leukaemia (n = 10). Lymphatic leukaemia was significantly related to carbon tetrachloride exposure (OR = 15.3). Lymphosarcoma showed similar but weaker associations with carbon tetrachloride exposure. Overlapping chemical exposures (strong associations with carbon disulphide were also noted for these two cancer types) limit the ability to draw conclusions regarding carbon tetrachloride exposure and cancer (Wilcosky et al., 1984).
A second case-control analysis was performed to evaluate the association of lymphocytic leukemia mortalities in 11 male workers and exposure to rubber industry solvents. Carbon tetrachloride (OR = 14.8) and carbon disulphide (OR = 8.7) showed the strongest associations with leukaemia mortality; however, due to small sample size and multi-solvent exposures, no conclusive associations between individual solvents and leukaemia mortality can be inferred (Checkoway et al., 1984).
A nested case-control study (Bond et al., 1986) of 308 lung cancer deaths in a cohort of chemical workers showed no association with exposure to carbon tetrachloride (OR < 1).
Although several epidemiological studies have explored a possible association between carbon tetrachloride and the incidence of cancer, these studies are all characterized by mixed exposures and a lack of carbon tetrachloride exposure data. Consequently, IPCS (1999) has concluded that any contribution from carbon tetrachloride cannot be reliably identified.
The effect of public drinking water contamination on birth outcomes was evaluated in an area of northern New Jersey by using the Birth Defects Registry data from 1985 to 1988 (including 80 958 live births and 594 fetal deaths) (Bove et al., 1995). Positive associations were found between exposure to >1 ppb carbon tetrachloride in drinking water and term low birth weight (OR = 2.26; 90% CI = 1.52-3.36), small for gestational age (OR = 1.75; 90% CI = 1.31-2.32), central nervous system defects (OR = 3.80; 90% CI = 1.14-10.63) and neural tube defects (OR = 5.39; 90% CI = 1.12-18.95). The authors concluded that this study did not resolve whether the drinking water contaminants caused the adverse birth outcomes, because drinking water databases were developed primarily for regulatory and enforcement purposes and are limited in their use for exposure assessment. Interpretation of this study is difficult due to simultaneous exposure to multiple chemicals in the drinking water, and the small number of cases observed; in the group exposed to >1 ppb carbon tetrachloride, only three cases of central nervous system defects and two cases of neural tube defects were observed.
No association was found between small for gestational age babies and exposure to carbon tetrachloride in 3418 out of 3946 women in Germany who had been exposed while pregnant (86.6%). The ORs for carbon tetrachloride in low- and moderate-exposure groups were 1.2 (95% CI = 0.6-2.7) and 2.4 (95% CI = 0.2-25.2), respectively, compared with 1.0 for the no-exposure group (Seidler et al., 1999).
Croen et al. (1997) investigated the association between maternal proximity to hazardous waste sites in California and selected congenital malformations using data from two populationbased case control studies. No association was found between conotruncal heart defects or oral cleft defects and maternal residential proximity to sites contaminated with carbon tetrachloride.
The acute toxicity of carbon tetrachloride has been widely studied in animals. In acute oral studies in rats, LD50 values of 4.7 mL/kg (equivalent to 7500 mg/kg bw; Pound et al., 1973), 6.4 mL/kg (equivalent to 10 200 mg/kg bw; McLean and McLean, 1966), and 10 054 mg/kg bw (Dashiell and Kennedy, 1984) have been reported.
In a study by Korsrud et al. (1972), single oral doses of carbon tetrachloride in corn oil were administered to male Wistar rats at 0, 0.001, 0.005, 0.025, 0.075, 0.125, 0.250, 0.750, or 2.50 mL/kg bw. The rats were killed 18 h after dosing. At 0.025 mL/kg bw (equivalent to 39.9 mg/kg bw) and above, liver weight and fat, serum urea, and serum enzyme activities were increased. In a second experiment, single oral doses of 0, 0.0125, 0.0250, 0.0500, or 0.1000 mL/kg bw in corn oil were administered to rats that were again sacrificed 18 h subsequent to exposure. Histopathological evidence of liver damage was seen in all treated animals and included loss of basophilic stippling of the cytoplasm, fat, and hydropic degeneration, with occasional single-cell necrosis at the highest dose level (Korsrud et al., 1972).
Kim et al. (1990b) investigated the effect of oral dosing vehicles on the acute hepatotoxicity of carbon tetrachloride . Male Sprague-Dawley rats were administered carbon tetrachloride (0, 10, 25, 50, 100, 250, 500, or 1000 mg/kg bw) by gavage in corn oil, as the undiluted chemical, as an aqueous emulsion, or in water (10 and 25 mg/kg bw doses only). Dosedependent increases in serum enzyme levels and histopathological changes were observed with each vehicle; however, hepatotoxicity was consistently less pronounced in groups given carbon tetrachloride in corn oil than in other vehicle groups. The lowest-observed-adverse-effect level (LOAEL) for carbon tetrachloride given in corn oil (25 mg/kg bw) was higher than that when carbon tetrachloride was given in the other vehicles (10 mg/kg bw) (Kim et al., 1990b).
The acute inhalation toxicity of carbon tetrachloride has also been studied in animals. Svirbely et al. (1947) reported a LC50 value of 9526 ppm (60 mg/L) for male and female Swiss mice following a 7-h inhalation exposure (8-h observation period). Adams et al. (1952) reported 100% mortality of Wistar rats following single inhalation exposures to 19 000 ppm carbon tetrachloride for 2.2 h, 12 000 ppm for 4 h, and 7300 ppm for 8 h. Exposure of male rats to 3000 ppm for 0.1 h, 800 ppm for 0.5 h, and 50 ppm for 7 h caused no adverse effects. In the same study, rats were repeatedly exposed to carbon tetrachloride for 7 h per day. At 10 ppm, 13 exposures in a 17-day period caused fatty degeneration of the liver, which increased in extent and severity with increasing concentration. Cirrhosis was also observed at 200 and 400 ppm following 10 exposures in a 12- to 13-day period.
Brondeau et al. (1983) exposed male Sprague-Dawley rats to carbon tetrachloride by inhalation at concentrations of 259, 531, 967, and 1459 ppm for 4 h. Twenty-four hours postexposure, serum glutamate dehydrogenase activity was increased at the lowest concentration. At the higher exposure levels, serum activities of sorbitol dehydrogenase (SDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were also increased. In a study by Boyd et al. (1980), male Swiss mice were exposed by inhalation to carbon tetrachloride concentrations of 0.46 or 0.92 mmol/L for 1 h, 1.84 mmol/L for 12 min, or 3.68 mmol/L for 2 min. All exposures produced marked Clara cell lesions in the lung and hepatic necrosis.
Acute toxicity following dermal exposure to carbon tetrachloride has also been observed in animals. LD50 values of >9.4 mL/kg were reported for rabbits and guinea pigs following a single dermal exposure to carbon tetrachloride (Roudabush et al., 1965). In guinea pigs, centrilobular hydropic change and necrosis were observed in the liver 16 h after dermal application of 1 mL carbon tetrachloride to a 3.1-cm² area of skin (Kronevi et al., 1979). A dose of 0.5 mL applied to the skin of guinea pigs (3.1 cm²) resulted in 25% mortality within 14 days; 65% mortality was reported 21 days following application of 2.0 mL (Wahlberg and Boman, 1979).
Several studies have investigated the effect of short-term oral exposure to carbon tetrachloride on animals. In a study by Bruckner et al. (1986), five male Sprague-Dawley rats (300-350 g) per dose level were administered carbon tetrachloride by gavage in corn oil (0, 20, 40, or 80 mg/kg bw/day) for 5 consecutive days, allowed 2 days without dosing, and dosed once daily for 4 additional days. In a second study, five rats (200-250 g) per dose level were gavaged with 0, 20, 80, or 160 mg/kg bw/day according to the same dosing schedule. In both studies, one group of rats at each dosage level was sacrificed 1, 4, and 11 days after initiation of dosing. Single doses of 20 and 40 mg/kg bw had no apparent toxic effects after 1 day. Significant increases in serum enzyme levels and hepatic vacuolization were observed 1 day after single doses of 80 and 160 mg/kg bw; hepatic necrosis was also observed at 160 mg/kg bw. Progressively severe hepatotoxicity at each dosage level was observed over the 11-day period.
In a subchronic experiment by Bruckner et al. (1986), carbon tetrachloride was administered to 15-16 male Sprague-Dawley rats per dose group by gavage in corn oil (0, 1, 10, or 33 mg/kg bw/day), 5 days per week for 12 weeks. At the end of the 12-week dosing period, 7-9 rats per group were sacrificed; the remaining animals were sacrificed 13 days after the last dose. The lowest dose (1 mg/kg bw/day) had no apparent adverse effects. At 10 mg/kg bw/day, serum SDH levels were modestly increased and mild centrilobular vacuolization was seen in the liver. Marked hepatotoxicity was observed at the highest dose (33 mg/kg bw/day). Serum levels of SDH, ornithine-carbamyl transferase, and ALT were significantly increased during the 12-week dosing period, but returned to normal within 13 days after the last dose. In rats sacrificed after 12 weeks, hepatic lesions observed included vacuolization, bile duct hyperplasia, periportal fibrosis, lobular distortion, parenchymal regeneration, hyperplastic nodules, and single-cell necrosis. The severity of the fibrosis and bile duct hyperplasia observed in rats sacrificed 13 days after the last dose was similar to that seen in rats sacrificed after 12 weeks. The no-observedadverse-effect level (NOAEL) was 1 mg/kg bw/day and the LOAEL was 10 mg/kg bw/day, based on increased serum SDH levels and mild centrilobular vacuolization observed at this dose.
Koporec et al. (1995) investigated the effect of oral dosing vehicles on the subchronic hepatotoxicity of carbon tetrachloride in rats. Carbon tetrachloride was administered to Sprague-Dawley rats at doses of 0, 25, or 100 mg/kg bw/day by gavage in either corn oil or a 1% Emulphor aqueous emulsion, 5 times per week for 13 weeks. Dose-dependent increases in serum SDH and ALT activities were observed for both vehicle groups. The incidence and severity of hepatic histopathological changes were dose dependent, however no differences between vehicle groups were observed. The majority of rats in the 25 mg/kg bw/day groups exhibited only minimal to slight vacuolation. At 100 mg/kg bw/day, hepatic lesions observed included vacuolation, cytomegaly, nodular hyperplasia and necrosis.
Smialowicz et al. (1991) administered carbon tetrachloride to male Fischer 344 rats by gavage in corn oil at 0, 5, 10, 20, or 40 mg/kg bw/day for 10 consecutive days. Rats were sacrificed 2 days following the last treatment. Relative liver weight was increased at 40 mg/kg bw/day. Increased serum levels of AST and ALT were observed at 20 and 40 mg/kg bw/day. Histopathological examination of livers showed minimal to moderate vacuolar degeneration at all doses other than the control. Minimal to mild hepatocellular necrosis was observed at 10 mg/kg bw/day and above.
In a study by Allis et al. (1990), 24 male F344 rats per dose group were administered carbon tetrachloride by gavage in corn oil (0, 20, or 40 mg/kg bw/day), 5 days per week for 12 weeks. Six rats from each dose level were sacrificed at 1, 3, 8, and 15 days post-exposure. At 1 day post-exposure, significant, dose-dependent increases in relative liver weight and serum levels of AST, ALT, and LDH were observed at both dose levels. Serum levels of alkaline phosphatase and cholesterol were significantly increased at the highest dose. At both doses, hepatic lesions observed included hepatocellular vacuolar degeneration, mild necrosis, and cirrhosis; cirrhosis was more severe at the higher dose. Recovery from the hepatotoxic effects was relatively rapid, with serum enzyme levels returning to normal and necrosis no longer evident at 8 days post-exposure. Cirrhosis and vacuolar degeneration were still evident at 15 days post-exposure, but had decreased in severity.
Hayes et al. (1986) administered carbon tetrachloride to 20 male and 20 female CD-1 mice per dose level by gavage in corn oil at 0, 625, 1250, or 2500 mg/kg bw/day for 14 days. A dosedependent decrease in body weight was observed in male mice. Mortality was dose dependent, and females appeared less sensitive than males. Serum levels of lactate dehydrogenase (LDH), ALT and AST were significantly increased at all doses other than the control in both sexes; serum alkaline phosphatase levels were significantly increased at the highest dose only. Liver weights were significantly increased at all doses other than the control in males and females. In addition, the reported LD50 of carbon tetrachloride for mice (12 000-14 000 mg/kg bw) was confirmed by administration of carbon tetrachloride to 10 CD-1 mice of each sex, at a dose of 14 000 mg/kg, by corn oil gavage.
In another experiment, Hayes et al. (1986) administered carbon tetrachloride to 20 male and 20 female CD-1 mice per dose group by gavage in corn oil (0, 12, 120, 540, or 1200 mg/kg bw/day) for 90 days. Increases in liver and spleen weights and serum levels of LDH, ALT, AST and alkaline phosphatase were observed at all dose levels in both sexes. Histopathological examination of the liver showed evidence of hepatotoxicity in all treated mice. Hepatic lesions including necrosis, chronic hepatitis, hepatocytomegaly, and fatty change were evident at all doses other than the control and tended to increase in severity at higher doses. A NOAEL was not obtained in this study.
Guo et al. (2000) administered carbon tetrachloride to eight female B6C3F1 mice per dose by gavage in corn oil at doses of 0, 50, 100, 500, or 1000 mg/kg bw/day for 14 days. Liver weight was significantly increased at all doses other than the control. Histopathological examination of the liver showed hydropic changes and necrosis. Significant increases in serum ALT levels were observed at all dose levels other than the control. Carbon tetrachloride was immunotoxic at all doses other than the control, causing a decrease in humoral immune response, compromising the mononuclear phagocyte system, and decreasing host resistance to pathogenic bacteria.
Condie et al. (1986) conducted a subchronic study in mice comparing the effects of different gavage vehicles on carbon tetrachloride hepatotoxicity. Carbon tetrachloride was administered to 12 male and 12 female CD-1 mice by gavage in either corn oil or 1% Tween-60 (0, 1.2, 12, or 120 mg/kg bw/day), 5 days per week for 90 days. Liver weights and liver to body weight ratios were increased at the high dose in both sexes with both vehicles. In mice receiving the corn oil vehicle, increases in serum enzyme activities (ALT, AST, LDH) were observed in the mid-dose groups, and substantial increases were seen in the high-dose groups. Significant increases in serum enzyme activities occurred only in the high-dose groups of mice that received carbon tetrachloride in Tween-60. Hepatocytomegaly was observed at the middle dose in mice receiving corn oil and in mice at the high dose with both vehicles. Moderate fat accumulation was observed at the middle dose in the livers of mice receiving corn oil only. Hepatic necrosis was observed in male mice at the middle and high doses with corn oil and at the high dose only with Tween-60. In female mice, necrosis was observed at the high dose with both vehicles. Necrosis and fatty infiltration were seen more frequently in male and female mice receiving carbon tetrachloride in corn oil. Fibrosis was detected at the high dose in both sexes with both vehicles. The NOAEL was 1.2 mg/kg bw/day when corn oil was used as the vehicle and 12 mg/kg bw/day when Tween-60 was used.
The toxicity of carbon tetrachloride following short-term inhalation exposure has also been studied in animals. Prendergast et al. (1967) exposed groups of Long-Evans or Sprague-Dawley rats (15 per group), Hartley guinea pigs (15 per group), squirrel monkeys (3 per group), New Zealand albino rabbits (3 per group), and beagle dogs (2 per group) to carbon tetrachloride by inhalation, either continuously (6.1 or 61 mg/m³) for 90 days or repeatedly (515 mg/m³) 8 h per day, 5 days per week, for 6 weeks. In animals exposed to 6.1 mg/m³ continuously, no visible signs of toxicity were seen, and all animals survived the exposure period. All species except the rat showed decreased body weight gain. Following continuous exposure to 61 mg/m³, three guinea pigs died on days 47, 63, and 71. All species showed decreased body weight gain, and all monkeys experienced hair loss and emaciation. In all species, liver changes observed included fatty changes associated with mononuclear cell infiltrates, fibroblastic proliferation, collagen deposition, hepatic cell degeneration and regeneration, and lobular alteration; these changes were more severe in rats and guinea pigs. After repeated exposure to 515 mg/m³, one monkey died after the 7th exposure, and three guinea pigs died after the 20th, 22nd, and 30th exposures. Body weight loss was seen in all species except the rat. All species exhibited pulmonary interstitial inflammation or pneumonitis. Histopathological examination showed fatty changes in the livers of all species. In the livers of guinea pigs, fibrosis, bile duct proliferation, hepatocellular degeneration and regeneration, focal inflammatory cell infiltration, lobular alteration, and early portal cirrhosis were also observed.
In a short-term inhalation study, BDF1 mice and F344 rats (10 per sex per group) were exposed to carbon tetrachloride by whole-body inhalation (0, 10, 30, 90, 270, or 810 ppm) 6 h per day, 5 days per week, for 13 weeks. In male mice, body weight gain was decreased at 30 ppm and above, and decreased haemoglobin levels and increased mean platelet volume were observed at the highest dose (810 ppm). In female mice, decreased haemoglobin, haematocrit, and red blood cells were observed at the two highest doses. In mice of both sexes, increased liver enzymes in the blood were observed at 90 ppm and above. Microscopic examination showed slight to moderate dose-related changes in the liver, even at the lowest dose level in males. At higher dose levels, more severe changes, described as collapse, deposit of ceroid, proliferative ducts, increase in mitosis, pleomorphism, and foci, were observed. In rats, a decrease in body weight gain was observed at the highest dose (810 ppm). Haematological changes were observed at 90 ppm and above in male rats and at 30 ppm and above in females. Increased liver enzymes in the blood and urinalysis changes were observed in male rats at 270 ppm and above and in females at 90 ppm and above. Microscopic examination showed slight to marked liver changes at all dose levels, including fatty change, cytological alterations, deposition of ceroid, proliferative ducts, increase in mitosis, pleomorphism, cirrhosis, and foci. At the two highest doses, vacuolar change of tubules, hyaline degeneration of the glomeruli, and protein casts were seen in the kidney (Japan Bioassay Research Centre, 1998).
A short-term inhalation study by Nagano et al. (2007) examined the subchronic toxicity of carbon tetrachloride in groups of 10 F344 rats and BDF1 mice (of both sexes) exposed to 0, 10, 30, 90, 270 or 810 ppm (v/v) of carbon tetrachloride vapour for 13 weeks (6 h/d and 5 d/wk). In the high exposure groups 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 carbon tetrachloride-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. Hematotoxicity and nephrotoxicity were observed in both rats and mice, but those toxicities were manifested at higher exposure concentrations than hepatotoxicity. A 6 hour inhalation exposure of rats and mice to 10 ppm carbon tetrachloride vapour corresponds to a daily uptake of 13 and 29 mg/kg bw, respectively, assuming a volume of 561 mL/min/kg bw for rats (Mauderly et al., 1979) and 1,239 mL/min/kg bw for mice (Guyton, 1947) and a lung absorption ratio of 100% for both rats and mice.
In long-term studies, hepatotoxicity and the development of liver tumours have been reported in mice, rats, and hamsters following oral and inhalation exposure to carbon tetrachloride.
Della Porta et al. (1961) administered carbon tetrachloride to 10 female and 10 male Syrian golden hamsters by gavage in weekly doses of 6.25-12.5 µL (equivalent to approximately 100-200 mg/kg bw/week) for 30 weeks. The livers of the nine animals dying during the treatment period and one female dying 11 weeks after treatment showed post-necrotic cirrhosis with regenerative hyperplastic nodules. All animals dying 13-24 weeks after treatment (two females) or sacrificed 25 weeks after treatment (three females and five males) had one or more liver cell carcinomas. No control groups were used in this study; however, the authors indicated that no liver cell tumours were observed in historical control groups.
Carbon tetrachloride was used as a positive control in National Cancer Institute carcinogenicity bioassays of chloroform and trichloroethylene. Doses of 47 and 94 mg/kg bw (males) and 80 and 159 mg/kg bw (females) were administered daily by gavage in corn oil 5 days per week to groups of 50 male and 50 female Osborne-Mendel rats for 78 weeks. Surviving rats were sacrificed at 110 weeks. Carbon tetrachloride treatment caused marked hepatotoxicity with resultant fibrosis, bile duct proliferation, and regeneration. A decrease in survival was also observed in treated rats. An increased incidence of hepatocellular carcinoma and neoplastic nodules was seen at both doses in both sexes. In the same study, doses of 1250 mg/kg bw and 2500 mg/kg bw were administered daily by gavage in corn oil 5 days per week to groups of 50 male and 50 female B6C3F1 mice for 78 weeks. Surviving mice were sacrificed at 90 weeks. Only 14% of treated mice survived to 78 weeks, and less than 1% survived to 90 weeks, compared with 66% of untreated controls surviving to 90 weeks. Hepatocellular carcinomas were found in almost all (>98%) treated mice, including those dying before termination of the test (NCI, 1976a,b).
In an inhalation study, groups of 50 male and 50 female BDF1 mice and F344 rats were exposed to 0, 5, 25, or 125 ppm carbon tetrachloride 6 h per day, 5 days per week, for 104 weeks. In mice, a significant decrease in survival was observed at 25 and 125 ppm. Decreases in body weight gain, as well as changes in haematology, blood biochemistry, including liver enzymes, and urinalysis were also observed at the two highest doses. In male mice, protein casts in the kidney and liver changes, including deposit of ceroid, cyst, and degeneration, were observed at 25 and 125 ppm. In the spleen, there was an increase in haemosiderin deposit at 25 ppm, and extramedullary haematopoeisis was observed at 125 ppm. In female mice, changes in the liver included deposit of ceroid, thrombus, necrosis, degeneration, and cyst at 25 and 125 ppm. At 25 ppm, an increased deposit of haemosiderin of the spleen was observed, whereas at 125 ppm, deposit of ceroid of the ovary was seen. The incidence of hepatocellular adenomas was significantly increased at 25 and 125 ppm in males and at 5 and 25 ppm in females. The incidence of hepatocellular carcinomas was significantly increased at 25 and 125 ppm in male and female mice. The incidence of pheochromocytomas of the adrenal gland was increased in male mice at 25 and 125 ppm and in female mice at 125 ppm (Japan Bioassay Research Centre, 1998; Nagano et al., 1998).
In rats, survival was significantly decreased at the highest dose, and body weight gain was decreased at the two highest doses. Changes in haematology, blood biochemistry, including liver enzymes, and urinalysis were observed at 25 ppm; changes in the nitrate and protein levels in the urine were also observed at 5 ppm. Liver changes observed in both sexes at the two highest doses included fatty change, deposit of ceroid, fibrosis, granulation, and cirrhosis. In males at all doses other than the control, an increased deposit of haemosiderin in the spleen was observed. Chronic nephropathy (progressive glomerulonephrosis) was observed in females at 25 ppm and in both sexes at 125 ppm. At 125 ppm, deposit of ceroid and granulation of the lymph nodes were observed in both sexes. The incidence of hepatocellular adenomas and hepatocellular carcinomas was significantly increased at 125 ppm in both sexes (Japan Bioassay Research Centre, 1998; Nagano et al., 1998).
Results of chronic studies in rodents exposed orally or by inhalation indicate that hepatic tumours were induced at hepatotoxic doses. The carcinogenicity of carbon tetrachloride appears to be secondary to its hepatotoxic effects, which suggests that a threshold for carbon tetrachloride carcinogenicity may exist.
A range of in vitro and in vivo assays has been performed to assess the possible genotoxic effects of carbon tetrachloride. DNA- or chromosome-damaging effects have been evaluated in bacteria, fungi, yeast, insects, rodents, and humans.
Negative results have been reported for the majority of mutagenicity assays with carbon tetrachloride in bacteria. Owing to the volatility of carbon tetrachloride, in some cases the use of unsealed vessels may have resulted in false negatives. In addition, carbon tetrachloride requires metabolic activation, and reactive metabolites produced by exogenous activation systems may not be able to cross cell membranes and reach DNA.
Although a few positive results have been reported, the majority of Salmonella typhimurium reversion assays of carbon tetrachloride have been negative with and without metabolic activation (McCann et al., 1975; Uehleke et al., 1977; Barber et al., 1981; De Flora, 1981; De Flora et al., 1984; Brams et al., 1987; Araki et al., 2004). Increased reversion frequency was observed in Escherichia coli strains WP2uvrA/pKM101 and WP2/pKM101, with and without activation (Araki et al., 2004). Negative results were reported for the Ara forward mutation assay in S. typhimurium strains BA13 and BAL13, as well as for SOS induction in S. typhimurium strain TA1535/psK1002 and E. coli strain PQ37 (Brams et al., 1987; Nakamura et al., 1987; Roldán-Arjona et al., 1991). Carbon tetrachloride did not induce differential DNA repair in E. coli strains K-12 343/113 (Hellmér and Bolcsfoldi, 1992). Induction of differential DNA repair was observed in E. coli strains WP2, WP67, and CM871 when sealed plates were used without activation; however, assays using sealed plates with activation, as well as spot tests without activation, were negative (De Flora et al., 1984).
Induction of mitotic, intrachromosomal, and interchromosomal recombination was observed in various strains of the yeast Saccharomyces cerevisiae exposed to toxic concentrations of carbon tetrachloride (Callen et al., 1980; Schiestl et al., 1989; Galli and Schiestl, 1995, 1996; Brennan and Schiestl, 1998). Carbon tetrachloride did not induce aneuploidy in S. cerevisiae (Whittaker et al., 1989). Assays in the mould Aspergillus nidulans were weakly positive for forward mutation and positive for somatic segregation at cytotoxic concentrations (Gualandi, 1984; Benigni et al., 1993).
Mixed results have been obtained from in vitro genotoxicity assays in mammalian cells. Carbon tetrachloride did not induce unscheduled DNA synthesis (UDS) in rat hepatocytes (Selden et al., 1994); however, weakly positive results were reported for UDS in human lymphocytes exposed to cytotoxic doses of carbon tetrachloride (Perocco and Prodi, 1981). Increases in DNA single-strand breaks were observed in rat hepatocytes and mouse lymphoma cells exposed to cytotoxic concentrations of carbon tetrachloride (Sina et al., 1983; Garberg et al., 1988; Beddowes et al., 2003). Negative results were reported for induction of DNA damage in human lymphocytes (Tafazoli et al., 1998). In one study, chromosomal aberrations were detected at anaphase following exposure of Chinese hamster ovary (CHO) cells to carbon tetrachloride; however, the results of other assays using CHO cells, ovine lymphocytes, human lymphocytes, and rat hepatocyte cell line RL1 were negative for chromosomal aberrations (Coutino, 1979; Dean and Hodson-Walker, 1979; Garry et al., 1990; Loveday et al., 1990; Šiviková et al., 2001). Carbon tetrachloride did not increase the frequency of sister chromatid exchange (SCE) in CHO cells or human lymphocytes; however, SCE was induced in ovine lymphocytes following exposure to carbon tetrachloride for 48 h (Garry et al., 1990; Loveday et al., 1990; Šiviková et al., 2001). Carbon tetrachloride also induced aneuploidy in Chinese hamster V79 lung cells (Önfelt, 1987). Micronucleus formation was induced by carbon tetrachloride in ovine lymphocytes, as well as in human lymphocytes from one of two donors (Tafazoli et al., 1998; Šiviková et al., 2001). Carbon tetrachloride induced micronuclei in human lymphoblastoid cell lines MCL-5, which expresses human cDNA for CYP1A2, 2A6, 3A4, 2E1, and microsomal epoxide hydrolase, and h2E1, which expresses cDNA for human CYP2E1 (Doherty et al., 1996).
The majority of in vivo genotoxicity assays with carbon tetrachloride have been negative. Although some positive results have been reported, these effects were observed only at cytotoxic doses. In studies in Drosophila melanogaster, no sex-linked recessive mutations were induced following exposure of males to carbon tetrachloride in the diet or by injection prior to mating (Foureman et al., 1994). There was no UDS in hepatocytes isolated from male Fischer 344 rats 2-48 h following carbon tetrachloride exposure by gavage (Mirsalis and Butterworth, 1980; Mirsalis et al., 1982). However, in a study by Craddock and Henderson (1978), UDS was observed 17 h after oral exposure of female Wistar rats to carbon tetrachloride. The authors suggested that the DNA damage may have been caused by an indirect process, such as DNase activity resulting from lysosomal damage (Craddock and Henderson, 1978). In most studies, no DNA damage was observed in mice or rats following carbon tetrachloride treatment by gavage or injection (Schwarz et al., 1979; Stewart, 1981; Bermudez et al., 1982; Barbin et al., 1983; Brambilla et al., 1983). Positive results have been reported for DNA damage in the liver of CD-1 mice; however, DNA damage was observed only at doses also resulting in liver necrosis (Gans and Korson, 1984; Sasaki et al., 1998). No increases in chromosomal aberrations, SCE, or micronuclei were detected in the liver of male F344 rats orally exposed to carbon tetrachloride at 1600 mg/kg bw by gavage 4-72 h prior to sacrifice (Sawada et al., 1991). In several studies, micronuclei were not induced in the bone marrow or peripheral blood of CD-1 or BDF1 mice treated with carbon tetrachloride at doses up to 3000 mg/kg bw (Morita et al., 1997; Suzuki et al., 1997; Crebelli et al., 1999).
Although results of in vivo genotoxicity assays were largely negative, carbon tetrachloride has been shown to produce an increase in DNA adducts following exposure in vivo. Covalent binding of reactive carbon tetrachloride metabolites to liver DNA and nuclear proteins was detected following treatment of mice, rats, and hamsters with carbon tetrachloride (Castro et al., 1989). Carbon tetrachloride treatment also resulted in an increase in lipid peroxidation induced DNA adducts. Following injection of F344 rats with carbon tetrachloride, an increase in hydroxynonenal-deoxyguanosine adducts has been observed in the liver, forestomach, lung, and colon (Chung et al., 2000; Wacker et al., 2001). Increases in malondialdehyde-DNA adducts have also been observed in the liver of Sprague-Dawley rats and liver and kidney of Syrian golden hamsters treated with carbon tetrachloride by gavage (Chaudhary et al., 1994; Wang and Liehr, 1995). DNA adducts have also been detected in mammalian cells following exposure to carbon tetrachloride in vitro. In rat hepatocytes treated with carbon tetrachloride, a dosedependent increase in malondialdehyde-deoxyguanosine adducts was observed, and increased 8-oxo-deoxyguanosine adducts were detected at the highest dose, which was also cytotoxic (Beddowes et al., 2003). In vitro, it has been demonstrated that reactive carbon tetrachloride metabolites are able to covalently bind to guanine, cytosine, and thymine, producing the altered bases 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxycytosine, and 5-hydroxymethyluracil (Castro et al., 1997). Increased covalent binding of carbon tetrachloride metabolites to calf thymus DNA has also been demonstrated following in vitro metabolic activation (DiRenzo et al., 1982).
In summary, while the genotoxicity data are not fully conclusive, there is some evidence that carbon tetrachloride exerts a weak genotoxic effect, likely secondary to cytotoxicity.
The reproductive and developmental effects of carbon tetrachloride have been studied in rats and mice. In an inhalation study by Adams et al. (1952), rats were exposed to 5, 10, 25, 50, 100, 200, or 400 ppm carbon tetrachloride for 7 h per day, 5 days per week, for 24-29 weeks. At 200 ppm, the weight of the testes was decreased, and some tubules showed complete atrophy of the germinal elements. Moderate to marked degeneration of the germinal elements of the testes was observed at 400 ppm (Adams et al., 1952). In a multigenerational study, groups of male and female rats were exposed to 50, 100, 200, or 400 ppm carbon tetrachloride by inhalation, 8 h per day, 5 days per week, for up to 10.5 months. A decrease in fertility was observed at 200 and 400 ppm; however, since both sexes were exposed, it not clear whether this was due to effects on the males or females (Smyth et al., 1936).
In a study by Alumot et al. (1976), male and female rats were fed fumigated mash with a residual carbon tetrachloride concentration of up to 200 ppm (10-18 mg/kg bw) for 2 years. There was no effect on fertility, litter size and weight, or pup mortality. Oral administration of carbon tetrachloride to pregnant B6D2F1 mice at 82.6 or 826 mg/kg bw/day for 5 days beginning on gestation day 1, 6, or 11 had no effect on maternal body weight, liver and kidney weight, or pregnancy. No malformations or effects on pup weight or crown-rump length were observed, and development of the pups was normal (Hamlin et al., 1993).
Narotsky et al. (1997a) administered carbon tetrachloride to pregnant Fischer 344 rats by gavage at dose levels of 0, 25, 50, or 75 mg/kg bw/day on gestation days 6-15 in either corn oil or an aqueous vehicle. No maternal or developmental toxicity occurred at 25 mg/kg bw/day. Full litter resorption was observed at 50 and 75 mg/kg bw/day with both vehicles, and the incidence of full litter resorption was significantly greater at 75 mg/kg bw/day with corn oil than with the aqueous vehicle. No effects on gestation length, pre- or postnatal survival, or pup morphology were observed in the surviving litters (Narotsky et al., 1997a). When a single dose of carbon tetrachloride (150 mg/kg bw) was administered to pregnant Fischer 344 rats on gestation day 6, 7, 8, 10, or 12, full litter resorption was observed in 36-72% of dams treated on gestation days 6-10; none was seen in dams treated on gestation day 12. No developmental toxicity was observed in the surviving litters (Narotsky et al., 1997b).
Inhalation exposure of pregnant Sprague-Dawley rats to 330 or 1000 ppm carbon tetrachloride for 7 h per day on gestation days 6-15 caused maternal hepatotoxicity and significant reductions in food consumption and maternal body weight. There was no effect on conception rate, number of implantations, litter size, or number of resorptions. No gross anomalies were observed; however, significant reductions in fetal body weight and crown-rump length were observed at both concentrations, and the incidence of sternebral anomalies was significantly increased at 1000 ppm (Schwetz et al., 1974).
In humans and laboratory animals, the major effect of exposure to carbon tetrachloride is hepatotoxicity, including fatty degeneration, necrosis, fibrosis, and cirrhosis. Hepatic tumours are also observed in rodents following chronic exposure to carbon tetrachloride. The mechanism of carbon tetrachloride carcinogenicity may involve both genotoxic and non-genotoxic processes. Hepatic tumours occur at doses higher than those inducing hepatotoxicity; therefore, the carcinogenicity of carbon tetrachloride may be secondary to its toxic effects (ATSDR, 2005).
The liver is particularly sensitive to carbon tetrachloride toxicity due to high levels of CYP2E1, the enzyme primarily responsible for the bioactivation of carbon tetrachloride to reactive metabolites (Raucy et al., 1993; Wong et al., 1998; Zangar et al., 2000). Carbon tetrachloride is bioactivated to form the trichloromethyl radical, which can react with oxygen, forming the trichloromethyl peroxyl radical (Pohl et al., 1984). Carbon tetrachloride-induced hepatic toxicity has been studied extensively, but similar cellular damage would be expected in other tissues with high levels of CYP2E1 (ATSDR, 2005).
Reactive carbon tetrachloride metabolites mediate hepatic injury by two initial processes: haloalkylation and lipid peroxidation (Weber et al., 2003). Haloalkylation of cellular macromolecules such as nucleic acids, proteins, and lipids can lead to impairment of cellular processes. Metabolism of carbon tetrachloride in vitro by rat liver microsomes and in vivo leads to covalent binding of trichloromethyl radicals to lipids (Link et al., 1984); lipid haloalkylation is involved in the early phases of impaired lipid secretion by the Golgi apparatus, which can lead to fatty degeneration (Poli et al., 1990). Covalent binding of reactive carbon tetrachloride metabolites to DNA and nuclear proteins in livers of rats, mice, and hamsters has also been detected (Castro et al., 1989).
Lipid peroxidation can be initiated by the trichloromethyl peroxyl radical and results in the destruction of polyunsaturated fatty acids, particularly membrane phospholipids. This affects the permeabilities of mitochondrial, endoplasmic reticulum, and plasma membranes, impairing cellular functions dependent on membrane integrity (Weber et al., 2003). Lipid peroxidation also results in the formation of reactive aldehydes, such as 4-hydroxynonenal and malondialdehyde, which can bind to proteins and DNA (Weber et al., 2003). 4-Hydroxynonenal and malondialdehyde protein adducts have been detected in the livers of rats treated with carbon tetrachloride (Hartley et al., 1999). Increased levels of 4-hydroxynonenal-deoxyguanosine adducts have also been observed in the liver DNA of carbon tetrachloride-treated rats (Chung et al., 2000; Wacker et al., 2001).
Several studies have suggested that the increased cytosolic calcium concentrations observed following carbon tetrachloride exposure may play a central role in the induction of cytotoxicity. Prolonged elevation of cytosolic calcium may activate calcium-dependent hydrolytic enzymes capable of causing irreversible cellular injury or death. Early studies showed that carbon tetrachloride exposure was associated with decreased calcium storage by the endoplasmic reticulum, and that this effect correlated well with decreased calcium pump activity (Long and Moore, 1986). More recently, carbon tetrachloride exposure has also been shown to inhibit calcium pumps in the mitochondria and plasma membrane (Hemmings et al., 2002).
The carcinogenicity of carbon tetrachloride appears to be secondary to its hepatotoxic effects, which suggests that a threshold for carbon tetrachloride carcinogenicity may exist. There is evidence that the mechanism involves both genotoxic and non-genotoxic processes. The ability of reactive carbon tetrachloride metabolites to bind to DNA (Castro et al., 1989) indicates that carbon tetrachloride is potentially genotoxic. However, the results of most in vivo studies in animals suggest that genotoxic effects occur only at cytotoxic doses. Since lipid peroxidation products such as 4-hydroxynonenal and malondialdehyde also have the ability to form adducts with DNA (Chaudhary et al., 1994; Chung et al., 2000; Wacker et al., 2001), the genotoxic effect of carbon tetrachloride may be indirect, secondary to lipid peroxidation. Carbon tetrachloride may also cause cancer by a non-genotoxic mechanism involving regenerative hyperplasia. Hepatic necrosis stimulates cellular regeneration; the resulting increase in cell proliferation increases the possibility that unrepaired DNA damage will become fixed mutations, possibly resulting in an initiated preneoplastic cell (ATSDR, 2005).
Report a problem or mistake on this page
- Date modified: