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Description of key information

Based on a weight of evidence approach, the NOAELs were considered to be :

- Repeated dose toxicity, oral:  NOAEL  is 1 mg/kg bw/day for the rat based on minimal to moderate hepatic effects in repeated dose toxicity studies at higher level exposures (increase of hepatic enzymes, histological changes - WOE approach)

- Repeated dose toxicity, dermal:      no study available

- Repeated dose toxicity, inhalation: NOAEC is 32 mg/m³  (= 5 ppm) for the rat, based on liver fatty changes at higher levels in the chronic toxicity study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
32 mg/m³
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In all repeat-dose toxicity studies with carbon tetrachloride in experimental animals, liver toxicity was observed regardless of the species studied. In humans, damage to the liver was observed in acute poisoning cases. Suggestive evidence of hepatotoxicity was also seen in workers exposed to carbon tetrachloride for an extended period of time in the workplace. Thus, carbon tetrachloride is regarded to be a classic hepatotoxic substance and often used as a model compound to study mechanisms of liver toxicity and regeneration. Furthermore, induction of hepatic tumor formation was reported in rats and mice exposed orally or by inhalation to carbon tetrachloride. However, liver tumors were only seen in the presence of overt hepatotoxicity.

Oral Exposure

No long-term toxicity data are available for humans with quantified oral exposures to carbon tetrachloride, but case reports identify the liver as the primary target organ following acute exposures[1]. Evidence of acute oral hepatotoxicity in humans comes from observations of liver enlargement, elevated serum enzyme levels (aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT)), bilirubin levels, or histopathology (hepatocyte degeneration). The prominence of hepatic injury in acutely exposed humans suggests that hepatic toxicity observed in repeat-dose toxicity studies in experimental animals is an important and relevant endpoint for human health risk assessment of carbon tetrachloride.

 

Studies in laboratory animals indicate that hepatic toxicity is the predominant non-cancer toxicity of sub-chronic or chronic oral exposure to carbon tetrachloride (see Table 1). In these studies, evidence of hepatic damage included liver histopathology (fatty degeneration, necrosis, fibrosis, cirrhosis, inflammation, and regenerative activity), along with increases in liver weight and serum markers for hepatotoxicity (ALT, AST, ornithine carbamoyltransferase (OCT), serine dehydratase (SDH), and bilirubin). Liver damage was produced at doses as low as 7–9 mg/kg/day in 90-day gavage studies in rats and mice using corn oil as vehicle. The corresponding NOAEL values were 0.7–0.9 mg/kg/day.

 

 Inhalation Exposure

Case reports of acute high-level exposures to carbon tetrachloride vapor or long-term occupational exposure provide evidence of mainly hepatotoxic and, to a lesser extent, nephrotoxic effects of carbon tetrachloride in humans. Observations indicative of an effect on the liver include jaundice, increased serum enzyme levels, and, in fatal cases, necrosis of the liver. A cross-sectional epidemiology study of hepatic function in workers exposed to carbon tetrachloride found suggestive evidence of an effect of occupational carbon tetrachloride exposure on serum enzymes indicative of hepatic effects at workplace concentrations in the range of 6.3-24.5 mg/m3. Serum enzyme activity levels are routinely employed as biochemical indices of this toxicity and serve as reliable indicators of hepatic damage.

 

The liver and kidney are the most prominent targets of carbon tetrachloride in subchronic and chronic inhalation studies of laboratory animals. Hepatic toxicity in these studies was demonstrated by histopathology (centrilobular fatty degeneration, necrosis, fibrosis, cirrhosis, hepatitis, and regenerative activity), as well as by increases in liver weight and serum markers for liver damage. Hepatic effects were observed in animals exposed to carbon tetrachloride concentrations as low as 60 mg/m3(adjusted to continuous exposure).

 

Mode of Action for Hepatotoxicity

Mechanistic studies demonstrate that metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites plays the predominant role in its mode of action (MoA). The primary metabolites, the trichloromethyl and trichloromethyl peroxy radicals, are highly reactive and are capable of covalently binding to cellular macromolecules such as proteins and lipids. Because carbon tetrachloride toxicity is secondary to its metabolism, the liver is expected to be the most important target organ for toxicity due to its high CYP2E1 activity.

 

The trichloromethyl peroxy and trichloromethyl radicals induce multiple cellular effects including lipid peroxidation, decreases in antioxidant levels, alterations in calcium homeostasis, and activation of calcium-dependent phospholipases. Additionally, secondary products of lipid peroxidation include reactive aldehydes that can form protein adducts, which may contribute to hepatotoxicity.

 

Furthermore, there is evidence that “suicide” inactivation of cytochrome P450 is triggered by a radical formed in the metabolism of carbon tetrachloride. The reduction of carbon tetrachloride can lead to irreversible inactivation of cytochrome P450 and subsequent loss of the prosthetic heme group; thus, carbon tetrachloride inhibits its own metabolism.

 

On the basis of available data, carbon tetrachloride can be considered as a non-genotoxic compound. Carbon tetrachloride induces hepatomas and hepatocellular carcinomas in mice and rats; however; the doses or concentrations required to induce hepatic tumors are higher than those inducing liver toxicity and the carcinogenicity of carbon tetrachloride likely is secondary to its hepatotoxic effects, suggesting that a threshold may exist. A hypothesized carcinogenic MoA for carbon tetrachloride-induced liver tumors has been proposed and includes the following key events: (1) reductive biotransformation of carbon tetrachloride to the trichloromethyl radical by CYP2E1 and subsequent formation of the trichloromethyl peroxy radical, (2) radical-induced mechanisms leading to hepatocellular cytotoxicity, and (3) sustained regenerative and proliferative changes in the liver in response to hepatotoxicity. This MoA appears to play a significant role at relatively high exposures, driving the steep increase in liver tumors in this exposure range.

 

NOAEL and NOAECs for carbon tetrachloride after repeated exposures:

 

Repeated dose toxicity

Target organs: liver, kidney

Oral route

NOAEL: 1 mg/kg bw/day

Inhalation route

NOAEC: 32 mg/m³ (5 ppm)

[1]Effects on the kidneys, which are a secondary target organ, will not be considered within this review. Generally, renal toxicity is usually delayed relative to hepatic toxicity, thus is considered secondary to hepatotoxicity.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

According to the directive 67/548/EEC, the substance is classified R48/23 (danger of serious damage to health by prolonged exposure through inhalation) in annex I. This classification is supported by a NOAEC of 32 mg/m3 after chronic exposure in rats, based on an increase of liver fatty changes at higher concentrations. In the CLP regulation, a STOT RE 1 (H372) is assigned.

There is a wide variability in observed NOAEL after subchronic oral exposures to CTC (NOAEL from 1 to 10 mg/kg and LOAEL from 12 to 120 mg/kg) and the proposed oral NOAEL is considered as very conservative. In conclusion no classification for repeated oral exposure is proposed.