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Chloroform (CAS No. 67-66-3)and other Trihalomethanes

Harmful Effects of the Agent Dose Response
Absorption, Distribution, Metabolism and Excretion Sites of Toxicity
Biomarkers of Disease Molecular Mechanism of Action
Risk Assessment Considerations

Harmful Effects of the Agent

There is no data from studies in humans that are adequate to assess the potential carcinogenicity of chloroform to humans. 9

In animals, studies involving chloroform have shown increased incidence of liver and kidney tumors in several species by several exposure routes. However, tumors and cancers are observed only at high dose levels that result in cytotoxicity. The carcinogenic responses observed in animals are associated with cell regeneration that occurs in response to cytolethality. 9

The table below summarizes some of the key chloroform data.

The Jorgenson et al rat study was for 104 weeks and the vehicle was drinking water. Only at the highest dosage was there a significant increase in kidney cancer and no evidence of liver tumors.9 EPA determined that the study was more appropriate than the corn oil gavage studies because exposure was via drinking water and they considered this the most applicable route of exposure for humans. 9

* considered statistically significant

The bromodichloromethane study (NTP, 1987) had the following response rates in a corn oil gavage study:

In the Heyword et al. 1979 study, dogs were exposed to chloroform in toothpaste base for 7.5 years. Chloroform doses of 15 mg/kg/day resulted in an increase in the severity of fatty cysts in liver.

Absorption, Distribution, Metabolism and Excretion

Chloroform is readily absorbed following oral, dermal, and inhalation exposure. Animals studies indicate that gastrointestinal absorption of chloroform is rapid (peak blood levels at about 1 hour) and extensive (64% to 98).9 Limited data indicate that gastrointestinal absorption of chloroform is also rapid and extensive in humans, with more than 90% of an oral dose recovered in expired air (chloroform or carbon dioxide) within 8 hours.9

A study on dermal and inhalation absorption of chloroform by humans during showering was conducted by Jo et al. (1990). Chloroform concentrations in exhaled breath were measured in six human subjects before and after a normal shower, and following inhalation-only shower exposure. Breath levels measured at 5 minutes following either exposure correlated with tap water levels of chloroform. Breath levels following inhalation exposure only were about half those following a normal shower (both inhalation and dermal contact). These data indicate that humans absorb chloroform by both the dermal and inhalation routes (U.S. EPA, 1994d). 9

Absorbed chloroform appears to distribute widely throughout the body. In postmortem samples from eight humans, the highest levels of chloroform were detected in the body fat (5–68 g/kg), with lower levels (1–10 g/kg) detected in the kidney, liver, and brain.9 Studies in animals indicate rapid uptake of chloroform by the liver and kidney. In mice receiving chloroform via gavage in either corn oil or water, the uptake of chloroform was achieved within 10 minutes in the liver and within 1 hour in the kidney. 9

Chloroform is metabolized mainly in the liver, but metabolism also occurs in other tissues such as the kidney. Metabolism may occur via two pathways, oxidative and reductive, which is illustrated in Figure 1.

Chloroform is metabolized, for both pathways, in humans and animals by cytochrome P450.

Evidence shows that the metabolism occurs mainly via the oxidative pathway. Reductive metabolism of chloroform is observed only in Phenobarbital induced animals, with negligible reducing activity observed in uninduced animals. 9

There is no clear evidence on the mechanism of carcinogencity for intestinal tumors, but this is currently being studied. 11

Reductive Pathway
Phenobarbital induces CYP2B1 and CYP2B2, which are virtually absent from the liver. This appears to shift the metabolism from an oxidative to a reductive pathway. 5 In the absence of oxygen (reductive metabolism), the chief metabolite is dichloromethyl free radical (CHCl 2). 9 Free radicals that are formed under conditions of low oxygen are also extremely reactive, forming covalent adducts with microsomal enzymes and the fatty acid tails of phospholipids, probably quite close to the site of free radical formation (cytochrome P450 in microsomal membranes). This results in a general loss of microsomal enzyme activity, and can also result in lipid peroxidation. 9

Oxidative Pathway

The phase I reaction occurs in the presence of oxygen (oxidative metabolism) and involves CYP2E1, producing trichloromethanol, which then eliminates hydrogen chloride to form phosgene, which is the toxic metabolite. 9

Phosgene is highly reactive and is electrophilic. The predominant Phase II reaction is hydrolysis by water, yielding carbon dioxide and hydrochloric acid. 9 The rate of phosgene hydrolysis is very rapid, with a half-time of less than 1 second. 9

Phosgene also reacts with glutathione (GSH), yielding S-chloro-carbonyl glutathione, which in turn can either interact further with glutathione to form diglutathionyl dithiocarbonate, or form glutathione disulfide and carbon monoxide, 9

Phosgene also undergoes attack by nucleophilic groups (-SH, -OH, -NH2) in cellular macromolecules such as enzymes, proteins, or the polar heads of phospholipids, resulting in formation of covalent adducts. Formation of these molecular adducts can interfere with molecular function (e.g., loss of enzyme activity), which in turn may lead to loss of cellular function and subsequent cell death. 9

Nearly all tissues of the body are capable of metabolizing chloroform, but the rate of metabolism is greatest in liver, kidney cortex, and nasal mucosa. 9 These tissues are also the principal sites of chloroform toxicity, indicating the importance of metabolism in the mode of action of chloroform toxicity. 9

At low chloroform concentrations, metabolism occurs primarily via cytochrome P450-2E1 (CYP2E1). The level of this isozyme, which affects the rate of chloroform metabolism, is induced by a variety of alcohols including ethanol and may be inhibited by phenobarbital. Under low dose-rate conditions, nearly all of a dose is metabolized . However, as the dose or the dose rate increases, metabolic capacity may become saturated and increasing amounts of the dose are excreted as the unmetabolized parent. 9

Excretion of chloroform occurs primarily via the lungs. Human studies indicate that approximately 90% of an oral dose of chloroform is exhaled (as chloroform or as carbon dioxide), with less than 0.01% of the dose excreted in the urine. In mice and rats, 45%–88% of an oral dose of chloroform was excreted from the lungs (as chloroform or carbon dioxide) and 1%–5% excreted in the urine. 9

Sites of Toxicity

The toxicity of chloroform on the liver and kidney is clearly related to the ability of tissues to metabolize chloroform. It has been shown that toxicity occurs in those tissues that have the greatest ability to metabolize chloroform, and the toxicity can be increased or decreased by agents that increase or decrease the activity of the metabolic enzymes. Also, there are differences between genders, strains, and species in their relative sensitivity to chloroform, and these differences in toxicity correlate with differences in metabolic capacity. For example, kidney toxicity is usually more severe in males than females, apparently because the cytochrome P450 (CYP2E1) responsible for the metabolism of chloroform is induced by testosterone. 9

There are three key steps in the sequence of events that lead to chloroform induced tumors in the liver and kidneys of rodents.

1. The oxidative metabolism of chloroform occurs primarily in the kidney and liver. Evidence shows that metabolism by CYP2E1 predominates at low exposures and chloroform has not been shown to cause tumors at low doses. 9

2. The next step is the resultant cytotoxicity and cell death caused by the oxidative metabolite, phosgene. 11 Regenerative cell proliferation follows the liver and kidney toxicity.

3. This increase in cell division can lead to an increased probability of cancer by of two different modes of action.

a) Cells that are undergoing cell division are more susceptible to initiation than are slowly growing or nondividing cells. This is because DNA undergoing replication is more exposed to nucleophilic attack. Also, any gene damage that occurs in a cell undergoing division has less time to be repaired before being converted into mutations. 9

b) Chemicals that promote cell division may provide a growth advantage to preexisting initiated cells in comparison with normal cells, allowing expansion of initiated cells. The ratio of cell birth to cell death of initiated cells increases compared with normal cells, leading to increased likelihood that a clone of initiated cells will form and survive. A key characteristic of this mode of action is that the effect is reversible: the clones of induced cells will tend to regress if the promoter (mitogen, cytotoxicant) is withdrawn. 9

Biomarkers of Disease

Dose does not measure the amount of the THMs absorbed, so a better measurement is the level of THMs in the blood.

The response to high levels of THMs is cell death and regeneration. This would be seen in tumor or cancer development.

Individuals may have a higher than average adverse response to the THMs for various reasons, such as increased absorption, decreased excretion, higher or lower metabolism (depending on whether metabolism increases or decreases toxicity), decreased cellular defense and repair mechanisms. 9

Molecular Mechanism of Action

The molecular mechanism by which chloroform metabolism results in cellular toxicity is not certain, but is probably due mainly to the formation of phosgene as a result of oxidative metabolism. Phosgene is highly reactive and can bind with and inactivate cellular molecules. This mode of toxicity is supported by the finding that glutathione protects against the toxic effects of chloroform and that toxicity occurs only after glutathione levels have been depleted. 9

Risk Assessment Considerations

Oral Reference Dose (RfD)
While the Heyword et al. study was for non-carcinogenic effects, the EPA refers to this study in the establishment of the oral cancer RfD (the RfD is an estimate of a daily exposure to the human population that is likely to be without an appreciable risk of health hazard effects during a lifetime). In a 1979 study, dogs were exposed to chloroform in toothpaste base for 7.5 years. Chloroform doses of 15 mg/kg/day resulted in an increase in the severity of fatty cysts in liver. Dose-response data from this study were evaluated using the benchmark dose (BMD) approach and a BMD of 1.0 mg/kg/day was established. An uncertainty factor of 100 was applied to the BMD to account for extrapolation from animals to humans and for potential variability in sensitivity between humans. The calculated RfD is 0.01 mg/kg/day. 9

Inhalation Reference Concentration (RfC)
The RfC for inhalation has not been established and this is a major weakness in the toxicological evaluation of chloroform. Inhalation is expected to be the major route of exposure. 10

Oral Cancer Risk
The EPA Proposed Guidelines for Carcinogenic Risk Assessment states that when available evidence indicates that a carcinogenic response is secondary to another toxicity that has a threshold, the Margin of Exposure (MOE) analysis performed for toxicity may be the same as is done for a noncancer endpoint. The weight of evidence indicates that chloroform-induced carcinogenicity is secondary to cytotoxicity and cell regeneration, and that doses below the RfD do not result in cytolethality and is unlikely to result in increased risk of cancer. Accordingly, the RfD of 0.01 mg/kg/day for protection against noncancer effects (including cell death and regeneration) can also be considered protective against increased risk of cancer. 9

Inhalation Cancer Risk
This has not been quantified by the EPA and is the major route of exposure for THMs.

Other Risk Assessment Considerations

Many of the studies for chloroform and the other THMs were conducted by corn oil gavage. Using corn oil as the vehicle has been shown to materially influence the toxicity and carcinogenicity of similar small halogenated molecules. 11 Larson et al. (1994, 1995) suggested that toxicity and carcinogenicity differences may have been due to the delivery of a higher dose to the liver from a single gavage dose, when compared to the delivery of smaller doses over a prolonged period, as would be the case when administered via drinking water. 9 Use of these studies may be overstating the cancer risk.

EPA concluded that the quantitative risk estimate for oral exposure to chloroform should be based on the 1985 Jorgenson et al. dog drinking water study. EPA determined that the study was more appropriate than the corn oil gavage studies because exposure was via drinking water and they considered this the most applicable route of exposure for humans. Also, EPA concluded that this study would eliminate the uncertainty regarding the potential impact of the corn oil vehicle. 9

Renal tumors were found in only two strains of mice. This seems to be related to the increased metabolic capability for chloroform bioactivation. 10 Is this increased bioactivation applicable for humans?

We found no data on the metabolism and pathway for intestinal tumors. The studies for intestinal tumors showed no toxicity in the male rat in the control group, but showed statistically significant incidences of tumors and/or polyps at the 50 and 100 mg/kg/day dosage. Additional testing at lower levels seems warranted to determine a NOAEL, especially since these dosages were much lower than the carcinogenic effects with chloroform. This seems to be important because the cytotoxicity may or may not be relevant for the intestinal tumors.

The total estimated mean intake of chloroform was estimated to be 2 ug/kg/day. Even in areas with the maximum chloroform levels in drinking water, the intake of chloroform was estimated to be 10 ug/kg/day. The maximum level of chloroform intake is at the reference dose level. Based on this information, the risk of excess exposure to chloroform seems low.

Figure 1.

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