Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 1 Chapter VII. Mechanism of Toxicity and Sensitive Populations A. Biochemical Basis of Toxicity No studies have been conducted describing the mechanism of toxicity for cyanogen chloride. However, since cyanogen chloride in the blood is almost immediately metabolized, it is likely that the systemic toxicity of cyanogen chloride is due to activity of metabolites rather than the parent compound. In contrast, irritation effects observed at the portal of entry following inhalation exposure may be due to cyanogen chloride itself. As discussed in Chapter 3, toxic effects of ingested cyanogen chloride and a plausible mode of action can be proposed based on consideration of cyanogen chloride biochemistry and the biochemistry of its known and putative metabolites.

Data from humans and laboratory animals shows that exposure to cyanogen chloride vapor results in irritation of the eyes, throat, and respiratory tract ( Flury and Zernick, 1931;

Prentiss, 1937; Michigan Department of Public Health, 1977; Reed, 1920; Aldridge and Evans,

1946; Haymaker et al., 1952). The irritation occurred within less than a minute after the initiation of exposure ( Flury and Zernick, 1931). Although it is possible that the irritation is due to HCl formed by glutathione mediated reduction to cyanide and HCl ( which is a very rapid reaction in blood), the glutathione concentration in the eyes, throat, and respiratory tract may not be sufficiently high to result in sufficiently rapid reduction of cyanogen chloride and production of HCl. Similarly, formation of HCl by cyanogen chloride hydrolysis or reactions with other Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 2 nucleophiles is slow at neutral pH. These considerations suggest that the irritation is due to the parent cyanogen chloride, rather than one of its metabolites.

Regardless of whether the parent or a metabolite is responsible for cyanogen chloride irritation, results from exposure to cyanogen chloride vapor indicate that ingestion of large quantities of cyanogen chloride may also cause irritation to the mucous membranes of the mouth and throat. Such irritant effects are likely to be concentration dependent ( rather than dosedependent and so unlikely to occur at environmentally relevant doses.

As discussed in Chapter 3, it is plausible that ingested cyanogen chloride is absorbed as the parent compound, in light of the pH dependence of cyanogen chloride hydrolysis and reaction with nucleophiles. It appears that formation of cyanate and cyanamide in the gastrointestinal tract would occur relatively slowly, although reaction with other physiological nucleophiles may occur.

Absorbed cyanogen chloride ( or its metabolites) would enter the blood stream either via the portal vein or directly from the intestine. Rapid glutathione mediated reduction of cyanogen chloride to cyanide and HCl would be expected to occur, based on injection and in vitro studies showing rapid reactivity of cyanogen chloride via this pathway in blood, and the high glutathione concentration in the liver. Cyanide produced in the portal vein or liver would likely be converted to thiocyanate by the rhodanese enzyme in the liver. Cyanide formed in the blood following intestinal absorption of cyanogen chloride would be metabolized by rhodanese found elsewhere in the body, or by mercaptopyruvate sulfurtransferase, which is concentrated in blood cells. This hypothesized metabolic pathway would suggest that HCl production would result in transient pH decreases, which are at least somewhat localized to the portal vein and liver. Systemic acidosis is Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 3 unlikely at environmental exposures, as shown by the calculations in Section III of Appendix E.

This hypothesis suggests that the primary effects of ingested cyanogen chloride would be: ( 1)

systemic effects due to cyanide and thiocyanate, ( 2) possible liver effects due to HCl and decreased pH, and ( 3) at very high concentrations, irritation of the mouth, throat, and possibly other portions of the gastrointestinal tract. As noted, no ingestion data are available to test the hypothesized endpoints. The observation of nervous system effects from injection and inhalation exposure to cyanogen chloride supports the prediction of systemic effects due to cyanide.

In contrast, the proposed liver effects would be specific to first pass metabolism following cyanogen chloride ingestion. Ideally, the potential for liver effects could be analyzed by evaluating the local buffering capacity of the liver and comparing that to HCl concentrations produced by cyanogen chloride metabolism, but data on the liver's buffering capacity were not located. In the absence of such data, a rough evaluation can be made by comparison with studies of HCl ingestion. In the only study of HCl ingestion that examined the liver ( Tober Meyer et al.,

1981), no effect was seen on liver weight or serum enzymes indicating liver injury ( SGOT,

SGPT), at drinking water doses of approximately 36 mg/ kg day in rats and 25 mg/ kg day in rabbits. Comparison of these results with cyanogen chloride ingestion is reasonable, since both involve exposure in drinking water and issues related to first pass metabolism. Uncertainties remain as to how differences in the rate or degree of absorption from the stomach would affect the peak hydrogen ion concentration in the liver, and the relative degree of absorption in the stomach versus intestine. In particular, the degree of HCl absorption at this relatively low dose is unknown. Liver effects were not investigated in the feed studies that provided higher doses of HCl ( Throssell et al., 1995, 1996), but liver damage is not one of the effects associated with Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 4 acidosis ( Bookallil, 2001), suggesting that the liver is resistant to moderate changes in blood pH.

Overall, the data suggest that HCl produced from metabolism of cyanogen chloride would not be sufficient to damage the liver, but there are a number of uncertainties in the data.

The primary uncertainties in the proposed cyanogen chloride metabolic pathway ( and associated predictions of toxicity) are the identity of other metabolites or reaction products, the degree to which other metabolites are formed, the form in which cyanogen chloride is absorbed,

the degree of absorption in the stomach versus the intestine, and the kinetics of HCl formation and neutralization. There are also uncertainties in the overall quantification of cyanogen chloride metabolism to cyanide and thiocyanate, and whether cyanogen chloride is metabolized directly to thiocyanate in vivo.

No in vitro studies evaluating cyanogen chloride toxicity were located. In the only in silico ( modeling) study evaluating cyanogen chloride toxicity, Moudgal et al. ( 2000) evaluated relationships between the structure of 244 disinfectant byproducts, including 21 nitriles, and potential developmental toxicity using a rat oral developmental toxicity submodel of TOPKAT ® , a quantitative structure toxicity relationship ( QSTR) prediction tool. Based on individual structural descriptors, model probabilities were used to derive qualitative estimates as follows: 0.0 to 0.3 negative, 0.3 to 0.7 indeterminate, 0.7 to 1.0 positive. The probability estimate is independent of the potency or severity of developmental effects that could be induced, and would be interpreted as the likelihood that the chemical can cause developmental toxicity in rats following oral dosing.

As a group, the nitrile disinfectant byproducts were characterized as having a high probability of developmental toxicity. Of the 21 individual nitrile compounds, 13 were positive, 5 were Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 5 negative, and for 3 the model did not make a prediction. Cyanogen chloride was predicted as positive for developmental toxicity using this system. The effects of individual structural moieties were also examined by the software for various structural classes of compounds. The nitrile moiety, chlorine atom, and bromine atom were identified as contributing most significantly to the developmental toxicity predictions for the group of 20 nitriles. The importance of the nitrile group in the developmental toxicity predictions is consistent with cyanide as a causal factor in the developmental toxicity of these compounds in vivo, although cyanide itself has not been shown to be a developmental toxicant.

One way to infer mechanism( s) of toxicity for cyanogen chloride is by comparing known toxic effects and target organs of cyanogen chloride with those of known or potential metabolites to find similarities. It appears that cyanogen chloride and cyanide produce similar effects in the central nervous system ( CNS). As will be discussed below, some of the effects produced by cyanogen chloride, namely severe weight loss and peripheral neurotoxicity, are also similar to those produced by cyanate. It is plausible that the eye and respiratory tract irritation and pulmonary edema observed following exposure to cyanogen chloride vapor are due to the parent chemical, although HCl formed from cyanogen chloride may also contribute. This initial examination of endpoints suggests that cyanide can account for the primary effects of cyanogen chloride, but not all of cyanogen chloride toxicity. In addition, no direct information is available on the effects of long term exposure to cyanogen chloride. In order to further aid in identifying the mechanism of toxicity for cyanogen chloride, mechanisms of the known and potential metabolites of cyanogen chloride are discussed in the following paragraphs. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 6 Cyanide. The mechanism of toxicity for cyanide has been described in detail by ATSDR ( 1997) and is summarized here. The CNS is the primary target of cyanide toxicity, particularly following acute exposure. Symptoms of CNS toxicity following cyanide exposure include respiratory depression, convulsions, coma, and death. Cyanide appears to exert its acute toxic effects by binding with cytochrome c oxidase, which then becomes unable to catalyze the reactions that transfer electrons from reduced cytochrome c to oxygen. As a result, cellular oxygen use is impaired and aerobic metabolism stops. As anaerobic metabolism proceeds, blood levels of pyruvic acid, lactic acid and NADPH rise; the ATP/ ADP ratio decreases. One of the reasons that the CNS is a primary target of cyanide toxicity may be the high energy demand in nervous tissue. The inhibition of oxygen use by cells causes oxygen tension to rise in the peripheral tissues, which results in a decrease in the unloading gradient for oxyhemoglobin. Thus,

oxyhemoglobin is present in the venous blood. In addition to cytochrome c oxidase, cyanide binds to other metalloenzymes that contain ferric iron, including catalase, peroxidase,

methemoglobin, and hydroxocobalmin; this binding also contributes to the symptoms of cyanide toxicity. Cyanide also stimulates the release of secondary neurotransmitters and catecholamines from the adrenal glands and adrenergic nerves. Thus, the cardiac effects and the peripheral autonomic responses observed following cyanide exposure appear to be due to the increase of plasma catecholamine levels. CNS necrosis and demyelination caused by cyanide may be due to vasoconstriction and low blood flow in the brain resulting from low carbon dioxide levels.

Alternatively, the decreased ATP/ ADP ratio may alter energy dependent calcium homeostasis in nerve cells. Thus, the acute effects of cyanide result primarily from the interruption of aerobic metabolism and from the release of secondary neurotransmitters and catecholamines, and include altered respiration, vomiting, nausea, and weakness; and ultimately, convulsions, coma, and death. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 7 Although the CNS effects of cyanide are well known, no definitive statement regarding whether cyanide also causes peripheral neuropathy is possible at this time. The weakness observed in the epidemiology study of El Ghawabi et al. ( 1975) could be due to either peripheral or central nervous system effects, and no objective measurements ( such as of nerve conduction velocity)

were performed.

Since cyanide is a known metabolite of cyanogen chloride, and the histological evidence of CNS toxicity, including necrosis and demyelination, is virtually identical for cyanogen chloride and cyanide, it is likely that cyanogen chloride toxicity to the CNS is mediated through cyanide.

The thyroid and male reproductive system are also target organs of cyanide toxicity,

particularly following longer term exposure. Cyanide's effects on the thyroid are mediated by its metabolite thiocyanate; the mechanism of thiocyanate toxicity is discussed below. No data were located that indicate that cyanide acts directly on the thyroid. No data were located that describe the mechanism of cyanide's toxicity on the male reproductive system, although NTP ( 1993)

suggested that the effects may be related to perturbations in hormonal balance. The available data are insufficient to determine whether cyanogen chloride affects the thyroid or the male reproductive system. Only minimal data were located on the potential developmental toxicity of cyanide, and, for that reason, extrapolation to cyanogen chloride is not possible. As discussed in greater detail in Section VII. C. 2, Tewe and Maner ( 1981) found no evidence of cyanide developmental toxicity at parental doses up to 21.6 mg CN/ kg day. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 8 Thiocyanate. There is no question that the thyroid is the primary target organ for thiocyanate toxicity; however, although no toxicity studies have thoroughly evaluated other organs and tissues, toxicity to other targets cannot be ruled out. Thiocyanate acts by blocking the active transport of iodide into the thyroid gland. Because its ionic size is similar to that of iodide,

thiocyanate is a competitive inhibitor with iodide for the Na+/ I symporter protein that concentrates iodide in the thyroid gland. In fact, the symporter has a greater affinity for thiocyanate than for iodide ( Wolff, 1998). In addition to blocking iodide uptake by the thyroid,

thiocyanate also causes iodide already accumulated in the thyroid to be discharged ( Wolff, 1998).

Once thiocyanate has blocked iodide uptake by the thyroid, the feedback mechanisms that regulate balance of the thyroid pituitary axis are disrupted, resulting in the classic symptoms of thyroid toxicity produced by thiocyanate. Specifically, by blocking iodide uptake, thiocyanate causes the thyroid to decrease production and secretion of the hormones triiodothyronine ( T3)

and thyroxine ( T4) ( Hill et al., 1989). As the blood levels of these hormones drop, the hypothalamus, through the release of thyrotropin releasing hormone ( TRH), stimulates the pituitary gland to produce thyroid stimulating hormone ( TSH). TSH then interacts with receptors on the cell surface of thyroid follicular cells to stimulate increased iodide uptake and production of thyroid hormones. Under normal circumstances, increasing blood levels of thyroid hormones serves as a negative feedback mechanism on the thyroid pituitary axis and the system equilibrates.

However, when thiocyanate is present at levels that completely block iodide uptake, the thyroid cannot produce T3 and T4, and so TSH levels in the blood remain elevated. As a result, the thyroid is subject to excessive TSH stimulation, causing an increase in thyroid weight and size as thyroid epithelial cells increase in volume and vascularity and colloid is resorbed from the follicular lumen ( Hill et al., 1989). With continued TSH stimulation, thyroid changes progress to Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 9 follicular cell hyperplasia, characterized by increased mitotic activity and in the number of follicular cells per gland. Eventually, prolonged TSH stimulation can lead to thyroid cancer in animals, although it is not clear if this progression occurs in humans ( Hill et al., 1989). Therefore,

the symptoms of thiocyanate toxicity, including decreased T3 and T4 blood levels, increased TSH blood levels, increased thyroid weight and thyroid hyperplasia, are all directly related to its ability to block iodide uptake by the thyroid.

Thiocyanate is a known metabolite of cyanogen chloride and of cyanide. The thyroid effects of cyanide, which are generally observed following longer term exposure, are mediated through its metabolism to thiocyanate. Since cyanogen chloride is also metabolized to both cyanide and thiocyanate, and these metabolites are available for distribution to the thyroid,

cyanogen chloride would also be expected to cause effects on the thyroid following longer term exposure. No data have been generated on the thyroid effects of cyanogen chloride; additional research is needed in this area.

Cyanate. The toxicological effects of cyanate are described in detail in Appendix C, but the biochemical mechanisms are summarized here to provide a context for cyanogen chloride toxicity. Cyanate is a biologically active molecule that forms irreversible carbamyl bonds with terminal amino groups of amino acids ( Nicholson et al., 1976). Although this action of cyanate is relatively nonspecific and can affect any protein, cyanate has a particular affinity for the terminal valine of hemoglobin, forming a carbamylated hemoglobin molecule that has greater affinity for oxygen than noncarbamylated hemoglobin. This property of cyanate led to its use in clinical trials in the 1970s as a therapeutic agent for sickle cell anemia. Carbamylated hemoglobin was thought Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 10 to reduce sickling by shifting the oxygen dissociation curve to the left, thereby increasing oxygen affinity and decreasing the formation of deoxyhemoglobin S ( Haut et al., 1975).

The primary toxic effects of cyanate include severe weight loss, cataracts, peripheral neuropathy, and liver toxicity characterized by altered glycogen metabolism. The mechanism of at least two of these, cataracts and liver toxicity, appear to be related to the ability of cyanate to carbamylate protein. Cyanic acid ( HOCN) is also described as highly irritating to the eyes, skin,

and mucous membranes ( O'Neil et al., 2001), an effect that might also be related to carbamylation. Charache et al. ( 1975) found that there was considerable retention of carbamylated residues in the eyes of rats administered a single dose of 300 mg potassium cyanate by gavage. In addition, the lenses of rabbits incubated with cyanate in vitro developed opacities and demonstrated inhibition of the lens cation pump ( Nicholson et al., 1976; Kern et al., 1977).

Therefore, it appears that carbamylation of either the lens structural proteins or enzymes that maintain ion fluxes is responsible for the development of cataracts. Haut et al. ( 1975)

demonstrated that cyanate inhibits several liver enzymes associated with glycogen metabolism,

including phosphorylase, glucose 6 phosphatase, G6PD, and UDPG pyrophosphorylase. This enzyme inhibition could account for the accumulation of glycogen deposits associated with cyanate exposure.

Less information is available regarding the mechanism of the weight loss and peripheral neuropathy caused by cyanate. The peripheral neuropathy is well characterized as such by objective measures such as decreased nerve conduction velocity and elevated sensation threshold ( Ohnishi et al., 1975; Charache et al., 1975). Charache et al. ( 1975) observed retention of Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 11 carbamylated residues in the spinal cord of rabbits administered a single dose of 300 mg potassium cyanate by gavage, but no information was provided on peripheral nerves. It is possible that cyanate directly carbamylates proteins in the myelin membrane or carbamylates enzymatic proteins associated with maintaining ion flux in the nerves, as was observed for lens tissue. However, no data have been located confirming these potential mechanisms.

Alternatively, the effects on weight loss and nervous tissue could be mediated through the cyanate induced increase in oxygen affinity of hemoglobin. Teisseire et al. ( 1986) found that rats treated with cyanate for three weeks developed hypoxia like effects characterized by decreased body weight, increased oxygen affinity of hemoglobin, and altered red blood cell morphology.

The authors noted that the weight loss was similar to that observed in animals maintained in a hypobaric chamber for 4 weeks, and that the mechanism could be an impairment of oxygen diffusion from capillaries to tissues.

Cyanamide. The toxicological effects of cyanamide are described in detail in Appendix D, but the biochemical mechanisms are summarized here to provide a context for cyanogen chloride toxicity. Little is known about the mechanism of toxicity of cyanamide. Cyanamide is described as having the potential to cause irritation of the eyes, skin, and respiratory system, as well as salivation ( O'Neil et al., 2001), but the mechanism of these effects is not known.

Cyanamide inhibits the enzyme aldehyde dehydrogenase ( ALDH) and has been widely used in Europe as a drug to treat chronic alcoholism. When taken with alcohol, cyanamide has an aversive effect caused by the increased levels of aldehyde in the blood ( Obach et al., 1986b; Valles et al.,

1987). Toxic effects observed in humans and animals following cyanamide exposure include severe weight loss, liver toxicity characterized by glycogen deposits, allergic dermatitis, and male Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 12 reproductive effects. Of these, only the male reproductive effects may be related to the action of cyanamide as an ALDH inhibitor. Valles et al. ( 1987) note that ALDH is widely distributed in the body, especially in the gonads. Increased levels of acetaldehyde have been shown to have a toxic effect on the gonads, and other compounds that inhibit ALDH have also been associated with testicular toxicity ( Valles et al., 1987). The effect of cyanamide on weight loss appears to be an anorectic effect on the CNS similar to that of amphetamine like drugs and unrelated to its action as an ALDH inhibitor. Obach et al. ( 1986b) observed that cyanamide exposure increased the brain concentration of MOPEG SO 4, a metabolite of noradrenaline, a change also see with amphetamines that induce anorexia. The mechanism of liver toxicity is unknown. Guillen and Vazquez ( 1984) observed the presence of inclusion bodies ( apparently containing glycogen) in the livers of rats that had been exposed to cyanamide, but had never been exposed to ethanol. The authors concluded that these findings exclude the possibility that liver toxicity seen in the human studies is due to accumulation of acetaldehyde or to chronic ethanol exposure. They speculated that cyanamide may also have an action, as yet unidentified, on enzymes involved with glycogen metabolism.

Relationship of Metabolite Data to Cyanogen Chloride. In summary, all of the toxic effects observed following cyanogen chloride exposure can be accounted for by the known or potential metabolites. The toxic effects observed, together with information about their mechanism, can also be used to address whether all of the toxic effects of cyanogen chloride are due to the known metabolites ( cyanide and thiocyanate), or whether other potential metabolites could also be contributing to cyanogen chloride toxicity. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 13 As described above, it is plausible that the eye and lung irritation observed following exposure to cyanogen chloride vapor is due to the parent chemical. It is also possible that these effects may be due to HCl, cyanate, or cyanamide formed from hydrolysis of cyanogen chloride or reaction with ammonia or other amines, since all of these latter chemicals are themselves irritating.

However, hydrolysis of cyanogen chloride to HCl and cyanate would likely be slow under the neutral to slightly acidic conditions at the contact sites of the eyes and respiratory tract.

Cyanogen chloride might also react with cellular constituents to produce HCl and substituted cyanamides, thiocyanates, or cyanates. However, it is unclear whether HCl, cyanate, and cyanamide would be formed quickly enough following exposure to cyanogen chloride vapor to explain the observed irritation. Similarly, the kinetics of their formation following ingestion exposure to cyanogen chloride is unknown, although increased local concentrations of HCl ( i. e.,

decreased pH) may occur in the liver and portal vein. Inhalation of hydrogen cyanide has not been reported to cause pulmonary edema or eye irritation, indicating that cyanogen chloride itself,

or a different metabolite, is responsible for the observed irritation.

Effects on the nervous system constitute the second major class of effects seen with cyanogen chloride. The observed symptoms include muscle weakness in humans ( Reed, 1920)

and tremors, muscle rigidity, and limb paralysis in dogs ( Haymaker et al., 1952). The available data and methods used were not specific enough to differentiate between effects on the central and peripheral nervous systems, although the limb paralysis suggests that the peripheral nervous system may have also been affected. Cyanide is known to affect the central nervous system;

effects on the peripheral nervous system might also occur, but the data are not specific enough to identify such effects. By contrast, cyanate is known to affect the peripheral nervous system, based Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 14 on objective measures such as nerve conduction velocity ( Ohnishi et al., 1975; Charache et al.,

1975). A finding that cyanogen chloride affects the peripheral nervous system would suggest that cyanate may be the toxic agent, but an effect of cyanide could not be ruled out. Production of cyanide would be expected following ingestion exposure to cyanogen chloride, but it is not known whether cyanate would be produced. Following ingestion of cyanogen chloride, cyanide that is not detoxified in the liver as part of first pass metabolism would be expected to be distributed widely in the body, including distribution to the nervous system.

Other effects associated with the known or putative metabolites of cyanogen chloride involve endpoints that are either nonspecific ( e. g., weight loss), or have not been investigated following cyanogen chloride exposure ( e. g., reproductive, thyroid effects). In the absence of data on distribution following cyanogen chloride exposure, it is plausible that metabolites of cyanogen chloride are widely distributed, and so reach the target tissues. The parent cyanogen chloride would be expected to react quickly once it reaches the blood, and so would not be distributed to the target tissues.

Severe weight loss and decreased appetite were also observed in workers chronically exposed to cyanogen chloride vapor ( Reed, 1920). Severe weight loss, accompanied by vomiting and diarrhea, was also observed in dogs exposed to cyanogen chloride via inhalation for 2 weeks ( Haymaker et al., 1952). Of the metabolites and potential metabolites described in this document,

severe weight loss is most closely associated with cyanate and cyanamide ( Haut et al., 1975; NCI,

1979). However, weight loss has also been associated with chronic exposure to cyanide ( Wolfsie and Shaffer, as cited by ACGIH, 1996), although cases of weight loss were not reported in the Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 15 cyanide studies reviewed for this document. In addition, the observation of Teisseire et al. ( 1986)

that hypoxia can cause weight loss supports the presumption that weight loss could also result from exposure to cyanide. Thus, the weight loss seen following repeated exposure to cyanogen chloride suggests that cyanate or cyanamide could be involved, but this effect might also be due to cyanide.

Male reproductive toxicity has also been observed in animals exposed to cyanide ( NTP,

1993) or cyanamide ( Valles et al., 1987), and thyroid effects result from chronic exposure to cyanide or thiocyanate. In addition, liver toxicity results from exposure to cyanate or cyanamide.

These effects have not been observed with cyanogen chloride, but only limited toxicity testing has been conducted to date, and specific studies to evaluate reproductive or thyroid effects of cyanogen chloride have not been conducted. Therefore, these endpoints do not shed further light on the issue of the toxic agents resulting from exposure to cyanogen chloride.

B. Mechanism of Carcinogenesis There are no cancer bioassays of cyanogen chloride or cyanide; therefore, no conclusion can be made regarding their carcinogencity, and any considerations of mode of action are speculative. The limited genotoxicity data for these chemicals ( De Flora 1981; De Flora et al.,

1984; Painter and Howard, 1982; Melzer et al., 1983; NTP, 1993) also indicate that genotoxicity is not of concern, although none of these chemicals has been fully evaluated. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 16 Moudgal et al. ( 2000) evaluated relationships between the structure of 244 disinfectant byproducts, including 21 nitriles, and potential carcinogenicity using mouse and rat oral submodels of TOPKAT ® , a quantitative structure toxicity relationship ( QSTR) prediction tool.

As a group, the nitrile disinfectant byproducts were characterized as having a low probability of carcinogenicity and cyanogen chloride was predicted as negative in male and female rats and mice.

The carcinogenic potential of thiocyanate is also unknown. Information on thiocyanate carcinogenicity is limited to two oral carcinogenicity studies in rats conducted in the same laboratory ( Lijinsky and Reuber, 1982; Lijinsky and Kovatch, 1989). The only effect in the first study was an increase in liver tumors, and this was not confirmed at the higher dose tested in the second study. The second study suggested an increase of thyroid tumors in treated animals.

Although the increase was not statistically significant, it is consistent with thiocyanate's mode of action. Limitations of these studies include the testing of only a single dose, use of an insufficient number of animals, and incomplete evaluation of key information. In addition, the thoroughness of the histopathological evaluation ( and whether the thyroid was evaluated) is unclear, since the authors only stated that major organs and all lesions were evaluated. Potassium thiocyanate does not have any structural alerts for genotoxicity ( Rosenkranz and Klopman, 1990), and genotoxicity data are limited to marginal results in a S. typhimurium mutagenicity assay ( Kier, 1988, as reported by Rosenkranz and Klopman, 1990).

However, the biochemical activity of thiocyanate suggests that, at sufficiently high doses,

thiocyanate could cause thyroid tumors. A similar supposition would apply to cyanide, since it is metabolized to thiocyanate. As described in greater detail in Section VII. A, thiocyanate acts by Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 17 blocking the active transport of iodide into the thyroid gland, resulting in decreased levels of the thyroid hormones T3 and T4, and increased production of TSH ( Hill et al., 1989). Since sufficiently high thiocyanate levels block the negative feedback regulation of TSH, the increased TSH levels in the blood can result in increased thyroid weight, follicular cell hyperplasia, and ultimately cancer. This mode of action is well established for chemicals that alter thyroid hormone homeostasis, and indicates that thiocyanate ( and, therefore, cyanide) can cause thyroid tumors in rats. It is unknown, however, whether they actually do cause tumors at nonlethal doses. In addition, because rats lack thyroxine binding globulin, resulting in a much shorter T4 half life than in humans ( 12 24 hours, in contrast to 6 7 days in humans), humans are less sensitive than rats. It is not clear if the progression from decreased levels of thyroid hormones to thyroid tumors occurs in humans ( Hill et al., 1989). Finally, if thiocyanate or cyanide did cause thyroid tumors via this mode of action, protection against changes in TSH levels would protect against cancer, and so extrapolation to low doses would be using the margin of exposure approach.

The carcinogenicity of cyanamide also cannot be determined. In the only standard cancer bioassay on cyanamide, NCI ( 1979) evaluated the carcinogenicity of calcium cyanamide in drinking water to F344 rats and B6C3F1 mice. No tumors related to treatment were observed in rats. The incidence of hemangiosarcomas was elevated in male mice, and the incidence of malignant lymphomas was elevated in female mice. However, NCI ( 1979) did not consider either of these tumors to be clearly related to administration of calcium cyanamide, because the incidence of hemangiosarcomas was not significantly different from controls, and the incidence of malignant lymphomas was within the range of historical controls. Based on negative results in Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 18 bacterial gene mutation assays ( Loveless et al., 1954; Zeiger 1987), a Drosophila sex linked recessive gene mutation assay ( Yoon et al., 1985), an assay of DNA damage in mammalian cells ( Sina et al., 1983), and a micronucleus assay in mice ( Menargues et al., 1984), cyanamide is not expected to be genotoxic.

C. Interactions and Susceptibilities This section focuses on the metabolic pathway in which cyanogen chloride is metabolized to cyanide and thiocyanate, since these are the only metabolites that have been identified in vivo.

Cyanate is also addressed, since cyanide can also be converted to cyanate.

1. Potential Interactions No data were located on the potential interactions between cyanogen chloride or its metabolites and other chemicals. However, since cyanide and thiocyanate are known metabolites of cyanogen chloride, it would be expected that the effects of cyanogen chloride would be additive with the effects of cyanide and thiocyanate. In addition, additive effects would be expected for any other chemicals that act through similar mechanisms.

Data are available to suggest that carbon monoxide, carbon dioxide and ascorbic acid may increase the toxicity of cyanide. Levin et al. ( 1987) evaluated the interactions between exposure to hydrogen cyanide and carbon monoxide or carbon dioxide in rats at exposure levels near the hydrogen cyanide LC 50. Co exposure to hydrogen cyanide and carbon monoxide resulted in approximately additive effects on lethality. Co exposure to hydrogen cyanide with carbon dioxide Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 19 at the nonlethal concentration of 5% decreased the LC 50 to 75 ppm, compared with 110 ppm for hydrogen cyanide alone, suggesting a more than additive effect on lethality. Basu et al. ( 1983)

reported that pretreatment of guinea pigs with ascorbic acid potentiated the toxic effects of oral dosing with potassium cyanide. Only 3/ 8 of the guinea pigs exhibited slight tremors following exposure to 3.2 mg/ kg cyanide as potassium cyanide alone, but severe tremors and convulsions were seen in all animals that were pretreated for 3 days with 300 mg ascorbic acid ( approximately 1.3 mg/ kg) and received the same dose of cyanide. The authors suggested that ascorbic acid competes with cyanide for cysteine, decreasing the detoxification of cyanide. Compounds that inhibit cytochrome c oxidase, such as sulfide or azide, would also be expected to interact with cyanide.

Chemicals that interfere with cyanide toxicity have been extensively studied as part of the identification of antidotes ( reviewed in ATSDR, 1997). Compounds that act as sulfane sulfur donors include thiosulfates and polythionates. These chemicals aid in the detoxification of cyanide by rhodanese. The other major class of cyanide antagonists produce compounds that compete with cytochrome c oxidase for binding to cyanide. Chemicals in this class include sodium nitrite, amyl nitrite, and hydroxylamine, all of which form methemoglobin.

Methemoglobin interacts with cyanide to form cyanomethemoglobin, preventing the cyanide from binding to cytochrome c oxidase and exerting its toxic effects. Cobalt may also act as an antagonist by forming a stable complex directly with cyanide. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 20 2. Childhood Susceptibility Cyanide. Data are limited, but there is no indication that children or fetuses are particularly susceptible to the acute effects of cyanide. Rather, as discussed in the next section,

the limited data suggest that young animals may be less susceptible than older animals to the acute effects of cyanide ( McMahon and Birnbaum, 1990). In support of this possibility, a study of an epidemic of konzo ( spastic paraparesis associated with cassava consumption and associated cyanide exposure) found no cases in children under 4 years of age out of 72 cases examined ( Cliff et al., 1997). It does not appear that the method of case identification ( through community notice) would have excluded young children, but the degree of exposure of young children was not addressed in the article. A prolonged period of breast feeding may have reduced exposure for the children. In contrast, Rosling ( 1987, as cited in ATSDR, 1997) reported that children and women may be more susceptible to spastic paraparesis. As described in Section VII. C. 3, the limited available data on age related differences in acute toxicity and in the expression of enzymes that metabolize cyanide suggest that susceptibility increases with old age, at least partially because of decreased activity of the detoxifying enzyme rhodanese ( McMahon and Birnbaum,

1990).

While higher levels of rhodanese in young animals would decrease the acute toxicity of cyanide, it would increase the thiocyanate levels in the body, and thus increase the susceptibility to thyroid effects, including neurodevelopmental effects secondary to thyroid effects. In addition,

the effects of decreased thyroid hormone levels can be more adverse in fetuses. While hypothyroidism in adults typically results in goiter ( an enlarged thyroid), congenital Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 21 hypothyroidism is associated with stunted bodily growth and mental development. Because the youngest animals evaluated in the McMahon and Birnbaum ( 1990) study were 2 3 months old,

and the late gestational and early neonatal periods constitute the critical time window for the effects of thyroid hormone levels on neurodevelopment ( Porterfield, 1994; Porterfield, 2000), the results of that study are not informative with regard to the effects of age related differences in metabolism on relative sensitivity to any neurodevelopmental effects of cyanide ( or cyanogen chloride). Since the thyroid effects of cyanide result from its conversion to thiocyanate, they are discussed below in further detail in the context of thiocyanate.

Data on the developmental toxicity of cyanide are limited to one dietary study in rats.

Tewe and Maner ( 1981) observed no effects on pup weights, litter size, or pup mortality in the offspring of dams exposed to 21.6 mg CN/ kg day as KCN throughout mating, gestation, and lactation. There was, however, a significant decrease in food consumption and growth rate, and an increase in the ratio of food consumption to body weight gain in weanling rats. The single dose tested in this study is approximately 5 fold the NOAEL of 4.5 mg CN/ kg day for male reproductive effects ( and almost 30 times the corresponding BMDL) that is the basis for the cyanide RfD presented in Chapter VIII. However, since no internal examination was conducted,

the teratogenic potential of cyanide is unknown, although, presumably, the authors would have noted overt external malformations. Although the NOAELs for other endpoints, such as the male reproductive toxicity seen with cyanide exposure, are lower than those identified for changes in serum levels of thyroid hormones in developmental toxicity studies, neurodevelopmental effects and sensitive markers of a hypothyroid state ( e. g., brain levels of thyroid hormones) have not been evaluated in any study of cyanide toxicity. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 22 Thiocyanate. Limited data were located that directly investigated age related differences in the systemic toxicity of thiocyanate. In the only study addressing this issue with dose response information, Kreutler et al. ( 1978) evaluated thyroid weight in rat pups and their dams in a study where the groups of 4 7 pregnant dams were administered sodium thiocyanate in drinking water during gestation through postpartum day 10. As described further in Section V. C, the estimated doses were 0, 6.3, 12.6, and 25.3 mg SCN/ kg day. Thyroid weights of the dams ( relative to body weight) were significantly increased at all doses at 5 days postpartum, but only at the high dose at 10 days postpartum. In the pups, there were dose related increases in thyroid weights that were statistically significant at all doses on day 5, but only at the two top doses on day 10. Some of the difference between the results in the dams on days 5 and 10 postpartum may be an artifact of the small sample size, since the control relative thyroid weight was much lower at day 5 than day 10.

However, the magnitude of the effect on thyroid weight was also higher in the pups at each dose level. These results suggest that the pup is more sensitive than the dam to the thyroid effects of thiocyanate, particularly since the plasma thiocyanate levels were much lower in the pups.

However, the absence of an evaluation of other endpoints of thyroid toxicity ( e. g., thyroid hormone levels, histopathology) makes it difficult to determine whether the increased thyroid weight in the pups was adaptive or adverse. T4 levels were evaluated by Bala et al. ( 1996) in dams exposed to thiocyanate in the diet for 8 weeks, from weaning through conception,

pregnancy, and lactation, and in their pups at weaning. Serum T4 levels were significantly decreased in both the dams and pups. The absolute T4 levels were similar in both groups,

although the control levels were lower in the pups, so the magnitude of the decrease was lower in the pups. Only one dietary level of thiocyanate was tested. In addition, the amount transferred to the pups is not known; therefore, comparisons cannot be done on a mg/ kg day basis. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 23 Limited human data support the suggestion that fetuses and neonates may be more susceptible to thyroid effects of cyanide and its metabolite thiocyanate. Ermans et al. ( 1980)

reported that congenital hypothyroidism occurs at an elevated incidence ( e. g., 5.5% in one region)

in newborns in areas where cassava is a staple food. Cassava contains cyanogenic glycosides, and can be a significant dietary source of cyanide. Although goiter is observed in adults under similar conditions, the effects of hypothyroidism in infants ( stunted growth and mental development) are more severe.

Developmental toxicity studies of thiocyanate have found that the primary effects were decreased litter weight ( Pyska, 1977) or growth retardation ( Heydens, 1985); no teratogenicity was observed ( Heydens, 1985). In one study ( Pyska, 1977), the maternal LOAEL for thyroid effects was the pup NOAEL for decreased litter weight, although neither thyroid effects in pups nor other standard developmental toxicity endpoints were evaluated. In another study ( Heydens,

1985), the dams were not evaluated and there was no pup NOAEL, but the pup LOAEL of 55 mg/ kg day was comparable to LOAELs reported in other studies for thyroid effects in adults.

Potential neurodevelopmental toxicity is a key unresolved issue for both thiocyanate and cyanide ( since cyanide is metabolized primarily to thiocyanate) because they have the potential for inducing hypothyroidism in fetuses and neonates. Hypothyroidism, defined by increased serum levels of TSH and decreased levels of T3 and T4, has been associated with neurodevelopmental delay in both humans and rats. In humans, congenital hypothyroidism, or cretinism, is characterized by long term effects on behavior, locomotor ability, speech, hearing and cognition ( Chan and Kilby, 2000). Prompt supplementation of neonates with thyroid hormone can restore Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 24 neurodevelopmental function. In rats, hypothyroidism has been associated with anatomical alterations in the cerebellum including reduction of growth and branching of Purkinje cells,

delayed proliferation and migration of granule cells, delayed myelination, and changes in synaptic connection among cerebellar neurons ( Koibuchi and Chin, 2000). These changes appear to be mediated by circulating T4, which is converted in the astrocytes to T3. It is not clear, however, if there is a direct correlation between serum T3 levels and brain T3 levels. Brain T3 then appears to bind to nuclear thyroid hormone receptors and regulate gene expression ( Koibuchi and Chin,

2000). However, it is not clear whether thyroid hormones exert their effect by directly regulating genes that encode for peptides crucial for cerebellar development, or whether thyroid hormones regulate the expression of genes encoding other transcription factors ( Koibuchi and Chin, 2000).

The degree of decrease in serum T4 levels that result in neurodevelopmental effects is also not known.

Maternal hypothyroidism during pregnancy can also affect the long term mental development of the child. Haddow et al. ( 1999) demonstrated that children whose mothers had diagnosed hypothyroidism during pregnancy were four times more likely to average 85 or less on I. Q. tests. Morreale de Escobar et al ( 2000) conducted a comparative analysis of 5 studies evaluating neurodevelopment in children of hypothyroid mothers by normalizing the IQ scores to a scale of 100 and estimating the frequency of IQ scores

85. For the control populations in all five studies, the frequency of IQ

85 ranged from 5 to 18% and the frequency of IQ

70 ranged from 1 to 2.3%. In contrast the frequency of IQ

85 ranged from 19 to 50% in children born to hypothyroid mothers. Frequency of IQ

70 ranged from 5 to 14% in these same children. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 25 Morreale de Escobar et al ( 2000) suggest that it is the levels of maternal T4 that are critical to proper neurodevelopment of child, not levels of T3 or TSH.

No information was located on age related differences in expression of thiocyanate oxidase, the enzyme that converts thiocyanate back to cyanide. Thiocyanate oxidase also converts thiocyanate to OSCN, which reacts nonenzymatically with cyanide to form cyanate.

Cyanate. No data were located on potential age related differences in the systemic toxicity of cyanate. Similarly, no standard developmental toxicity studies of cyanate were located.

Graziano et al. ( 1973) conducted a study investigating the effects of cyanate on reproductive capacity of mice ( 6 male/ female pairs per group) fed either 0 mg or approximately 1,200 1300 ( depending on the sex) mg cyanate/ kg day. This study is limited by the small sample size and the limited number of endpoints evaluated. Nevertheless, there was no effect on pup birth weight and no gross abnormalities, although the cyanate exposure altered the estrus cycle after the first pregnancy. No information was located on age related differences in expression of cyanase, the enzyme that hydrolyzes cyanate to ammonia and carbon dioxide.

Implications for Cyanogen Chloride. There are no data on potential age related differences in the toxicity or toxicokinetics of cyanogen chloride itself. Data on age related differences of the metabolites is also limited, but suggest that acute toxicity and developmental toxicity ( excluding neurodevelopmental toxicity) would not occur at doses below those that cause systemic toxicity in adults. The data suggest that young rats and animals ( and possibly humans)

are more susceptible to effects of thiocyanate on thyroid weight, but there was insufficient Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 26 associated information on thyroid hormone changes to determine if the effects on thyroid weight were adaptive or adverse. Epidemiology data support the suggestion that cyanide exposure of fetuses from maternal consumption of cassava can lead to altered physical and mental development at doses that only cause goiter in adults. It has been long known that decreased T4 levels in neonates, if left untreated, lead to severe neurological deficits. A newly emerging body of data demonstrates that children born to women who had decreased T4 during pregnancy also suffer long term, permanent cognitive impairment. The data strongly demonstrate that decreased T4 in both pregnant women and fetuses/ neonates results in neurodevelopmental effects. The key outstanding issue is whether thiocyanate ( and, by implication, cyanide and cyanogen chloride) can cause enough of a decrease in T4 to result in neurodevelopmental effects, and if so, at what doses those effects occur. This endpoint is of concern because neurodevelopmental effects are known to result from decreased T4 levels.

3. Other Potential Susceptible Populations People with a defect in the enzyme systems that convert cyanide to thiocyanate ( rhodanese and mercaptopyruvate sulfurtransferase) may be more susceptible to the toxic effects of cyanide.

For example, people with amyotrophic lateral sclerosis possess a disorder in cyanide metabolism that may result in an increased susceptibility to cyanide ( Kato et al., 1985, cited in ATSDR,

1997). Similarly, a deficiency in rhodanese has been associated with Leber's hereditary optic atrophy ( Wilson, 1983). Vitamin B 12 deficiency increases the susceptibility to tobacco amblyopia,

a disease associated with abnormalities in both cyanide and vitamin B 12 metabolism ( Wilson, Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 27 1983). As noted in Section III, cyanide can be metabolized by conversion to vitamin B 12 ,

cyanocobalamin.

People with protein or iodine deficiency may be more sensitive to the thyroid effects of cyanide that are caused by the metabolite thiocyanate. Kreutler et al. ( 1978) observed that rats on a low protein diet ( 2% casein) demonstrated increased plasma TSH and thyroid weights following potassium cyanide administration; rats on a normal protein diet ( 20% casein) that were exposed to the same concentrations of cyanide did not develop these effects. When the rats were administered iodine concurrently with cyanide, the thyroid effects were not observed. Studies in human populations that eat cyanide containing foods, such as cassava, also suggest that an increased susceptibility to thyroid effects may be associated with deficiencies of protein, iodine,

vitamin B 12, or other vitamins, although an etiological agent other than cyanide has been proposed as the cause of the tropical neuropathies ( reviewed in ATSDR, 1997).

Protein deficiency is not common in the Western world, although it does occur in Third World countries ( U. S. FDA, 1999). By contrast, iodine deficiency is more common in the United States. Hollowell et al. ( 1998) evaluated the iodine excretion data from the National Health and Nutrition Examination Surveys III ( NHANES III) conducted in 1988 1994, and determined that the median urinary iodine ( I) excretion in the total U. S. population was 124.6  g I/ g creatinine.

They also found that 7.5% of the total U. S. population had urinary iodine excretion lower than 50  g I/ g creatinine, a value that the National Research Council ( NRC, 1989) determined to be " adequate" for normal thyroid function. Hollowell et al. ( 1998) reported that urinary iodine excretion below the level determined by the NRC to be " adequate" was observed in 9.0% of U. S. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 28 males and 6.1% of U. S. females. Among U. S. women, urinary iodine excretion below " adequate"

levels was observed in 5.1% of pregnant women, and 8.2% of women of child bearing age ( 15

44 years). People with less than adequate levels of iodine excretion are at increased risk of hypothyroidism. Note that the " adequacy" is reported here in terms of excretion, which is easier to measure than intake. In order to maintain an " adequate" level of iodine excretion, a minimum dietary iodine of 50 to 75  g/ day is needed for members of the general population. To provide an extra margin of safety, the recommended iodine allowance for adults of both sexes is set at 150  g/ day. An extra 25  g/ day intake is recommended for pregnant women, and an extra 50  g/ day is recommended for lactating women, in order to cover the extra demands of the fetus and infant.

Since the extra iodine is provided to the fetus or infant through the placenta or mother milk, only iodine used by the mother will be excreted in the mother's urine. Therefore, urinary iodine excretion is a good indicator of an " adequate" intake, even for pregnant or lactating women.

Age related differences in the amounts of rhodanese and the kinetics of cyanide suggest that the elderly have an increased sensitivity to the acute toxic effects of cyanide. McMahon and Birnbaum ( 1990) evaluated age related differences in cyanide toxicity in male C57Bl/ 6N mice aged 2 3 months, 10 12 months, and 25 30 months. Mice ( 10/ age group) were treated with single doses of 0, 1, 2, 4, or 6 mg/ kg potassium cyanide by gavage and were monitored for clinical signs of toxicity ( prostration, labored breathing, tremors) for 2 hours. The same animals were used for each dose level with a 2 week recovery period between exposures. After treatment with the high dose, the mice were allowed to recover, were sacrificed, and the liver and brain were evaluated for activity of rhodanese, mercaptopyruvate sulfurtransferase ( MPST), and cytochrome oxidase. In a separate experiment, 2 3 month and 10 12 month old mice were treated Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 29 orally with a single dose of 6 mg/ kg potassium cyanide, and groups of 3 4 mice were evaluated for blood, liver, and brain levels of cyanide at time points ranging from 2.5 minutes to 25 minutes after exposure.

At a dose of 6 mg/ kg, the incidence of labored breathing was statistically significantly increased in both the 10 12 month mice and the 25 30 month mice compared with the 2 3 month mice, suggesting that cyanide was more toxic in the older animals. Similar age dependent toxicity was observed in the 4 mg/ kg dose group; lower doses were apparently not toxic. To evaluate the reasons for the age dependent differences, the authors evaluated the levels of rhodanese, the primary enzyme responsible for cyanide metabolism, in the livers and brains of the mice. Liver rhodanese exhibited the highest activity ( both on a protein weight basis and on a tissue weight basis) in the 10 12 month mice. By contrast, brain rhodanese, as measured by activity/ g tissue,

was significantly lower in 10 12 month mice and 25 30 month mice, compared with the 2 3 month mice. In addition, both the peak concentration of cyanide in the brain and the area under the curve ( AUC) were significantly higher, and the time to peak concentration in the brain was shorter in 10 12 month mice compared with the 2 3 month mice. No significant effects of age on cyanide levels in the blood or liver were seen. ( Tissue levels of cyanide were not measured in the aged mice.) The authors also investigated age related changes in the inhibition of cytochrome oxidase ( the primary cellular target of cyanide) in the liver and brain, but found no significant effect. Activity of MPST in the brain and liver was higher in the 10 12 month and 25 20 month mice. Because MPST supplies the sulfur for detoxification of cyanide by rhodanese, one might have expected increased toxicity to be associated with decreased MPST activity. Since increased toxicity was instead associated with increased MPST activity, the authors concluded that the Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 30 older mice were more sensitive to the acute toxic effects of cyanide, and suggested that this resulted from slower reactivation of cytochrome oxidase due to lower rhodanese levels in the brain. Other possible mechanisms suggested by the authors included age related decreases in enzymes involved in antioxidant defense, or alterations in the " sink" of reversible cyanide binding to unknown tissue constituents. The study authors ( McMahon and Birnbaum, 1990) noted that other authors have reported that the experimental manipulation of hepatic rhodanese level does not always result in the expected change in cyanide toxicity. Therefore, the implications of this study are unclear. There is much more liver rhodanese than brain rhodanese, so liver accounts for a much higher percentage of the total body detoxifying capacity, but it is possible that brain rhodanese is important in determining acute neurotoxicity.

People with hypothyroidism may also represent a susceptible population because their thyroid status is already compromised and they have less capacity to adapt to an additional challenge to the thyroid. As described by the American Association of Clinical Endocrinologists ( 2002), hypothyroidism is of primary concern in two populations: neonates and women over 35.

Congenital hypothyroidism in the US occurs in about 1 in 4,000 children. Women are 5 to 8 times more likely than men to develop thyroid disease. Incidence of hypothyroidism increases with age; peak onset of hypothyroidism occurs between ages 35 and 60. One in ten women aged 65 or older has hypothyroidism. In addition, hypothyroidism appears to be linked with diabetes:

hypothyroidism occurs in about 36% of the diabetic population, compared with about 6% of the general population. Finally, pregnancy appears to alter thyroid function resulting in an increased likelihood of developing hypothyroidism. One of 50 women in the U. S. is diagnosed with hypothyroidism during pregnancy. Up to 10% of women are diagnosed with postpartum Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 31 thyroiditis within 1 year following delivery and about 50% of these will develop permanent hypothyroidism within 5 years of diagnosis.

No data were located on polymorphisms in any genes related to the metabolism of cyanide, thiocyanate, or cyanate.

D. Summary Since data on cyanogen chloride toxicity and mechanism of action are very limited, data on the known toxic effects and target organs of cyanogen chloride were compared with those of its known or potential metabolites ( along with the available information on cyanogen chloride biochemistry and toxicokinetics) to provide information on the potential mechanism( s) of action of cyanogen chloride toxicity. The primary target of acute exposure to cyanide is the CNS.

Cyanide acts by binding with cytochrome c oxidase, interfering with cellular oxygen use. It is likely that cyanogen chloride toxicity to the CNS is mediated through cyanide. Chronic exposure to cyanide results in effects on the thyroid and the male reproductive system. The mechanism for the reproductive effects is not known, but the thyroid effects result from chronic exposure to the cyanide metabolite thiocyanate. Thiocyanate acts by blocking the active transport of iodide into the thyroid gland, disrupting the feedback mechanisms that regulate balance of the thyroidpituitary axis. As documented for a number of other chemicals exerting similar effects on the thyroid, this results in decreased production and secretion of the hormones T3 and T4, and increased levels of TRH and TSH. Prolonged increases in TSH can result in increased thyroid weight, follicular cell hyperplasia, and cancer. Decreased T3 and T4 levels, and increased thyroid Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 32 weight, have been observed in toxicity studies with thiocyanate. However, follicular cell hyperplasia and cancer have not been observed following exposure to thiocyanate, although it is unclear whether the relevant studies fully evaluated the thyroid. Decreased T4 levels in pregnant women and in fetuses/ neonates have been demonstrated to result in neurodevelopmental effects in children. Based on the mode of action of thiocyanate, and altered hormone levels in exposed pups, it would also be useful to evaluate the potential neurodevelopmental toxicity of thiocyanate,

but such studies have not been conducted. Based on the observed in vivo metabolism of cyanogen chloride to thiocyanate, the effects seen with thiocyanate may also occur following chronic exposure to cyanogen chloride.

Cyanate is also produced from the metabolism of cyanide, although cyanate has not been identified as a metabolite of cyanogen chloride. The primary toxic effects of cyanate include irritation of the eyes, skin, and respiratory tract ( following inhalation exposure), severe weight loss, cataracts, peripheral neuropathy, and liver toxicity characterized by altered glycogen metabolism. The mechanism of at least two of these, cataracts and liver toxicity, appear to be related to the ability of cyanate to carbamylate protein. Taking advantage of the affinity of cyanate for hemoglobin, cyanate was used in clinical trials as a therapeutic agent for sickle cell anemia. Peripheral neuropathy caused by cyanate may be due to carbamylation of proteins in the myelin membrane or proteins associated with maintaining ion flux in the nerves. It has also been proposed that the effects on weight loss and nervous tissue could be mediated through the increased oxygen affinity of hemoglobin induced by cyanate. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 33 Cyanamide inhibits the enzyme aldehyde dehydrogenase ( ALDH) and has been widely used in Europe as a drug to treat chronic alcoholism. Toxic effects observed in humans and animals following cyanamide exposure include irritation of the eyes, skin, and respiratory system ( following inhalation exposure), severe weight loss, liver toxicity characterized by glycogen deposits, allergic dermatitis, and male reproductive effects, but only the male reproductive effects may be related to the action of cyanamide as an ALDH inhibitor.

All of the toxic effects observed following cyanogen chloride exposure can be accounted for by the known or potential metabolites. It is plausible that the eye and lung irritation observed following exposure to cyanogen chloride vapor is due to the parent chemical. It is also possible that these effects may be due to HCl, cyanate, or cyanamide formed from hydrolysis of cyanogen chloride or reaction with ammonia or other amines, although it appears less likely that these reactions occur sufficiently rapidly in vivo following inhalation exposure. Similarly, the kinetics of formation of these metabolites following ingestion exposure to cyanogen chloride is unknown,

although transient increased concentrations of HCl ( decreased pH) may occur, particularly in the liver and portal vein. It is plausible to expect that ingested cyanogen chloride will be reduced by glutathione to cyanide and HCl as part of first pass metabolism, resulting in elevated HCl levels in the blood that is part of enterohepatic circulation, and in the liver.

Nervous system effects of cyanogen chloride can be attributed to cyanide. If cyanogen chloride were also to cause peripheral nervous system effects, this could suggest a role of cyanate,

although such effects might also be due to cyanide. Severe weight loss and decreased appetite have been seen in workers chronically exposed to cyanogen chloride ( Reed, 1920) and in exposed Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 34 animals ( Haymaker et al., 1952). This rather nonspecific effect is most closely associated with cyanate and cyanamide ( Haut et al., 1975; NCI, 1979), but has also been seen following chronic exposure to cyanide ( Wolfsie and Shaffer, 1958, as cited by ACGIH, 1996).

The carcinogenic potential of cyanogen chloride, cyanide, and thiocyanate cannot be determined from available data. QSTR analysis suggests that cyanogen chloride is not carcinogenic ( Moudgal et al., 2000). Adequate carcinogenicity studies are lacking for most of these chemicals, although the mode of action of thiocyanate on the thyroid suggests that it could cause thyroid cancer in rats via a nongenotoxic mode of action. Although none of these compounds have been completely tested, the available data do not indicate that any of the compounds are genotoxic.

Additive interactions would be expected between cyanogen chloride and its metabolites,

cyanide and thiocyanate. Cyanide also exhibited additive interactions with carbon monoxide, and synergism with ascorbic acid, and there is some evidence for synergism with carbon dioxide. A number of compounds have been identified that inhibit the toxicity of cyanide.

The only information on populations particularly susceptible to cyanogen chloride is based on extrapolation from its metabolites. The limited data do not indicate that children or fetuses are particularly susceptible to the acute effects of cyanide; rather, the limited data suggest that young animals are less susceptible than older animals to the acute effects of cyanide ( McMahon and Birnbaum, 1990). A QSTR analysis ( Moudgal et al., 2000) suggests that cyanogen chloride is a developmental toxicant, but the model does not provide information on the potency of the Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 35 chemical or the severity of effects predicted. Although the limited data indicate that standard developmental effects of cyanide occur at doses higher than those that cause thyroid effects, the potential for neurodevelopmental effects resulting from the thyroid effects of thiocyanate is of concern, and has not been investigated. Congenital hypothyroidism is associated with stunted bodily growth and mental development. In addition, young animals have higher levels of rhodanese, decreasing the acute toxicity of cyanide, but increasing the thiocyanate levels in the body, and thus potentially the susceptibility to thyroid effects. Limited data from a study in which thyroid hormone levels were measured in pups and dams following exposure of pregnant and lactating dams ( Kreutler et al., 1978) suggests that rat pups are more sensitive than dams to the thyroid effects of thiocyanate.

Other potentially susceptible populations include people with a defect in the enzyme systems that convert cyanide to thiocyanate ( rhodanese and mercaptopyruvate sulfurtransferase),

who may be more susceptible to the acute effects of cyanide. Vitamin B 12 deficiency would also increase susceptibility to cyanide, while hypothyroid disorders and protein or iodine deficiency may increase sensitivity to cyanide and its metabolite thiocyanate. Hypothyroidism occurs at a higher incidence among the elderly, diabetics, and pregnant women. The elderly may also be more susceptible to the acute toxicity of cyanide, possibly due to slower reactivation of cytochrome oxidase, due to lower rhodanese levels in the brain; liver rhodanese level was not correlated with acute toxicity.

Other than the age related differences in rhodanese levels, no information was located on age related differences in the expression of any of the genes related to the metabolism of cyanide Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD Final draft VII 36 or thiocyanate. No data were located on polymorphisms in any genes related to the metabolism of cyanide or thiocyanate.