Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 1 Final draft Chapter V. Health Effects in Animals A. Short Term Exposures Cyanogen chloride. Toxicity studies on cyanogen chloride are summarized in Table VIII 1. Very few studies are available on the toxicity of cyanogen chloride, and the existing studies are generally old and incompletely reported. No short term studies of cyanogen chloride by the oral or dermal route were located. The available studies ( Reed, 1920; Haymaker, 1952;

Aldridge and Evans, 1946; Flury and Zernik, 1931) are primarily useful for hazard identification,

rather than dose response assessment, because they were conducted with small numbers of animals, and typically investigated only one exposure level. However, they may provide some useful quantitative comparisons between cyanogen chloride and hydrogen cyanide.

Aldridge and Evans ( 1946) report the effects of acute inhalation exposures of various species to cyanogen chloride. The reported air concentrations ranged from 50 to 120 mg/ m3, but only single exposure levels were tested, so the studies are not useful for exposure response assessment. However, the studies do provide some useful hazard identification information.

Effects of acute, high level exposure to cyanogen chloride included irritation of the respiratory passages, lachrimation, and severe blepharospasm ( spasmodic winking). The authors noted that the initial symptoms of exposure to 100 mg/ m3 decreased after the first minute or two, indicating some degree of adaptation. The authors reported that vomiting, deep respiration, and loss of consciousness occurred at high levels, and convulsions may occur after cessation of exposure.

Coughing and increased ventilation rate were considered reflex responses, and the coughing was Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 2 Final draft eliminated by cutting the vagus nerve. Concentrations of 50 mg/ m3 were reported to be fatal in dogs, yet other dogs appeared to survive exposures to 120 mg/ m3. Death was preceded by lung edema, and respiration failed before circulation did. Necropsy revealed inflammation of upper respiratory tract, lung edema, and dilated right ventricle. Exposure of one dog for 3 hours to 40

50 mg/ m3, followed immediately by exposure to 81 105 mg/ m3 for approximately 2.75 hours,

resulted in the dog being unconscious for several days. After the animal recovered, it was blind,

and had an altered ( less friendly) personality.

Aldridge and Evans ( 1946) exposed anesthetized cats to 3000 mg/ m3 and anesthetized rabbits to 2000 mg/ m3 cyanogen chloride by inhalation for 1 2 minutes. Arterial pressure demonstrated a transitory rise, followed by a decrease. The heart rate ultimately slowed, after a brief period of acceleration. The initial reaction of the animals was violent with rapid coughing,

followed by deep, sometimes rapid breathing, which became reduced in depth and frequency, with intermittent gasping breaths. Circulation continued to function for some period after respiration failed. If the animals were exposed a second time, the amount of coughing was reduced. The authors concluded that this effect was " probably because of damage to sensory surfaces." A second exposure also resulted in a less evident increase in ventilation; the authors concluded that this was due to damage to respiratory center or various chemoreceptors. The general course of events following acute inhalation exposure to cyanogen chloride was identified as stimulation of upper respiratory passages, resulting in coughing. This was followed by stimulation of chemoreceptors, resulting in hyperpnea, followed by paralysis of the respiratory center, with gasping breathing. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 3 Final draft Haymaker et al. ( 1952) examined the neurological effects in dogs of acute exposure to both cyanogen chloride and cyanide following inhalation and intravenous injection. Eleven adult mongrel dogs ( sex not specified) were exposed by inhalation to concentrations of cyanogen chloride ranging from 2 to 3.8 mg/ L ( 2000 to 3800 mg/ m3) for approximately 2 minutes ( one dog was exposed for 49 minutes). Three of the animals were pretreated with amyl nitrate just prior to exposure, and two were given p aminopropiophenone 3 to 4 hours before exposure to cyanogen chloride. In a separate experiment, one dog was administered cyanogen chloride intravenously at a dose of 3 mg/ kg. In order to compare the toxicity of cyanogen chloride and HCN, six adult mongrel dogs were exposed by inhalation to HCN concentrations ranging from 0.2 to 0.7 mg/ L ( 200 to 700 mg/ m3) for approximately 2 minutes. Converted to cyanide equivalents, the dogs were exposed to 850 1600 mg CN/ m3 as cyanogen chloride, and 190 680 mg CN/ m3 as hydrogen cyanide. Five dogs were also administered sodium cyanide intravenously at doses ranging from 1.5 to 2.25 mg/ kg. All dogs either died or were sacrificed. The brains and abdominal and thoracic viscera were evaluated histopathologically. Survival in the animals exposed by inhalation to cyanogen chloride ranged from 5 minutes to 7 days; 5 animals died within 21 minutes of exposure. In contrast, survival in the animals exposed to HCN ranged from 16 to 28 hours.

Clinical signs observed in animals treated with cyanogen chloride include coma, apnea, rigidity and weakness in limbs, and incoordination. One animal receiving cyanogen chloride by inhalation and the dog receiving cyanogen chloride intravenously had convulsive seizures during the period of administration. In contrast, 4 of 6 dogs receiving HCN by inhalation had convulsive seizures. The authors note that " with the exception of severe pulmonary edema in a few of the dogs, the only lesions of significance were in the CNS ( central nervous system)." However, the authors do not indicate whether the edema was observed following exposure to cyanogen Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 4 Final draft chloride, HCN, or both. Also, the authors note that the pathological changes in the CNS were essentially the same following exposure to both cyanogen chloride and HCN. No CNS effects were observed in dogs that died within 3 hours. The CNS effects in the remaining animals consisted of necrosis of the cerebral cortex, the caudate nucleus, putamen, substantia nigra,

globus pallidus, pulvinar of the thalamus, and cerebellar cortex. Although many of the dogs exposed to cyanogen chloride died at earlier post exposure timepoints than the dogs exposed to HCN, the higher exposure level ( in cyanide equivalents) in the former group and the lack of quantitative effect measures precludes a quantitative comparison of toxicity of these two compounds. It is interesting to note, however, that the survival time of the dogs exposed to HCN was rather narrow ( 16 28 hours), while that for the dogs exposed to cyanogen chloride ranged from 5 minutes to 7 days. The latter range could have been due to a higher variability in that group of dogs, but the results suggest that there may be multiple mechanisms by which cyanogen chloride exerts its toxic effects, and that these mechanisms occur at different rates.

Flury and Zernik ( 1931) reported the effects of exposing various species to cyanogen chloride for durations ranging from a few breaths to 6 hours. Most of the exposure levels were fatal. The observed effects were reported to be similar to those of cyanide, except that irritation was also observed, and the lower concentrations only caused irritation. Pathology analysis showed severe irritation of the bronchioles and respiratory tubes, and bloody lung edema.

Reed ( 1920) evaluated the short term toxicity of cyanogen chloride vapor in animals. Five dogs and a goat ( sex and strain not specified) were exposed to sublethal concentrations ( concentration not reported) of vaporized cyanogen for 30 minutes to 2 hours/ day for two weeks. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 5 Final draft No mortality was observed following exposure. The dogs lost an average of 13% of their body weight; the goat lost 17% of its body weight. The symptoms observed during the exposure period included convulsions, irritation, excitement, nausea, vomiting, urination, defecation,

tearing, and salivation. Subsequent symptoms included muscular tremors, weakness, listlessness,

diarrhea, depression of reflexes, cardiac irregularities, conjunctivitis, and rhinitis. Necropsy revealed that all animals developed severe pulmonary congestion. These symptoms were similar to those observed in dogs administered a single sublethal injection of cyanogen chloride,

suggesting that the toxicity of cyanogen chloride is not route dependent. The author compared these results to those he obtained in a similar study of cyanide gas and reported that the symptoms observed following exposure to cyanide gas were less severe than those observed following exposure to cyanogen chloride vapor ( although it is unclear whether comparable exposure levels were tested). The author also suggested that some of the symptoms observed following cyanogen chloride exposure, such as the lung congestion, are due to the presence of " chloride," presumably the chlorine moiety in cyanogen chloride.

Reed ( 1920) also investigated the effects of cyanogen chloride in dogs following exposure by injection. Six dogs ( strain and sex not specified) were injected with a single sublethal dose of cyanogen chloride ( dose not specified) in 0.9% sodium chloride and were observed for 10 to 12 days. The dogs lost up to 15% of their body weight and demonstrated symptoms of listlessness,

diarrhea, cachexia, depressed reflexes, cardiac irregularity, conjunctivitis, and rhinitis. Autopsy revealed no gross lesions. In a second study, eight dogs ( sex and strain not specified) first Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites 1It is now known that sodium thiosulfate acts as a sulfur donor, thus enhancing the conversion of cyanide to thiocyanate by the enzyme rhodanese ( ATSDR, 1997). Therefore, if the toxic effects of cyanogen chloride were due to its metabolism to cyanide, pretreatment with thiosulfate should reduce the toxicity of cyanogen chloride by enhancing conversion of its primary metabolite to a less toxic compound.

EPA/ OW/ OST/ HECD V 6 Final draft received a protective subcutaneous injection of sodium thiosulfate1 and then received a lethal injection of cyanogen chloride ( dose not reported). The dogs receiving the thiosulfate lost up to 14% of their body weight and showed greater general depression and greater depression of reflexes than the dogs in the first study. Necropsy revealed several dogs had varying degrees of pulmonary congestion. Although the dogs demonstrated less cardiac irregularity, the author concluded that, in general, the symptoms in the second study were much more severe than those in the first study, despite the administration of a protective dose of thiosulfate. This apparent inconsistency can be explained by the assumption that the dogs in the second study received a higher dose of cyanogen chloride than the dogs in the first study. In fact, the author described the dose in the second study as lethal, and yet no mortality was reported. This suggests that the sodium thiosulfate was indeed having a protective effect against the acute toxicity of cyanogen chloride, and further suggests that at least some of the acute toxicity of cyanogen chloride results from its metabolism to cyanide.

Cyanide. Oral toxicity studies for cyanide are summarized in Table VIII 2 and inhalation studies are summarized in Table VIII 3. Oral LD 50 values for cyanide in rats range from 3 mg CN/ kg day ( Ballantyne, 1988) to 8 mg CN/ kg day ( Smyth et al., 1969) for cyanide administered as sodium cyanide, and have been reported as 22 mg CN/ kg day for cyanide administered as calcium cyanide ( Smyth et al., 1969). In contrast, single doses of 4 mg CN/ kg day in rats and 6 Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 7 Final draft mg CN/ kg day in mice as potassium cyanide resulted in 95% mortality ( Ferguson, 1962).

ATSDR ( 1997) summarizes the acute dermal studies of cyanide. Dermal LD 50 s in rabbits range from 4.1 to 8.9 mg CN/ kg. Clinical signs observed following single dermal doses ranging from 0.9 to 2.5 mg CN/ kg include rapid breathing, dizziness, weakness, convulsions and loss of consciousness. ATSDR ( 1997) also summarizes the acute inhalation studies of cyanide. LC 50 s in animals range from 154 to 543 mg CN/ m3.

In evaluating oral toxicity of cyanide, both the total amount administered and the rate of absorption are important. This is because toxicity results from exceeding the body's capacity for detoxification of cyanide ( which occurs primarily in the liver). Like any other enzymaticallycatalyzed reaction, this detoxification reaches a maximum rate in the presence of excess substrate.

If absorption of ingested cyanide proceeds too quickly, the capacity of the liver to form thiocyanate upon first pass of mesenteric blood through the organ may be exceeded. In contrast,

slow absorption of the same total oral load of cyanide may allow complete metabolism by the liver. For the most part, cyanide is readily absorbed from the GI tract, especially since at physiological pH ( and in acidic solution) it is present mostly in the highly diffusible nonionized form. However, the rate of absorption may be influenced by factors such as the composition and volume of the intestinal contents and by the rate of peristalsis. This influence of the rate of absorption is why the LD 50 values for sodium cyanide presented above are lower than the acute and chronic LOAELs.

Palmer and Olson ( 1979) administered potassium cyanide to groups of 7 male Sprague

Dawley rats at 0 or 200 ppm in drinking water, or at 0 or 200 ppm in feed for 21 days. These Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 8 Final draft doses correspond to approximately 14 mg CN/ kg day in drinking water and 8 mg CN/ kg day in feed. ( Dose conversions were conducted using an average body weight of 0.12 kg, water consumption of 0.17 L/ day, and food consumption of 0.10 kg/ day, calculated using the methods of U. S. EPA, 1988). The only endpoints evaluated were body weight gain and liver weight. No effects were seen in the rats administered cyanide in feed, but a statistically significant 17%

increase in absolute liver weight was observed in the group administered cyanide in drinking water. The study authors noted that the analysis of the feed resulted in < 20% of the theoretical value for cyanide, compared to 95% recovery for cyanide added to the feed immediately before analysis. The study authors suggested that cyanide added to the diet was lost or fixated ( and therefore unavailable), suggesting that the NOAEL for cyanide in the diet in this study was < 20%

of 8 mg CN/ kg day, or < 1.6 mg CN/ kg day; the actual administered dose in uncertain. Some of the toxicity difference may have also been due to the difference in doses, or to differences in absorption rate from food and water. Benchmark dose ( BMD) modeling could not be conducted for this study, because no measure of variability was provided.

Kreutler et al. ( 1978) evaluated the short term effects on the thyroid of oral exposure to cyanide. Male albino rats ( strain not specified; 10 24 animals/ group) were fed diets containing either 2% or 20% casein; treated rats received the same diets supplemented with 0.2% potassium cyanide for two weeks ( equivalent to 87 mg CN/ kg day, using an average body weight of 85 g and the allometric equation of U. S. EPA [ 1988], and adjusting by the molecular weight ratio of cyanide to potassium cyanide). In addition, one group receiving 2% casein diet with or without potassium cyanide, and with or without iodide supplementation. Body weights and food consumption were recorded. Blood was collected and evaluated for plasma thyroid stimulating Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 9 Final draft hormone ( TSH) levels. Thyroids were removed and weighed. No difference in body weight was observed between cyanide treated rats and their respective controls. Rats treated with cyanide on the 2% casein diet had significantly elevated plasma TSH levels and increased thyroid weights compared to the 2% casein controls; supplementation with iodide in addition to cyanide eliminated these effects. There was no effect on TSH or thyroid weight in the cyanide treated rats on the 20% casein diet. This study suggests that people with protein and iodine deficient diets may be a sensitive population. No consistent guidance is available on the interpretation of thyroid effects and the determination of which thyroid effects constitute an adverse effect rather than an adaptive effect. A recent peer review panel evaluating this issue ( Research Triangle Institute,

1999) concluded that hormone changes, increased thyroid weight, and thyroid hypertrophy were all considered to be adaptive effects, although these effects are necessary precursors to adverse effects. Thyroid hyperplasia was considered to be an adverse effect in the thyroid. Based on this analysis, the increased TSH levels and thyroid weight observed in Kreutler et al ( 1978) could be considered adaptive effects; although they are precursors to adverse effects. However, because thyroid histopathology was not conducted, it is not known whether these effects were accompanied by thyroid hyperplasia. Therefore, the dose level tested in this study is considered to be a minimal LOAEL. The BMDL ( 95% lower confidence limit on the benchmark dose) for this study was 2.1 mg/ kg day, based on increased plasma TSH, but confidence in this value is limited,

because only one nonzero dose was tested and significant extrapolation below the data was required to reach the BMDL. Details of the modeling are provided in Appendix A, and the modeling output is provided in Appendix B. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites 2BMD modeling was not conducted for any of the thiocyanate studies, since thiocyanate was not chosen as the surrogate for calculation of effect levels for cyanogen chloride.

EPA/ OW/ OST/ HECD V 10 Final draft Purser et al. ( 1984) exposed Cynomolgus monkeys individually to 100, 102, 123, 147, or 156 ppm HCN for up to 30 minutes. These concentrations correspond to 106, 108, 131, 156, and 166 mg CN/ m3. A single monkey was exposed per concentration, with one monkey exposed to 100 ppm and 147 ppm in separate experiments. There was no control. The time to incapacitation decreased with increasing exposure levels, and ranged from 8 minutes to 19 minutes. The authors noted that three of the exposures ( exposure levels not reported) were terminated prior to 30 minutes due to the severity of the symptoms. The observed symptoms included hyperventilation,

decreased and arrhythmic heart rate, loss of muscle tone and reflexes, and convulsions. Blood cyanide levels reached steady state within 10 minutes. There was no correlation between air concentration and blood cyanide levels.

Bhattacharya et al. ( 1994) investigated the effects of the inhalation of 55 ppm HCN ( 58 mg CN/ m3) for 30 minutes on the pulmonary mechanics of six male Wistar rats. The air flow was increased, accompanied by increased transthoracic pressure and tidal volume. The respiratory rate, compliance, and minute volume decreased, accompanied by a decrease in pulmonary lipids.

Other effects of cyanide were not evaluated.

Thiocyanate. Toxicity studies for thiocyanate are summarized in VIII 4. No short term inhalation or dermal toxicity studies of thiocyanate in animals were identified. 2 Most of the acute toxicity studies with thiocyanate were conducted prior to the advent of modern toxicologicaltesting methods, and concentrated on identifying lethal doses. For example, Gorman et al. ( 1949) Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 11 Final draft reported that the minimal lethal dose of sodium thiocyanate in guinea pigs is 200 400 mg/ kg ( 143

286 mg SCN/ kg). Anderson and Chen ( 1940) reviewed the literature on acute toxicity of thiocyanate in animals. They found that 600 800 mg/ kg of potassium, sodium, or ammonium thiocyanate was lethal to guinea pigs ( dose conversion not possible because form not specified).

They also reported that 1000 mg/ kg potassium thiocyanate was always fatal to rabbits, and a single dose of 500 mg/ kg was " never fatal." These doses correspond to 600 and 300 mg SCN/ kg,

respectively.

Taubman and Heilborn ( 1930; as cited in Lindberg et al., 1941; study in German) reported that administration of 2 doses of 200 mg/ kg potassium thiocyanate ( 120 mg SCN/ kg) to 15 guinea pigs ( sex and strain not specified) resulted in a 25% decrease in hemoglobin and erythrocytes. Information was not available as to whether the doses were administered on the same day. Smaller doses given over longer periods resulted in greater decreases in these parameters ( dosing regimen not specified). No NOAEL was determined in this study. Given the information provided in the study, the LOAEL was 120 mg SCN/ kg, but the likely target organ ( thyroid) was apparently not evaluated.

Wolff et al. ( 1946) exposed rats ( 4 6/ group; sex and strain not specified) to 0.5%

potassium thiocyanate in the diet for 18 or 24 days. Assuming a food factor of 0.096 ( based on the average across strains and sexes, U. S. EPA, 1988), this corresponds to a dose of approximately 287 mg SCN/ kg day. Controls received a basal diet. Only body weight and thyroid parameters were evaluated. There was no clear effect on body weight, although this was difficult to evaluate, since several of the control and exposed animals lost weight. Treated rats Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 12 Final draft had increased thyroid weights and decreased thyroxine iodine and total iodine in the thyroid.

Protein bound iodine in the plasma ( a measure of triiodothyronine [ T3] and thyroxine [ T4]) was also decreased in the treated rats. No NOAEL was determined, and the LOAEL was 287 mg SCN/ kg day, based on the thyroid effects.

Rawson et al. ( 1944) exposed male Sherman rats ( 25/ group) to 0.25% potassium thiocyanate in drinking water for 28 days. Controls received drinking water without added potassium thiocyanate. Using the average drinking water consumption across male rats of 0.145 L/ kg day ( calculated from U. S. EPA, 1988), this corresponds to doses of 0 and 217 mg SCN/

kgday The authors noted that the intake of iodine was borderline deficient, based on the amount in the diet, together with iodine supplementation of the drinking water. Thyroid weight was increased in the potassium thiocyanate treated rats. At necropsy, the thyroids of treated rats were dark red, highly vascularized, and grossly enlarged; histopathology revealed marked hyperplasia and loss of colloid. Uptake of radiolabeled iodine by the thyroid was increased. No NOAEL was determined in this study, and the LOAEL was 217 mg SCN/ kg day, based on thyroid toxicity.

De Groot et al. ( 1991) exposed male F344 rats ( 10/ group) to 0 or 0.2% potassium thiocyanate in the diet for 4 weeks. Based on the average of the initial and final body weights ( 124 g) and the reported food intake of 12.8 g/ rat/ day, the daily dose was 123 mg SCN/ kg day.

There was no effect on relative liver or kidney weight. A statistically significant decrease in hemoglobin and increase in prothrombin time was observed. However, the most marked effects were on the thyroid, with marked and statistically significant decreases in T4 and increased TSH and thyroid weight. ( T3 levels were not measured.) A blind evaluation of the thyroid also found Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 13 Final draft moderate activation of the thyroid follicles ( 20 50% of follicles activated). The LOAEL in this study was 123 mg SCN/ kg day.

Summary of Short Term Exposures Only four old short term studies of cyanogen chloride were located ( Reed, 1920; Aldridge and Evans, 1946; Haymaker et al., 1952; Flury and Zernik, 1931). Rats, cats, dogs, and a goat were exposed by either inhalation or injection. These studies are unsuitable for risk assessment because key information about doses and study design are not reported. However, the studies do help to identify the potential health effects of cyanogen chloride, which include neurotoxicity ( depressed reflexes, muscular tremors, rigidity and weakness of limbs, convulsions, necrosis in brain), weight loss, cardiac irregularities, and pulmonary congestion and edema. Cyanogen chloride also irritates the respiratory passages and eyes.

Only limited information regarding the short term effects of cyanide in animals is available.

LD 50 s ( either oral or dermal) for cyanide range from about 3 to 9 mg/ kg; inhalation LC 50 s range from 154 to 543 mg/ m3. Inhalation exposure of rats or monkeys for 30 minutes resulted in signs of neurotoxicity ( loss of muscle tone and reflexes, convulsions), decreased and arrhythmic heart rate, and alterations in respiration ( increased air flow, decreased respiratory rate, compliance, and minute volume) ( Purser et al., 1984; Bhattacharya et al., 1994). Two week oral exposure of rats to 87 mg CN/ kg day resulted in thyroid toxicity ( decreased TSH and increased thyroid weights)

in animals that were protein and iodine deficient ( Kreutler et al., 1978). No effects were observed in animals with an adequate protein diet. Supplementation of protein deficient rats with Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 14 Final draft iodine also eliminated the effects of cyanide on thyroid. Protein deficiency is rare in Western populations ( U. S. FDA, 1999), but iodine deficiency occurs in approximately 5 8% of the U. S.

population ( Hollowell et al., 1998), as described further in Chapter 7.

Although there is a relatively large number of animal studies on the effects of thiocyanate in animals, most of the studies concentrated on the key target organ, the thyroid, rather than evaluating a range of endpoints. More importantly, many of the studies tested only a single dose,

so a dose response could not be evaluated, and often a NOAEL could not be identified. The animal studies consistently reported decreased plasma T4 levels and increased thyroid weight;

effects on T3 were inconsistent. Other studies reported decreased mammary development at similar doses ( possibly secondary to thyroid effects), and decreased hemoglobin. Short term animal toxicity data are available from rats ( Wolff et al., 1946; Rawson et al., 1944; De Groot et al., 1991) and guinea pigs ( Taubman and Heilborn, 1930). No NOAEL was identified in any of the studies. The lowest reliable LOAEL was 123 mg SCN/ kg day, based on decreased T4,

increased thyroid weight, and activation of thyroid follicles ( De Groot et al., 1991). Decreased hemoglobin was also observed in guinea pigs following 2 doses of 120 mg SCN/ kg/ dose ( Taubman and Heilborn, 1930).

B. Long Term Exposures Cyanogen chloride. No long term toxicity studies of cyanogen chloride via any route were located. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 15 Final draft Cyanide. No long term toxicity studies of cyanide by the inhalation or dermal routes were located. NTP ( 1993) reported the results of a subchronic bioassay of sodium cyanide administered in drinking water to rats and mice. F344 rats and B6C3F1 mice ( 10/ sex/ group) were administered sodium cyanide in drinking water at concentrations of 0, 3, 10, 30, 100, or 300 ppm for 13 weeks. These concentrations are equivalent to the following doses, estimated by the study authors ( and converted to CN equivalents) based on measured body weights and water consumption: male rats  0, 0.16, 0.48, 1.4, 4.5, and 12.5 mg CN/ kg day; female rats  0, 0.16,

0.53, 1.7, 4.9, and 12.5 mg CN/ kg day; male mice  0, 0.26, 0.96, 2.7, 8.6, and 24.4 mg CN/

kgday female mice  0, 0.32, 1.1, 3.3, 10.1, and 28.8 mg CN/ kg day. The parameters evaluated included body weight, clinical signs, water consumption, clinical chemistry, hematology,

urinalysis, selected organ weights ( heart, kidney, liver, lungs, testis, and thymus), histopathology,

sperm motility, and vaginal cytology. NTP did not measure thyroid hormones.

In rats, there were no treatment related effects on mortality, body weight, or clinical signs in either males or females. There was a dose related decrease in water consumption that was greater than 10% in both sexes receiving 100 or 300 ppm, compared with controls. Decreased urine volume and increased urine specific gravity were observed in the high dose male rats ( these endpoints were apparently not measured in females) but were attributed to the decrease in water consumption rather than a specific adverse effect of cyanide. Urinary thiocyanate concentration increased in all animals at concentrations of 30 ppm and higher. There was no effect on organ weight in males, but there was a small ( 16% for absolute weight), statistically significant increase in absolute and relative liver weight in high dose females. The study authors did not consider any effects on organ weights to be chemical related. There were no histopathological changes that Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 16 Final draft were attributed to cyanide exposure. In particular, no effects were observed in either the thyroid or the brain. In male rats, there were significant decreases in left cauda epididymis weight and sperm motility at concentrations

30 ppm ( 1.4 mg CN/ kg day) ( Table V 1). However, the authors considered that the changes in sperm motility were not biologically Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 17 Final draft Table V 1. Reproductive Effects in Rats and Mice Administered Sodium Cyanide in Drinking Water for 13 Weeks.

Study Parameter 0 ppm 30 ppm 100 ppm 300 ppm Male Rats Dose ( mg CN/ kg day) 0 1.4 4.5 12.5 Body weight ( g) 338 ± 5a 335 ± 5 338 ± 4 319 ± 5*

Left epididymis ( g) 0.448 ± 0.006 0.437 ± 0.005 0.425 ± 0.007 0.417 ± 0.005**

Left cauda epididymis ( g) 0.162 ± 0.003 0.150 ± 0.004* 0.148 ± 0.004* 0.141 ± 0.003**

Left testis ( g) 1.58 ± 0.03 1.56 ± 0.02 1.52 ± 0.02 1.46 ± 0.02**

Spermatid heads ( 107/ testis) 17.86 ± 0.61 16.94 ± 0.81 16.58 ± 0.63 15.42 ± 0.44*

Spermatid count ( mean/ 10 4 mL suspension) 89.28 ± 3.05 84.68 ± 4.03 82.90 ± 3.16 77.10 ± 2.20*

Motility (%) 94.24 ± 0.58 90.67 ± 1.25* 92.09 ± 0.85* 90.66 ± 1.46*

Male Mice Dose ( mg CN/ kg day) 0 2.7 8.6 24.3 Body weight ( g) 37 ± 1.0 39.2 ± 1.3 38.6 ± 1.1 35.5 ± 1.1 Left epididymis ( g) 0.049 ± 0.001 0.047 ± 0.002 0.047 ± 0.001 0.044 ± 0.001*

Left cauda epididymis ( g) 0.017 ± 0.001 0.016 ± 0.000 0.015 ± 0.001 0.014 ± 0.001*

Left testis ( g) 0.121 ± 0.002 0.113 ± 0.008 0.117 ± 0.002 0.118 ± 0.003 Spermatid heads ( 107/ testis) 2.24 ± 0.14 2.26 ± 0.14 2.03 ± 0.15 2.11 ± 0.16 Spermatid count ( mean/ 10 4 mL suspension) 69.94 ± 4.34 70.80 ± 4.25 63.28 ± 4.53 66.06 ± 4.87 Motility (%) 92.38 ± 0.81 90.63 ± 1.34 91.43 ± 0.55 89.52 ± 0.96 Adapted from NTP ( 1993) a. Mean ± S. E. * Significantly different from control p

0.05 ** Significantly different from control p

0.01 Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 18 Final draft significant, because they were small and within the range of historical controls. Dose related decreases in the left epididymis weight, left testis weight, number of spermatid heads ( per testis),

and spermatid count ( per mL suspension) were observed, with the decreases at the high dose ( 12.5 mg CN/ kg day) statistically significant. The study authors considered the effect at the high dose to be consistent with a small but measurable adverse effect on male reproduction, although they suggested that the observed effects alone are insufficient to decrease fertility in rats. Female rats in the 100 and 300 ppm groups ( 4.9 and 12.5 mg CN/ kg day) spent significantly more time in proestrus and diestrus compared with controls, but there was no clear dose response and the authors did not consider these results to be chemical related. Although 1.4 mg CN/ kg day might be considered a minimal LOAEL based on a statistically significant decrease in left caudal epididymis weight, the dose response at this dose and the next higher dose ( 4.5 mg/ kg day) was very shallow and the magnitude of the decrease was only 6.5% and 9%, respectively. Based on the minimal degree of effect at the dose of 4.5 mg/ kg day, the high dose of 12.5 mg CN/ kg day is the LOAEL in this study, based on male reproductive effects in several related endpoints ( decreased epididymal weight, decreased sperm levels, decreased testis weight), and the NOAEL is 4.5 mg CN/ kg day. Benchmark dose modeling was conducted for several endpoints in this study, as described in Appendix A. The most sensitive BMDL was 0.79 mg CN/ kg day,

calculated based on decreased left epididymis weight, supported by a BMDL of 1.3 mg/ kg day for both decreased spermatid heads/ testis and decreased spermatid count.

In mice, there were no significant treatment related effects on mortality, body weight, or clinical signs. Water consumption in both males and females was decreased in the 100 and 300

ppm groups. No treatment related effects were observed in clinical chemistry, hematology, Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 19 Final draft urinalysis, organ weights, or histopathology. In males, the weights of the left epididymis and left cauda epididymis were significantly decreased in the high dose group ( 24.4 mg CN/ kg day).

However, there were no effects on sperm motility or spermatid density in males, or on estrous cycle length in females. Absolute and relative liver weights were statistically significantly increased by 18% ( absolute weight) in the high dose group, and there was an indication of a doseresponse The study authors considered the increased organ weights sporadic and not chemicalrelated Based on male reproductive effects ( decreased epididymal weight), this study identified a NOAEL of 8.6 mg CN/ kg day and a LOAEL of 24.4 mg CN/ kg day in mice. Benchmark dose modeling was conducted for several endpoints in this study, as described in Appendix A. The most sensitive BMDL was 12 mg CN/ kg day, calculated based on decreased left caudal epididymis weight.

Kamalu ( 1993) evaluated the short term toxicity of inorganic cyanide administered in diet to dogs. Six male dogs per group ( strain not specified) were administered sodium cyanide in diet for 14 weeks at doses of 0 or 1.04 mg CN/ kg day ( based on a reported food consumption of 0.1 kg food/ kg body weight). Blood was obtained from each dog at weeks 1, 3, and 14; urine was collected at weeks 1, 3, 5, 7, and 14. Plasma and urinary thiocyanate concentrations were determined. In addition, serum enzymes, total serum protein, serum albumin, serum globulin, and urinary protein were measured. A histopathological evaluation was performed on the liver,

kidney, myocardium, testis and adrenal gland of each dog. Both plasma and urinary thiocyanate concentrations were significantly increased in the treated dogs compared with controls at all time points evaluated. In addition, treated dogs had significantly increased urinary protein concentrations compared with controls at weeks 5 and 14. No treatment related effects were Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 20 Final draft observed in serum enzymes, total serum protein, albumin, or globulin. No histopathological changes were observed in the liver or myocardium of treated dogs; however, treatment related effects were observed in kidney, testis, and adrenal gland. Kidneys of the treated dogs demonstrated casts in the lumina of the tubules and occasional desquamation. In testis, the treated dogs had a significantly decreased percentage of tubules in stage 8 of the spermatogenic cycle ( characterized by elongated spermatids lining the seminiferous tubule lumens) compared with controls. In addition, treated dogs had an increased incidence compared to the controls of animals with abnormal cells and sloughing of germ cells in the seminiferous tubules. Finally, the zona glomerulosa of the adrenal gland was significantly wider in treated dogs compared with controls. This study indicates that cyanide may be a reproductive toxicant in male dogs. Based on histopathological changes in kidney, testis, and adrenal gland, the dose of 1.04 mg CN/ kg day is considered to be a LOAEL. However, this LOAEL is not relevant for the assessment of effects in humans. Dogs are not a suitable model for the toxicity of cyanide in humans, because levels of rhodanese ( the enzyme which detoxifies cyanide) in dogs are much lower than the levels in humans ( ATSDR, 1997). Therefore, dogs are more sensitive than humans to the effects of cyanide. Similarly, BMD modeling was not done on this study, since dogs are not an appropriate model for cyanide risk assessment.

Jackson ( 1988) evaluated the effects of oral administration of potassium cyanide on thyroid function and behavior in miniature pigs. Doses of 0, 0.4, 0.7, or 1.2 mg CN/ kg day potassium cyanide were administered to 3 pigs/ group using a plastic syringe placed at the back of the animals' throats. The solutions were administered once daily for 24 weeks, just before the daily meal to increase the absorption of cyanide. A total of five females and seven males were Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 21 Final draft used; each group contained both male and female animals. Serum levels of T3, T4, glucose, and thiocyanate were measured every six weeks. Behavioral evaluations were conducted daily. The scope of behaviors evaluated fell into two major categories: performance measures included innate behavior, and learning measures included the acquisition and retention of new behaviors.

No other endpoints were evaluated. Both T3 and T4 demonstrated a dose related decrease that was statistically significant by week 18 of the study. Serum thiocyanate levels were positively correlated with cyanide dose, demonstrating that the cyanide was being metabolized by the animals. A variety of behaviors were significantly altered in treated animals, including a decrease in dominance behavior ( high dose group), decrease in fighting ( mid and high dose group),

increase in victimization ( all treated groups), decrease in exploratory behaviors ( all groups), and less aggressive feeding patterns ( high dose group). The authors concluded that the overall pattern of behavioral changes in the 1.2 mg CN/ kg day group was different from the untreated animals, but that the changes at the lower doses were too inconsistent to be considered adverse.

This study is limited by the small number of animals ( only 3/ group). Because an uneven number of males and females were used, at least one group would have had to include one male and two females, while the remaining groups would have had two males and one female. The small size and mixed sexes of the groups would contribute to high variability in the results, particularly because the endpoints evaluated, hormone levels and behavior, tend to differ between sexes.

Also, no information is provided on the biological significance of the altered hormone levels.

Although the hormones showed statistically significant differences between groups, they could still be within the range of normal for this species. In addition, the study did not evaluate thyroid histopathology to determine if cyanide's effects were adverse or adaptive. Although this study is incomplete for risk assessment purposes, it does confirm that the thyroid and nervous system are Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 22 Final draft target organs for cyanide. Based on behavioral changes and decreased thyroid hormones, the 1.2 mg CN/ kg day dose level is considered to be a LOAEL and the 0.7 mg CN/ kg day dose is considered to be a NOAEL. BMD modeling was not done on this study, due to the uncertainties regarding the data reporting, particularly reporting of averages across males and females in group despite differences in response by sex and differences in the numbers of males/ females per group.

Philbrick et al. ( 1979) evaluated the long term health effects of oral exposure to cyanide in rats. Male rats ( 10/ group, strain not specified) received diets ( 10% casein supplemented with 0.3% methionine, potassium iodide and vitamin B 12) containing either 0 or 1500 ppm potassium cyanide for 11.5 months. Parallel studies were conducted with rats provided a diet deficient in methionine, iodine, and vitamin B 12, containing 0 or 1500 ppm potassium cyanide. Based on the average food factor across rat strains of 0.073 kg/ kg body weight/ day for a chronic study ( U. S.

EPA, 1988) and adjusting for the molecular weight ratio of cyanide to potassium cyanide, the rats received a dose of 0 or 44 mg CN/ kg day. At 4 and 11 months, plasma T4 levels, T4 secretion rates, and urinary thiocyanate levels were measured in 5 animals/ group. After sacrifice, brain,

heart, liver, and thyroid weights were recorded. Histopathologic evaluation was conducted on the brains, optic and sciatic nerves, spinal cords, and thyroid glands. Body weight gains of the treated animals were significantly lower than controls beginning at week 8. Administration of cyanide altered thyroid function at both 4 and 11 months. Compared with control animals, rats in the cyanide exposure groups had significantly decreased plasma T4 levels, decreased T4 secretion rates, and increased thyroid weights at 4 months. At 11.5 months, thyroid weight was increased and T4 secretion rate was decreased. Similar, but more severe effects were observed in the rats provided the deficient diet. No histopathologic effects were observed in optic or sciatic nerve, or Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 23 Final draft thyroid. However, vacuolation and demyelination was observed in the spinal cord white matter of treated animals compared to controls. As discussed above for Kreutler et al. ( 1978), altered thyroid hormones and increased thyroid weight without accompanying thyroid hyperplasia could be considered adaptive rather than adverse. Based on neurotoxicity, the dose of 44 mg CN/

kgday is considered to be a LOAEL. BMD modeling could not be done on the neurological effects in this study, because no quantitative results were available. A BMDL of 19 mg CN/ kg day was calculated for decreased thyroxine ( T4) secretion in the rats with normal diets, and a BMDL of 12 mg CN/ kg day was calculated for the same endpoint in rats fed the deficient diets. However, this endpoint may be adaptive, rather than adverse.

Howard and Hanzal ( 1955) conducted a 2 year dietary study in which 10 rats/ sex/ group were administered food fumigated with hydrogen cyanide ( HCN). The average daily concentrations were 73 and 183 mg CN/ kg diet. From the data reported on food consumption and body weight, daily estimated doses were 4.3 mg and 10.8 mg CN/ kg body weight. The average concentrations of cyanide in the food were estimated based on the authors' data for concentrations at the beginning and end of each food preparation period and by assuming a firstorder rate of loss for the intervening period. There were no treatment related effects on growth rate, no gross signs of toxicity, and no histopathologic lesions. BMD modeling could not be done for this study, since no adverse effects were observed.

Thiocyanate. Several studies in animals examined the effects of subchronic oral exposure to thiocyanate ( Kanno et al., 1990; Lindberg et al., 1941; Nagasawa et al., 1980; Philbrick et al.,

1979; Pyska, 1977). Most of these studies have concentrated on further elucidating the known Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 24 Final draft thyroid effects of thiocyanate. BMD modeling was not conducted for any of the thiocyanate studies, since thiocyanate was not chosen as the surrogate for calculation of effect levels for cyanogen chloride.

Kanno et al. ( 1990) exposed male Fischer 344/ DuCrj rats ( 28 30/ group) to 0.5%

potassium thiocyanate in the drinking water for 25 weeks. Assuming a reference body weight of 0.18 kg and water consumption of 0.028 L/ day ( U. S. EPA, 1988), and adjusting for the molecular weight of thiocyanate and potassium thiocyanate, the estimated dose is 465 mg SCN/ kg day.

Controls received drinking water without added potassium thiocyanate. Only thyroid related parameters were evaluated. Treated rats showed a significant increase in thyroid weight.

Histopathologic analysis of the thyroids showed slight diffuse hyperplasia and increased colloid,

so that the percent area occupied by the follicular epithelial cells relative to total thyroid area was decreased relative to controls. Treated rats had significantly increased serum levels of thyroid stimulating hormone and T3, and decreased serum levels of T4. No neoplasias were observed in the thyroids of control or treated rats. A NOAEL was not determined in this study. The LOAEL for this study was 465 mg SCN/ kg day, based on thyroid toxicity.

Nagasawa et al. ( 1980) exposed female SHN mice ( 18/ group) to 0.1% or 0.3% potassium thiocyanate in the drinking water for 12 weeks. Controls received tap water. Using the average water consumption across strains of 0.2718 L/ kg day ( calculated from U. S. EPA, 1988), this corresponds to doses of 0, 163, and 488 mg SCN/ kg day. Plasma T4 was statistically significantly decreased at both doses of potassium thiocyanate, and T3 was decreased at the high Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 25 Final draft dose. Other systemic toxicity endpoints were not evaluated. A NOAEL was not determined in this study. The LOAEL was 163 mg SCN/ kg day, based on thyroid toxicity.

Pyska ( 1977) exposed 3 week old female Wistar rats ( 17   19/ group) to 0 or 0.1%

potassium thiocyanate in drinking water for approximately 10 weeks ( until the rats were 3 months old). Using the allometric equation of U. S. EPA ( 1988) and a body weight of 0.14 kg, a food factor of 0.167 can be calculated, resulting in an estimated dose of 100 mg/ kg day. However, the actual dose may have been lower, in light of the lower drinking water consumption in the accompanying developmental toxicity study ( see Section V. C). Higher doses were reported to cause mortality, but no supporting data were provided. There was a statistically significant decrease in final body weight of the dosed group compared to the controls, although the magnitude of the decrease ( only 8%) did not reach biological significance. There was also a statistically significant decrease in plasma protein bound iodine ( a measure of T3 and T4). The only dose tested, 100 mg SCN/ kg day, was a LOAEL, based on thyroid toxicity.

Philbrick et al. ( 1979) fed male weanling rats ( 10/ group; strain not specified) for 11.5 months with control casein diets ( supplemented with 0.3% DL methionine, potassium iodide, and vitamin B 12) or with similarly supplemented casein diets containing 2240 ppm potassium thiocyanate. Assuming a food factor of 0.073 ( average across strains, U. S. EPA, 1988), this corresponds to a dose of 98 mg SCN/ kg day. No deaths, clinical signs, or adverse effects on body weight gain were observed in the potassium thiocyanate treated rats. However, the rats treated with potassium thiocyanate had significantly decreased T4 secretion rates and plasma T4 levels after 4 months; after 11 months of exposure, thyroid weight was significantly increased and Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 26 Final draft plasma T4 was significantly decreased, but T4 secretion rate was unaffected. Histopathological analysis showed no effects on the thyroid, optic nerve, sciatic nerve, or other neural tissues. The effects on the thyroid were seen with or without potassium iodide supplementation. In addition,

modest myelin degeneration ( compared to the corresponding unsupplemented control) was observed in the group receiving thiocyanate but no iodide supplement. No NOAEL was determined in this study. The LOAEL was 98 mg SCN/ kg day, based on thyroid toxicity.

Lindberg et al. ( 1941) gave 12 dogs ( breed and sex not specified) potassium thiocyanate orally ( 325 mg/ dose, corresponding to 194 mg SCN/ dose) and examined effects on erythrocyte count, hematocrit, serum proteins, and plasma cholesterol. Neither the frequency nor the duration of dosing was reported, although the authors reported that the dosing was for a " long period."

Thiocyanate levels in blood and hematology were monitored for 4 27 weeks, but it is unclear whether dosing continued for that long. Thyroid related endpoints were not evaluated. Blood thiocyanate levels ranged from 13 mg/ 100 mL to 75 mg/ 100 mL. Decreases in serum cholesterol,

total protein, erythrocyte count, and hematocrit were observed. Histopathological analyses showed bone marrow acellularity and fatty vacuolation of the liver. Insufficient information was provided to determine a NOAEL or LOAEL for these effects.

Summary of Long Term Exposures No long term toxicity studies of cyanogen chloride are available. Subchronic toxicity data for cyanide by oral exposure are available in rats ( NTP, 1993; Philbrick et al., 1979), mice ( NTP, 1993), dogs ( Kamalu, 1993), and miniature pigs ( Jackson, 1988). The only chronic study Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 27 Final draft ( Howard and Hanzal, 1955) of cyanide identified a free standing NOAEL of 10.8 mg CN/ kg day.

No data on the effects of subchronic or chronic exposure to cyanide by the inhalation or dermal routes were located. The older studies of cyanide ( Philbrick et al., 1979; Jackson, 1988)

established that the CNS and thyroid are targets of long term cyanide toxicity. Philbrick et al.

( 1979) observed demyelination of the spinal cord, decreased plasma T4 levels, and increased thyroid weights in rats ingesting cyanide ( 44 mg CN/ kg day) in the diet for 11.5 months. Jackson ( 1988) observed behavioral changes and decreased levels of T3 and T4 in pigs that received cyanide in water for 24 weeks, with a NOAEL of 0.7 mg CN/ kg day and a LOAEL of 1.2 mg CN/ kg day. However, this study is limited by the small group size, the lack of information on the biological significance of the hormone changes, and the lack of thyroid histopathology data that would confirm that cyanide is having an adverse, rather than adaptive, effect on the thyroid at these doses. Kamalu ( 1993) observed histopathological changes in kidney, adrenal, and testis of dogs that received cyanide ( 1.04 mg CN/ kg day) in the diet for 14 weeks. In particular, this study suggests that cyanide may be a male reproductive toxicant, based on the presence of abnormal cells and sloughing of germ cells in the seminiferous tubules and a decreased percentage of tubules in stage 8 of the spermatogenic cycle. However, Kamalu ( 1993) did not conduct histopathology on the CNS or thyroid tissue and did not measure thyroid hormones. Therefore, it is not possible to rule out the CNS and thyroid as target organs based on this study, or to evaluate the relative sensitivities of the thyroid, CNS, and male reproductive tract. In addition, dogs have low levels of rhodanese, so they are not good models for human toxicity ( ATSDR, 1997). Male reproductive effects, characterized by decreased epididymis and testis weights, were also observed in rats and mice exposed to cyanide in drinking water for 13 weeks ( NTP, 1993). The NOAEL was 4.5 mg CN/ kg day in rats, and 8.6 mg CN/ kg day in mice. This study did not Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 28 Final draft observe histopathological changes in brain or thyroid tissue, but did not measure plasma levels of thyroid hormones or measure thyroid weight. Although evidence of thyroid hyperplasia would be needed to determine that a given dose of cyanide is adverse to the thyroid, it is possible that this study did not detect early signs of adaptive effects in the thyroid.

Long term toxicity data for thiocyanate are available from mice ( Nagasawa et al., 1980)

and rats ( Pyska, 1977; Philbrick et al., 1979; Kanno et al., 1990) exposed to thiocyanate via the diet and drinking water. No data on the effects of inhalation or dermal exposure were located.

As for the short term studies, no subchronic or chronic studies were located that meet EPA test guidelines ( including serum biochemistry for a number of endpoints and histopathology of an extensive list of tissues and organs). Nonetheless, there is fairly good support that the target organ ( the thyroid) has been identified, and a number of studies have focused on effects on that organ. None of the studies that evaluated thyroid endpoints identified a NOAEL. The lowest LOAEL was identified by Philbrick et al. ( 1979), who reported a statistically significant decrease in plasma T4 and increased thyroid weight ( but no thyroid histopathology) in rats administered 98 mg SCN/ kg day in diet for 11.5 months; plasma T4 was increased at this dose after 4 months of exposure. Pyska et al. ( 1977) also reported decreased plasma protein bound iodine ( a measure of plasma T3 and T4) in rats administered 100 mg SCN/ kg day for approximately 10 weeks in drinking water. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 29 Final draft C. Reproductive/ Developmental Toxicity Cyanogen chloride. No studies on the reproductive or developmental toxicity of cyanogen chloride by any route of exposure were located in the literature. The remainder of this section presents the data available on metabolites of cyanogen chloride.

Cyanide. NTP ( 1993) conducted a subchronic bioassay on sodium cyanide administered in drinking water to F344 rats and B6C3F1 mice. Decreases in epididymis weight, testis weight,

and sperm count were observed at 12.5 mg CN/ kg day in rats ( NOAEL of 4.5 mg CN/ kg day)

and 24.4 mg CN/ kg day in mice ( NOAEL of 8.6 mg CN/ kg day). Female rats treated with 4.9 or 12.5 mg CN/ kg day had altered estrous cycles, but the authors suggested that this effect may not have been chemical related, in the absence of a clear dose response. This study was described in detail in Section V. B. Kamalu ( 1993) observed histopathological changes in the testis of dogs that received cyanide ( 1.04 mg CN/ kg day) in the diet for 14 weeks. Although dogs have low levels of rhodanese and are not good models for human cyanide toxicity ( ATSDR, 1997), this study suggests that cyanide can be a male reproductive toxicant, based on the presence of abnormal cells and sloughing of germ cells in the seminiferous tubules and a decreased percentage of tubules in stage 8 of the spermatogenic cycle.

In the only available study that evaluated the developmental effects of cyanide exposure,

Tewe and Maner ( 1981) evaluated the effects of cyanide in the diet of rats. Female rats ( 20/ group, strain not specified) were offered either a basal diet prepared from low HCN cassava meal or the basal diet supplemented with 500 ppm of potassium cyanide throughout mating, Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 30 Final draft gestation, and lactation. In addition, two female weanling rats per litter were maintained on each diet for an additional 28 days. Rats receiving the basal diet alone received a dose of 1.2 mg CN/ kg day ( based on a dietary HCN concentration of 12 mg/ kg and an average food consumption across female rats of 0.102 kg/ kg body weight). Adult rats receiving the KCN treatment received a total CN dose of 21.6 mg CN/ kg day ( including the 1.2 from the basal diet and 20.4 mg CN/

kgday as KCN). For the weanling rats, the corresponding doses were approximately 1.9 mg CN/

kgday for the basal diet and 34.3 mg CN/ kg day for the KCN diet, based on an average food factor of 0.162 for female weanling rats ( U. S. EPA, 1988). Supplementation with KCN had no effect on body weight of pregnant rats, food consumption, maternal liver or kidney weights, litter size,

birth weight of pups, or pup mortality. In the weanling rats, the high cyanide diet resulted in significant decreases in food consumption and growth rate, and an increase in the ratio of food consumption to body weight gain. The high cyanide diet also resulted in significant increase in serum thiocyanate in both dams and weanlings compared with animals on the basal diet alone.

However, the activity of rhodanese, the enzyme that metabolizes cyanide to thiocyanate, in the liver and kidney was comparable in all groups. A LOAEL of 34.3 mg CN/ kg day was identified in this study, based on decreased daily weight gain in weanlings; the corresponding BMDL was 15 mg CN/ kg day. However, confidence in the BMDL is limited, because only one positive dose was tested, and so no information was available on the shape of the dose response curve.

Thiocyanate. Nagasawa et al. ( 1980) exposed female 8 week old SHN ( 18/ group) or 5 week old GR/ A ( 32/ group) mice to 0%, 0.1% or 0.3% potassium thiocyanate in the drinking water. Using an average water consumption of 0.27 L/ kg day, this corresponds to doses of 0,

163, and 488 mg SCN/ kg day. In the SHN mice, normal mammary development was significantly Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 31 Final draft decreased after exposure to the high dose for 12 weeks, based on a staged rating of end bud and lobulo alveolar growth. However, no effect on the weight of the pituitary or adrenals or ovarian histology was observed. In the GR/ A mice, exposure for 5 weeks prior to mating and then for 4 additional weeks had no effect on fertility, litter size, fetal viability, or pup weight. In addition,

maternal pituitary levels of prolactin and growth hormone were unaffected when measured on lactation day 4. No LOAEL for developmental toxicity was determined in the GR/ A mice. The developmental NOAEL in that strain was 488 mg SCN/ kg day, but the study did not include a full assessment of developmental toxicity endpoints. The effect on mammary development in the SHN mice could be considered evidence of developmental toxicity of thiocyanate. However, the significance of this finding is unclear, since the strain was chosen based on its high incidence of mammary tumors. In addition, the effect on mammary development occurred at a higher dose than the thyroid effects in the same study ( described in the section on long term exposure). The study authors attributed the inhibition of normal mammary development ( as well as the inhibition of mammary tumor development, described in the section on carcinogenicity) to decreased circulating thyroid hormones.

Pyska ( 1977) exposed 3 week old female Wistar rats ( 17   19/ group) to 0.1% potassium thiocyanate in the drinking water for approximately 10 weeks ( until the rats were 3 months old).

Controls received tap water. Water consumption data were not reported for this phase of the study. Based on a body weight of 0.14 kg and the allometric equation of U. S. EPA ( 1988) for calculating water consumption, the dose can be estimated as 100 mg/ kg day. However, the actual dose may have been lower, in light of the lower maternal drinking water consumption described below. Mammary weight and DNA content ( which is a measure related to cell number) were Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 32 Final draft significantly decreased in the treated rats, indicating that the thiocyanate inhibited mammary gland development. In another experiment, female rats ( 12   27/ group) were exposed to 0, 0.1%, 0.3%,

or 0.5% potassium thiocyanate in the drinking water during pregnancy and 14 days of lactation.

The daily intake of drinking water was reported as 20   30 mL. Based on the reported maternal body weight and the lower drinking water intake ( as more conservative), the daily doses can be estimated at 0, 52, 156, and 260 mg SCN/ kg day. There was a statistically significant,

doserelated decrease in plasma protein bound iodine at all doses. Maternal mammary gland dry weight, total DNA, and RNA content were also significantly decreased at all doses, indicating depressed mammary growth. The authors suggested that the depressed mammary growth was associated with the depressed thyroid activity. There was also a marked and statistically significant decrease compared to controls in maternal body weight at the high dose, and in the weight of 14 day litters at the mid and high doses. It is unclear whether the effects on litter weight were due to pre or post natal exposure to thiocyanate. Based on thyroid toxicity and inhibited mammary gland development, the maternal LOAEL was 52 mg/ kg day; no NOAEL was identified. Based on decreased litter weight, the NOAEL for developmental toxicity was 52 mg SCN/ kg day and the LOAEL was 156 mg SCN/ kg day. However, the study did not evaluate thyroid effects in the pups or other standard developmental toxicity endpoints.

Bala et al. ( 1996) fed groups of 6 female Wistar/ NIN rats a semi synthetic diet sufficient in iodine and containing 0 or 25 mg potassium thiocyanate/ rat. The diet was provided for 8 weeks before mating, and during gestation and lactation. Additional groups were fed the control diet until conception, and then provided the thiocyanate during gestation and lactation, or during lactation only. Based on the reported body weight of 139 g after 8 weeks, the dose can be Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 33 Final draft estimated as 108 mg SCN/ kg day. Increased maternal thyroid weight and urinary excretion of iodine, and decreased serum T4 were reported in the dams. Body weight and T3 levels were not affected. Pups also had significantly decreased serum T4 under all of the treatment scenarios.

Weanling pup body weight was also significantly decreased in the group provided thiocyanate during gestation and lactation, but not prior to mating. Other markers of developmental toxicity were not assessed. This study identifies a maternal and developmental LOAEL of 108 mg SCN/ kg day, based on thyroid effects.

In a related study from the same laboratory, Raghunath and Bala ( 1998) measured the effect of potassium thiocyanate on T4 in Wistar/ NIN rats fed through two generations. The number of rats exposed was not reported, although it appears that only 6 8 pups from the F1 and F2 generations were evaluated. Female weanling rats were fed either a nutrient sufficient caseinbased semisynthetic diet ( control) or the same diet with added potassium thiocyanate. The authors did not provide the concentration of the added KSCN in the diet, but stated that the treatment animals received about 25 mg KSCN/ rat/ day. Using a body weight of 0.14 kg ( based on the data from Bala et al., 1996), the daily intake can be estimated at about 108 mg SCN/

kgday After eight weeks on the diet, the treated female rats became hypothyroidic, were mated with control males, and continued on their respective diets throughout the subsequent gestation and lactation periods. The females from the F1 offspring were weaned and fed their respective dam's diet for eight additional weeks and, likewise, mated with control males and continued through to gestation and lactation. At weaning, the F2 pups were assessed for thyroid status.

Continued feeding of KSCN over two generations had no effect on the body weights of F1 or F2 pups at birth or at weaning as compared to corresponding controls. Serum T4 levels were Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 34 Final draft significantly decreased in F1 and F2 pups compared to their respective controls. The decrease of T4 levels was more pronounced in the F2 generation. There was, however, no effect on pup body weight or brain weight in the F2 generation ( the only generation in which these endpoints were assessed). Other reproductive and developmental toxicity endpoints were not evaluated. Overall,

the single dose tested, 108 mg SCN/ kg day, was a maternal and developmental LOAEL.

Kreutler et al. ( 1978) administered sodium thiocyanate in drinking water at 0, 40, 80, or 160 mg SCN/ L to groups of 4 7 pregnant rats ( strain not reported, but source was Charles River)

during gestation through postpartum day 10. The authors reported that the rats drank about 40 mL water daily, but did not report the body weight. Using the average water consumption of 0.158 L/ kg day for female rats in a subchronic study ( calculated from U. S. EPA, 1988), the doses can be estimated as 0, 6.3, 12.6, and 25.3 mg SCN/ kg day. The rats were allowed to deliver, and the litters were culled to 6 pups at birth. The only index of toxicity evaluated was increased thyroid weight; other developmental endpoints were not evaluated. In the dams, thyroid weights ( relative to body weight) were significantly increased at all doses at 5 days postpartum, but only at the high dose at 10 days postpartum. However, this difference may be an artifact of the small sample size, since the control relative thyroid weight was much lower at day 5 than at day 10. 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. These results suggest that the pup is more sensitive than the dam to effects of thiocyanate, particularly since the plasma thiocyanate levels were much lower in the pups. 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. Increased thyroid weight can be an Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 35 Final draft adaptive homeostatic response to changes in thyroid hormone levels, and can be followed by a return of thyroid hormones to basal levels. In the absence of data on other thyroid endpoints, the increase in thyroid weight at the low dose indicates that 6.3 mg SCN/ kg day is an minimal developmental and maternal LOAEL.

Heydens ( 1985) exposed female rats ( number/ dose and strain not specified) to 55 or 220 mg/ kg of thiocyanate on gestation days 6   15 or to 150 mg/ kg of thiocyanate throughout gestation ( route of exposure not specified). No teratogenicity was observed, but postnatal growth and development were reported to have been retarded. Continued exposure throughout the period of lactation did not result in substantially greater growth retardation, indicating that the effects observed were caused primarily during gestation. Limited experimental details and results were presented in this report. No NOAEL was reported. Based on the information provided, the LOAEL for this study was 55 mg thiocyanate/ kg day, based on developmental retardation.

Summary of Reproductive/ Developmental Toxicity No studies on the reproductive or developmental toxicity of cyanogen chloride by any route of exposure were located. No standard multigeneration or developmental toxicity studies of cyanide are available. Cyanide appears to be a male reproductive toxicant in rats, mice, and dogs as discussed in the previous section ( NTP, 1993; Kamalu, 1993). Tewe and Maner ( 1981) found no effects on litter size, birth weight of pups, or pup mortality in the offspring of female rats receiving cyanide in the diet throughout mating, gestation, and lactation, although weanling body weight gain was decreased. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 36 Final draft No standard multigeneration reproduction study of thiocyanate by any route was located.

A multigeneration study ( Raghunath and Bala, 1998) was conducted in rats administered 108 mg SCN/ kg day in feed, and decreased serum T4 was observed at the single dose tested, with a somewhat stronger effect in the F2 generation. In addition, there was no developmental toxicity study that included morphological evaluation of the pups. Two studies reported, however, that mammary gland development of young females is inhibited at doses at or above those that cause thyroid effects ( Pyska, 1977; Nagasawa et al., 1980). No standard developmental toxicity studies were identified, although Nagasawa et al. ( 1980) found no effect on fertility, litter size, fetal viability, or pup weight when mice were administered thiocyanate in drinking water up to 488 mg SCN/ kg day. Decreased litter weight was observed in the offspring of rats administered approximately 156 or 260 mg SCN/ kg day in drinking water, but not 52 mg SCN/ kg day ( Pyska,

1977). Several studies evaluated thyroid effects in pups that were exposed to thiocyanate during gestation and lactation ( Bala et al., 1996; Raghunath and Bala, 1998; Kreutler et al., 1978), but no NOAEL was identified. Kreutler et al. ( 1978) included the lowest doses, testing rats down to 6.3 mg SCN/ kg day in drinking water. Unfortunately, these authors only evaluated relative thyroid weight, and increases in thyroid weight may be adaptive or adverse, depending on the effects on thyroid hormones and histopathology. Using the most conservative interpretation, the low dose of 6.3 mg SCN/ kg day in the Kreutler et al. ( 1978) study can be considered a minimal LOAEL. The next lowest developmental LOAEL was 108 mg SCN/ kg day, identified by Bala et al. ( 1996) and Raghunath and Bala ( 1998), based on decreased serum T4 in pups.

In general, the reproductive and developmental database for cyanogen chloride and its metabolites is incomplete: standard 2 generation reproductive and developmental toxicity studies Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 37 Final draft are missing for all three chemicals. Of particular concern for cyanide is the fact that two subchronic studies found altered epididymis weights, testis weights, and sperm counts in rats and altered testis weight in dogs. Therefore, a significant data gap is a reproductive study that evaluates male reproductive function. Although limited data suggest that neither cyanide nor thiocyanate affect some endpoints evaluated in developmental toxicity studies, such as litter size,

pup weights, or pup viability, none of the studies evaluated the full spectrum of developmental toxicity endpoints, particularly morphological evaluation of pups. Thus, a significant data gap is the lack of studies that evaluate the potential of cyanogen chloride or its metabolites to have teratogenic effects. Thiocyanate appears to alter thyroid function in pups exposed during gestation and lactation, including altered thyroid weights and thyroid hormone levels. However,

the existing studies did not do a complete analysis of thyroid to determine if the effects observed were adaptive or adverse. In addition, decreased T4 levels in mothers and fetuses/ neonates has been associated with significant neurological deficit in children. Therefore, lack of neurodevelopmental toxicity studies on cyanogen chloride or its metabolites also represents a significant data gap.

D. Mutagenicity and Genotoxicity Cyanogen chloride. No studies regarding the mutagenicity or genotoxicity of cyanogen chloride were located.

Cyanide. The limited information available on the mutagenicity of cyanide is summarized by ATSDR ( 1997). Potassium cyanide was not mutagenic in strains TA82, TA102, TA98, Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 38 Final draft TA100, TA1535, TA1537, and TA1538 of the Salmonella typhimurium reverse mutation assay with or without metabolic activation ( De Flora, 1981; De Flora et al., 1984). Sodium cyanide was not mutagenic in strains TA97, TA98, TA100, and TA1535 with or without metabolic activation ( NTP, 1993). However, a positive response was reported for hydrogen cyanide in strain TA100 without metabolic activation; adding metabolic activation reduced the magnitude of the positive response to 40% of what it had been without metabolic activation ( Kushi et al.,

1983). Negative results were also obtained in the DNA repair test in Escherichia coli strains WP67, CM871, and WP2 ( De Flora et al., 1984) and in a test for inhibition of DNA synthesis in HeLa cells ( Painter and Howard, 1982).

A report of U. S. EPA's Gene Tox program summarized data on the Arabidopsis plant assay for chemical mutagens ( Redei, 1982). This assay allows the screening of thousands of gene loci for recessive forward mutations. This summary report cited an earlier unpublished study by Müller ( 1965), in which potassium cyanide was negative up to 10 mM. Further details were not provided, but the studies presented met some ( unspecified) criteria for acceptability.

Kihlman ( 1957) conducted a chromosome aberration assay with potassium cyanide using the Vicia faba, a broad bean plant. Exposure was to potassium cyanide in air. Both exchanges and isolocus breaks were observed, with the concentration in air being more important than the exposure duration. Production of aberrations increased as the oxygen concentration increased from 0 to 100%. Noting that cyanide inhibits peroxidase and catalase, other authors have hypothesized that cyanide induced chromosome aberrations result from the accumulation of hydrogen peroxide. Noting, however, that peroxide alone does not cause chromosome Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 39 Final draft aberrations in the Vicia test system, Kihlman ( 1957) proposed that cyanide acts by forming a complex with iron or other heavy metals bound to chromosomes, and that the metals act to mediate redox reactions.

Overall, cyanide has been negative in bacterial mutagenicity studies with and without S9 activation ( De Flora, 1981; De Flora et al., 1984; NTP, 1993), although a positive result was obtained in strain TA100 with and without S9 activation in one study ( Kushi et al., 1983).

Mammalian gene mutation studies of cyanide are not available, but a negative result was obtained in a study of Arabidopsis ( Müller, 1965, as cited by Redei, 1982). Standard chromosome aberration assays of cyanide are also not available. Potassium cyanide in air caused chromosome aberrations in the broad bean plant, apparently by a mechanism related to the production of reactive oxygen species ( Kihlman, 1957). Cyanide was negative in assays for the production of DNA damage and repair ( De Flora et al., 1984; Painter and Howard, 1982).

Thiocyanate. The data on the genotoxicity of thiocyanate are very limited. Marginal results were reported in the S. typhimurium mutagenicity assay ( Kier, 1988, as reported by Rosenkranz and Klopman, 1990). Further study details were not available. No other standard gene mutation or chromosome aberration assays were located. Rosenkranz and Klopman ( 1990)

reported that potassium thiocyanate does not have any structural alerts for genotoxicity. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 40 Final draft Summary of Genotoxicity No data are available on the genotoxicity of cyanogen chloride. Although the data for some known or potential metabolites of cyanogen chloride are limited, the available data suggest,

overall, that none of the metabolites is genotoxic. Overall, cyanide has been negative in bacterial mutagenicity assays ( De Flora, 1981; De Flora et al., 1984; NTP, 1993) and assays of DNA damage and repair ( De Flora et al., 1984; Painter and Howard, 1982), although a positive result was obtained in one strain of Salmonella with and without S9 activation ( Kushi et al., 1983). No assays of mammalian gene mutation or chromosome aberration induction by cyanide are available.

Genotoxicity data on thiocyanate are limited to marginal results in a S. typhimurium mutagenicity assay ( Kier, 1988, as reported by Rosenkranz and Klopman, 1990). Potassium thiocyanate does not have any structural alerts for genotoxicity ( Rosenkranz and Klopman, 1990).

E. Carcinogenicity Cyanogen chloride. No carcinogenicity studies of cyanogen chloride via any route were located.

Cyanide. No carcinogenicity studies of cyanide via any route of exposure were located.

Thiocyanate. In a cancer bioassay, Lijinsky and Reuber ( 1982) fed male Fischer rats ( 20/ group) a powdered diet with or without sodium thiocyanate at 800 mg/ kg for 130 weeks.

Using a food factor of 0.079 for a chronic study in male Fischer rats ( U. S. EPA, 1988), this Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 41 Final draft corresponds to doses of 0 and 45 mg SCN/ kg day. The purpose of the study was to examine the effect of in vivo transnitrosation of nitrosamines. SCN was used as a catalyst of nitrosation when administered with other nitroso compounds; the group receiving SCN alone was essentially considered a control group by the study authors. Therefore, this study was not conducted with the intent of evaluating SCN carcinogenicity and the reporting of SCN health effects is limited.

All animals were necropsied and the major organs and all lesions were fixed for histological examination. Food consumption was not reported by the authors, nor was the composition of the diet. Survival of the rats given SCN was comparable to the untreated control at all time points.

Survival at 1 year was 100% in both groups; survival at 100 weeks ( approximately 2 years) was 65% in untreated controls compared with 85% in SCN treated animals. By week 120, survival was 45% in both groups. The number of hepatic tumors was elevated in the SCN treated group ( 5/ 20) as compared to the control group ( 0/ 20). There were no differences in tumor incidences between SCN treated and control rats at all other sites examined. No other information was presented regarding the type of tumor, the time of first tumor appearance, or the presence of any nonneoplastic lesions. This study is limited by the small size of the dose groups, and the incomplete evaluation of SCN carcinogenicity. It is unclear whether a sufficiently high dose was tested, although higher doses were tested in the follow up study described in the next paragraph.

Nonetheless, it is of interest that the number of thyroid tumors ( 2/ 20) was the same in the exposed group as in the controls.

In a follow up study, Lijinsky and Kovatch ( 1989) exposed Fischer 344 rats ( 20

24/ sex/ dose) to 0% or 0.32% sodium thiocyanate in drinking water 5 days/ week for 112 weeks.

The study continued until all of the animals died, or until 130 weeks. The study authors Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 42 Final draft determined that the total dose of sodium thiocyanate ingested was approximately 1 mole/ kg in males and 1.7 mole/ kg in females. The authors estimated that this was roughly equivalent to a daily dose of 250 mg/ kg of sodium thiocyanate in females ( 147 mg/ kg of sodium thiocyanate in males, by extrapolation). Thus, the thiocyanate doses were 75 mg SCN/ kg day for males and 128 mg SCN/ kg day for females, after adjusting for dosing 5/ 7 days/ week. No increase in mortality,

noticeably adverse effects, or tumor formation was reported in the treated rats. It is unclear from the report whether the incidence of any nonneoplastic lesions was increased in treated rats. The increase in liver tumors seen by Lijinsky and Reuber ( 1982) was not confirmed. However, the incidence of thyroid tumors doubled for both treated males ( 3/ 20 or 15%) and females ( 2/ 20 or 10%) compared to controls ( 2/ 24 or 8% for males, 1/ 24 or 4% for females). The study authors did not conduct statistical analysis of thyroid tumor incidence; EPA conducted a Fisher's Exact test, which indicated that the increase was not statistically significant. No additional information is provided on the type of tumors, the time of first tumor appearance, or other nonneoplastic lesions associated with thyroid toxicity. This study is limited by the small sample size and the incomplete reporting of information needed to completely assess the carcinogenicity of SCN.

In a short term test of carcinogenicity, Nagasawa et al. ( 1980) exposed female SHN mice ( 18/ group) to 0.1% or 0.3% potassium thiocyanate in the drinking water for 12 weeks. Controls received tap water. Using the average water consumption of 0.27 L/ kg day across mouse strains ( U. S. EPA, 1988), this corresponds to doses of 0, 163, and 488 mg/ kg day. The strain was chosen due to its high incidence of mammary tumors, but thiocyanate did not increase the incidence of mammary tumors. Instead, there was a dose related decrease in mammary tumor incidence and in the incidence of mammary hyperplastic alveolar nodules. Significantly decreased Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 43 Final draft T3 ( at the high dose) and T4 ( at both doses) indicated that sufficiently high doses were tested. In a related study, female inbred GR/ A mice ( 19 22/ group) received the same doses for 5 weeks prior to mating and during gestation. There were dose related decreases in the incidence and size of pregnancy dependent mammary tumors, although the effect on tumor size was not statistically significant. The authors attributed the inhibition of mammary tumor development by thiocyanate,

as well as the inhibition of normal mammary development ( described above in the section on reproductive/ developmental toxicity) to decreased thyroid hormones, since body weight and weights of the pituitary and adrenals were not affected. Other tumor endpoints were not evaluated.

Summary of Carcinogenicity There are no cancer bioassays of cyanogen chloride or cyanide. There are no wellconducted standard cancer bioassays of thiocyanate. Two oral carcinogenicity studies in rats were 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 higher doses of SCN may be associated with increased incidence of thyroid tumors. Although the increase of thyroid tumors was not statistically significant, the observation of thyroid tumors in consistent with what is known about SCN mode of action on the thyroid. However, these studies suffer from a number of limitations. Only a single dose was tested in each study, limited numbers of animals were tested ( 20/ group first study; 20/ sex/ group second study), and the studies did not provide a complete report of the information needed to adequately assess SCN carcinogenicity. Drinking Water Criteria Document for Cyanogen Chloride and Potential Metabolites EPA/ OW/ OST/ HECD V 44 Final draft Based on these considerations, cyanogen chloride, cyanide, and thiocyanate would all be classified as Group D, Not Classifiable as to Human Carcinogenicity, using the U. S. EPA ( 1986)

guidelines for carcinogen risk assessment. Using the U. S. EPA ( 1999) Draft Guidelines for Carcinogen Risk Assessment, the data are inadequate for an assessment of the human carcinogenic potential of these compounds.


[Federal Register Volume 87, Number 6 (Monday, January 10, 2022)]
[Rules and Regulations]
[Pages 1076-1078]
[FR Doc No: 2022-00168]

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DEPARTMENT OF HOMELAND SECURITY

Coast Guard

33 CFR Part 165

[Docket Number USCG-2022-0011]
RIN 1625-AA00

Safety Zone; Deep Creek, Elizabeth River, Chesapeake, VA

AGENCY: Coast Guard, DHS.

ACTION: Temporary final rule.

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SUMMARY: The Coast Guard is establishing a temporary safety zone for
certain navigable waters of Deep Creek and the Elizabeth River. The
safey zone is needed to safeguard personnel and vessels from potential
hazards associated with an incident involving an adrift barge that has
struck a power transmission tower in the waterway. Entry of vessels or
persons into this zone is prohibited unless specifically authorized by
the Captain of the Port Sector Virginia or designated respresentative.

DATES: This rule is effective without actual notice from January 10,
2022 until January 19, 2022. For the purposes of enforcement, actual
notice will be used from January 4, 2022, until January 10, 2022.

ADDRESSES: To view documents mentioned in this preamble as being
available in the docket, go to https://www.regulations.gov, type USCG-
2022-0011 in the search box and click ``Search.'' Next, in the Document
Type column, select ``Supporting & Related Material.''

FOR FURTHER INFORMATION CONTACT: If you have questions on this rule,
call or email LCDR Ashley Holm, Sector Virginia, Waterways Management
Division, U.S. Coast Guard, Telephone:<PHONE_NUMBER>, email:
[email protected].

SUPPLEMENTARY INFORMATION:

I. Table of Abbreviations

CFR Code of Federal Regulations
DHS Department of Homeland Security
FR Federal Register
NPRM Notice of proposed rulemaking
Sec.  Section
U.S.C. United States Code

II. Background Information and Regulatory History

    On January 3, 2022, an adrift barge struck a power transmission
tower in the navigable waters of Deep Creek and the Elizabeth River
causing the tower to lean. The structural integrity of the transmission
tower is unknown at this time creating a potential hazard to navigation
including the potential for de-energized power lines to enter the
waterway. Every effort is being made to ensure the structure is
supported until power lines can be disconnected and the tower is
removed. The Coast Guard is issuing this temporary rule without prior
notice and opportunity to comment pursuant to authority under section
4(a) of the Administrative Procedure Act (APA) (5 U.S.C. 553(b)). This
provision authorizes an agency to issue a rule without prior notice and
opportunity to comment when the agency for good cause finds that those
procedures are ``impracticable, unnecessary, or contrary to the public
interest.'' Under 5 U.S.C. 553(b)(B), the Coast Guard finds that good
cause exists for not publishing a notice of proposed rulemaking (NPRM)
with respect to this rule because it would be impracticable and
contrary to public interest. The Coast Guard received information about
this potential hazard to navigation on January 3, 2022. Immediate
action is needed to protect transiting vessels from the damaged
transmission tower causing a potential hazard to navigation which
includes the potential for de-energized power lines to enter the
waterway.
    For those same reasons, under 5 U.S.C. 553(d)(3), the Coast Guard
finds that good cause exists for making this rule effective less than
30 days after publication in the Federal Register. Delaying the
effective date of this rule would be impracticable and contrary to the
public interest because immediate action to restrict vessel traffic is
needed to protect life, property and the environment. Delaying the
effective date would be contrary to the safety zone's intended
objectives of protecting personnel and vessel from the immediate
potential hazard, enhancing maritime safety.

III. Legal Authority and Need for Rule

    The Coast Guard is issuing this rule under authority in 46 U.S.C.
70034 (previously 33 U.S.C. 1231). The Captain of the Port Virginia
(COTP) has determined a potential hazardous situation in Deep Creek,
Southern Branch of the Elizabeth River, requires the establishment of a
safety zone to protect personnel and vessels transiting the area.

IV. Discussion of the Rule

    The U.S. Coast Guard is establishing a temporary safety zone on
certain navigable waters in the vicinity of Deep Creek, Southern Branch
of the Elizabeth River north of the I-64/High Rise Bridge. This rule
will be in effect from January 4, 2022, through January 19, 2022. The
duration of the zone is intended to protect vessels from a damaged
power transmission tower causing potential hazard to navigation in the
waterway and to protect personnel performing repair and recovery. No
vessel or person will be permitted to enter the safety zone without
obtaining permission from the COTP or a designated representative.

V. Regulatory Analyses

    We developed this rule after considering numerous statutes and
Executive orders related to rulemaking. Below we summarize our analyses
based on a number of these statutes and Executive orders, and we
discuss First Amendment rights of protestors.

A. Regulatory Planning and Review

    Executive Orders 12866 and 13563 direct agencies to assess the
costs and benefits of available regulatory alternatives and, if
regulation is necessary, to select regulatory approaches that maximize
net benefits. This rule has not been designated a ``significant
regulatory action,'' under Executive Order 12866. Accordingly, this
rule has not been reviewed by the Office of Management and Budget
(OMB).
    This regulatory action determination is based on the safety zone
being in effect for a limited duration; this is a portion of the
waterway with minimal vessel traffic; and the Coast Guard will continue
to make notifications via maritime broadcasts.

B. Impact on Small Entities

    The Regulatory Flexibility Act of 1980, 5 U.S.C. 601-612, as
amended, requires Federal agencies to consider the potential impact of
regulations on small entities during rulemaking. The term ``small
entities'' comprises small businesses, not-for-profit organizations
that are independently owned and operated and are not dominant in their
fields, and governmental jurisdictions with populations of less than
50,000. The Coast Guard certifies under 5 U.S.C. 605(b) that this rule
will not have a significant economic impact on a substantial number of
small entities.
    While some owners or operators of vessels intending to transit the
safety zone may be small entities, for the reasons stated in section
V.A above, this rule will not have a significant economic impact on any
vessel owner or operator.
    Under section 213(a) of the Small Business Regulatory Enforcement
Fairness Act of 1996 (Pub. L. 104-121), we want to assist small
entities in understanding this rule. If the rule would affect your
small business, organization, or governmental jurisdiction and you have
questions concerning its provisions or options for compliance, please
call or email the person listed in the FOR FURTHER INFORMATION CONTACT
section.
    Small businesses may send comments on the actions of Federal
employees who enforce, or otherwise determine compliance with, Federal
regulations to the Small Business and Agriculture Regulatory
Enforcement Ombudsman and the Regional Small Business Regulatory
Fairness Boards. The Ombudsman evaluates these actions annually and
rates each agency's responsiveness to small business. If you wish to
comment on actions by employees of the Coast Guard, call 1-888-REG-FAIR
(1-888-734-3247). The Coast Guard will not retaliate against small
entities that question or complain about this rule or any policy or
action of the Coast Guard.

C. Collection of Information

    This rule will not call for a new collection of information under
the Paperwork Reduction Act of 1995 (44 U.S.C. 3501-3520).

D. Federalism and Indian Tribal Governments

    A rule has implications for federalism under Executive Order 13132,
Federalism, if it has a substantial direct effect on the States, on the
relationship between the National Government and the States, or on the
distribution of power and responsibilities among the various levels of
government. We have analyzed this rule under that Order and have
determined that it is consistent with the fundamental federalism
principles and preemption requirements described in Executive Order
13132.
    Also, this rule does not have tribal implications under Executive
Order 13175, Consultation and Coordination with Indian Tribal
Governments, because it does not have a substantial direct effect on
one or more Indian tribes, on the relationship between the Federal
Government and Indian tribes, or on the distribution of power and
responsibilities between the Federal Government and Indian tribes.

E. Unfunded Mandates Reform Act

    The Unfunded Mandates Reform Act of 1995 (2 U.S.C. 1531-1538)
requires Federal agencies to assess the effects of their discretionary
regulatory actions. In particular, the Act addresses actions that may
result in the expenditure by a State, local, or tribal government, in
the aggregate, or by the private sector of $100,000,000 (adjusted for
inflation) or more in any one year. Though this rule will not result in
such an expenditure, we do discuss the effects of this rule elsewhere
in this preamble.

F. Environment

    We have analyzed this rule under Department of Homeland Security
Directive 023-01, Rev. 1, associated implementing instructions, and
Environmental Planning COMDTINST 5090.1 (series), which guide the Coast
Guard in complying with the National Environmental Policy Act of 1969
(42 U.S.C. 4321-4370f), and have determined that this action is one of
a category of actions that do not individually or cumulatively have a
significant effect on the human environment. This rule involves a
safety zone lasting for 15 days that will prohibit entry within certain
navigable waters of the Southern Branch of the Elizabeth River. It is
categorically excluded from further review under paragraph L60(c) of
Appendix A, Table 1 of DHS Instruction Manual 023-01-001-01, Rev. 1.

G. Protest Activities

    The Coast Guard respects the First Amendment rights of protesters.
Protesters are asked to call or email the person listed in the FOR
FURTHER INFORMATION CONTACT section to coordinate protest activities so
that your message can be received without jeopardizing the safety or
security of people, places or vessels.

List of Subjects in 33 CFR Part 165

    Harbors, Marine safety, Navigation (water), Reporting and
recordkeeping requirements, Security measures, Waterways.

    For the reasons discussed in the preamble, the Coast Guard amends
33 CFR part 165 as follows:

PART 165--REGULATED NAVIGATION AREAS AND LIMITED ACCESS AREAS

0
1. The authority citation for part 165 continues to read as follows:

    Authority:  46 U.S.C. 70034, 70051; 33 CFR 1.05-1, 6.04-1, 6.04-
6, and 160.5; Department of Homeland Security Delegation No.
00170.1, Revision No. 01.2.

0
2. Add Sec.  165.T05-0861to read as follows:

Sec.  165.T05-0861  Safety Zone; Deep Creek, Elizabeth River,
Chesapeake, VA.

    (a) Location. The following area is a safety zone: The waters
enclosed by the shoreline and the following lines: A line drawn across
Deep Creek, Elizabeth River from 36-45.71N 076-18.52W to 36-45.64N 076
18.52W and a line drawn across Deep Creek, Elizabeth River from 36-
45.74N 076-18.30W to 36-45.66N 076-18.30W.
    (b) Definitions. As used in this section, designated representative
means a Coast Guard Patrol Commander, including a Coast Guard coxswain,
petty officer, or other officer operating a Coast Guard vessel and a
Federal, State, and local officer designated by or assisting the
Captain of the Port Sector Virginia (COTP) in the enforcement of the
safety zone.
    (c) Regulations. (1) Under the general safety zone regulations in
subpart C of this part, you may not enter the safety zone described in
paragraph (a) of this section unless authorized by the COTP or the
COTP's designated representative.
    (2) To seek permission to enter, contact the COTP or the COTP's
representative by VHF/FM Chanel 16. Those in the safety zone must
comply with all lawful orders or directions given to them by the COTP
or the COTP's designated representative.
    (d) Enforcement period. This section will be enforced January 4,
2022, through January 19, 2022, unless canceled sooner by the COTP.

    Dated: January 4, 2022.
Samson C. Stevens,
Captain, U.S. Coast Guard, Captain of the Port, Sector Virginia.
[FR Doc. 2022-00168 Filed 1-7-22; 8:45 am]
BILLING CODE 9110-04-P