Appendix A
Executive Summaries of Reports on Toxicological or Biological Agents

   

 1.  Bacillus globigii (BG)

 

 

   

 2.  Betapropiolactone (beta-propiolactone; BPL)

 

 

   

 3.  Bis Hydrogen Phosphite (BHP)

 

 

   

 4.  Calcofluor

 

 

   

 5.  Coxiella burnetii (CB; Q fever)

 

 

   

 6.  Diethylphthalate (DEP or D)

 

 

   

 7.  Escherichia coli [E. coli]

 

 

   

 8.  Methyl Acetoacetate (MAA)

 

 

   

 9.  Phosphorus-32 [32P]

 

 

   

 10.  Sarin

 

 

   

 11.  Serratia marcescens (SM)

 

 

   

 12.  Staphylococcal Enterotoxin Type B (SEB)

 

 

   

 13.  Sulfur Dioxide (SO2)

 

 

   

 14.  Trioctyl Phosphate (TEHP or TOF)

 

 

   

 15.  Pasteurella tularensis (Francisella tularensis)

 

 

   

 16.  Uranine

 

 

   

 17.  VX Nerve Agent (VX)

 

 

   

 18.  Zinc Cadmium Sulfide (ZnCdS)

 

 

   

 References

 

 



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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Appendix A Executive Summaries of Reports on Toxicological or Biological Agents      1.  Bacillus globigii (BG)          2.  Betapropiolactone (beta-propiolactone; BPL)          3.  Bis Hydrogen Phosphite (BHP)          4.  Calcofluor          5.  Coxiella burnetii (CB; Q fever)          6.  Diethylphthalate (DEP or D)          7.  Escherichia coli [E. coli]          8.  Methyl Acetoacetate (MAA)          9.  Phosphorus-32 [32P]          10.  Sarin          11.  Serratia marcescens (SM)          12.  Staphylococcal Enterotoxin Type B (SEB)          13.  Sulfur Dioxide (SO2)          14.  Trioctyl Phosphate (TEHP or TOF)          15.  Pasteurella tularensis (Francisella tularensis)          16.  Uranine          17.  VX Nerve Agent (VX)          18.  Zinc Cadmium Sulfide (ZnCdS)          References    

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Bacillus globigii Bacillus globigii (BG) has been called B. subtilis var niger, B. licheniformis and, most recently, B. atrophaeus. It is a Gram-positive, spore-forming, facultative anaerobe commonly found in dust, soil, and water. It is widely used as a biological tracer and has been shown to produce substances that exhibit antimicrobial activity. In Project SHAD, B.globigii was used to simulate biological warfare agents, because it was then considered a contaminant with little health consequence to humans. BG is now considered a pathogen for humans. Most infections are associated with the experience of invasive trauma (e.g., catheters, surgery) and/or a debilitated health state; thus it is often encountered as a nosocomial pathogen. BG is also a well-known cause of food poisoning, resulting in diarrhea and vomiting. Infections are rarely known to be fatal, although fatal food poisoning has been reported. Ocular infections, bacteremia, sepsis/septicemia, ventriculitis, and peritonitis are the reported types of infection, and they are usually treated with antibiotics. Cases of long-term persistence or recurrence, or of extended latency, have not been found. Psychogenic effects specifically of BG exposure are not reported. General psychogenic effects of perceived exposure to biological and chemical weapons are found in the supplement under this contract entitled “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Prevention of exposure is conscientious hospital and food hygiene. Treatment involves various regimens of antibiotics; the literature provides inconsistent reports on resistance and efficacy of various antimicrobial agents. Betapropiolactone Betapropiolactone (beta-propiolactone; BPL) bears the chemical formula C3-H4-O2 and is identified by the Chemical Abstracts Service Registry Number 57-57-8. It normally appears as a colorless liquid with a pungent irritating odor. Beta-propiolactone is soluble in water and miscible with acetone, chloroform, and ethanol. Beta-propiolactone has been used as a disinfectant. Capable of sporicidal action, it has been employed in the making of vaccines and in the sterilization of surgical instruments and tissue grafts. Other medical sterilization uses have included the sterilization of blood plasma, water, nutrient broth, and milk. Beta-propiolactone has also served as a versatile intermediate in organic synthesis (acrylic acid and esters). In Project SHAD, it was used as a decontaminant. Beta-propiolactone is quickly hydrolyzed, metabolized, and excreted by mammals. The hydrolysis products excrete rapidly as well. The main metabolite of beta-propiolactone is lactic acid; its main hydrolysis product is hydracrylic acid. The alkylating action of beta-propiolactone reacts with polynucleotides and DNA to form carboxylethyl derivatives, and this process is regarded as responsible for the genotoxicity characteristic of the compound. Beta-propiolactone is a significant irritant to several systems and has shown permanent effects on the eye, liver, and kidney. Since the 1960s awareness has grown of the compound’s high tumorigenic, genotoxic, and carcinogenic toxicity in animals, which have been observed to occur even from single-dose administration. Human epidemiological, case-study, and in vivo experimental reports have not been found, however, except for reports of a series of Henry Ford Hospital volunteer experiments in the 1950s using beta-propiolactone as an anti-hepatitis blood plasma disinfectant and the testing in 1968 of beta-propiolactone as a disinfectant in reaginic sera administered for allergy studies. The Henry Ford studies reported that human acute and chronic risks from intravenous administration are negligible; the reaginic sera study found minor irritations, displayed vesicles, discoloration, and papules in the areas of human skin inoculation. Related animal studies at Henry Ford did find chronic cumulative toxicity in animals, manifested as weight loss and necrosis of kidney tubules and the liver. In acute administration in animals, beta-propiolactone has proven an irritant to skin, eyes, and the respiratory and digestive systems. Dermal contact can elicit blisters and burns. Scarring, erythema, and hair loss have been found on mouse skin after 1–6 administrations of 0.8–100 mg of beta-propiolactone. Ocular administration in rabbits has resulted in pain, miosis, and corneal opacity, which can become permanent. Respiratory exposure is associated with inflammation of the respiratory tract. Oral ingestion can cause stomach and mouth burns. Acute intravenous administration has resulted in liver necrosis and kidney tubular damage. Systemic

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) absorption may result in twitching and gasping, with convulsion and death at higher doses. Frequent urination, dysuria, and hematuria may also attend higher systemic doses. Degradation products from the hydrolysis of beta-propiolactone have been tested. They have been found to be significantly less toxic than beta-propiolactone. A comparison of their LD50s shows toxicity levels of the degradation products to be as much as 5–10 times less toxic than beta-propiolactone. Beta-propiolactone is rated a confirmed animal carcinogen with unknown relevance to humans (Group A3) by the ACGIH (American Conference of Governmental Industrial Hygienists). The Threshold Limit Value (TLV) recommended by the ACGIH is 0.5 ppm (1.5 mg/m3). The NIOSH Pocket Guide to Chemical Hazards considers beta-propiolactone to be a potential occupational carcinogen. The International Agency for Research on Cancer (IARC) regards beta-propiolactone as a possible human carcinogen (Group 2B) and cautions that a single-dose exposure is enough to pose a significant risk of cancer Probably as a result of the fact that beta-propiolactone degrades rapidly in water and plasma, its tumorigenic effects appear to occur primarily around the initial site of exposure. Thus, in tested animals, benign and malignant skin tumors (papillomas, squamous cell carcinomas, keratocanthomas, melanomas; subcutaneous injection-site sarcomas, fibrosarcomas, adenocarcinomas, squamous cell carcinomas), nasal tumors, and forestomach tumors (squamous cell carcinomas) are the observed effects, related to dermal/subcutaneous, inhalational, and oral administration respectively. Meanwhile, beta-propiolactone has been ruled out as an agent causing central nervous system cancer in rats. Single-dose administration has resulted in cancer induction in experimental animals. After single-dose administration of 100 mg beta-propiolactone on suckling mice 9–11 days after birth, lymphomas and hepatomas were induced. Single-dose exposures also have been genotoxic. The genotoxicity of beta-propiolactone has been well studied. Genotoxicity testing indicates a wide range of effects, both in vivo and in vitro. Cell transformation and gene mutations have been observed in human cells in vitro. Bacterial testing has induced gene conversion, aneuploidy, and mutations. In Drosophila, beta-propiolactone produced translocations and sex-linked recessive lethal mutations. In vivo, gene mutations in the stomach and liver of mice and DNA strand breaks in rat bone marrow cells have been reported, along with covalent binding to mouse skin DNA and RNA. The treatment for acute exposure to beta-propiolactone is the standard emergency treatment for a highly irritant chemical, including avoiding emesis and diluting the chemical in the stomach after oral consumption. A possibility for chemoprevention of cancer effects is sodium thiosulfate, which may inhibit beta-propiolactone’s capacity for stomach tumorigenesis. Psychogenic effects of exposure specifically to beta-propiolactone were not found in the literature. General psychogenic effects of perceived exposure to agents involved in chemical and biological warfare are examined in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” An online “glossary” of Project SHAD agents suggests that beta-propiolactone’s carcinogenicity is subject to question due to the absence of adequate controls in experiments. That appears to derive from a comment by the National Toxicology Program (2002) referring to one prior study’s finding of beta-propiolactone induction of keratocanthomas and melanoma in one species. Controls, however, are reported in many studies, and the studies have been generally evaluated as adequate; beta-propiolactone’s animal carcinogenicity is regarded as confirmed by the IARC; the chemical is regularly used to induce animal cancer in controlled tests. Bis Hydrogen Phosphite Bis hydrogen phosphite (BHP), more commonly termed bis(2-ethylhexyl) hydrogen phosphite in the scientific literature, bears the chemical formula C16-H35-O3-P. It is identified by the Chemical Abstracts Service Registry Number 3658-48-8. Bis hydrogen phosphite appears as a colorless liquid with a faint odor. It is commonly used as a lubricant additive to prevent corrosion. In Project SHAD, it served as a chemical warfare agent simulant. No published human studies of any kind, or experimental studies of carcinogenicity, genotoxicity, and reproductive toxicity of bis hydrogen phosphite are known. (There is a 1986 study suggesting that compounds with a 2-ethylhexyl moiety may have a tendency to cause liver cancer in female mice but it did not specifically address

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) bis hydrogen phosphite.) Nevertheless, the Toxicology Division of the U.S. Army Chemical Warfare Laboratories performed several animal studies in acute and subacute exposure to bis hydrogen phosphite in the late 1950s. The tests also evaluated cholinesterase inhibition through a red blood cell assay. The study concluded overall that acute oral and ocular exposure was “relatively innocuous” as also was a cumulative oral exposure of 70 days (Joffe et al., 1958). It found, however, a significant degree of toxic reaction to inhalational, cutaneous, and intraperitoneal and intravenous exposure. The study nevertheless dismissed concerns regarding the latter two pathways because of the unlikely administration of the chemical through those routes into humans. Inhalational exposure of rats and guinea pigs to saturated vapor and mist suggested that both one-time and cumulative exposure could cause significant respiratory distress and tissue injury. Dermal exposure caused a coagulative necrosis on the epidermis and dermis, with repeated exposure inhibiting regeneration. Human skin exposure, reported from accidental hand contact with bis hydrogen phosphite, also induced cases of dermatitis. An assay aimed at cholinesterase inhibition was also performed, testing for any inhibition in exposed rabbits and dogs. No effect was found. Psychogenic effects specifically of bis hydrogen phosphite are not reported. General psychogenic effects of perceived exposure to agents of chemical and biological warfare are examined in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” There is no reported antidote to any of the effects of bis hydrogen phosphite. The Registry of Toxic Effects of Chemical Substances (RTECS) categorizes bis hydrogen phosphite as a “primary irritant,” for which standard medical emergency procedures should be performed, e.g., removal from the area of contact; monitoring and ventilating the victim; irrigating or washing the locus of contact, etc., as appropriate (RTECS, 2004). Bis hydrogen phosphite is barely treated in secondary sources. Where it is, the discussion may be overly dismissive of risk. One Project SHAD information site declares flatly and conclusively that the substance is not carcinogenic. Actually, published studies of human carcinogenicity are unknown, as are animal studies on the same subject. Nor are there found published genotoxicity studies. A commercial distributor advertises for sale bis hydrogen phosphite as “harmless” despite its irritant qualities and absent long-term data (Pfaltz & Bauer Co., 1997). The Hazardous Substances Data Bank (HSDB) does not even report the main animal toxicology studies that have been published. Calcofluor Calcofluor is a member of the class of fluorescent whitening agent. Its chemical formula is C40H42N12O10S2.2Na, and its Chemical Abstracts Service Registry Number is 4193-55-9. Calcofluor has a binding affinity specifically to both cellulose and chitin. Calcofluor is used as a brightening agent for white-colored objects, such as paper, detergents, and textiles. Calcofluor’s chitin-binding specificity makes it a good laboratory stain to detect, identify, and quantitate fungi. Another use for Calcofluor is as a groundwater tracer. In Project SHAD, Calcofluor was used as a fluorescent tracer, along with Bacillus globigii. The toxicity of Calcofluor is low. Oral and dermal toxicity studies show Calcoflour to have relatively low toxicity to fish, mammals, and humans. There is moderate irritation to the eye, as evidenced in rabbit testing. The acute toxicity potential, as indicated by the Lethal Dose/Concentration levels for several species, appears very low. Mice were found to have no abnormalities in body weight, food consumption, survival, appearance, behavior, hematology, clinical chemistry, urinalysis, organ weights, gross pathology, microscopic pathology, or increases in neoplasms after chronic administration of oral Calcofluor. Topical application of Calcofluor to mice, rats, and rabbits elicited no irritation or sensitization. Human volunteers experienced no skin reactions and no mucous membrane reactions at concentrations of up to 1% (Burg et al., 1977). Calcofluor has not been found to be carcinogenic or mutagenic to humans. Phototoxicity studies were also performed, with no adverse reactions found. Psychogenic effects specifically of exposure to Calcofluor have not been found in the literature. General psychogenic effects of perceived exposure to agents of chemical and biological warfare are examined in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.”

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Discrepancies in nomenclature and identification exist. Alternate names (e.g., Fluorescent Brightener 28) appear in the literature but are not consistently applied to the same compound. Coxiella burnetii Coxiella burnetii (CB), the etiologic agent of Q fever, is a pleomorphic, Gram-negative, obligate intracellular coccobacillus, typically 0.2–0.4 μm wide and 0.4-1.0 μm long. In the 1950s, CB was investigated as a potential biowarfare agent and a stock of the microbe was maintained as part of the United States’ biological warfare arsenal until the arsenal was destroyed in the early 1970s. The term “Q fever” was first proposed in 1937, by Edward Holbrook Derrick, the Director of the Laboratory of Microbiology and Pathology of the Queensland Health Department, to describe an outbreak of febrile illness among abattoir workers in Queensland, Australia. Derrick provided infectious material to F. Macfarlane Burnet (who would later win a Nobel Prize in Medicine for work in immunology) who with Mavis Freeman was able to reproduce the disease in guinea pigs, mice, and monkeys as well as visualize an intracellular organism that appeared rickettsial in nature (Burnet and Freeman, 1937). Independently, in 1936 Herald Cox, working at the Rocky Mountain Labs in Hamilton Montana was able to transmit a febrile illness to guinea pigs from ticks collected at Nine Mile, Montana. Cox also demonstrated that the organism displayed properties consistent with a virus or rickettsia and was able to propagate the infectious agent in embryonated eggs. The agent isolated by both groups was shown to be the same microorganism after R. Eugene Dyer, the Director of NIH, became infected with the organism while working at the Rocky Mountain Laboratory. Dyer received material from Burnet and demonstrated that animals infected with Burnet’s Q-fever strain were protected from challenge by strains isolated for his own blood (Maurin and Raoult, 1999). CB is incapable of axenic growth but can be grown in vitro in a number of cell lines including macrophage-like cells, fibroblasts, and Vero cells. Monocytes-macrophages, however, are the only cells CB appears to target in vivo. CB entry into monocytes-macrophages is mediated through interactions between the bacteria’s lipopolysaccharide (LPS) and an integrin complex consisting of alpha(v)beta(3) integrin and CR3, a complement receptor. CB initially enters phagosomes that then fuse rapidly with lysosomes to form large acidic vacuoles. CB appears to require acidic vacuoles for replication. The replication of CB is very slow for a bacterium with a doubling time of approximately 20 hours (Maurin and Raoult, 1999). CB has a complex intracellular lifecycle leading to the formation of both small-cell and large-cell variants. Small-cell variants (SCV; spore-like), the extracellular form of CB, are metabolically inactive and resistant to both chemical and physical inactivation. The bacterium will remain infectious in natural environments for several weeks (Scott and Williams, 1990; McCaul, 1991). In addition to a spore-like transformation, CB undergoes phase variation akin to the smooth-to-rough transition of other Gram-negative bacteria. During acute Q fever the predominant antibody response is to phase II antigens and during chronic Q fever the predominant response is to phase I antigens. There are no morphological differences between phase I and phase II bacteria, but there are differences in the composition of LPS, buoyant density, and affinity for basic dyes. Q fever is a zoonosis with a large reservoir that includes domestic and wild mammals, birds, and ticks. Ruminants, such as goats, sheep, and cattle are the most frequent source of human exposure, but domestic dogs and cats can be a source in urban environments. Many animals appear to be chronically infected but asymptomatic. Chronically infected animals constantly shed CB in their feces, urine, and milk with substantial shedding occurring during parturition. Human transmission occurs principally from aerosols of shedded bacteria, but ingestion of high doses of CB can also result in infection. Q fever is geographically diverse in spread, with epidemics seen throughout the world with the exception of New Zealand (Greenslade et al., 2003). Persons who work with animals, particularly goats or sheep or animal products, are at highest risk of infection. There is an increasing awareness that the prevalence of Q fever is underreported and underestimated (Besalgic et al., 2002, 2003). The study of natural human exposures indicate that approximately 60% of patients infected with CB sero-convert without any clinical manifestations and only 2% are hospitalized after primary infection (Scheld et al., 2001). There are three major clinical manifestations seen in acute Q fever: a self-limited or isolated febrile illness, pneumonia, and hepatitis, and more than one of these manifestations can be seen during a single exposure. The

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) incubation time between exposure and acute clinical manifestations can range from 13–32 days. Both the incubation time and type of manifestation appear to be related to dose, route of exposure, and strain (Williams, 1991). Self-limited febrile illness caused by CB usually consists of a high fever accompanied by a severe headache. Fever typically increases to a plateau of 39 to 40° C over 2–4 days and then rapidly disappears after 5–14 days. Almost all patients who present with Q-fever pneumonia also have fevers and headaches. Fatigue, myalgic and arthralgic pain, chest pains, dry cough, and moderate gastroenteric disturbances are also seen. Q-fever hepatitis is often only detected by increases in liver enzymes. Hepatitis is also frequently accompanied by fever and increases in several cytokines, less frequently by abdominal pain, anorexia, nausea, vomiting, and diarrhea, and occasionally by progressive jaundice. Myocarditis, pericarditis, meningoencephalitis, bone marrow necrosis, hemophagocytosis, hemolytic anemia, transient hypoplastic anemia, erythema nodosum, and skin rash can also be manifestations of acute Q fever. Autoantibodies are also frequently seen during acute Q fever. The route of exposure may also influence the clinical presentation with pneumonia being more common following aerosol exposure and hepatitis being more common following ingestion (Maurin and Raoult, 1999). Although complications such as pyrurria, spleen rupture, rapid fatal pneumonia, encephalitis, acute renal failure, acute respiratory distress, multiple organ failure, and congestive heart failure are occasionally seen, mortality from acute infection is nevertheless low (approximately 1%) (Kazar, 1999; Raoult et al., 2000). Pregnancy can also be compromised; CB causes placentitis with resultant spontaneous abortion, premature birth, and low birth weight commonly seen (Maurin and Raoult, 1999; Raoult et al., 2000; Hellmeyer et al., 2002). Several studies have also indicated that chronic fatigue syndrome is more frequently in acute Q-fever patients 5 years post-infection than in case controls (Ayres et al., 1991). T-cell immunity appears to be largely responsible for the control of CB infections although it is not clear if eradication is achieved in most cases. CB is able to survive within macrophages withstanding low pH and reactive oxygen intermediates. Persistence, recurrence, or reemergence of CB is a constant worry following acute infection. A significant decrease in CD4+ T-cells has been associated with chronic Q-fever endocarditis (Sabatier et al., 1997). A recent large study in France indicates that chronic Q fever will evolve in 1.5% of patients with acute Q fever. Q-fever endocarditis is the most frequent manifestation of chronic Q fever. Vascular and osteoarticular infection, chronic hepatitis and pericarditis, adenopathies, hepatomegaly, splenomegaly, clubbing of digits, purpuric rash, and arterial embolisms are also seen in chronic Q fever. The shift to chronic Q fever is favored in patients with heart valve disease and/or immunosuppression. The death rate for Q-fever endocarditis can be as high as 60% but is substantially reduced if diagnosed and treated early (Maurin and Raoult, 1999; Raoult et al., 2000). Diagnosis is usually performed by serology after a culture-negative presentation of a fever when other possible Q-fever symptoms, exposure risks (e.g., animal contact), and biomarkers are present. Commercial kits that detect antibodies to different phases of CB are available using complement fixation, immunofluorescence, or ELISA formats. Electron microscopy and DNA detection schemes are used in research laboratories. Several of the immunodominant epitopes of CB have now been identified and cloned (Zhang et al., 2004). Doxycycline administered at 200 mg daily over 14 days is the standard therapy for acute Q fever. Fluoroquinolones, macrolides, and co-trimoxazole are also effective alternatives. CB is resistant to both β-lactams and aminoglycosides. The treatment of chronic Q fever is more problematic. Doxycycline/fluoroquinolones combination therapy over an extended period of time was shown to be effective, but relapse rates of over 50% prompted a need for a new therapy. Chloroquine, which raises the pH of acidic vesicles, was chosen to be combined with doxycycline. An 18-month regimen of 100 mg b.i.d. of doxycycline and 200 mg t.i.d. of chloroquine is the currently recommended for the treatment of chronic Q fever (Maurin and Raoult, 1999). A safe, efficacious vaccine against Q fever has been developed in Australia. A formalin inactivated preparation of CB commonly referred to as Q-Vax, prepared from the phase I form of C. burnetii Henzerling strain, appears to provide 100% protection from natural exposure over a period of 5 years (Ackland et al., 1994). This health effects report details the microbiology, epidemiology, clinical course, and treatment of Coxiella burnetii Q-fever infection. (Given the diffuse and evolving state of study of Q fever, it is likely, however, that the last word on CB infection is far from being written.) A presentation of some of the deficiencies in the secondary health effects literature, including federal government advisories on Project SHAD agents, will also be provided. A supplementary table listing CB health effects also follows. A bibliography containing abstracts and some anno-

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) tation concludes this review. A review of possible psychogenic effects arising from the subjective perception of exposure to biological or chemical warfare agents will supplement this report. Diethylphthalate Diethylphthalate (more commonly rendered in the scientific literature as two words “diethyl phthalate”) is a phthalic acid ester with the chemical formula C12H14O4 and commonly identified by Chemical Abstracts Service Registry Number 84-66-2. It ordinarily appears as a bitter-tasting colorless or water-white liquid with no odor, or a slight aromatic odor. It is slightly soluble in water, while also soluble in alcohol, ether, benzene, and acetone. Diethylphthalate is miscible with vegetable oils, esters, and aromatic hydrocarbons. It is manufactured by refluxing one equivalent of phthalic anhydride with a greater than two-fold excess of ethanol in the presence of 1% of concentrated sulfuric acid. It is also classed as a phthalic anhydride ester (PAE). Diethylphthalate is a widely encountered compound in daily life. Automobile parts, toothbrushes, tools, and food packaging are ordinary products in which one can frequently find diethylphthalate. Aspirin, insecticides, and cosmetics can also contain it. The most common industrial use for diethylphthalate is as a “plasticizer”—an agent for making plastics more flexible. In Project SHAD, diethylphthalate was used as a simulant for VX Nerve Agent. Because of its common use in so many household and personal consumer products, exposure through many pathways (oral, dermal, respiratory) has been studied. The Threshold Limit Value for diethylphthalate of the American Conference of Governmental Industrial Hygienists (ACGIH) is 5 mg/m3 based on an 8-hour workday time-weighted average. The pharmacology and kinetics of diethylphthalate exposure indicate slow absorption by the skin, the metabolic conversion of absorbed diethyphthalate into ethanol and the monoester monoethyl phthalate, followed by rapid excretion, mostly in the urine. The effects of diethylphthalate are fairly extensively studied. The chemical shares with other phthalates the characteristic of being among the least toxic of substances in industrial use. In vivo human studies or case reports of serious direct physiological insult as a result of diethylphthalate exposure are not to be found, with the exception of mucous membrane/pulmonary irritation, or a general anesthetic effect at very high concentrations/doses, along with unusual sensitive skin reactions in exceptional sensitized individual cases. An in vitro study on a human skin model did produce a strong cytotoxic reaction, but this has not been duplicated in vivo. Animal studies provide powerful corroboration of diethylphthalate’s low toxicity. Only very high acute oral doses have produced lethality in animals. Otherwise, nontoxic systemic effects usually seen in animal testing are decreased weight gain with alterations in liver and kidney size, likely attributable to hypertrophy. Animal studies indicate that diethylphthalate is only mildly or moderately irritating when applied to the skin or the eye. Evidence of carinogenicity is at best equivocal. In rodent studies a carcinoma/adenoma positive dose-response versus control results was found in only one sex of one species, and the response did not differ significantly from a historical mean for the species and gender. Evidence of genotoxicity is also weak, with only in vitro sister-chromatid exchanges (SCEs) a confirmed effect, but these occurred only in the presence of an S9 fraction from a sensitive species in which a correlation between SCEs and carcinogenicity is regarded as tenuous. Both the Environmental Protection Agency (EPA) and ACGIH regard diethylphthalate to be a substance without evidence of cancer risk [EPA class D; ACGIH class A4]; human case reports or epidemiological study of carcinogenesis from diethylphthalate have not been found. Some concern may exist for toxicity in the reproductive/developmental area. Skeletal abnormalities in rodent offspring have been seen after maternal administration of high doses. Chicken embryos die at a faster rate after direct injection of diethylphthalate. A lowering of testosterone levels in rodents has been seen following diethylphthalate exposure, though no fertility or testicular damage was seen. A lowering of human sperm motility was observed after direct in vitro administration of diethylphthalate. Concerns have been raised on risks to pregnant human females and offspring in light of the detected presence of significant amounts of diethylphthalate in the blood of pregnant women in urban areas. One comprehensive and relatively recent (2001) review of diethylphthalate toxicity concludes that there are ultimately “no toxic endpoints of concern” for the substance in regard to acute toxicity, eye irritation, dermal irrita-

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) tion, dermal sensitization, phototoxicity, photoallergenicity, percutaneous absorption, subchronic toxicity, teratogenicity, reproductive toxicity, genetic toxicity, chronic toxicity, carcinogenicity, and potential human exposure. Psychogenic effects specifically of diethylphthalate exposure have not been found in the literature, but the general effects of a perceived exposure to chemical warfare agents are treated in the supplement provided under this contract entitled “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Secondary literature tends to be comprehensive. It appears that the similarity in names and characteristics of the PAE class may cause confusion in reportage of effects, however. Escherichia coli Escherichia coli (E. coli) is a Gram-negative, rod-shaped, facultatively anaerobic bacterium of the Enterobacteriacae family whose members are sometimes referred to simply as enteric bacteria. Discovered in 1884 by Theodor Escherich, E. coli and its strains are probably the most widely studied of microorganisms. The species is well known as part of the normal human intestinal microflora, where its presence is typically harmless or benignly symbiotic. It has abundant uses in the laboratory, lately finding a new role as a useful cloning host in recombinant DNA technology. In Project SHAD, it was released atmospherically as a simulant to study biological decay rates; the strain is unspecified. Many strains of E. coli, along with nonintestinal exposure to “commensal” bacteria from the intestines, can be harmful, even deadly, however. The microbiology and molecular pathology of the microbe’s virulence is an ongoing subject of intensive study. The effects of one factor in E. coli virulence, the endotoxin liposaccharide (LPS), is an area of particular note as it has, in some studies, shown the potential for long-term effects related to autoimmunity and fever regulation. Currently, classification of the various infectious strains of E. coli is based on a mixture of several considerations—areas of colonization, clinical effects, serotype, and determinants of virulence. For strains of E. coli with intraintestinal pathogenicity, the following are the most noted classes of strains and the pathogenic activity with which they are associated: Enterotoxigenic E. coli (ETEC) —contaminates foods and water, causing diarrhea Enterohemorrhagic (EHEC) strain—synthesizes verotoxin (VT) (shiga-like toxin), which damage the intestinal lining, causing hemorrhagic colitis with its uniquely severe bloody diarrhea (CFSAN, 2003) Enteroinvasive E. coli (EIEC)—creates manifestations similar to the dysentery caused by Shigella, but does not synthesize shiga toxin Enteropathogenic E. coli (EPEC)—damages intestines by adhering to and altering the cellular structure of the lining Enteroaggregative E. coli (EAggEC)—also adheres to the intestinal lining and produces a toxin Enteroadherent E. coli (EAEC)—colonizes and adheres to the small intestine and causes “traveler’s diarrhea” The relatively new strain E. coli O157:H7 has been of special interest over the past two decades. The Centers for Disease Control and Prevention (CDC) devotes a special notice to that strain. Strains that are pathogenic outside the intestinal tracts are called extra intestinal or uropathogenic E. coli. E. coli is also a common nosocomial infection risk. Definitive diagnosis is by culturing the body fluid of the infected area. The effects of E. coli are well-characterized. Acute food poisoning, manifested as nausea and diarrhea, is the most commonly noted effect. “Traveler’s diarrhea” and the Mexican water-borne “Montezuma’s revenge” diarrhea are two very familiar examples. These conditions are usually self-limiting and last a few days. Areas of greater concern in terms of seriousness and duration of effects include infections of the urinary tract and the abdomen and related complications. The spectrum of urinary tract infections (UTIs) ranges from asymptomatic bacteriuria to cystitis to acute and chronic pyelonephritis and renal abscess. The kidney infection pyelonephritis may lead to temporary or chronic renal insufficiency. Although urinary tract infections are more commonly seen in women (where they can be chronic but not serious to overall life and health), they can also occur in men. They are often nosocomial, related to the use of

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) catheters and other invasive/manipulative procedures. Acute and chronic prostatitis, the latter being difficult to treat, are possible manifestations and consequences of E. coli UTIs. Pathogenic E. coli may progress into other systems from the area of colonization. This spread can happen through the blood as bacteremia, and then proceed into sepsis and septic shock. Blood dissemination can lead to infection in other areas of the host. In UTIs, E. coli have been known to proceed to the kidney and induce pyelonephritis. This kidney infection can lead to acute or chronic renal challenge. Severe, complicated pyelonephritis is mainly seen among alcoholic, diabetic, and immunocompromised patients. E. coli pneumonia is usually encountered also as a secondary infection of UTI. Rarely is it known to have arisen from direct exposure, though there have been cases of community-acquired E. coli pneumonia. The nervous system may be directly invaded. Meningitis in neonates is a well-observed effect of E. coli activity; meningitis in adults is, however, far rarer and usually connected with neuroinvasive procedures. (One case study, nevertheless, reports aspergillar sinusitis being associated with recurrent E. coli meningitis episodes.) The appearance of strain O157:H7 (EHEC) since about 1982 has given rise to a new concern over E. coli exposure: namely the complications hemolytic uremic syndrome (HUS) and thrombocytopenic purpura (TTP). Strain O157:H7 infection usually involves a gastrointestinal episode of severe diarrhea with blood in the stool. But in about 10% of these cases, in a matter of days or weeks, endothelial damage further induces microvascular lesions with platelet-fibrin hyaline microthrombi that occlude arterioles and capillaries. The aggregation of the platelets then causes consumptive thrombocytopenia. In the HUS manifestation, the health effects are primarily limited to the kidneys with some possible central nervous system effects. TTP’s effects are primarily of the central nervous system type; they typically include seizures arising from hypertensive encephalopathy. End stage failure and death are possible consequences of HUS; the overall death rate from HUS is 5–15%. Untreated TTP can have a mortality rate of 95%. Symptoms may include thrombocytopenia, fever, renal insufficiency, neurological deficit, microangiopathic hemolytic anemia (MAHA), headache, fatigue/malaise, altered mental status, and hemiplegia. A lesser chronic complication of EHEC strain infection is the risk of irritable bowel syndrome after uncomplicated gastrointestinal infection. Intra-abdominal effects tend to follow puncturing of the peritoneum. These effects, which often are polymicrobial, can lead to abscesses, which are usually accompanied by a low-grade fever and may proceed to septic shock, pylephlebitis of the portal vein, and liver abscess, as well as cholecystitis and cholangitis. Partial obstructions in the biliary system can be a greater risk for infection than full obstructions. Peritonitis is a common consequence of E. coli penetration of the peritoneum. Other noted E. coli infection effects include endophthalmitis (usually associated with diabetic patients suffering from UTI or pyelonephritis), osteomyelitis, endocarditis, septic arthritis, and skin, soft tissue, and surgical wound injuries. Special attention is called to lipopolysaccharide (LPS) endotoxin activity and possible associations it may have with long-term effects on the immune and immune regulatory systems. (LPS forms part of the outer cell wall of Gram-negative bacteria, including nonpathogenic laboratory strains like K-12.) Animal tests suggest that neonate exposure can lead to a diminution of fever response to a subsequent adult challenge from LPS. LPS has also been shown to have possible associations with the initiation of autoimmune joint disorders and in the induction of autoimmune diabetes. Studies or reports of clinical psychogenic health effects resulting specifically from exposure to E. coli have not been found. General psychogenic effects of perceived exposure to agents of biological (and chemical) warfare are examined in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Preventive measures center on proper hygiene. No standardized treatment for E. coli infections exist; treatment is site and severity specific. Infection management usually includes intravenous hydration. The employment of antimicrobials in strain O157:H7 infection are not recommended because they may worsen the condition. Under development is the use of neutralizing human antitoxin antibodies, which appear to have a protective role in HUS.

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Methyl Acetoacetate Methyl acetoacetate bears the chemical formula C5H8O3 (structured CH3COCH2COOCH3) and has a molecular weight of 116.11. Its Chemical Abstracts Service Registry Number is 105-45-3. Its common alternative name is “Acetoacetic acid, methyl ester.” Its density/specific gravity is 1.0762. At room temperature, the chemical is a colorless liquid with an agreeable odor. Its most common use is in the fragrance industry. Methyl acetoacetate was used as a simulant for sarin in at least two tests over the course of Project SHAD. Methyl acetoacetate is generally regarded as being a mild to moderate irritant to the skin and mucous membrane, but with the capability (sometimes overlooked in secondary sources) of severe corrosive effect on the eye if directly contacted. The ocular exposure effect has been demonstrated in one earlier rabbit study. Secondary sources indicate gastrointestinal difficulties (nausea, vomiting, diarrhea) if it is swallowed, based upon the general characteristics of irritant toxic chemicals. Other effects extrapolated from general effects of irritant substances include swelling, redness, and pain at any dermal site of exposure, but also particularly on mucous membranes. Mouth, nose, and eyes are especially susceptible. Irritants also commonly cause cough, tachypnea, and wheezing after inhalation exposure. There do not appear to have been any published studies of chronic exposure. Recently, however, two Japanese research laboratories have examined methyl acetoacetate’s toxicity with greater thoroughness and a more updated focus on mutagenicity and carcinogenicity. They obtained results generally consistent with earlier studies on the questions of acute exposure. They also found no indication of mutagenicity or carcinogenicity. One mutagenicity test did yield a tentative finding of genotoxicity, but this was explained, after failure to replicate the effect in confirmation testing, to be the result of methyl acetoacetate’s alteration of the test medium’s pH. Methyl acetoacetate is also nowhere reported as a carcinogen. One noticeable aspect of the literature on methyl acetoacetate has been the presence of significant discrepancies or omissions in the major secondary sources when compared with the primary studies or earlier secondary studies. These include (1) listing the wrong animal species used in a study, (2) providing dose figures not stated in the study being reported on, (3) offering a possibly misleading description of the animal lethality of one inhalation test, (4) omitting note of the substantial ocular toxicity of methyl acetoacetate, and (5) failing to update with later studies, including especially the recent Japanese laboratory studies. Issues of this type exist in the sources on methyl acetoacetate toxicity recommended by the Department of Defense and extend to such standard or authoritative sources as Toxnet’s HSDB (Hazardous Substances Data Bank), Patty’s Toxicology, RTECS (during its existence as a publicly-owned resource), and the Merck Index. Phosphorus-32 Phosphorus-32 [32P] was first synthesized in the 1930s. It has a physical half-life of 14.3 days and emits a relatively high-energy β particle. It was the first synthetic radionuclide to be used for human therapy. The isotope has found wide use as a tracer element in both biological and chemical studies. 32P is one of only six radionuclides classified as a human carcinogen. The classification is primarily due to its ability to cause leukemia in polycythemia (PV) patients. Sodium [32P] phosphate is currently a treatment of choice for PV and essential thrombocythaemia (ET) in the elderly; it is also used to treat bone pain from metastatic disease. Chromic [32P] phosphate and other forms of 32P have been used to treat a number of conditions. Sodium [32P] phosphate tends to concentrate in the bone, liver, and spleen and has a whole-body biological half-life of 39.2 days. Overdoses of sodium [32P] phosphate result in haematological disorders such as aplasia, agranulocytosis, and severe thrombocytopenia. 32P has been shown to cause cancer when locally deposited in animals. Sodium [32P] phosphate has also been shown to cause low sperm counts and thyroid and blood disorders in animals. There is still controversy on whether a dose-response exists for the induction of leukemia and whether 32P would cause leukemia in the general population. Leukemia is typically seen 5–15 years after exposure. Single exposures can result in chronic effects. Occasional side effects of intraperitoneal instillation of chromic [32P] phosphate have included bone marrow depression, pleuritis, nausea, and abdominal cramping.

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Acute high-exposure responses are nonstochastic. These acute effects usually appear quickly and can result in burns and radiation sickness. The symptoms of radiation sickness can include nausea, weakness, hair loss, skin burns, and diminished organ function. At higher levels and exposure durations, system collapse, intestinal lining destruction, bleeding, and death can occur. Eye lens damage from external exposure also can occur, as indicated by the 15 rem yearly limit on eye radiation exposure. Sarin In 1936 German chemist Gerhard Schrader discovered that an organophosphate compound, ethyl dimethylphosphoramidocyanidate (later called tabun), was a potent insecticide. Dr. Schrader reported his discovery to German authorities, who then set up a laboratory for Schrader to further pursue toxic nerve agents for military purposes. In 1938, Schrader along with some associates, synthesized 1-methylethyl methylphosphonofluoridate. It was named sarin, after the chemists Schrader, Ambrose, Rüdige, and van der Linde, who were responsible for its synthesis. Sarin is a chemical warfare nerve agent, which is described by the chemical formula C4H10FO2P and is identified by Chemical Abstracts Service Registry Number 107-44-8. Under normal conditions it is a colorless and odorless liquid. It is miscible in both polar and nonpolar solvents, and it hydrolyzes slowly in water at neutral or slightly acidic pH. Sarin is significantly less stable to hydrolysis than VX. Sarin’s hydrolysis products are considerably less toxic than the original agent. The synthesis of sarin’s chemical class, the organophosphosphates, dates back to 1820. Widespread poisoning by organophosphates was first seen in the United States in early 1930, when many people developed a strange paralytic illness traced to a Prohibition-era alcohol substitute, called Jamaican Ginger or Jake, which had been adulterated with tri-ortho-cresyl phosphate (TOCP). TOCP was the first chemical proven to show a delayed type of neurotoxicity. The use of chemical warfare agents is ancient, but their most extensive use occurred during World War I when chlorine and mustard gas inflicted over 1 million casualties. Nazi Germany later produced large amounts of the organophosphate agent tabun along with far lesser amounts of sarin (1,000 lb) throughout World War II, but they were not known to be used. In 1950, the U.S. Army’s Chemical Corp began the construction of plants for the full-scale production of sarin but ceased in 1957 because stockpile requirements were met. The only confirmed military use of nerve agents in history was by Iraq, which used tabun and sarin aerial bombs to repel Iranian troops. In the latter part of the war, Iraq’s extensive use of chemical warfare agents is believed to have brought an end to the conflict. Reports claim that between 5,500 to 10,000 Iranian troops were killed by nerve agents and mustard gas, and up to 100,000 soldiers were exposed. In March of 1988, Iraq used a combination of chemical weapons, including mustard gas, tabun, sarin, VX, and possibly even cyanide to kill as many as 5,000 people in the Kurdish town of Halabja. Iraq is believed to have produced between 790 to 810 tons of sarin, which degraded or were destroyed after the Gulf War. The first known terrorist use of a nerve agent involved sarin and occurred in Matsumoto City, Japan, on the evening of June 27, 1994. About 12 liters of sarin were released using a heater and a fan from the window of a delivery truck. The attack was undertaken to kill four judges involved in a dispute with the Aum Shinrikyo cult. There were 471 victims of sarin poisoning; 54 were hospitalized and 253 were treated at outpatient facilities. Seven died. On March 20, 1995, Aum Shinrikyo launched an even bolder attack on the subway system in Tokyo. At 8:00 a.m., at the height of rush hour, sarin was released. Twelve subway passengers were killed. About 980 persons suffered mild to moderate exposure, and 500 persons were hospitalized. Over 5,000 people, many of whom were not actually exposed, sought medical attention. The largest experimental use of sarin on humans appears to have occurred at Porton Down in the United Kingdom in the 1950s. The purpose of the studies was to obtain precise information on the toxic properties of these agents. Certain experiments went terribly wrong. One man died 45 minutes after 200 mg of sarin were dripped onto a uniform patch on his forearm.

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) The lungs are particularly susceptible to both the chronic and acute effects of SO2. Acute reactions to the compound, which typically occur at levels higher than the odor threshold and standard permissible levels, include irritation, bronchoconstriction, asthma-like symptoms, and respiratory distress. Asthmatics can be particularly susceptible to the pulmonary effects of SO2. Permanent impairment of lung function, particularly in the form of reactive airways dysfunction syndrome (RADS), chronic pulmonary disease, or Chronic Obstructive Pulmonary Disease (COPD), can result from exposures to high enough levels; asthmatics may suffer enhanced sensitivity. SO2 may also cause damage to developing fetuses and to the reproductive system. The testes in particular appear to be especially vulnerable to permanent toxic effects, indicated from both animal and human data. Chronic exposures to elevated SO2 levels are associated with increases in cerebrovascular and heart disease, pulmonary disorders, increased morbidity and mortality, and low birth weights. At the cellular/molecular level, SO2 decreases levels of antioxidant enzymes, increases membrane permeability, causes chromosome breakage, and is mutagenic or comutagenic. There exists evidence of a possible correlation between elevated SO2 levels and increases in cancer. While evidence suggests sulfur dioxide to be a co-carcinogen, there is insufficient evidence to show that it causes cancer directly. (The International Agency for Research on Cancer (IARC) finds SO2 to be not classifiable as to its carcinogenicity to humans [IARC Group 3], citing inadequate or limited evidence of carcinogenicity from either human or animal studies.) Psychogenic health effects of perceived exposure to sulfur dioxide have been speculated to have occurred during pollution scares. Respiratory and cardiovascular diseases were proportionately increased in one incident although it could not be ruled out that the increase was from other causes. Information on the general psychogenic issues and effects of perceived exposure to biological or chemical warfare agents is contained in the supplement report under this contract, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Recommended treatments for sulfur dioxide exposure include 2% sodium bicarbonate sprayed into the air as well as inhaled into the lungs to neutralize its effects. Other treatments for SO2 exposure include s-carboxymethylcysteine for asthmatics; theophylline, zafirlukast (a leukotriene receptor antagonist), and albuterol for patients with a specific allergy. Trioctyl Phosphate Trioctyl phosphate (TEHP), more commonly known as Tris(2-ethylhexyl) phosphate, bears the chemical formula C24-H51-O4-P and is identified by the Chemical Abstracts Service Registry Number 78-42-2. It normally appears as a colorless viscous liquid possessing a low vapor pressure. It is soluble in alcohol, acetone, and ether but insoluble in water. Tricotyl phosphate is ordinarily used as a plasticizer or fire retardant. It is commonly employed as a component of vinyl stabilizers. More than 10 million pounds of TEHP is produced worldwide each year. In Project SHAD, TEHP was used as a simulant for the chemical warfare nerve agent VX. A National Toxicology Program (NTP) set of studies on TEHP was performed in 1984 and serves as the main source for TEHP toxicology. Its overall profile was of a substance with little toxic risk, though with some areas of concern. Those areas related to positive carcinogenic indications from certain chronic animal tests, and to mild acute irriation effects. The report also included a subchronic dog and rhesus monkey study that suggests chronic lung injury is possible due to continuous inhalation exposure. Mammalian acute toxicity of TEHP tends to be very low, with median lethal oral animal doses exceeding testing levels in rats and mice. Acute findings indicate TEHP induces mild temporary irritation on the skin, eye, and respiratory systems. Moderate erythema on shaved skin has been reported for rabbits. Effects on the eye are usually mild, with animal studies showing very mild irritant effects or a causing temporary and moderate conjunctivitis in rabbit (Draize) testing. Acute inhalation exposure is only harmful at high doses with continuous exposure. Wistar rats experienced no mortality at a concentration of 287–460 mg/m3 for 30 minutes. Guinea pigs experienced about 30% mortality at the same concentration after 60 minutes, which increased to 80% after 2 hours. Human studies and case reports are not found in the published literature, with the exception of an NTP skin test on human volunteers, which resulted in no signs of significant skin irritation. Chronic and subchronic studies in

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) animals did show a mild chronic inflammation in dog lungs after 3 months of regular exposure to up to 85 mg/m3. Other than that effect, which was restricted to dogs, no dogs or rhesus monkeys (the other tested animal) showed any signs of toxic effect. Neurotoxicology testing indicates no inhibition of cholinesterase activity, and no signs of delayed neurotoxicity. Cytotoxicity and micronucleation was not found in a series of rat exposures to aerosolized trioctyl phosphate. Trioctyl phosphate is not classified anywhere as a human carcinogen. There is also no evidence of genotoxicity. Bacterial tests (Salmonella tester strains TA 98, TA 100, TA 1535, TA 1537) showed no signs of mutagenicity regardless of the presence of S9 liver fraction. Tests for sister-chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells have also been negative for genotoxicity. Hyperplasia in thyroid follicular cells has been observed in rodents in a 2-year study. Weight loss was also reported in rats and mice after long-term exposure, but it was not found to be harmful or to have resulted from toxic action. Some evidence of possible carcinogenicity has been found in the increase of hepatocellular carcinomas in female-only B6C3F1 mice in one NTP 2-year gavage test. Equivocal evidence has also been found in the dose-related presence of pheochromocytomas appearing in some male rats. The evidence from the studies has been deemed insufficient to establish a significant risk of human carcinogenicity. Four factors were decisive in that assessment: the neoplastic tumors occured in only one sex of one species, hepatocellular carcinoma tumors are considered rare in general, genotoxicity evidence is absent, and the background incidence of pheochromocytomas in rats is too variable to establish the significance of the tumor’s appearance in the non-control rodents. (Some studies suggest that 2-ethylhexanol, a metabolite of TEHP, as well as an ingredient of its manufacture and a characteristic component of chemicals with the 2-ethylhexyl moiety, may constitute a factor in any TEHP carcinogenic potential.) Psychogenic effects specifically of trioctyl phosphate are not known. General psychogenic effects of perceived exposure to agents of chemical and biological warfare are examined in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Treatment of exposure to trioctyl phosphate is the standard regimen of assistance to anyone exposed to a general or unknown toxic substance. Laboratory facilities involved in caregiving ought to monitor the affected person’s complete blood count and perform urinalysis if necessary. Liver and kidney function tests are suggested for patients with significant exposure. In cases of respiratory tract irritation or respiratory depression, the caregiver should monitor arterial blood gases, and chest x-rays, and perform pulmonary function tests. Secondary sources do not appear to contain significant errors or oversights in treating the toxicology of TEHP, although Patty’s Toxicology contains no separate monograph on trioctyl phosphate. The Hazardous Substances Data Bank of Toxnet at the National Library of Medicine contains a scattering of not clearly organized or updated information. For example, at one point it cites in all capital letters an outdated assertion that there are no long-term toxicity studies of trioctyl phosphate. Pasteurella tularensis Pasteurella tularensis is currently known as Francisella tularensis, which is the term employed throughout this report. Its newer name derives from the one developed by U.S. Public Health Service physician and scientist Edward Francis who pioneered the study of the microbe and its associated affliction, tularemia. Francis’s work on infectious pathology would result in his nomination for a Nobel Prize, as well as his failure to follow through on that nomination process due to his hospitalization from the effects of another infectious agent he acquired from his dogged field and laboratory research. Ultimately considered a possible pathogen for Cold War–era biowarfare, Pasteurella tularensis was eventually renamed Francisella tularensis to give Francis his due, an effort initiated and pursued by admiring scientists from America’s Cold War enemy, the Soviet Union. Francisella tularensis is a Gram-negative small pleomorphic facultative intracellular coccobacillus. It is a zoonosis, most associated with tick bites or being in contact with infected animal carcasses or meats. Transmission of the pathogen to humans in an aerosol or dust form is also possible and is the likely method for bioterrorism or biowarfare use. Culturing a sample of F. tularensis can be dangerous (biosafety level 3 is the usual laboratory

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) requirement), and so determination of the pathogen’s presence is typically performed by serology. An agglutin titer greater than 1:160 is the standard determinant. Generally, however, those levels are not reached until close to the second week of infection. A skin test developed by the U.S. Army (Active E-rosette test) has a high degree of specificity but also can yield a positive result over 3 decades after infection and illness by Francisella tularensis. The incubation period of tularemia normally falls within a 3–6-day range but shorter and longer periods have been observed. When not asymptomatic, the infection usually presents as an acute febrile illness, along with some or all of the following generalized symptoms: chills, headaches, weight loss, emesis, diarrhea, muscle aches, joint pains, dry cough, hepatitis, and jaundice in serious cases. The fever is often biphasic, peaking twice in the first month of debilitation. In general, the full course of the illness is 1 month of fever, 1 month of complete weakness, and 1.5 months of gradual but complete recovery. More extended infections have been reported to have durations lasting for several months to a few years. Only one case exists in the literature, however, in which a person continued to manifest recurrences (fever and ulcerations) over a clearly observed period (by the National Institutes of Health) lasting over a decade, and with no complete recovery ever recorded. Another older report also exists of an acute and atypical case that involved a peripheral neuropathy in which the elderly patient could no longer dorsflex his foot, and this ability was not known to have subsequently returned. No case has been found of a person who first manifested symptoms many months to years after initial infection. This is so despite the likelihood of a long-term persistence of some Francisella tularensis pathogens in previously diseased individuals. In light of this, it is not surprising that tularemia is normally considered a strictly acute disease granting extraordinary immunity, if one survives it. In pre-antibiotic times, death rates of about 20% were reported, associated particularly with pre-existent health debilitations, delays in seeking treatment, and septicemia. In more recent times, this rate has been reduced to less than 4% through therapeutic intervention. Locally and systemically, tularemia manifests acutely in several syndromes, often related to the manner of contact and inoculation. These syndromes are the ulceroglandular, the glandular, the oculoglandular, the pneumonic, the oropharyngeal, and the typhoidal. The rare typhoidal form is more deadly than the others, and also the most likely to result from aerosol contact. Respiratory involvement and lymphadenitis is very common in all varieties, however, though patients may not always present overt respiratory troubles. In the most common syndome, the ulceroglandular (as well as the oculoglandular and glandular syndromes), local lymphadenopathies, skin eruptions, and ulcerations are the common manifestations of tularemia in addition to the generalized symptoms. The manifestations in those syndromes typically occur at the place of initial inoculation (e.g., the eye in the case of oculoglandular tularemia). F. tularensis has an affinity for the skin, lymph system, lungs and, to a lesser extent, liver. Differential diagnoses include “ulcer node” syndrome, rat-bite fever, cat-scratch disease, mycobacterial infection, chancroid, chancre, nocardiesis, sporotrichosis, cutaneous anthrax, inhalational anthrax, Erysipelothrix, pneumonic plague, influenza, mycplasma pneumonia, staphylococcal/streptococcal lymphadenitis, Legionnaire’s disease, Q fever, bacterial pneumonia, brucellosis, Listeria, syphilis, lymphogranuloma venereum, scrub typhus, and plague. Reflecting tularemia’s protean manifestations, cases have been known to also present atypical signs and effects involving systems beyond the more common ones described above. Some neuropathies (peripheral and central) are reported, and meningeal and meningoencephalitic involvements have occurred (especially among children). Pericarditis, typically among those with pre-existing cardiac impairments, is an unusual but nevertheless well-known complication of tularemia. Cardiac complications tend to resolve with recovery from tularemia infection. Recovery tends to be complete after the acute period of about 3.5 months, but cases of greater duration are known. Abdominal involvement is rare, but liver and spleen enlargement, sometimes with systemic jaundice, does occur. Tularemic disorders of the gastrointestinal tract are relatively rare; enteritis and appendicitis are mentioned in the literature but not as significant effects. Psychogenic effects specific to F. tularensis exposure have not been reported. The general question of possible psychogenic effects arising from the awareness of exposure to chemical and biological warfare agents is contained in the supplement, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Treatment for tularemia is usually the early administration of aminoglycoside antibiotics. Streptomycin and gentamicin are the common therapeutic agents. A vaccine (LVS—“live vaccine strain”) was developed at Ft. Detrick

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) in the 1960s but it has proved only of limited effectiveness, primarily against the typhoidal form of tularemia and a weaker strain of F. tularensis. The secondary literature, including that of the Department of Defense, does not offer significant contradictions to each other or to the information in the literature on tularemia. They acknowledge that it is an acute disease with no significant demonstrated long-term or late developing effects, but nevertheless they note that it can be serious and life-threatening, especially if untreated. Uranine When Johann Strauss composed the classic waltz, the “Beautiful Blue Danube” in 1867, he could not have known that, only 10 years later, a famous part of the blue Danube—its “sinks” in the upper river region—would turn green. The color change would be temporary and artificial, however, as it was the result of one of the first uses of fluorescein, a fluorescent tracer dye. Soon thereafter its more water-soluble sodium salt would circulate under the industrial name Uranin or Uranine. The dye would go on to have enormous and still-continuing important medical and environmental uses. Uranine dye is known in more scientific circles as sodium fluorescein (or fluorescein sodium), as well as disodium fluorescein. It has the chemical formula C20-H12-O5-2Na and the Chemical Abstracts Service (CAS) Registry Number 518-47-8. The name fluorescein is often used carelessly and interchangeably with the various compounds derived from fluorescein, including sodium fluorescein/uranine. In this report, therefore, the term uranine is used to mean specifically the sodium salt of fluorescein (CAS #518-47-8). The term fluorescein is used to mean the acid compound, fluorescein, which is identified by the CAS #2321-07-5 and has the formula C20-H12-O5. Uranine is freely soluble in water and alcohol; after dissolution it emits a bright yellowish-green fluorescence, especially under blue light. This indicator dye tends to appear more green the more alkaline the medium. Its use in ocular therapy is long-established: first synthesized in 1871, by 1882 uranine was being used as an injected dye for examining ocular fluid dynamics in cases of glaucoma. In 1959, it saw its first use in its most widespread application, intravenous fluorescein angiography, considered a vital advance in the examination of the pathophysiology of retinal diseases. Uranine is also used in topical ocular diagnostic and therapeutic applications. Uranine is useful as a dye to trace cerebrospinal fluid leaks during surgery. Outside of medical uses, uranine is also used to trace the flow of subterranean waters. It functions also as a dye in cosmetics. In Project SHAD, uranine dye was used as a tracer for the biological agent Stapphylococcal Enterotoxin Type B. Increasingly, it is used as a tracer for the activity of inhaled particulates. Typically, injected uranine takes less than 20 seconds to circulate in the blood stream. When absorbed, uranine is rapidly metabolized thorough glucuronidation in the liver. 80% of the dose is usually metabolized within in 1 hour. The pharmacodynamics and toxicodynamics of fluorescein are not well understood. Animal studies show very low toxicity. At very high doses, death occurs from CNS depression, and one study suggests sensitivity to light exposure. There exists a great deal of clinical data on the effects of injected uranine. Systemically, the common responses to injection range from a nontoxic yellowing of the skin to acute severe reactions up to and including (in very rare circumstances) mortality. The adverse effects of fluorescein angiography are usually grouped into three broad categories: mild, moderate, and severe. Males appear to be more susceptible to adverse effects than females. The main mild adverse effects are transient nausea, vomiting, local pruritus, extravasation, and some allergic reactions. Urticaria, lowered pulse rate. syncope, dyspnea, and local effects at the injection site and region (thrombophlebitis, subcutaneous granuloma, neuritis) are among the more moderate reactions. The more severe reactions include respiratory effects like laryngeal edema, pulmonary edema, bronchospasms, anaphylaxis along with certain cardiac effects like basilar artery ischemia, circulatory shock, myocardial infarction and cardiac arrest. Tonic-clonic seizure is a noted neurologic reaction. Death can occur, though very rarely, about one case being reported per year. The main risk factor in such reactions appears to be a prior adverse reaction to uranine treatment. The main noted risk factor in a fluorescein angiography appears to be a prior adverse event. Local administration affects certain systems in observed ways. Topical ocular administration has produced transient discoloration and conjunctival chemosis. This occurred only when accompanied by active inflammatory

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) disease. When uranine has been employed intrathecally as a tracer for cerebrospinal fluid leaks in surgery, suboccipital punctures have resulted in cases of grand mal seizure, which did not seem to occur when suboccipital punctures were stopped. Lumbar administration has yielded severe neurotoxic signs: temperature elevation, headache, nausea, vomiting, dizziness, nuchal pain, and grand mal seizures. Increasing interest in inhalation drug therapy has resulted in the use of uranine in pulmonary exposure experiments. The kinetics of such exposure include very rapid absorption by the lung and without any significant metabolism inside the lung. No studies or reports of toxic effects from this type of exposure have been found. A recent correspondence from a leading investigator in the field reports that although there is an absence of existing studies on the toxicity of inhalation exposure to uranine, studies with aerosolized uranine have been ongoing for several years in European hospitals with no untoward clinical effects of any kind known. The only known studies of carcinogenicity go back to two tests in Japan in the 1950s. Cancerous tumors at the application site were elicited after chronic application of large concentrations of uranine. These results have been deemed equivocal evidence only of tumorigenicity by the Registry of Toxic Effects of Chemical Substances (RTECS). A screen for the carcinogenic/mutagenic potential of compounds using DNA cell binding assay gave inconclusive results for uranine. Other results from a genetic toxicity screen to predict carcinogenicity, Salmonella/ microsome mutagenesis, chromosome aberration, sister-chromatid exchange, and mouse lympoma mutagenesis assay were compared for consistency to assess DNA damage from chemicals. Uranine yielded either negative or equivocal results for tumorigenicity and genetic toxicity and positive activity both with and without exogenous metabolic activation for sister-chromatid exchange. Neither uranine nor fluorescein has been found by the International Agency for Research on Cancer (IARC), or any other authoritative agency, to be carcinogenic. No human cancer effects reports or studies have been found. Psychogenic reactions brought on by the manner of uranine administration have been suggested to explain some adverse effects. A variation in response to fluorescein angiographies by gender has been noted in that regard. Observed reactions like syncope, hypotension, and lowered pulse rate (vasovagal effects) have been suggested to arise from the nature of the treatment, which is the internal injection of a discoloring and glowing substance while cameras are brought to peer into the inner eye along with strange bodily effects (e.g., skin discoloration) that can occur. Other psychogenic issues, such as the general stressor reactions to perceived exposure to a contaminant in biological and chemical warfare testing, are treated in the supplement under this contract, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Standard prophylaxis is to have an emergency tray and oxygen supply handy when a uranine procedure is performed. It has been shown that persons with allergic sensitivities benefit from a prophylactic administration of antihistamines. Epinephrine followed by diphenhydramine hydrochloride may be necessary for patients who have a hypotensive reaction. Secondary sources (outside the field of ophthalmology) do not contain a great deal of data on uranine except in the context of fluorescein angiography. The confusing and careless interchangeable use among fluorescein, sodium fluorescein, and the term uranine (dye) can render research problematic. The Hazardous Substances Data Bank conflates acid fluorescein with the disodium salt fluorescein (i.e., uranine) in the same entry. Patty’s Toxicology contains only a brief reference to fluorescein angiography and no section on fluorescein or sodium fluorescein (uranine). VX Nerve Agent VX nerve agent (VX) is a chemical warfare nerve agent. Its chemical formula is C11H26NO2PS. Its formal chemical name is O-Ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate. Due to the existence of several isomers, VX has several Chemical Abstracts Service Registry Numbers: 50782-69-9, 51848-47-6, 53800-40-1, and 70938-84-0. VX is an organophosphate compound and it belongs to the specific class of compounds known as the phosphonothiocholines. The “V” in VX stands for “venom,” a tribute to this compound class having high potency and a characteristic ability to penetrate the skin. At normal temperatures, it is an oily liquid of low volatility with viscosity similar to motor oil.

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Ranaji Ghosh first synthesized VX in the early 1950s. The British government noted VX’s potential as a warfare agent and shared its research with the U.S. Army Edgewood facility. Eventually large quantities of VX were produced through the 1960s at a Newport Indiana facility. Some stocks still remain there and on other bases and were slated for destruction in 2004. The Soviet Union developed a related compound called Russian VX [O-Isobutyl S-(2-diethylamino) methylphosphonothioate]. VX has been the subject of accidental releases and controlled releases, and has been used as a weapon. The largest reported accidental release occurred at Utah’s Dugway Proving Grounds on March 13, 1968, when approximately 9 kg of VX drifted over adjacent grazing land, killing over 6,000 sheep. There was also an accidental release of a nerve agent (sources conflict on whether VX was involved) at a storage facility in Okinawa in 1969, which resulted in the hospitalization of 23 military personnel and 1 civilian. In Project SHAD at least two test releases on ships have been reported. In addition to releases by the U.S. Army, VX was used by the Aum Shinrikyo cult in Japan to kill several dissident members and others opposed to the cult. It may have also been used by Iraq as part of a cocktail in the Iran–Iraq war and to quell Kurdish uprisings in the 1980s. U.S. troops were exposed to nerve agents during destruction and disposal operations in the Gulf War, though VX is not reported to be among those agents. VX is a potent and selective inhibitor of acetylcholinesterases (AChE), which results in the accumulation of acetylcholine in the synapses of both central and peripheral nerves. VX, in contrast to other nerve agents inhibits AChE significantly more than plasma cholinesterases. VX exposure and action results in intense stimulation of nicotinic, muscarinic, and central nervous system (CNS) receptors. Toxic effects are generally seen when over 50% of the AChE enzyme is inhibited. Death typically occurs when over 90% of the AChE enzyme is inhibited. Death is usually due to inhibition of the enzyme in the brain and diaphragm. The increased amounts of acetylcholine in the brain produced by VX exposure leads to the release of large amounts of excitory amino acids, which stimulate NMDA receptors and result in neuronal toxicity. Seizures typically occur when 25–75% of AChE is inhibited and always occur during exposure to supralethal doses. Convulsions without treatment can lead to permanent neurological damage. In addition to the inhibition of acetylcholinesterase, VX has been shown to bind to and block postjunctional glutamate receptors, nicotinic acetylcholine receptor-ion channels, and muscarinic acetylcholine receptors. The role of receptor binding and inhibition in toxicity is not clear. Studies in mice in which the acetylcholinesterase gene has been knocked out indicate that other targets of organophosphates may play a major role in toxicity and lethality. The toxic effects of VX can be grouped around the types of nerve receptors overstimulated by acetylcholine. The muscarinic effects are typically miosis, headaches, blurring of vision, rhinorrhea, bradycardia, anorexia, nausea, vomiting, diarrhea, increased sweating, and lacrimation. The nicotinic effects are typically fatigue, muscular twitching, cramps, and paralysis of muscles (including respiratory muscles). The acute CNS effects are typically generalized weakness, cyanosis, hypotension, convulsions, loss of consciousness, coma, and death. Longer-term CNS effects including anxiety, insomnia, tremor, headaches, drowsiness, difficulty in concentration, memory problems, confusion, slurred speech, and ataxia have been associated with organophosphate poisoning but not VX specifically. There currently are no commercial test kits that diagnose VX exposure. Diagnosis is from signs and symptoms. Gas chromatography coupled with mass spectrometry (GC-MS) can detect metabolites of VX in both urine and serum. Several tests have been developed that attempt to identify nerve agent poisoning through the quantification of cholinesterase activity in blood. The monitoring of AChE activity is a reliable marker for systemic toxicity. Systemic toxic effects are seen in approximately 50% of subjects when 75% of red blood cell AChE is inhibited. A more recent test relies on the ability of potassium fluoride to reactivate enzymes such as butyryl-cholinesterase and release fluorinated compounds. This technique can be used to monitor low levels of exposures and unambiguously identify both nerve agents and pesticides. VX is considered to be the most toxic of the nerve agents developed for chemical warfare. Course, symptoms, and relative toxicity, however, can vary considerably by exposure route and dose. The human dermal LD50 (Lethal Dose) is estimated to be as low as 0.04 mg/kg; human inhalation LCt50 (Lethal Concentration) is estimated to be 36 mg · min/m3. By inhalation, it is twice as lethal as sarin. It is also 10 times more toxic in inducing miosis. VX

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) is at least 100 times more toxic than sarin as a percutaneous agent due to its low volatility, its stability, and its lipophilicity. The effects of exposure by inhalation usually occur within minutes. Miosis, rhinorrhea, and airway constriction are initially seen at low to moderate concentrations. Larger doses of VX result in loss of consciousness, seizures, cessation of cardiac and respiratory activity, and death in the absence of medical treatment. Neuropsychiatric effects including loss of memory and depression are also seen but are relatively short-lived following exposure to VX. The onset of symptoms can take hours when sublethal doses are applied to the skin. A small drop may initially cause localized muscle twitching and sweating, followed by nausea, vomiting, diarrhea, and generalized weakness. These symptoms typically last for several hours. Systematic dermal studies in humans showed vomiting occurred in 33% and 67% of subjects when red blood cholinesterase activity was 30–39% and less than 20% of control activity. Other studies have shown that a dose of 5 mg/kg of VX resulted in systemic toxicity in roughly half of the subjects. Persons whose skin is exposed to higher doses of VX may show no symptoms for up to 30 minutes, but then rapidly suffer loss of consciousness, convulsions, difficulty breathing, profuse secretions from nose and mouth, generalized muscle twitching, paralysis, and death. At lethal and near-lethal levels of exposure loss of consciousness, convulsions, flaccid paralysis, and apnea are seen. At high doses there is also a more rapid onset of signs and symptoms. Clothing, site of skin exposure, and temperature can greatly affect the nature and toxicity of dermal exposure. Animal studies have indicated VX can cause cardiac effects, although these effects have not been seen in human volunteer studies. Arrhythmias were seen both in rats and dogs at doses that did not result in convulsions. Electrophysiological studies using guinea pig heart tissue showed that VX exposure led to a positive inotropic effect, two contractile events in response to each stimulation, and the development of delayed after-depolarizations. VX cardiac toxicity has been attributed to the inhibition of the rat cardiac Na+,K(+)-ATPase alpha 1 isoform. Few studies have addressed long-term toxicity or effects of nerve agents in general and VX in particular. Textbooks indicate that most if not all of the effects of nerve agents dissipate within months after exposure. A recent telephone survey of over 4,000 volunteers who had participated in programs that involved exposure to chemical agents between 1955 and 1975 at the Edgewood facility found fewer attention problems as the only statistically significant differences between those exposed to nerve agents and those exposed to other chemical agents but it also found greater sleep disturbances in volunteers who had been exposed to nerve agents. VX differs from other nerve agents in that it does not appear to undergo aging or stabilization but does undergo spontaneous reactivation. Unlike many other organophosphates, VX also has not been shown to induce a syndrome called organophosphorus-induced delayed neuropathy (OPIDN). OPIDN results from the inhibition of the enzyme neuropathy target esterase (NTE; also termed neurotoxic esterase). VX has been reported to be at least 1,000 times less effective than sarin in inhibiting NTE. The failure of VX to inhibit neuropathy target esterase and cause organophosphorus-induced delayed neuropathy together with the inability to “age” when bound to AChE or other proteins indicates that VX may not cause much of the long-term toxicity associated with other organophosphates. VX has tested negative in a number of assays for mutagenicity, with and without metabolic activation. Human studies in personnel working with VX on a daily basis found no increased incidence of cancer. The teratogenic potential of VX has also been evaluated in sheep, rats, and rabbits; all have all been negative for teratogenicity. VX has not been deemed a carcinogen by any authority. In regard to long-term neurotoxicity, VX has not been shown to have delayed or persistent psychological effects or to result in any long-term EEG changes. OPIDN has not resulted from VX exposure. Convulsions without treatment can lead to permanent neuropathogical damage. Brain damage has been seen in animals injected with VX. Microinjections of VX into the amygdala resulted in convulsions and resultant neuropathology. Much of the brain damage that has been observed is believed to be due from the induction of convulsions and not the direct toxic actions of VX. Studies on neuroblastoma cells have indicated that VX displays some toxicity presumably through binding to muscarinic receptors. No studies have been found addressing purely psychogenic effects arising from an awareness of, or a perception of, exposure to VX specifically. But the use of another organophosphate agent (sarin) in terror attacks in Japan in the 1990s has led to some investigation and consideration of the possible psychogenic effects of exposure to a nerve agent. Discussion of those reports appear in the review prepared under this contract for the health effects

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) of sarin. Information on the general psychogenic effects of perceived exposure to biological or chemical warfare agents is contained in the supplement report under this contract, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” There have been several approaches towards the treatment of, and protection against, VX exposure. Barrier methods, including garments, respirators, and even protective creams have been developed that will protect against even high levels of VX exposure. The use of reversible inhibitors of AChE to protect against subsequent exposure to nerve agents has been pursued extensively by the U.S. military. Pyridostigmine bromide was used by a large number of troops during the Gulf War to protect against possible exposure to soman and other nerve agents. Several studies since then have implicated pyridostigmine as a potential contributory factor in the induction of Gulf War Syndrome, a multi-symptom illness found in a number of veterans who served in Iraq. Other reports have since questioned its utility in protecting against VX exposure. Several other agents have also been proposed for prophylaxis against nerve agent exposure. Both physostigmine and hyoscine has been reported superior to pyridostigmine in preventing the death of animals following VX exposure. Huperzine has also been found to be a more effective prophylactic agent than pyridostigmine. In contrast to other prophylactic agents, huperzine does not inhibit butyryl-cholinesterase (plasma), which can then still act to scavenge nerve agents. To prevent mortality and minimize morbidity, aggressive medical intervention should be pursued following nerve agent exposure. Thorough decontamination should occur immediately following suspected exposure. Casualties should be decontaminated as fast as possible but should not be moved into clean treatment areas until decontamination is complete. Bleach should be used extensively to decontaminate any area or material where exposure has occurred. Atropine sulfate, an anticholinergic agent, should be administered as soon as possible following decontamination. Oxygen or oxygen-rich air should be used for ventilation if available. Oximes, such as pralidoxime salts, should also be administered as soon as possible to regenerate AChE enzymes. Early intervention to prevent or treat convulsions is also an essential component in the treatment of nerve agent poisoning. Imidazenil, a partial selective allosteric modulator of GABA action, has been shown to be more effective than diazepam in protecting rats against organophosphate-induced convulsions and death. Secondary literature on VX generally adequately covers its well-known lethality and toxicity. Researchers ought to be cautioned to note that VX, due to varied isomers, has multiple CAS Registry Numbers. Zinc Cadmium Sulfide Zinc cadmium sulfide (ZnCdS) is a brightly fluorescent, stable compound formed by sintering ZnS (zinc sulfide) and CdS (cadmium sulfide). ZnCdS has several CAS (Chemical Abstracts Service) Registry Numbers. It is used in pigments, and its fluorescence is employed as a visualization agent for applications such as histology and nanotechnology. It was used in Project SHAD as a tracer for chemical and biological warfare agents because it was regarded to be a harmless dye. Very little is published about its health effects. What little there is suggests minimal toxicity. Older studies found that ZnCdS did not induce deaths in dogs or rats despite extraordinarily high oral doses. No epidemiological, clinical, or case studies demonstrating deleterious effects from exposures were found. Personnel who had been most exposed during tests of the compound in the United Kingdom did not show unusual or discernible health consequences. The National Research Council (NRC) published a book-length report in 1997 on ZnCdS toxicity arising out of public concern over the exposure of civilian populations to the compound during U.S. Army biological warfare testing in the 1950s and 1960s. The NRC found little literature existing on the subject and concluded that toxic effects of the compound are highly unlikely as the substance is insoluble and very unlikely to become bioavailable. Nonetheless, the NRC proceeded on a “worst-case” assumption that if ZnCdS were to degrade into its original sintered components, the most harmful product would be CdS. The report then focused upon the toxicological effects of CdS. It concluded that the amount of cadmium that people were exposed to in the trials was too low to pose a significant health risk. A follow-up study by the U.S. Army concluded that particulate ZnCdS remained intact in rats after pulmonary

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) exposure and supported the NRC supposition that the compound was poorly bioavailable. ZnCdS was found to pass through the alveolar walls via macrophage action, but Zn and Cd were found present in the kidneys only in small amounts, were barely present in the liver, with no significant increase found in the blood. Proportionate (though slow, over 14 weeks) removal of Zn and Cd from the lungs indicated that the compound did not fragment; low liver and no significant blood levels of ZnCdS further argued against bioavailability. Some lung clearance was mucociliary in nature. Some minor local and transitory toxic effects were noted: lung and lymph node inflammations, accumulations of foreign bodies in the lung, and altered enzyme, protein, and cell count levels. The experimental doses tested (on a body weight relative basis) far exceeded (at least by a factor of 500) the highest level of human exposure in previous U.S. Army tests. No other health effects were reported. Other literature and uses of ZnCdS indicates that it lodges in capillaries after administration into the bloodstream. This is perhaps a factor to consider if ZnCdS is capable of passing into the bloodstream through the alveolar epithelium or other means. A review of cadmium toxicity as a “worst-case” scenario (rendered less likely in light of the finding of zinc cadmium sulfate’s lack of degradation and lack of bioavailabilty in the rat) reveals concerns over cancer, particularly lung cancer, although the high level of human carcinogenic potential of conventionally assumed to be the case has lately been challenged. CdS, the main toxic component of ZnCdS, has been shown to be genotoxic, and recent studies show clastogenesis. Possible effects of acute exposure to cadmium include acute chemical pneumonitis or metal fume fever. There is typically no inflammatory response to cadmium sulfide (in contrast to observed effects of ZnCdS in the rat lung). Renal toxicity has been noted and long-term exposure to cadmium, even at low doses, damages kidney tubules and results in renal dysfunction. No psychogenic effects of exposure to ZnCdS are reported. General reactions to perceived exposure to agents in biological and chemical warfare uses can be found in the supplement under this contract, “Psychogenic Effects of Perceived Exposure to Biochemical Warfare Agents.” Secondary source literature is sparse and multiple CAS numbers and terminological variations complicate searching. The CAS number used by the NRC is used by NIOSH to identify a product called “Cadmium Sulfide, Solid Soln. With Zinc Sulfide Silver Chloride-Doped” while “Cadmium Zinc Sulfide” is identified as 12442-27-2. British documents prefer to render “sulfide” as “sulphide.” No published specific handling instructions for ZnCdS were found. REFERENCES Ackland, J. R., D. A. Worswick, and B. P. Marmion. 1994. Vaccine prophylaxis of Q fever. A follow-up study of the efficacy of Q-Vax (CSL) 1985-1990. Medical Journal of Australia 160:704-708. Ayres, J. G., N. Flint, E. G. Smith, W. S. Tunnicliffe, T. J. Fletcher, K. Hammond, D. Ward, and B. P. Marmion. 1991. Post-infection fatigue syndrome following Q fever. QJM: Monthly Journal of the Associations of Physicians 91:105-123. Beslagic, E., S. Hamzic, S. Zvizdic, T. Bajrovic, and R. Velic. 2002. Laboratory diagnosis of Q-fever with the indirect immunofluorescence test. Medicinski Arhiv 56(2):89-92. Beslagic, E., S. Hamzic, S. Puvacic, and S. Cavaljuga-Hotic. 2003. Q-fever serologic diagnostics with inhabitants of Canton of Sarajevo 2001 year. Medicinski Arhiv 57(2):71-74. Burg, A. W., M. W. Rohovsky, and C. J. Kensler. 1977. Current status of human safety and environmental aspects of fluorescent whitening agents used in detergents in the United States. CRC Critical Reviews in Environmental Control 7:91-120. Burnet, F. M., and M. Freeman. 1937. Experimental studies on the virus of “Q” fever. Medical Journal of Australia 2:299-305. CFSAN (Center for Food Safety and Applied Nutrition). 2004. Escherichia coli O157:H7. http://vm.cfsan.fda.gov/~mow/chap15.html (accessed January 23, 2007). Greenslade, E., R. Beasley, L. Jennings, A. Woodward, and P. Weinstein. 2003. Has Coxiella burnetii (Q fever) been introduced into New Zealand? Emerging Infectious Diseases 9:138-140. Hellmeyer, L., G. Schmitz-Ziegler, W. Slenczka, and S. Schmidt. 2002. Q fever in pregnancy: A case report and review of the literature. Zeitschrift fur Geburtshilfe und Neonatologie 206:193-198. Joffe, M. H., L. E. Gongwer, and C. L. Punte. 1958. Studies on the acute and subacute toxicity of bis(2-ethylhexyl) hydrogen phosphite. AMA Archives of Industrial Health 18(6):464-469.

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Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense) Kazar, J. 1999. Q-fever current concept. In Rickettsia and rickettsial diseases at the turn of the third millenium, D. Raoult and P. Brouqui (eds.). Paris, France: Elsevier. Pp. 304-319. Lee, E. C. 2003. Clinical manifestations of sarin nerve gas exposure. Journal of the American Medical Association 290(5):659-662. Maurin, M., and D. Raoult. 1999. Q fever. Clinical Microbiology Review 12(4):518-553. McCaul, T. F. 1991. The developmental cycle of Coxiella burnetii. In Q-fever: The biology of Coxiella burnetii, J. C. Williams and H. A. Thompson (eds.). Boca Raton, FL: CRC Press, 223-258. NRC (National Research Council). 1997. Review of acute human-toxicity estimates for selected chemical-warfare agents. Washington, DC: National Academy Press. http://www.nap.edu/readingroom/books/toxicity/ (accessed January 25, 2007). National Toxicology Program. 2002. Beta-Propiolactone. Report on Carcinogens 10:207-208. Pfaltz & Bauer Co. 1997. Bis(2-ethylhexyl) hydrogen phosphite 94%. http://www.pfaltzandbauer.com/cgi-bin/details.pl?type=chemname&order=item&chem=&category=&item=B13840/ (accessed January 23, 2007). Raoult, D., H. Tissot-Dupont, C. Foucault, J. Gouvernet, P. E. Fournier, E. Bernit, A. Stein, M. Nesri, J. R. Harie, and P. J. Weiller. 2000. Q fever 1985-1998. Clinical and epidemiologic features of 1,383 infections. Medicine (Baltimore) 79(2):109-123. RTECS (Registry of Toxic Effects of Chemical Substances). 1997. Phosphonic acid, bis (2-ethylhexyl) ester. RTECS # SZ6840000. http://www.cdc.gov/niosh/rtecs/sz685ec0.html (accessed January 23, 2007). Sabatier, F., F. Dignat-George, J. L. Mege, C. Brunet, D. Raoult, and J. Sampol. 1997. CD4+ T-cell lymphopenia in Q fever endocarditis. Clinical and Diagnostic Laboratory Immunology 4:89-92. Scheld, W. M., W. A. Craig, and J. M. Hughes. 2001. Q fever: Queries remaining after decades of research. In Emerging infections, W. M. Scheld, W. A. Craig, Hughes (eds.). Washington, DC: ASM Press. Pp. 29-56. Scott, G. H., and J. C. Williams. 1990. Susceptibility of Coxiella burnetii to chemical disinfectants. Annals of the New York Academy of Science 590:291-296. Williams, J. C. 1991. Infectivity, virulence and pathogenicity of Coxiella burnetii for various hosts. In Q-fever: The biology of Coxiella burnetii, J. C. Williams and H. A. Thompson (eds.). Boca Raton, FL: CRC Press. Pp. 21-64. Zhang, G., K. Kiss, R. Seshadri, L. R. Hendrix, and J. E. Samuel. 2004. Identification and cloning of immunodominant antigens of Coxiella burnetii. Infection and Immunity 72:844-852.

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