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Guidelines for Organophosphorus Nerve Agents

INTRODUCTION

The organophosphorus nerve agents have been used as chemical warfare agents for over 50 years (Dunn and Sidell, 1989; Somani, 1992). This class of compounds includes tabun (Agent GA), sarin (Agent GB), soman (Agent GD), and Agent VX. These agents are lipid-soluble organic compounds that rapidly inhibit the enzyme acetylcholinesterase (AChE), and AChE inhibition leads to inhibition of nerve-muscle impulse transmission. In addition to phosphorus, organophosphorus nerve agents contain cyanide (GA), fluoride (GB and GD), or sulfur (VX).

Daniels (1990a) compiled a detailed review of the properties and fate of organophosphorus nerve agents and their health effects in animals and humans. Because of its volatility, GB is an effective toxicant by the inhalation route, whereas the relatively low volatility of VX makes it more effective following dermal exposures. All four compounds are toxic by ingestion, but only GB and VX have been thoroughly investigated for their fate in water. GB is soluble in water and dissolves rapidly (Epstein, 1974). GB hydrolysis to two strong acids, isopropyl methylphosphonic acid and hydrofluoric acid, is dependent on pH. At increased pH levels (between 6.5 and 14), hydrolysis is mediated by hydroxide-ion catalysis. However, the hydrolysis rate of GB is at a



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Guidelines for Chemical Warfare Agents in Military Field Drinking Water 3 Guidelines for Organophosphorus Nerve Agents INTRODUCTION The organophosphorus nerve agents have been used as chemical warfare agents for over 50 years (Dunn and Sidell, 1989; Somani, 1992). This class of compounds includes tabun (Agent GA), sarin (Agent GB), soman (Agent GD), and Agent VX. These agents are lipid-soluble organic compounds that rapidly inhibit the enzyme acetylcholinesterase (AChE), and AChE inhibition leads to inhibition of nerve-muscle impulse transmission. In addition to phosphorus, organophosphorus nerve agents contain cyanide (GA), fluoride (GB and GD), or sulfur (VX). Daniels (1990a) compiled a detailed review of the properties and fate of organophosphorus nerve agents and their health effects in animals and humans. Because of its volatility, GB is an effective toxicant by the inhalation route, whereas the relatively low volatility of VX makes it more effective following dermal exposures. All four compounds are toxic by ingestion, but only GB and VX have been thoroughly investigated for their fate in water. GB is soluble in water and dissolves rapidly (Epstein, 1974). GB hydrolysis to two strong acids, isopropyl methylphosphonic acid and hydrofluoric acid, is dependent on pH. At increased pH levels (between 6.5 and 14), hydrolysis is mediated by hydroxide-ion catalysis. However, the hydrolysis rate of GB is at a

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water minimum at pH levels of 4-6.5, where the reaction occurs between GB and water molecules. Increasing the temperature accelerates the hydrolysis process; however, the temperature effect will be greater for hydrolysis under alkaline conditions than for hydrolysis under acidic conditions (Epstein, 1974). Somani (1992) lists the half-lives of VX and GB (at 25°C) as 350 days and 5.4 hr, respectively. VX hydrolyzes in water very slowly and therefore is more stable in water than GB. Information on the solubility of GD is limited; it might be more persistent in water than GB. Like that of GB, a principal hydrolysis product of GD is reported to be hydrofluoric acid. There is little information on the solubility of GA. Data on the toxicity of the hydrolysis products of GB suggest that the toxicity is negligible; at 200 ppm, the products were not toxic. Performance degradation would be unlikely to occur in military personnel consuming field water containing these products over a 7-day exposure period (Epstein, 1974; Daniels, 1990a). The toxicity of the hydrolysis products of VX has not been studied in detail. A report prepared by Small (1983) summarizes the toxicological data of the two hydrolysis products of VX that might occur in water (i.e., bisdiisopropylaminoethylaminosulfide and S-diisopropylaminoethyl methylphosphonic acid). The limited data suggest that hydrolysis of VX to these products does not necessarily mean that water containing these products will be potable. Consequently, water that has been contaminated with VX should always be treated before consumption by military personnel. Data on the toxicity of the hydrolysis products of GA or GD are not available. TOXICITY The organophosphorus nerve agents are among the most toxic synthetic substances. The 24-hr LD50s (via the subcutaneous route) range from 20-165 µg/kg for soman to 43-158 µg/kg for sarin in laboratory animals, such as rabbits, guinea pigs, and mice. By comparison, organophosphorus insecticides, such as parathion, guthion, and malathion, are toxic only at doses exceeding tens to thousands of milligrams per kilograms. Toxic effects observed in humans and animals acutely exposed to organophosphorus nerve agents are excessive bronchial, salivary, ocu-

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water lar, and intestinal secretions. Other responses are sweating, bronchospasm, intestinal hypermotility, bradycardia, muscle fasiculations, twitching, weakness, paralysis, loss of consciousness, tension, anxiety, restlessness, insomnia, convulsions, and depression of central respiratory drive (Namba et al., 1971; Murphy, 1975; Rickett et al., 1987; Dunn and Sidell, 1989). The route and rate of exposure strongly influence the intensity and duration of action of the organophosphorus nerve agents. The inhalation route is associated with the greatest toxicity. Inhalation of vapors or aerosols can result in toxic effects within seconds to 5 min of exposure (Somani, 1992). After percutaneous exposure to a large amount of agent (an LD50 or greater), enzyme inhibition and onset of effects occur within 1-30 min—the time being inversely related to the amount of agent (Somani, 1992). EXPOSURE AND BIOLOGICAL MONITORING There is a relationship between the toxicity of organophosphorus nerve agents and AChE inhibition. However, data are insufficient to predict the risk accurately in humans exposed to low doses of these nerve agents, and since most investigators use AChE inhibition as a measure of exposure rather than more direct measures, there are multiple sources of uncertainty. The World Health Organization reports that a 50-70% reduction of plasma or red-blood-cell-AChE activity in workers exposed to organophosphate pesticides justifies the removal of workers from further exposure (WHO, 1975). Gage (1967) argues that, regardless of the health of an individual, if an individual's AChE activity falls below a certain percentage of normal, further exposure should be prevented. The use of AChE measurements to signal the healthfulness of the work environment is well established and provides useful data concerning worker exposures to anticholinesterase agents (Lauwerys and Hoet, 1993). The abundant data on AChE activity permit the study of the relationship between clinical toxicity, AChE inhibition, and exposure. AChE measurements alone, however, are not sufficient to predict toxic thresholds. There are several factors that affect AChE inhibition.

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water Among these are the form of AChE affected (Sussman et al., 1991), route of administration of the inhibitor (Grob and Harvey, 1953), alterations in blood flow to target tissues (Maxwell et al., 1987), stereochemistry of the inhibitor (Benschop and de Jong, 1989), pharmacokinetics of the inhibitor (Reynolds et al., 1985), species (Martin, 1985), and health (Dillon and Ho, 1987). In general terms, the AChE status of a population can be used as an indicator of the magnitude and duration of exposure, but there is a need to augment enzyme-inhibition data with direct chemical measures of exposure to develop more protective health standards. In addition, any military field drinking-water standard must be more conservative than the standards applied in industry and agriculture because the nature of the chemicals and their potential use as chemical warfare agents is in direct contrast with the goals and objectives of a workplace hygiene program. For example, AChE monitoring in agriculture is designed to protect workers from excessive exposure and AChE monitoring in the military is designed to protect against impaired performance by personnel in a situation that might already be life-threatening. FIELD DRINKING-WATER STANDARDS McNamara et al. (1973) and McNamara and Leitnaker (1971) investigated the toxicity and fate of the chemical warfare agent VX. Variables considered in their model included the initial concentration, the removal rate, and the time to reach 50% concentration in the blood (effective biological half-life). Estimation of either the daily dose of an organophosphorus nerve agent or the accumulated effective dose is expressed as the percentage decrease in AChE activity. Daniels (1990a) reviewed the removal rates and potencies of the organophosphorus nerve agents. The resulting interim maximum permissible concentrations (MPCs) for organophosphorus nerve agents in field drinking water were based on the relationship between AChE inhibition and toxicity. Table 3-1 contains Daniels's estimated MPCs for 50% inhibition as well as the subcommittee's MPCs for 25% inhibition for each of the four organophosphorus nerve agents. The concentrations were calculated on the basis of depression of red-blood-cell AChE not exceeding either 50% or 25% of normal red-blood-cell-AChE activity. The

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water TABLE 3-1 Maximum Permissible Concentrations for Organophosphorus Nerve Agents a   Consumption Rate and Corresponding MPC, µg/L Agent 5 L/day 15 L/day     AChE depression not > 50% VX 15.0 5.0 GD 12.0 4.0 GB 28.0 9.3 GA 140.0 46.0     AChE depression not >25% VX 7.5 2.5 GD 6.0 2.0 GB 13.8 4.6 GA 70.0 22.5 aAssumes a 70-kg person consuming field drinking water at 5 L/day or 15 L/day for up to 7 days. Sources: Daniels (1990a) and the subcommittee's estimate of 25% AChE inhibition. MPCs assume a 70-kg person consuming field drinking water at 5 L/day or 15 L/day for up to 7 days. The MPCs for 25% inhibition of AChE are advocated as standards at this time based on the limited dose-response data available and on the likelihood that AChE inhibitions adversely affecting performance are smaller than previously thought. Use of AChE to develop a standard results in an extremely conservative value; however, it is justified, given the need to protect health and assure against decreased battlefield performance. Using available data, Saady (1991) computed equivalent dose estimates of the organophosphorus nerve agents that would be expected to be immediately dangerous to life or health (IDLH) via inhalation exposure in humans (Table 3-2). IDLH concentrations are defined as 30-min air concentrations that would produce such signs and symptoms as tight-

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water TABLE 3-2 Comparison of Estimated Doses and Proposed Field Drinking-Water Standards Agent 30-Min IDLH Inhalation Dose, mg/m3 Estimated IDLH Oral Dose, µg/kga 24-hr Water Dose, µg/kgb VX 0.04 0.7 0.5 GD 0.06 1.1 0.4 GB 0.18 3.2 1.0 GA 0.18 3.2 5.0 aMinute volume (2.5 m3/hr) × time (0.5 hr) × IDLH (mg/m3)/70 kg. bDrinking-water volume (L/day) × drinking-water concentration (µg/L)/70 kg. Sources: Saady (1991) and subcommittee calculations. ness of the chest, headache, runny nose, and miosis. For comparative purposes, inhalation exposures are considered more analogous to intravenous exposures than to ingestion or dermal contact (Somani, 1992). With respect to route and rate of exposure, absorption of organophosphorus nerve agents from the gastrointestinal tract is expected to be intermittent during waking hours. Thus, the extent of absorption from the gastrointestinal tract is considered to be substantially less than the extent of absorption following inhalation exposures (Grob and Harvey, 1953). To compare the total body dose allowed by the 30-min IDLH to the total dose allowed by the proposed drinking-water standards for 1 day, the subcommittee converted both the air-concentration IDLH (in milligrams per cubic meter, Table 3-2) and the proposed drinking-water standard (in micrograms per liter, Table 3-1) to equivalent dose estimates in micrograms per kilogram. To convert the IDLH to dose equivalents, assume a moderate minute volume (2.5 m3/hr) for 30 min for a 70-kg person. Assuming that 100% of the inhaled chemical is retained and absorbed, the dose equivalent can be estimated; these values are provided in Table 3-2, column 3. Please note that in the absence of agent-specific data characterizing retention and absorption of the human lung, the subcommittee has made the protective default assumption of 100% retention and absorption for purposes of calculation. It is likely that some fraction <100% is actually retained during inhalation exposure.

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water To change the corresponding proposed drinking-water standards to dose equivalents, the subcommittee used the proposed MPC at 25% AChE inhibition (Table 3-1) times the ingested water volume per day (either 5 or 15 L) for a 70-kg person. Assuming that 100% of the orally ingested chemical is absorbed, the dose equivalent can be estimated; these values are provided in Table 3-2, column 4. The dose equivalent provided by the proposed drinking-water standard is less than the IDLH dose equivalent for VX, GD, and GB by 29%, 64%, and 69%, respectively (Table 3-2, columns 3 and 4). The proposed drinking-water-standard dose equivalent for GA is higher than the IDLH dose equivalent for two reasons. First, the IDLH estimate is conservative because GA and GB are considered to be equally toxic (Saady, 1991). Available data for GA are sparse. Chresthull (1957) reported the LCt50 (the product of concentration and time that produces 50% mortality) of GA to be 71%, 63%, and 40% of the LCt50 of GB in the mouse, rat, and monkey, respectively. Therefore, in the three animals studied, GA was found to be 29-60% less toxic than GB by inhalation. Comparable human data are not available. Second, Daniels (1990a) describes the estimated value for potency (k) used in the pharmacokinetic model for GA (kGA = 1) to be 20% of the value for GB (kGB = 5). Based on the animal studies of Chresthull (1957), the subcommittee observed that the Daniels (1990a) assumed value for kGA is low, resulting in an inflated MPC estimate for GA in water. Based on the work of Chresthull (1957), the subcommittee estimates that the k value for GA likely approximates 60% of the k value for GB (i.e., kGA = 3). Because oral ingestion of GA is considered less toxic than inhalation of GA and because the assumed exposure time is 24 hr per day (for a maximum of 7 days), as opposed to 30 min for the IDLH exposure time, it is the subcommittee's view that the proposed field drinking-water standards for organophosphorus nerve agents (Table 3-1) are sufficiently protective. CONCLUSIONS AND RECOMMENDATIONS It has been over 20 years since Hayes (1975) suggested that concurrent biomonitoring of AChE activity and urinary metabolites could yield a complimentary analysis of the status of persons exposed to organophosphorus chemicals. Measurements of AChE inhibition are indicative of

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water recent exposure experiences. Similarly, urinalysis would reveal day-to-day exposure experience because of the rapid clearance of metabolites in the urine. Until data similar to those called for by Hayes (1975) are developed for the organophosphorus nerve agents, the subcommittee recommends that guidelines for exposure to organophosphorus nerve agents in field drinking water be based on an estimated 25% inhibition of AChE. Hayes (1982) pointed out that the lower limit of statistical reliability in measuring changes in red-blood-cell-AChE activity is 20%; changes that are less than 20% cannot be detected reliably. As shown in Table 3-1, use of AChE to develop standards results in conservative interim standards. However, conservative standards might be justified since drinking-water standards are based on data obtained from human exposures (Daniels, 1990a) and are designed to protect the health and combat readiness of troops in the field. The Army's proposed standards for organophosphorus nerve agents are based on 50% AChE inhibition. It proposed standards of 4 µg/L and 12 µg/L for a water consumption of 15 L/day and 5 L/day, respectively. The subcommittee, however, disagrees with the Army's approach of using 50% AChE inhibition as the basis for the standards. Clinical signs and symptoms of toxicity of organophosphorus nerve agents have been reported to occur in some individuals at 50% inhibition of AChE. In addition, a 50% inhibition of AChE might be associated with performance degradation in healthy adults. To accommodate for the biological variability inherent in red-blood-cell acetylcholine measurements (up to a 2-fold difference) and the possibility of confounding effects from exposure to other anticholinesterase chemicals, to assure against decreased battlefield performance, and to protect previously sensitized individuals, the subcommittee selected an AChE inhibition level of 25% as a definite NOAEL. It should be noted that the lowest level of statistical reliability in measuring AChE changes is approximately 20%; changes that are less than 20% cannot be detected reliably. Based on the available data, the subcommittee recommends that the 25% AChE inhibition level be used as the basis for the field drinking-water guidelines for organophosphorus nerve agents and recommends the following guidelines for the organophosphorus nerve agents: GA, 22.5 µg/L and 70.0 µg/L; GB, 4.6 and 13.8 µg/L; GD, 2.0 and 6.0 µg/L; and VX, 2.5 and 7.5 µg/L—assuming a water consumption of 15

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water and 5 L/day, respectively. The subcommittee concludes that these guidelines are appropriate until the results of future research indicate that 25% AChE inhibition is inadequate or overly conservative. In addition, the subcommittee identifies the following research needs: Dose-Response Relationship. The use of AChE inhibition alone is not an adequate quantitative indicator of exposure to organophosphorus nerve agents. Daniels (1990a) reviewed the multiple factors affecting the measurement of AChE activity in animals and humans. Based on that review, only the most conservative field drinking-water standards are acceptable pending development of more dose-response data directly relating the toxicity of organophosphorus nerve agents to exposure. Extensive studies during the past 15 years with structurally related methyl and ethyl organophosphate insecticides have shown the corresponding dialkyl phosphates to be suitable biomarkers in 24-hr urine specimens. The biomarkers are stable and readily derivable to analytes, which can be measured in parts-per-billion amounts. The use of those biomarkers as indicators of exposure needs further investigation. Evaluation of Model. Daniels (1990a) noted that the calculated MPCs must be evaluated further. Supporting data are needed to (1) confirm the use of AChE activity as an indicator of the potential for an individual to develop adverse health effects, (2) ascertain the uncertainties about individual variability, and (3) confirm the use of butyryl cholinesterase recovery data both for indicating tissue recovery and for deriving permissible exposure levels. Those three factors should be evaluated using enzyme and chemical biomarkers (see “Dose-Response Relationship” above). Medical Doctrine for Pretreatment. The current military medical doctrine requires that military personnel with potential for exposure to organophosphorus nerve agents undergo treatment with pyridostigmine bromide both before and during a battlefield exposure (Dunn and Sidell, 1989; U.S. Army, 1990b). Pyridostigmine bromide is a carbamate that binds reversibly to AChE. It is administered prophylactically and is intended to preserve enough AChE to allow a person to survive exposure to organophosphorus nerve agents. The effectiveness of pyridostigmine bromide should be investigated further. Performance Criteria. It is recognized that the present set of

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Guidelines for Chemical Warfare Agents in Military Field Drinking Water interim standards are conservative, and it is only speculative that they could represent the threshold above which significant impaired performance could occur (Daniels, 1990a). However, performance criteria for military personnel operating complicated equipment, including aircraft, weapons systems, and heavy machinery, are critical to the subcommittee's recommendation that field drinking-water guidelines based on 25% AChE inhibition be adopted for the entire class of organophosphorus nerve agents.