Review of the Department of Defense Research Program on Low-Level Exposures to Chemical Warfare Agents (2005)

Historically, research related to chemical warfare agents (CWAs) has focused on life-threatening battlefield effects caused by high-level exposure to CWAs. In contrast, there are limited data on the adverse health effects associated with exposure to low concentrations of CWAs. Concerns have been raised about the potential health effects of longer-term exposure to CWAs at concentrations lower than those needed to produce effects associated with high concentrations. Such concerns have become a priority of the U.S. Department of Defense (DOD). The threat of low-level CWA exposure includes the following:

• Downwind from or at the periphery of a CWA attack or release.

• Entry or reentry into an area after a CWA release.

• Exposures that occur during decontamination operations or from secondary contamination due to incompletely decontaminated material, supplies, and so forth.

• Exposures after planned but inadequate or improper destruction of CWA munitions.

In response to those concerns, the DOD Low-Level Chemical Working Group was formed to develop research programs within the DOD Chemical and Biological Defense Program to understand the adverse health effects of low-level exposure to CWAs, to defend against low-level exposure, to prevent unnecessary duplication of research efforts, and to focus and direct scientific investigations to address operational issues. The DOD Master Research Plan (Research Plan) devel-

oped for this effort addresses research on operationally relevant performance decrements and delayed adverse health effects that potentially might be associated with low-level exposure to CWAs.

The stated objective of the Research Plan is to characterize the toxicity of CWAs to enable rational military decision making for issues related to doctrine, training, materiel, leadership, personnel, and facilities. The Research Plan includes consideration of the following: (1) decision making in operational risk management for the range of sensitivities needed for detectors, sensors, and alarms; (2) efficiency needed for individual and collective protection systems; (3) effectiveness of decontamination measures and procedures; (4) restoration of normal military operations; (5) return of previously contaminated materiel to “normal” use; (6) operationally relevant performance decrements and adverse long-term health sequelae; (7) as yet unrecognized outcomes of exposure; and (8) medical diagnostics, prophylaxes, pretreatments, and treatments.

The Research Plan describes DOD’s planned research on low-level exposure from Fiscal Year 2002¹ (FY 2002) to FY 2007 that has been or is to be conducted within the existing framework of the DOD Chemical and Biological Defense Program. Every study proposed under the Research Plan is designed to answer one mandatory question: How do data from this work contribute materially toward a quantitative refinement of the human health risk assessment for low-level CWA exposure? The Research Plan is based on three major research thrusts:

• Characterize concentration-time relationships for low-level and longer-time CWA vapor exposures.

• Identify alternative, but physiologically significant, toxicologic end points.

• Conduct appropriate integration studies linking experimental data sets with predictive human health-effect risk assessments.

In section 247 of the 1999 Defense Authorization Act, the U.S. Congress directed the Secretary of Defense to review and modify DOD policies and doctrines that relate to protecting personnel from low-level exposure to CWAs. In response to that congressional mandate, the Secretary of Defense directed DOD’s Nuclear, Chemical, and Biological Defense Program to develop a Research Plan to obtain toxicologic and other relevant data to assess risk to military personnel. According to DOD, the Research Plan is intended to accomplish two objectives. The first is to support operational commanders in the field with information for real-time decision making required to accomplish their missions while not unduly jeopardizing the health and performance capability of their forces. The second is to understand and prevent or reduce operationally relevant performance decrements as well as the potential health consequences of low-level exposure that may not manifest immediately but may become evident months or years after the exposure.

For the Research Plan, CWAs of initial concern are nerve and vesicant agents. Nerve agents include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX. The vesicant agent of concern is sulfur mustard (HD). Exposure duration and frequencies to be considered are those likely to be experienced by deployed military personnel. Concentrations of interest are those at which no observable adverse health effects (immediate or delayed) are expected for healthy military personnel, as determined by accepted toxicologic tests and standard medical practices. Performance decrements significant to personnel carrying out their military duties are of greater concern than delayed health effects and therefore are assigned a higher priority.

The committee’s tasks were to review research proposed in the Research Plan, to provide recommendations about that research, and to develop recommendations for additional research as appropriate. The committee’s tasks also include providing guidance to DOD on appropriate risk assessment methods for assessing toxicologic risk to military personnel from low-level exposure to CWAs.

The committee’s major conclusions and recommendations concerning the Research Plan are presented below.

• The committee concludes that DOD’s master research plan on low-level exposure to CWAs, in general, is well planned and many of the proposed research tasks are likely to provide valuable information in protecting the military personnel from low-level exposure to CWAs and avoiding performance decrements. The Research Plan includes some studies that have some potential to identify delayed adverse health effects, but those studies should be assigned lower priority in the context of DOD’s primary objectives. Available information to date do not provide a sound basis for anticipation of delayed adverse health effects following low-level (in particular, short-term) exposure to nerve agents. However, the committee recommends that a small portion of DOD research budget be allocated to some research tasks to rule out the possibility of delayed health effects.

• For DOD, an important task is also to identify the highest concentration of CWA to which an unprotected person can be intermittently or continuously exposed without immediate or delayed health effects. Exposures should range from 1 hour to 1 year, the focus being on acute (short-term, high-level) single exposures and those repeated over 2 weeks.

• Although miosis (pupil contraction) has typically been considered the critical effect and most frequently used indicator of toxicity, questions about other adverse effects at low concentrations of exposure remain. DOD should conduct research to identify whether there are more sensitive toxicity end points from exposure to low concentrations of CWAs.

• If miosis is selected as the critical adverse response, studies must determine what level of pupillary constriction in miosis is operationally relevant. This information is needed for operational risk management and risk-risk comparison, which must consider the range of exposure limits and the probability of adverse outcomes for exceedances of those exposure limits for various levels of toxicity severity and operationally relevant exposure durations. Miosis studies in laboratory animals should include instillation of CWAs in eyes, using appropriate dose ranges, for correlation with cholinesterase inhibition in eyes. Because

ambient light levels confound pupillary effects of CWAs, the committee recommends that the effects of CWAs be determined under known lighting conditions. Furthermore, a rapid and accurate sensor of both miosis and ambient light will be needed to provide information necessary for field commanders to make decisions. There is also a need to review data on decrements in task performance related to changes in pupil size under various conditions of ambient light, including the magnitude and durations of these changes.

• The committee recommends that studies be undertaken to determine whether miosis is the sole cause of operationally relevant performance decrements in humans after low-dose CWA exposure. This research could be done safely in humans with Food and Drug Administration (FDA) approved organophosphate (OP) therapeutic agents and in nonhuman primates with both CWAs and FDA-approved OP agents.

• If cholinesterase levels in the sphincter muscle controlling pupillary constriction do not correlate with functional pupillary changes, then some other mechanism might be responsible for miosis, and a search for alternative macromolecular targets in the ocular tissue might be warranted. The committee recommends that DOD study toxicity end points in addition to cholinesterase inhibition, although tissues (e.g., blood, brain, and eye) should be routinely collected for cholinesterase determinations when animals are killed. Noncholinesterase end points might include fluoride regenerated agent, Na⁺/K⁺-ATPase, the Comet assay for DNA damage in lymphocytes, toxicogenomics, and cardiac effects (but not a computer model for cardiac arrythmias).

• One of the tasks of the Research Plan is to study neurobehavioral and cognitive changes at CWA doses below those causing overt toxicity in rodents. According to DOD, neurobehavioral tests, such as the functional observational battery, identify changes in central and peripheral nervous system function beyond those observable as clinical signs of intoxication. The committee understands that animal studies focusing on alterations in behavior are difficult to use when extrapolations must be made across species and to humans. The committee concludes that nonhuman primates are better animal models than rodents for subtle behavior changes in humans. The committee recommends that DOD conduct neurobehavior studies in nonhuman primates. In such animal studies and other studies proposed in the Research Plan, good laboratory practice (GLP) standards must be followed, but no evidence of GLP documentation was presented to the committee. The committee also recommends that DOD identify a behavior, if one exists, that is associated with miosis

and that occurs after systemic (inhalation) or topical (ophthalmic) exposure to CWAs. Review of existing data collected from experiments in humans or nonhuman primates would be of value. Regardless of the particular behavior tests to be used, the committee recommends that consideration be given to evaluating adaptive changes in behavior after repeated low-level exposure to CWAs.

• The committee assigns a high priority to those research tasks that study performance degradation. Performance decrements in humans could be caused by miosis or by more subtle neurophysiologic changes unrelated to miosis. Conducting accurate range-finding studies in a nonhuman primate model will be more useful than extrapolating a maximum tolerated dose from a rodent model or using a dose of CWA that shows an effect in rodents, the method recommended in the Research Plan.

• The committee recommends that data from previous studies on the effects of CWAs on military personnel be reevaluated as potentially useful sources with respect to end points of importance in humans. Between 1958 and 1975, the U.S. Army undertook a human volunteer study of 4,826 subjects to investigate the immediate and long-term effects of various classes of chemicals with warfare potential. The results of that study were perviously reviewed by the National Research Council (NRC) Committee on Toxicology. The committee’s first report (NRC 1982) found no evidence to support a finding of adverse long-term or delayed health effects after exposure to nerve agents. However, that report was unable to rule out the possibility that some nerve agents produced long-term adverse health effects in some individuals. In the follow-up NRC study (NRC 1985), 4,000 subjects were sent a health questionnaire. The results indicated that subjects who received nerve agents as a group did not differ from controls who did not receive that chemical treatment, but mortality was lower than expected in the exposed population. DOD also should review the database used to derive acute exposure guideline levels (AEGLs) for nerve agents and sulfur mustard recently published by NRC (2003).

• One of the major tasks of the Research Plan is to develop a method to generate consistent vapor atmospheres for GB across a range of relevant concentrations and exposure durations and relate fairly robust historical vapor generation data to modern, validated chamber exposure methodology. The committee is aware that conducting experiments with potent, volatile CWAs having very steep dose-response curves is exceedingly difficult. The committee concludes that DOD’s proposed research to overcome technical challenges to generate consistent vapor atmos-

pheres for CWAs and the deployment of sampling and analytical systems is appropriate, and the committee recommends that such research be continued.

• The military commander or decision maker must have the ability to know if a CWA is present in the ambient air, at what concentration, and whether this concentration can induce adverse operational impairments (performance decrements) or adverse delayed health effects in exposed personnel. However, there are limitations in the detection and measurement of CWAs. The committee recommends that DOD strive to develop more sensitive monitors capable of detecting concentrations of CWAs that can cause performance decrements or potential delayed health effects in an operational setting. The committee also recommends that the degree of detection sensitivity required should be driven by an understanding of CWA toxicology—that is, field-operation detector sensitivity that can identify CWA concentrations that are expected to result in operationally relevant performance decrements or immediate health effects.

• The methodology developed for deriving AEGLs is very pertinent. The generalization of Haber’s law to adverse effects related to Cⁿt (ten Berge method) is a valuable contribution. The committee recommends that DOD utilize information and techniques developed for deriving AEGLs.

• In a deployed military setting, being too protective can be as lethal as being insufficiently protected. Protective equipment not only interferes with an individual’s ability to fight, it can also cause significant heat stress. Full protective gear—donning mission-oriented protective posture, level 4 (MOPP4)—can restrict movement and vision and causes dehydration and hyperthermia when used in hot climates. It is critical that operational doctrine not require maximal physically protective measures at exposure concentrations significantly below those likely to produce casualties or long-term disabilities. Therefore, the committee recommends that science-based exposure standards (human toxicity estimates) be determined as accurately as possible and that appropriate toxicologic data be developed to minimize the uncertainty around those values.

• One goal of the Research Plan is to develop biomarkers of CWA exposure and to develop consistent measures directly related to the absorbed dose and physiologic effectiveness of a given chemical agent. The goal is also to demonstrate that the dose-metric profile for exposures in animal models enables direct extrapolation to human physiology.

Proposed biomarkers and dose metrics include alkyl phosphonates, regenerated nerve agent, acetylcholinesterase activity, and butyrylcholinesterase activity. The committee concludes that the proposed research on biomarkers is appropriate and recommends that such studies focus on effects from low-level exposure, paying particular attention to fluoride regeneration of the agent.

• One of the tasks of the Research Plan is to characterize the concentration-time relationship (dose-response) for low-level and longer-time CWA exposures (primarily for miosis and mortality end points). The committee recommends that relevant routes of exposure (e.g., inhalation and local exposure of the eye) be specifically evaluated. The setting of exposure limits for CWAs usually involves extrapolation to concentrations or durations of interest by invoking Haber’s law—adverse response is related to the product of agent concentration and exposure duration—or the approach of ten Berge (ten Berge et al. 1986), a generalization of Haber’s law stating that adverse response is related to Cⁿt. That expression weighs (1) concentration more than time (when n > 1), (2) concentration less than time (when n < 1), and (3) concentration and time equally (when n = 1). The committee recommends that Haber’s law not be applied in the absence of data, because naive applications of Haber’s law can lead to erroneous risk estimates. Physiologically based pharmacokinetic modeling can be used to improve the evaluation of concentration-duration-response relationships. Mechanistic considerations also would be informative regarding the conditions and dose metrics for which Haber’s law is and is not applicable. In the absence of such mechanistic information, the committee recommends that studies be conducted to test and validate duration-exposure models.

• The committee recommends that the DOD develop and apply appropriate statistical models that include concentration of the agent and duration of exposure as predictor variables along with important covariates that allow for testing various extrapolation methods (e.g., Haber’s law and ten Berge’s law). To ensure that studies include sufficient sample size, statistical principles of design should be used. Exposure concentrations, durations, and routes of exposure should be selected to include realistic scenarios. To facilitate evaluation of the concentration-time relationship, the committee recommends that exposure-response studies be conducted with a minimum of nine concentration-time treatments (three exposure concentrations crossed with three exposure durations).

• To carry out the operational risk management concept, the risk assessor will require robust information on response probabilities from the experimental study. For CWAs, for each critical response (e.g., miosis), the committee recommends that access to recently generated experimental data sets and contact with investigators be sought to develop response probabilities needed in operational risk assessment beyond single values, such as ECt₅₀ (the concentration and time that causes an effect in 50% of subjects).

• Field commanders ultimately must be able to use the information generated by this Research Plan to make decisions. The committee recommends that the DOD risk assessors confer with operations personnel to determine the nature and form of information that would be most useful to field commanders. The committee also recommends consultation with risk communication specialists to further assist with this task.

• One of the tasks of the Research Plan is to identify polymorphisms in human blood esterases and other enzymes or genes to identify susceptible subpopulations. Some proportion of the U.S. population might have genetically determined low levels of plasma cholinesterase and thus unusual susceptibility to some anticholinesterase compounds. Several studies indicate that plasma and red-blood-cell cholinesterase activity is significantly lower in women than in men. Therefore, women might be at a greater risk from systemic exposure to OP compounds. Other enzymes, such as paraoxonase and carboxylesterase, involved in nerve agent toxicity also show polymorphisms, which might result in greater susceptibility in personnel with particular genotypes. This research might be helpful in protecting such individuals using proper risk management strategies.

The committee recognizes that a considerable amount of research has been done, and much information is available on the acute and subchronic toxicities (delayed effects) of nerve agents and sulfur mustard. Genetic testing, neurotoxicity testing, metabolic studies, and other research studies have been done. The committee recommends that such studies not be repeated. The committee recommends that the Research Plan not attempt to fill in all the data gaps—that is, investigation in numerous species using multiple dosages by various routes of administra-

tion. The time, money, and effort could be better used in focusing on the most important and promising animal models and toxicity end points. The operational relevance of the research in terms of relevant durations of exposure and CWA concentrations must be considered in establishing research priorities.

To obtain the information most valuable in protecting military personnel from operationally significant performance decrements or potential delayed adverse health effects after short-term exposure to low concnetrations of CWAs, DOD should ensure that the total database from previous human and animal studies has been fully examined to fill data gaps. These studies include human studies, nonhuman primate studies, toxicokinetic studies, and the studies used to derive NRC’s AEGLs.