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Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents (1999)

Chapter: D: Health Risk Assessment for the Nerve Agent VX

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Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX D
Health Risk Assessment for The Nerve Agent VX

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×
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Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

HEALTH RISK ASSESSMENT FOR THE NERVE AGENT VX

DRAFT REPORT

September 1996

(editorial corrections made April 1997)

Prepared for

U.S. Department of the Army

Army Environmental Center

under Interagency Agreement No. 1769-1769-A1

Prepared by

Life Sciences Division

OAK RIDGE NATIONAL LABORATORY*

Oak Ridge, Tennessee 37831

Submitted to

Material Chemical Risk Assessment Working Group

Advisory and Coordinating Committee

Environmental Risk Assessment Program

*  

Managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract No. DE-AC05-96OR22464

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

DISCLAIMER

This document is an internal review draft for review purposes only and does not constitute U.S. Government policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

PREFACE

This report assesses the potential non-cancer and cancer effects of chemical agent VX (CAS No. 50782-69-9).

This document supports the activities of the Material/Chemical Risk Assessment Working Group of the Environmental Risk Assessment Program, a cooperative endeavor of the Department of Defense, Department of Energy, and Environmental Protection Agency. This working group is developing toxicity values for selected chemicals of concern at federal facilities. Toxicity values will be submitted for consideration by the EPA's IRIS Consensus Process for inclusion on IRIS (EPA's Integrated Risk Information System). The Material/Chemical Risk Assessment Working Group consists of Drs. Jim Cogliano (chair) and Harlal Choudhury (U.S. EPA), Dr. Bruce Briggs (Geo-Centers); Lt. Cmdr. Warren Jederberg and Dr. Robert L. Carpenter (U.S. Naval Medical Research Institute); Dr. Elizabeth Maull and Mr. John Hinz (U.S. Air Force Occupational and Environmental Health Directorate); Drs. Glenn Leach and Winnie Palmer (U.S. Army Center for Health Promotion and Preventive Medicine); Drs. Robert Young and Po-Yung Lu (Oak Ridge National Laboratory).

This document was written by Dr. Dennis M. Opresko, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN. Internal peer review was provided by Dr. Robert Young, Dr. Annetta Watson, and Mr. Robert Ross. External review of the toxicity data was provided by Dr. Thomas J. Bucci, Integrated Services, White Hall, AR and Dr. I.K Ho of the U. of Mississippi Medical Center, Jackson MS. External review of the derivation of the RfDs was provided by Drs. Michael Dourson and Susan Velazquez of Toxicology Excellence for Risk Assessment, Cincinnati, OH, and Dr. William Hartley of Tulane Medical Center, New Orleans LA. Additional reviews were provided by Mr. Joe King, Dr. Jack Heller, Ms. Veronique Hauschild, Ms. Bonnie Gaborek, Mr. Maurice Weeks, Maj. Robert Gum, and Mr Kenneth Williams of the U.S Army.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×
Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

LIST OF TABLES

Table 1.

 

RBC-ChE activity in different species

 

5

Table 2.

 

RBC-ChE activity in sheep fed VX

 

9

Table 3.

 

RBC-ChE activity in rats injected subcutaneously with VX

 

11

Table 4.

 

Regression analysis of Rice et al. (1971) data for sheep dosed with VX

 

19

Table 5.

 

Comparison of RfD with human toxicity data for VX

 

20

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

1. INTRODUCTION

Military nerve agents are organophosphate compounds containing either a fluorine, sulfur, or cyanide substituent group (Dacre, 1984). VX contains a sulfur substituent group (for comparison GB contains fluorine and GA contains a cyanide group). The chemical synonyms, Chemical Abstract Service (CAS), Army identification numbers (DA, 1974, 1992; Dacre, 1984), and chemical formula for VX are as follows:

Phosphonothioic acid, methyl-, S-[2-[bis(1-methylethylamino)ethyl] O-ethyl ester;

Phosphonothioic acid, methyl, S-(2-(diisopropylamino)ethyl) O-ethyl ester;

O-Ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate;

S-2-Diisopropylaminoethyl O-ethyl methylphosphonothiolate;

O-Ethyl S-(2-diisopropylaminoethyl) methylthiolphosphonate;

TX60;

CAS No. 50782-69-9;

Edgewood Arsenal No. 1701

1.1. PHYSICAL/CHEMICAL PROPERTIES

Agent VX is a colorless to straw-colored liquid with a molecular weight of 267.4 (DA, 1974, MacNaughton and Brewer, 1994); it has a vapor density of 9.2 (air = 1) and a liquid density of 1.0083 g/ml at 25°C (DA, 1974). The vapor pressure of VX is 0.0007 mm Hg at 25°C; its water solubility is 30 g/L per 100 g at 25°C and 7.5 g per 100 g at 15°C (DA, 1974).

1.2. ENVIRONMENTAL FATE

1.2.1 Air

The volatility of agent VX is relatively low (vapor pressure 0.0007 mm HG (DA, 1974; MacNaughton and Brewer, 1994). A vapor concentration of 10.5 mg/m3 has been reported for a temperature of 25°C (DA, 1974) (although not adequately described in the reference, this is presumably the saturation concentration above a pure liquid). Because VX does not absorb UV radiation above 290 nm (Rewick et al., 1986), photodegradation is not a significant environmental fate process. Based on structure-activity relationships, VX is predicted to react in the troposphere with photochemically produced hydroxyl radicals, with a half-life estimated to be 0.24 days (Atkinson, 1987).

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×
1.2.2 Water

VX has a water solubility of 3 g per 100 g solvent at 25°C and 7.5 g per 100 g solvent at 15°C (DA, 1974). It's Henry's Law Constant has been estimated to be 3.5×10-9 atm m3/mol, indicating a low potential for evaporation from water (MacNaughton and Brewer, 1994); its evaporation rate is about 1/1,500 that of water (Rosenblatt et al., 1995). The agent is relatively resistant to hydrolysis (Franke, 1982); reported half-lives in water at 25°C and pH 7 range from 400 to 1000 hours (Clark, 1989); half-life increases under acidic conditions [(100 days at pH 2–3 (DA, 1974)]. Although solubility is increased at lower temperatures, low temperatures decrease the rate of hydrolysis (Clark, 1989). VX in surface waters may sink and be adsorbed by sediment (Trapp, 1985).

1.2.3 Soil

VX is moderately persistent on bare ground and may remain in significant concentrations for varying time periods, depending on temperature, organic carbon content of the soil, and moisture (Sage and Howard, 1989). Its volatility potential (slope of the vapor pressure vs. concentration in soil organics) of 3.0×10-11 mm Hg/mg/kg and its air-soil partition coefficient (for a soil density of 1.4 g/cm3) of 1.5×10-8 mg/m3 (MacNaughton and Brewer, 1994), indicate that relatively little will evaporate into air. In the laboratory, unstabilized VX of 95% purity decomposed at a rate of 5% per month at 22°C (DA, 1992). In contrast, VX in soils from Carroll Island, MD (a chemical agent test site) decreased to 2.5–7.2% of initial levels (10 mg/g soil) after 14 days storage at room temperature in closed containers (studies reviewed by Small, 1984). In similar studies conducted with soil from Dugway Proving Ground, VX levels (initially 1 mg/g of soil) decreased 79% after 3 days and 90% after 15 days. In other laboratory studies, a VX concentration of 0.2 mg/g in humic sand decreased by 78% after one day, and the same concentration in humic loam and clayey peat decreased by 98% in one day; only 0.1% of the applied amount was detected after 3 weeks in either soil type (Kaaijk and Frijlink, 1977; Verweij and Boter, 1976).

Degradation of VX in soil has also been evaluated in several field studies (see review by Small, 1984). At Carroll Island, MD, VX sprayed on soil decreased by about three orders of magnitude within 17 to 52 days. In an area of Dugway Proving Ground, where VX soil levels prior to 1969 were as high as 6 mg/g, no VX was detected (detection limit 0.4 µg/g) 10 years later. The degradation product, methyl phosphonic acid, was detected at concentrations ranging from 14.9 to 23 µg/g. Approximately three weeks after an accidental release of VX near the Dugway Proving Ground snow samples contained 7–9 ng VX per 400–500 gm of water and grass samples contained 4 µg VX per 900 gm of solid material [estimates based on an assumed 100% extraction efficiency (Sass et al., 1970)].

2. MECHANISM OF ACTION

Nerve agents are inhibitors of acetylcholinesterase (AChE), an enzyme responsible for deactivating the neurotransmitter acetylcholine at some neuronal synapses and myoneural junctions. By a mechanism of phosphorylation, nerve agents act as substrates for the enzyme, thereby preventing deactivation of acetylcholine. The organophosphate-inhibited enzyme can be reactivated by dephosphorylation, but this occurs at a rate that is slower than the rate of reactivation of acetylcholine. Consequently, there is a depletion of acetylcholinesterase and a buildup of acetylcholine. In addition, the nerve agent-enzyme complex can also undergo an ''aging" process (thought to be due to a loss of an alkyl or alkoxy group),

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

whereby it becomes resistant to dephosphorylation (see review by Munro et al., 1994). Differences in rates of aging and reactivation may be important in evaluating toxicity data especially when extrapolating from animal studies to humans. In vitro tests conducted by Grob and Harvey (1958) indicate that both GA and GB combine with cholinesterase almost irreversibly during the first hour of their reaction. Sidell and Groff (1974) reported that the GB-ChE complex ages very rapidly in vivo, with 45–70% completion by 5 hours after infusion. In contrast, the complex formed between ChE and the nerve agent VX does not age significantly, and the rate of spontaneous reactivation can be as fast as 1%/hr in humans (Sidell and Groff, 1974).

2.1 Effects of Organophosphate Compounds on the Nervous System

The anticholinesterase effects of the organophosphate nerve agents can be characterized as being muscarinic, nicotinic, or central nervous system (CNS)-related. Muscarinic effects occur in the parasympathetic system (bronchi, heart, pupils of the eyes; and salivary, lacrimal and sweat glands) and result in signs of pulmonary edema, bradycardia, miosis, tearing, and sweating. Nicotinic effects occur in somatic (skeletal/motor) and sympathetic systems, and result in muscle fasciculation, muscle weakness, tachycardia, and diarrhea. Effects on the CNS by organophosphates are manifested as giddiness, anxiety, emotional lability, ataxia, confusion, and depression (O'Brien, 1960).

Although the inhibition of cholinesterase within neuro-effector junctions or the effector itself is thought to be responsible for the major toxic effects of organophosphate chemical agents, these compounds can apparently affect nerve-impulse transmission by more direct processes as well. In addition to cholinesterase inhibition, VX reacts directly with ACh receptors and receptors of other neurotransmitters (e.g., norepinephrine, dopamine, gamma-aminobutyric acid) (Zhao et al., 1983; Ho and Hoskins, 1983; Chen and Chi, 1986; Idriss et al., 1986). According to Somani et al. (1992), the direct action of nerve agents on nicotinic and muscarinic ACh receptors may occur when concentrations in the blood rise above micromolar levels, whereas at lower levels the action is mainly the result of inhibition of AChE; however, nanomolar blood concentrations of VX"may directly affect a small population of muscarinic ACh receptors that have a high affinity for [3H]-cis-methyldioxalane binding" (Somani et al., 1992). VX may also counteract the effects of ACh by acting as an open channel blocker at the neuromuscular junction, thereby interrupting neuromuscular function (Rickett et al., 1987).

Exposure to some organophosphate cholinesterase inhibitors results in a delayed neuropathy characterized by degeneration of axons and myelin. This effect is not associated with the inhibition of acetylcholinesterase, but rather with the inhibition of an enzyme described as neuropathy target esterase (NTE); however, the exact mechanism of toxicity is not yet fully understood (Munro et al., 1994). For some organophosphate compounds, delayed neuropathy can be induced in experimental animals at relatively low exposure levels, whereas for others the effect is only seen following exposure to supralethal doses when the animal is protected from the acute toxic effects caused by cholinesterase inhibition. Although there is the potential for nerve agents to have direct toxic effects on the nervous system, there is no evidence that such effects occur in humans at doses lower than those causing cholinesterase inhibition. For the purpose of evaluating potential health effects, inhibition of blood cholinesterase is generally considered the most useful biological endpoint.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

2.2 Effect on Blood Cholinesterases

Acetylcholinesterase is a natural component of human blood, where it is found on the surface of red blood cells (RBC-AChE). RBC-AChE activity, as well as the activity of a second type of cholinesterase found in blood plasma (butyrylcholinesterase, or plasma cholinesterase), have been used to monitor exposure to organophosphate compounds (pesticides and nerve agents). Both RBC-AChE and plasma-ChE have been used as bioindicators of potential toxic effects of organophosphate cholinesterase inhibitors. There is some evidence that RBC-AChE is as sensitive as brain ChE to the effects of nerve agents. Grob and Harvey (1958) reported that the in vitro concentrations producing 50% depression of brain-ChE and RBC-AChE activity were the same in the case of GA (1.5 × 10-8 mol/L), and only slightly different (3 × 10-9 mol/L and 3.3 × 10-9 mol/L) in the case of GB. However, in vivo animal studies indicate a poor correlation between brain and RBC-AChE in cases of acute exposures (Jimmerson et al., 1989), and this is reflected in the fact that blood cholinesterase activity may not always be correlated with exposure or with signs and symptoms of toxicity (Holmstedt, 1959). Acute exposures to high concentrations may cause immediate toxic effects before significant changes occur in blood ChE activity, and repeated exposures over a period of several days may result in a sudden appearance of symptoms due to cumulative effects (Grob and Harvey, 1958). Conversely, blood ChE activity can become very low without overt signs or symptoms during chronic exposures to low concentrations of organophosphates. This may be due to a slower rate of recovery of RBC-ChE compared to tissue ChE, or to a noncholinesterase-dependent recovery pathway for neural tissue (Grob and Harvey, 1958). Sumerford et al. (1953) reported that orchard workers exposed to organophosphate insecticides had RBC-AChE values as low as 13% of preexposure values without any other signs or symptoms of exposure. Animal studies have demonstrated that chronic exposures to low concentrations of organophosphate insecticides can also result in increased tolerance levels (Barnes, 1954; Rider et al., 1952; Dulaney et al., 1985). Similarly, Sumerford et al. (1953) reported increased levels of tolerance to organophosphate insecticides in people living near orchards subject to insecticide applications. Such adaptation may result from increased rates of formation of blood ChE, or from increased rates of detoxification. Additional information on the development of tolerance to organophosphate cholinesterase inhibitors can be found in a review paper by Hoskins and Ho (1992).

The blood cholinesterases may, to some degree, provide a protective effect by binding with some fraction of the anticholinesterase compound (Wills, 1972). However, not all nerve agents bind equally well with all cholinesterases. In tests conducted on dogs, Holmstedt (1951) found that GA affected RBC and plasma cholinesterase to a nearly equal degree. In contrast, VX preferentially inhibits RBC-ChE; 70% compared with about 20% inhibition of plasma ChE (Sidell and Groff, 1974). Rodents (but not humans) have other enzymes in the blood, termed aliesterases, which can bind with organophosphates, thereby reducing the amount available for binding with acetylcholinesterase (Fonnum and Sterri, 1981). Agent GB binds with aliesterases; however, according to Fonnum and Sterri (1981), VX has a quaternary ammonium group which prevents it from being a substrate for aliesterases. The strong specificity of agent VX to AChE may account, in part, for the fact that it is more acutely toxic than agents GA, GB, or GD (see Appendix A).

2.2.1 Intra- and Interspecies Variation in Blood Cholinesterase Activity

Although blood cholinesterase activity is used as a measure of exposure to organophosphate compounds, baseline activity levels can vary between individuals and between species. According to Wills (1972), both plasma- and RBC-ChE activity are generally lower in women than in men. Sidell and Kaminskis (1975) reported that, for a test population of 22 human subjects, the highest coefficient of

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

variation of RBC-ChE was 4.1% per single subject; the average range of variation was ± 2.1% for men and ± 3.1% for women. In individuals studied for one year, the RBC-ChE activity varied by 11% in men and 16% in women. Yager et al. (1976) reported a 10.0% intra-individual coefficient of variation for RBC-ChE and 14.4% for plasma-ChE. Callaway et al. (1951) estimated that with only one pre-exposure measurement, the smallest measurable decrease was 15% of the baseline value for RBC-ChE activity and 20% of the baseline for plasma-ChE.

A small subpopulation of men and women have a genetic defect causing their blood cholinesterase activity to be abnormally low (Evans et al., 1952; Harris and Whittaker, 1962). For homozygous individuals, the activity can be as low as 8–21% of the normal mean (Bonderman and Bonderman, 1971). Morgan (1989) suggests that these individuals may be unusually sensitive to organophosphate anticholinesterase compounds.

Data compiled by Ellin (1981) reveal that the RBC-ChE activity for humans is slightly higher than that for monkeys and much higher than that for rats and other laboratory animals (Table 1).

Table 1. RBC-ChE activity in different species

Species

RBC-ChE activity (µmol/mL/min)

Optimum substratea concentration (M)

Human

12.6

2 × 10-3

Monkey

7.1

2 × 10-3

Pig

4.7

1 × 10-3

Goat

4.0

2 × 10-3

Sheep

2.9

2 × 10-3

Mouse

2.4

2 × 10-3

Dog

2.0

2 × 10-2

Guinea pig

2.7

2 × 10-3

Rabbit

1.7

5 × 10-3

Rat

1.7

5 × 10-3

Cat

1.5

5 × 10-3

Source: Ellin, 1981

a Acetylthiocholine iodide concentration for maximum RBC-ChE activity.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

These differences in RBC-ChE activity may affect a species' sensitivity to a particular organophosphate compound. At the same time, the relative amount of plasma cholinesterase and other compounds in the blood that can bind to the organophosphate agents must also be considered. As noted above, rodents, but not humans, have high levels of aliesterases in the blood (Cohen et al., 1971). These compounds may provide rats and mice with a higher level of resistance to anticholinesterase compounds to which they bind, such as GB, but not to others such as VX (Fonnum and Sterri, 1981).

2.2.2 Potency of Nerve Agents as Cholinesterase Inhibitors

The potency of the anticholinesterase activity of nerve agents and other organophosphates is measured by either the bimolecular rate constant (ki) for the reaction of the phosphate compound with the enzyme or by the molar concentration causing 50% inhibition of the enzyme (I50) in vitro. I50 data for several organophosphate nerve agents have been tabulated by Dacre (1984). The pI50 (negative log of the molar concentration causing 50% inhibition) for VX was reported to be 8.8. The relationship between I50 and ki as a function of time (t) is expressed by the following equation (Eto, 1974):

Relative potency of nerve agents can also be expressed in terms of the in vivo dose necessary to produce the same level of cholinesterase inhibition by a specific exposure route. As would be expected, the effectiveness of the agents in inhibiting cholinesterase is closely correlated with their acute toxicity (see Appendix A).

2.2.3 Cholinesterase Inhibition by VX

The potency of VX in a given species can be expressed in terms of the dose necessary to produce 50% inhibition of AChE (AChE50). In humans, the RBC-AChE50 for VX is 0.001 mg/kg for an intravenous (i.v.) dose (Sidell and Groff, 1974), 0.0023 mg/kg for an oral dose (Sidell and Groff, 1974), and 0.034 mg/kg (12 hr) and 0.029 mg/kg (24 hr) for a dermal dose of an undiluted liquid (Sim and Stubbs, 1960). These data indicate that an oral dose about 2 times greater than an i.v. dose is needed to produce the same amount of AChE inhibition in the blood. Similiar information is available from animal studies. Goldman et al. (1988) dosed Sprague-Dawley rats with 4 µg/kg VX by various routes of exposure and measured RBC-AChE activity after 3 and 24 hr. The i.v. and s.c. routes resulted in the greatest decreases in RBC-AChE. RBC-AChE levels (expressed as fraction of control values) were 0.14 ± 0.07 at 3 hr and 0.20 after 24 hr for i.v., and 0.13 ± 0.07 at 3 hr and 0.20 after 24 hr for s.c. In contrast, RBC-AChE levels after intragastric administration were 0.48 ± 0.14 of controls at 3 hr and 0.46 after 24 hr, and after intraperitoneal (i.p.) administration 0.35 ± 0.19 at 3 hr and 0.35 after 24 hr. These data indicate that the s.c. and i.v. routes produced a similar level of inhibition, with an estimated RBC-AChE50 of about 1 µg/kg. The intragastric route was much less effective in reducing RBC-AChE, possibly due to hydrolysis and detoxification in the stomach and/or limited absorption through the gastrointestinal tract.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

3. TOXICOLOGY

3.1 Introduction

Health and environmental impacts of nerve agents and related compounds (i.e., organophosphate insecticides) have been reviewed by O'Brien (1960), Matsumura (1976), Dacre (1984), Carnes and Watson (1989), Watson et al. (1989), and Munro et al. (1994). A brief discussion of the general toxicology of nerve agents and related organophosphate pesticides is given below.

Nerve agents are toxic by all routes of exposure. Initial symptoms of acute poisoning are fatigue, headache, mild vertigo, weakness, and loss of concentration. Moderate exposures result in miosis and excessive sweating, tearing, and salivation. Acidosis and hyperglycemia may also occur in addition to muscular weakness, muscular twitching, lacrimation, urination, and defecation. Acute poisoning can result in prostration, clonic convulsions (rapid repetitive movements), and tonic convulsions (limbs stretched and rigid) (Matsumura, 1976). Exposures sufficiently high to cause convulsions have resulted in brain lesions and cardiomyopathy in laboratory animals (Singer et al., 1987).

In addition to the immediate toxicity of the nerve agents, there is concern that exposures may lead to chronic neurological effects similar to those reported for some related organophosphate insecticides. Included among these possible effects are organophosphate-induced delayed neuropathy (OPIDN), EEG changes, and long-term psychological disturbances (Munro et al., 1994). OPIDN, which appears 5–30 days after exposure, manifests itself as muscle weakness, tingling, and twitching followed by paralysis (Munro et al., 1994). Histopathological changes, which consist of degeneration of axons and myelin of the nervous system, can be correlated, not with inhibition of acetylcholinesterase, but rather with inhibition of an enzyme described as neuropathy target esterase (NTE); however, the exact mechanism of toxicity is not yet fully understood (Munro et al., 1994). There is no clinical or experimental evidence to suggest that VX causes OPIDN in humans. In addition, in studies conducted with antidote-protected animals dosed with supralethal amounts of VX no signs of OPIDN were observed (see Section 3.5). The available data, therefore, indicate that OPIDN is unlikely to occur in humans exposed to VX.

Acute exposures to nerve agents are known to result in EEG changes and psychological effects (Grob and Harvey, 1958; Sidell, 1992). Some studies have indicated that changes in EEG patterns may persist for long periods of time after exposure (Metcalf and Holmes, 1969; Burchfiel et al., 1976; Duffy et al., 1979; Duffy and Burchfiel, 1980); however, the reported changes have been considered to be clinically insignificant and not correlated with behavioral or physiological changes (DHHS, 1988). Although acute exposures can also induce neuropsychological changes, there is no evidence of these effects persisting for months or years as has been reported for some organophosphate insecticides (Savage et al., 1988; Gershon and Shaw, 1961; Mick, 1974; Rodnitzky, 1974; Wagner, 1983; Tabershaw and Cooper, 1966). The available data for the organophosphate insecticides suggest that chronic neuropsychological effects (excluding OPIDN) do not occur in the absence of significant changes in blood cholinesterase. The same conclusion may apply to the organophosphate nerve agents.

3.2 Acute Toxicity

Limited information is available on the oral toxicity of VX to humans. In clinical studies conducted by Sidell and Groff (1974), single oral doses of 2–4.5 µg VX/kg produced gastrointestinal symptoms in 5 of 32 test subjects. Regression analysis of the dose-response data indicated that the RBC

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

ChE50 was 2.3 µg/kg. Sim et al. (1964) reported no signs of toxicity in human volunteers receiving 1.43 µg VX/kg/day for seven days (in four daily doses of 500 mL drinking water); however, average RBC-ChE activity was reduced 60% (to 40% of baseline values).

Data compiled by Sidell (1992) revealed that for individuals exposed to VX dermally, gastrointestinal symptoms (vomiting) occurred in 0.6% (1/166) when RBC-ChE activity was 50% of control values, and in 8% (2/24), 33% (9/27), 45% (19/42) and 67% (16/24) when RBC-ChE levels were 40–49%, 30–39%, 20–29%, and less than 20% of control values, respectively. Sim (1962) reported that a dose of 5 µg VX/kg applied to the cheeks or ear lobes resulted in symptoms of systemic toxicity in about half of the test subjects.

Several studies have been conducted in which human volunteers were injected intravenously with VX. Kimura et al. (1960) reported that a 30-sec i.v. injection of 0.04 µg/kg in one adult test subject caused headaches, tiredness and irritability, but no change in RBC or whole blood cholinesterase activity. A subsequent 30-sec i.v. injection of 0.08 µg/kg 3.5 hr later resulted in headaches, lightheadedness and abdominal cramps as well as an increase in airway resistance, a decrease in respiratory rate, a decrease in pulse rate and an increase in minute volume, but no change in cholinesterase activity. A single 30-sec i.v. dose of 0.225 µg/kg resulted in a 27% decrease in baseline RBC-ChE activity within 15 min and frontal retrobulbar headaches in one test subject. Six subjects receiving 1 µg VX/kg by i.v. infusion over 1.75–4 hr periods exhibited 50–60% depression in cholinesterase activity but no signs of toxicity (except for one 84 kg individual who reported headaches). Sidell and Groff (1974) reported that an i.v. dose of 1.5 µg VX/kg in 18 test subjects resulted in dizziness, nausea, and vomiting in 11, 4, and 6 individuals, respectively; RBC-ChE was depressed 55–90% from baseline values (average about 75%). Regression analysis of dose response data (doses of 1.2–1.7 µg/kg) indicated that the ChE50 was 1.1 µg VX/kg. Using the data provided by Kimura et al. (1960), McNamara et al. (1973) concluded that an i.v dose of 0.1 µg/kg would have no effect on RBC-ChE activity.

Based on inhalation data for agent GB, McNamara et al. (1973) calculated the no-effect dose for VX-induced tremors in humans to be 0.34 µg/kg. Carnes et al. (1986) suggested that the threshold for muscular tremors in sensitive subpopulations, such as infants, may be 0.16 µg/kg. McNamara et al. (1973) estimated that the human LD50 and no-death levels for VX were 7.5 µg/kg and 0.94 µg/kg, respectively. These estimates were based on extrapolations of LCt50 data for GB.

The short-term toxicity of VX to Sprague-Dawley rats was investigated by Goldman et al. (1988). In one series of tests, a single subcutaneous (s.c) injection of 1 µg VX/kg resulted in a 50% inhibition of RBC-AChE relative to controls and a dose of 4 µg/kg resulted in a 87% inhibition relative to controls. In a second test series, male rats were dosed with 4 µg VX/kg by different exposure routes and RBC-AChE levels relative to controls were determined at 3 hr and at 24 hr. High variability in response was seen in animals dosed by intratracheal instillation, intragastric lavage, and i.p. injection. RBC-AChE levels were reduced a similar amount for s.c. injections (13 ± 7% of control values at 3 hr and 21% at 24 hr) and i.v. injections (14 ± 0.07% of control values at 3 hr and 20% at 24 hr). In another pilot study, male and female rats (8–10 weeks old) were injected subcutaneously with 0, 0.25, 0.63, 1.56, 3.91, 9.77, or 14.65 µg VX/kg/day, 5 days/week for 14 days. Each dose group consisted of eight males and eight females except for the high-dose group which had two males and two females. All the animals in the two highest dose groups died as a result of the exposures, but none of the animals in the three other dose groups died as a result of the exposures. Of the remaining animals, one-half was sacrificed at 7 days and the other half at 14 days. RBC-AChE activity levels were depressed in all dose groups in a dose-dependent manner (the results for the 0.25 and 1.56 µg/kg/day dose levels are included in Table 3).

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

3.3 Subchronic Toxicity

Rice et al. (1971) fed VX to healthy yearling ewes (Columbian, or Columbian crossed with Shropshire, or Rambouillet) for 56 days. The dose levels were 0, 3, 9, or 15 µg/day (five animals per dose group and ten controls). The agent was mixed with Pillsbury 16% rabbit pellets and hand-fed to the test animals. The animals were checked periodically during the feeding period for clinical signs of toxicity (i.e., slowed pupil response, slowed pain reflex, and profuse salivation). There were no reported data identifying changes in clinical chemistry (except whole blood cholinesterase), hematology, body and organ weights, or gross or microscopic pathology. Whole blood ChE activity levels were monitored prior to the first exposure (one determination per animal) and then 16 times during the 56-day test period. Each whole blood ChE determination was first normalized to the average control value for the same time period to give an adjusted daily value. The normalized values for each time period were then compared to the average normalized pre-exposure (baseline) ChE value for each dose group (Table 2). According to Rice et al. (1971), whole blood ChE was significantly depressed at all dose levels (statistical analysis and levels of significance not reported). It was also reported that at the lowest dose (3 µg/day) the decrease in ChE was statistically significant by the twenty-first day. Whole blood ChE stabilized at about 62% of the

Table 2. RBC-ChE activity in sheep fed VXa

 

VX dose

Time (hr)a

3 µg/day

9 µg/day

15 µg/day

 

Fraction controlb

Fraction baselinec

Fraction controlb

Fraction baselinec

Fraction controlb

Fraction baselinec

0

1.15

1.00

1.03

1.00

1.17

1.00

24

1.05

0.91

0.84

0.81

0.86

0.74

240

1.16

1.01

0.58

0.57

0.51

0.44

360

0.92

0.80

0.55

0.53

0.29

0.24

744

0.72

0.62

0.26

0.25

0.09

0.07

912

0.73

0.64

0.12

0.11

0.05

0.04

1320

0.72

0.63

0.20

0.19

0.08

0.07

Source: Rice et al., 1971

a Selected data points presented

b Data expressed as a fraction of the control value ChE.

c Data expressed as fraction of adjusted baseline ChE

adjusted baseline by the thirty-first day and remained at this level for the remainder of the 56-day feeding period (see Appendix B). The sheep used in this study had an average weight of 52.7 kg; therefore, the weight-normalized dose for 3 µg/day was 0.06 µg/kg/day. None of the sheep dosed with 3, 9, or 15 µg/day exhibited any physical signs of clinical toxicity, even though the whole blood ChE in the highest dose group was reduced to 5% of the normalized baseline values for the last three weeks of the feeding period. In additional studies (also reported in Rice et al., 1971), physical signs of VX toxicity (not

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

described in detail) were observed in "culled" or weakened ewes dosed with 30 µg VX/day for about 4 weeks and in healthy sheep dosed with 75 µg VX/day for about 3 weeks. Rice et al. (1971) noted that the culled animals surviving the exposure recovered fully without developing any permanent signs of toxicity. Because of the significant reduction in whole blood ChE (38% relative to pre-exposure values), the test dose (3 µg/day) in the 8-week study is considered a lowest-observed-adverse-effect level (LOAEL). Alternately, based solely on the reported physical signs of clinical toxicity, the dose of 75 µg/day from the 3-week study is a LOAEL (for healthy animals), and 15 µg/day (from the 8-week study) is a no-observed-adverse-effect level (NOAEL), even though there was significant reduction in whole blood ChE at this dose (i.e., 96% reduction relative to pre-exposure values by day 38).

The subchronic toxicity of VX to animals was also investigated by Goldman et al. (1988) who injected rats (25/sex/dose group) subcutaneously with VX 5 days/week, for up to 90 days. The administered doses were 0 (saline controls), 0.25, 1.0, or 4.0 µg VX/kg/day. Five animals of each sex were sacrificed at 30, 60, and 120 days (includes a 30-day recovery period), and 10 animals of each sex were sacrificed at 90 days. RBC-AChE activity levels were monitored in 2 of 5 or in 3 of 10 animals per sex at each of the sacrifice times; blood chemistry was evaluated in the remaining animals. The tissues of all sacrificed animals were processed for histological analysis. Urinalysis were conducted on samples collected during weeks 8 and 12. Animals in the highest dose group exhibited body weight loss and behavioral changes (increased irritability and aggressiveness by week 2, followed by decreased grooming and lethargy at week 8). There were periodic cases of diarrhea in this group. By week 5, some of the animals dosed with 1.0 µg/kg/day exhibited some irritability.

Relative brain weight (ratio of brain to body weight) was elevated in the 4.0 µg/kg/day group. There were no significant changes in clinical chemistry or urinalysis parameters that were dose-related. Histopathological examination did not indicate a VX-associated pathology. In a separate 3-generation study in which hematological parameters were evaluated in rats maintained for 120 days under the same exposure protocol, there were no significant effects in male rats; however, in F0 females dosed with 4.0 µg/kg/day, statistically significant decreases occurred in hemogloblin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin (Goldman et al., 1988).

Plasma cholinesterase was significantly reduced at 30 days (p <0.05) in males and females given 1.0 µg/kg/day and at 30, 60, and 90 days for males and females given 4.0 µg/kg/day. RBC-AChE levels in males and females were reduced in a dose-dependent manner when compared to control values for the same time period (Table 3); however, the study did not include baseline or preexposure RBC-AChE levels for each test group. Goldman et al. (1988) reported that the observed decreases were significant but the level of statistical significance was not reported. The 30-day data for both males and females were reanalyzed using ANOVA and Dunnett's and Scheffe's Comparisons, and RBC-AChE activity in all dose groups for both sexes was significantly lower (p <0.05) than control values for the same time period (Appendix C). RBC-AChE levels in both males and females returned to 83–98% of control values in the test groups allowed a 30-day recovery period. Daily exposure to VX by s.c. injection for 30 days resulted in statistically significant depression of RBC-AChE at all dose levels.

3.4 Chronic Toxicity

Data on the chronic toxicity of VX were not found in the available literature.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

Table 3. RBC-ChE activity in rats injected subcutaneously with VXa

 

 

 

VX dose (µg/kg/day)b

Time of sacrifice

Sex

No.

0.25

1.0

1.56

4.0

7 daysc

M

4

0.85 ± 0.06

0.46 ± 0.07

0.31 ± 0.03d

 

F

4

0.90 ± 0.05

0.36 ± 0.09

0.34 ± 0.13d

14 daysc

M

4

0.72 ± 0.07

0.34 ± 0.12

0.29 ± 0.03d

 

F

4

0.64 ± 0.12

0.28 ± 0.13

0.31 ± 0.08d

30 dayse

M

2

0.46 ± 0.04

0.22 ± 0.00

0.04 ± 0.02

 

F

2

0.48 ± 0.06

0.20 ± 0.01

0.10 ± 0.05

60 dayse

M

2

0.33 ± 0.17

0.23 ± 0.06

0.14 ± 0.00

 

F

2

0.53 ± 0.04

0.34 ± 0.02

0.23 ± 0.02

90 dayse

M

3

0.34 ± 0.02

0.37 ± 0.03

,:—

,,0.23 ± 0.08

 

F

3

0.64 ± 0.13

0.51 ± 0.17

,:0.27 ± 0.07

120 daysf

M

5

0.91 ± 0.07

0.88 ± 0.11

,:—

,,0.83 ± 0.11

 

F

5

0.98 ± 0.12

0.93 ± 0.13

,:0.88 ± 0.16

Source: Goldman et al., 1988

a Data expressed as fraction of control value; mean ± SD; includes data from 14-day pilot study and 90-day study.

b Dosing schedule once per day, five days per week.

c Fourteen-day study.

d Dose was 3.91 µg/kg/day.

e Ninety-day subchronic study

f Includes recovery period of 30 days.

3.5 Nervous System Toxicity

Sidell and Groff (1974) reported that volunteers dosed with VX (1.5 µg/kg, i.v.) exhibited a significant decrement in performance on a number facility test within 1 hr after treatment. Bowers et al. (1964) reported anxiety, psychomotor depression, intellectual impairment, and unusual dreaming in volunteers exposed to VX dermally and in whom RBC-ChE was depressed 70% or greater.

No clinical or experimental evidence is available to indicate that VX causes delayed neuropathy in humans (Munro et al., 1994). Chickens injected subcutaneously with supralethal doses of VX (10, 100, or 150 µg/kg, following treatment with antidotes to protect against acute toxicity) exhibited no signs of a delayed neurotoxic response (Goldman et al., 1988). However, QL, a chemical intermediate of VX, has been reported to cause delayed neurotoxic effects in hens dosed at ≥635 mg/kg (Olajos et al., 1986). The available data indicate that delayed neuropathy in humans exposed to VX is unlikely.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

3.6 Developmental and Reproductive Effects

In studies conducted by Schreider et al. (1984), pregnant rats were dosed with 0.25, 1.0, or 4.0 µg VX/kg by s.c. injection on days 6–15 of gestation. The animals were sacrificed on day 20 of gestation. The examined fetuses showed no evidence of malformations. Fetal body weight, litter size, and sex ratio were within normal limits.

The effects of VX on the development and reproduction of sheep were evaluated by Van Kampen et al. (1970) following an accidental release of the nerve agent VX in Skull Valley, Utah. Of some 6,300 affected animals, about 4,500 died or were killed (Van Kampen et al., 1970). Seventy-nine surviving animals that had been pregnant at the time of exposure and their lambs were evaluated for changes in RBC-AChE activity and for signs of toxicity over a 6-month post-exposure period. RBC-AChE activity in the ewes remained significantly depressed for about four months and then returned to normal. Ewes that were sacrificed at 2-week intervals had no gross or microscopic evidence of damage to the central nervous system. Torticollis (wryneck) developed in one ewe one week following exposure and persisted for nine months (a similar effect was seen in 1 of 38 ewes dosed in the laboratory with an undisclosed amount of VX). Of the lambs born 2–3 months after exposure of the ewes, only one (total number examined not reported) exhibited deformities (extra oral opening below the right ear), but these were not considered to be agent related. None of the lambs displayed neurotoxic signs or symptoms, and their whole blood cholinesterase activity was not reduced even when suckling from exposed and affected ewes. Five months after exposure, the ewes exposed in the field as well as ewes dosed with an undisclosed amount of VX four months previously, were mated to unexposed males. Examination four months later indicated that fetal growth and development were normal except for one fetus that appeared stunted (total number examined not reported). The investigators concluded that VX had little or no effect on fetal growth or development.

Goldman et al. (1988) administered VX subcutaneously to Sprague-Dawley rats on days 6–15 of gestation. The administered doses were 0, 0.25, 1.0, or 4.0 µg/kg/day. Body weight, frequency of visceral and skeletal abnormalites, litter size, and sex ratios were evaluated. There was no statistical evidence that VX affected any of the parameters studied. Blood cholinesterase levels were not monitored.

Goldman et al. (1988) administered s.c. doses of 0, 0.25, 1.0 and 4.0 µg VX/kg/day to New Zealand white rabbits on days 6–19 of gestation. Animals were also observed daily for signs of toxicity. The does were sacrificed on day 29 of gestation. Body weight, fetal weights, fetal deaths, frequency of visceral and skeletal abnormalites, litter size, and sex ratios were evaluated. There was no statistical evidence that VX affected any of the parameters studied. Blood cholinesterase levels were monitored in a 7-day pilot study which also included a dose of 8 µg/kg. The 8 µg/kg dose was severely toxic to the rabbits (1/3 died, 2/3 ataxic). The dose of 0.25 µg/kg resulted in a level of RBC-AChE inhibition equal to 0.71 of the control value, but produced no signs of toxicity.

In a modified dominant lethal study, Goldman et al. (1988) administered VX by subcutaneous injection to male and/or female Sprague-Dawley rats and observed the effects on various parameters including terminal body weight, testes weight, testicular histopathology, maternal weight, implantation sites, resorptions, and total corpora lutea. The test animals were dosed with 0 (saline control), 0.25, 1.0, or 4 µg VX/kg/day for ten weeks. Triethylenemelamine was used as a positive control. Exposure to VX produced no significant changes in body or organ weights. VX had no adverse effects on preimplantation losses as evaluated by number of implants, live fetuses, dead fetuses, and resorptions. Microscopic examination of the testes did not reveal any abnormalities that could be attributed to VX exposure.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

In a 3-generation study, male and female Sprague-Dawley rats were dosed subcutaneously with 0 (saline controls), 0.25, 1.0, or 4.0 µg VX/kg/day, 5 days/week (Goldman et al., 1988). The F0 generation (11–12 males and 24 females per dose group) was dosed for about 105 days after which they were mated and the dosing continued through gestation and weaning (total duration of dosing 21–25 weeks). Dosing of the F1 generation began after weaning and continued for approximately 126 days after which they were mated and dosing continued through gestation and weaning (total duration 24–27 weeks). Five males and 5 females of each dose group of the F2 generation were sacrificed at weaning. The study included analysis of pup mortality in each of the generations, body and organ weight changes and hematological parameters in the F0 generation, and histopathological examination of tissues (including nervous system, reproductive system, gastrointestinal tract, lung, liver, and kidney) of the F1 parental males and females, the F1 weanlings, and the F2 weanlings. Blood cholinesterase activity levels were not monitored during the study. VX exposure had no adverse effect on the number of pups born in the F1 or F2 generation. Perinatal mortality (i.e., percent of pups born dead or dying within 24 hr of birth) was not significantly different among dose levels for both generations; however, perinatal mortality in the high-dose group (5.7%) was considerably higher than that in the lower dose groups (1.2%). Pup mortality from birth to weaning was significantly (p <0.01) related to VX exposure primarily for the F1 generation pups in the 4.0 µg/kg/day dose group. Goldman et al. (1988) attributed this increase to the effect of VX on the dams which resulted in the increased incidence of cannibalism of the pups; the investigators concluded that under the conditions of the test, there was no evidence of direct VX reproductive toxicity. The hematological studies conducted on dosed males of the F0 generation revealed no significant VX-associated effects. In females dosed with 4.0 µg VX/kg/day, statistically significant decreases occurred in hemogloblin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin. Body and organ weight analysis and histopathological examination revealed three effects that may have been dose related - changes in brain weight, incidence of eosinophilic gastritis, and incidence of pituitary cysts; however, Goldman et al. (1988) attributed the first two effects to statistical chance and considered the third as not being biologically significant. The overall conclusion of the investigators was that there were no organ weight or microscopic changes that could be attributed specifically to the action of VX.

3.7 Carcinogenicity

No information is available regarding the potential carcinogenicity of VX in humans. Standard long-term carcinogenicity studies have not been conducted on laboratory animals exposed to VX. Neoplastic lesions were not observed in male and female CD rats injected subcutaneously with up to 0.25, 1.0, or 4.0 µg VX/kg/day for 90 days (Goldman et al., 1988). No other animal data are available to assess the potential carcinogenicity of VX.

3.8 Genotoxicity

No information is available regarding the genotoxicity of VX in humans. In tests on microorganisms and mammalian cell cultures, VX was not found to be mutagenic or was only weakly mutagenic (Goldman et al., 1988). VX did not induce biologically significant increases in mutations when tested in the Ames Salmonella assay using five revertant strains (TA135, TA100, TA98, TA1537, and TA1538) with and without metabolic activation (Goldman et al., 1988). In tests using the yeast Saccharomyces cerevisiae, VX did not induce recombinants following exposures to concentrations as high as 100 µg/mL (Goldman et al., 1988). VX also failed to induce forward mutations when tested on mouse L5178Y lymphoma cells at concentrations less than 50 µg/mL (Goldman et al., 1988). Although doses of 50 and 100 µg VX/mL resulted in increased numbers of mutations; these were not more than 1.5 times the control level (a 2-fold increase was considered the minimum required to establish a positive result).

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

4. ORAL REFERENCE DOSE FOR VX

4.1 Cholinesterase Inhibition as an RfD Endpoint

The critical endpoint for defining a maximum acceptable exposure level for nerve agents such as VX is considered to be the level at which no significant depression in blood cholinesterase activity occurs. In humans, 15% inhibition is generally considered to be the minimal change that can be observed with any statistical reliability (Callaway et al., 1951). Existing human response data (Marquis, 1988) indicate that human RBC-AChE inhibition of as much as 20% is not associated with adverse clinical signs or symptoms and should be considered only as evidence of organophosphate exposure. This contention is supported by the U.S. EPA (1995a) which reports scientific agreement that statistically significant inhibition of cholinesterase in multiple organs and tissues accompanied by clinical effects constitutes a hazard; however, in the absence of clinical effects, such inhibition may not be of biological significance. It is generally agreed that inhibition of RBC and/or plasma cholinesterase contributes to the overall hazard identification of cholinesterase inhibiting agents by serving as biomarkers (U.S. EPA, 1995a). In addition, animal data have shown that exposure to low doses of nerve agents for extended periods of time can result in low blood ChE activity levels without signs of toxicity. Bucci et al. (1992) found no evidence of toxicity in rats dosed i.p. with GA (up to 112 μg/kg), even though RBC-AChE activity was reduced about 37% in females (relative to controls). In oral toxicity studies conducted on GB, Bucci and Parker (1992) found that gavage doses of 0.3 mg/kg/day to rats caused nearly a 50% reduction in RBC-AChE activity without signs of toxicity. Goldman et al. (1988) observed no physical signs of clinical toxicity in Sprague-Dawley rats dosed subcutaneously with 1.0 μg VX/kg/day over 30 days, even though RBC-AChE activity was reduced 78–80% relative to controls. Rice et al. (1971) reported that whole blood cholinesterase of sheep dosed orally with 15 μg VX/day was reduced to 5% of the normalized baseline values (during the last 3 weeks of a 8-week dosing period) without any physical signs of clinical toxicity. Rice et al. (1971) also found that sheep showing signs of toxicity at higher dose levels recovered fully after the exposures ended. Further complicating the evaluation is the extreme variability in ChE levels of individual animals and different sexes and ages of the same species (Halbrook et al., 1992). Possible changes in blood ChE that may occur with increasing age of the animals requires comparisons with concurrent controls, because the absence of a significant difference from pre-exposure value may be due to age-related increases in ChE in the dosed animals.

Blood ChE activity has been used by EPA as the critical endpoint in the establishment of oral RfDs for organophosphate insecticides. In the case of malathion (U.S. EPA, 1995b), the no-observed-effect level (NOEL) was identified as the highest oral dose level at which no significant change in RBC-AChE or plasma-ChE activity was recorded in five human volunteers who received the compound orally for 47 days (Moeller and Rider, 1962). The next highest dose was associated with a depression of about 25% in both RBC-AChE and plasma-ChE, but no clinical signs of toxicity. The EPA approach, also used for other organophosphate pesticides, is, therefore, to identify the lowest-effect level (LEL) as the dose at which statistically significant decreases in ChE levels (RBC-AChE, plasma-ChE, or brain-ChE) occur, and then to base an RfD on the dose level where the change in ChE is not statistically significant. This approach is also used in this report so that the RfDs developed for the nerve agents will not be disproportionally different from those for organophosphate insecticides; however, it should be emphasized that these values may be overly conservative. Furthermore, in evaluating the experimental data for the nerve agents, added weight was given to those cases where significant changes in ChE occurred relative to both control and pre-exposure values and where there was evidence of a dose-response relationship.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

4.2 Derivation of the Oral RfD for VX

For the derivation of an oral RfD, chronic or subchronic human oral exposure data are preferred; however, the only available human dose-response data for VX pertain to acute exposures. Although such data can be used to establish short-term exposure limits, they are generally not used for establishing subchronic or chronic reference doses (for comparative purposes, an RfD for VX was derived using short-term human exposure data; see Appendix D).

No chronic animal toxicity studies have been conducted on VX; however, there are two subchronic studies which can be used for developing an RfD. In one study, rats were dosed by s.c. injection 5 days per week for 90 days (Goldman et al., 1988). In the second study, sheep received daily doses of VX in feed for 56 days (Rice et al., 1971). Both of these studies identify blood cholinesterase as the most sensitive endpoint. Data are available indicating that sheep are more sensitive than rats to the toxic effects of VX. Ivanov et al. (1993) reported that the oral LD50 in sheep is 6 μg/kg whereas that for rats is 66 μg/kg. In addition, Ivanov et al. (1993) suggested that this increased susceptibility in sheep may be due, in part, to the lower concentration of catalytic sites for serum ChE in sheep (7.098 × 10-10 mol/L vs. 1.704 × 10-9 mol/L in rats). The Rice et al. (1971) study is selected here for deriving an oral RfD because it utilized an exposure route that is more relevant for an oral RfD, and also because the experimental evidence indicates that sheep are the more sensitive of the species tested.

Rice et al. (1971) fed VX to healthy yearling ewes for 56 days and measured changes in whole blood cholinesterase over this time period (see Section 3.3 for a more complete discussion of this study). The dose levels were 0, 3, 9, and 15 μg VX/day. Whole blood ChE was significantly depressed at all dose levels (levels of significance not reported) without any physical signs of clinical toxicity in any dose group. At the lowest dose, the decrease in whole blood ChE was statistically significant by the twenty-first day. Because of this significant reduction in ChE (38% relative to pre-exposure values), this dose (3 μg/day) is considered a LOAEL. The sheep used in this study had an average weight of 52.7 kg; therefore, the weight-normalized dose for 3 μg/day is 0.06 μg/kg/day.

The LOAEL of 0.06 μg/kg/day can be used to estimate a human oral reference dose (RfD) by using the following formula:

where:

LOAEL

=

0.06 μg/kg/day

UF1

=

10 (sensitive subpopulations)

UF2

=

1 (animal to human extrapolation)

UF3

=

3 (extrapolation from subchronic to chronic exposures)

UF4

=

3 (LOAEL to a NOAEL extrapolation)

UF5

=

1 (data base completeness)

MF

=

1 (Modifying factor)

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

An uncertainty factor of 10 for sensitive subpopulations is considered necessary because some individuals have a genetic defect causing their blood cholinesterase activity to be abnormally low (Evans et al., 1952; Harris and Whitaker, 1962). For homozygous individuals, the activity can be as low as 821% of the normal mean (Bondenan and Bonderman, 1971). These individuals may be unusually sensitive to organophosphate anticholinesterase compounds (Morgan, 1989).

An uncertainty factor is not used to extrapolate from the animals to humans because there is sufficient evidence that humans are not more sensitive to VX than sheep. The following evidence is available to support this position:

  1. Sheep have a much lower RBC-AChE activity level compared to humans, 2.9 μmol/mL/min versus 12.6 μmol/mL/mi (see Table 1). If ChE activity in the blood acts as a buffer to the effects of anticholinesterase compounds, than the lower activity level in sheep may cause them to have a higher susceptibility to agents such as VX.

  2. In humans, a daily oral dose of 1.43 μg/kg for seven days resulted in a 60% reduction in RBC-AChE activity (Sim et al., 1964), whereas in sheep a nearly equivalent reduction in whole blood ChE (56% inhibition) resulted from a dose of only 0.28 μg/kg/day administered for 8–13 days (Rice et al., 1971). [Note: in sheep, about 90% of the blood ChE activity is in the RBC fraction (Osweiler et al., 1985); therefore, sheep whole blood ChE measurements can be reasonably compared to human RBC-AChE values].

  3. The whole blood ChE50 in sheep is about 2.4 μg VX/kg (estimated from data presented in Rice et al., 1971), and the oral RBC-AChE50 in humans is about 2.3 μg/kg (Sidell and Groff, 1974).

  4. The similarities in VX sensitivity between sheep and humans may, in part, be accounted for by similarities in rates of metabolic detoxification of the agent. The latter can be estimated from a comparison of body surface areas (based on body weight raised to the 3/4ths power) as is done for animal-to-human extrapolations used in EPA cancer risk assessments(U.S. EPA, 1996). Using this approach, the human equivalent dose for the 0.06 μg/kg/day sheep LOAEL in the Rice et al. study can be calculated as:

where:

BWhuman

= default body weight of 70 kg for humans

BWsheep

= average body weight of 52.7 kg for sheep in the Rice et al. (1971) study

LOAELsheep

= the experimental dose of 0.06 μg/kg/day for sheep

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

therefore:

This calculation indicates that the human dose to produce a similar level of effect would not be substantially different from that in sheep. Considered together with the blood ChE activity and toxicity values mentioned above, the evidence is considered sufficient to support the use of an Uncertainty Factor of 1 for animal-to-human extrapolation.

An uncertainty factor of 3 is used to extrapolate from a subchronic to chronic exposure. In the derivation of oral RfDs for other organophosphate compounds, EPA has used NOAELs for cholinesterase inhibition following short-term exposures without adjustment for a more prolonged exposure period because of the unlikelihood that the endpoint would change over time (i.e., a subchronic-to-chronic UF of 1 was used). In addition, animal data indicate that maximum ChE inhibition may occur 30–60 days or more after exposure begins after which it levels off or even shows signs of recovery. This pattern can be seen in the data for the Rice et al. (1971) study (see Appendix B). However, an uncertainty factor of 3 is used here because chronic studies are not available to verify the unlikelihood that additional effects would occur following chronic exposures.

A LOAEL-to-NOAEL uncertainty factor of 3 is used instead of 10 because the endpoint, cholinesterase inhibition, was not associated with any physical signs of clinical toxicity. Furthermore, regression analysis of the Rice et al. (1971) data (see Table 4) indicates that 30% inhibition of ChE which is considered to be the threshold for a biological significant level of inhibition by EPA (pers. commun. H. Choudhury) would have occurred at about 2 μg/kg/day, substantially above the NOAEL value of 0.3 μg/kg/day that would result from applying a full UF of 10 to the LOAEL.

The data base requirements have been met in that there are subchronic toxicity studies in two species (sheep and rats), teratology studies in two species (rats and rabbits), a modified dominant lethal study in rats, a delayed neuropathy study in chickens, and a multigeneration study in rats. In addition, there are substantial human data supporting the RfD. The uncertainty associated with the absence of a chronic toxicity study is accounted for in UF3 above.

No modifying factor is required in the derivation of the RfD for VX.

The RfD for VX is therefore:

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

Table 4. Regression analysis of Rice et al. (1971) data for sheep dosed with VX

 

Linear Analysis

Log Transformation

Time (hr)a

Mean Doseb for 30% ChE Inhibition (μg/kg/day)

r2

Mean Doseb for 30% ChE Inhibition (μg/kg/day)

r2

744

0.88

0.961742

1.94

0.993047

912

1.69

0.836172

2.26

0.940522

1176

0.97

0.930412

1.97

0.980956

1320

1.21

0.90184

2.08

0.969214

Overall meanc

1.33

 

2.15

 

Standard deviation

0.46

 

0.24

 

Lower 95% CL

0.76

 

1.86

 

Upper 95% CL

1.90

 

2.45

 

a Data analysis based on four time points at which ChE inhibition stabilized

b Based on %ChE inhibition at 3, 9 and 15 μg/kg/day (see Appendix B)

c Derived from means for 744, 912, 1176, and 1320 hr

4.3 Overall Confidence in the RfD

Study: Medium

Data Base: High

RfD: High

The data base for VX consists of subchronic studies in sheep and rats, teratology studies in rats and rabbits, a modified dominant lethal study in rats, a delayed neuropathy study in chickens, and a multigeneration study in rats. In addition, there are also data available evaluating the effects of VX in humans following acute and short-term exposures. Although the principal study did not report on clinical chemistry, hematology, body and organ weight changes, or gross or histological pathology, there are supporting studies to indicate that cholinesterase inhibition is the appropriate endpoint. There is also evidence that sheep are one of the most sensitive species in their response to cholinesterase inhibitors. Therefore, the overall confidence in the RfD is high.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

4.4 Comparison of RfD with Human Toxicity Data

The proposed RfD is compared to the available human toxicity data in Table 5. One study in humans indicated that an oral dose of 1.43 µg/kg/day for 7 days resulted in a 60% RBC-AChE inhibition but no toxic effects (Sim et al., 1964). This dose is over 2000 times greater than the RfD. For an adverse effect level (i.e., mild toxic effect at 2–4.5 µg/kg/day), the ''margin of safety" would be larger. The results of the Sim et al. (1964) study indicating a LOAEL of 1.43 µg/kg/day for a 7-day exposure, were used to calculate an oral RfD for comparison with the RfD derived from the animal data (see Appendix D). Using a UF of 10 for sensitive subpopulations, a UF of 10 for a LOAEL-to-NOAEL extrapolation (10 is chosen because the dose of 1.43 µg/kg/day produced a 60% inhibition of RBC-ChE, which could be close to the toxic effect level), and a UF of 3 for protecting against longer exposures (animal data indicate that maximum ChE inhibition may occur 30–60 days after exposure begins and the Sim et al. study was for only a 7-day duration), the resulting estimated oral RfD is 0.005 µg/kg/day, a value approximately one order of magnitude greater than the RfD of 0.0006 µg/kg/day estimated from the Rice et al. (1971) sheep data.

Table 5. Comparison of RfD with human toxicity data of VX

Dose (µg/kg)

Exposure Route

Endpoint

References

0.0006a

oral

RfD - no inhibition of RBC-ChE

This report

0.1

intravenous

Estimated no effect level for RBC-ChE inhibition

McNamara et al., 1973

0.24

oral

Estimated no effect level for RBC-ChE inhibition, based on ration of oral to i.v. doses required for 50% RBC-ChE inhibition.

This report

0.34

inhalation

Estimated threshold for tremors based on inhalation data for GB

McNamara et al., 1973

1.0

intravenous

50% inhibition of RBC-ChE

Sidell and Groff, 1974

1.43

oral; once per day for 7 days

60% inhibition of RBC-ChE; no signs or symptoms of toxicity

Sime et al., 1964

2.4

oral

50% inhibition of RBC-ChE

Sidell and Groff, 1974

2-4.5

oral

Gastrointestinal symptoms in 5/32

Sidell and Groff, 1974

a Daily dose for chronic exposure

5. CARCINOGENICITY ASSESSMENT

The potential carcinogenicity of VX cannot be determined. Data are inadequate for performing a quantitative assessment of agent VX. The results of tests on bacteria, yeast and mammalian cell cultures (see Section 3.8) indicate that VX is not mutagenic or is only weakly mutagenic. These data provide supporting evidence that VX is not likely to be carcinogenic.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

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Yager, J., H. McLean, M. Hudes and R.C. Spear. 1976. Components of variability in blood cholinesterase assay results. J. Occup. Med. 18:242–244.


Zhao, D.-L., Z.-X Wang, S,-Q. Pei and C.-H. Liu. 1983. Effects of soman, sarin, and VX on the specific binding of 3H-QNB in rat cerebral cortex homogenates. Acta Pharmacol. Sinica 4:225–228.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX A Comparison of RfDs, ChE Inhibition and Toxicity Data for GA, GB, GD and VX

Endpoint

GA (µg/kg/day)

GB (µg/kg/day)

GD (µg/kg/day)

VX (µg/kg/day)

Ref.

RfD

0.04

0.02

0.004

0.0006

This report

Estimated no-effect level for RBC-AChE inhibition

-

1.0

-

0.24

GBd

VXa

27–33% inhibition of RBC-AChE in humans/oral dose

-

2.3 (3 days)

-

0.2–2.0

GB - Grob and Harvey, 1958;

VX-this report

RBC-AChE inhibition in humans/i.v. dose

-

-

1.5–2.0

(30%)

1.0

(50%)

DA, 1974;

Sidell and Groff, 1974

50–60% RBC-AChE inhibition in humans/oral dose

-

10

-

2.4

GB - Grob and Harvey, 1958;

VX-Sidell and Groff, 1974

50% brain ChE inhibition

in vitro

1.5 × 10-8 (c)

0.3 × 10-8 (c)

-

-

Grob and Harvey, 1958

Acute toxic effects in humans/oral dose

-

20–30

-

2–4.5

GB - Thienes and Haley 1972; Grob and Harvey, 1958;

VX-Sidell and Groff, 1974

human oral LD50 (estimated)

25–50b

5–20b

5–20

3–10b

Somani et al., 1992

rat oral LD50

3700

870–1060

600

400

77–128

DA, 1974

Grob & Harvey, 1958

monkey i.v. LD50

50

20

-

6–11

DA, 1974

rat i.v. LD50

70

45–63

50

6.9–10.1

Dacre, 1984

rat i.p. LD50

490, 800

250

218

-

37–55

DA, 1974

RTECS, 1995

a Based on ratio of oral to i.v. doses (2.4 and 1.0 µg/kg, respectively) required for 50% RbC-ChE inhibition and the estimated i.v. no effect dose of 0.1 µg/kg.

b Values were estimated from animal data.

c Molar concentration

d Estimated from RBC-ChE50 values for GB and VX.

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX B Graphical Analysis of Rice et al. (1971) Data for Sheep Dosed with VX

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX C Statistical Analysis of RBC-AChE Inhibition in Male Rats Dosed With VX

Study: Goldman et al., 1988

Species/sex: Sprague-Dawley rats/males

Endpoint: RBC-cholinesterase inhibition

Analysis: Comparisons made with controls (0 µ/kg/day) at 30 days

Dose (µ/kg)

0

0.25

1.0

4.0

 

 

 

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Mean

3.36

1.546

0.74

0.13

 

 

Std

0

0.06

0.0007

0.003

 

 

N

2

2

2

2

 

 

Bartlett's Test for homogeniety indicates the data is suitable for ANOVA.

ANOVA

SS Between

11.807

df =

3

 

F =

4361.593

SS Among

0.004

df =

4

 

p =

<0.001

MS Among

3.936

 

 

 

 

 

MS Between

0.001

 

 

 

 

 

Scheffe's Comparison

Comparison with:

 

Group 2

Group 3

Group 4

 

 

Group 1

 

p<0.05

p<0.05

p<0.05

 

 

Group 2

 

 

p<0.05

p<0.05

 

 

Group 3

 

 

 

p<0.05

 

 

Dunnett's Comparison

Comparison with:

 

Group 2

Group 3

Group 4

 

 

Group 1

 

p<0.05

p<0.05

p<0.05

 

 

Group 2

 

 

p<0.05

p<0.05

 

 

Group 3

 

 

 

p<0.05

 

 

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX C Statistical Analysis of RBC-AChE Inhibition in Female Rats Dosed With VX

Study: Goldman et al., 1988

Species/sex: Sprague-Dawley rats/females

Endpoint: RBC-cholinesterase inhibition

Analysis: Comparisons made with controls (0 µg/kg/day) at 30 days

Dose (µg/kg)

0

0.25

1.0

4.0

 

 

 

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Mean

2.86

1.37

0.57

0.29

 

 

Std

0

0.08

0.006

0.014

 

 

N

2

2

2

2

 

 

Bartlett's Test for homogeniety indicates the data is suitable for ANOVA.

ANOVA

SS Between

7.977

df =

3

 

F =

1603.729

SS Among

0.007

df =

4

 

p =

<0.001

MS Among

2.659

 

 

 

 

 

MS Between

0.002

 

 

 

 

 

Scheffe's Comparison

Comparison with:

Group 2

Group 3

Group 4

 

 

 

Group 1

p<0.05

p<0.05

p<0.05

 

 

 

Group 2

 

p<0.05

p<0.05

 

 

 

Group 3

 

 

p<0.05

 

 

 

Dunnett's Comparison

Comparison with:

Group 2

Group 3

Group 4

 

 

 

Group 1

p<0.05

p<0.05

p<0.05

 

 

 

Group 2

 

p<0.05

p<0.05

 

 

 

Group 3

 

 

p<0.05

 

 

 

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×

APPENDIX D CALCULATION OF ORAL RFD FROM HUMAN DATA

Study: Sim et al., 1964

Dose: 1.43 µg/kg/day orally for 7 days

Effect: 60% RBC-ChE inhibition but no toxic effects

LOAEL: 1.43 µg/kg/day Reference Dose:

where:

UF1

=

10 (sensitive subpopulations).

UF2

=

1 (animal to human extrapolation), not needed.

UF3

=

3 (extrapolation from 7-day exposure to subchronic exposures). Animal data suggest that ChE effects may increase over the first 30–60 days of exposure and then follow a slow rate of recovery.

UF4

=

10 (LOAEL to a NOAEL extrapolation); 60% inhibition is near a level where physical signs of clinical toxicity may occur.

UF5

=

1 the data base requirements are met and cholinesterase inhibition is considered to be the mechanism of toxicity.

MF

=

1 no modifying factor is needed.

therefore:

Suggested Citation:"D: Health Risk Assessment for the Nerve Agent VX." National Research Council. 1999. Review of the U.S. Army's Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents. Washington, DC: The National Academies Press. doi: 10.17226/9644.
×
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