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

Chapter: A: Health Risk Assessment for the Nerve Agent GA

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Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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
Health Risk Assessment for The Nerve Agent GA

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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 GA

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:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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 GA (CAS No. 77-81-6).

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:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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.

 

LD50 values for agent GA

 

8

Table 3.

 

RBC-AChE levels in 90-day subchronic rat study using agent GA

 

9

Table 4.

 

Plasma-ChE levels in 90-day subchronic rat study using agent GA

 

10

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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). GA contains a cyanide substituent group (VX contains a sulfur group and GB a fluorine group). The chemical synonyms, Chemical Abstract Service (CAS) and Army identification numbers (DA, 1974, 1992; Dacre, 1984), and chemical formula for GA are as follows:

Dimethylphosphoramidocyanidic acid, ethyl ester;

Dimethylaminoethoxy-cyanophosphine oxide;

Dimethylamidoethoxyphosphoryl cyanide;

Ethyl N, N-dimethylphosphoramidocyanidate;

Ethyl N, N-dimethylaminocyanophosphate

Ethyl dimethylphosphoramidocyanidate;

Ethyl dimethylamidocyanophosphate;

Ethylphosphorodimethylamidocyanidate;

Tabun;

CAS No. 77-81-6;

Edgewood Arsenal No. 1205

1.1. PHYSICAL/CHEMICAL PROPERTIES

Agent GA is a colorless to brown-colored liquid with a molecular weight of 162.1 (DA, 1974; MacNaughton and Brewer, 1994); it has a vapor density of 5.6 (air = 1) and a liquid density of 1.08 g/mL at 25°C (DA, 1974). The vapor pressure of GA is 0.07 mm Hg at 25°C; its solubility in distilled water is 9.8 g per 100 g at 25°C and 7.2 g per 100 g at 20°C (DA, 1974).

1.2. ENVIRONMENTAL FATE

1.2.1 Air

The vapor pressure for GA is 0.07 mm Hg at 25°C indicating a moderate potential for volatilization. A vapor concentration of 610 mg/m3 has been reported for a temperature of 25°C (DA, 1974) (although not adequately described in the reference, this presumably is the saturation concentration above a pure liquid).

1.2.2 Water
Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

GA has a water solubility of 50–100 mg/L (MacNaughton and Brewer, 1994)); therefore, it is a potential water contaminant. However, because it is subject to hydrolysis, it is not expected to be very persistent in aqueous systems. The half-life of GA is less than 10 min at pH levels greater than 9, 2–3 hr at pH 8–9, and about 6 hr at a pH of 4 (MacNaughton and Brewer, 1994).

The Henry's Law Constant for GA has been estimated to be 1.3 × 10-6 atm m3/mol (MacNaughton and Brewer, 1994), indicating that GA may volatilize slowly from water.

1.2.3 Soil

Although a soil half-life of 1 to 1.5 days has been reported for GA (DA, 1974), information was not provided on the temperature, pH, or moisture content and other environmental conditions for which this estimate was made.

The volatility potential (slope of the vapor pressure vs. concentration in soil organics) of GA is 2.4 × 10-7 mm Hg/mg/kg and its air-soil partition coefficient (for a soil density of 1.4 g/cm3) of 1 × 10-4 mg/m3 (MacNaughton and Brewer, 1994), indicate that GA will evaporate from soil into the air. Results of a field trial with GA showed 10% evaporation in 0.27 hours and 90% evaporation in 4.66 hours (Morrill et al., 1985).

Binding of GA to soil organics is likely to be limited considering the relatively low log Kow of 0.11 and low Koc values of 25 (MacNaughton and Brewer, 1994); therefore, a potential exists for leaching and groundwater contamination. MacNaughton and Brewer (1994) calculated a leaching index of 2 for GA, (i.e., the number of leachings required to reduce the GA soil concentration to one-tenth of the original amount, assuming that for each leaching one kilogram of soil is in equilibrium with one liter of water). However, the amount of GA reaching ground water is likely to be limited by hydrolysis.

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 (deactivated by acetylcholinesterase). 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), 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).

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

2.1 Effects of Organophosphate Agents 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 agents, these compounds can apparently affect nerve-impulse transmission by more direct processes as well. Direct effects may occur on excitable tissues, receptors, and ionic channels. 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. Albuquerque et al. (1985) have shown that agent GA, as well as agents GB and GD are capable of changing receptor sites in a manner similar to that exhibited by acetylcholine, which promotes the conductance of electrophysiological signals associated with stimulation of neuromuscular function. 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.

2.2 Effect on Blood Cholinesterases

In addition to being found in the nervous system, acetylcholinesterase also occurs in the blood where it is bound to the surface of red blood cells (termed RBC-ChE or 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 activity have been used as bioindicators of potential toxic effects. 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),

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

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 or more 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-AChE compared to tissue ChE, or to noncholinesterase-dependent recovery pathways 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 toxicity. Animal studies have demonstrated that chronic exposures to low concentrations of organophosphate insecticides and nerve agents can 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 treated with organophosphate insecticides. 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, agent VX preferentially inhibits RBC-AChE (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 to 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 much more acutely toxic than agents GA and GB (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-AChE 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 variation of RBC-AChE 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-AChE 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-AChE activity and 14.4% for plasma-ChE activity. Callaway et al. (1951) estimated that with only one pre-exposure measurement, the smallest measurable decrease was 15% of the baseline value for RBC-AChE activity and 20% of the baseline for plasma-ChE activity.

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).

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

Morgan (1989) suggests that these individuals may be unusually sensitive to organophosphate anticholinesterase compounds.

Data compiled by Ellin (1981) reveal that the RBC-AChE activity for humans is slightly higher than that for monkeys and much higher than that for rats and other laboratory animals (Table 1). These differences in RBC-AChE 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. For example, rodents, but not humans, have high levels of aliesterases (AE) in the blood, and these compounds may provide rats and mice with a higher level of resistance to some anticholinesterase compounds (McNamara and Leitnaker, 1971).

Table 1. RBC-AChE activity in different species

Species

RBC-AChE 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-AChE activity.

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×
2.2.2 Potency of Nerve Agents as Cholinesterase Inhibitors

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

(1)

The pI50 (negative log of the molar concentration causing 50% inhibition) for GA was reported to be 8.4 by Holmstedt (1959), 8.6 by Dacre (1984), and calculated as 7.8 from an I50 of 1.5 × 10-8 mol/L reported by Grob and Harvey (1958). Grob and Harvey (1958) reported that the potency of GA in inhibiting RBC-AChE was only one-fifth of that for GB (I50 = 0.3 × 10-8 mol/L).

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).

3. Toxicology

3.1 Introduction

Health and environmental impacts of nerve agents and related compounds (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 general discussion of the 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 acute 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

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

by paralysis (see review by 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 NTE; however, the exact mechanism of toxicity is not yet fully understood (Munro et al., 1994). There is no evidence that GA causes OPIDN in humans. The limited animal data indicate that GA may cause some inhibition of NTE and slight signs of OPIDN in some species, but OPIDN has only been observed in antidote-protected chickens dosed with 120 times the LD50 for GA (see section 3.5). The likelihood that GA would induce OPIDN in humans at dose levels below those causing acute toxicity or cholinesterase inhibition is very small.

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 nerve agents can induce neuropsychological changes in acutely exposed individuals, there is no evidence of effects persisting for months or years as has been reported for some organophosphate insecticides (Savage et al., 1988). Although some studies have identified neurologic and psychological changes in workers occupationally exposed to organophosphate insecticides, it is unclear to what degree these effects may have been caused by intermittent acute exposures (Gershon and Shaw, 1961; Mick, 1974; Rodnitzky, 1974; Wagner, 1983; Tabershaw and Cooper, 1966). However, the available data on the organophosphate insecticides suggest that long-term toxicological effects do not occur in the absence of significant changes in blood cholinesterase activities, and a similar conclusion is likely to apply to the nerve agents.

3.2 Acute Toxicity

GA lethality data for animals and estimates of human LD50 values are given in Table 2. Oral LD50 values for humans have been estimated to be 357–714 µg/kg (Somani et al., 1992). A subcutaneous injection of 0.43 µmol/kg (0.0698 mg/kg) in dogs resulted in a depression in erythrocyte cholinesterase activity to about 30% of its baseline value; however, no overt toxic effects were observed (Holmstedt, 1951). In cats, a slow drip intravenous (i.v.) infusion of 0.05 mg/kg or more resulted in increased bronchial constriction and labored respiration (Holmstedt, 1951). In rabbits, an i.v. dose of about 0.04 mg/kg (25% of the lethal dose) caused a steady decrease in cardiac output (Holmstedt, 1951).

3.3 Subchronic Toxicity

The National Center for Toxicological Research evaluated the subchronic toxicity of agent GA on male and female CD rats (Bucci et al., 1992). The test animals (12/sex/dose group) were injected intraperitoneally with GA at dose levels equivalent to 0, 28.13, 56.25, or 112.5 µg/kg/day. The injections were given once per day, 5 days per week for 13 weeks. Animals were observed daily for clinical signs of toxicity and weighed weekly. Necropsy examination was performed on all animals. Terminal body and organ weights were recorded. Microscopic evaluation was performed on all high-dose and control animals as well as on all gross lesions and on animals dying or sacrificed before the end of the test period. Blood samples were taken from 6 rats/sex/dose during weeks -1, 1, 3, 7 and at necropsy. Hematological analyses consisted of blood cell counts, hemoglobin, hematocrit, and mean corpuscular volume, hemoglobin and hemoglobin concentration. Clinical chemistry included measurements of alanine

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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 2. LD50 values for agent GA

Exposure route

Speciesa

LD50(µg/kg)

References

Intravenous

human

14b

Robinson, 1967

 

monkey

~50

DA, 1974

 

pig

 

rat

70

DA, 1974

Oral

human

357–714c

Somani et al., 1992

 

monkey

 

rat

3700

RTECS, 1995

Subcutaneous

human

 

monkey

70

RTECS, 1995

 

rat

162

RTECS, 1995

 

rat

~300

DA, 1974

Percutaneous

human

14,000–21,000

DA, 1974

 

human

2,857–14,286c

Somani et al., 1992

 

monkey

9,300

RTECS, 1995

 

pig

 

rat

18,000

RTECS, 1995

 

rat

12,600

Crook et al., 1983

Intramuscular

human

 

monkey

34

RTECS, 1995

 

rat

800

RTECS, 1995

Intraperitoneal

human

 

monkey

 

rat

~800

DA, 1974

 

 

490

RTECS, 1995

a Values for humans estimated from animal data

b LDLo

c Estimated for 70 kg individuals

aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, creatinine kinase, and RBC and plasma cholinesterase. In addition, at necropsy, brain samples were tested for NTE.

The results of the clinical chemistry tests indicated no adverse effects on liver, kidney, or muscle. Hematological parameters for the dosed animals were generally within the normal range, and brain NTE activity was not affected by GA administration. There were no GA-related neoplastic or non-neoplastic lesions.

Cholinesterase levels in the dosed rats were compared to control values for the same sampling times (Tables 3, 4). There was considerable variability in the RBC-AChE data. Mean baseline values for both male and female rats were elevated substantially (5029 versus 1848 IU/L in females and 4045 versus 1552 IU/L in males) when compared to control levels recorded in previous nerve agent studies conducted at the same laboratory. The elevated pre-exposure RBC-AChE readings in the current study were attributed to faulty reagents. Mean RBC-AChE activity levels in dosed and concurrent control animals also showed irregular fluctuations over time, with unusually high readings occurring in females at week 3 and in males at week 7. It was reported that the percent reduction in RBC-AChE at week 3

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

was about 37% in dosed females and 18% in males. Statistical analysis of the RBC-AChE data indicated significant reductions in RBC-AChE activity (relative to controls) in only a few cases; i.e., in high-dose females at week 3; in mid-dose females at week 7; and in males of all dose groups at week 1.

Table 3. RBC-AChE levels in 90-day subchronic rat study using agent GAa

 

 

Week of treatment

Dose (µg/kg/d)

Sex

-1

1

%b

3

%b

7

%b

13

%b

0

F

4604 (1451)

2949 (370)

64

5472 (801)

119

4483 (441)

97

4457 (261)

97

28.13

F

5272 (589)

3181 (331)

60

4360 (179)

83

3723 (397)

71

4386 (414)

83

56.25

F

5168 (623)

2975 (180)

58

4315 (514)

83

3169 (381)c

61

4205 (545)

81

112.50

F

5073 (428)

2421 (184)

48

3428 (174)c

68

3404 (364)

67

4229 (721)

83

0

M

3939 (434)

3588 (297)

91

4427 (422)

112

5582 (455)

142

4789 (290)

122

28.13

M

4115 (356)

2549 (311)c

62

4106 (557)

99

5025 (98)

>100

4058 (465)

99

56.25

M

4369 (499)

2409 (788)c

55

3630 (417)

83

5200 (340)

>100

4601 (553)

>100

112.50

M

3760 (541)

2367 (534)c

63

3612 (582)

96

5145 (281)

>100

4126 (491)

>100

Source: Bucci et al., 1992

a Results given in IU/L, mean and (SEM)

b Percent of baseline.

c p <0.05, different from control value (analysis by Bucci et al., 1992)

The RBC-AChE data were re-analyzed by ORNL (using standard deviations) with ANOVA, and Dunnett's Comparison (see Appendix B). The ORNL analysis indicated that RBC-AChE levels in males at week 1 were not significantly lower than control values, but were significantly lower than pre-exposure values (p <0.05) for the two lowest dose groups, but not the high-dose group. Similar re-analysis of the female RBC-AChE data indicated that the high-dose group had significant reductions from pre-exposure values at weeks 1, 3, and 7 (Appendix B).

Changes in plasma-ChE activity in dosed and control animals are shown in Table 4. Over the course of the study, plasma-ChE activity levels in dosed and control animals appear to be more stable than RBC-AChE activity. It was reported that plasma-ChE activity was decreased by about 55% in dosed females at week 7, and by 37.5% in dosed males at week 3. Mean plasma-ChE activity in the female controls exhibited a slow increase over the 13-week test period (from 1743 IU/L at week -1 to 2891 IU/L at week 13). A similar response was seen in the two lowest dose groups of females. In males, mean plasma-ChE activity in controls was lower than pre-exposure levels (401 IU/L at week -1) at all weeks except week 3 (413 IU/L). In the dosed groups of males, mean plasma-ChE levels were lower than pre-exposure values at all sampling times. Statistical analysis of the plasma-ChE activity indicated that mean values were significantly lower than controls in the mid- and high-dose females at weeks -1, 1, 3, and 7 but not at week 13, and in the high-dose males at weeks 3 and 7.

The plasma-ChE data were re-analyzed by ORNL (using standard deviations) with ANOVA, and

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

Dunnett's Comparison (see Appendix C). The ORNL analysis indicated that plasma-ChE levels in males were significantly lower (p <0.05) than pre-exposure values at week 1, 3, 7 and 13 in the mid-dose group; and at weeks 1, 3, and 7 in the high-dose group. The two highest dose groups were also significantly lower (p <0.05) than control values at weeks 3 and 7 (and at week 1 for the high-dose group). In females, plasma-ChE levels were not significantly lower than preexposure values, but were significantly lower (p <0.05) than controls at weeks 1, 3 and 7 for all dose groups (Appendix C).

Table 4 Plasma-ChE levels in 90-day subchronic rat study using agent GAa

 

 

Week of treatment

Dose (µg/kg/d)

Sex

-1

1

%b

3

%b

7

%b

13

%b

0

F

1743(273)

2033(284)

116

2204(303)

126

2544(334)

146

2891(425)

166

28.13

F

1369(111)

1416(125)

103

1540(138)

112

1794(188)

131

2311(245)

169

56.25

F

1233(135)c

1288(164)c

104

1424(186)c

115

1548(194)c

125

1928(297)

156

112.50

F

1449(122)c

1202(113)c

82

1136(133)c

78

1148(121)c

79

1755(190)

121

0

M

401(25)

397(25)

99

413(37)

103

383(23)

96

344(26)

86

28.13

M

426(28)

394(25)

92

361(22)

85

362(17)

85

375(21)

88

56.25

M

415(13)

351(13)

85

319(12)

77

323(14)

78

330(18)

79

112.50

M

405(31)

311(16)

77

258(11)c

64

268(13)c

66

360(32)

89

Source: Bucci et al., 1992

a Results given in IU/L, mean and (SEM)

b Percent of baseline.

c p<0.05, different from control value (analysis by Bucci et al., 1992).

Dulaney et al. (1985) evaluated the effects of GA on the growth rates of rats given daily subcutaneous doses of 100 µg/kg for 85 days. The dosed animals exhibited reduced growth rates (42% of controls in the first 15 days, 82% of controls in the next 22 days, and 95% of controls from the 38th day to the end of the study). AChE activity was determined in the striatum and the remainder of the brain 24 hr after the last exposure. Mean brain striatal AChE activity was only 13% of the control value; in the remaining parts of the brain the AChE activity was 22% of the control. In cumulative mortality studies, rats (8–11/dose group) were dosed with 75 or 100 µg/kg/day for 25 days. In the low-dose group one of 8 animals died on day 10; in the high-dose group, one animal died on day 15 and another on day 20. Dosing was continued for an additional 60 days without any further mortality. Because of the observed mortality, the subcutaneous dose of 75 µg/kg/day can be considered a lowest-observed-adverse-effect level (LOAEL) under the conditions of the study.

3.4 Chronic Toxicity

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

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

3.5 Nervous System Toxicity

There has been concern that organophosphate compounds like GA may have direct toxic effects on the nervous system. Some organophosphate compounds cause a neurotoxic effect (organophosphate-induced delayed neuropathy or OPIDN) that is not associated with AChE inhibition but rather with inhibition of NTE. Agent GA has not been shown to produce OPIDN in humans. Some animal data suggest that it might have the potential to do so, but only following extremely high doses (Munro et al., 1994). In vitro and in vivo studies have demonstrated that supralethal doses of GA can cause NTE inhibition in antidote-protected chickens (Lotti and Johnson, 1978; Vranken et al., 1982); however, small daily doses given over a prolonged time period do not appear to induce this effect. In studies conducted by Bucci et al. (1992), intraperitoneal injections of GA into CD rats at dose levels up to 112.5 µg/kg/day, 5 days per week for 13 weeks did not result in a significant change in brain NTE. Furthermore, there was no clinical evidence of neuropathology, even though blood cholinesterase activity decreased significantly in the dosed animals. Although a number of studies have been conducted on chickens, in only a few cases have signs of OPIDN been observed. Henderson et al. (1989, 1992) reported no signs of OPIDN in chickens receiving one 0.125 mg/kg dose by intramuscular injection or repeated injections of 0.07 mg/kg, 5 days/week for 90 days. OPIDN was also not observed in antidote-protected chickens receiving one 12 or 15 mg/kg dose or two 12 mg/kg doses (120–150 times the LD50) by intramuscular injection (Willems et al., 1984; Johnson et al., 1988); however, mild signs of neuropathy occurred in one of two surviving chickens receiving two 6 mg/kg doses (Willems et al., 1984). Overall, the data suggest that it is unlikely that OPIDN would occur in humans at less-than-lethal doses (see Munro et al., 1994).

3.6 Developmental and Reproductive Effects

There are no data evaluating the potential developmental and reproductive toxicity of GA in humans. Limited animal data indicate that such effects are unlikely. In studies in which CD rats were injected intraperitoneally with 0, 75, 150, or 300 µg GA/kg/day on gestation days 6–15, no fetal malformations or developmental effects (with the exception of increased pre-implantation losses relative to controls) were observed (Bucci et al., 1993). Because dosing began on gestation day 6, which was near the end or after the time of implantation, the observed pre-implantation losses were not considered to be agent-related (Bucci et al., 1993). Blood cholinesterase levels were not monitored during the study. Signs of maternal toxicity (salivation and lacrimation) were seen at all dose levels, and the highest dose produced tremors in some animals. Mean maternal weight gain was also reduced in the high-dose animals when compared to that of controls. Maternal mortality rates were 1/31, 2/32, and 12/33 in the low-, mid-, and high-dose groups and all were considered to be agent-related. Therefore, the lowest dose of 75 µg/kg/day can be considered a LOAEL for maternal effects in rats under the conditions of the study.

In the tests in which agent GA (28.1, 56.3, and 112.5 µg/kg/day) was administered subcutaneously to New Zealand white rabbits on gestation days 6–19, no adverse effects on fetal implantations, fetal weight, and fetal malformations were observed (Bucci et al., 1993). However, maternal toxicity (indicated by salivation, diarrhea, and nasal discharge) was evident in the high-dose group which also experienced a mortality rate of 13.3% (4/30). Therefore, the highest dose of 112.5 µg/kg/day can be considered a LOAEL for maternal effects in rabbits.

3.7 Carcinogenicity

No information is available regarding the potential carcinogenicity of GA in humans. No long-

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

term animal carcinogenicity studies have been carried out on GA. Neoplastic lesions were not observed in male and female CD rats injected intraperitoneally with up to 28.13, 56.25, or 112.5 µg GA/kg/day for 90 days (Bucci et al., 1992); however, this subchronic study was of insufficient duration to fully evaluate tumor incidence rates. No other animal data are available to assess the potential carcinogenicity of GA.

3.8 Genotoxicity

No information is available regarding the genotoxicity of GA in humans; however, genotoxicity and mutagenicity data are available from microbial assays, and in vitro and in vivo tests on laboratory animals (Wilson et al., 1994). GA was found to be weakly mutagenic in eight of 11 Ames Salmonella assays using the revertant strains TA98, TA100, TA1535, and TA1538. GA also induced dose-related increases in mutation rates when tested on mouse L5178Y lymphoma cells without metabolic activation; the increase observed at a test concentration of 100 µg/mL was nearly three-times that of the control. An increase in sister chromatid exchanges (SCE) was observed in Chinese hamster ovary cells exposed in vitro to GA concentrations of 25–200 µg/mL. Dose-responses were linear and highly statistically significant; however, the number of SCEs did not exceed twice the control value at any of the concentrations tested. C57B1/6 mice treated in vivo with a maximally tolerated intraperitoneal dose of 700 µg GA/kg did not exhibit a significant increase in SCE in splenic lymphocytes. Exposure of rat hepatocytes to GA concentrations as high as 200 µg/mL resulted in inhibition of unscheduled DNA synthesis. From the results of these studies (i.e., three positive responses in five assays), Wilson et al. (1994) concluded that GA was a weakly acting mutagen.

4. ORAL REFERENCE DOSE FOR GA

4.1 Cholinesterase Inhibition as an RfD Endpoint

The endpoint for defining a maximum acceptable exposure level for nerve agents such as GA is considered to be the level at which no significant depression in blood cholinesterase activity occurs. In humans, 15% inhibition of RBC-AChE is generally considered to be the minimum 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). Animal data have shown that exposure to low doses of nerve agents for extended periods of time

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

can result in low blood ChE activity levels without signs of toxicity. Bucci et al. (1992) found no evidence of toxicity in rats dosed intraperitoneally 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 et al. (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) reported no signs of toxicity, but 78–80% reduction in RBC-AChE activity, in Sprague-Dawley rats dosed subcutaneously with 1.0 µg VX/kg/day over 30 days. Rice et al. (1971) reported that whole blood cholinesterase of sheep dosed with 15 µg VX/day was reduced to 4–5% of the normalized baseline values (during the last 3 weeks of the dosing period) without any signs of toxicity. Rice et al. (1971) also found that sheep showing signs of toxicity (not described) 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 (U.S. EPA 1995a,b). In the case of malathion (U.S. EPA, 1995a), 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 5 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 (U.S. EPA, 1995b), 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.

4.2 Derivation of the Oral RfD for GA

For the derivation of a chronic oral RfD, chronic or subchronic human oral exposure data are preferred; however, the only available human data for GA pertain to acute exposures. Although such data can be used to establish short-term exposure limits, acute toxicity endpoints are generally not used for developing subchronic or chronic reference values since they do not provide information on the possibility of cumulative effects following prolonged exposures. There are no subchronic or chronic animal studies on GA using the oral exposure route. Subchronic exposure data are available for GA from one rat study in which the animals were injected intraperitoneally once per day for 90 days (Bucci et al., 1992) and from another rat study in which rats were injected subcutaneously for 85 days (Dulaney et al., 1985). Non-oral exposure data are not normally used by EPA for deriving an oral RfD because of difficulties in estimating equivalent dose levels for oral exposures. EPA has used non-oral exposure data to derive an oral RfD for silver, but only because adequate data were available to estimate equivalent oral doses from experimental intravenous data by using information on the absorption of the element through the gastrointestinal tract (U.S. EPA, 1991a). The Bucci et al. (1992) study is used here to derive an oral RfD for GA because it included a series of doses and a more comprehensive evaluation of the potential toxicity

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

than did the Dulaney et al. (1985) study; however, an oral dosing study would have been preferred. Ideally, information on the relative absorption of GA through both the oral and i.p. routes should be used to estimate an equivalent oral dose from i.p. data. Because such data are not available, a very provisional estimate of the equivalent oral dose was derived from a comparison of oral and i.p. LD50 values for the rat. The reported rat oral LD50 value of 3700 µg/kg is 4.6 and 7.6 times larger than the reported i.p. LD50s of 800 and 490 µg/kg, respectively. The assumption is made that a similar relationship would occur under a subchronic exposure protocol.

The use of a rat study for developing an RfD for GA is complicated by the fact that rodents have a much lower RBC-AChE activity level compared to humans (Ellin, 1981). By itself, this could cause rats to be relatively more sensitive than humans to anticholinesterase compounds; however, the lower RBC-AChE activity may be offset by the presence of aliesterases in the blood of rats. Aliesterases, which are not found in human blood plasma, are known to bind to and, therefore, reduce the toxicity of GB, and a similar mechanism may operate in the case of GA. Other species differences, such as in the rates of aging of the GA-ChE complex, in the rates of synthesis of plasma-ChE in the liver, and in the levels of AChE in the nervous system (see Ivanov et al., 1993) may also result in difference between species in sensitivity to GA. Data are insufficient to more fully evaluate these possibilities. There is little human acute toxicity data that can be compared with the available rat data; however, acute toxicity data for primates in general (see Table 2) suggests that humans are likely to be more sensitive than rats. Therefore, for the purpose of this assessment, the standard EPA method will be followed which assumes that humans can be as much as ten times more sensitive to a chemical than laboratory animals.

In the Bucci et al. (1992) study, 12 CD rats/sex/dose group were injected intraperitoneally with GA at dose levels equivalent to 0, 28.13, 56.25, or 112.5 µg/kg/day. The injections were given once per day, 5 days per week for 13 weeks. Details of the study are given in section 3.3. The only significant changes observed in the dosed animals were decreases in blood ChE activity levels. In the case of RBC-AChE levels, considerable fluctuations occurred between and within test and control groups (0 µg/kg/day) (see Table 3). The variability in the RBC-AChE data in the male and female control groups makes these data sets less reliable for identifying a LOAEL and a no-observed-adverse-effect level (NOAEL) for ChE inhibition (Appendix B).

Of the parameters measured in the Bucci et al. study, changes in plasma-ChE values in male rats provided the least variable indication of a LOAEL and NOAEL for GA (see Appendix C); in the two highest dose groups plasma-ChE was significantly lower (p <0.05) than both pre-exposure values and control levels at weeks 3 and 7, and significantly (p <0.05) lower than pre-exposure values at week 1 (also significantly lower than controls in the high-dose group at week 1). There is also evidence (based on mean plasma ChE values) of a dose-response relationship for all sampling times (i.e., plasma-ChE was lower at the higher doses). Maximum depression of plasma-ChE occurred at 3–7 weeks, a condition also seen in a study of rats dosed with the nerve agent VX (Goldman et al., 1988). Therefore, because of the significantly lower levels of plasma-ChE in male rats (relative to both controls and pre-exposure vales), the mid-dose of 56.25 µg/kg/day is considered a LOAEL for plasma-ChE inhibition, and because of the lack of consistent change in plasma and RBC-AChE (relative to controls or preexposure values), the dose of 28.13 µg/kg/day is considered a NOAEL.

The equivalent oral NOAEL is estimated by comparing oral and intraperitoneal LD50 values for the rat and assuming that about the same ratio would apply for longer term exposures. A rat oral LD50 of 3700 µg/kg, and i.p. LD50 values of 490 and 800 µg/kg (average 645 µg/kg) have been reported. Therefore the equivalent oral NOAEL is:

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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)

The estimated oral NOAEL of 161 µg/kg/day can be used to estimate a human oral reference dose (RfD) by first adjusting the NOAEL for a 7 days/week exposure period by using a factor of 5/7; i.e., 5/7 × 161 µg/kg = 115 µg/kg/day, and then applying the result to the following EPA formula:

(3)

where:

UF1

=

10 (sensitive subpopulations)

UF2

=

10 (animal to human extrapolation)

UF3

=

3 (extrapolation from subchronic to chronic exposures).

UF4

=

1 (LOAEL to NOAEL extrapolation)

UF5

=

3 (data base incomplete)

MF

=

3 (modifying factor).

A total uncertainty factor of 1000 was applied, accounting for protection of sensitive subpopulations (10), subchronic-to-chronic extrapolation (3), animal-to-human extrapolation (10), and lack of a complete data base (3). In addition, a Modifying Factor of 3 was used because the RfD was based on a non-oral study.

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 Whittaker, 1962). For homozygous individuals, the activity can be as low as 8–821% of the normal mean (Bonderman and Bonderman, 1971). These individuals may be unusually sensitive to organophosphate anticholinesterase compounds (Morgan, 1989).

The standard uncertainty factor of 10 is used for animal-to-human extrapolation because there is no evidence to suggest that humans are less sensitive to GA than animals.

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 for other organophosphate cholinesterase inhibitors such as agent VX indicate that maximum ChE inhibition usually occurs 30–60 days after exposure begins and then levels off or even shows signs of recovery. However, an uncertainty factor of 3 is used here because chronic studies are not available to verify that additional effects would not occur following chronic exposures.

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

The data base for GA consists of two subchronic toxicity studies in rats, teratology studies in two species (rats and rabbits), and delayed neuropathy studies in chickens. These studies generally support the use of cholinesterase inhibition as the critical endpoint for deriving an oral RfD. Deficiencies in the data base include the lack of a multi-generation reproductive toxicity study, a standard toxicity study in a second species, and toxicity studies by the oral exposure route. Because studies on other organophosphate cholinesterase inhibitors, including a multi-generational study on agent VX, indicate that reproductive effects are unlikely, a full Uncertainty Factor of 10 is not considered necessary for data base deficiencies.

The principal study involved a non-oral exposure route (intraperitoneal) and required route-to-route extrapolation using acute toxicity data. Because of uncertainties associated with the use of this nonstandard methodology, a Modifying Factor of 3 was applied to the RfD.

Therefore:

(4)

(5)

4.3 Overall Confidence in the Oral RfD

Study: Medium

Data Base: Low

RfD: Low

The data base for GA consists of intraperitoneal and subcutaneous subchronic studies in rats, teratology studies in rats and rabbits, and delayed neuropathy studies in rats and chickens. Deficiencies in the data base include the lack of a multi-generation reproductive toxicity study, a standard toxicity study in a second species, and adequate toxicity studies by the oral exposure route. Although well-designed and well-conducted, the principal study involved a non-oral (intraperitoneal) exposure route. Consequently, overall confidence in the RfD is low.

4.4 Comparison of the Oral RfD with Human Toxicity Data

There are no human data available for GA for the oral exposure route. The intravenous LD50 was estimated to be 0.014 mg/kg (Robinson, 1967). Inhalation data indicate that severe effects would occur

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

at a concentration of 50 mg-min/m3 (Reutter, unpublished data). The LCt50 is 135 mg min/m3 for time periods of 0.5–2.0 min (DA, 1974). DHHS (1988) has set an inhalation maximum control limit of 0.000003 mg/m3 for the general public (72 hr time-weighted average).

5. CARCINOGENICITY ASSESSMENT

The potential carcinogenicity of GA cannot be determined. Data are inadequate for performing a quantitative assessment of agent GA.

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

6. REFERENCES CITED

Albuquerque, E.X., S.S. Deshpande, M. Kawabuchi, Y. Aracava, M. Idriss, D.L. Rickett, and A.F. Boyne. 1985. Multiple actions of anticholinesterase agents on chemosensitive synapses: Molecular basis for prophylaxis and treatment of organophosphate poisoning. Fund. Appl. Toxicol. 5:S182–S203.


Barnes, J.M. 1954. Organo-phosphorus insecticides. The toxic action of organo-phosphorus insecticides in mammals. Chem. and Ind. January 2, 1954, pp. 478–480.

Bonderman, R.P. and D.P. Bonderman. 1971. A titrimetric method for differentiating between atypical and inhibited human serum pseudocholinesterase. Arch. Environ. Health 22:578–581. (Cited in Hayes, 1982)

Bucci, T.J., R.M. Parker, J.A. Crowell, J.D. Thurman and P.A. Gosnell. 1992. Toxicity Studies on Agent GA (Phase II): 90 Day Subchronic Study of GA (Tabun) in CD Rats. Final Report. Prepared for the U.S. Army Biomedical Research and Development Laboratory, Fort Detrick, MD. DTIC AD-A258020.

Bucci, T.J., J.D. Fikes, R.M. Parker, K.H. Denny and J.C. Dacre. 1993. Developmental Toxicity Study (Segment II Teratology) of Tabun in CD Rats and in New Zealand White Rabbits. National Center for Toxicological Research, Final Report Nos. E515 and E516. Prepared for the U.S. Army Medical Research and Development Command, Fort Detrick, MD.

Burchfiel, J.L., F.H. Duffy and V.M. Sim. 1976. Persistent effects of sarin and dieldrin upon the primate electroencephalogram. Toxicol. Appl. Pharmacol. 35: 365–369.


Callaway, S., D.R. Davies and J.P. Rutland. 1951. Blood cholinesterase levels and range of personal variation in a healthy adult population. Br. Med. J. 2:812–816. (Cited in Hayes, 1982)

Carnes, S.A. and A.P. Watson. 1989. Disposing of the U.S. chemical weapons stockpile: An approaching reality. JAMA 262:653–659.

Crook, J.W., P. Hott, E.J. Owens, E.G. Cummings, R.L. Farrand, and A.E. Cooper. 1983. The Effects of Subacute Exposures of the Mouse, Rat, Guinea Pig, and Rabbit to Low-Level VX Concentrations. Technical Report ARCSL-TR-82038, AD BO86567L. Chemical Systems Laboratory, U.S. Army Armament Research and Development Command, Aberdeen Proving Ground, MD.


DA (U.S. Department of the Army). 1974. Chemical Agent Data Sheets, vol. 1. Edgewood Arsenal Special Report, EO-SR 74001. Defense Tech. Inform. Center, Alexandria, VA.

DA (U.S. Department of the Army). 1992. Material Safety Data Sheets: GA. Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD.

Dacre, J.C. 1984. Toxicology of some anticholinesterases used as chemical warfare agents - a review. In: Cholinesterases: Fundamental and Applied Aspects, M. Brzin, E.A. Barnard and D. Sket, eds., Walter de Gruyter, New York. pp. 415–426.

DHHS (U. S. Department of Health and Human Services, Centers for Disease Control). 1988. Final

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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.
×

recommendations for protecting the health and safety against potential adverse effects of long-term exposure to low doses of agents: GA, GB, VX, Mustard Agent (H, HD, T), and Lewisite (L). Federal Register 53:8504–8507.

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Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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

800–1060

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:"A: Health Risk Assessment for the Nerve Agent GA." 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 STATISTICAL ANALYSIS OF GA-INDUCED RBC-AChE INHIBITION IN RATS (Bucci et al., 1992)

GA - RBC-AChE Inhibition in Female Rats (ANOVA and Dunnett's)

 

Week

I.P. Dosea (µg/kg/day)

-1

1

3

7

13

0

 

ns/

ns/

ns/

ns/

28.13

_/ns

S/ns

ns/ns

S/ns

ns/ns

56.25

_/ns

S/ns

ns/ns

S/ns

ns/ns

112.5

_/ns

S/ns

S/ns

S/ns

ns/ns

a Six animals/dose group

b Comparison to pre-exposure value (week -1)/comparison to control (0 µg/kg/day) value.

ns. Not statistically significant.

S. Statistically significant at p <0.05.

GA - RBC-AChE Inhibition in Male Rats (ANOVA and Dunnett's)

 

Week

I.P. Dosea (µg/kg/day)

-1

1

3

7

13

0

 

ns/

ns/

S/

ns/

28.13

_/ns

S/ns

ns/ns

ns/ns

ns/ns

56.25

_/ns

S/ns

ns/ns

ns/ns

ns/ns

112.5

_/ns

ns/ns

ns/ns

ns/ns

ns/ns

a Six animals/dose group

b Comparison to pre-exposure value (week -1)/comparison to control (0 µg/kg/day) value.

ns. Not statistically significant.

S. Statistically significant at p <0.05.

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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 GA-INDUCED PLASMA-ChE INHIBITION IN RATS (Bucci et al., 1992)

GA - Plasma-ChE Inhibition in Female Rats (ANOVA and Dunnett's)

 

Week

I.P. Dosea (µg/kg/day)

-1

1

3

7

13

0

 

ns/

ns/

ns/

ns/

28.13

_/ns

ns/S

ns/S

ns/S

S/ns

56.25

_/ns

ns/S

ns/S

ns/S

ns/ns

112.5

_/ns

ns/S

ns/S

ns/S

ns/ns

a Six animals/dose group

b Comparison to pre-exposure value (week-1)/comparison to control (0 µg/kg/day) value.

ns. Not statistically significant.

S. Statistically significant at p <0.05.

GA - Plasma-ChE Inhibition in Male Rats (ANOVA and Dunnett's)

 

Week

I.P. Dosea (µg/kg/day)

-1

1

3

7

13

0

 

ns/

ns/

ns/

ns/

28.13

_/ns

ns/ns

ns/ns

ns/ns

ns/ns

56.25

_/ns

S/ns

S/S

S/S

S/ns

112.5

_/ns

S/S

S/S

S/S

ns/ns

a Six animals/dose group

b Comparison to pre-exposure value (week -1)/comparison to control (0 µg/kg/day) value.

ns. Not statistically significant.

S. Statistically significant at p <0.05.

Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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:"A: Health Risk Assessment for the Nerve Agent GA." 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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Page 127
Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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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Page 128
Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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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Page 129
Suggested Citation:"A: Health Risk Assessment for the Nerve Agent GA." 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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Page 130
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