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APPENDIX D
Health Risk Assessment for The Nerve Agent VX
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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
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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.
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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.
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TABLE OF CONTENTS
1. Introduction
1
1.1 Physical/Chemical Properties
1
1.2 Environmental Fate
1
2. Mechanism of Action
2
2.1 Effects of Organophosphate Agents on the Nervous System
3
2.2 Effect on Blood Cholinesterases
4
2.2.1 Intra- and Interspecies Variation in Blood Cholinesterase Activity
4
2.2.2 Potency of Nerve Agents as Cholinesterase Inhibitors
6
2.2.3 Cholinesterase Inhibition by VX
6
3. Toxicology
7
3.1 Introduction
7
3.2 Acute Toxicity
7
3.3 Subchronic Toxicity
9
3.4 Chronic Toxicity
10
3.5 Nervous System Toxicity
11
3.6 Developmental and Reproductive Effects
12
3.7 Carcinogenicity
13
3.8 Genotoxicity
13
4. Oral Reference Dose for VX
14
4.1 Cholinesterase Inhibition as an RfD Endpoint
14
4.2 Derivation of the Oral RfD
15
4.3 Overall Confidence in the Oral RfD
18
4.4 Comparison of the RfD with Human Toxicity Data
19
5. Carcinogenicity Assessment
19
6. References Cited
20
Appendix A. Comparison of RfDs, ChE Inhibition and Toxicity Data for GA, GB, GD and VX
A-1
Appendix B. Graphical Analysis of Rice et al. (1971) Data for Sheep Dosed with VX
B-1
Appendix C. Statistical Analysis of RBC-AChE Inhibition in Rats Dosed with VX
C-1
Appendix D. Oral RfD Estimated from Human Data
D-1
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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
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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).
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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),
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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.
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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
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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.
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APPENDIX B Graphical Analysis of Rice et al. (1971) Data for Sheep Dosed with VX
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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
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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
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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:
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Representative terms from entire chapter:
blood cholinesterase
