Recent events in the Middle East focused attention on the renewed threat
of chemical warfare. The relative ease of warfare agent production from
readily available industrial chemicals, the documented use of chemical weapons
by Iraq against Kurdish civilians and Iranian military personnel (1-3),
and the widespread possession of such weapons, raises the issue of chemical
warfare proliferation to other conflicts (4) or to terrorist activity. Domestic
attention to chemical munitions has also been sparked by the congressional
mandate to destroy the U.S. unitary stockpile via incineration (PL 99-145
and PL 100-456); congressional directives to examine safe disposal of nonstockpile
chemical material thought to be present in 32 states, the District of Columbia,
and the Virgin Islands (PL 102-484; 5); and the January 1993 signing of
the Chemical Weapons Convention banning the manufacture, use, stockpiling,
and transfer of chemical weapons (6).
The Chemical Stockpile Disposal Program (CSDP) of the U. S. Army will
carry out the intent of Congress regarding the unitary stockpile; details
are provided in the CSDP-Final Programmatic Environmental Impact Statement
(FPEIS), (7) and summarized by Carnes (8) and Carnes and Watson (9). Workplans,
budgets, and decision criteria for nonstockpile agents and munitions are
currently under development (5).
Two nerve agents [GB (sarin), methylphosphonofluoridate isopropyl ester
and VX, S-(diisopropylaminoethyl) methylphosphonothiolate o-ethyl ester],
are stockpiled at six continental U. S. military installations. These installations
include Anniston Army Depot (ANAD), near Anniston, Alabama; Blue Grass Army
Depot (BGAD), near Richmond, Kentucky; Newport Army Ammunition Plant (NAAP),
near Newport, Indiana; Pine Bluff Arsenal (PBA), near Pine Bluff, Arkansas;
Tooele Army Depot (TEAD), near Tooele, Utah; and Umatilla Depot Activity
(UMDA), near Umatilla, Oregon (see Table 1 for location of individual agent
stockpiles) (7). A third nerve agent, GA (tabun; N,N-dimethyl phosphoroamidocyanidate
ethyl ester), is present in small quantities only at TEAD (7,10). Physical
and chemical characteristics of these agents are presented in Table 1 and
their chemical structures are shown in Figure 1.
Figure 1. Molecular structures of the OP nerve
agents GA, GB, and VX [adapted from Dacre (13) and Gordon et al. (14)].
Organophosphate (OP) nerve agents were designed specifically to cause
incapacitation or death in military use and are particularly effective because
of their extremely high acute toxicity. This acute toxicity is three to
four orders of magnitude greater than most of the chemically similar OP
pesticides, whose acute toxicological endpoints are much the same (15).
The probability of an inadvertent release with off-site consequences during
current storage or any disposal alternative considered in the CSDP-FPEIS
is extremely low, being estimated to range from 1 in 10-4 to
1 in 10-10 (7,9). A credible risk, for purposes of CSDP planning,
is conservatively considered to be one with a probability of one in 100
million or greater (>= 10-8) (16). Some of the release scenarios
considered in the CSDP-FPEIS include exposure of Army personnel and a few
extend to off-site populations. Effects on individuals could range from
none to life threatening, depending on factors such as the type and concentration
of agent released, the duration of exposure, individual variations in sensitivity,
and the availability of antidotes, decontamination, and treatment capability.
Some low-probability scenarios could result in catastrophic aggregate effects
(i.e., > 1000 fatalities). One alternative considered, and rejected in
the FPEIS, was continued storage of the agents for 25 years. This option
is estimated to entail a higher number of potential fatalities from credible
accidents than on-site disposal (17). An analysis of the toxicity of each
nerve agent in the stockpile was performed as part of evaluating the on-
or off-site destruction options (7). Watson et al. (18) and Carnes and Watson
(9) summarized the results of that analysis. This review is the third in
a series of articles in EHP addressing health effects issues related to
stockpile destruction. In the first, Munro et al. (10) evaluated nerve and
vesicant agent antidotes, decontamination procedures, and treatment protocols
for use in a civilian context. In the second, Watson and Griffin (19) detailed
the toxicity of vesicant agents, with particular attention to mustard agent
carcinogenicity. The present review documents essential information on nerve
agent toxicity that is useful to civilian medical personnel and emergency
planners involved in preparation for stockpile disposal at each community.
We first review briefly the general features of the nerve agents, signs
and symptoms of exposure and mechanism of action, biochemical indicators
of exposure, and metabolism. We then present indices of acute toxicity of
the nerve agents alone and in combination, followed by information on potential
delayed and persistent effects of acute exposure. These endpoints include
delayed neuropathy, psychological and EEG changes, and cardiac effects.
We next review results of studies on chronic or subchronic systemic toxicity,
carcinogenicity, genotoxicity, teratogenicity, and reproductive toxicity.
Finally, we discuss the implications of the varied acute toxic effects of
nerve agent exposure for protection of the general public, as well as emergency
and medical personnel.
According to Harris and Paxton (20), GA, or tabun, was the first
nerve agent developed for chemical warfare; it was discovered in late 1936
and produced in large scale by 1942 (20,21). Subsequent G agents
such as GB are both more toxic than GA and also more resistant to hydrolysis.
GA contains cyanide instead of fluoride (see Fig. 1). It is more volatile
than VX (see Table 1). Agent GA is stored in relatively small quantities
in bulk at only one rather remote continental U. S. site (TEAD) (Table 1).
Thus, concerns about public health hazards presented by GA in the course
of the CSDP are relatively minor compared with those of GB and VX.
Because of its volatility, GA is primarily an inhalation hazard; it tends
to disperse rapidly and is not likely to be a contact or ingestion hazard.
However, GA is less volatile than GB (Table 1) and would be expected to
remain on the skin and in the environment somewhat longer. Although it is
not as persistent as VX, under certain weather conditions (light breeze,
20°C or 68°F) GA can remain in the environment from 1 to 4 days
(22). Agent GA differs from other G nerve agents in some of its biochemical
effects on the brains of exposed animals and also in the rarity of GA-induced
convulsions, even at lethal doses (23). Agent GB, or sarin, a fluorine-containing
OP (Fig. 1), is the most studied of the three nerve agents considered in
this analysis. Because of its high volatility and expected rapid dispersion,
GB is the agent of greatest concern for acute inhalation exposures in an
unplanned release at those sites housing it (ANAD, BGAD, PBA, TEAD, and
UMDA; Table 1). Because of its high volatility, GB is not a great concern
from the standpoint of reentry to a previously contaminated area. GB is
somewhat less effective as a skin penetrant than as an inhalant because
it evaporates so rapidly from the skin. Agent VX, a sulfur-containing OP
(Fig. 1), is, by any route of exposure, the most potent of all the nerve
agents discussed here (Table 2). When compared to the G agents, VX is more
stable, more resistant to detoxification, less volatile, more efficient
at skin penetration, and more environmentally persistent. Because of these
characteristics, VX is more effective as a skin penetrant and lethal contact
agent rather than as an inhalation threat.
The nerve agents are among the most potent of all chemical warfare agents
and are highly toxic in both liquid and vapor form. In vapor or aerosol
form, nerve agents can be inhaled or absorbed through the skin or the conjunctiva
of the eye; as a liquid, they can be absorbed through the skin, conjunctiva,
and upper gastrointestinal tract (38). Because they are essentially colorless,
odorless, tasteless, and nonirritating to the skin, their entry into the
body may not be perceived by the individual until grave signs and symptoms
appear.
Within seconds after exposure to low levels of nerve agent vapor, local
effects may be observed in the eyes and the respiratory system of humans.
Depending on the agent and the dosage, the local ocular effects may be a
constriction of the pupils of the eye (miosis) lasting only several days
or a prolonged miosis persisting for many weeks (39,40), pain, and/or dim
vision (29). Respiratory effects may include bronchoconstriction, excess
secretion in airways, wheezing, and labored breathing (29). The time of
onset of moderate systemic effects depends in part on the route of exposure;
it is within seconds to a minute or two after inhalation, within 45 min
after ingestion, and within 2-18 hr after application on the skin (38,41).
Exposure to lethal doses, however, can lead to collapse within seconds and
death within 10 min after a single deep inhalation (21). After short-term
(acute) exposure, mild systemic effects may last for several hours to days,
whereas moderately severe symptoms can last for 1-6 days. During the recovery
period, symptoms may recur intermittently, particularly after physical exertion
(38).
The toxic actions of nerve agents are due primarily to their ability
to inhibit acetylcholinesterase (AChE), an enzyme responsible for the breakdown
of the neurotransmitter acetylcholine (ACh). The result is excessive ACh
accumulation at synapses, where only minute quantities of ACh are needed
for transmission. Acetylcholine overstimulation of the portions of the nervous
system that control smooth muscle, cardiac muscle, and exocrine glandular
function results in the following signs: drooling, increased bronchial secretions,
bronchoconstriction, miosis, excessive sweating, vomiting, diarrhea, abdominal
cramping, involuntary urination, and cardiac arrhythmias. In addition, ACh
overstimulation of the central nervous system (CNS) may result in headache,
anxiety, restlessness, irritability, giddiness, insomnia, nightmares, EEG
changes, or even convulsions and coma, depending on the agent and the dosage
(38). Finally, ACh accumulation affects the nerves controlling skeletal
muscle, resulting in a dose-dependent generalized weakness that increases
with exertion, as well as muscle twitching and fasciculation, cramping,
and even flaccid paralysis.
Respiratory failure, the immediate cause of death in nerve agent exposure,
is an example of an effect that occurs as the result of ACh accumulation
at several sites in the nervous system. Depression of the brain's respiratory
center, neuromuscular block of the respiratory muscles, bronchial constriction,
and increased lung secretions are all factors contributing to nerve agent-induced
respiratory failure; the relative importance of each depends on the species
studied, the nerve agent, and the dosage used (42-48).
Recent interest has developed in the acute behavioral toxicity of nerve
agents. In this relatively new field of investigation, animals are tested
for changes in motor and learning behavior after exposure to the compound
of interest. Karczmar (49) has listed the CNS effects, including behavioral
and mental health effects, that have been observed with several anticholinesterase
chemicals. To date, the number of these effects that can be ascribed to
nerve agent exposure is limited. In most cases, motor effects in animals
appeared at levels of exposure that caused mild (some salivation, fine tremors)
to moderate (excessive salivation and weeping, generalized tremors) toxic
effects (36,50-54). However, some animal tests indicate acute effects on
learning behavior at exposure levels below those that cause signs of nerve
agent poisoning (51,53). Results of such studies must be interpreted with
much caution (55). Applicability to humans is suggested by preliminary work
in volunteers exposed to VX, demonstrating that performance on a number
facility test was impaired to a statistically significant extent (p <
0.01) in conjunction with minimal or absent physical signs and symptoms
(56).
Mild to severe human exposures to nerve agents have been associated with
other mental and emotional effects. These range from giddiness and loss
of ability to concentrate through anxiety, tension, and irritability, to
withdrawal, depression, insomnia, and nightmares (56,57). Such effects and
associated EEG changes were experienced in concert with the onset of nausea
and other symptoms in the case of GB, or earlier in the case of VX. Some
mental and emotional effects may persist for hours, days, or weeks, depending
partly on the severity of exposure.
Agent-induced ACh accumulation generates side effects that involve action
on other CNS neurotransmitter systems (e.g., norepinephrine, dopamine, 
aminobutyric
acid). Numerous biological effects result (58-61) including hypothermia
in rats (61,62), prolonged analgesia in mice (63), and brain and cardiac
lesions in animals surviving high doses of nerve agents (64,65). The interplay
of the various neurotransmitters within the nervous system probably results
in these varied side effects (45,66), although nerve agents may exert direct
effects on these same noncholinergic systems (67-69).
Despite new knowledge derived in animals as to the novel cholinergic
and noncholinergic effects of nerve agents and related organophosphates,
it is still widely accepted that inhibition of AChE is the primary cause
of acute toxic responses to nerve agent exposure in humans. For this reason,
attempts have been made to measure blood cholinesterase (ChE) activity as
an indicator of the magnitude of nerve agent exposure and/or the severity
of clinical signs and symptoms or to monitor the return of blood ChE function
as an index of recovery. Only in the case of systemic effects is there a
reasonably good correlation with the degree of ChE inhibition. Two types
of ChE activity can be measured in blood, red blood cell AChE (RBC-ChE),
and nonspecific plasma ChE (butyryl ChE or pseudocholinesterese). Systemic
effects are seen in about 50% of exposed volunteers when RBC-ChE is 20-25%
of normal baseline (a depression of 75-80%) (56,57,70-72). Monitoring of
RBC-ChE activity is theoretically preferred because this cholinesterase
is similar to the AChE found at the nerve synapses. RBC-ChE, however, is
replenished only with the formation of new RBCs in the case of GB (57),
while it spontaneously reactivates (1% per hour) in the case of VX (54).
Furthermore, recovery of function or cessation of signs and symptoms occurs
well before RBC-ChE levels show much recovery, especially after GB exposure
(57). Thus, recovery of RBC-ChE activity does not reflect the time course
of recovery of AChE activity in the tissues. As a result, monitoring of
RBC-ChE activity is of questionable utility in assessing recovery from nerve
agent (particularly GB) exposure in individuals for whom baseline RBC-ChE
values are unavailable (see below) (71).
Plasma ChE measurement is less relevant than RBC-ChE activity; the inhibition
of plasma ChE, which has no known biological function, may not reflect actual
AChE inhibition (73). For example, agent VX causes significantly less inhibition
of plasma ChE than AChE (57,72,74). Agent GB also preferentially inhibits
RBC-ChE (75), although not to the same extent as VX. Furthermore, plasma
ChE is more labile than RBC-ChE, being affected by gender, age, and oral
contraceptive use (76), as well as genetic determinants, disease states,
nutritional status, hormonal changes, race, and circadian patterns (77,78).
Plasma and RBC-ChE do serve a protective function, complexing with nerve
agent and thus reducing the concentration of free nerve agent available
to complex with tissue AChE (Fig. 2).
Figure 2. Plasma and RBC-cholinesterase (ChE)
provide a buffer to neurotoxic action by complexing with nerve agent, thus
reducing the concentration of free nerve agent available to move into tissue
compartments and inactivate synaptic or myoneural acetylcholinesterase (AChE).
PChE, plasma cholinesterase; NA, nerve agent; NS, nervous system.
Because of the variability of blood ChE activity (both plasma and RBC)
in unexposed individuals, it is difficult to determine conclusively from
a single test whether a person has had a recent exposure to a cholinesterase
inhibitor, especially if the exposure is minor (79,80). Yager et al. (81)
found the RBC-ChE intraindividual coefficient of variation to be 10.0% and
that of plasma ChE to be 14.4%. With one prior measurement of baseline ChE
activity in an individual, a 15% RBC-ChE depression is the least that can
be reliably detected compared to a 20% decrease from baseline for plasma
ChE (80) (Table 3). When individual baseline blood AChE activity is known
and is compared with the post-exposure activity level, a dose-dependent
relationship can be demonstrated between AChE inhibition and dosage at a
limited range of acute sublethal doses (56,57). Doses of GB and VX required
to depress human RBC-ChE activities by 50% (ChE50) are presented
in Table 4. Brain AChE inhibition and the degree of toxicity show a better
correlation in that GA and GB injected into rats produced a dose-dependent
inhibition of brain AChE, with lethal doses producing >90% inhibition
(61). Although brain AChE activity may reflect the dose response to nerve
agent exposure more closely than blood AChE, human brain AChE monitoring
can be done only in an invasive manner and thus has no practical application
in assessing human exposure.
The detoxification or breakdown of GA within the body proceeds at a low
rate (27), by way of the enzyme diisopropyl-fluorophosphatase (formerly
termed tabunase), which has been identified in several species including
man (83). Agent GB is detoxified in certain animal species by the plasma
enzyme carboxylesterase, formerly called aliesterase. Carboxylesterase combines
rapidly with GB and prevents it from interacting with AChE. In rats, 10
min after intravenous (IV) injection of radiolabeled GB, approximately 70%
of the plasma activity was bound to large protein molecules identical to
carboxylesterase (84). Pretreatment of rats with triorthocresylphosphate
(TOCP), a weakly anti-ChE organophosphorus compound that irreversibly blocks
carboxylesterase, resulted in a six- to eightfold enhancement of GB toxicity
(85). Additionally, more GB was found in the brain, muscles, kidneys, and
lungs and less GB in the plasma of TOCP-pretreated rats as compared to rats
that received no pretreatment. Similar carboxylesterase modification of
GB toxicity has been observed in guinea pigs and mice, although guinea pig
plasma carboxylesterase binding capacity for GB is lower than that of rat
plasma (86,87). The presence of carboxylesterase in rodent plasma may by
itself account for the relative resistance of mice and rats to GB toxicity
compared with other animal species (see Table 2 for LD50 values).
Human plasma does not contain carboxylesterase. Grob and Harvey (57) calculated
that there is very little detoxification when GB is injected into the human
bloodstream. This major difference in the detoxification of GB between rodents
and humans highlights the uncertainty of estimating human LD50
values from data obtained for rodents.
Metabolism studies of GB have been carried out in dogs and mice. Metabolism
studies in dogs demonstrated that the nearly exclusive product of GB detoxification
is isopropyl methylphosphonic acid (88). This compound accounted for the
majority of GB activity found not only in plasma and urine, but also in
brain tissue, suggesting that brain, like a variety of tissues from several
mammalian species, can hydrolyze GB (88). A rapid hydrolysis of intravenously
injected GB occurred in mice, such that less than 10% of the GB found in
the tissues was nonhydrolyzed within 1 min (36). This rapid hydrolysis of
GB may be again due to plasma AE, but this hypothesis has not been established.
A question remains as to the relevance of extrapolating to humans from any
metabolism studies using a species (mouse) that is resistant to the toxic
effects of GB.
Because human exposure data are not available on the lethal doses of
the nerve agents discussed here, animal toxicity data have historically
been extrapolated to develop human dose estimates. The dose or exposure
levels of GA, GB, and VX that result in 50% lethality (LD50,
LCt50) in several species by various routes of exposure are presented
in Table 2. The route of exposure is important because there are differences
in absorption and/or degradation with different avenues of entry into the
body. Although the IV route is not relevant to accidental exposure of man,
inclusion of these data in Table 2 illustrates the intrinsic toxicity of
the agents without individual or species variations in absorption and is
useful for comparison. Table 2 also includes estimates of doses, based on
animal data, that could cause 50% mortality in human populations exposed
by some of these same routes. Comparisons between species (particularly
comparisons of animal data with human estimates) are possible since the
LD50 values (or the estimated values) are given on a milligram
of agent administered per kilogram of body weight (mg/kg) basis. Table 2
also contains the estimated human median incapacitating concentration-time
product (ICt50), effective dose or concentration-time product
(ED50, ECt50) [also termed minimum effective dose
in the U. S. Army Chemical Agent Data Sheets (11)], and no-effects dose
for each nerve agent. Unfortunately, the source document (9) does not define
what is meant by incapacitation.
Agent GA
As mentioned previously, the human doses for lethality (LD50)
or incapacitation (ICt50) provided in Table 2 are merely estimates
(11,22). This is evident in a comparison of the inhalation incapacitating
Ct product (ICt50) of 300 mg-min/m3 and the range
of 200-400 mg-min/m3 for the lethal dose (LCt50) for
GA in resting humans (breathing 10 l/min); the incapacitating dose falls
within the range for the lethal dose. The degree of incapacitation associated
with this dose is not defined in the source (11), but likely is severe,
meaning unconscious and convulsing.
Agent GB
GB is a very rapidly acting toxicant; there is little difference between
the 15-min and the 24-hr lethal dose for animals by IV injection (Table
2) (28). GB has been thought by some to act primarily on the peripheral
nervous system; however, respiratory arrest induced in cats by an IV dose
equivalent to one-half the feline LD50 (48) was mediated through
effects on the central nervous system. GB is very efficient at producing
central respiratory arrest in guinea pigs and cats at IV doses too low to
cause an effect on the respiratory muscles (32). Thus, the primary effects
of GB appear to be on the CNS.
Like all other nerve agents, GB combines with and inhibits AChE, resulting
in the accumulation of ACh. From studies in which small quantities of GB
were injected directly into the bloodstream of human volunteers, Grob and
Harvey (57) calculated that about 75% of GB combined with AChE in the muscle,
about 22% with blood ChE, and about 3% with AChE in brain and liver. The
inhibition of blood ChE results in no toxic effect; rather, it is the GB
inhibition of brain and muscle AChE that causes the symptoms of nerve agent
exposure. Within the muscles of cats, GB caused a dose-dependent inhibition
of AChE activity; however, no simple relationship existed between AChE inhibition
and alteration of muscle function (89).
Once GB is in the blood, it can penetrate the blood-brain barrier. Cholinesterase
inhibitors vary in their ability to pass through this barrier, a property
that has been related to the lipid solubility of the compound (90). Within
the brains of mice injected intramuscularly with GB, Bajgar (91,92) observed
regional differences in AChE inhibition. He concluded that the differences
in AChE inhibition were due to regional differences in GB penetration rather
than to a differential selectivity of GB for AChE in specific parts of the
brain. Studies of isolated, blood-perfused in situ dog brains administered
GB via intracarotid arterial injection (93) and of the brains of dogs after
IV injection of GB (88) also showed regional differences in AChE activity.
Studies in rats demonstrated that more than 94% of apparent GB bound to
AChE in the brains 30 min after injection is actually the GB metabolite
isopropyl methylphosphonic acid (94).
Mechanisms other than (or in addition to) AChE inhibition appear to be
responsible for the observed toxicity of GB to the brain. In rats, Harris
et al. (95) reported that 51% of the GB found in the brain was bound to
sites other than AChE. In studies of spontaneous recovery from central respiratory
failure in guinea pigs, respiratory recovery did not correlate with recovery
of brainstem AChE levels (44). Adams and his colleagues concluded that the
recovery occurred through a desensitization of the ACh receptors to the
excess ACh, but it is also possible that AChE inhibition was not actually
responsible for the initial respiratory failure. GB causes a number of noncholinergic
effects in the brain, including effects on other neurotransmitters and enzymes.
Most effects are too detailed to discuss individually in this analysis,
but all emphasize the point that GB does much more than simply inhibit AChE
in the brain (23,58,59,61,67,96,97).
Data on human responses to GB come from accidental exposures and from
limited studies on low doses of GB given to volunteers. In one incident
of accidental exposure to GB vapors (estimated at 0.09 mg/m3
for an undefined duration), two men had significantly lowered RBC-ChE for
80-90 days (one showed depression to 19% of baseline activity, the other
to 84% of baseline) and extreme miosis that persisted for 30-45 days, but
no other signs or symptoms of nerve agent poisoning (39). Other accidental
inhalation exposures to GB with similar recovery times for RBC-ChE activity
and miosis were described by Sidell (40). In one, the individual manifested
severe symptoms and required respiratory assistance and extended hospitalization
after cleaning a GB-contaminated area while wearing defective protective
gear. In the other case, three workers who were in an area with a leaky
GB storage container suffered temporary symptoms, such as transient mild
respiratory distress, together with marked miosis and RBC-ChE activity depression.
The RBC-ChE depression required 3 months for full recovery; the miosis (measured
in the dark) recovered in 30-60 days.
Grob and Harvey (57) reported the effects in humans of administered low
doses of GB. When either 0.003 or 0.005 mg/kg of GB was injected directly
into an artery in the arm of one volunteer, Grob and Harvey observed some
initial local effects (reduction in grip strength, tremors after exercise)
followed by systemic effects, including many of the symptoms listed earlier.
These doses, which correspond to 21% and 36% of the estimated human IV LD50,
resulted in RBC-ChE activity reductions to 52% and 28% of original activity
(i.e., depressions of 48% and 72%) and plasma ChE activity reductions to
61% and 42% (i.e., depressions of 39% and 58%), respectively.
After combining with a ChE molecule, the agent-ChE complex may either
spontaneously dissociate (resulting in reactivation of the ChE) or "age,"
in which case the agent-ChE complex becomes resistant to reactivation by
an oxime antidote. Aging is thought to result from stabilization of the
nerve agent-ChE complex by loss of an alkyl or alkoxy group (Fig. 3). In
agreement with Grob and Harvey's work (57), Sidell and Groff (56) observed
little, if any, spontaneous reactivation of RBC-ChE after GB administration
to volunteers. Furthermore, the GB-ChE complex aged at a moderate pace,
with aging 50-60% complete 5 hr after GB infusion (56).
Figure 3. The nerve agent-acetylcholinesterase (AChE) complex
may undergo either spontaneous reactivation by hydrolysis or stabilization
("aging") by loss of an alkyl or alkoxy group; stabilization proceeds
at a faster rate than hydrolysis and therefore predominates. In humans,
the GB-AChE complex is 50-60% aged by 5 hr, whereas VX ages more slowly,
with only 40% aged at 48 hr after exposure (56).
When GB was given orally to 10 volunteers, approximately 3.5 times as
much GB (in mg/kg) was needed to produce the same degree of plasma or RBC-ChE
activity depression as previously observed with intra-arterial injection
(see Table 4) (57). With the oral administration, Grob and Harvey noted
a narrow margin between doses that produce mild signs and symptoms and those
that produce moderately severe effects. They also noted that, after the
disappearance of signs and symptoms, an increased susceptibility (in terms
of type and severity of responses) remains to further GB exposure within
24 hr of the first exposure. Anorexia, nausea, and chest tightness were
among the first symptoms reported; abdominal cramping, vomiting, and diarrhea
were among later effects; miosis was not observed after oral administration.
The possibility of oral exposure of the population to GB is remote because
GB dissipates rapidly under most environmental conditions. Only when temperatures
are 0° or less can GB persist for a few hours as a ground contaminant
(22,98).
As mentioned previously, GB vapor is less effective as a toxic skin penetrant
than as an inhalant. The estimated human LCt50 (clothed, resting)
for dermal toxicity is 150 times higher than the estimated human LCt50
for inhalation (Table 2). Fielding (28) summarizes information from several
sources, some still classified. Rapid evaporation from the skin is the primary
factor in the relatively low dermal toxicity of GB; if evaporation is prevented
(i.e., by covering the exposed skin with a cup), the toxicity of GB increases
almost 100-fold (99). Another factor limiting the dermal toxicity is the
reaction of GB with skin constituents, which attenuates the amount of GB
that reaches target tissues (100). Fats such as lanolin and lard have been
shown to enhance the skin penetration of GB, probably by dissolving the
agent and by preventing evaporation (28). Mechanical abrasion of rabbit
skin increased GB dermal toxicity 100-fold (101). Fielding (28) relates
a tragic incident that illustrates the wide individual variability in dermal
sensitivity to GB. Seventeen of 18 men exposed dermally to 200 mg of GB
(12% of the estimated dermal LD50 for a 70 kg man) through two
layers of clothing showed no signs or symptoms of GB poisoning; the eighteenth
man died shortly after the onset of exposure, despite immediate treatment
when signs of nerve agent poisoning appeared.
In a review of GB toxicity, McNamara and Leitnaker (25) state: "Absorption
through the conjunctiva causes local effects but negligible systemic effects."
Grob and Harvey (57) instilled 0.0003 mg GB in the eyes (conjunctival sacs)
of volunteers and noted a marked miosis that began at 10 min and slowly
diminished over a period of 60 hr. At a dose of 0.0009 mg, the pupillary
constriction that occurred was near maximal for 72 hr and did not disappear
until after 90 hr. In this study, miosis was measured in the light; other
studies in which it was measured in the dark showed it persisted for weeks.
No depression of blood ChE activity was noted at either dose level. In studies
on GB applied to the conjunctival sac of guinea pigs, a rapid dose-dependent
depression of AChE activity in the iris and cornea was noted with a lesser
inhibition of AChE in the retina (retina required 10 times the iris dose
to achieve the same AChE inhibition), but no examination was made of RBC-ChE
depression or other systemic effects in the treated guinea pigs (102). However,
ocular LD50 values are available for several animal species that
are equivalent to the LD50 values for subcutaneous injection
(11). This suggests that systemic effects are possible with GB absorption
through the conjunctiva and possibly the cornea of the eye.
Studies of the retention and absorption of GB vapors by resting or exercising
men demonstrated that the inactive men retained a higher percentage of the
inhaled GB (82). Under similar exposure conditions of time and concentration,
however, the active men received a larger dose of GB because of their greater
air intake.
In determining the lowest concentration of GB that produces a biological
effect, miosis provides a sensitive indicator for nerve agent exposure in
humans. Questions, however, cloud the validity of the estimated no effects
(0.5 mg-min/m3) concentration-time product (Ct) for miosis by
GB (Table 2). The basis for this determination by McNamara and Leitnaker
(25) is found in a report by Johns (103) of pupil diameter response in volunteers
exposed to low atmospheric concentrations of GB (maximum Ct = 6 mg-min/m3
where tmax = 20 min). We consider the data insufficient to confidently
predict concentrations of GB that would cause miosis in none of the population
(no-effects level). The Johns study (103) was not designed to determine
a no-effects level; it is not clear how McNamara and Leitnaker (25) derived
their no-effects value of 0.5 mg-min/m3 from Johns's data. We
consider the true no-effects level likely to lie below 0.5 mg-min/m3.
The lack of raw data and absence of measures of variability in Johns's (103)
report hinder precise reanalysis. Estimates of a human no-effects level
for VX, as discussed below, were based in part on these for GB; the VX estimate
consequently suffers from a similar question of reliability.
Agent VX
A contributing factor to the high toxicity of VX may be its preferential
reaction with AChE. Unlike the G agents, VX depresses RBC-ChE activity significantly
more than plasma ChE in humans (56); the result is that more VX is available
to react specifically with the target enzyme, AChE. Less VX is required
than GB to reduce RBC-ChE 50% below baseline levels in humans by all routes
of administration for which data are available (see Table 4).
Once inside the body, VX not only inhibits AChE activity but also reacts
directly with the ACh receptors and other neurotransmitter receptors (68,97,104).
Rickett et al. (105) have briefly reviewed some of the evidence for effects
at the receptor level. Although GA and GB may react with the ACh receptor
in a manner similar to ACh itself (106), results of preliminary studies
suggest that VX may counteract the effects of ACh, acting as an open channel
blocker at the neuromuscular junction (105). The clinical significance of
these effects is doubtful, however, because the concentrations of anticholinesterase
needed to exert effects in ionic channels in vitro are many times
the LD50 in vivo.
Two other features of VX toxicity are worthy of mention. First, in contrast
to observations on GB, the VX-RBC-ChE complex has been found to undergo
a significant degree of spontaneous reactivation in humans. In a study by
Sidell and Groff (56), spontaneous reactivation of human RBC-ChE proceeded
at a rate of about 1%/hr over the first 70 hr after IV administration of
VX.
A second feature of VX toxicity is the lack of aging or stabilization
of the agent-ChE complex and the relative ease of reactivation of VX-poisoned
enzyme by oxime antidotes in humans (56). By 48 hr after exposure, no aging
was observed. The VX-ChE complex was more easily reactivated by oxime antidote
at all times up to 48 hr after exposure (when the experiment was terminated)
than was GB-ChE.
Estimates are available for human lethal inhalation doses of VX in both
aerosol (small particles) and vapor (gas) phases (Table 2). Animal inhalation
data are available primarily for VX aerosol. In most cases, only the animals'
heads and not their total bodies were exposed, so as to limit the skin absorption
of VX. The mouse LCt50 values for both vapor and aerosol were
obtained with total-body exposure; in the case of VX aerosol, skin absorption
appears to contribute to the total toxicity. The estimated human LCt50
values are equivalent for vapor and aerosol. However, it would probably
be difficult to achieve a high vapor concentration of VX because of its
low volatility; therefore, it is likely that a longer exposure to VX vapor
would be necessary to achieve the same endpoint.
It should be borne in mind that the VX LD50 values for humans
have been derived from mathematical models, extrapolations from animal data,
and estimates from sublethal experimentation in humans. Many of the original
reports in which these human values were derived are confidential and unavailable
for open-literature review. In general, AChE activity levels have been used
as indicators of VX toxicity in humans. However, extrapolation to LD50
estimates from AChE activity determinations may have little meaning because
of the poor correlation between AChE activity and toxicity in animals and
wide variations in RBC-ChE levels at which toxic effects occurred in human
studies (56,74).
Dermal absorption is a more likely route of VX exposure than inhalation;
moreover, dermal toxicity is more likely to occur from the absorption of
VX aerosol or liquid than from the vapor. The LCt50 estimates
for dermal absorption are established by exposure of animals to VX vapor
or aerosol in a special chamber in which only the body is exposed. The animals
were often shaved, clipped, or depilated before exposure to approximate
human skin exposure, and the wind speed within the chamber was varied to
simulate a range of meteorological conditions.
Although wind speeds of 20 mph may never be encountered in an unplanned
release of VX, it is important to realize that wind speed can significantly
increase the dermal toxicity of VX. A 20-mph wind speed resulted in a 20-fold
reduction in the dog LCt50 values when compared with tests conducted
in still air (89 versus 4.6 mg-min/m3; Table 2), and the rabbit
LCt50, when determined with an 8-mph wind speed (8.3 mg-min/m3),
was 3.4 times lower than that obtained in still air (28 mg-min/m3)
(Table 2). Another way of determining VX dermal toxicity in animals is to
apply liquid VX directly to the bare skin. The LD50 values for
skin absorption are similar for monkey, pig, dog, cat, rabbit, and mouse
(Table 2).
Although the animal data summarized in Table 2 primarily show the influence
of wind speed, other factors affecting the dermal LCt50 values
include particle size and degree of skin exposure (clothed or bare). Krackow
(24) calculated dermal LCt50 values for men wearing gas masks
so that only the neck, ears, hands, and wrists were exposed to aerosol particles
ranging in size from 5 to 15 µm at wind velocities from 0 to 10 mph.
Under these conditions, a lower LCt50 (75 mg-min/m3)
was estimated for the larger particles with the higher wind velocity, while
an LCt50 of 300 mg-min/m3 was for the smaller particles
at the lowest wind speed. Other data of Krackow suggest that, with 10-µm
particles and 20-mph winds (or 20-µm particles and 10-mph winds),
the LCt50 might be as low as 10 mg-min/m3.
The human dermal LCt50 values listed in Table 2 for VX vapor
are based on VX vapor containing 2-µm particles (11). These exposure
conditions represent a hybrid vapor/aerosol situation. Fielding (28) considers
that the dermal LCt50 values for VX vapor and VX aerosol would
be similar under conditions of high concentrations in air and short exposure
times. With lower vapor concentrations, Fielding (28) notes that "doubt
has been expressed regarding the dermal lethality of VX vapor to humans
in view of the possibility that the (systemic metabolism and) excretion
rate may be greater than the skin-absorption rate."
The amount of skin surface exposed to VX vapor or aerosol is of obvious
importance. Clothing is estimated to reduce by 10-fold the dermal vapor
toxicity of VX (see Table 2). Not all body areas, however, are equally permeable
to VX. The doses of VX necessary to cause 70% inhibition of AChE when applied
to equal areas of the human cheek, forehead, abdomen, and volar surface
(i.e., palm side) of the forearm have been estimated to be 0.0051, 0.0112,
0.0318, and 0.04 mg/kg, respectively (107,108). The differences in absorption
are important in evaluating studies in which the forearm is exposed and
extrapolations are made to total-body exposure. Craig et al. (108) measured
the dermal absorption of liquid VX through the cheeks and the forearms men
at environmental temperatures ranging from -18° to 46°C. The fraction
of the applied dose that penetrated in 3 hr ranged from 3.5% at -18°C
to 31.9% at 46°C for the cheek and from 0.4% at +18°C to 2.9% at
46°C for the forearm. Wide individual differences in RBC-ChE depression
were seen for both skin sites and most doses tested; in some cases, responses
ranged from 0 to 100% depression (108). The wide range of individual responses
to dermal VX exposure, caused in part by differences in penetrability of
the skin in various parts of the body, makes the prediction of a human dermal
VX LD50 value difficult. Thus, ranges are given rather than single
values.
Because skin can act as a storage depot for VX with movement from this
depot promoted by increased temperature, the authors suggest that cooling
of the skin surface after dermal exposure to VX can delay absorption until
treatment is possible. Also note that immediate decontamination should be
a particularly effective procedure for dermal VX exposure because of the
slowness of its skin penetration. However, if decontamination is delayed
until 3 hr after exposure, significant lowering of RBC-ChE continues after
decontamination (108). Specific decontamination procedures following nerve
agent exposure can be found in Munro et al. (10).
The estimated IV LD50 for humans (0.008 mg/kg) is similar
to that determined for many animal species, with the major exception of
the mouse, which is less sensitive. Low doses of VX (0.001 mg/kg, IV) were
administered to volunteers to assess correlation of dose with the degree
of AChE inhibition or the presence of clinical signs and symptoms. By injecting
VX directly into the bloodstream, the wide differences in individual skin
absorption observed in other studies (108) can be bypassed. In four men
who received 0.001 mg/kg VX in a 4-hr infusion, good agreement was observed
between the individual percent decrease (50%) in RBC-ChE activity compared
with preinfusion AChE levels (74). (It is not possible to compare the range
of the absolute AChE values because these were not given.) In another study,
reported by Sidell and Groff (56), a group of 34 men were given doses ranging
from 0.0012 to 0.0017 mg/kg in an attempt to find an IV dose of VX that
would cause 75% inhibition of RBC-ChE levels. In this dose range, most subjects
had transient symptoms of lightheadedness and some experienced nausea and
vomiting, with the most prominent effects occurring 1 hr after injection,
when the RBC-ChE inhibition was maximal. No miosis was observed, even with
90% inhibition of RBC-ChE activity. A dose of 0.0015 mg/kg produced 75%
inhibition of the baseline AChE levels; linear regression analysis of the
dose- response curve gave 0.0011 mg/kg as the estimated dose causing 50%
decrease in RBC-ChE activity (see Table 4).
There is a paucity of data on the oral toxicity of VX, despite the fact
that the demonstrated environmental persistence of this agent makes ingestion
a relevant route for human exposure. VX can persist on leaf surfaces in
an undegraded form, so that animals grazing on contaminated vegetation can
ingest VX. In a study on VX persistence in soil after shell bursts, 46 days
after contamination sufficient VX remained to kill 4 of 10 guinea pigs fed
grass from the contaminated area [Dewey and Fish (28,109). In sheep accidentally
exposed in winter to VX-contaminated vegetation, clinical signs of toxicity
persisted for at least 3 weeks (110). Slight ChE depression was noted in
newly introduced sheep grazing the suspect area 2 months after the VX release
(111). The only animal oral LD50 value available for VX is 0.1
mg/kg for rats (Table 2).
In 32 human volunteers given single oral doses of VX in water, a dose
of 0.004 mg/kg caused a 70% reduction in RBC-ChE levels (56). This oral
dose is about three times the human IV dose needed to cause a similar level
of RBC-ChE inhibition. The oral ChE50 value calculated by Sidell
and Groff (56) from the dose- response curve obtained in their study is
0.0023 mg/kg (see Table 4). At oral doses ranging from 0.002 to 0.0045 mg/kg,
only a few subjects (5/32) suffered any gastrointestinal signs or symptoms,
and there were no changes in heart rate, blood pressure, or pupil size in
any of the subjects. Eating 30 min before drinking the VX solution appeared
to enhance the RBC-ChE inhibition; tap water (as compared with a 5% dextrose
solution) seemed to retard the anti-RBC-ChE activity. In an earlier study,
volunteers were given four oral doses/day of VX in drinking water for 7
days (concentration about 0.05 mg/l in four 500-ml portions; individual
daily dose of 0.00143 mg/kg) (112). No signs or symptoms of toxicity were
observed although RBC-ChE was 40% of baseline by day 7. Estimates have also
been made for human ICt50 either by inhalation or by skin absorption
of VX aerosol or vapor (Table 2). The ranges in these estimates are due
in part to different test conditions (i.e., varied particle sizes in the
case of aerosols and different wind velocities). Fielding (28) notes that
these ranges may also depend on what is meant by incapacitation.
Estimates have been made for the lowest air concentration of VX that
produces miosis, one of the more sensitive indices of human exposure to
the vapors of anticholinesterase compounds. The estimated ECt50
found in Table 2 for pupillary constriction by VX is an extrapolation from
the derived value for GB in humans (25). To obtain the VX ECt50
for humans, the estimated ECt50 for miosis in humans exposed
to GB was first compared with the minimum dose of GB that causes miosis
in rabbits, and the assumption was made that man is twice as sensitive as
the rabbit. This factor of two was then applied to the concentration of
VX that produces pupillary constriction in rabbits to arrive at the minimum
concentration of VX that would be expected to cause miosis in humans (37).
Similarly, the VX no-effects doses for miosis and for tremors are based
on extrapolations from derived values for GB. Because these VX estimates
are used to determine presumed safe levels for human exposure to VX, more
research is needed to determine whether these minimum and no-effects values
are credible.
The effects of acute VX exposure on mood and mental function are similar
to those of GB. Kimura et al. (74) reported the results of the first experimental
human exposures to VX. One subject became irritable, reported headache,
spoke less clearly, and became confused and then irrational and agitated
after receiving 0.00212 mg/kg VX IV over 5.5 hr when whole-blood ChE activity
reached about 10% of baseline. Six others received 0.001 mg/kg VX IV over
periods of 1.75-4 hr; only one subject reported headache and discomfort.
Bowers et al. (41) reported transient depressive effects on mental functioning
and mood (as correlated with ChE depression and gastrointestinal symptoms)
in 93 adult male volunteers percutaneously to VX. [Bowers et al. (41) identify
the agent only as EA-1701; Krackow (24) identifies this code with VX.] The
psychological effects were usually seen well before the onset of gastrointestinal
symptoms in those subjects who experienced both types of effects.
Sidell and Groff (56) reported a study in which 66 volunteers received
VX either IV or orally. Doses ranged from 0.0012 to 0.0017 mg/kg IV and
0.0020 to 0.0035 mg/kg orally. The 0.0015 mg/kg IV group suffered a significant
decrement in mental performance on a number facility test (the higher dose
groups had been pretreated with scopolamine and were not monitored for changes
other than in RBC-ChE activity). The effect was seen only in the first hour
after VX injection. Those receiving VX by the oral route showed little or
no indications of CNS effects and fewer gastrointestinal effects despite
generally lower RBC-ChE activities. Thus, the authors suggested that the
nausea and vomiting in the IV group were probably centrally mediated.
The relative potency of GA, GB, and VX varies with the route of exposure.
Inhalation or percutaneous absorption of vapor or aerosol demonstrates that
VX is more toxic than GB, which is more toxic than GA (i.e., VX > GB
> GA). The dermal toxicity ranking is VX > GA > GB, while the ranking
based on estimates of IV toxicity is VX > GA = GB. These differences
relate to varying physical, chemical, and toxicological properties among
the nerve agents. Agent VX, for example, is not only much less volatile
than the G agents, it is not detoxified in the skin and combines little,
if at all, with plasma cholinesterases. Thus, VX is more readily available
to inactivate tissue AChE.
The human inhalation toxicity of GA vapor is approximately half that
of GB (Table 2); this difference is well supported by the animal data. GA
appears to be more toxic to the ciliary muscles of the eyes than GB because
constriction of pupils occurs at a lower concentration of GA [i.e., minimum
effective doses of 0.9 and 2-4 mg-min/m3, respectively (11)].
The estimated LD50 for GA toxicity in humans by skin absorption
is roughly equivalent to the estimate for GB, and the human IV LD50
estimate for GA is equal to that for GB. The equivalencies of these estimates
are not necessarily supported by the animal data, but no discussions of
the bases for the human estimates are given in the source documents (11,21).
Although GB is less toxic than VX by a variety of exposure routes, GB
may actually be more toxic than VX at the neuromuscular junction. When GB
or any one of several V agents related to VX was applied directly to the
isolated rat diaphragm at the neuromuscular junction (thereby eliminating
factors such as absorption efficiencies and attenuation differences), GB
was found to be twice as potent as the V agents (113). Intravenous infusion
of GA, GB, or VX in cats at the rate of one LD50 per 15 min demonstrated
that 0.5 LD50 of GB was sufficient to induce respiratory arrest,
whereas 1.25 and 15 LD50 doses were needed for GA and VX, respectively
(48). These differences reflect the rapidity of the toxic action of GB compared
with VX and the somewhat higher toxicity compared with GA.
In comparison with GB human exposure estimates, VX is estimated to be
approximately twice as toxic by inhalation, 10 times as toxic by oral administration,
and approximately 170 times as toxic after skin exposure (114). Under conditions
favorable for skin penetration, VX can be about 1000 times as toxic as GB
in rabbits (28). The evaporation of VX from the skin is almost negligible,
whereas GB evaporates in a matter of minutes (see Table 1 for comparative
volatility data). Agent GB penetrates the skin more rapidly than VX, but
VX undergoes virtually no degradation as it slowly penetrates the skin;
thus, more of this compound is able to reach the bloodstream (115). Whereas
GB skin penetration in rabbits appeared to be complete by 30 min (100) [with
a penetration efficiency of nonevaporating GB calculated to be only 0.04%
(28)], complete penetration by VX, with essentially 100% of the skin dose
reaching the circulatory system, required about 4.5 hr (116). In vitro
studies suggest that VX can penetrate in unaltered form through the epidermis
and dermis of the skin, penetrate through the nerve membranes, and can accumulate
within the nerve cells (117).
A number of investigators have reported the distinctly slower toxic action
of VX as compared with the G agents, as well as a slower rate of recovery
(105,118). This delay cannot be attributed only to slower skin penetration
because a slower response is also observed when VX is administered intravenously
(48,56). With GB there is essentially no difference between the 15-min and
24-hr lethal IV dose; with VX there is an approximate twofold difference
(28). It is important, therefore, in determining LD50 and LCt50
levels for VX to allow enough time to accurately assess the toxicity. Although
the biological basis for this delay is not fully understood, Fielding (28)
hypothesized that the larger molecular size (see Fig. 1) and different solubility
characteristics of VX may cause it to diffuse more slowly than G agents
through tissues and cell membranes to the target tissues.
Both GB and VX are stored at several of the stockpile sites (LBAD, ANAD,
PBA, TEAD, and UMDA). While munitions containing a given agent are placed
in segregated bunkers, igloos, or storage buildings and, likewise, ton containers
are segregated by agent type, the stockpile sites contain limited areas
of contiguous rows of bunkers or other storage units containing unlike agents.
While the probability of an accident such as an airplane crash into one
of these areas of adjacent storage units resulting in release of more than
one agent type is extremely low, such a release is considered here for the
sake of completeness.
Thus, the question arises as to toxic effects of a GB-VX mixture if these
agents were simultaneously released. In the only study found to date that
addresses this issue, GB and VX were administered simultaneously and sequentially
to mice (119). When the agents were administered as a mixture of 0.5 LD50
of each agent (GB = 95 µg/kg, VX = 9 µg/kg), the resulting mixture
had an LD50 lower than one LD50 of either agent alone,
meaning that the total effect was more than additive. When a 0.5 LD50
dose of VX was administered 1 hr before a 0.5 LD50 dose of GB,
brain and blood AChE were depressed less than by sequential administration
of two 0.5 LD50 doses of GB given 1 hr apart. Thus, VX had a
protective effect on blood and brain AChE depression produced by GB. However,
when the nerve agents were administered in reverse order (0.5 LD50
of GB before 0.5 LD50 of VX), blood AChE inhibition was greater,
but brain AChE inhibition was less than that induced by two serial 0.5 LD50
doses of VX.
Approximately 50-fold potentiation of toxicity with the administration
of certain combinations of OP insecticides [EPN, O-ethyl O-(4-nitrophenyl)
phenylphosphonothioate and malathion] has been described (120); however,
with other insecticide combinations [malathion and ronnel; compound 4072
(Dermaton) and dichlorvos; ronnel and dichlorvos] there was no potentiation
of AChE inhibition in the dog (121). Further investigation is needed to
quantify the possible interactions of toxic mixtures of nerve agents or
combinations of nerve agents and pesticides, especially those relevant to
the CSDP.
Public concern has been expressed regarding the induction of organophosphorus-induced
delayed neuropathy (OPIDN) by the OP nerve agents in the U. S. stockpile.
Other possible delayed or persistent effects of concern include cardiac
dysfunction, psychological effects, and EEG abnormalities.
The OPIDN syndrome is characterized by a delay of 5-30 days, followed
by some initially mild symptoms, such as weakness, tingling, and muscle
twitching in the legs. A flaccid paralysis eventually develops, first in
the toes and then progressing to the hands and thighs. Depending on dose,
the paralysis is usually persistent; recovery is generally slow and incomplete.
Some 16,000 cases of OPIDN were reported in 1930-31 among individuals in
the southern United States who drank an illicit alcoholic extract ("Jamaica
Ginger" or "Ginger Jake") that was contaminated with TOCP,
a weak anticholinesterase OP ester (122,123). Thousands of others have suffered
from TOCP-induced OPIDN as a consequence of ingesting contaminated food
oils (124,125). A limited number of people have also developed delayed neuropathy
in response to other OP compounds, mainly the OP insecticides malathion,
parathion (122), methamidophos (126), mipafox, a fluorine-containing OP
(127), isophenfos (128), and probably leptophos (129). Other OPs implicated
causally in human OPIDN induction are listed in a recent review by Lotti
(130) and include dichlorvos, EPN, trichlorfon, and trichlornat. Delayed
neuropathy induction is associated with 70-80% inhibition of a specific
protein, neuropathy target esterase (NTE; formerly termed neurotoxic esterase).
The function of NTE and role, if any, in the mechanism of OPIDN induction
is unknown. Recent reviews by Johnson (131), Abou-Donia and Lapadula (132),
and Lotti (130) summarize much of what is known about NTE. Abou-Donia and
Lapadula (132) propose a mechanism involving phosphorylation of Ca2+/calmodulin
kinase II, increasing its activity and causing disruptive effects on cytoskeletal
proteins in neuronal tissue. Thus, although NTE inhibition may be a marker
of OPIDN-inducing potential, it may play no role in OPIDN induction.
Human beings are one of the most sensitive species for OPIDN induction;
hens are equivalently sensitive and have been used widely to test chemicals
for OPIDN-inducing potential (133). To date, human beings are known to be
significantly more sensitive than hens to OPIDN induction by only one chemical,
methamidophos (126,130). Lotti (130) attributed this greater human sensitivity
to a higher rate of spontaneous reactivation of methamidophos-inhibited
human AChE, and the availability of assisted ventilation to human beings
(but not hens).
Ratios of anticholinesterase activity and NTE-inhibiting activity in
vitro for hen tissue (AChE I50/NTE I50) and human
tissue are similar (133,134). In vivo toxicity ratios (LD50/neurotoxic
dose) for the hen correspond well with the in vitro AChE I50/NTE
I50 ratios. Thus, it is likely that in vivo assays in
hens and in vitro human and hen enzyme activity ratios are at least
qualitatively predictive for human OPIDN-inducing potential (133).
Although the data are often sparse on the other delayed health effects
of nerve agents per se, an expanding field of literature on delayed health
effects of OPs, particularly insecticides, exists (49,66,35). Information
from this literature is included, particularly where there are data gaps
for nerve agents.
Agent GA
OPIDN. Agent GA has not been shown to produce OPIDN, but it appears that
it may have the potential to do so under conditions highly unlikely for
human exposure. Agent GA at extremely high doses inhibits NTE both in
vitro (136) and in vivo in antidote-protected chickens (134).
Johnson et al. (137) showed that GA produces the aged or unreactivatable
form of inhibited NTE that is often associated with induction of OPIDN.
Results of in vitro assays of GA potency against bovine AChE and
hen NTE suggested that doses of 100-150 times the LD50 would
be necessary to induce OPIDN in vivo in hens (14). Tests of GA in
antidote-protected chickens at 120 times the subcutaneous (SC) LD50
dose of 0.610 µmol/kg (14) [two 6 mg/kg doses/day intramuscular (IM);
total of 12 mg/kg/day] elicited mild neuropathic signs in one of two surviving
hens (138). Despite this observation, neuropathy was not observed in survivors
of a single dose (12 or 15 mg/kg, IM) in that same study (138) nor in survivors
of single doses (12 mg/kg, IM) or two 12-mg/kg doses (total dose, 24 mg/kg,
IM) (one surviving hen) (137). Willems et al. (138) concluded that even
higher doses of GA would be needed to produce the fully developed clinical
signs of delayed peripheral neuropathy in chickens. Henderson et al. (139)
found no effect of GA on NTE activity in chicken with or without atropine
protection given single IM injections of 0.125 mg/kg; results of 90-day
studies using 0.07 mg/kg IM 5 days/week in atropine-protected hens were
negative for behavioral or histopathologic signs of OPIDN. Furthermore,
dosing male and female CD rats without atropine protection at 1, 1/2, and
1/4 times the MTD (0.1125, 0.05625, and 0.02813 mg/kg IP, respectively)
5 days/week for 13 weekss (90-day study) revealed no effect on brain NTE
activity (140) and no clinical evidence of neuropathy (141). The higher
two doses used were sufficient to depress plama and RBC-ChE activities significantly
and result in transient clinical signs of OP toxicity in some animals (141).
It appears that GA doses many times the LD50 may be necessary
to induce OPIDN in humans. If unprotected human populations were ever exposed
to doses this great, there would be few survivors. There are no data to
support OPIDN induction in humans at less-than-lethal doses of GA. Thus,
OPIDN induction is not a relevant concern for GA exposure.
Agent GB
OPIDN. Agent GB at high doses has been shown to cause OPIDN in
chickens, a particularly sensitive species for this endpoint (14,142-144).
This effect required doses 30-60 times the chicken IM LD50 (0.025-0.05
mg/kg) (142,143) in birds protected from death by prior injection of antidotes.
A review by Abou-Donia (129) documents the induction of OPIDN by GB only
in chickens and only at supralethal doses (1 mg/kg). More recently, Crowell
et al. (145) failed to observe significant decreases in brain, spinal cord,
or lymphocyte NTE in atropine-protected hens 24 hr after single administration
of GB as sarin I by gavage at 1, 3.3, and 6.6 times the maximum tolerated
dose (MTD) (0.61, 0.2, and 0.4 mg/kg) or sarin II at 1, 1/2, and 1/4 the
MTD (0.28, 0.14, and 0.07 mg/kg). The positive control, TOCP, was effective
at lowering all types of NTE. Repeated doses of GB (by gavage at 1/3, 1/6,
and 1/12 MTD for 42 days) in atropine-protected hens failed to result in
signs of OPIDN (146), despite exposure sufficient to cause signs of acute
toxicity.
Little evidence for GB-induced neuropathy has accrued from studies in
mammals. For example, agent GB failed to induce OPIDN in cats either at
a supralethal dose, 1 mg/kg, SC, in atropine-physostigmine-protected animals
(compared to the LD50 dose of 0.035 mg/kg, SC), or at multiple
low-dose exposures adding up to the LD50 (147,148). The low doses
(0.0035 mg/kg/day for 10 days or 0.007 mg/kg/day for 5 days, SC) generated
no signs of cholinesterase poisoning. Agent GB (sarin I) also failed to
induce OPIDN in CD rats exposed by gavage five times per week for 13 weeks
(90 days) at doses ranging from 0 to 0.3 mg/kg/day (the MTD) (149). A 15%
decrease (p < 0.05) in NTE was seen only in the high-dose female
group. Sarin II in similar experiments also failed to induce neuropathy
in rats at doses up to the MTD, and no effects on NTE were seen at any dose
(150). It should be noted that the rat, unlike the cat, is relatively insensitive
to full OPIDN induction (130,132) and variably sensitive to histopathological
damage. A study of the effects of high (convulsive, 5 µg/kg) or multiple
small-dose GB exposures of rhesus monkeys on EEG patterns showed no difference
in behavior between exposed and control animals examined both at 24 hr and
1 year after dosing (151). No signs of atoxia were noted. Some primates
are considered resistant to OPIDN induction, but others are susceptible
(129).
A recent report (152) of a small study in eight female Swiss albino mice
suggests early changes characteristic of OPIDN induction resulting from
low-dose GB exposure. The animals were exposed 20 min/day for 10 days, whole-body,
to 5 mg/m3 GB (17% of LC50, 600 mg/m3/min,
for this strain). This exposure regimen resulted in 27% inhibition of blood
AChE and 19% inhibition of brain AChE but caused no signs of anti-AChE toxicity.
By day 14 after onset of exposure, the mice displayed mild signs (slight
ataxia, muscle weakness of limbs, twitching), NTE inhibition of brain, spinal
cord, and platelets, and light or moderate axonal degeneration in the spinal
cord. Mipafox caused more pronounced changes in positive control animals.
At present we know of no reports indicating that other groups have tried
to replicate these results in this or any other mouse strain. Further work
should be done to verify these results in view of the lesser sensitivity
of the mouse to OPIDN induction by TOCP (153).
Bidstrup et al. (127) reported that another fluorine-containing OP, mipafox,
induced delayed neuropathy in two chemists (a male and a female) after occupational
exposure sufficient to cause severe acute toxic effects. A third worker
who developed less severe acute symptoms failed to exhibit OPIDN. No dose
information was presented, but substantial exposure over a period of several
days was documented. Davies et al. (142) tested 36 alkyl OPs and found that
the 17 compounds that caused delayed neuropathy contained fluorine. However,
the possession of a fluorine atom by an OP compound does not, by itself,
indicate neurotoxic potency, as a number of fluorine OP compounds have been
tested and found to lack such activity (144).
Although many human volunteers (246 individuals) (144) have been exposed
to GB by a variety of routes, no reported instances of OPIDN are known,
either from the experimental studies (144,154) or from accidental exposures
of more than 200 individuals (Leffingwell SS, personal communication). The
doses ranged from those causing no signs and symptoms and no detectable
decrease in RBC-ChE activity to doses causing moderate or severe signs and
symptoms and RBC-ChE activity depression of as much as 90% below normal
baseline levels (57,155). Most of the accidental GB exposures were very
mild and, while formal long-term follow-up was not done, no employee reported
signs of OPIDN after returning to work, nor did they report such symptoms
on subsequent contacts with the medical staff. Six severe GB exposures are
known, and the U. S. Army Medical Research Institute of Chemical Defense
is not aware of any evidence for OPIDN having developed in any of those
cases (Sidell FR, personal communication).
In summary, while the possibility of a human developing OPIDN in response
to a supralethal dose of GB cannot be ruled out, the major concern would
be immediate treatment to prevent death. There is no evidence of GB causing
OPIDN in humans, nor is there current evidence for this effect resulting
from low-dose GB exposure (lower than those resulting in acute toxic effects)
in any species other than the mouse.
Psychological effects. Acute exposure to GB has been shown to
cause both transient and prolonged changes in psychological function. Evidence
is available from several cases of accidental exposure in which the doses
are unknown but effects can be categorized as severe or moderate (56,156,
157). At least some of the persistent changes may have resulted from brain
damage caused by GB-induced convulsions (156). Agent GB induction of transient
depressive emotions, insomnia, excessive dreaming, and nightmares have been
observed in volunteers in the absence of seizure activity (155). Grob and
Harvey (57) reported similar effects, as well as EEG changes that persisted
for 4-18 days after oral administration of GB to 10 volunteers for 1-4 days.
Repeated doses were sufficient to produce 85% depression of plasma ChE activity
and more than 97% depression of RBC-ChE activity. Associated physical signs
and symptoms were described as moderately severe but fell short of convulsions.
Occupationally exposed workers exhibited similar signs and symptoms after
low-level exposures to G agents; in some cases, effects persisted beyond
3 days (158,159).
Sidell (71) considers that mild psychological changes resulting from
nerve agent exposure to be more common than ordinarily recognized and to
occur even in a small fraction of individuals experiencing few or no other
signs of exposure; effects may persist from days to weeks. Sidell (71) also
points out that the psychological effects can delay fitness for return to
any work requiring full cognitive function and rapid decision-making.
That GB doses sufficient to cause acute toxic effects may also result
in long-term psychological changes is further suggested by a recent study
of workers previously acutely intoxicated by OP insecticides. This study
documents persistent insecticide effects similar to those of nerve agent
exposure on mental function and emotional state. Savage and colleagues (135)
evaluated 100 individuals who had experienced 1 or more documented episodes
of acute poisoning on average 9 years earlier (in at least 80% of the cases
by parathion, methyl parathion, or malathion, dose unknown). These individuals
showed mild but statistically significant deficits in intellectual ability,
academic skills, abstract thinking ability, and speed and coordination on
motor skill tests in comparison to matched controls. They evidenced more
depression, irritability, confusion, and tendency to withdrawal than controls
on an inventory by relatives and perceived themselves to have areas of difficulty
with memory, thinking ability, and use of language.
Organophosphate insecticides are sequestered in body fat and gradually
mobilized from these depots to a greater extent than the OP nerve agents
as evidenced by their longer time course of recovery and need for repeated
treatment with atropine (160,161). Thus, OP insecticides may be more likely
than nerve agents to cause CNS effects and to induce changes persisting
longer than those possibly induced by OP nerve agents.
EEG effects. Duffy and colleagues reported subtle long-term changes
in human brain function after acute GB exposure (162,163). In these studies,
exceedingly subtle changes in EEG patterns and increases in rapid eye movement
(REM) sleep were observed at 1-6 years after accidental exposure to GB sufficient
to cause acute signs and symptoms and to lower RBC-ChE by at least 25% below
baseline. Statistically significant EEG changes were detected only by computer
analysis in a group comparison of exposed workers with control subjects;
trained neurologists were unable to distinguish by visual inspection between
EEGs of exposed and unexposed individuals. Thus, the EEG changes are not
considered clinically significant. Some of the workers studied by Duffy
and colleagues (162,163) had been studied earlier by Metcalf and Holmes
(164), who also reported on EEG, psychological, and neurological changes
in persons exposed to OPs including insecticides and nerve agents. When
the EEG patterns of the exposed OP worker group were compared with the EEGs
of a control group of workers who had no exposure or access to OPs, minimal
group differences were observed, consisting mainly of increased medium-voltage
irregular 
waves, usually during drowsiness [for
details of EEG spectra differences, see Duffy et al. (162)]. Note that,
as in the study by Duffy and collegues (162,163), these EEG changes were
evident a year or more after exposure--during this time the workers had
no other known OP exposure and showed no blood ChE activity depression.
Comparing the "highly exposed" worker group to the "minimally
exposed" worker group, Metcalf and Holmes (164) found memory, concentration,
and sleep disturbances, as well as subtle EEG changes (not clinically significant)
and minor motor coordination deficits. The Metcalf and Holmes report does
not clarify whether the GB exposures of the subjects were recent, nor whether
the persistent EEG changes could be correlated with the observed persistent
psychological changes.
After the observation of EEG changes in GB-exposed workers, a study was
carried out in monkeys in an attempt to substantiate these long-term EEG
effects (151). The monkeys were given either a single dose of GB (0.005
mg/kg, IV) that produced overt toxic signs or 10 smaller doses (0.001 mg/kg,
IM, at weekly intervals) that resulted in no clinical signs. In both the
acute and subchronic exposure groups, increases in ß activity were
observed in the spontaneous cortical EEG patterns up to 1 year after exposure.
No difference in gross behavior was observed between treated and control
animals. Another important finding from this study was that, at 1 year,
there were greater differences in the EEG patterns of the animals that received
the series of smaller doses (with no resulting clinical symptoms) than in
the animals receiving the single dose. Because the total dose in the series
was twice that of the single dose, this suggests it is the total amount
of GB received and not the induction of clinical effects that determined
the degree of EEG alteration.
In summary, clinically insignificant EEG changes and increases in REM
sleep were observed in the worker group exposed 1-6 years earlier to levels
of GB sufficient to cause signs of toxicity. Changes were more evident in
the worker group with more recent exposures or more than one episode of
exposure (163). Although some workers in the same population had experienced
psychological changes, this study did not address any possible correlation
between EEG changes and psychological effects. Thus, the meaning of subtle
persistent EEG changes after GB exposure is unclear; there may be no meaningful
behavioral or physiological correlates. Levels of GB exposure too low to
cause acute toxic signs and symptons have not been tested for the ability
to induce persistent EEG changes.
Cardiac effects. Another potential delayed effect of GB exposure
is cardiac damage. In a study of OP insecticide poisonings, certain clinical
effects such as cardiac problems often showed a delay in their onset (160).
Agent GB has been shown to cause cardiomyopathy in rats in doses sufficient
to cause convulsions in many of the animals (0.111-0.17 mg/kg, SC) (65).
Cardiac lesions were seen only in animals that had convulsed with resulting
brain lesions. The cardiomyopathy may result from CNS damage and consequent
sympathetic overstimulation.
Agent VX
OPIDN. Agent VX shows no potential for inducing OPIDN (Table 5).
In tests of the ability of nerve agents to inhibit NTE in vitro,
VX was at least 1000-fold less active than agent GB (14,136). Three VX-related
thiolates were also ineffective at in vitro inhibition of NTE (14).
Single IM or SC injections of VX at 0.15 mg/kg (5 times the LD50,
IM) in atropine-protected chickens produced neither inhibition of NTE nor
histological or behavioral evidence of OPIDN (165). A structurally unrelated
fluoridothionate compound was neuropathic in an acute test in the chicken
at 5 mg/kg, IM (142). The ability of VX to induce OPIDN has also been tested
in antidote-protected chickens when injected IM at 0.04 mg/kg for 90-100
days. The results of this subchronic exposure test were negative; no behavioral
signs or histological degeneration of spinal cord or muscles were produced,
in contrast to effects seen in the positive controls exposed to diisopropylfluorophosphonate
(DFP) (166). In summary, there is no indication that VX has any potential
at low or high doses for the induction of OPIDN in human beings or other
species either with acute or long-term exposure.
Psychological effects and EEG changes. Delayed or persistent psychological
effects of VX have not been reported; however, no accidental exposures such
as those with GB are known (156), and evidence of long-term, low-dose exposures
such as the occupational exposures to GB has not been found for comparison.
It is not known whether long-term psychological effects could be produced
by acute or chronic exposure. The potential for VX to induce long-term EEG
changes has not been tested.
Cardiac effects. Acute exposure to agent VX has been shown to
cause cardiac arrhythmias in rats (0.012 mg/kg, SC) (167) and beagle dogs
(0.0015, 0.003, or 0.006 mg/kg, SC; 0.25, 0.5, and 1.0 LD50,
respectively) (168) at doses too low to cause convulsions. The arrhythmias
in rats were associated with a high incidence of mortality. The ventricular
arrhythmias seen in beagles included a form (Torsade de pointes, a rapid,
multifocal ventricular arrhythmia) that is rare but characteristic of cardiac
abnormalities seen in OP insecticide-poisoned humans. Histological examination
to evaluate the induction of cardiomyopathy was not performed in these studies.
Whether VX has the potential to cause fatal arrhythmias in humans or long-term
cardiac damage at high doses is not known, although cardiac arrhythmias
were not observed in experimental studies on volunteers reported above (56,74).
Agent GA
Information on the toxicological effects of GA is limited in comparison
to that for GB and VX, but a number of studies have been sponsored by the
U.S. Army Medical Bioengineering Research and Development Laboratory; results
are summarized in Table 6. These include studies of subchronic toxicity
in rats, teratogenesis testing in rabbits, and several types of short-term
genotoxicity.
Subchronic toxicity. Male and female CD rats were given GA without
atropine protection at 0.1125, 0.05625, 0.02813, and 0 mg/kg/day, 5 days/week,
for 13 weeks (90-day study). Plasma and RBC-ChE activities were significantly
depressed in the two higher dose groups. No evidence of systemic toxicity
was observed at any dose other than the effects on cholinesterase activity.
Clinical chemistry results and hitopathology examinations revealed no other
toxicity (140,141).
Genotoxicity. In tests of mutagenicity, GA was weakly mutagenic
in the mouse lymphoma assay and in the Ames test using S. typhimurium
(170). Agent GA induced sister chromatid exchanges (SCE) in vitro
in mouse cells but not in vivo in mice (170). Agent GA failed to
induce unscheduled DNA synthesis (UDS) in rat hepatocytes and, in fact,
depressed UDS with no evidence of cytotoxicity (170). Wilson et al. (170)
concluded that GA is a weakly active mutagen.
Teratogenicity. New Zealand white rabbits were used to test GA
for teratogenic activity and fetotoxicity. GA was administered SC at 0.1125,
0.05625, 0.02813 mg/kg on days 6-19 of gestation. The results were negative
for teratogenic activity, and no fetal toxicity of any kind was seen at
doses below those causing maternal toxicity (Bucci TJ, personal communication).
Agent GB
Chronic and subchronic toxicity. Weimer et al. (169) exposed beagle
dogs, Sprague-Dawley/Wistar rats, ICR Swiss mice, and tumor-sensitive Fischer
344 rats and strain A mice to low concentrations (0.001 and 0.0001 mg/m3)
of airborne GB for 6 hr/day, 5 days/week for 4-52 weeks. Animals were observed
daily for toxic signs; blood chemistry was monitored monthly in the dogs
and at the time of euthanasia of rodents; gross and microscopic examination
of tissue samples from all major organ systems was performed; body weights
were monitored throughout the exposure period and body and organ weights
for heart, lung, liver, kidney, and testes or ovary were recorded at necropsy.
Cardiovascular function was monitored in the dogs. No evidence of acute
or chronic toxicity was found at these low intermittent exposure levels.
Blood activity of RBC-ChE was not depressed in any species at either GB
concentration.
Bucci and Parker (149,150) reported the results of subchronic toxicity
testing in which male and female CD rats were exposed to GB together with
the stabilizers at the concentrations used in unitary weapons systems [tributylamine
(GB type I or sarin I) and diisopropylcarbodiimide (GB type II or sarin
II)]. The rats were administered GB at 0.3, 0.15, and 0.075 mg/kg by gavage
5 days/week for 13 weeks. Body weights were monitored throughout the study;
blood was drawn at 1, 3, and 7 weeks and at euthanasia for hematology and
clinical chemistry measures. At necropsy, gross and histopathological examination
of 144 tissues and all lesions was performed. Although plasma and RBC-ChE
activities were significantly depressed at all dose levels, investigators
saw no evidence of hematologic abnormalities; they noted no liver, kidney,
or muscle damage, nor effects on body weight gain or organ weight. Neither
form of GB was associated with any type of neoplastic or nonneoplastic lesion
except for infrequent evidence of sarin I-induced cerebral necrosis. This
effect was not related to dose.
Carcinogenicity and genotoxicity. In the studies by Weimer et al. (169),
groups of each rodent strain were held for an additional 6 months for observation
of carcinogenicity. No increase in tumors was detected in either the tumor-sensitive
rodent strain or any other test animals in response to 6 hr/day, 5 days/week
exposure for up to 52 weeks. Although the results suggest that GB is not
carcinogenic, the low doses and less-than-lifetime exposure and observation
period preclude definitive interpretation of the study.
Negative results in genotoxicity studies of GB as summarized in Table
6 support the likelihood that GB is not carcinogenic. Agent GB did not induce
mutations in the Ames test (171) nor in mouse lymphoma cells (171); it failed
to induce SCE (174) or DNA repair as indicated by UDS (175). Like GA, GB
actually inhibited UDS (175). It is not known whether this inhibition reflects
an ability of G-agent metabolites to scavenge free radicals and thus reduce
DNA damage resulting in decreased need for repair. Alternatively, DNA repair
capacity may be blocked by the agents with the result that permanent mutations
could be produced (180).
Teratogenicity and reproductive toxicity. Tests for teratogenic
effects of GB in the rabbit and rat were negative (176) (Table 6). Agent
GB as sarin I and sarin II was tested via oral exposure in pregnant New
Zealand White rabbits and CD rats. The number and status of fetal implants,
individual fetal weights, and fetal malformations were evaluated; no evidence
of developmental toxicity in the first 20 days of pregnancy was seen in
either species, even at doses of GB that resulted in maternal toxicity or
mortality.
Definitive tests of the effects of GB on reproduction have not been performed,
but some data are available from a chronic exposure study of low levels
of GB in rats. Denk (179) [as reviewed by Weimer et al. (169)] reported
that no dominant lethal mutations or adverse effects on reproductive performance
occurred in rats through three generations after exposure for 10 months
to airborne GB at concentrations of 0.001 or 0.0001 mg/m3 (see
Table 6). These levels were so low that no overt signs of toxicity were
produced. Another study evaluated testicular atrophy in Fischer rats after
a 6-month exposure via SC or intraperitoneal injections of low doses of
GB; no differences were found between treated and nontreated animals (181).
Weimer et al. (169) reported no reproductive effects of long-term GB inhalation
on any group of mice or rats except for the Fischer rats, which exhibited
an increased incidence of testicular atrophy, a condition to which this
strain is genetically susceptible. It was noted that this group of Fischer
rats had undergone heat stress during the experiment. A follow-up study
of unstressed Fischer rats exposed to the same concentrations (0, 0.01,
and 0.0001 mg/m3) for 12 or 24 weeks showed no testicular atrophy.
The investigators concluded that the increase in testicular atrophy in the
first experiment was due solely to heat stress during several weeks of the
exposure period.
Agent VX
Chronic and subchronic toxicity. Goldman, et al. (173) reported
the results of exposing male and female Sprague-Dawley rats to VX (0.00025,
0.001, or 0.004 mg/kg, SC) daily, 5 days/week, for 30, 60 and 90 days. Blood
was assayed for RBC-ChE and plasma ChE activity, and a standard battery
of clinical chemistry tests was performed. At 60 and 90 days, creatinine
phosphokinase activity, an index of muscular injury, was also determined.
Urine was collected for analysis during weeks 8 and 12 of the study. Body
and organ weights were recorded and histopathological examination was performed
on tissues. Hematological parameters were observed in a separate group of
male and female rats exposed identically to VX in the first generation of
a three-generation reproduction study (173).
RBC-ChE activity was significantly depressed in male and female rats
at all VX doses for 30, 60, and 90 days. Plasma ChE was significantly depressed
in both genders of rats given 0.001 mg/kg VX for 30 days and in both genders
of the high-dose group at all exposure periods.
A slight decrease in body weight was observed in the high-dose group,
but no consistent effects on organ weights were seen that could be ascribed
to VX exposure. No dose-related changes were reported in clinical chemistry
or urinalysis parameters. No histopathologic lesions were reported. Overall,
the authors concluded that VX exposure sufficient to significantly depress
RBC-ChE activity produced no subchronic toxic effects. No studies of long-term
(chronic) VX exposure have been reported.
Carcinogenicity. As with the other nerve agents, the majority
of animal studies on VX have dealt with the acute toxicity of this chemical
agent. We have found no reports of carcinogenesis studies with VX. McNamara
et al. (37) reported that there was no association of increased cancer in
personnel working daily with VX; however, as with the other nerve agents,
definitive studies on the carcinogenic potential of VX are lacking.
Genotoxicity. The potential of VX to cause genetic effects has
been addressed in several studies supported by the U.S. Army Medical Bioengineering
Research and Development Laboratory (172,173) (see Table 6). These studies
include mutagenicity in bacteria (S. typhimurium, Ames assay), yeast
(Saccharomyces cerevisiae), fruitflies (Drosophila melanogaster),
and in a mammalian cell line (mouse lymphoma L5178Y). In the bacteria and
yeast studies, VX was tested with and without metabolic enzyme activation
to determine if VX metabolites might be mutagenic. The range of concentrations
in the Ames assay included concentrations (1.093 mg/plate) that corresponded
to approximately 40,000 times the estimated IV LD50 for humans
(172). Results were negative in both the Ames assay (172,173) and the S.
cerevisiae assays (173). In the Drosophila sex-linked, recessive
lethal mutation test, only one mutation was observed at the higher VX concentration
(0.004 mg/m3), for a mutation percentage of 0.5% (172). A repeat
test at the same concentration yielded no mutations. Thus, results were
also negative for VX in this mutagenicity assay.
The fourth assay for mutagenic activity involved the use of mouse lymphoma
cells, which may provide better health risk estimates for humans than tests
using bacteria or yeast. Again, in this assay, VX was tested with and without
metabolic activation. At lower concentrations (0.001-0.02 mg/ml), there
was no statistically significant increase in the mutation frequency; at
the higher test concentrations (0.02-0.1 mg/ml), there was a small but statistically
significant increase in the number of mutants that appeared to be related
to dose but not to activation (173). Compared with controls, this increase
in mutations was less than the twofold increase set as a criterion for a
positive mutagen (182,183); thus VX was considered by the authors to be
a nonmutagen. Agent VX also gave negative results in the SCE assay, which
tests for chromosomal damage rather than mutations.
Teratogenicity. Data on the potential of VX to affect fetal development
(teratogenesis) come from the accidental exposure of sheep to lethal concentrations
of VX and from controlled studies in rats and rabbits (see Table 6). Van
Kampen et al. (111) reported studies on 79 surviving ewes in an accidental
1968 VX exposure in Skull Valley, Utah, in which 4500 of the 6300 affected
sheep died or were killed. The dose that the exposed pregnant ewes received
is not known, and the dose given another group of purposely exposed pregnant
ewes is classified, making it difficult to evaluate this study. The accidentally
exposed animals demonstrated clinical signs of toxicity; their RBC-ChE activities
were depressed for up to 4 months after the initial intoxication, suggesting
significant VX exposure. Under the conditions of both accidental and intentional
exposure, no evidence of any significant developmental effects were noted
in the offspring of the ewes.
The teratogenic potential of VX in rats and rabbits was tested by SC
injection of 0.00025 to 0.004 mg/kg/day on gestational days 6 through 15
in rats and days 6 through 19 in rabbits. The pregnant animals were killed
on day 20; the fetuses were removed and examined for body weight and for
skeletal and organ abnormalities. Results of the studies in rats showed
no statistically significant relationship between the dose of VX and any
of the parameters studied (173). Results of the teratogenic studies in rabbits
were also negative.
A preliminary study (177) suggested embryotoxicity of VX to rat fetuses
after 0.03 mg SC doses to the mother and embryolethality to chick embryos
at 0.032 mg/egg. Another preliminary study (178) suggested that VX may exert
behavioral toxicity effects on the rat fetus after repeated SC doses of
0.005 mg/kg at varying times during fetal development. These results indicate
that further study may be warranted.
Reproductive toxicity. Information to date suggests that neither
acute nor chronic VX exposure has deleterious effects on reproductive potential.
One data set comes from the 1968 Skull Valley incident of acute accidental
exposure of sheep to unknown levels of VX (111). The exposed ewes were evaluated
for their reproductive capacity by breeding them 5-6 months after exposure.
Although the dose of VX received by the ewes is unknown, it was sufficient
to cause signs of acute toxicity. No effects on reproductive capacity were
found in these animals. The results of a three-generation assay for reproductive
potential were reported to be negative in Sprague-Dawley rats (171
) (Table 6). The doses of VX (0.00025-0.004 mg/kg, SC) were administered
for 5 days per week for 21-25 weeks in the F0 generation and
for 24-27 weeks in the F1 generation.
Several aspects of nerve agent physicochemical characteristics and toxicity
have ramifications of particular importance in planning for public protection
during continued storage and the active phase of the Chemical Stockpile
Disposal
