6. Pesticide Metabolism and Potential Cumulative Effects
Two of the most important determinants of the potential for cumulative effects to occur are whether or not exposures to different chemicals (pesticide active ingredients and daughter products) are "concurrent," and the extent to which binding of the chemical with the critical enzyme acetylcholinesterase is reversible. "Concurrent," as used here, means that active forms of the different chemicals are available in the body at the same time to exert toxic action on the biological target(s), such as cholinesterases. "Reversible," as used here, is a relative term. If the time from initial chemical binding with acetylcholinesterase to regeneration of active enzyme is short, the reaction is said to be reversible. If the time is long, or if no regeneration occurs, the reaction is said to be irreversible. Many, but not all, organophosphate pesticides have the potential to bind irreversibly with acetylcholinesterase, due to a process known as "aging" of the enzyme-pesticide complex. Carbamates bind reversibly. In any case, some nervous system acetylcholinesterase can be inhibited, reversibly or irreversibly, without other observable adverse effects. Beyond a certain point, however, adverse effects will be evident.
All the POPCs are subject to mechanisms in the body which may activate, inactivate and/or otherwise remove them, mainly by metabolism ("biotransformation") and excretion. In general, the body acts to make the chemicals more water-soluble in order to facilitate excretion, and individuals vary widely in their ability to metabolize pesticide active ingredients. Some pesticide active ingredients may undergo a process called "bioactivation" in which a form of the chemical generated in the body is more toxic to the target than the original form of the pesticide active ingredient. The major organ for pesticide active ingredient metabolism is the liver, the site of the highest levels of detoxifying enzyme systems. There is a vast amount of literature on the complicated pathways of pesticide active ingredient metabolism, and it is not feasible to cover the subject in detail here; details are readily available elsewhere.[593] Instead, the following text will address a few additional key points, relying on three important organophosphates (OPs) and one carbamate for illustrative purposes.
Another important consideration of pesticide active ingredient toxicity is its elimination half-life. This is particularly true in the case of repeated exposures. Elimination half-life is the time it takes for the plasma concentration of the pesticide active ingredient to decrease by one-half. Frequently, if the half-life is short in relation to the time between exposures, the pesticide may be almost completely eliminated by the time a subsequent dose is absorbed, and there will be little or no cumulative effect.
More than half of an absorbed dose of chlorpyrifos (an OP) is typically eliminated within 62 hours.[594] Thus, less than 1/8 of the starting dose would typically remain in the body after 8 days. It is safe to conclude that following a single asymptomatic exposure, little of the dose remaining after 8 days would be an active form of chlorpyrifos; most would be in relatively inactive forms. The binding of chlorpyrifos and its bioactivated metabolite chlorpyrifos oxon with cholinesterase can be irreversible.
Inhaled dichlorvos (an OP) is probably absorbed fairly rapidly, although definitive data are lacking. However, once absorbed, dichlorvos is rapidly broken down and excreted. It is expected that asymptomatic inhalation exposures over the course of several days would be unlikely to lead to detectable levels of dichlorvos in the body within hours of discontinuing exposure. The binding of dichlorvos with cholinesterase is reversible.[595]
Diazinon (an OP) is rapidly broken down and eliminated from the body, and almost all of the dose received during asymptomatic exposure would be eliminated from the body in 12 days. In non-fatal exposures, the effects of diazinon are transient and recovery is rapid and complete following cessation of exposure. The binding of diazinon and its bioactivated metabolite diazoxon with cholinesterase can be irreversible.[596]
Propoxur (a carbamate) is rapidly broken down and excreted. Humans given a single dose of propoxur excreted 38% within 24 hours.[597] Assuming this rate of excretion is constant, about 1/7 of the initial dose should remain after 4 days. Given the rapid metabolism, there should be little active residue left after 4 days. One source stated that no adverse cumulative effects on cholinesterase activity were demonstrated.[598] The binding of propoxur with cholinesterase is reversible.
Thus, sequential asymptomatic exposures to the above pesticide active ingredients, separated by a few days each, are unlikely to contribute to cumulative effects, as manifested by frank effects. Nervous system acetylcholinesterase levels may or may not be reduced in such cases. Conversely, repeated exposures to one or more of the above pesticides occurring within a few hours have the potential to contribute to cumulative effects on nervous system acetylcholinesterase activity. At high enough exposures, frank effects may be evident.
7. Potential Pesticide Interactions with Pyridostigmine Bromide
Many scientists have expressed concern about the potential for pyridostigmine bromide (PB) to interact with other pesticide active ingredients, particularly organophosphates, carbamates, permethrin, and DEET. At present, there is no evidence that untoward interactions occurred in veterans, only various hypotheses regarding what could have occurred. Thorough discussions of potential PB - pesticide interactions are provided in the RAND literature reviews for PB and pesticides.[599,] [600] A few salient points from these sources and some newer information is provided below.
Potential interaction between PB and pesticide active ingredients may be a concern because:
Several specific items of note are presented in the RAND pesticides literature review.[602] RAND describes two 1996 studies by Abou-Donia et al. in which adult hens were exposed to the combination of DEET, PB, and either permethrin or chlorpyrifos. The hens exhibited greater than additive effects (not synergistic) when exposed to two of the three compounds, and there was an even greater effect when all three compounds were present. However, given that extremely high doses of some compounds were used, and some were injected, the relevance of these findings to veterans may be limited. The doses used in the hens, if extrapolated to humans, would amount to 467 tablets of PB, 1,667 cans of permethrin, and 76 tubes of 33% DEET. Also, RAND states that, while DEET does not exhibit cholinergic effects, it may enhance the effect of anticholinesterases.
RAND also described a 1997 study by McCain et al., which evaluated the lethal action of DEET, PB, and permethrin when given orally to rats via gastric gavage.[603] A gavage is an instrument for delivering a substance directly into the stomach via the esophagus. Again, the combination of compounds produced significantly greater than additive toxicity. Similar to the Abou-Donia et al. studies, extremely high doses and the route of administration limit the relevance. Greater than additive effects from combinations of PB and various pesticide active ingredients were also found in mice and cockroaches.[604] The mouse study utilized intraperitoneal injection to deliver the compounds, while RAND did not report the route of administration for the cockroaches. Thus, relevance remains an issue for the latter two studies due to route of administration. Also, the relevance of a cockroach study is inherently questionable.
A 2002 study by Abdel-Rahman, Shetty, and Abou-Donia appears to have greater potential relevance to veterans than the studies noted above. This study investigated the effects of a combined exposure to restraint stress and low doses of PB, DEET, and permethrin in adult male rats. The authors concluded that the combination of stress, PB, DEET, and permethrin leads to significant brain injury, and that the various neurological symptoms reported by Gulf War veterans could be linked to this kind of injury.[605]
The findings of the Abdel-Rahman et al. study may be particularly relevant because: 1) adult male mammals were the test organism; 2) the doses of all chemicals were administered to whole, live animals, and are close to or within the range of doses we estimated for veterans in this HRA; 3) the exposure routes were realistic (oral for PB; dermal for DEET and permethrin); 4) the exposure duration was realistic (28 days); 5) the stress induced was moderate (5 minutes restraint per day); 6) the combination of the chemicals and stress caused significant liver damage (thereby interfering with normal detoxification processes); 7) the combination of the chemicals and stress significantly increased the permeability of the blood-brain barrier; and 8) the combination of the chemicals and stress reportedly caused substantial damage to multiple regions of the brain.
It is important to note that the highest levels of pesticide use would not have occurred simultaneously with the highest levels of PB use. In fact, for the majority of personnel, there would have been little if any overlap in exposure. The highest levels of pesticide use, in general, would have been during warm months when the main pest populations were highest; that is, roughly August through November 1990, and March through June 1991. Most servicemembers who took PB would have done so only within the interval of January 17 through February 28, 1991, while offensive operations were in progress. Exposure to pesticides weeks before exposure to PB would be unlikely to produce an adverse interaction. Similarly, exposure to PB at least several days prior to pesticide exposure would be unlikely to produce an adverse interaction.
8. Regulatory Status
The EPA registered all the POPC active ingredients for use at the time of deployment, with the exception of azamethiphos. Most of the POPC active ingredients are still registered, although the EPA has cut back on many uses since the Gulf War. Azamethiphos has never been registered. The following are currently registered: DEET,[606] d-phenothrin, methomyl,[607] and propoxur.[608] The following are currently registered, and are scheduled for interim or final reregistration eligibility decisions during FY2002 or FY2003: lindane, permethrin, diazinon, dichlorvos, and malathion.[609] The EPA reregistration decision documents are a good source of information on regulatory status; they are cited here as available.
The EPA has taken steps described below to severely curtail or eliminate the use of five of the eleven active ingredients in the POPCs due to concerns about adverse health effects. All five of these active ingredients were once used in a wide variety of products in addition to those mentioned below.
9. Uncertainty and Variability Analysis
"Uncertainty" refers to those aspects of the data and exposure factors which are not presently known, and/or are unknowable. "Variability" refers to the variation within a population which may or may not be presently known and/or well characterized. For example, the actual levels of pesticide active ingredients to which veterans were exposed can never be known because no real-time monitoring was conducted at the time, and it is impossible to collect the data today. Thus, there may be a high level of uncertainty associated with some of the estimates of exposure levels calculated. In contrast, the data on body weights among veterans who served in the Gulf War exists and either has been or can be quantitatively described, if desired, and such a description would be a description of variability. What follows, however, is predominantly a qualitative description of uncertainty and variability.
a. Uncertainty in the Selection of POPCs
The group of all pesticide products identified as definitely or possibly having been used during the Gulf War was assembled from many sources with varying shortcomings. There is solid documentation regarding the pesticide products ordered by deploying units through the military supply system. However, no documentation was identified detailing what was actually shipped, received, used, disposed, and/or returned. Military guidance consulted from the period indicate only which pesticide formulations were recommended under various circumstances. Discussions with the Armed Forces Pest Management Board helped fill in many gaps about what was probably used. The survey and interviews of the veterans provided much additional information, but were also based on years-old recollections and had many inaccuracies identified.
The criteria used to limit the group of all pesticides and related products to a manageable subset, known as the POPCs, while logical and defensible, are imperfect. For one thing, both the survey and interviews have varying degrees of bias. In the case of the survey, recall bias is an issue. Among other things, veterans have had much potential exposure to media reports on Gulf War illness issues. In the case of the PM interviews, while these individuals are presumed by investigators to be the most knowledgeable about pesticide application, they bore some of the responsibility for application, and some may have avoided conveying information with the potential to reflect negatively on them.
b. Uncertainty in the Exposure Assessment
The most significant uncertainties in the exposure assessment, and in this risk assessment as a whole, are associated with the estimation of exposure concentrations and doses of POPCs active ingredients. No documentation from the period has been identified which records, at the unit level, what pesticide products were applied, how they were applied, and in what quantities they were applied. There are also no real-time monitoring data for pesticides. Thus, it was necessary to employ many location-specific and generic assumptions in order to estimate concentrations. While many of these assumptions originated with the EPA, are widely used, and are based on credible field and laboratory studies, they have varying degrees of applicability in this HRA exposure assessment. All attempts were made to develop realistic exposure scenarios and assumptions, based for example on veteran survey and interviews, yet varying degrees of uncertainty remain.
In many cases, pesticide product exposures during deployment would have been very similar to exposures normally occurring in the US at the time. On the other hand, there were certainly conditions existing some of the time that would have contributed to higher-than-normal, or otherwise unusual exposures for some servicemembers.
c. Uncertainty in the Toxicity Assessment
Some uncertainty is inherent in the toxicity values used to quantify potential systemic and carcinogenic effects. The EPA toxicity values for systemic effects are derived using available toxicity information and standard uncertainty factors. Uncertainty factors are assigned based on the quality of the available data. Consequently, the less information that is known about a given constituent, the more conservative the systemic toxicity value. Carcinogenic toxicity values (slope factors) are based on the 95% upper confidence limit on lifetime risk and, therefore, will tend to overestimate cancer risks.
A full set of ideal toxicity values for the HRA do not exist. The ideal reference dose is a verified duration-specific and route-specific value. For example, the ideal subchronic dermal reference dose is based on a reliable subchronic dermal no-observed-adverse-effect level. Since ideal values do not exist in many cases, provisional values were identified or developed, and used. Published toxicity values from various sources were compiled and modified to provide the most comprehensive and appropriate values available. Route-to-route extrapolation was carried out; for example, oral toxicity values were modified to be used for dermal exposure risk assessment.
All the POPC active ingredients were registered for use by the EPA and/or FDA at the time of deployment, with the exception of azamethiphos. Most of the POPC active ingredients are still registered for at least some uses, although the EPA and the FDA have made a number of changes in specific uses since the Gulf War, and plan future changes. Azamethiphos has never been registered. The following are currently registered: DEET,[615] d-phenothrin, methomyl,[616] and propoxur.[617] The following are currently registered, and are scheduled for reregistration eligibility decisions by the end of FY2002: dichlorvos, malathion, and lindane.[618] The EPA currently registers lindane only for pre-plant seed treatment; there are no homeowner uses or above-ground uses.[619] FDA currently registers lindane for prescription-only treatments for lice and scabies on humans, and encourages patients to use non-lindane treatments where possible.[620] FDA currently registers permethrin for creams used in the treatment of lice and scabies on humans.[621] The EPA has announced the cancellation of registrations for all pet collars and all indoor end-use products containing diazinon. Retail sale of such diazinon products will be prohibited after December 31, 2002.[622] In 1995, the EPA proposed to cancel most uses of dichlorvos, including all uses in or on residences, but has not made the final decision on this.[623] All bendiocarb products were canceled as of December 31, 2001.[624]
d. Uncertainty in the Risk Characterization
Most of the hazard quotients (HQs) calculated for individual pesticide active ingredients should be considered reasonably reliable, depending on the reliability of the specific components. As HQs are added together across exposure routes and different chemicals, to yield a hazard index (HI), certainty declines. In the HRA, organophosphates and carbamates were grouped together because investigators presumed a common mechanism of action. This particular grouping is widely practiced and accepted. The effects of additional pesticide active ingredients (e.g., permethrin, d-phenothrin, lindane) are unknown. It seems more likely that those pesticide active ingredients that potentially affect the central nervous system are likely to have equal to or greater than additive effects (synergism), rather than less than additive effects (antagonism). Additional uncertainty is associated with the assumption that all toxicity values used in the evaluation have an equal degree of reliability, which in reality is not the case.
There are many factors that would have significantly reduced actual exposure, while there are many conservative assumptions used in the HRA that significantly increase apparent (estimated) exposure. Pesticide applicators who followed the required procedures would have reduced their hazards and risks substantially. For example, proper use of PPE would have reduced associated hazards and risks by factors ranging from 10 to 100. However, a key assumption in the HRA was that, "the most exposed were the least protected," which probably tends to exaggerate hazards and risks.
The hazard quotients, hazard indices, and risks calculated are based on the specific endpoints of animal and human testing, such as the suppression of cholinesterase within a specified time frame. Normally, the most sensitive endpoints are used to establish the toxicity values; however, there are other potential endpoints and effects which the HRA does not take into account, such as:
Investigators do not believe that OPIDN due to pesticide exposure plays any significant role in veteran health. First, investigators did not uncover any reports of OPIDN symptoms. Second, chlorpyrifos and dichlorvos are the only pesticide active ingredient identified which have been associated at all with OPIDN, and they are only weakly associated. Additionally, RAND concluded that the relevance of OPIDN symptoms to the issue of undiagnosed illnesses in veterans is limited.[625] OPIDN is a very well-characterized syndrome, with very specific sequelae. A major problem with studying unexplained illnesses in veterans has been the lack of objective measures. OPIDN begins with acute symptoms, and later presents clearly recognizable clinical and histopathological manifestations (e.g., peripheral neuropathy). Investigators uncovered no reports of the occurrence of organophosphate intermediate syndrome.
Delayed cognitive toxicity (DCT) is a condition of delayed signs and symptoms that is not akin to OPIDN. In two studies, industrial workers exposed to OPs during sarin manufacture reportedly showed electroencephalogram (EEG), sleep, memory, and personality changes sometimes 2 years after exposure.[626] Given that literature reports describing DCT are rare, and it has only been associated with sarin exposure, it too is of questionable relevance to pesticide exposure during deployment.
Perhaps most importantly, the HRA does not account for the potential combined effects of some pesticide active ingredients and other chemicals. The HRA addresses to some extent the combined effects of the specific pesticide formulations identified as having had a fairly high level of use during the Gulf War, but does not fully account for any additional effects due to the following:
One unanswered, and probably unanswerable, question then is, "What were the individual body burdens of all like-acting chemicals, such as cholinesterase inhibitors, and what is the full spectrum of health effects that may have resulted?" Possible interaction of organophosphate and carbamate pesticide active ingredients with PB is of particular interest, since PB is a carbamate, and all act by a similar toxic mechanism, namely, inhibition of various forms of cholinesterase.
Discussion of uncertainties related to the other human benchmarks is presented in Tab J.
e. Variability
Some of the exposure factors and assumptions used in the HRA are associated with no significant variability. For example, one can say with a high level of certainty that the floor area of a GP large tent used during the Gulf War was 900 ft2, and that there is little variability associated with this datum. On the other hand, each of many of the other exposure factors and assumptions used in the HRA is associated with an underlying variability which may be well known and well characterized, or may be unknown but knowable, or unknowable. In this risk assessment investigators assumed that all receptors weigh 70 kg. This is a commonly used assumption for adult body weight, but in reality the weights of the veterans vary substantially, and the data exist to characterize the data distribution with a high degree of certainty. Investigators selected the value of 70 kg for BW because it is close to the mean adult body weight for males and females combined, and because of the precedent for its use.
The HRA took the traditional "deterministic" risk assessment approach of using point estimates for each input rather than the "probabilistic" approach using data distributions for each, and then applying a technique such as Monte Carlo Analysis. While we acknowledge that there are more rigorous quantitative methods for evaluating and describing data distributions, and that it would be possible to do this for some exposure factors, we decided not to employ the more rigorous approach. The reasons for our decision to use the deterministic approach are that 1) we can meet the objectives of the HRA by doing so; 2) it would be a much larger effort to use the probabilistic approach; 3) we lack sufficient data to truly characterize the distributions for many exposure factors; 4) the results used to draw conclusions would likely not change dramatically. During the course of an in-depth review of the HRA, neither the EPA nor peer reviewers recommended that we should use the probabilistic approach.
F. CONCLUSIONS FROM THE HEALTH RISK ASSESSMENT
The HRA drew from a number of sources along with the balance of this Environmental Exposure Report to provide the most comprehensive analysis to date of pesticide exposures during the Gulf War. While the HRA alone does not prove or disprove that pesticide overexposures occurred during the Gulf War, or that any connection exists between pesticide exposures and chronic health effects reported by some veterans months or years after exposure, we can still draw the following important conclusions:
One important result of the HRA is the identification and prioritization of a small number of pesticides of potential concern, and the elimination of the many pesticides which posed little or no risk. This will help focus future work.
Investigators employed a toxicological approach and an epidemiological approach to evaluate the potential health threat. The results of the toxicological approach identified more cases where estimated doses exceeded levels of concern; while the epidemiological approach found that most of the estimated doses were well below the benchmarks described. Under the present circumstances, the toxicological approach is more thorough and reliable in terms of establishing levels of concern.
The investigators believe the biological indices used and the assumptions made to establish the likelihood of exceeding risk-based levels are conservative. The probability that exceeding the levels of concern defined gives rise to any of the symptoms associated with the undiagnosed illnesses in Gulf War veterans remains unknown. For example, even if some veterans were exposed to pesticide active ingredients at the highest levels estimated, and even if biological changes such as mild to moderate suppression of cholinesterase occurred, it is uncertain if such changes could be associated with any long-term health problems, particularly given that so few serious acute reactions to pesticide products were documented.