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A Set of Scientific Issues Being Considered Concerning the Office of Pesticide Programs (OPP) Cholinesterase Inhibition Policy

Scientific Advisory Panel (SAP) June 1997 Meeting

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) has completed its review of the Office of Pesticide Programs (OPP) review of Cholinesterase. The review was conducted in an open meeting held in Arlington, Virginia, on June 4, 1997. The meeting was chaired by Dr. Ernest E. McConnell. Other panel members present were: Dr. William S. Brimijoin (Mayo Clinic); Dr. Janice E. Chambers (Mississippi State University); Dr. Amira T. Eldefrawi (University of Maryland); Dr. Richard Fenske (University of Washington); Dr. Ernest Hodgson (North Carolina State University); Dr. Ronald J. Kendall (Texas Tech University/Texas Tech University Health Sciences Center); Dr. Harihara M. Mehendale (Northeast Louisiana University); Dr. Genevieve M. Matanoski (Johns Hopkins University); Dr. Carey Pope (Northeast Louisiana University); Dr. Stephen Saunders (Frito-Lay Corporation); Dr. Edward Stein (U.S. Department of Labor); Dr. Mary Anna Thrall (Colorado State University).

Public Notice of the meeting was published in the Federal Register on April 22, 1997.

Oral statements were received from:
Dr. Barry Astroff, Bayer Corporation
Dr. Sir Colin Berry, Royal London Hospital
Dr. William Chen, Dow Elanco
Dr. David Clegg, American Crop Protection Association
Dr. Donald Grant, Pest Management Regulatory Agency, Health Canada
Dr. Carolyn Lewis, State of California EPA
Dr. Marcello Lotti, University of Padua, Italy
Dr. Larry Sheets, Bayer Corporation
Mr. R. Thomas Van Arsdall, National Council of Farmer Cooperatives
Ms. Carolyn Van Pelt, DuPont Agricultural Products
Dr. David Wallinga, Natural Resources Defense Council
Dr. Chris Wilkinson, Technology Sciences Group, Inc.

Written statements were received from:
American Crop Protection Association
Acute Cholinesterase Risk Assessment Work Group
Ricerca, Inc.
Dr. Brian Dementi, Environmental Protection Agency


Literature Review

1. Does the review include the major concepts and citations from the literature and present an overall objective analysis consistent with the proposed policy?

The Panel gave a strongly positive answer. A question was raised about the relation between part A and part B. It was noted that part A did not reflect a broad consensus within the Agency. Another Panel Member commented on the discussion of the epidemiologic data and offered the opinion that present studies do not allow a conclusion as to whether long term effects in pesticide workers might represent a persistent effect of acute overexposure or chronic low dose exposure. Additional information on a number of points was presented by the Panel, including the likelihood that some organophosphate pesticides bind with nanomolar affinity to muscarinic or nicotinic receptors. Overall, however, the review was judged to comprise an excellent survey of the relevant data, and the Panel was quite satisfied by the review. The weight of evidence approach seems like an especially rational approach for a group of compounds which display so much inter-compound variability in response (qualitative, quantitative and time course). The complexities of metabolism and the differences in acetylcholinesterase potencies for inhibition among compounds results in great differences in both time course and magnitude of effect among various anti-cholinesterases. All of these diverse factors would be expected to yield different responses, qualitatively and quantitatively, among different compounds. Therefore a very rigid approach to risk assessment of all anticholinesterases might lead to the missing of important, critical biological responses.

2. ChE methodology. Does the paper accurately lay out the state of the science and the limitations regarding the measurement of cholinesterase inhibition?"

Again the answer was very positive. It was pointed out that the section does not describe a standard operating procedure and attention was focused on factors that promote variability in assays of red blood cell AChE, especially when there has been exposure to carbamates. However, it was recognized that many of these issues were treated in depth in earlier EPA documents and workshops and are receiving continued attention inside EPA.

3. Case studies. Do the case studies help to illustrate an adequate variety of data sets and how in the recent past the EPA has been using the Weight of Evidence" (WOE) approach to assess ChE inhibiting chemicals in accordance with the proposed science policy?"

The Panel agreed that these case studies did illustrate fairly how EPA has used and might use the WOE approach in this area with a few possible exceptions. One Panel Member offered the opinion that the case studies also point out the difficulties that would likely be encountered in trying to apply a more rigid, algorithmic approach to the same problem. It was noted that none of the presented cases demonstrated how an assessment might use blood cholinesterase data when there was a large difference in dose required to inhibit that activity relative to other endpoints such as brain AChE inhibition.

4. Science Policy. Is a weight of evidence approach a reasonable means of evaluating the overall significance of: clinical signs and overall behavioral or functional effects in humans and animals; symptoms in humans, central or peripheral nervous tissue measures of ChE inhibition; and blood measures of ChE inhibition?"

This question was deemed by the Panel to be of major importance. There was a consensus that the weight of evidence approach is indeed reasonable and justified on the basis of the available scientific data so long as these data are derived from rigorous experiments with standardized methods and proper controls. In particular, this approach allows flexibility to weight heavily inhibition in non-target tissues when the overall toxicologic context suggests that other approaches pose danger of serious risk from overexposure.

Careful study of the counterproposal" in the Acute Cholinesterase Risk Assessment Work Group (ACRA) document reveals that the industry work group also favors a weight of evidence approach in most respects. Thus, ACRA proposes to discount data that are out of context" in the sense of representing effects that do not appear to be clearly dose-related or occur sporadically rather than consistently across time. Other examples of industry consensus with WOE include the recommendation to give priority to human over animal data (where of equivalent quality) and to emphasize effects on target vs non-target tissues (where data are available). The major difference between the ACRA position and the EPA position with regard to WOE is that ACRA would not use data on effects falling below an arbitrarily designated level of 20%. This cutoff value seems reasonable on the surface but, when dose-response curves are steep, it could lead to RfDs uncomfortably close to those that actually cause toxicity.

5. Recognizing that people disagree as to the significance of blood cholinesterase values, is it supportable to use them as a matter of science policy in certain cases where;

a. there is a steep dose-effect curve for ChEI toxicity and blood ChE is the most sensitive endpoint?
b. the NOELs and LOELs for various effects are essentially the same?
c. the pesticide poorly penetrates the blood brain barrier, and blood ChE is the only indicator of adverse effect for the peripheral nervous system other than clinical signs?
d. human data indicate that blood ChE is the most sensitive endpoint?"

There was unanimous support for the notion that, under SOME circumstances, measurements of SOME blood-borne cholinesterases would be appropriate to consider in establishing RfDs for anticholinesterases. Several panel members pointed out that generic measurements of total ChE activity in whole blood were unsuitable from this point of view. With human blood samples, where plasma contains almost exclusively BChE, it would be acceptable to measure separately red cell ChE (entirely AChE) and plasma ChE. With animal samples, where plasma contains a variable proportion of BChE and AChE (about 1:1 in rat), it would be better to divide plasma activity into specific types by using selective enzyme inhibitors in the assay (eg., iso-OMPA or ethopropazine to block BChE, BW284C51 to block AChE).

It was recognized that measured inhibition of cholinesterase activities in any of the blood fractions is best regarded as an imperfect mirror of enzyme inhibition in the true target tissues: brain, neuromuscular junctions, autonomic ganglia, and autonomic synapses. When, or if, direct measurements at the probable target sites become available, data from the blood might be under-weighted or even ignored. The best course would be to use blood cholinesterase values" as a matter of science policy in cases a) and c) above. This course of action is readily justified if the discrepancy between blood ChE and functional endpoints is not too great. One Panel Member pointed out that such use simply introduces a safety factor. It becomes more difficult to justify permanent reliance on blood ChE as the relevant endpoint when the discrepancy is very wide (e.g., 100 fold or more). This situation, however, is the subject of the next question, number 6.

6. There is uncertainty and disagreement in interpreting cases where blood ChE is perturbed at doses far below those showing concern from other effects. As a means of prompting the development of further information to resolve the issue (as described below), OPP is proposing to use the blood ChE measurements on an interim basis for RfD determination, awaiting further data. Is this proposed science policy a reasonable way of helping to resolve these cases?"

As implied above, the Panel felt it reasonable to use blood cholinesterase measurements on an interim basis, awaiting further information pertaining to cholinesterase inhibition in the peripheral tissues (e.g., heart, diaphragm). Another way of stating this view is to say that the registrant who wishes to see an agent regulated on some basis other than cholinesterase inhibition n the blood faces a burden of proof. This burden would be difficult to meet without generating data on cholinesterases in the presumed target tissues.

7. Following the selection of critical endpoint, the program will generally apply the traditional uncertainty factors of 10X for inter-species variations and 10X for intra-species variations. Is this approach reasonable?"

The committee generally felt that the common 10X factors for intra- and inter-species extrapolation were appropriate following the selection of the critical effect. It was argued that, even though we understand at a molecular level the structural basis for AChE inhibition in red cells, for example, this is no reason to use a smaller safety factor when extrapolating from animal species to humans. For one thing, experiments with purified enzymes from rat and human tissue show that inhibitory potency of some anticholinesterases is species dependent. For another, it is well known that some species, as compared with humans, have different concentrations of blood-borne or hepatic enzymes that represent "sinks" or different levels of metabolic pathways which bioactivate or degrade certain pesticides.

In considering intra-species safety factors, it was emphasized that, not only must one take into account genetic differences in enzyme and receptor levels, and developmental changes from infancy to adulthood, but also variations that might stem from drug interactions in patients treated with cholinergic drugs (e.g., for neurologic disease, ulcerative colitis, glaucoma) as well as smokers whose blood has high concentrations of nicotine. After discussion, the Panel concluded that a 10X intra-species safety factor remains appropriate.

In the current testing paradigm, effects of cholinesterase inhibitors on the peripheral nervous system (PNS) have not been systematically examined. Generally, the only measures available are clinical signs and other neurobehavioral endpoints, which are often rather gross and insensitive measures of adverse effects. The Panel believes that it is important that joint efforts be mounted to evaluate ChEI in the PNS per se and in the neuroeffector junctions.

8. Is the collection of data from peripheral nervous tissues and/or neuroeffector organs technically feasible?"

There was some discussion of the difficulties in obtaining homogeneous, consistent tissue preparations (e.g., skeletal muscle, diaphragm) for measuring cholinesterase activity. Several members of the Panel did consider it technically feasible to routinely conduct cholinesterase assays in such tissues, however. This information would be extremely important in establishing the value of blood cholinesterase information in predicting peripheral effects of anticholinesterases or replacing that information, at least in animal tests.

9. What factors are important to the conduct of that testing"

The most important factors identified by the Panel were a) standardized and reproducible dissection and homogenization of the tissue; b) use of assays that can be conducted with minimal tissue dilution (critical in dealing with carbamate inhibitors), c) selection of tissues representing the most toxicologically relevant targets; d) t me elapsing between collection and assay; e) standardization of tissue storage conditions. It is important that the Agency move to develop a required or recommended standard testing protocol.

10. Which nerves or tissues should be measured?"

Several suggestions were offered by the Panel. Skeletal muscles, heart, lung, salivary glands, diaphragm and autonomic ganglia (e.g., superior cervical ganglia) are particularly appropriate. Consistent dissection of any of these tissues would be necessary. Perhaps at least two or more of these tissues could be agreed upon to pursue as peripheral targets of anticholinesterases. One tissue not believed to be particularly useful in this sense was the ma n trunk of peripheral nerve itself. Sciatic nerve, for example, is easy to dissect and assay. However, it is protected by an efficient blood nerve barrier (unlike the autonomic ganglia which are fairly open to circulating compounds). Thus, nerve trunks are expected to behave more like brain than like the tissues that represent true peripheral targets of pesticides.

11. Along with the PNS ChE measures, what other endpoints should be included?"

Two Panel members felt strongly that cholinergic receptor binding assays should be incorporated into long-term exposure studies, and the rest of the Panel concurred. The development of tolerance during long-term exposures can mask" neurochemical changes induced by the anticholinesterases. Changes in receptor populations may therefore be able to explain discrepancies in studies wherein cholinesterase inhibition in target tissues does not appear to correlate with signs of toxicity, in particular when the target tissue assays are only performed at the end of the study. The ultimate regulatory significance and use of this information is speculative at present.

12. Should elements of this proposal become a research priority?"

This idea was endorsed enthusiastically and it was agreed that both acute and chronic studies on PNS ChEI are needed. Several Panel members noted that the importance of blood cholinesterase values in the regulation of organophosphate and carbamate pesticides has been a point of debate for decades. This conflict might be resolved by comparing the relative sensitivity of acetylcholinesterase inhibition in peripheral tissues to that noted in plasma and erythrocytes. Support for such research could be an excellent investment, since we may need to continue relying on blood cholinesterase values as the only biomarker of exposure/effect in humans. Therefore, more definitive knowledge on the utility of these markers will be essential to provide a sound scientific basis for hazard assessment and regulation.

One Panel Member suggested that research on the direct action of organophosphates on muscarinic and nicotinic receptor subtypes in vitro might have some value. Such action could exacerbate or ameliorate organophosphate toxicity depending on the organophosphate, the receptor subtype and its location (presynaptic or postsynaptic). Anticholinesterases may produce excessive receptor activation in acute exposure, but change receptor numbers in chronic exposure to produce tolerance.

Related research priorities would address the developing brain (prenatal and postnatal), which undergoes many changes, including cell migration and consolidation and elimination of synapses. Therefore, it is potentially more sensitive than an adult brain to disruptions caused by a toxicant. If an anticholinesterase did affect brain development, there would be potential for permanent deficits.

Additional Comments related to Medical Surveillance by Cholinesterase Monitoring

As the Agency considers policies associated with cholinesterase inhibition, it is important to keep in mind the role of cholinesterase monitoring in medical surveillance programs in the United States and throughout the world. There are literally thousands of farm operators who are collecting periodic measurements of plasma or erythrocyte activity cholinesterase levels from exposed workers with the belief that such monitoring is an effective means of preventing pesticide-related illness. They have come to this belief through an effective campaign mounted by public health scientists. This campaign, in turn, was based on clinical evidence that workers with significantly depressed cholinesterase were at greater risk for acute intoxications than were workers without notable depression.

The State of California requires removal of workers from pesticide handling activities on the basis of plasma and erythrocyte activity cholinesterase monitoring. The regulation states specifically: "If plasma cholinesterase falls to 60 percent or less of the baseline, or if red blood cell cholinesterase falls to 70 percent or less of baseline, the employee shall be removed from further exposure until cholinesterase values return to 80 percent or more of their respective baseline values."

In a recent review of the California program, researchers found that plasma cholinesterase inhibition was predictive of pesticide-related illness. They state this point as follows: "The relative risk of pesticide poisoning was increased in workers whose initial baseline plasma levels were low, or if their levels had already dropped to 60-80 percent of their baseline previously in the season. (Fillmore C., Lessinger J.E. A cholinesterase testing program for pesticide applicators. Journal of Occupational Medicine, Volume 35, January 1993)


Certified as an accurate report of findings:

Larry C. Dorsey
Designated Federal Official
FIFRA/Scientific Advisory Panel

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