United States
Environmental
Protection Agency
Science Advisory
Board (A-101)
EPA-SAB-EHC-33-011
April 1933
&EPA AN SAB REPORT:
CHOLINESTERASE INHIBITION
AND RISK ASSESSMENT
REVIEW OF THE RISK
ASSESSMENT FORUM'S
DRAFT GUIDANCE ON THE
USE OF DATA ON
CHOLINESTERASE
INHIBITION IN RISK
ASSESSMENT BY THE
SAB/SAP JOINT COMMITTEE
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C, 20460
April 23, 1993
OFFICE OF WE ADMINISTRATOR
SCIENCE ADVISORY BOARD
EPA-SAB-EHC-93-011
Honorable Carol M, Browner
Administrator
U.S. Environmental Protection Agency
401 M Street, S,W.
Washington, D.C. 20460
Subject: Science Advisory Board/Scientific Advisory Panel's review of the Risk
Assessment Forum's document Guidance on the Use of Data on Cholinester-
ase Inhibition in Risk Assessment (August, 1992).
Dear Ms. Browner:
Cholinesterase inhibition resulting from exposure to drugs and chemicals
(especially carbamate and organophosphorus pesticides) has long been an issue of
concern to EPA and others involved in assessing environmental health risks, EPA
currently evaluates the risks from cholinesterase inhibitors using clinical effects, brain
cholinesterase inhibition, and/or blood cholinesterase inhibition to define hazard and
set Reference Doses (RfDs). This policy stems from the efforts of an EPA Technical
Panel which reviewed the literature on cholinesterase inhibition and proposed some
science policy positions (19S8) and the comments of a Science Advisory
Board/Scientific Advisory Panel Special Joint Study Group which reviewed the
Agency's position in 1989.
In August, 1992, the Risk Assessment Forum prepared a new draft policy
document addressing the key issues identified in the earlier review, with particular
regard to their application to risk assessment. The Agency asked that a new Joint Co-
mmittee of the Science Advisory Board and the Scientific Advisory Panel (SAB/SAP)
review critically the Forum's new draft document; consequently, the Committee met on
November 5, 1992 in Washington, DC, The primary issues addressed at this review,
and the Committee's findings and comments follow below:
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a) Does the document accurately represent the relevant data and constitute
a credible analysis of the scientific information on cholinesterase inhibi-
tion?
The Committee found that the positions presented in the draft document are, in
general, well supported by the underlying scientific data, and considers it to be a
credible document As with any such undertaking, however, the Committee noted
areas in which improvements could be made. In particular, the section of the docu-
ment addressing red blood cell (RBC) measures of inhibition should be rewritten for
clarity, and the document should be revised to stress the need for better studies on
several critical areas — the relevance of cholinesterase inhibition (erythrocyte, plasma
and brain) measurements; methods to compare results of such methods among
laboratories; and the subsequent use of these measurements as biomarkers of expo-
sure or as correlates to data on clinical signs and symptoms. The Forum document
also needs to consider the peripheral effects of anticholinesterases, in addition to the
focus on the effects of these agents on the central nervous system. The Committee
was also concerned that, in general, the EPA document did not give adequate weight
to the problems associated with the inhibition of peripheral nervous system cholines-
terase.
b) Are the following Agency positions consistent with available scientific
information:
1) Clinical effects associated with exposure to cholinesterase inhibi-
tors can be used in risk assessment to define hazard and to
calculate benchmark doses and RfDs.
The Committee agrees that clinical effects associated with exposure to cholin-
esterase inhibitors can be used to establish benchmark doses and reference doses
(RfD), but only in conjunction with other relevant toxicological information. The
inclusion of biochemical data regarding cholinesterase inhibition in conjunction with
these signs and symptoms is considered essential for the complete hazard evaluation
for these compounds.
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2) Cholinesterase inhibition in plasma and in red blood cells consti-
tutes a biomarker of exposure.
We recommend that the Agency's policy continue to include the use of blood
cholinesterase data in the risk assessment process. The Committee agrees that blood
cholinesterase inhibition is a biomarker of exposure which offers crucial supporting
data for confirming exposures and corroborating clinical signs.
3) Statistically significant cholinesterase inhibition in brain tissue,of
animals can be used in risk assessment to define hazard and to
calculate benchmark doses and RfDs.
The Committee noted several problems with the use of cholinesterase inhibition
in animal brain tissue as a means of defining hazard and calculating benchmark or
reference doses. These issues will have to be resolved if brain cholinesterase
inhibition data are to be used with confidence in regulatory decision-making.
4) To date, analyses of studies of cholinesterase inhibition in plasma
and in red blood cells do not provide information useful for eval-
uating potential hazards and risks in the nervous system. This
finding justifies a new science policy against the use of blood
cholinesterase inhibition data for risk assessment purposes.
Extant animal studies (cited in the enclosed report) provide conflicting evidence
on the issue of using RBC cholinesterase inhibition data by itself (Le„ in the absence
of clinical symptoms) for risk assessment purposes. Measurement of plasma and
RBC cholinesterase inhibition should not be used by itself, but should be used in
conjunction with the clinical data.
5) Should data emerge of a consistent predictive relationship be-
tween red blood cell cholinesterase and neurotoxicity, those data
will be evaluated on a case-by-case basis to determine the utility
of red blood cell acetyl cholinesterase inhibition in risk assessment
to define hazard and to calculate benchmark doses and RfDs,
The Committee recommends that EPA evaluate the possibility that an RfD
could be set based on clinical signs and symptoms that would be associated with a
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significant inhibition of cholinesterase occurring at a specified dose. The Committee
also recommends that EPA continue research aimed at examining carefully the
correlation of clinical signs and erythrocyte cholinesterase inhibition, particularly
regarding the correlations with respect to dose, time, and linearity. Such studies will
be very important in future decisions on the usefulness of erythrocyte cholinesterase
inhibition in regulatory decision-making.
We appreciate the opportunity to review this document, and look forward to
your response to the issues we have raised.
Dr, Raymond C. Loehr, Chair
Science Advisory Board
(p
Dr. G# P. Carlson, Chair
Science Advisory Board/Scientific Advisory Panel
Joint Committee
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NOTICE
This report has been written as a part of the activities of the Science Advisory
Board, a public advisory group providing extramural scientific information and advice
to the Administrator and other officials of the Environmental Protection Agency. The
Board is structured to provide balanced, expert assessment of scientific matters
related to problems facing the Agency. This report has not been reviewed for
approval by the Agency and, hence, the contents of this report do not necessarily
represent the views and policies of the Environmental Protection Agency, nor of other
agencies in the Executive Branch of the Federal government, nor does mention of
trade names or commercial products constitute a recommendation for use.
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ROSTER
SAB/SAP Special Joint Committfe
November 5-6,1992
Chair
Dr. Gary P. Carlson, Department of Pharmacology and Toxicology, Purdue University, West
Lafayette, IN
Members and Consultants
Dr. S. Brimijoin, Department of Pharmacology, Mayo Clinic, Rochester, MN
Dr. William B. Bunn, Mobil Oil Company, Princeton, NJ *"
Dr. George B. Koelle, School of Medicine, University of Pennsylvania, Philadelphia, PA
Dr. David E. Lenz, Medical Research Institute of Chemical Defense, Department of the Army,
Aberdeen, MD
Dr. Fumio Matsumura, Institute of Toxicology & Environmental Health, University of California,
Davis, CA
Dr. Harihara M, Mehendale, School of Pharmacy, Northeast Louisiana University, Monroe, IA
Dr. Carey N. Pope, School of Pharmacy, Northeast Louisiana University, Monroe, LA
Dr. Peter S. Spencer, Center for Research on Occupational & Environmental Toxicology, Ore.
Health Sciences Univ., Portland, OR
Dr. Toshio Narahashi, Department of Pharmacology, Northwestern University, Chicago, IL
Dr. Barry Wilson, Professor, Dept. of En v. Toxicology, University of California, Davis, CA
Dr. John T. Wilson, Professor of Pharmacology, Louisiana State University Medical Center,
Shreveport, LA
Designated Federal Officials
Mr. Samuel Rondberg, Science Advisory Board (A101F), U.S. EPA, 401 M St., SW, Washington
DC 20460
Mr. Bruce Jaeger, Scientific Advisory Panel (H7509C), U.S. EPA, 401 M St., SW
Washington DC 20460
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ABSTRACT
In August, 1992, the EPA Risk Assessment Forum prepared a new draft policy
document addressing key issues in assessing the risks from cholinesterase inhibitors.
A Joint Committee of the Science Advisory Board and the Scientific Advisory Panel
reviewed the document on November 5,1992 in Washington, DC.
The Committee found that the draft document is generally supported by the
underlying scientific data. Improvements could be made in the material addressing red
blood cell (RBC) inhibition and the document revised to stress the need for better
studies on the relevance of cholinesterase inhibition (erythrocyte, plasma and brain)
measurements; methods to compare measurement results methods among laboratories;
and the use of these measurements as biomarkers of exposure and correlates to data
on clinical signs and symptoms. The document should consider the peripheral effects
of anticholinesterases.
The Committee agrees that clinical effects associated with exposure to cholines-
terase inhibitors can be used to establish benchmark doses and reference doses (RfD),
but only in conjunction with other relevant toxicological information. The Committee
also recommends that the Agency's policy continue to include the use of blood cholin-
esterase data in the risk assessment process, and agrees that blood cholinesterase
inhibition is a biomarker of exposure which offers crucial supporting data for confirming
exposures and clinical signs.
EPA should evaluate the possibility that an RfD coutd be set based on clinical
signs and symptoms associated with a significant inhibition of cholinesterase occurring
at a specified dose. EPA should continue research to examine the correlation of
clinical signs and erythrocyte cholinesterase Inhibition.
KEYWORDS: Cholinesterase; cholinesterase inhibition; anticholinesterases;
organophosphates; neurological; myopathy; pesticides.
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TABLE OF CONTENTS
1. EXECUTIVE SUMMARY .... 1
2. INTRODUCTION 3
2.1 Background 3
2.2 Charge 4
3. DETAILED FINDINGS ^ . 6
3.1 Analysis of Relevant Data on Cholinesteras# Inhibition 6
3.2 Agency Positions Vis-a-vis Available Data 8
3.2.1 Use of Clinical Effects In Risk Assessment 8
3.2.2 ChEl in Plasma And RBC As A Biomarker of Exposure 10
3.2.3 ChEl in Brain Tissue And Benchmark/Reference Doses 11.
3.2.4 Using RBC ChEl Data for Risk Assessment 13
3.2.5 Utility of RBC Acetylcholinesterase Inhibition in Risk Assess-
ment-Case by Case Basis . 15
4. CONCLUSIONS 17
5. -^GLOSSARY G-1
6. REFERENCES R-1
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f. EXECUTIVE SUMMARY
The positions presented in the draft Guidance on the Use of Data on Cholines-
terase Inhibition in Risk Assessment are, in general, well supported by the underlying
scientific data, and the Committee considers it to be a credible document. As noted in
the following report (section 3,2.5), however, the Guidance may misinterpret the findings
reported in the studies of Kaptovitz (1984) and of Blick (1989) on red blood cell (RBC)
measures of inhibition; this section of the document should be rewritten for clarity. The
document should be revised to stress the need for better studies on the relevance of
cholinesterase inhibition (erythrocyte, plasma and brain) measurements, better methods
to compare results among laboratories, and the use of these measurements either as
bromarkers of exposure or as correlates to data on clinical signs and symptoms. The
focus of the document on the central nervous system should be broadened to consider
the peripheral effects of anticholinesterases as well. The Committee was concerned
that, in general, the EPA document did not give adequate weight to the problems
associated with the inhibition of peripheral nervous system cholinesterase.
The Committee agrees that clinical effects (as defined in section 3,2,1 of this
report) associated with exposure to cholinesterase Inhibitors can be used to establish
benchmark doses and reference doses (RfD), but only in conjunction with other relevant
toxieological information. The Committee finds that the sole use of clinical signs of
toxicity, or the lack thereof, (especially in long-term exposure studies) for the assess-
ment of hazard and determination of reference or benchmark doses may not be
justified, The inclusion of biochemical data regarding cholinesterase inhibition in
conjunction with these signs and symptoms is considered essential for the complete
hazard evaluation for these compounds.
We recommend that the Agency's policy continue to include-the use of blood
cholinesterase data in the risk assessment process. The Committee agrees that blood
cholinesterase inhibition is a biomarker of exposure which offers crucial supporting data
for confirming exposures and corroborating clinical signs.
The Committee noted several problems with the use of cholinesterase inhibition
in animal brain tissue as a means of defining hazard and calculating benchmark or
reference doses, stemming from our lack of knowledge about the most sensitive brain
regions and their physiological functions. These issues will have to be resolved if brain
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cholinesterase inhibition data are to be used with confidence in regulatory decision-
making.
Extant animal studies provide conflicting evidence on the issue of using RBC
cholinesterase inhibition data by themselves (i.e., in the absence of clinical symptoms)
for risk assessment purposes. There are strong theoretical reasons why the correlation
should exist for agents that traverse the blood-brain barrier. Although this is difficult to
demonstrate empirically, it is nevertheless true, for such agents, that blood enzyme
inhibition precedes (and therefore predicts) brain enzyme inhibition. In the absence of
any other test of proven greater reliability, measurement of plasma and RBC cholines-
terase inhibition should be retained in EPA's policy and used in conjunction with clinical
data. The Committee recommends that EPA evaluate the possibility that an RfD could
be set based on clinical signs and symptoms that would be associated with a significant
inhibition of cholinesterase occurring at a specified dose.
The Committee also recommends that EPA continue research aimed at examin-
ing carefully the correlation of clinical signs and erythrocyte cholinesterase inhibition,
particularly regarding the correlations with respect to dose, time, and linearity. Such
studies will be very important in future decisions on the usefulness of erythrocyte
cholinesterase inhibition in regulatory decision-making.
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2. INTRODUCTION
2.1 Background
Cholinesterase inhibition resulting from exposure to drugs and chemicals
{especially carbamate and organophosphorus pesticides) has long been an issue of
concern to EPA and others involved in assessing environmental health risks. Questions
exist as to which measures (e.g., inhibition in red blood cells, plasma, the brain, etc.)
best correlate with neurotoxic effects and are most appropriate for use in hazard
identification and risk assessment The major issue has centered on the relevance of
data on cholinesterase inhibition in either plasma or red blood cells in establishing a
predictive or causative relationship with effects on the nervous system, both central and
peripheral.
For some years EPA has been evaluating the risks from cholinesterase inhibitors
using clinical effect, brain cholinesterase inhibition, and/or blood cholinesterase
inhibition to define hazard and set Reference Doses (RfDs). In 1988, an EPA Technical
Panel reviewed the literature on cholinesterase inhibition and proposed some science
policy positions. External reviews of that paper confirmed the existence of areas of
both controversy and of consensus (U.S. EPA, 1988). A Special Joint Study Group of
the Science Advisory Boartf'and the Scientific Advisory Panel reviewed the Agency's
position in 1989, expressing concern with regard to using plasma and red blood cell
inhibition as a measure of toxicity, especially in the absence of other effects, but
concurring that inhibition was a biomarker for exposure and absorption (U.S. EPA,
1990). The Study Group also noted that the relationship between the degree of
inhibition noted and toxicity remained unclear, because of the complexities of the dose
and time relationship, and differences among individual chemicals.
The Risk Assessment Forum's present paper replaces the 1988 draft, and
reflects Agency consideration of comments on ..that draft. In summary, the Agency
continues its policy to recognize clinical effects of cholinesterase inhibition as biological-
ly significant, and to use them to set Reference Doses (RfD). The Agency will also
continue to use statistically significant inhibition of cholinesterase in brain, with or
without accompanying clinical manifestations, to set RfDs. The Agency proposes
changing its position on the use of red blood cell/plasma cholinesterase inhibition in risk
assessment. Plasma and red blood cell inhibition data will be used as biomarkers of
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exposure to cholinesterase inhibiting chemicals, but, except for special cases, the
Agency will no longer define hazard or compute RfDs based on such data alone,
2.2 Charge
The Agency asked the Joint Committee of the Science Advisory Board and the
Scientific Advisory Panel (3AB/SAP) to review critically the Risk Assessment Forum's
document Guidance on the Use of Data on Cholinesterase Inhibition in Risk Assess-
ment Although EPA is interested in the Committee's comments on all aspects.cf the
document, there is special interest in the Committee's response to the following specific
questions:
a) The science policy position articulated in the Risk Assessment Forum
document is based in part upon an analysis of scientific data bearing on
cholinesterase inhibition. , Does the document accurately represent the
relevant data and constitute a credible analysis of the scientific informa-
tion?
b) Are the following Agency positions consistent with available scientific infor-
mation:
1) Clinical effects associated with exposure to cholinesterase inhibitors
can be used in risk assessment to define hazard and to calculate
benchmark doses and reference doses (RfDs).
2) Cholinesterase inhibition in plasma and in red blood cells consti-
tutes a biomarker of exposure.
3) Statistically significant cholinesterase inhibition in brain tissue of
animals can be used in risk assessment to define hazard and to
calculate benchmark doses and reference doses (RfDs).
4) To date, analyses of studies of cholinesterase inhibition in plasma
and in red blood cells do not provide information useful for eval-
uating potential hazards and risks in the nervous system. This
finding justifies a new science policy against the use of blood
cholinesterase inhibition data for risk assessment purposes.
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5} Should data emerge of a consistent predictive relationship between
red blood cell cholinesterase and neurotoxicity, those data will be
evaluated on a case-by-case basis to determine the utility of red
blood cell acetylcholinesterase inhibition in risk assessment to
define hazard and to calculate benchmark doses and RfDs.
The Committee met on November 5, 1S92, in Washington DC to address the
issues noted above.
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3. DETAILED FINDINGS
3.1 Analysis of Relevant Data on Cholinesterase Inhibition
Within the physical limitations and constraints of the document, the material
presented in Guidance on the Use of Data on Cholinesterase Inhibition in Risk Assess-
ment represents, in general, the critical scientific data and is credible. It presents a
simple but accurate assessment of the role of cholinesterase and the problems involved
with its inhibition. Furthermore, it gives a clear picture of the differences between
inhibition by organophosphorus, and by carbamate, pesticides. However, as noted
below in section 3.2.5, the document may misinterpret the studies of Kaplovitz et aL
(1984) and Blick et a/. (1989)
Although the document adequately addresses some of the difficulties in the
measurement of cholinesterase and its inhibition, it would be strengthened by empha-
sizing even more the findings of the Workshop on Cholinesterase Methodologies (U.S.
EPA, 1992), particularly the need for more and better studies on the relevance of
cholinesterase inhibition (erythrocyte, plasma and brain) measurements. This includes
not only the need for better and more standardized methods to compare results among
laboratories but also the use of these measurements either as biomarkers of exposure
or as correlates to data on clinical signs and symptoms.
As noted throughout this review, the Committee is especially concerned about
species differences-the effects of which must be clearly understood in making extrapo-
lations from animals to humans in risk assessment. In this regard, additional informa-
tion could be added, such as that provided in the National Academy of Sciences (NAS)
review of anticholinesterases and anticholinergics (NAS, 1982).
One other area needs to be addressed in the Forum document related to the
peripheral effects of anticholinesterases. The document seems to conclude that
important effects of these agents lay primarily within the central nervous system. It
has been known for some time, however, that organophosphorus agents and other
anticholinergics bring about immediate damage to motor endplates, an adverse effect of
these agents outside of the central nervous system. This peripheral effect takes days-
to-weeks to disappear, and may damage an appreciable number of muscle fibers
(Laskowski et al.t 1975; Wecker and Dettbarn, 1976; Leonard and Salpeter, 1979;
Dettbarn, 1984; De Bleecker etal. 1992).
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In agonist-induced myopathy in rodents, the muscle necrosis is first noted near
the neuromusele junction. There is an increase in large-diameter vesicles in the
cytoplasm beneath the junction, dissolution of 2-disks, dilatation of mitochondria and
destruction of the sarcoplasmic reticulum (Leonard and Salpeter, 1979), Less than ten
percent of muscle fibers may appear to be affected at the light microscope level under
conditions where many more show damage under the electron microscope (Dettbarn,
ibid.). The situation is repaired within one-to-two weeks. Damage is evident as early
as 30 minutes after exposure to paraoxon. Exposures of two hours with a loss of
approximately 85% of AChE activity causes severe muscle fiber necrosis.
Several lines of evidence indicate the damage is caused by inhibition of
cholinesterases at the motor end plate, producing an excess of acetylcholine, which in
turn leads to an increase in intracellular CA++ that presumably activates proteolytic
enzymes leading to muscle necrosis For example; (a) Botulinum toxin type A blocks
quantal release of acetylcholine and protects muscles from DFP (Diisopropyl Fluoro
Phosphate) -induced lesions (Sket et al, 1991; Salpeter et a/. 1979); (b) the extent of
muscle damage induced by pyridostigmine is reduced by pretreatment with the calcium
blocker diltiazem (Meshul, 1989); (c) the myopathy js blocked in vitro by removing Ca"
with EGTA (Ethylene Glycol-bis (beta-aminoethyI ether) N.N.N'.N', Tetra acetic Acid).
Several workers report the severity of the necrosis is correlated with the degree of
AChE inhibition (Wecker et al. 1978; De Bleecker et ai. 1991). Many of the studies
were conducted with levels of anticholinesterase agents that produced major cholinergic
symptoms requiring treatment with antidotes, but a recent study found necrotic fibers in
low-dose poisoned rats as well (De Bleecker et al. 1991). Such findings suggest that
. peripheral "adverse" effects produced by anticholinesterase agents might be monitored
by blood cholinesterase measurements. The degree to which this agonist-induced
muscle necrosis in rodents is found in the human is not known.
Recently, OP poisoning cases loosely classed under the rubric of "Intermediate
Syndrome" have been described and studied in the human. The first reports were from
Sri Lanka where symptoms of neuromusele damage appeared 1-4 days after poisoning
with fenthion, dimethoate, monocrotophos and methamidophos (Senanayake and
Karalliede, 1987). Several other cases have been reported and studied since then (e.g.
Samal and Sahu, 1990; Karademir et a/. 1990; De Wilde et ai. 1991, Van den Neucker
et ai. 1991) The "syndrome" Is characterized by respiratory paralysis, cranial motor
nerve palsies, proximal limb muscle, and neck flexor, weakness. In addition to the OPs
mentioned above, a mixture of parathion and methyl parathion, diazinon, diclofenthion,
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fenitrothion, dicrotophos (Bidrin) and malathion have all been associated with such
symptoms. Although antidote treatments (atropine and praidoxime) have not been
considered a factor by some, Benson (1992) raises the question of whether the closes
used were too low. Blood cholinesterase levels have been used to monitor the time
course of the 'Intermediate Syndrome;" symptoms have been reported to subside as
ChE levels return to normal. The extent of damage at low levels of pesticides is not
known. Those studying these poisonings usually distinguish the "Intermediate Syn-
drome" from organophosphate-induced delayed neuropathy (OPIDN).
3.2 Agency Positions Vis-a-vis Available Dam
3,2,1 Use of Clinical Effects in Risk Assessment
Because it means different things to differant people, the definition of "clinical *
effects" was first clarified by the Committee to include both overt clinical signs and
symptoms in humans and behavioral changes m animals. In general, an observable
"clinical" effect following a chemical exposure starts with the interaction between that
chemical and a specific macromolecular "receptor" within the organism. In the case of
cholinesterase-inhibiting pesticides, the specific "receptor" involved in initiating toxicity is
generally agreed to be the enzyme acetylcholinesterase (AChE). Cholinergic neurons
rely on AChE for the degradation and rapid termination of the synaptic transmitter
(acetylcholine) in the dynamic regulation of cholinergic neurotransmission. Following
extensive inhibition of AChE, acetylcholine accumulates at synaptic terminals of the
central and peripheral nervous system, with subsequent over-activation or blockade of
those cholinergic pathways, Through elevation of synaptic acetylcholine levels,
cholinesterase-inhibiting pesticides produce typical signs of acute toxicity, including
autonomic dysfunction such as excessive salivation, lacrimation, urination and defeca-
tion (SLUD signs), and evidence of neuromuscular disorder such as muscle
fasciculations and motor weakness.
In general, it was the Committee's belief that such clinical effects associated with
exposure to cholinesterase inhibitors could indeed be used within the context of risk
assessment to establish benchmark doses and reference doses, but not without also
considering other relevant toxicologicat information. The characteristic clinical effects
produced by exposure to cholinesterase Inhibitors are the toxic manifestations which
typically warrant strict regulation of these pesticides. Therefore, exhibition of such
typical signs associated with exposure to cholinesterase inhibitors, either in controlled
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human or animal studies, should undoubtedly be useful indicators of toxicity for
establishment of reference doses when other supportive toxicological data are available,
and when the clinical effects are reproducible, quantifable and statistically significant,
and exhibit a logical cause-effect, dose-response relationship.
A caveat to this view is that other clinical changes can occur after exposure to
chemicals, including cholinesterase inhibitors, which may or may not be considered as
adverse effects. In addition, as evidenced by some of the reports reviewed by the
Committee, dose-related changes in such clinical effects are sometimes not evident;
often the statistical estimates of treatment-related clinical effects, in particular with low
dose exposures, fail to reach significance. The problems associated with using clinical
signs for the establishment of reference doses for cholinesterase inhibitors, especially
when the clinical endpoints cannot undoubtedly be associated with cholinesterase
inhibitor exposure (e.g., a transient change in blood pressure) introduce additional
uncertainty to the risk assessment process. Furthermore, there are practical problems.
For example, a) the signs and symptoms associated with different cholinesterase
inhibitors may not always occur in the same order; b) they depend on the rate of
exposure to the toxicant and whether or not the toxicant needs to undergo metabolic
activation; and c) they tend to be qualitative in nature and therefore difficult,to grade as
to severity (although the Committee notes that EFA is working on this problem and
encourages this endeavor).-.
Although the sequence of events outlined above is generally adequate to explain
acute toxic manifestations following exposures to cholinesterase-inhibiting pesticides, ,
repeated exposure to these chemicals can produce dramatically different toxic sequel-
ae. Cellular compensatory responses (e.g., modulation of the density of cholinergic
receptors) can also occur in response to AChE inhibition, within hours of exposure, and
these changes can modify the degree of clinical dysfunction, (for a review see Costa et
a!., 1982). It should be stressed that changes in cholinergic receptor populations and
changes in response to cholinergic agonists or antagonists (both indirect indicators of
tolerance) can occur even in the absence of overt signs of toxicity (Costa et a/., 1982,
Pope et ah, 1992). These compensatory responses, while thought to be prominent in
the development of tolerance to the AChE inhibitor, can thus alter sensitivity to subse-
quent environmental or therapeutic challenges. Repeated exposures to cholinest-
erase-inhibiting pesticides could, therefore, fail to produce the typical overt signs of
acute toxicity in animals or persons exhibiting extensive changes in neurochemistry.
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In addition, the available literature suggests that all cholinesierase inhibitors do
not act the same, i.e., different inhibitors may produce somewhat different spectra of
toxic effects through the relative predominance of either central or peripheral involve-
ment or through interaction with other macromoiecular targets. For example, as
detailed In section 3.2.3, some cholinesterase inhibitors can readily cross the blood-
brain barrier while others have considerable difficulty entering the central nervous
system; this differential central/peripheral involvement can dramatically affect the type
and degree of "clinical" effects associated with exposure. Recent reports also suggest
that some organophosphorus agents or their active metabolites (oxons) may bind
directly to muscarinic receptors at concentrations significantly lower than those required
to inhibit AChE (Volpe et al., 1985; Bakry et a/., 1988; Jett et sK, 1991), Therefore,
while the specific molecular target and the mechanism of acute toxicity for this class of
compounds is well known, the ultimate expression of clinical signs following exposure to
a cholinesterase inhibitor can be under complex regulation by biochemita! events
simultaneous or subsequent to binding to and inactivating the molecular target mole-
cule, AChE. The conclusion is that clinical signs may be limited or ev«n masked, in a
time-dependent manner, in animals or persons severely poisoned by cholinesterase-
inhibiting pesticides through the activity of alternate biochemical mechanisms. There-
fore, the sole use of clinical signs of toxicity (especially in long-term exposure studies)
or peitiaps more importantly, the lack of such signs for the assessment of hazard and
determination of reference or benchmark doses, is not justified. Inclusion of biochemi-
cal data regarding cholinesterase inhibition in conjunction with these signs and symp-
toms is considered essential for the complete hazard evaluation for these compounds.
3,2.2 ChEl in Plasma And RBC As A Blomarker of Exposure
Originally defined by their relative sensitivity to different inhibitors, the cholines-
terases can be operationally divided into two types: acetylcholinesterase or "true"
cholinesterase and butyrylcholinesterase or "pseudo" cholinesterase. Although AChE,
the target for cholinesterase inhibitors pertinent to induction of acute toxicity, is located
within the terminal regions of the central and peripheral nervous system, it is also found
in tissues where no cholinergic function is apparent (e.g., erythrocyte membrane).
Pseudocholinesterase (PChE) is found in the nervous system but typically In very low
concentrations. PChE is also abundant in the plasma and serum, but there is no
known function for PChE in any of these tissues.
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In the sdluble fraction of blood (i.e., either serum or plasma) in humans PChE
is the overwhelmingly predominant cholinesterase, whereas rat plasma contains AChE
and PChE activities in approximately equal amounts. As stated before, although there
are no known functions for either PChE or AChE in the blood, inhibition of these
enzymes has been historically used as a biomarker of exposure to cholinesterase
inhibitors. It is generally thought that blood AChE, whether in the erythrocyte or in the
plasma (in species which express that activity), responds similarly to inhibitors as does
the AChE in the nervous system. In contrast, PChE may exhibit marked differences in
sensitivity to some inhibitors compared to AChE. This dichotomy in response to
inhibitors between the PChE and AChE has suggested to some that erythrocyte AChE
activity may be a better marker for sensitivity to inhibition in "target" tissues. However,
very few good correlative studies have been performed to examine the relationship
between sensitivity of blood AChE, blood PChE and target AChE activities in vivo and
therefore the relative Importance of these two blood cholinesterases for prediction o*
target effects is unknown.
There was full agreement among the Committee members that Inhibition of blood
cholinesterase activity (either in plasma or erythrocytes) provides an indication of
previous exposure to a cholinesterase-inhibiting pesticide, l.e.» that blood cholinesterase
inhibition is a biomarker of exposure. As indicated above, information regarding
inhibition of the blood enzymes is often crucial supporting data for confirming exposures
and corroboration of clinical signs. In addition, inhibition of ChE is regarded as a sign
of a depleted enzyme reserve. Although probably not deserving to be considered an
adverse effect, such depletion is itself a sign of heightened vulnerability to adverse
effects from subsequent exposures. There was strong support for the continued
inclusion of blood cholinesterase data in the risk assessment process, in particular In
human studies where cholinesterase data from other target tissues are unavailable.
3.2.3 ChEl in Brain Tissue And Benchmark/Reference Doses
Although the concept itself is very attractive, there are several problems with the
use of cholinesterase inhibition in animal brain tissue as a means of defining hazard
and calculating benchmark or reference doses;
a) ChE inhibitors may be neurotoxic, even lethal, without significant entry into
brain tissue
11
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b) Only certain laboratory animals (rodents) are practically useful for con-
trolled experiments
c) Inbred rodents matched for all significant factors show a marked variability
in brain response to ChE inhibitors (Jimmerson et al.t 1989)
d) Although neuro-behaviora! abnormalities may be related to CNS ChE
inhibition, the correlation may be imperfect, presumably because the
status of spinal cord and, in particular, peripheral ChE plays a significant
role.
Toxic agents circulating in the blood have immediate access to certain regions of
the nervous system that normally lack a regulatory interface ("barrier") between Wood
and neural tissue. These regions include the segmental spinal ganglia (containing
sensory neurons), autonomic ganglia (some containing cholinergic receptors), and the
circumvetricular organs of the brain (subfornical organ, area postrema, etc.). Additional-
ly, the absence of a perineural barrier at the neuromuscular junction may increase
access of blood-borne agents to this ChE-critical site. Certain ChE inhibitors are unable
to traverse the blood brain and blood-nerve barriers that confront them in other regions
of the nervous system, yet these agents nevertheless may produce severe neurotoxic
effects, presumably through actions at peripheral sites. In these cases, measurement
of CNS AChE activity would be of little or no relevance, and measurement of activity at
functionally significant peripheral sites poses severe technical hurdles. The Committee
was concerned that, In general, the EPA document did not give adequate weight to the
problems associated with the inhibition of peripheral nervous system cholinesterase.
ChE inhibitors that traverse the blood-brain and blood-nerve barriers may inhibit
AChE variably and with regional specificity. For example, studies with soman demon-
strate that the cortex and hippocampus are more vulnerable than the striatum to
systemic exposure (Jimmerson et sL, 1989). Studies with soman and other ChE
inhibitors show variability in the degree of enzyme inhibition in a uniform group of
animals and a variable correlation between the degree of enzyme inhibition and neuro-
behavioral sequelae or lethality. Furthermore, the data base is too small to determine
whether these results are compound-specific or dose-specific, or related to other
factors, such as the rate of drug administration to the animal type or the investigator
undertaking the experiment.
12
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There is presently limited understanding of the relationship between regional
(brain, spinal cord) choiinesterase inhibition and neuro-behavioral sequelae. Effects on
learning and memory are under study, but it is presently unknown if these will prove
sensitive indicators of functional brain (e.g. hippocampus, cortex) choiinesterase
perturbation. There is much more complete understanding of the peripheral somatic
and autonomic targets of choiinesterase inhibition and the functional consequences for
the animal (and for humans).
A major challenge in resolving the relation between brain choiinesterase inhibi-
tion and behavioral outcome is to determine the most sensitive region and the physio-
logical functions for which it Is responsible. While this alone would be a significant
advance, it will provide no information on the enzyme levels at sub-regional sites
(specific synapses) sensitive to choiinesterase inhibitors. Resolution of these issues
and other applications to risk assessment will require the combined expertise of the
neurophysiologist, morphologist, pharmacologist/toxicologist and neuro-behavioral *-
scientist working in concert. For their results to be of use in the regulatory climate, they
will need to show the relationship between agent dose, extra-cellular concentration,
sub-regional choiinesterase depletion and functional change.
Although the foregoing discussion outlines the uncertainties surrounding the
relationship of ChE inhibition In blood and brain tissues of animals treated with anticho-
linesterase agents, for certain agents there are nevertheless extant data for brain ChE
inhibition in the presence or absence of corresponding data for the blood enzyme. In
the risk assessment process for those anticholinesterase chemicals/metabolites known
-to cross the blood/brain barrier and to inhibit brain ChE, the Agency may continue to
consider brain ChE data if corresponding information on blood ChE inhibition indicates
the brain enzyme is a more sensitive marker of toxicity. However, the Agency should
not use brain ChE inhibition data if information on the blood enzyme is unavailable;
rather the Agency should take steps to obtain these data,
3.2.4 Using RBC ChE! Data for Risk Assessment
The Committee reached no simple "yes" or "no" answer on the question of using
choiinesterase inhibition, by itself, for risk assessment purposes. The Committee would
be very much concerned about the need to set an RfD for a chemical in which the only
data available were choiinesterase inhibition measurements. Conflicting evidence exists
from recent studies of neonatal and adult rats treated with common organophosphorous
-------
pesticides. Pope and Chakraborti (1992) reported (for methyl parathion, parathion, and
chlorpyrifos) that under defined conditions plasma choiinesterase inhibition may be a
useful quantitative index for the degree of brain choiinesterase inhibition following
exposure to organophosphorous agents. However, the degree of correlation would be
inhibitor specific and could be significantly affected by factors such as the route of
exposure and the lapsed time between treatment and choiinesterase inhibition mea-
surement. A priori, the clearest window of opportunity to measure the effects in blood
would be shortly after exposure, the degree of correlation falling off with time. A more
complex picture would exist for agents that are metabolized to active ChE inhibitors.
«r
Additional factors affecting the correlation between blood and brain choiinesterase
activity would exist for carbamates.
The correlation between plasma (or red blood cell) and brain choiinesterase
activity may also be poor, for the reasons outlined above. The Jimmerson et aL (ibid?
study of soman-treated rats found a poor correlation over that portion of the dose-re-
sponse curve where acute toxicity occurs. They attributed the result to differences in
the magnitude of change of choiinesterase in blood versus brain. They also noted that
differences in the rate of enzyme inactivation may be an important variable in dictating
behavioral signs of toxicity. A similar conclusion may be apparent in human subjects
treated with potent nerve agents under controlled experimental conditions, as in the
1958-1975 U.S. Army program (NAS, 1982), The EPA is encouraged to review the
original data from this study since they represent the largest body of information
collected from human subjects treated with potent AChE inhibitors.
Although broadly applicable correlations between blood ChE and brain ChE
inhibition have yet to be established, there are strong theoretical reasons why the
correlation should exist for agents that traverse the blood-brain barrier. Simply stated, a
direct-acting ChE inhibitor that traverses the blood-brain barrier should first inhibit
plasma and RBC enzyme and, somewhat later, the brain enzyme. Provided regenera-
tion of enzyme does not occur instantaneously, a time should exist shortly after dosing
when brain enzyme inhibition is related to blood enzyme inhibition. Even if this is
difficult to demonstrate empirically, it is nevertheless true that blood enzyme inhibition
precedes (and therefore predicts) brain enzyme inhibition. Given the absence of any
other test of proven greater reliability, measurement of plasma and red blood cell
choiinesterase inhibition should be retained. Attempts should be made to standardize
the conditions for these tests in order to exploit the window of opportunity when blood
and brain choiinesterase inhibition are well correlated.
14
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The Committee emphasized two additional points in its discussions. One is that
when cholinesterase data are considered, one needs to use judgment in what consti-
tutes "significance". This becomes a question of biological versus statistical differences.
If the variations within groups are small and a very small decrease in cholinesterase
activity is judged as statistically significant, what is the biological relevance? On the
other hand, if there is a large decrease in activity but the variances are great, there
would still be some concern even though the findings did not reach significance
because of the biological (individual variation and/or small sample size) or technical
(poor quality of the measurements) differences. A second concern is that, depending
on the timing of the measurement, cholinesterase inhibition in the absence of clinical
signs and symptoms may act as a portent of things to come or, as pointed out by the
EPA, may show that the cholinesterase which is acting as a protective sink for the in-
hibitor is compromised. The Committee is concerned that, if the EPA were to base an
RfD solely on clinical signs and symptoms, the possibility arises that at that dose there
would be a large inhibition of cholinesterase. The Committee advises EPA against
such an action uNI this question is resolved. We suggest that the current data sets
available to the Agency be reviewed to evaluate the likelihood of such an outcome.
3.2,5 Utility of RBC Acetylcholinesterase Inhibition In Risk Assessment-Case
by Case Basis
This element of the Charge postulates an assessment situation in which there is
an assumption of a consistent, predictive relationship between red blood cell cholines-
terase and neurotoxicity. As it is posed, the reasoning behind this question appears to
be circular. If indeed one has the data on neurotoxicity to directly evaluate the dose
response relationship for the chemical of interest and to determine the RfD or bench-
mark dose based on clinical endpoints, why go to a surrogate? However, at the
Committee's meeting, the EPA clarified this to suggest that these factors - clinical
effects and cholinesterase inhibition - would be used in conjunction with each other and
that it was not simply a replacement of the clinical data with the erythrocyte cholinester-
ase inhibition data. This is, of course, the main point that was made by the Committee,
i.e., analysis of all the appropriate data, Thus, there is genera! agreement on the basic
usefulness of the erythrocyte cholinesterase data.
The Committee did express some concerns. The cholinesterase data must have
a similar time frame to the clinical data. That is, for many of the very fast acting and
readily reversible inhibitors, particularly the carbamates, the blood samples must be
15
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obtained soon after exposure. For organophosphorus agents, the prolonged inhibition
of the erythrocyte cholinesterase beyond the period of signs and symptoms of cholines-
terase Inhibition needs to be kept In mind.
The document stems to suggest that there is no direct correlation between the
Inhibition of erythrocyte cholinesterase and neurotoxicity. As noted above, this is not
wholly true (although there is a certain window in time wherein it is correct). Part of the
problem may stem from an inaccurate interpretation of the Kaplovitz et al. (1984) and
Bliek ef a/. (1989} studies cited in the Policy document. The Forum document states
that these studies "lend support to the concept that blood ChEl is not predictive of
neurological effects," but it is not clear how the interpretation was reached. The
f ocument may be suggesting that 40-50% inhibition is reached before there are signs
of neurotoxicity, or it may fail to take into account the fact that the reversible nature of
carbamate inhibition protected against the irreversible agents. Furthermore, interpreta-
tion of the cited experiments is complicated by the presence of supportive therapy given
at the same time. The Committee recommends that this section of the Forum docu-
ment be rewritten for clarity.
The Committee also recommends that EPA look carefully, both through the
literature and via further experimentation, at this correlation of clinical signs and
erythrocyte cholinesterase inhibition. How good are the correlations with respect to
dose? With respect to time? With regard to linearity? Can the results be generalized
either narrowly (some organophosphorus pesticides but not others) or broadly (e.g.
organophosphorus agents vs. carbamate) or are they going to be very highly compound
specific? The Committee was pleased to note that such research has already been
started at EPA's Health Effects Research Laboratory in Research Triangle Park.
16
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4. CONCLUSIONS
The positions presented in the draft Guidance on the Use of Data on Cholines'
terase Inhibition in Risk Assessment reflect, in the main, the critical scientific data, and
the Committee considers it to be a credible document. As noted in the Detailed
Findings section of this report (section 3*2.5), addressing the use of RBC acetylcholin-
esterase inhibition in risk assessment, however, the Guidance may misinterpret the
findings reported in the studies of Kaplovitz et aL {1984} and of Blick (1989); this
section of the document should be rewritten for clarity. The Comrnittee also recom-
mends that the document emphasize more the findings of the Workshop on Cholines-
terase Methodologies, particularly a) the need for more and better studies on the
relevance of cholinesterase inhibition (erythrocyte, plasma and brain) measurements; b)
the need for better methods to compare results among laboratories; and c) the use of
these measurements either as biomarkers of exposure or as correlates to data on
clinical signs and symptoms. The Forum document also needs to consider the ;
peripheral effects of anticholinesterases, In addition to the focus on the effects of these
agents on the central nervous system.
The Agency needs to be aware that the rodent myopathy studies and the
"Intermediate Syndrome" case reports support the importance of blood cholinesterase
measurements as a part of monitoring the risk of OP damage to: the peripheral
neuromuscle system. It should begin to look for data associated with the myopathy and
the "Intermediate Syndrome." There are two major data gaps;
a) Relation to other disorders: Whether or not the human and rodent phe-
nomena are due to the same or to different mechanisms and whether
some of the "Intermediate Syndrome" cases are OPIDN waits upon future
study,
b) No-Effect Levels; More dose/response studies are needed to establish
quantitative relationships between organophosphate levels, extent of
muscle damage, type of organophosphate, and muscle and blood levels of
cholinesterases. With regard to the matter at hand, such studies are
needed on rats, quantifying cholinesterase levels and extent of muscle
damage for several selected organophosphates.
-------
The Committee agrees that clinical effects (as defined in section 3.2.1 of this
report) associated with exposure to cholinesterase inhibitors can be used to establish
benchmark doses and reference doses, but not without also considering other relevant
toxicological information. In addition, the clinical effects must be reproducible,
quantifable and statistically significant, and exhibit a logical cause-effect, dose-response
relationship..
Also, some clinical changes can occur after exposure to cholinesterase inhibitors,
as is the case with other toxicants, which may or may not be considered as adverse
effects. The problems associated with using clinical signs for the establishment of
reference doses for cholinesterase inhibitors, especially when the clinical endpoints
cannot undoubtedly be associated with cholinesterase inhibitor exposure (e.g., a
transient change in blood pressure) introduce additional uncertainty to the risk assess-
ment process. Given these reasons, as well as other related issues addressed in detail
in this report, the Committee finds that the sole use of clinical signs of toxicity (especial-;
ly'in long-term exposure studies) or perhaps more importantly, the lack of such signs,
for the assessment of hazard and determination of reference or benchmark doses may
not be justified. The inclusion of biochemical data regarding cholinesterase inhibition in
conjunction with these signs and symptoms is considered essential for the complete
hazard evaluation for these compounds.
There was full agreement among the Committee members that blood cholinester-
ase inhibition is a biomarker of exposure, and that data regarding inhibition of the blood
enzymes are often crucial supporting data for confirming exposures and corroborating
clinical signs. We recommend that the Agency's policy continue to include the use of
blood cholinesterase data in the risk assessment process, particularly in human studies
where cholinesterase data from the target tissues of most concern (i.e., brain and
peripheral nervous system) are unavailable.
The Committee noted several problems (detailed in the report) with the use of
cholinesterase inhibition in animal brain tissue as a means of defining hazard and
calculating benchmark or reference doses. Certain cholinesterase inhibitors are unable
to traverse the blood-brain barrier, yet these agents may produce severe neurotoxicity,
presumably through actions at peripheral sites. In these cases, measurement of CNS
AChE activity would be of little or no relevance. The Committee was concerned that, in
general, the EPA document did not give adequate weight to the problems associated
with the inhibition of peripheral nervous system cholinesterase. A major obstacle to
18
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[
resolving the relation between QNS cholinesterase inhibition and behavioral outcome is
the current inability to determine the most sensitive brain regions and the physiological
functions for which they are responsible. Resolution of this and other relevant issues
will be required to show the relationship between agent dose, extra-cellular concentra-
tion, sub-regional cholinesterase depletion and functional change in order to support the
use of brain cholinesterase inhibition data in the regulatory climate with confidence.
These issues not withstanding, there are nevertheless extant data for certain agents on
brain ChE inhibition in the presence or absence of corresponding data for the blood
enzyme. In the risk assessment process for those anticholinesterase chemicals or
metabolites known to cross the blood/brain barrier and to inhibit brain ChE, the Agency
may continue to consider brain ChE data if corresponding information on blood ChE
inhibition indicates the brain enzyme ",s a more sensitive marker of toxicity.
The Committee could not provide a simple yes or, no answer to the issue of
using RBC cholinesterase inhibition data by itself (i.e., in the absence of clinical symp-.
toms) for risk assessment purposes. Extant animal studies provide conflicting evidence.
Although broadly applicable correlations between blood ChE and brain ChE inhibition
have yet to be established, there are strong theoretical reasons why the correlation
should exist for agents that traverse the blood-brain barrier. However, even if this is
difficult to demonstrate empirically, it is nevertheless true, for such agents, that blood
enzyme inhibition precedes {and therefore predicts) brain enzyme inhibition. Given the
absence of any other test of proven greater reliability, measurement of plasma and red
blood cell cholinesterase inhibition should be retained in ERA'S policy, not as the sole
basis for standard setting, but in conjunction with clinical signs and symptoms.
--Depending on the timing of the measurement, cholinesterase inhibition in the absence
of clinical signs and symptoms may predict impending adverse clinical effects. The
Committee recommends that EPA evaluate the possibility that an RfD could be set
based on clinical signs and symptoms that would be associated with a significant
inhibition of cholinesterase occurring at a specified dose. This question could be exam-
ined with current data sets available to the Agency.
The issue of using red blood cell cholinesterase inhibition for risk assessment, as
posed originally in the Charge to the Committee, was difficult to address because it
appeared to be a circular argument. If there are data on neurotoxicity to evaluate
directly the dose response relationship for the chemical of interest and to determine the
RfD or benchmark dose based on clinical endpoints, why go to a surrogate? However,
at the Committee's meeting the EPA clarified this to suggest that these factors - clinical
19
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effects and choiinesterase Inhibition - would be used in conjunction with each other
and that it was not simply a replacement of the clinical data with the erythrocyte
choiinesterase inhibition data. This is, of course, the main point that was made by the
panel, i.e., analysis of all the appropriate data. Thus, there is general agreement on the
basic usefulness of the erythrocyte choiinesterase data in this context.
The Committee also recommends that EPA continue research aimed at examin-
ing carefully the con-elation of clinical signs and erythrocyte choiinesterase inhibition,
particularly regarding the correlations with respect to dose, time, and linearity. Such
studies will be very important in future decisions on the usefulness of erythrocyte
choiinesterase inhibition in regulatory decision-making.
20
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5. GLOSSARY
AChE Acetylcholinesterase
ChE Cholinesterase
ChEl Cholinesterase Inhibition
CNS Central Nervous System
DFP Diisopropyl Fluoro Phosphate
EGTA Ethylene Glycoi-bis (beta-aminoethyl ether) N.N.N'.N', Tetra acetic Acad
EPA Environmental Protection Agency
NAS National Academy of Science
OP Organophosphorous
OPIDN Organophosphate-induced Delayed Neuropathy
PChE Pseudo Cholinesterase
RBC Red Blood Cell
RfD Reference Dose
SAB Science Advisory Board
SAP Science Advisory Panel
SLUD Salivation, Lacrimation, Urination and Defecation
G-1
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