EPA
U.S. Environmental
Protection Agency
Washington, DC
EP A~S AB-EC- 90-014
Report of theSAB/SAP Joint Study
Group on Cholinesterase
i
Review of Cholinesterase
Inhibition And Its Effects
A SCIENCE ADVISORY BOARD REPORT
May, 1990

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D, S. EHVIRONMEMTM, PROTECTION AGENCY
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,
henee, 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 pro-
ducts constitute a recommendation for use.
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 204SO
EPA-SAB-EC-90-014
May 23, 1990
of»i« flt
THE »DM|NUTniTOB
Honorable William K. Reilly
Administrator
U.S. Environmental Protection Agency
401 M Street, S.w.
Washington, d.c. 204S0
subject} Joint Science Advisory Board/Scientific Advisory Panel's
review of Cholinesterase Inhibition and Its Effects
Dear Mr. Reilly;
Inhibition of cholinesterase enzyme activity is a mechanism
by which an important class of insecticides exerts its effects.
Compounds of this class exert toxic effects in mammals, including
humans. Because of their widespread application in agriculture
they arouse concerns about hazards to agricultural workers, and to
consumers as well, who may be exposed to residues in or on
agricultural products. EPA is responsible for setting standards
for general population exposure to cholinesterase (ChE) inhibitors.
To do so, it must evaluate several risk assessment issues whose
resolution poses numerous complex and difficult questions.
Agency scientists and consultants have assembled and surveyed
the available information and offered a set of provisional
recommendations. The Science Advisory Board (SAB) and the
Scientific Advisory Panel (SAP) were asked* to review these
recommendations, which addressed the major issues confronting the
Agency. The SAB/SAP Joint study Group was formed, and subsequently
met on September 27, 1989 in Crystal City, Virginia where it was
briefed by Agency staff and received comments from members of the
public.
The Joint Study Group wishes to recognize the immense amount
of work devoted to this effort by the Agency and commends EPA for
the clarity with which it has characterized the core issues.
Although we feel that a clear and full resolution of these issues
can not be accomplished at this time, we welcome the opportunity

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to offer our comments on, and to extend the recommendations of, the
original EPA Risk Assessment Forum Technical panel, which reviewed
and discussed these issues in a 1988 report, Cholinesterase
Inhibition as an Indicator of Adverse Toxicolocrical Effect.
The issues posed to the Joint Study Group and the Group's
responses are:
1.	Is chE inhibition (ChEI) in blood (plasma and/or
erythrocytes) or brain an adverse effect?
The Joint Group expressed doubt about the validity of plasma and
red blood cell (RBC) cholinesterase inhibition (ChEI) as indicators
of toxicity. Members pointed out that these measures could not be
correlated with recognized adverse effects. In fact, such measures
may indicate that the organism's defenses against toxicity are.
intact.
2.	What are the appropriate uncertainty factors for
estimating reference doses (RfDs)?
The Group did not propose appropriate uncertainty factors
(UP)r rather it proposed an improvement over the current approach.
The improvement called for fitting a tolerance distribution model
to the ChEI-dose response data to determine the lower confidence
level of the dose at which the change in ChEI level is just
statistically significant, and to adjust that dose level with the
UF, The of should be chosen with the understanding that the lower
confidence level dose already accounts, in some degree, for
intraspecies variations.
3.	Is the developing organism at special risk?
The Group agrees with the Technical Panel conclusion that the
evidence for enhanced susceptibility is ambiguous*
4.	Can ChEI be considered a valid biomarKer of exposure?
The Group expressed unanimity that ChEI is a biomarker for
exposure and, whether in blood or nervous system tissue, indicates
absorption of the enzyme-inhibiting agent.

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5. Related issues involving quantitative models, inter-species
differences# neurobehavioral measures, and statistical
variability.
The Group concluded that for interspecies predictions, rats appear
to be no less accurate than dogs. The crucial element in
extrapolation to humans is the similarity in processes determining
ChEl levels; these, inl turn, depend upon many interacting
processes. Complete duplication of human processes in other
species is unlikely. Models and other issues are discussed in the
report.
The Joint study Group recommends research to enhance the basis
for risk evaluation. The research should include exploitation of
currently available data, work on techniques for detecting
organophosphate exposure, study of the neurological consequences
of chEl, structure-activity studies, and should address ecological
concerns and other subjects noted in the report.
We appreciate the opportunity to review these issues, and look
forward to your response.

Dr. Bernard Weiss, Chairman
Joint SAB/SAP Study Group

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United States Environmental Protection Agency-
Science Advisory Board/Scientific Advisory Panel
CHAIRMAN"
Dr. Bernard Weiss
Division of Toxicology
University of Rochester
School of Medicine
Members and Consultants
Dr. Zoltan Annau
The Johns Hopkins University
School of Hygiene & Public
Health
Dr. Leo Abood
Department of Pharmacology
University of Rochester
School of Medicine
Dr. Robert Anthony
Department of Fisheries and
Wildlife
Oregon State University
Dr. Gary Carlson
Department of Pharmacology
and Toxicology
Purdue University
Dr. Janice Chambers
Department of Biological
Sciences
Mississippi state University
Dr. Daniel Icobichon
Department of Pharmacology
and Therapeutics
McGill University
Dr. Herbert Lowndes
Department of Pharmacology
and Toxicology
College of Pharmacy
Rutgers University
Dr. Donald P. Morgan
Iowa City, Iowa
Dr. John 0*Donoghue
Health & Environment
Laboratories
Eastman Kodak
Dr. Gary Sprague
Smithkline Beecham
Philadelphia, PA
Dr. Barry w. Wilson
Department of Environmental
Toxicology
University of California
Dr. Ronald Wyzga
Electric Power Research
Institute
Executive Secretary
Mr. Bruce Jaeger
Scientific Advisory Panel
U.S. Environmental
Protection Agency
Washington, D.C. 20460
Executive Secretary
Mr. Samuel Rondberg
Science Advisory Board
U.S. Environmental
Protection Agency
Washington, D.C. 20460
Staff Secretary
Mary L. Winston
science Advisory Board
U. S. Environmental
Protection Agency
Washington, D.C. 20460
Director, Science Advisory
Board
Donald G. Barnes
U.S. Environmental
Protection Agency
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TAIL! OF CONTENTS
1.0 Executive Summary			 1
2.0 Background . . 		.	. . . 2
3.0 Charge to The SAB/SAP Joint Review Group ........ 4
3.1	Interpretation Of Choiinesterase Inhibition: An
Adverse Effect? ...... 	 ....	4
3.2	Recommended Uncertainty Factors For RfD
Estimation 		5
3.3	The Developing Organism: A Special Case? .....	5
3.4	Choiinesterase Inhibition: A Biomarker? 		5
3.5	Additional Issues ......... 		6
3.6	Research Needs				 . .	6
4,0 Detailed Findings 	 ...	6
4.1	ChEl As An Adverse effect		7
4.2	Uncertainty Factors for Calculating RfDs .....	8
4.3	Vulnerability of The developing organism to ChEl
Agents ......................	10
4.4	chll As A Valid Biological Marker of Exposure ...	11
4.5	Related Issues 		12
4.6	Research Needs . . 				13
4.6.1	Exploitation of currently Available Data . .	13
4.6.2	Detection of OP Exposure ..........	13
4.6.3	Neurobehavioral Consequences of ChE
Inhibition 			14
4.6.4	Sources of Individual Variability 		15
4.6.5	Temporal Focus for Studies		 . . .	15
4.6.6	Ecological Concerns				16
4.6.7	Structure-activity studies 	 ...	16
5.0 Recommendations 					16
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1.0 executing gumma r-y Inhibition of chol inesterase enzyme
activity is a mechanism by which an important class of insecticides
exerts its effects* Compounds of this class exert toxic effects
in mammals, including humans. Because of their widespread
application in agriculture they arouse health concerns, not only
about exposure of agricultural workers, but also about exposure of
consumers who may be exposed to minute residues in or on
agricultural products.
EPA is responsible for setting standards for general
population exposure to cholinesterase (chs) inhibitors. To do so,
it must evaluate several risk assessment issues whose resolution
poses numerous complex and difficult questions. Many of the
difficulties stem from the lack of crucial data, so that answers
to those questions necessarily are based on scientific judgment.
Agency scientists and consultants have assembled and surveyed the
available information and offered a set of provisional
recommendations. The Science Advisory Board/Scientific Advisory
Panel (SAB/SAP) Joint Study Group was asked to review these
recommendations, which were framed to respond to the major issues
confronting the Agency, The Joint Study Group recognizes the
immense amount of work devoted to this effort and commends epa for
the clarity with which it has characterized the core issues.
Although we feel that a clear and full resolution of these issues
cannot be accomplished at this time, we welcomed the opportunity
to offer our comments on, and to extend the recommendations of the
original EPA Risk Assessment Forum Technical, Panel, which reviewed
and discussed these issues in a 1988 report, Choiinesterase
Inhibition as an Indicator of Adverse Toxicoloarical Effect.
The issues posed to the Joint Study Group were described.as
follows;
a)	Is ChE inhibition (chEI) in blood (plasma and/or
erythrocytes) or brain an adverse effect?
b)	What are the appropriate uncertainty factors for
estimating reference doses (RfDs)?
c)	Is the developing organism at special risk?
d)	Can chEI be considered a valid bioaarkar of exposure?
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e) How should the following related issues be treated?
1.	Other quantitative models to assess dose-
response
2.	Species differences and surrogates for humans
3.	Neurofaehavioral measures of ChEI
4.	Appropriate test methods for ChEI
5.	Statistical Procedures pertinent to variability
The Joint Study Group found several of the issues, as posed,
almost inseparable, so that its responses to each individually also
bear upon the others.
First, the Group expressed doubt about the validity of plasma
and red blood cell (RBC) ChEI as indicators of toxicity- In
addressing the issue of vulnerability of the developing organism,
we note that, although maternal exposure to ChEI agents may also
expose the fetus, and alter brain Chi levels, the consequences for
nervous system development and postnatal function have received no
more than minimal study. The Group agrees with the Technical Panel
conclusion that the evidence for enhanced developmental
susceptibility is ambiguous. The Joint Study Group expressed
unanimity that ChEI is a biomarker for exposure, and, whether in
blood or nervous tissue, indicates absorption. The relationship
between degree of ChEI and toxicity remains unclear, and
correlations between exposure indices and neurotoxic manifestations
tend to be weak.
2.0 Background Over the past several years, the Agency has sought
to develop and implement risk assessment methodologies through a
consensus-building process reflecting participation by scientists
in all parts of the Agency, and to apply these methodologies in a
consistent manner across all EPA program offices and regions. The
primary institutional vehicles to accomplish this goal are the Risk
Assessment Forum, administratively housed in the Office of Research
and Development, and the Risk Assessment Council, an assembly of
senior EPA managers chaired by the Deputy Administrator of the
Agency.
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Both of these organizations have been concerned with issues
relating to cholinesterase inhibition, since it seems to represent
an important endpoint for assessing exposure and may have potential
for assessing health effects of certain types of, agents (e.g.,
monitoring the exposure of human populations to organophosphate
pesticides). Measurements of blood cholinesterase are "conducted
as a surrogate for the observation of changes occurring in the
central and peripheral somatic and autonomic nervous systems* This
is necessary because of the technical difficulties inherent in
attempting to measure directly these effects in tissues innervated
by cholinergic neurons. Exposure to ChE inhibiting agents can be
inferred from blood ChEI because most such compounds cause an
irreversible inhibition with only art extremely slow reactivation,
Ensyrae inhibition by most carbamates is so readily reversible
that its usefulness as an indicator of absorption "of this pesticide
class is dubious except under controlled experimental conditions.
Some carbamates and thiocarbaiuates, however, can produce ChEI
evoking clinical manifestations similar to those produced by QPs.
Interpretation of the biological significance of
cholinesterase inhibition and its use in assessing human risk has
been hampered by the lack of a consensus as to what level of which
kind of cholinesterase inhibition {i.e. plasma, RBC, brain) is
associated with overt' toxicity. Extensive cholinesterase
inhibition may be observed in the absence of any distinct signs of
overt toxicity. A critical or "threshold" level of enzyme
inhibition below which there is no biologically relevant or truly
adverse health effect has not been established for any of the
possible loci cited above. This may, in part, be attributable to
our insufficient knowledge about the effects of cholinesterase
inhibition on various neurotransmitter or other physiological
systems or it may be a consequence of biological variability. Use
of animal data and uncertainties associated with cross-species
extrapolation pose additional problems in assessing human risk.
Because there were several organizations within the Agency
developing risk assessment and/or regulatory positions on
substances shown to produce cholinesterase inhibition (ChEI), a
Technical Panel was formed in 1988, under the auspices of the Risk
Assessment Forum, to address the relevant "issues and provide a
consistent approach. The Technical Panel was charged with
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reviewing the relevant literature and preparing a report which
would identify the appropriate uncertainty factor(s) to employ in
the quantitative assessment of this endpoint (e.g. the derivation
of a Reference Dose) and with assessing the consequences to the
developing organism of pre- and perinatal exposure to
cholinesterase inhibitors.
3-0 charge to Tha SAB/SAP Joint Review Group The Forum Technical
Panel, and a subsequent ,Colloquium, made a number of specific
recommendations and developed a set of "working principles" which
can be applied to the interpretation and use of cholinesterase
data. These "working principles" are outlined below. The SAB/SAP
Joint Panel was asked to evaluate the scientific bases for these
principles and their relevance to assessing human risk. The Risk
Assessment Forum and the Risk Assessment Council were particularly
interested in the Joint Group"s views on four major issues which
devolve from the Technical Panel report and are discussed below.
In addition, two other issues, not specifically addressed in the
Technical Panel report were also posed to the Joint Study Group for
consideration. The issues follow below?
3•1 interpretation of cholinesterase inhibition? An adverse
Effect? A major focus of the Technical Panel report reviewed by
the Joint Study Group was on the interpretation of ChE inhibition
and its relevance to adverse outcomes. The Panel reached the
following conclusions;
a)	Although correlation of CHE inhibition with the nature
and severity of an overt response is difficult to predict
and is dependent upon many variables, statistically
significant inhibition of cholinesterase (i.e., plasma,
RBC, or CNS) is usually considered a potentially adverse
effect.
b)	Statistically significant inhibition of cholinesterase
in the CNS should always be considered an adverse effect.
c)	Statistically significant plasma or RBC cholinesterase
inhibition should be considered biologically significant
unless an exception can be made on a case^by-case basis as
reflected by such factors as dose-response relationships,
comparative pharmacokinetics and elements of study design.
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The Joint Panel was asked to comment on the validity of the
recommendations and findings above, and suggest any revisions it
thought appropriate.
3•2 Recommended Uncertainty Pactora For RfD Estimation The
Technical Panel recommended use of the following uncertainty
factors for estimation of RfDsi
a)	A factor of 10 for a NOAEL (No Observed Adverse Effects
Level) based upon in vivo human RBC AChE or plasma BuChE
data.
b)	A factor of 100 for a NOAEL based upon animal brain
AChE, RBC AChE, or plasma BuChE data.
The SAB/SAP study Group was asked to comment on the merits
of Recommendations a") and b). In addition, the Panel was invited
to comment on interspecies extrapolation, specifically concerning
use of the dog, and as to whether or not a 10-fold uncertainty
factor is adequate to account for human variability.
3.3	The -Developing oraaai3gi A Special ease? Although the
consequences of exposure to cholinesterase inhibitors during
development remain, in large measure, unknown, the developing
organism may be especially vulnerable to the effects of Chi
inhibitors. The Technical Panel noted that while some ChE
inhibitors can cause alterations in ChE levels in the developing
brain and peripheral nervous system, a direct relationship between
decreased ChE activity and abnormal development has not been
established. The Technical Panel also noted that this does not
preclude special concern for the developing organism- The Panel
recommended additional research to determine whether an equivalent
level of Chi inhibition places the developing organism at risk for
more severe effects than would occur in the adult.
The views of the Joint Study Group were sought to assess the
merits of the Colloquium Panel1s suggestions, given current
scientific understanding of the developing nervous system and the
potential effects of agents which may inhibit cholinesterase
levels*
3.4	cholinesterase Inhibition? A Biomarker? Cholinesterase
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enzymes are marker enzymes which reflect exposure to, and the
degree of absorption of, chE inhibitors and, in addition, may
reflect impact on biological systems. ChE inhibition should,
therefore, be addressed as a biomarker of both exposure and also
as of a potential neurological effect. The SAB/SAP Panel was asked
to comment on this issue.
3*5 Additional issues There were a number of other issues which
were not specifically addressed or which received less attention
in the Technical Panel Report. The Joint Group was asked to
comment on the following questions:
a)	Are there other quantitative approaches (e.g.,
biologically based or statistical models) which can
provide a better assessment of dose response than the RfD?
b)	If the RfD method is used, are there specific classes
of agents to which the dog (or other species such as the
non-human primate) may be more sensitive and therefore
warrant a reduced uncertainty factor (e.g, 10)?
c)	Are there other neurobehavioral measures which have
been found to be sensitive and reliable measures of effects
associated with cholinesterase inhibition?
d)	If the operational criteria for adversity is
statistical inhibition of ChE, study conduct will be of
critical importance. Is there sufficient scientific
consensus as to the most appropriate test methods to
utilize?
e)	Given the variability of cholinesterase levels observed
even in the absence of exposure to cholinesterase
inhibitors, are there statistical procedures that the Study
Group would recommend to deal with this problem?
3-6	Research Heeds The Technical Panel identified a number of
research needs. Comment was invited on the merits of the
identified research needs,- identification of appropriate additional
areas of research was also requested.
4-0	Detailed Findings This report is not a detailed review of the
available literature, but a response to the recommendations of the
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Technical Panel. Literature citations' are provided 'sparingly, and
only to amplify certain singular points.
4.1 ChEI aa &n Adverse effect The relevant questions addressed
here are: Is a "statistically significant" reduction in ChE a
criterion for establishing a NOEL or NOAEL?? Is a reduction of
brain GhE, without exception, an adverse effect?? What role should
be played by reversibility? Although the Technical Panel
recommended reliance on statistical significance, it also noted
that it is difficult to predict the correlation of ChEI with the
nature and severity of an overt response which depends on many
variables.
The Group expressed doubt about the validity of plasma and
red blood cell (RBC) ChEI as indicators of toxicity. Members
pointed out that these measures could not be correlated with
recognized adverse effects. In fact, such measures may indicate
that the organism's defenses against toxicity are intact? perhaps
this is the reason blood enzymes sometimes can be severely
depressed in the absence of signs of poisoning. Another,
explanation for the apparent lack of toxic signs, despite
substantial ChEI, is the high turnover rate for AChE, amounting to
300,000 moles/minute for ACh. Under such circumstances, a high
level of inhibition may not markedly influence the rate of ACh
hydrolysis. In addition, the body contains many esterases,
typically in abundance, offering many possibilities for interaction
without adverse effects. It is difficult to argue, moreover, that
reduced brain ChE, given the widespreaid distribution of chEs, is
adverse in itself although it should be taken more seriously than
ChEI in blood.. Even so, the large functional reserve in brain
provides an intrinsic protective mechanism, and compensatory
processes are also operative. Receptor populations undergo up- and
down-regulation, and normal behavior can occur in animals
concurrently experiencing marked brain ChEI.
If statistically significant (or, alternatively, 20%) ChEI is
lacking in cogency and validity as a measure of toxicity, what
alternatives might be pursued? The one making the most scientific
sense is to define toxicity on the basis of functional (e.g.,
behavioral, electrophysiological) or morphological indices. Then,
proceed to determine what degree of ChEI, in a critical tissue,
predicts the neurotoxic response,' and set the exposure level
accordingly. Such an approach is common in toxicology.
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Reliance on statistical significance is a separate issue to
be discussed later. Note here that Group members expressed
considerable skepticism about the validity of such an approach.
4.2 Uncertainty Factors for Calculating RfDs This question
divides into many ramifications, some of them deriving from current
Agency practices. In essence, the Technical Panel proposed to
compute a NOAEL or NOEL on the basis of statistically significant
chEI (blood or brain), then apply a specified uncertainty factor
(UF) large enough to include a NOAEL derived from the most
sensitive members of the human population. The question posed in
section 4.1 above, according to the majority of the Joint Group,
is premature because we view the "statistically significant"
criterion as seriously flawed. The objections arise from two
problems? first, with the intrinsic statistical assumptions? and
second, as described above, the absence of a firm criterion founded
in toxicology.
Risk assessment is acknowledged as the basis for EPA actions
and initiatives. The approach advocated by the Technical Panel,
however, is not based on risk estimation. It is, instead, a
convenient management tool. It does not incorporate the
fundamental measure of toxicology, the dose-consequence
relationship. It does not take into account statistical power,
dose spacing, variability, or trends. Most notably, it is an
arbitrary captive of the sensitivity of a particular method and
its variability. As the SAB has noted repeatedly, the established
NOAEL/UF approach may reward low sensitivity and expanded
variability because these properties lead to inflated NOAELs.
For example, an experiment with a small number of animals in
each dose group might yield a NOAEL significantly higher than a
loaf.l based on an experiment with a larger number of animals in
each dose group. If ChEI variability (which seems high in many
circumstances) is broadened even more by aberrant observations,
additional NOAEL inflation may result.
The most attractive alternative to the current approach would
involve the estimation of a dose-response surface that relates
exposure to both incidence and severity of nerotoxicity, and that,
given appropriate tolerance distribution models and a specified
ChEI level, would yield actual estimates of the risk of adverse
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effects. Unfortunately, available information and data do not
currently allow such an approach, however. An improvement over the
current approach however, would be to estimate a dose-activity
relationship by fitting a dose-tolerance model such as the probit
model or expressions such as y = a + Bx or y = a + Blog x, or
another function, where x = dose or exposure, and y « ChEl activity
level in selected tissues such as blood and brain, if these
reasonably approximate the probit model over the range of
extrapolation. For estimated values of a and b, determine the
value for x at which the value of y becomes significantly different
from zero, or is associated with 20% chEI in blood. The latter
(20% ChEI) is most closely associated with the current approach,
the former with the proposed approach. The application of UFs to
this dose level, which takes into account the total dose-response
data, can be combined with statistical power calculations. The UF
can be applied to the lower statistical confidence limit of the
estimate of dose (associated with a statistically significant
change in activity level or with a 20% ChEI) to penalize small
samples or large variability. Or the OF could be applied to the
dose inducing any alternate response, such as, say, 30% ChEI.
Even such statistically attractive options founder without
cogent toxicological endpoints, as noted earlier. The basis for
statistical modeling should be appropriate functional measures
based on both the central and peripheral nervous systems. Markers
of exposure (see 4.4 below), even with the current risk assessment
protocol, are interpretable only with parallel indicators of
toxicity. Using one index to serve as both a measure of exposure
and a measure of toxicity poses problems that are highlighted by
the risk assessment process.
The appropriate UFs, then, have to be seen in that context.
Large UFs seem generally unwarranted because of inherent margins
of safety between ChEI and detectable neurotoxicity? that is,
substantial ChEI may be measured in the absence of detectably
adverse functional or morphological effects. However, given our
elementary grasp of the correlations between measures of ChEI in
blood and neurotoxicity, we should be wary from another direction
about how we apply UFs? they are not applied to the appropriate
endpoint. We need to define more direct endpoints. Behavioral
measures, for example, may offer useful indicators because they can
be used for monitoring adverse effects and, also, dispense with the
intervening, indirect variable of ChEI. With both neurotoxicity
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and ChEI measures available concurrently,, together with dose-
response (effect) information, the Agency can proceed to apply UFs
on a much more rational basis,
New experiments on adult humans, with proper safeguards, and
a thorough review of the available data, might be considered for
some purposes* Volunteers are used by some manufacturers, and of
course, Food and Drug Administration protocols make provisions for
observations in humans during drug development. The doses chosen,
of course, would be very low, but could provide guidance about the
dose sufficient to inhibit ChE in blood. Such observations,
however, would provide minimal information about possible chronic
effects and potential hazard to the fetus.
4.3 vulnerability of The developing Organism to ChEI &aent3
For many environmental agents, the developing brain offers an
especially vulnerable target. Lead, methylmercury, ethanol, and
FCBs are among the agents identified as posing enhanced risks to
the developing brain. Not only are nervous system elements
undergoing rapid growth, an important dimension of vulnerability,
but the immature blood-brain barrier permits entrance of agents
that ordinarily would be excluded*Also, the drug-metabolizing
enzymes that later function to detoxify many chemicals are not yet
available. At the same time, this biochemical immaturity could
also afford some protection against OPs such as the
phashorothionate insecticides. These compounds represent a
subclass that requires metabolic activation by cytochrome P-450
dependent monooxygenases for conversion to the final anti-chE
metabolite.
For other agents inhibiting ChE, the available information is
less compelling. Although maternal exposure to ChEI agents may
also expose the fetus, and alter brain ChE levels, the consequences
for nervous system development and postnatal function have received
no more than minimal study. Even the comparative sensitivity of
fetal and adult brain, as measured by ChEI, is the subject of
conflicting findings. But conflicts would be the predicted outcome
of assessments that differ in agent, dose, and timing of exposure
and stage of development. The Group agrees with the Technical
Panel conclusion that the evidence for enhanced developmental
susceptibility is ambiguous.
The Joint Study Group, however, believes that the Agency
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should also consider the evidence implicating advanced age as a
period of enhanced vulnerability to ChEI agents. Research
indicates, for example, that older rats exhibit more pronounced
toxic syndromes than young rats, "Developmental toxicity" should
not be confined to early development, especially given the •
incidence of neurodegenerative disorders in our aging population.
A more scientifically rigorous point of view is that variations in
susceptibility occur through the total life-span.
4.4 ChEI As A Valid Biological Marker of Exposure The Joint Study ,
Group expressed unanimity on the answer to this question, and
agreed that ChEI provides an index of exposure, or more
specifically, absorption, In fact, it is exactly the lack of
correspondence between exposure markers and neurotoxic measures
that fosters the Group1s skepticism about collapsing them into a
single index. That is, ChEI, whether in blood or in nervous
tissue, indicates exposure rather than toxicity. The amount of
observable inhibition, in fact, could just as well be interpreted
as* a measure of detoxification'efficiency, to the extent that it
reflects scavenging and inactivation of the agent. Although, with
some carbamates, substantial ChEI and serious toxicity may occur
concurrently, with others,, the rapid reactivation of ChE makes ChEI
less useful as a measure of exposure. Reactivation, however, is
a process that depends on both rate of inhibition and rate of
recovery. With these agents, also, plasma ChE activity may be
reduced to undetectable levels without causing lethal outcomes, or
even signs of poisoning.
In accepting the validity of ChEI as a measure of exposure,
the Group also remains cautious about direct quantitative
interpretation. Plasma (butyryl) ChE assays are not standardized,
but, instead, are conducted with a variety of methods, leading to
a highly variable literature. Perhaps standardization could be
expedited by adopting commercial kits for spectrophotometric
assays. However, because most contain proprietary buffers, their
constituents cannot be reproduced and tested in research
laboratories. Also, for detection of exposure at environmental
levels, if such data are deemed necessary, High Pressure Liquid
chromatography (HPLC) and Mass Spectroscopy (MS) may be required.
These techniques possess the advantage that they can detect
hydrolytic products at OP dose'levels insufficient to inhibit Chi.
Assays of brain Chi, as noted above, arouse additional questions.
Should the various forms, at least those so far identified, be
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assayed individually?	Should anatomical distribution be
considered, and regional brain analyses performed? The SAB/SAP
Joint Study Group endorses the need for clearly defined standard
procedures for all tissues and views as feasible, even
advantageous, a protocol that included several methods, each
contributing a different facet of information.
Underlying questions such as these are, again, the
undefendable connections between blood ChE, brain ChE and
neurotoxicity.. With some agents, Chll of 20% in blood may be
associated with toxic manifestations* With others 90% chEI is
required. Predictions based on structural chemical features might
help in the future, but are still not reliable enough to screen
compounds that evoke a unique response. Although i&onMss consistent
relationships might arise by relying on erythrocyte rather than
plasma ChE in assays of many OPs, the measurements are more
laborious and difficult. Furthermore, even the correlations
between brain ChEI and functional disturbances may be abysmally low
within a single experiment, as in the recent report by Jimmersoh
et al1, so that generalizations about effects and biochemical
lesions are of limited accuracy. In essence, then, Chll serves
primarily as a biomarker of exposure, not of neurotoxicity? the
latter is defined independently.
4.5 Belated Issues Most categories of what were classified as
related issues have already been discussed. Species variations
remain a fulcrum of debate. They were addressed by the Joint Study
Group and by the Technical Panel from the standpoint of a suitable
surrogate species for humans. At least in the past, some
investigators have advocated the dog as providing the closest
correspondence with humans. Few data support such a position, even
though dog data are routinely collected for pesticides. For
interspecies predictions# however# rats appear no less accurate
(nor more innacurate) than dogs. The crucial element in
extrapolation to humans is the similarity in processes determining
chl levels and these, in turn, depend upon many interacting
processes. They include bioactivation and degradation of liver
enzymes, breakdown by plasma enzymes, and absorption and
distribution parameters. It is unlikely that any combination of
such processes found in the human body will be duplicated
'Toxicology 57:241-254, 1989
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completely in any other species,
The questions of quantitative models to assess dose response,
and statistical procedures pertinent to variability were included
in the discussion of UFs; appropriate test" methods for ChEI were
discussed above with section 3.4? and the issue of neurobebavioral
methods will be discussed under Research Needs and Recommendations
(sections 3-S and 3.7 below)*
4.6 Research Needs Risk assessment is ultimately an extension
of, and an extrapolation from, scientific data. Because both
inflated and inadequate risk estimates engender costs to society,
research to enhance the basis for risk evaluation is a sound
investment. The recommendations for research support listed below
are designed to support such an enhancement. They are not intended
to delay actions by the Agency on the questions posed to the Joint
Study Group. The Group recognizes that perfection in regulatory
decisions will always remain an elusive target.
4.6.1	Exploitation of currently Available Data Existing data on
correlations between ChEI and manifestations of toxicity should be
examined more thoroughly. An enormous amount of information,
especially on humans, may be available from classified military
files that probably could safely be declassified. The Chi data
base generated by industry research, although unavailable to the
general public, could also be surveyed by EPA scientists, as could
the Agency's own data from study populations such as applicators,
formulators, and others. Both sources of information would
amplify, for example, the relative contributions of intra- and
interindividual variability.
4.6.2	Detection of OP Exposure New .techniques for detecting op
exposure are under investigation, e.g., transdermal patches
permitting the analysis of parent OP compounds and their metabolic
products in tissue fluids. An expansion of this research, which
could also profit from making use of exposed agricultural workers,
would directly benefit the Agency. Leukocytes and other blood
elements contain a variety of neurotransmitter receptors, including
those mediating cholinergic function. Receptor assays based on
these blood cells might provide another means of detecting antiChE
exposure. Direct measures of ChEI, however, remain the most
dependable estimates.
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4.6.3 wetirobehavioral Consequences of chE Inhibition Although the
neurobehavioral consequences of ChEI have been the focus of many
publications, immense gaps in knowledge remain. These take several
forms;
a)	We possess inadequate information about the long-term
consequences of exposure, especially at levels insufficient
to elicit overt signs of poisoning. For example, are there
subtle adverse effects associated with chronic exposure
that are not severe enough to induce clinical signs? some
studies indicate that psychological testing can uncover
such corollaries2. Are there forms of what has been called
silent toxicity, such as aberrant responses to certain
drugs? Published data suggest this to be an important
question3. Are there effects remote in time from early
developmental exposure? Findings based upon many other
classes of toxicants and drugs underscore the need to
explore this question in comprehensive fashion.
b)	To what degree is acute poisoning reversible? Is there
subtle neurobehavioral impairment that lingers once ChE
levels return to normal and the acute symptoms subside?
In part, such a question arises from observations of
workers exposed in the past to high levels of OPs during
accidents in the manufacture or field application of such
agents for chemical warfare , and in part from animal
studies documenting lesions following exposure. For both
chronic exposures and acute aftermaths, a detailed series
of laboratory investigations on processes such as memory,
because of their connection with cholinergic neurotrans-
mission, is warranted. A recent paper by Savage jgt al,
which noted impaired psychological test performance long
after acute poisoning episodes, supports the need to ask
such questions5.
zLevin et al, Arch, Gen. Psychiat* 33:225, 1976
3See Bignami et al, Fund. Appl. Toxicol. S:S213-S224, 1985
4Duffy e£ al, Neurotoxicology It667, 1980? Whorton and
obrinsky, j. Toxicol. Environ. Health 11:347, 1983.
5Arch. Environ. Health 43:38-45, 1988
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(The next two items not only embody research needs, but are also
important components of standard setting,)
c)	Given the lengthy history of anti-ChE agents/ the lack
of information on the correlation between central and
peripheral nervous system effects and ChEI is surprising.
For example, are the temporal properties similar? Might
peripheral nervous system ChEI and associated functional
measures better reflect acute toxicity?
d)	Cholinergic mechanisms in the brain do not act in
isolation. Numerous interactions between cholinergic and
other neurotransmitter and neuromodulator systems are now
recognized, as is the coupling of receptors with second
messengers and other steps in the chain of events by which
neurotransmission occurs. How do these entwined mechanisms
modify or control the action of anti-ChE agents?
e)	Measurement of whole-brain ChE, especially given the.
many varieties of esterases present in brain, could prove
misleading and, in addition, represent one of the sources
of the low correlations with functional endpoints.
Regional analyses, based upon biochemical measures,
receptor assays, and, possibly, histochemistry, when
correlated with specific functional endpoints, could help
resolve several puzzling discrepancies* such research
would also help clarify the contributions of various
cholinergic pathways in the brain whose functional roles
are not well understood.
4-6** sources of individual Variability The causes of
variability, although mostly obscure at present, surely arise, in
part, from genetic predispositions. At least one phenotypic
variant of butyrlChE has been identified that increases
vulnerability to antiChE agents* The defective gene underwent a
100-fold amplification in a farmer chronically exposed to an op
insecticide6. Current techniques of molecular biology can be used
to identify other variants whose bearers may be at greater risk.
6See Prody al* *NAS 86;690-694, 1989
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4.6.5	Temporal Focu3 for studies The total life cycle, not just
early development, should be examined for relative vulnerability
to ChEI and its consequences, especially because of the association
between cholinergic function and certain facets of nerodegen-
erative disease,
4.6.6	Ecological Concerns Given the growing concern within the
Agency over ecological consequences and ecological risk
assessments, the impact of antichE agents on wild populations
requires careful study. One example; minimally impaired alertness
or motor function in a specific wild animal population might result
in greater losses to predators or to accidents, and alter a large
range of ecological inter-relationships and balances.
4.6.7	Structure-activity studies These studies, although expen-
sive, should be expanded in conjunction with the other research
needs described above.
5*0 Recommendations The Joint Study Group is distinctly aware of
EPA's responsibilities and the dilemmas posed by lacunae in those
data sets upon which reasonable risk reduction policies must be
founded. The Group also recognizes EPA's eagerness to move beyond
current, often improvisational practices. At the same time, the
members of the Group remain unconvinced by the Technical Panel's
arguments for changing these current practices, imperfect as they
are, and substituting another batch of equally unsatisfactory
criteria.
Four recommendations addressing this situation arise from the
Joint study Group's deliberations;
a) Base the criteria for adverse effects upon adverse effects.
That is, define an adverse effect on the basis of functional
(behavioral, electrophysiological) measures, accompanied,
where feasible, by morphological indices such as those
provided by both newer and established histochemical
techniques. Determine the associated biochemical indices
(plasma, rbc ChEI), and, for certain animal studies, brain
ChEI values. Include a range of exposure levels to encompass
minimal to marked neurotoxicity and preserve individual
organic data to facilitate statistical analysis. An ironic
counterpoint to the Joint Study Group agenda was the meeting,
on the following day, devoted to the opp/FXFRA Guidelines for
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Neurotoxicity. Such criteria should be implemented quickly
and a research, program adopted to insure continued improvement
in sensitivity and specificity.
b)	Replace the NOAEL/UF strategy with one based on the kinds
of dose-consequence data available from the information in (1)
above. Use the exposure data (such as plasma or RBC ChEI) in
company with the performance data to construct a specified
function derived from a recognized tolerance distribution, or,
better yet, a dose-response correlation surface (See Section
4.2), From these, distill a specified level of ChEI based
upon, say, a 10% decrement of performance. To the S5% lower
bound, attach a UF to yield the RfD.
c)	Reexamine structure-activity relationships on the basis of
the kinds of information sought in (1) above, A more complete
grasp of toxicity should permit higher correlations between
structural features and potential toxicity.
d)	Devote more support to peripheral nervous systems
assessments? although it poses some difficult technical
problems, using such assessments, based on correlations of
ChEI and functional measures, may offer an improved basis for
setting exposure standards.
Such an approach as is outlined above is also some distance
from what might be regarded as optimal, and also requires
scientific judgement because of variations in pharmacokinetic
parameters, metabolic transformation, and other associated factors.
It comes closer, however, to scientific rigor than what so far has
been implemented. It may also be more demanding of .both industry
and EPA resources than the current practices, but, given how much
both have invested because of other uncertainties, that assertion
could he argued. Finally, it is laboratory rather than debate
intensive, so that it is more likely to nurture improved practices
than what in essence is a minor change in the status quo and that
does not s&em to the Joint Study Group to offer a significant
advance in the protection of public health.
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