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Telephone: (202)401-0527
Item: 6022
Office of Pesticide Programs
Science Policy on
The Use of Data on
Cholinesterase Inhibition
for Risk Assessments
of Organophosphate and Carbamate
Pesticides
Prepared by William F. Sette, Ph.D. for the Office of Pesticide Programs
U.S. Environmental Protection Agency
October 27,1998

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Tolerance Reassessment Advisory Committee Review Draft1
ACKNOWLEDGMENTS
This policy statement is a product of an HED Cholinesterase work group that has met
over the last 2 years. It was chaired by William Burnam, and included Karl Baetcke, Brian
Dementi, Karen Hamernik, and William Sette. Kerry Dearfield, now in ORD, co-authored the
first four versions of this statement.
Other members of OPP who provided written comments on one or more drafts include
Stephanie Irene, Margaret Stasikowski, and Penelope Fenner-Crisp. Internal EPA peer reviewers
included Edward Ohanian of the Office of Water, Richard Hill of OPPTS, and Suzanne
McMaster, Stephanie Padilla, Robert MacPhail, and Hugh Tilson of ORD. We thank them for
their time and efforts at improving this proposal.
1 This draft is essentially the same as that presented for SAP review and dated April 30,
1997, except that certain editorial changes have been made, outdated general material removed
and an update added to reflect the Agency's presentation to the SAP in June, 1997.

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OPP ChE Policy
October 1998
I. Introduction
The purpose of this document is to describe a science policy in the Office of Pesticide
Programs (OPP) for the selection of appropriate endpoints for assessing potential risks to humans
exposed to cholinesterase inhibiting pesticides. In addition, it will propose a series of steps for
conducting risk characterizations for these chemicals.
Regulatory decision making in EPA is described in two major steps, risk assessment and
risk management. Risk assessments define the potential adverse health effects which may occur in
individuals or populations, while risk management weighs regulatory alternatives and integrates
the risk assessment with social, economic, and political concerns. (NAS, 1983).
Risk assessment contains four steps: hazard identification, dose response assessment,
exposure analysis, and risk characterization.
Risk assessments for systemic toxicity are generally based on the derivation of reference
doses (RfDs)2. Reference doses are calculated by dividing the no effect level (NOEL), or other
point of departure (e.g., an ED10 or other benchmark dose), usually for the most sensitive
endpoint, called the critical effect, by uncertainty factors (UF). These values are then compared
to the potential exposure levels in the risk characterization, which fully describes the nature and
extent of the risks posed, and the limitations and uncertainties involved.
Cholinesterase inhibition (ChEI) and cholinergic effects resulting from exposure to
organophosphate and carbamate pesticides have long been prominent effects of concern to the
USEPA in assessing environmental health risks. The Office of Pesticide Programs (OPP)
Reference Dose Tracking Report (3/28/97) lists over 50 Reference Doses for chronic exposure
alone based in whole or in part on cholinesterase inhibition. If we consider that acute dietary
exposure endpoints, short term and intermediate exposure endpoints are also generally needed for
risk assessments, and that ChEI is most often the critical effect for those exposure categories,
there are probably over 100 specific risk assessments based on this endpoint for roughly 50
chemicals.
For at least the last ten years, OPP has based these reference doses on the critical effects
2 A Reference dose is an estimate, with uncertainty spanning perhaps an order of
magnitude, of a daily exposure to human populations, including sensitive subgroups, that is likely
to be without appreciable risk of deleterious effects during a lifetime. RfD = NOEL/UF.
Application of uncertainty factors typically involves use of 10 for intra-species differences and 10
for inter-species extrapolation, a total of 100, standard factors for systemic toxicity. Comparable
evaluations for acute, short term, or intermediate exposures are derived in exactly the same way.
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of plasma, red blood cell, and brain ChE inhibition, or functional effects, and has used the same
uncertainty factors, e.g., 10 for inter-species and 10 for intra-species extrapolation, for all of those
endpoints. Further, OPP has used statistical significance, rather than a fixed generic difference
from baseline, e.g., 20% inhibition, as the primary, but not exclusive determinant of toxicological
significance. Both the use of uncertainty factors and this use of statistical significance are
consistent with EPA practice for most systemic toxicity endpoints.
A. Previous EPA Policy Proposals and SAP/SAB Reviews
There have been four major external groups in the last 10 years that have been asked by
EPA to review proposed science policy positions for this type of neurotoxic effect. One peer
review colloquium (US EPA, 1988) and two SAB/SAP meetings (US EPA, 1990, 1993)
considered EPA reports in this area. A SAP/SAB review in 1992 of a proposed reference dose on
aldicarb also addressed these issues (US EPA, 1992). Each of these groups provided somewhat
different recommendations, based in part on the different policy proposals, as well on their
differing judgments. The area of greatest divergence among these reports and in these
recommendations, involves the interpretation and use of blood measures of ChE inhibition for
deriving reference doses.
Two other federal groups have issued general guidelines on neurotoxicity risk assessment
that have mentioned this area.
Outside of EPA, two major regulatory groups have published their views of the use of this
type of neurotoxicity data. A group of experts, on behalf of the United Nations Environment
Programme, the International Labour Organisation, and the World Health Organization, have
described the evolution of positions in various groups meeting for those bodies on the use of
cholinesterase inhibition data (WHO, 1990). A manual of The Department of Pesticide
Regulations of the state of California also provides draft guidance on the use of cholinesterase
inhibition data in risk assessments for pesticides (Lewis, 1993). The sections quoted below are
focused on the discussions related to the interpretation and use of blood measures, and some
considerations regarding uncertainty factors and statistical analysis.
1. 1988 RAF Peer Review Colloquium
In 1988 a Peer Review Panel for the EPA Risk Assessment Forum reviewed an EPA
Technical Panel Report on Cholinesterase Inhibition as an Indication of Adverse Toxicologic
Effect (June 1988).
On the adversity of ChE inhibition the following consensus conclusion was reported:
"After considerable discussion, the Review Panel agreed with the conclusion of the
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Technical Panel that inhibition of brain ChEs is an adverse effect. Statistically significant
inhibition of blood ChEs is sufficient indication of a potential adverse biological effect, and
reversible effects should be taken as seriously as irreversible effects. Concern however, was
expressed that there are no data to support a simple correlation between a particular ChE
inhibition level and an observable biological effect."
In response to a question about what level of ChE inhibition constitutes toxicological
significance, they concluded: "In general, the Review Panel agreed with the Technical Panel's
conclusion that baseline, pre-exposure ChE levels provide the best basis for statistical
comparisons." (This was, in part, in contrast to use of a generic value of 20% as a threshold for
toxicological significance).
2.	1990 SAB/SAP Meeting
In 1990, an SAP/SAB panel considered a revised EPA Technical Panel report and
recommendations. On the issue of adversity of blood measures they concluded:
"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."
On the issue of uncertainty factors they concluded:
"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..."
"Replace the NOAEL/UF strategy with one based on the kinds of dose-consequence data
available..." "From these, distill a specified level of ChEI, based on say, a 10% decrement of
performance. To the 95% lower bound, attach a UF to yield the RfD."
3.	1992 SAB/SAP Meeting
Another EPA ChE policy report was reviewed by another SAB/SAP Panel in November
of 1992 (US EPA, 1993). On the use of blood measures alone for risk assessment, they
concluded,
"The Committee reached no simple "yes" or "no" answer on the question of using
cholinesterase inhibition, by itself, for risk assessment purposes."
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"There was full agreement among the Committee members that blood cholinesterase
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, in particular in human studies where cholinesterase data from the
target tissues of most concern (i.e., brain and peripheral nervous system) are unavailable."
This Committee also emphasized that "The inclusion of biochemical data regarding
cholinesterase inhibition with these signs and symptoms is considered essential for the complete
hazard evaluation for these compounds." Last, they supported the use of statistically significant
brain ChE inhibition for setting reference doses, but noted the importance of regional measures
and correlative blood measures.
4.	1992 SAB/SAP Aldicarb RfD Review
On the next day, in response to a question concerning the use of blood cholinesterase data
for aldicarb and in general, another SAP/SAB panel gave the following response.
"The committee felt in general that blood ChEI data are highly relevant to determination
of NOELs, NOAELs, and RfDs. As detailed in a separate report, it was felt appropriate to
emphasize functional data that are obviously related to toxic effects, that are quantitative and
demonstrate a dose response. It was expected that ChE would usually be a sensitive and relevant
variable, both as a quantitative predictor, as a measure of exposure per sc\ as an index of the
depletion of what may represent a protective buffer or biological site for ChE inhibitors and as a
biomarker of effects occurring outside of the central nervous system. Finally, this variable is the
only one which is directly comparable from animal studies to human studies. The final consensus
was that both cholinesterase data and clinical/functional findings be used where appropriate and
that regardless of how derived, RfDs should ensure that there would be no significant
cholinesterase inhibition. The committee recommended the submissions contain cholinesterase
data but that those consisting solely of cholinesterase data not be considered."
5.	June 1997 SAP Meeting
In June, 1997, the Agency made a presentation to the SAP which included a literature
review, a series of case studies, a summary of activities related to methodology of ChE
measurement, and a briefing of the Agency's science policy on cholinesterase inhibition for risk
assessment. This briefing covered the April 30, 1997 draft of this paper, provided EPA's
analysis of the issues and options, and proposed to use a weight of evidence approach considering
all of the data that might result in the use of ChE measures in plasma, red blood cells or brain for
defining critical effects. The SAP firmly supported the Agency's weight of evidence approach for
cholinesterase inhibitors as "reasonable and justified" as long as the data used "are derived from
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from rigorous experiments with standardized methods and proper controls." The SAP also
agreed that the Agency should continue evaluating data on cholinesterase inhibition in the
peripheral nervous system.
B.	1994 FCCSET and 1995 EPA Neurotoxicity Risk Assessment Guidelines
Both a Federal Coordinating Council for Science, Engineering, and Technology
(FCCSET) and EPA have published guidelines for neurotoxicity risk assessment that address in
part, the issue of ChE inhibition.
The FCCSET document (US EPA, 1994) rather tersely notes "Inhibition of this enzyme
(AChE) in brain may be considered evidence of neurotoxicity, whereas decreases in AChE in
blood, which can easily be determined in humans, are only suggestive of a neurotoxic effect."
A proposed EPA Neurotoxicity Risk Assessment Guideline (US EPA, 1995c) also briefly
reviews the issue of ChE inhibition. It concludes that "statistically significant decreases in brain
cholinesterase inhibition could be considered to be a biologically significant effect" but describes a
lack of consensus about "whether RBC and/or plasma cholinesterase represent biologically
significant events." An SAB meeting in 1996 (US EPA 1997b) to review the draft guideline was
asked to address 2 issues: "the use of blood and/or brain acetylcholinesterase activity as an
indication of neurotoxicity for risk assessment"; and "Considering the available data and the state
of the science, does the SAB agree with the recommendation that inhibition of RBC and/or
plasma cholinesterase can serve only as a biomarker of exposure? (Drs. Pfitzer, Weiss)."
Their response was, "The Committee addressed these two issues together because of their
close relationship. The EHC concurred with the findings of previous SAB reviews regarding the
consideration of data on the inhibition of RBC and/or plasma cholinesterase. In the absence of
clinical signs in humans or animals or the absence of morphological data in animals, the
quantitative nature of the inhibition of red blood cell (RBC) and/or plasma cholinesterase
inhibition is considered unreliable for assessing significant biological adverse changes, but can be
used as a biomarker of exposure. The Committee also recommended that a noted decline in brain
ChE should be evaluated by risk assessors in terms of possible effects that are biologically
significant, and that the term "statistically significant" needed to be better explicated - perhaps in
terms of the benchmark dose or by some measure which reflected information about the
distribution of the effect under study. The Committee also suggested that further details
concerning reversibility and possible tolerance effects (which could enhance sensitivity to other
agents) be provided."
C.	1990 Experts for UNEP. ILO. and WHO
A group of experts, on behalf of the United Nations Environment Programme, the
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International Labour Organisation, and the World Health Organization, have described the
evolution of positions in various groups meeting for those bodies on the use of cholinesterase
inhibition data (WHO, 1990).
From 1967-1982, their review groups used plasma and RBC ChE inhibition in their risk
assessment documents, but noted that blood measures were not useful "as an invariable guide to
the degree of intoxication present or predicted."
In 1982, they reconsidered their position and focussed on the use of RBC AChE since it
contained AChE, and pronounced as biologically significant a "reduction of >20% of pretest
levels in the same animals in short duration studies, or in concurrent controls in longer studies."
No further rationale for this 20% level is provided.
In 1988, they noted "the correlation between acetylcholinesterase inhibition in
erythrocytes and in the nervous system is usually unknown" and found brain levels of ChEI to be
of greater value, but noted that RBC ChEI was still better than plasma. They also noted that "in
vitro kinetic studies may be necessary for pesticides with anti-esterase activity."
Last, they noted concerns about ChE methodology for carbamates, and the adequacy of
reporting of assay details, and concluded that "The results obtained in in vivo studies should be
interpreted cautiously until more satisfactory methods are available."
D. 1993 California DPR Guidance
Lewis (1993), for the Department of Pesticide Regulation of the state of California, has
also written draft guidance on the use of cholinesterase inhibition data in risk assessments for
pesticides. While brief, this document contains a detailed review of literature related to the
interpretation of changes in ChEI measures in the absence of clinical signs.
While noting the species differences in the amount of AChE in rats and humans, they
conclude that blood ChE of any kind will not be regarded as an adverse effect. They cite a
number of animal studies to indicate that a wide range of levels of brain ChEI may be associated
with overt signs, i.e., 15-80%). They conclude that "if there is statistically significant inhibition of
brain ChE inhibition (sic), there is probably some deleterious effect on the neurological system."
They note that ChE decreases in peripheral tissues should also be regarded as adverse. After
review of studies on variations in ChE measures within and between individuals, they endorse the
use of concurrent controls for long term studies, or the use of individual pre-exposure measures
for acute and subchronic studies, if available.
While not supporting the general use of blood measures of ChEI for risk assessments, they
go on to note a number of instances where that might be done:
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first, in animal or human studies where brain ChE was not measured, using the blood
measure, plasma or RBCs, which best correlated with brain ChEI in other studies;
second, "if there is strong evidence... that the chemical does not penetrate the blood brain
barrier and therefore the cholinergic effects are predominantly peripheral in origin."
They would also use the blood measures if peripheral tissue levels were not available and
if the cholinergic effects correlated with the blood measures.
E. Discussion
Until the 1997 SAP meeting, the Agency had been unable to define a consensus policy on
cholinesterase inhibition. However, with EPA's proposed policy emphasizing the weight of
evidence analysis, the SAP agreed that the Agency was taking a reasonable and scientifically
sound approach.
The remainder of this paper consists of a summary science policy statement and more
detailed guidance on evaluation of functional data, i.e., clinical signs, human symptoms, and
behavioral effects, ChE measures in brain and blood; and a series of steps and analyses to conduct
risk characterizations to support completion of the risk assessments and to provide a framework
for consistent risk management decisions.
II. Science Policy Statement
A.	For an adequate evaluation of a ChE inhibitor, the essential elements of a critical study
or a data base should include:
	data on clinical signs (and symptoms in humans);
	other functional effects related to ChE inhibition;
	measurements of CNS and PNS AChE inhibition;
	plasma and RBC ChE inhibition.
	data on the time of peak functional and biochemical effects.
B.	Clinical signs and other behavioral or neurophysiological effects related to
cholinesterase inhibition in humans and animals, and symptoms in humans provide direct evidence
of adverse effects3.
3 Adverse effects include alterations from baseline that diminish an organism's ability to
survive, reproduce, or adapt to the environment. (U.S. EPA 1995)
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Most commonly reported in humans are headache, nausea, and dizziness. Anxiety and
restlessness are prominent. Worsening may result in muscle twitching, weakness, tremor,
incoordination, vomiting, abdominal cramps, diarrhea. Often prominent are sweating, salivation,
tearing, rhinorrhea, and bronchorrhea. Blurred and/or dark vision, and miosis may also be seen.
Tightness in the chest, wheezing and productive cough may progress to frank pulmonary edema.
Bradycardia may progress to sinus arrest, or tachycardia and hypertension. Confusion, bizarre
behavior, and toxic psychosis may occur. In severe poisonings, toxic myocardiopathy,
unconsciousness, incontinence, convulsions, respiratory depression and death may be seen.
Repeated absorption, but not enough to cause acute poisoning may result in persistent anorexia,
weakness, and malaise.
(U.S. EPA, 1989)
C.	Inhibition of acetylcholinesterase in the central nervous system is an indicator of an
adverse effect because it interferes with the timely de-activation of neuronal acetylcholine, which
prolongs the actions of cholinergic neurons which results in the adverse effects associated with
these chemicals.
D.	Inhibition of acetylcholinesterase in the peripheral nervous system or at neuroeffector
junctions is, by the same mechanism, an indicator of adverse effects on the peripheral nervous
system.
E.	Blood cholinesterase inhibition4'5 represents an indirect indicator of adverse effects on
the nervous system. While blood measures of ChEI are not adverse in themselves, they are
generally the only available estimator of ChEI potential in the peripheral nervous system, since
data on ChEI in peripheral nervous tissues or target organs are rarely available. In humans,
blood ChEI measures serve as the essential estimators of ChEI potential in both the central and
peripheral nervous systems, since neither CNS nor PNS or related organ ChEI measures are
available.
F.	OPP will use a weight of evidence (WOE) approach to select the appropriate endpoint
for risk assessment. This includes analysis and comparison of the dose effect data from all
available studies, a description of the strengths, weaknesses, and limitations of the data,
identification of data needs that might be needed to refine the data base, and finally the application
4	Blood cholinesterase in this document refers to both plasma cholinesterases and red
blood cell acetylcholinesterase (AChE).
5	While human plasma is predominantly butyrlcholinesterase (BuChE), (AChE:BuChE,
1:1000), in rats, plasma contains a considerable amount of AChE (AchE:BuChE, 3:1;
males)(Brimijoin, 1991).
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of our best scientific judgments. Based on this weight of the evidence analysis for any ChE
inhibiting pesticide, OPP may select as critical effects:
	clinical signs and other behavioral or neurophysiological effects in humans and animals;
	symptoms in humans;
	central or peripheral nervous tissue measures of ChE inhibition; or
	blood measures of ChE inhibition.
G.	There are a number of instances where the use of blood ChE inhibition can be more
readily justified as a critical effect for a risk assessment based on the weight of evidence analysis
of the available data. Examples include but are not limited to:
1.	A pesticide which, based on animal data, exhibits a steep dose effect curve for the
development of progressively more severe toxic effects and where blood ChEI is the most
sensitive effect;
2.	A pesticide for which, the LOELs and NOELs for various indicators of ChEI are
essentially the same;
3.	An OP for which there is evidence from toxicity, metabolism, or pharmacokinetic
studies, or other sources, to indicate that it poorly penetrates the blood-brain barrier such that its
potential effects would be expected to be mediated largely through the peripheral nervous system;
4.	An OP for which the available human data are judged to be the most critical data for
risk assessment and where blood ChEI is the most sensitive effect. In the absence of brain ChE
activity measurements, which are not made in human studies, the inhibition of blood ChE activity
can serve as an indirect indicator of potential adverse effects in the CNS.
5.	When there is a wide disparity in doses between those affecting blood ChE and other
parameters and when there is an absence of other data (see I. below).
H.	The primary objective of the WOE analysis is to determine the critical effect and
calculate a reference dose (RfD) or margin of exposure (MOE)6. Evaluation of statistical and
toxicological significance7 and application of uncertainty factors, e.g., 10 for inter-species and 10
6	A margin of exposure is the ratio of the no effect level to the exposure level,
NOEL/EXPOSURE LEVEL = MOE.
7	While statistical significance is the primary empirical measure inherent in the
experimental design, it is recognized that judgment is important in considering the toxicological
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for intra-species extrapolation, will follow the established procedures for assessing potential
human risk.
I. Where there are significant uncertainties regarding the available data or the resulting
risk characterization, an iterative process for refining the risk characterization should be followed.
Refinements of the risk characterizations are intended to provide risk managers with analyses that
will allow them to evaluate potential risks, including evaluation of risk mitigation options in a way
that is clear, transparent, and consistent. These steps may include:
	Collection of additional data, to address a number of issues such as:
to provide better dose effect data and to refine NOELs;
to provide data on PNS ChEI;
to provide data on metabolism, pharmacokinetics and pharmacodynamics; or
to provide direct data on exposure routes of interest;
	Refinement of dose response assessments by evaluating LOELs and NOELs for all
compartments, and by defining as completely as possible dose effect data for all critical effects
and/or compartments
	Repeating the risk characterizations for the expanded data base and refined dose effect
data.
III. Guidance For Evaluating Chemically Induced ChE Inhibition
There are 2 major divisions of the nervous system, both of which contain cholinergic
pathways that may be affected by cholinesterase inhibitors:
"the peripheral nervous system (PNS) consisting of skeletal muscle, and tissues of the
autonomic nervous system, consisting of ganglia of the sympathetic and parasympathetic nervous
systems, smooth muscles, cardiac muscle, and glands;" "and ...the central nervous system (CNS),
consisting of brain and spinal cord." (Dementi, 1997).
Access of chemicals to the central nervous system is limited by the blood brain barrier.
Lacking such a fine barrier, the peripheral nervous system is more accessible to many chemicals.
Many of the typical adverse effects of ChE inhibitors may be peripherally mediated.
significance of very small changes in a data set with small variability, or the lack of statistical
significance of large changes. Historical control data or additional analyses may help address such
issues. (See also, US EPA 1993, p 15)
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A.	Functional Effects
Clinical signs and symptoms in humans, clinical signs in animals, and neurobehavioral
effects in both humans and animals are the physiological and behavioral effects typically
associated with exposure to ChE inhibitors and are most generally regarded as adverse and
therefore considered first in defining critical effects. In addition to the common physiological
cholinergic signs, many more complex functions may be impaired by ChE inhibition in the
peripheral or central nervous system. They may be produced following acute or repeated
exposures, and would require exhaustive and specialized testing for complete evaluation (See
Dementi, 1997).
Generally, evaluation of clinical signs and behavioral effects depends on the scale of
measurement (descriptive versus quantitative), number of subjects, the power of the study and
statistical significance, and toxicological significance. The spectrum of effects that can be
evaluated in humans is greater than the effects generally studied in animals, but is often limited in
available studies of either species. Learning and memory evaluations, for example, are rare,
though they are a potential target of ChE inhibitors due to the role of cholinergic systems in these
functions. Repeated exposure, due to the development of tolerance for clinical signs, sometimes
can fail to produce typical signs of acute toxicity in the presence of extensive changes in
neurochemistry (See Dementi, 1997).
Different chemicals may and generally do produce different spectra of clinical signs and
behavioral effects. This complexity in part may arise from differences in distribution between the
CNS and PNS, differential binding in those compartments, or differential interactions with the 2
major types of cholinergic receptors, muscarinic and nicotinic receptors. The nature and temporal
pattern of effects may also depend on the rate of exposure and whether metabolic activation is
needed.
Due to our limited understanding of the precise relationships between behavioral and
neurochemical measures of ChEI, we seek both types of measures for an broader perspective on
potential effects. This has been a general tenet in testing for neurotoxicity, i.e., to examine effects
at different levels of organization of the nervous system, and mirrors the broad efforts in
toxicology at proceeding from identification of hazards to an understanding of the biochemical
mechanism of action. Since the generally accepted mechanism of action of these chemicals is the
neurochemical inhibition of AChE, these measures are the presumptive choice as the biochemical
correlate of the functional effects.
B.	Neurochemical Effects
1. CNS AChE Inhibition
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Data on CNS acetylcholinesterase inhibition typically come from animal studies, in which
whole brain homogenates (or brain regions) are assayed periodically or, more commonly, at the
end of exposure. Statistically significant decreases in brain ChE are generally considered
toxicologically significant because they define a change in nervous system functions, by blocking
the degradation of ACh and prolonging the action of the nerve cells. This effect in the brain and
peripheral nervous system is the generally accepted mechanism by which the expected overt and
adverse effects are caused. Brain cholinesterase inhibition provides direct evidence of adverse
effects on the nervous system and may be used to define a critical effect.
As noted earlier, concomitant evaluation of clinical signs, behavioral effects, and blood
ChE inhibition are considered essential for an overall evaluation of a pesticide. Reductions in
brain ChE activity may or may not be accompanied by overt clinical signs or symptoms because
many behavioral and physiological effects, including death, may be predominantly mediated
through the peripheral nervous system. Further, the CNS functions potentially affected may not
be sufficiently assessed. Whole brain measurements may also mask changes in specific brain
regions associated with particular functions (e.g., hippocampus and memory). Time of
assessment as well as other factors generally affecting these neurotoxicity studies may also
contribute to the lack of concordance.
2.	Peripheral Nervous System and Neuroeffector ChE Inhibition
As with the CNS, inhibition of acetylcholinesterase in the PNS and neuroeffector junctions
is an indicator of adverse effects in the PNS. Although PNS ChEI has rarely been evaluated in
toxicological studies submitted to EPA, there have long been recognition of the potential value of
such data, and there is merit in developing and standardizing techniques to assess this
compartment. Many of the adverse signs and symptoms associated with exposure to ChE
inhibiting pesticides, e.g. diarrhea, excess salivation, are peripherally mediated.
3.	Blood ChE Inhibition
Blood cholinesterase inhibition provides direct evidence of exposure but only indirect
evidence of neurotoxicity or adverse effects. There are many reasons why the blood measures of
ChE should be considered as an appropriate endpoint for derivation of reference doses as a matter
of prudent science policy. Blood measures are generally the only available estimator of ChEI
potential in the peripheral nervous system, so while they are not adverse in themselves, they can
be a unique indirect measure of potential PNS toxicity. In humans, neither CNS nor PNS ChEI
measures are available, so blood ChEI measures serve as the essential estimators of both central
and peripheral nervous system ChEI potential.
Blood ChEI is not only a measure of exposure, but also a measure of a pesticide's ability
to bind to AChE. This is because the binding of a pesticide to the neural and blood enzyme AChE
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is essentially the same, or in the case of BuChE at least somewhat similar. Pharmacokinetically,
both the blood and the peripheral nervous system are outside the CNS. So for pesticides with
limited penetration of the blood-brain barrier, blood ChE measures may be much better indicators
of PNS ChEI activity than brain measures.
RBC AChE is typically regarded as all AChE in humans and animals, and plasma ChE is
often viewed as BuChE. Since in neurons AChE is the active ChE, it is naturally considered that
RBC AChE more closely reflects neuronal activity, and that plasma BuChEs appear less relevant.
But the composition of plasma ChEs vary widely between humans and rats4. Plasma ChEs in rats
may contain mostly AChE, the neuronal form. Thus, for rat studies, the most common test
species, a significant portion of plasma ChEI may be due to AChEI, the form most directly
relevant to neural functions. In other cases, for unclear reasons, empirical correlations between
plasma and brain ChE may exceed those between RBCs and brain ChE
(see Dementi, 1997; also ACRA Case study 1).
Demonstration of inhibition of both plasma and RBC ChEs in workers (in the absence of
signs, symptoms, or other behavioral effects) are rightly considered as providing sufficient
grounds for companies/agencies to remove workers from the exposure environment.
As for CNS effects, functional evaluation of the peripheral nervous system may be quite
limited, e.g., for cardiovascular effects.
Further, measurement of ChE activity at peripheral target sites is rarely done. So, for
most pesticide data bases, the only available estimate of ChE inhibition in the PNS will be the
blood measures of ChE.
Limited reporting of methodological details or of assay
conduct are very common in available studies. In some cases, the methods as used may
underestimate red blood cell AChEI. Increased variability (coefficients of variation) related to
assay conduct can decrease the sensitivity of the assay, i.e., will require larger levels of inhibition
to achieve statistical significance. Further details and current efforts in OPP directed at these
issues are discussed in other parts of this package (Hamernik, 1995).
Many other factors can influence the observed pattern of blood ChE inhibition. These
include a) the time course and reversibility of inhibition, b) time of measurement with respect to
the time course of inhibition, c) whether or not the inhibitor is metabolically activated, d)
analytical methodology used, and e) whether comparisons are made with pre-exposure
measurements in the same subjects or separate control groups (See Dementi for further
discussion).
IV. Guidance for Risk Assessments of Cholinesterase Inhibitors
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A. Dose Response Assessment: Weight of Evidence Analysis for selection of critical
effects.
A weight of the evidence approach for evaluation of any ChE inhibitor should consider all
of the available data from animal and human studies, and human exposures to identify the hazards
and the exposure levels at which they occur. First the individual studies are evaluated, then all
studies and their relation to one another are examined in concert.
1.	Analysis of Individual Studies
Each study may include ChE measures in blood and brain, PNS (though rarely), clinical
signs and symptoms (from humans, if available), and other functional data. Following critical
evaluation of the validity of a study, No-observed-effect-levels (NOELs) and/or lowest-observed-
effect-levels (LOELs) are determined. The evaluation of each study involves consideration of the
study design including dose spacing, the analytical and behavioral methods used, whether pre-
exposure data were obtained, the conduct of the study, the statistical analysis and significance
(both statistical and biological) of the results, the slope of the dose response and dose effect
curve(s), the consistency of the findings within the study when repeated measures are taken, and
the relation of the effects seen to one another.
2.	Analysis of the Data Base
When evaluating the entire database, consistency of LOELs and NOELs for clinical signs,
behavioral effects, ChE inhibition in the various compartments, in different studies within a
species, across species, across durations of exposure, and across routes all may contribute to the
weight of the evidence for the critical endpoints needed. Pharmacokinetic data may also be
important.
3.	Selection of the Critical Effect
Typically, a critical effect level is selected for a route and duration of exposure that
represents the most sensitive effect seen. Based on considerations of the weight of the evidence
from all of the studies as a group, this level may or may not be the lowest one in which an effect
was seen. Valid and reliable human data, when available, take precedence.
An RfD for chronic dietary exposures is then derived by division of the critical effect
NOEL by uncertainty factors to account for potential inter-species (animals to humans) and intra-
species (among all people at risk) variability. For acute, short term, and intermediate exposures,
the critical effect NOEL is identified from the appropriate study(ies). This is typically the end of
the dose response assessment stage in the risk assessment process.
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B. Risk Characterization
Risk characterization has been described as the interface between risk assessment and risk
management (US EPA, 1995). This stage involves the integration of the exposure analysis with
the reference dose or critical effects data to derive estimates of the potential risks for each
exposure and population of concern. This stage may include both an iterative approach to risk
assessment and presenting multiple risk descriptors. It also should describe the limitations and
uncertainties in all of the earlier steps in the risk assessment process. What follows is guidance for
risk characterizations for ChE inhibitors. It is an iterative process using multiple RfDs (or other
available data) as risk descriptors to facilitate risk management decisions.
After analysis of the exposure data, calculate margins of exposure or compare the
anticipated exposure levels to the reference dose for each situation of interest. A margin of
exposure (MOE) is a comparison made by dividing the NOEL by an anticipated human exposure
level. This is the procedure used for acute, short term and intermediate exposures, by tradition.
Comparison of exposures to the RfD or calculated MOEs are then used in risk characterizations
to evaluate exposures of concern. If reviews of RfDs or MOEs lead to significant concerns, an
iterative sequence of actions may be considered to refine the risk assessment (including refining
the exposure issues which is not described here).
1. Collect additional data
A comprehensive risk assessment should describe the dose effect curves for each
compartment (plasma, RBC, and brain) or other endpoints (e.g., clinical signs) and would allow
estimation of e.g., ED 10s, so that simple and consistent comparisons could be made between
different compartments. Given that toxicology guideline studies usually have 3 doses, and that
one dose is usually chosen to be a NOEL, the dose effect relationship may be quite difficult to
ascertain. Limited knowledge may thus result in NOELs and associated RfDs that over or
underestimate the true potency of the pesticide. In this situation, data on intermediate dose levels
or replication of a key finding may be needed to better define the dose effect curves and to more
clearly establish critical effect levels. Benchmark dose estimation or other curve fitting may be
helpful in some cases.
In some cases, study by the dermal or inhalation route may provide a better and direct
means of risk assessment for those routes of potential exposure, reducing or eliminating the
uncertainties that may arise from route to route extrapolation.
When most or all LOELs for different measures are seen within a narrow dose range, as in
our experience is generally the case, there is greater confidence in the selection of their associated
NOELs for use in the derivation of RfDs or MOEs. And there will be less debate about the
adversity of the endpoints if direct measures are involved. On the other hand, if significant
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inhibition in blood compartments is seen at much lower doses than in other ChE compartments or
than in functional measures, there is less coherence in the data set, and there may be more concern
about the selection of the critical effects. This may reflect data seen in one study, one species, or
be a consistent finding across the database for a chemical.
In some cases, direct measurement of ChEI in peripheral neural or neuroeffector target
tissues may be considered. If those tissues are assayed, they would provide direct evidence of
ChEI in peripheral tissues, and would potentially be more relevant than the indirect measures of
the blood. While current methods for measuring ChEI in peripheral tissues have not been
required and may pose some technical difficulties, they offer a potential scientific means to clarify
the meaning of blood ChE measures in animals.
2.	Broaden the scope of critical doses and effects examined and the risk
characterization for the expanded data base and dose effect data
Expand the analysis beyond the use of one critical RfD, by defining RfDs for all
compartments, and as completely as possible defining the dose effect data for all critical effects
and/or compartments. An attempt to illustrate this idea is provided in Figure 1. This graph plots
Exposure incidence (as a % of exposures) against dose in mg/kg. Reference doses for blood
ChEI, brain ChEI, and clinical signs are indicated by broad bars. A theoretical exposure
distribution is plotted as a curve. Risks of any exposure and for each effect then can be seen
visually. This approach can then be used for different exposure distributions, which may represent
different commodities, or different application rates, etc. One could also graph dose effect curves
similarly for comparisons, if exposures were high enough (not common). A similar approach
could be generated to evaluate margins of exposure.
The relationship between exposures and different effects, can be one factor in defining the
level of concern for a pattern of toxicity. For example, exposure to a chemical at levels greater
than an RfD of 0.01 mg/kg based on, e.g., blood measures may be of greater concern when the
RfD based on brain measures is only 3 times that level, than when the RfD based on brain
measures is at 50 times that level.
Other critical factors in this broader description of the pattern of observed toxicity may
include the nature and severity of effects seen; the slope of the dose effect curves for different
effects, and the completeness of the effects evaluated. Other factors important to consider in the
total data base are the number of human incidents reported, and the scope of the effects evaluated.
Last, the strengths and weaknesses in the data base should be summarized and the uncertainties in
defining the critical effects should be clearly documented.
3.	Evaluate exposures in terms of risk characterization.
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The objective of the expanded risk characterization is to provide as detailed a means as
possible for describing the relation between exposures and ChE-related effects of all kinds in
qualitative and quantitative terms. This in turn is aimed at providing risk managers with analyses
that are clear and transparent and that will serve as a basis for defining consistent risk
management decisions.
V. References
Brimijoin, S. 1991. Enzymology and Biology of Cholinesterases. In: Proceedings of the U.S. EPA
Workshop on Cholinesterase Methodologies. U.S. EPA, 1992.
Dementi B. 1997. Cholinesterase Literature Review and Comment. 4/29/97. 175 pp.
Hamernik, K. 1995. Assay of Cholinesterase Activity in Toxicology Studies submitted to the
Office of Pesticide Programs, August 22, 1995. 3 pp. with attachments.
Lewis, C. 1993 Use of Cholinesterase Inhibition Data in Risk Assessments for Pesticides .
6/14/93. State of California, Department of Pesticide Regulations. DPR/MT/HAS Manual Sec
V.C. pages 1-7.
NAS 1983. (National Academy of Sciences). Risk Assessment in the Federal Government:
Managing the Process. Washington DC: National Academy Press.
US EPA 1988. Colloquium on Cholinesterase Inhibition. Risk Assessment Forum, Office of
Research and Development. 6/30/88.
5 pp.
US EPA 1989 Recognition and Management of Pesticide Poisonings.
4th Edition. D.P. Morgan. EPA 540/9-88-001.
US EPA 1990. Report of the SAB/SAP Joint Study Group on Cholinesterase. Review of
Cholinesterase Inhibition and its Effects. EPA-SAB-EC-90-014. 17 pp.
US EPA 1992. Report of the SAB/SAP Meeting 11/6/92. A Set of Scientific Issues Being
Considered by the Agency in Connection with Aldicarb and Aldicarb Sulfone. 11/25/92.
US EPA 1993. 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 Joint SAB/SAP Joint Committee. EPA-SAB-EHC-93-011. 20 pp.
US EPA 1994. Final Report: Principles of Neurotoxicity Risk Assessment. Federal Register 59,
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No. 158. pp. 42360-42404.
US EPA 1995a. Proposed Guidelines for Neurotoxicity Risk Assessment. 10/04/95 Federal
Register 60, 192. pp 52032-52056
USEPA 1995b. Policy for Risk Characterization at the U.S. Environmental Protection Agency.
Approved by CM Browner, 3/21/95.
US EPA 1995c. Proposed Guidelines for Neurotoxicity Risk Assessment. Federal Register 60,
No. 192 pp 52032-52056.
US EPA 1997a. Office of Pesticide Programs Reference Dose Tracking Report (3/28/97)
US EPA 1997b. Science Advisory Board's review of the revised Guideline for Neurotoxicity Risk
Assessment. EPA SAB EHC 97-XXX April XX, 1997
WHO 1990. Environmental Health Criteria 104: Principles for the Toxicological Assessment of
Pesticide Residues in Foods. 1990 WHO Geneva, pp 63-5.
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