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**Item No.: 6065**

Office of Pesticide Programs

Science Policy on

The Use of Data on

Cholinesterase Inhibition

for Risk Assessments

of Organophosphorous and Carbamate

Pesticides

**August 18, 2000**

Office of Pesticide Programs

US Environmental Protection Agency

Washington DC 20460

**TABLE OF CONTENTS**

**EXECUTIVE SUMMARY** 1

**LIST OF ABBREVIATIONS** 4

**ORGANIZATION OF POLICY DOCUMENT** 5

**1. INTRODUCTION** 7

1.1 RISK ASSESSMENT FRAMEWORK 7

1.2 BIOLOGY AND TOXICOLOGY OF CHOLINESTERASE INHIBITION 9

**2. HISTORICAL BACKGROUND** 14

2.1 OPP=s HISTORICAL APPROACH 14

2.2 REVIEWS OF PROPOSED AGENCY/OPP SCIENCE POLICY POSITIONS 15

**3. IDENTIFICATION OF THE TOXICOLOGICAL ENDPOINTS FOR ASSESSMENT OF
CHOLINESTERASE INHIBITORS** 18

3.1 EVALUATION OF EFFECTS ON CHOLINERGIC FUNCTIONS 19

3.1.1 CONCLUSIONS 19

3.1.2 RATIONALE 20

3.2 NERVOUS SYSTEM CHOLINESTERASE INHIBITION 25

3.2.1 CONCLUSIONS 25

3.2.2 RATIONALE 26

3.3 BLOOD CHOLINESTERASE INHIBITION 28

3.3.1 CONCLUSIONS 28

3.3.2 RATIONALE 30

**4. WEIGHT-OF-THE-EVIDENCE ANALYSIS FOR SELECTION OF CRITICAL EFFECTS**
34

4.1 ANALYSIS OF INDIVIDUAL STUDIES 37

4.2 INTEGRATIVE ANALYSIS OF THE DATA BASE 38

4.3 COLLECTION OF PERIPHERAL NERVOUS SYSTEM CHOLINESTERASE INHIBITION
DATA 41

**5. CONCLUSION** 43

**6. REFERENCES** 45

**EXECUTIVE SUMMARYEXECUTIVE SUMMARY**

The Office of Pesticide Programs (OPP) published a policy statement on
the use of data on cholinesterase inhibition (and other events
associated with cholinergic effects related to nervous system function)
in human health risk assessment of certain classes of pesticide
chemicals for review by the FIFRA Scientific Advisory Panel (SAP) in
1997 and for public comment in 1997 and 1998 (US EPA, 1997b). The 1997
science policy document described the approaches OPP would employ in
assessing the potential for human health hazard from the cholinergic
effects on nervous system function following exposure to
cholinesterase-inhibiting pesticides.

The 1997 policy document has been reorganized and revised, taking into
consideration, as appropriate, comments offered by the public, the SAP,
other EPA offices and other government agencies. As did the 1997 policy,
this revised science policy emphasizes the weighing of all relevant
evidence when selecting endpoints for the hazard assessment of
anticholinesterase pesticides. This \"weight-of-the-evidence\" review,
conducted on a case-by-case, chemical-by-chemical basis, is accomplished
by performing an integrative analysis after assessing all the individual
lines of evidence (including all available data on cholinesterase
inhibition in all compartments \-- central nervous system, peripheral
nervous system, red blood cells, and plasma \-- as well as data on
clinical signs, symptoms and other physiological or behavioral effects).
Weighing of the evidence must include considerations of many factors,
including the adequacy of study protocols; quality of data; number of
studies on each endpoint; dose-dependency of responses; time course and
duration of effects; and similarities or differences of responses
observed in all the species, strains, and sexes tested for each duration
and route of exposure evaluated.

In a weight-of-the-evidence assessment of cholinesterase-inhibiting
substances, acetylcholinesterase inhibition in the nervous system is
viewed as a key event in the mechanism of toxicity of these compounds
and an important critical effect to consider in the hazard assessment.
Evaluations of the cholinergic effects (i.e., physiological and
behavioral changes and measures of cholinesterase inhibition in the
central and peripheral nervous systems) caused by exposure to the
cholinesterase-inhibiting organophosphorous and carbamate pesticides
provide direct evidence for characterizing potential human health
hazard. Because of likely differences in both the chemicals= and the
cholinesterases= pharmacodynamic properties, measures of cholinesterase
inhibition in both the central and peripheral nervous systems are
important for a thorough evaluation of potential hazard. However, direct
measurement of cholinesterase activity in peripheral nervous system
tissues are rarely available at the present time. When these data are
not available, as a matter of prudent science policy protective of human
health, EPA will treat cholinesterase inhibition in the blood as a
surrogate measure for the peripheral nervous system in animals and for
both the peripheral and central nervous systems in humans. Information
from blood cholinesterase inhibition data is considered to provide
important insights into potential hazard. Red blood cell (RBC) measures
of acetylcholinesterase (AChE) are generally preferred over plasma
measures of cholinesterase activity because data on red blood cells may
provide a better representation of the inhibition of the neural target
enzyme, acetylcholinesterase. OPP, however, may use plasma
cholinesterase inhibition data under certain circumstances, such as if
red blood cell data are insufficient, of poor quality, or unavailable;
if there is a lack of dose-dependency for the red blood cell
acetylcholinesterase inhibition; or, if the dose responses for
inhibition of plasma cholinesterase more closely approximate those for
AChE inhibition in the nervous system than do the dose responses for RBC
acetylcholinesterase inhibition.

It should be noted that the present policy provides guidance *only* on
how to deal with data as they relate to the cholinergic endpoints
associated with nervous system *function* following exposure to
organophosphorous and carbamate pesticides. This scope is consistent
with all earlier descriptions of Agency assessment approaches as well as
that of other organizations with regard to the evaluation of
cholinesterase-inhibiting substances (e.g., WHO JMPR (1990,1999),
DPR-CalEPA (1997) and other national authorities such as Canada\'s PMRA
(Franklin, 1999). When applying the weight-of-the-evidence approach for
selecting critical effect(s) for derivation of a reference dose (RfD) or
concentration (RfC), however, the entire toxicological data base on a
pesticide must be evaluated (i.e., there also must be consideration of
endpoints not related to the cholinergic consequences of
anticholinesterase activity, for instance, liver or developmental
toxicity or carcinogenicity). It is possible that, for one or more of
the exposure scenarios being evaluated, the non-cholinergic effects will
be identified as critical or co-critical, and they may become a more
appropriate basis for deriving RfDs or RfCs.

Finally, OPP policy documents are meant to be Aliving documents,@ that
is, they are open to periodic updating and revision to reflect advances
in the science. Thus, this policy, too, will be updated to incorporate
important new scientific knowledge as it becomes available. For example,
the routine availability of data on acetylcholinesterase activity in the
peripheral nervous system may allow for refinements in the hazard
assessment approach for anticholinesterase chemicals. Also, as knowledge
increases about the potential roles of the different cholinesterases in
the developing organism, particularly as they impact the development of
the nervous system, it may allow for refinements in evaluating the
potential differential sensitivity and susceptibility of the young
versus adults. In fact, a substantial research effort has been, and
continues to be, made to determine what roles acetylcholine-,
butyrylcholine- and other esterases may play in the development of the
nervous system and in cell growth, proliferation and death in other
tissues. OPP encourages further discussion of the possible implications
of the research findings, both for future research planning and for the
Agency=s regulation of cholinesterase-inhibiting pesticides.

**LIST OF ABBREVIATIONSLIST OF ABBREVIATIONS**

**[Scientific Terms]{.underline}:**

**ACHE** Acetylcholinesterase

**BMD** Benchmark Dose

**BuChE** Butyrylcholinesterase

**LOAEL** Lowest-Observed-Adverse-Effect Level

**MF** Modifying factor

**NOAEL** No-Observed-Adverse-Effect Level

**PoD** Point of Departure

**RBC** Red Blood Cell (or erythrocyte)

**RfC** Reference Concentration

**RfD** Reference Dose

**UF** Uncertainty Factor

**[Organizational Terms:]{.underline}**

**DPR-CalEPA** Department of Pesticide Regulation-California
Environmental Protection Agency

**FIFRA SAP** EPA=s FIFRA Scientific Advisory Panel

**ILSI** International Life Sciences Institute

**PMRA Canada** Pesticide Management Regulatory Agency-Canada

**NRC/NAS** National Research Council-National Academy of Sciences

**OPP** Office of Pesticide Programs

**SAB** EPA=s Science Advisory Board

**SAP** EPA=s FIFRA Scientific Advisory Panel

**TRAC** Tolerance Reassessment Advisory Committee

**WHO/FAO JMPR** World Health Organization/Food and Agricultural
Organization Joint Meeting on Pesticide Residues

**ORGANIZATION OF POLICY DOCUMENTORGANIZATION OF POLICY DOCUMENT**

This science policy document describes the approaches that the Office of
Pesticide Programs (OPP) employs when evaluating data on cholinesterase
inhibition and other cholinergic effects related to nervous system
function that are the consequences of acetycholinesterase inhibition
(i.e., physiological or behavioral changes) in assessing the potential
hazard following exposure to organophosphorous or carbamate pesticides
that inhibit cholinesterase.

 Section 1(**Introduction)** presents a very brief description of the
Agency=s general approach to non-cancer risk assessment. This chapter
also includes a brief introduction to the biology of the cholinergic
nervous system, description of cholinesterase enzymes and their
distribution, the inhibition of acetylcholinesterase as a mechanism of
toxicity for cholinesterase-inhibiting pesticides, and the consequences
of acetylcholinesterase inhibition in the body.

 Section 2 (**Historical Background**) presents the history that has
led to the development of this policy document.

-   Section 3 (**Identification of the Toxicological Endpoints for
    Assessment of Cholinesterase Inhibitors**) describes the scientific
    rationale for selection of endpoints for anticholinesterase
    pesticides, their toxicological significance and relevance to hazard
    assessment.

-   Section 4 (**Weight-of-the-Evidence Analysis for Selection of
    Critical Effects**) describes the weight-of-the-evidence approach
    that is used to select the critical effect(s) for the risk
    assessment of anticholinesterase pesticides.

 Section 5 (**Conclusions**) provides a summary of the key elements of
the weight-of-the-evidence approach that OPP uses when evaluating
anticholinesterase organophosphorous and carbamate pesticides.

 Section 6 **(References)** contains the literature citations and other
references used as source material for the policy document.

> **1. INTRODUCTION1. INTRODUCTION**

The purpose of this document is to set forth the principles and
procedures, including a weight-of-the-evidence approach, used by OPP for
the selection of appropriate endpoints for assessing potential hazards
to humans exposed to anticholinesterase pesticides. In addition, this
science policy document also describes science policy approaches
specific to effects related to cholinesterase inhibition that will be
used to address inadequacies in data or lack of knowledge. The Agency=s
policy which addresses the potential for pre- and postnatal effects and
the completeness of databases with respect to toxicity and exposure as
they relate to infants and children when conducting risk assessments and
making regulatory decisions regarding the setting of tolerances
(residues in food) under the 1996 Food Quality Protection Act can be
found in the draft guidance document entitled AThe Office of Pesticide
Programs= Policy on Determination of the Appropriate FQPA Safety
Factor(s) for Use in the Tolerance-setting Process@ (US EPA, 1999b).

**1.1 RISK ASSESSMENT FRAMEWORK.1 RISK ASSESSMENT FRAMEWORK**

Regulatory decision making in EPA is described as consisting of two
major steps\--risk assessment and risk managementBwhich are closely
related but different processes. Risk assessment defines the potential
for adverse effects[^1] to occur in individuals or populations, while
risk management weighs risk reduction alternatives and integrates the
risk assessment with social, economic, and other factors, as
appropriate. The Agency uses the paradigm put forward by the National
Research Council of the National Academy of Sciences in 1983 and
modified in 1994 (NRC/NAS, 1983; 1994) that defines and organizes risk
assessment into four phases: hazard identification, dose-response
assessment, exposure assessment, and risk characterization. Risk
assessment for noncancer effects including those addressed in this
policy is generally based on identifying a
no-observed-adverse-effect-level (NOAEL) or calculating a benchmark dose
(BMD) for a critical effect[^2], which is usually determined from
laboratory animal studies, for use as a Point of Departure (PoD) when
deriving a reference dose (RfD) or reference concentration (RfC). The
point of departure (PoD) is generally defined as a point estimate of an
empirically-measured or modeled dose or exposure level that is used as
the Ajumping-off@ point for extrapolation to exposure levels below those
tested, where actual human exposures are actually likely to be
occurring. The PoD can be a dose at which no effects are found or a dose
level which is associated with some percent of response relative to the
control or baseline level of response. The PoD is divided by one or more
uncertainty factors (UF) or modifying factors (MF). The UFs (typically
3‑ or 10‑fold in magnitude) reflect uncertainties inherent in the
extrapolation from laboratory animal species to humans (the interspecies
UF), in the variations in sensitivity among members of the human
population (the intraspecies UF), for the use of subchronic rather than
chronic data (the subchronic to chronic UF), the use of a
lowest-observed-adverse-effect level (LOAEL) rather than a NOAEL (the
LOAEL to NOAEL UF), and the comprehensiveness and quality of the
database available, i.e*.*, whether or not all potential endpoints of
concern are identified and evaluated in acceptable studies (the database
UF). A modifying factor may be used to address scientific uncertainties
in the principal study used for RfD/C derivation which are not
explicitly addressed by the other standard Ufs.

The result of dividing a PoD by the appropriate uncertainty factors
and/or modifying factor is a reference dose (RfD) for oral or dermal
exposuresBor reference concentration (RfC) for inhalation exposure(s).
The RfD or RfC is defined as an estimate, within an order of magnitude,
of exposure assumed to be without appreciable risk for adverse noncancer
health effects. In the risk characterization step, the RfD and RfC
values are compared to potential or known exposure levels. Risk
characterization also fully describes the nature and extent of the risks
posed, and how well the data support the conclusions, including a
discussion of the limitations and uncertainties involved. Sometimes,
because of these limitations and uncertainties, further data may be
collected to reduce the uncertainties and refine the risk assessment.

The Agency has acknowledged that the historical approach to defining a
NOAEL and calculating RFDs and RfCs has limitations (see USEPA, 1994;
1995; 1996). In response, the Agency has developed draft guidance on an
alternative methodBthe Benchmark Dose (BMD) Approach (USEPA, 1996). The
BMD is defined as the statistical lower confidence limit on the dose
producing a predetermined level of change in response compared with the
background response. A BMD is derived by fitting a mathematical model to
the dose-response data.[^3] The Agency is still gaining experience with
BMD analyses and has not yet formally finalized standard operating
procedures. OPP, however, will use the BMD approach for derivation of
RfDs and RfCs to the extent possible.

**1.2 BIOLOGY AND TOXICOLOGY OF CHOLINESTERASE INHIBITION.2 BIOLOGY AND
TOXICOLOGY OF CHOLINESTERASE INHIBITION**

Acetylcholine plays an important role in the functioning of the nervous
system.[^4]

Acetylcholine is a neurotransmitter which enables chemical communication
to occur between a nerve cell and a target cell. This target cell may be
another nerve cell, muscle fiber or gland. Upon stimulation, the nerve
cell releases acetylcholine into the synapse (or space) between the two
cells. This released acetylcholine binds to receptors on a target cell,
thereby passing the signal on to that nerve cell, muscle or gland. The
end result of the stimulation of cholinergic pathway(s) includes, for
example, the contraction of smooth (e.g*.*, in the gastrointestinal
tract) or skeletal muscle, changes in heart rate or glandular secretion
(e.g*.*, sweat glands) or communication between nerve cells in the brain
or in the autonomic ganglia of the peripheral nervous system.
Cholinergic pathways innervate virtually every organ in the body,
including the brain and peripheral nervous system.

There are two major divisions of the nervous system, both of which
contain cholinergic pathways that may be affected by
cholinesterase-inhibiting chemicals:

 the peripheral nervous system, consisting of neuromuscular junctions
in 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, consisting of brain and spinal cord.

The distribution of cholinergic receptors in the central nervous system
and the peripheral nervous system is not uniform (Brimijoin, 1992). For
example, certain brain regions of the mature organism are rich in
cholinergic neurons (e.g., the striatum, hippocampus, cerebral cortex),
while other regions are less well innervated and have less
cholinesterase activity (e.g., cerebellum). There are two major types of
cholinergic receptors \-- muscarinic and nicotinic \-- and there are
several subtypes of each. These receptor types also are differentially
distributed in different regions of the central and peripheral nervous
systems, thus contributing to the complexity of effects that may occur.

Acetylcholinesterase (AChE) is found in cholinergic neurons, in the
vicinity of synapses, and in other, non-neural tissues. It is highly
concentrated at the neuromuscular and other neuroeffector junctions. It
is the enzyme that breaks down acetylcholine and terminates its action
in the synapses between neurons and between neurons and muscle fibers or
glands. Inhibition of AChE leads to an accumulation of acetylcholine and
a prolongation of the action of acetylcholine at the nerve-nerve,
nerve-muscle or nerve-gland interface. Peripherally, the accumulation of
acetylcholine can result in cholinergic responses such as smooth muscle
contractions (e.g., abdominal cramps), glandular secretions (e.g.,
sweating), skeletal muscle twitching, and, at higher concentrations,
flaccid paralysis. In addition, there may be centrally-mediated effects
on learning, memory and other behavioral parameters. Thus, the
inhibition of AChE potentially results in a broad range of adverse
effects, having an impact on most bodily functions, and depending on the
magnitude and half-life of an exposure dose, these effects can be
serious, even fatal.

Effects caused by AChE inhibition may be a result of action on neurons
in the central nervous system and/or the peripheral nervous system.
Access of chemicals to the central nervous system and the peripheral
nervous system may be different because of differences in
pharmacokinetic properties of these two compartments (e.g.*,*
differences in absorption, distribution, metabolism, elimination). These
differences may be due to chemical specific characteristics as well as
the characteristics of the exposed organism (e.g., degree of maturation
of the blood-brain barrier). The pattern of effects seen may also depend
upon factors such as the pharmacodynamic characteristics (i.e*.*,
binding potency, rate of reversal) of the cholinesterase-inhibiting
chemical and the molecular form of cholinesterase with which it is
interacting (e.g., see Scarsella, et al., 1979).

Butyrylcholinesterase (BuChE) is similar in structure to AChE, but it is
encoded by a separate gene. BuChE, which is synthesized primarily in the
liver and found in plasma and other tissues, is generally distinguished
from AChE by BuChE=s slower rate of hydrolysis of acetylcholine, by
function and by localization using histochemical techniques after
subjecting the experimental model to inhibitors which selectively block
the activity of one but not the other enzyme (Taylor and Radic, 1994).
Furthermore, the binding affinity of anticholinesterase chemicals for
each enzyme can differ among these substances (Silver, 1974; Taylor and
Radic, 1994). Both enzymes are present during development of the nervous
system, with the ratios of one to the other changing substantially over
time and with location (Hoffman, et al., 1996). While no neurological
function has been shown definitively for BuChE in the developing or
mature nervous system, the BuChE present in the plasma will catalyze the
hydrolysis and inactivation of ingested esters from plant sources (e.g.,
cocaine and related synthetic local anesthetics (Hoffman, et al.,1996)
and neuromuscular blocking agents such as succinylcholine (Taylor,
1996b). Likewise, there is no known physiological or biochemical
function for erythrocyte acetylcholinesterase (Brimijoin, 1992; Dementi,
1997).

As discussed later, the blood cholinesterase enzymes are regarded, as a
matter of policy, as surrogate measures of neuronal cholinesterase
activity. Of the two common blood elements measured, red blood cells
(RBC) contain AChE exclusively, while the ratio of AChE to BuChE in
plasma varies widely among humans, dogs, and rats, the species in which
these measures are most typically made for risk assessment and
regulatory purposes. While human plasma is overwhelmingly BuChE, the
plasma of dogs and rats contains both AChE and BuChE (Scarsella, 1979;
Edwards and Brimijoin, 1983).

The question of whether, and, if so, how, BuChE plays a role in the
development and/or functioning of the nervous system still awaits
resolution. Research has been, and continues to be, conducted to
determine if butyrylcholinesterase plays a role in nervous system
morphogenesis (development) and function, and whether, and if so, how,
butyryl- and/or acetylcholinesterase and other esterases play a more
general role in cell growth and death, including in carcinogenesis. In
addition, the dose response relationships attendant to
acetylcholinesterase=s function(s) in the development of the nervous
system remain to be described and compared with those of the endpoints
currently used in the evaluation of nervous system function. OPP is
preparing a brief summary of the available literature on the role of the
cholinesterases (and, perhaps, other esterases) in these areas. OPP also
is preparing a series of questions to serve as a starting point for
discussion for addressing whether or not this information may justify
further revisions to the present policy. This effort is being conducted
separately from the revision of the current policy document.

**2. HISTORICAL BACKGROUND2. HISTORICAL BACKGROUND**

**2.1 OPP=s HISTORICAL APPROACH.1 OPP=s HISTORICAL APPROACH**

Cholinesterase inhibition and the cholinergic effects (i.e.*,* the
physiological or behavioral changes) caused by organophosphorous and
carbamate pesticides have long been endpoints that OPP has used in
assessing potential human health hazards. For well over a decade, OPP
has regarded data showing cholinesterase inhibition in brain, RBC, or
plasma, and data on physiological or behavioral changes as critical
effects (i.e., effects that should be considered for use in the
derivation of an RfD or RfC). OPP has used statistical significance,
rather than a fixed percentage of response from baseline, as the
primary, but not exclusive, determinant of toxicological and biological
significance in selecting Points of Departure (e.g., NOAELs or LOAELs or
Benchmark Doses). This approach treats cholinesterase activity data like
most continuous endpoints (*i.e.,* graded measures of response such as
changes in organ weight, hormone levels or enzyme activity), where no
fixed generic percentage of change from the baseline is considered to
separate adverse from non-adverse effects (US EPA, 1995). OPP believes
that a fixed percentage for describing adversity that would apply to all
cholinesterase-inhibiting pesticides or to all compartments (i.e.,
blood, central nervous system, peripheral nervous system) cannot be
determined realistically or scientifically justified. Each data set must
be judged on its own merits, consistent with the weight-of-the-evidence
approach that OPP is implementing. The use of uncertainty factors and
the use of statistical significance are consistent with Agency practice
for all non-cancer, systemic toxicity endpoints.

OPP=s Reference Dose Tracking Report (US EPA, 1997a) lists chronic
Reference Doses for over 50 chemicals based in whole, or in part, on
cholinesterase inhibition. There are, however, many more than 50 risk
assessments that make use of this endpoint in acute and chronic dietary
exposure/risk assessments and in other, non-dietary scenarios
representing both short-term and intermediate-term exposure(s).

**2.2 REVIEWS OF PROPOSED AGENCY/OPP SCIENCE POLICY POSITIONS.2 REVIEWS
OF PROPOSED AGENCY/OPP SCIENCE POLICY POSITIONS**

Prior to 1997, one internal Agency colloquium (US EPA, 1988) and two
public Science Advisory Board (SAB)/Scientific Advisory Panel (SAP)
meetings ( SAB/SAP, 1990; 1993) considered draft Agency guidance on the
use of cholinesterase data in risk assessment. An additional SAP/SAB
review in 1992 of a proposed reference dose for aldicarb also addressed
the issue of cholinesterase inhibition as an endpoint in risk assessment
(SAB/SAP, 1992). Each of these reviews yielded somewhat different
perspectives and recommendations, based in part on somewhat differing
proposed policies, but primarily on differing points of view of each
peer review group. The area of greatest divergence among these reports
and in their recommendations involved the interpretation and use of
blood measures of cholinesterase inhibition, particularly in plasma, for
deriving reference doses. Some reviewers and panels placed less (or no)
reliance on plasma measures of cholinesterase inhibition and/or less
reliance on red blood cell measures of AChE inhibition as a critical
effect than OPP traditionally has placed on each. EPA has never
finalized guidance on this topic for use Agency-wide.

In 1997, OPP published its own policy statement on the use of data on
cholinesterase inhibition for risk assessments, accompanied by case
studies illustrating the application of this policy and a review of
pertinent literature on cholinesterase inhibition prepared by OPP staff
for public comment and SAP review (US EPA, 1997b; Dementi, 1997). In
1998, as part of the OPP review process for science policy issues agreed
upon in conjunction with the Tolerance Reassessment Advisory Committee
(TRAC), OPP again made the 1997 policy paper available for broader
public comment (US EPA, 1998b).

The 1997 OPP policy statement described a weight-of-the-evidence
approach for use when evaluating the data on cholinesterase inhibition
and its consequent potential adverse cholinergic effects. The SAP
expressed support for the use of such an approach (SAP, 1997). Briefly,
the SAP stated that:

> \... the weight of evidence approach is indeed reasonable and
> justified on the basis of the available scientific data [so long as
> these data are derived from rigorous experiments with standardized
> methods and proper controls]{.underline}. In particular, this approach
> allows flexibility to weight heavily inhibition in non-target tissue
> when the overall toxicologic context suggests that other approaches
> pose danger of serious risk from overexposure. (Emphasis added in
> original.)

The 1997 policy paper also proposed that the differences of opinion with
respect to the use of blood measures in risk assessment could be reduced
or resolved by the collection of peripheral nervous system tissue
measurements of AChE inhibition in animal studies which might serve
instead of the blood measures as critical effects for use in hazard
assessment. The 1997 SAP concluded that the use of blood measures Ais
readily justified if the discrepancy between blood cholinesterase and
functional endpoints is not too great@ and recommended that data on AChE
inhibition in the peripheral nervous system be collected. On these
points, the SAP also noted that:

 There was unanimous support for the notion that, under SOME
circumstances, measurement of SOME blood-borne cholinesterases would be
appropriate to consider in establishing RfDs for
anticholinesterases\....(Emphasis added in original.)

and

 Measured inhibition of cholinesterase activities in any of the blood
fractions is best regarded as an imperfect mirror of enzyme inhibition
in the true target tissues\...

OPP subsequently asked the International Life Sciences Institute
(ILSI)/Risk Science Institute to convene a workgroup to help further
define the feasibility and details for collecting these data. This
workgroup\'s report concluded that it was currently feasible to measure
AChE inhibition in the peripheral nervous system (Mileson, et. al.*,*
1999b). The ILSI workgroup further concluded, \"Methods and techniques
currently available are adequate to characterize the AChE activity in
the peripheral nervous system, but additional studies would help to
improve these methods.\"

**3. IDENTIFICATION OF THE TOXICOLOGICAL ENDPOINTS FOR ASSESSMENT OF
CHOLINESTERASE INHIBITORS3. IDENTIFICATION OF THE TOXICOLOGICAL
ENDPOINTS FOR ASSESSMENT OF CHOLINESTERASE INHIBITORS**

This Section explains the science policy decisions and rationale
specific to the evaluation of the various cholinergic effects on nervous
system function caused by anticholinesterase pesticides. This rationale
forms the basis of the weight-of-the-evidence approach described later
in Section 4. The general principles, including definitions of key
terms, and approaches used by OPP and EPA for evaluating the neurotoxic
potential of environmental agents can be found in the Agency\'s
Guidelines for Neurotoxicity Risk Assessment (US EPA, 1998a).

This Section is organized around conclusions followed by a rationale
addressing three key types of endpoints generally assessed currently for
cholinesterase-inhibiting pesticides:

1\) evaluations of physiological and behavioral/functional effects;

2\) measures of acetylcholinesterase inhibition in the neural tissues
(i.e., brain and peripheral nervous system); and

3\) measures of cholinesterase inhibition in the blood (i.e., red blood
cells and plasma).

**3.1 EVALUATION OF EFFECTS ON CHOLINERGIC FUNCTIONS.1 EVALUATION OF
EFFECTS ON CHOLINERGIC FUNCTIONS**

**3.1.1 CONCLUSIONS.1.1 CONCLUSIONS**

! Clinical signs/symptoms[^5] in humans and behavioral or physiological
effects in humans and animals provide the most direct evidence of the
potential adverse consequences of human exposure to anticholinesterase
pesticides.

! Effects observable in humans[^6] can cover a broader range than those
that can be observed in animal studies, including psychological
complaints, cognitive complaints and other subjective effects, and
performance measures of learning and memory. Human studies following
either deliberate or inadvertent exposure, nevertheless, are currently
quite limited in the scope of the evaluations made and scale of the
measurements used. As for animal studies, it is possible that one or
more effects of concern may be occurring but measures for their
evaluation were not or could not be incorporated in the study design.
Also, the generally small numbers of subjects may limit the power of the
study to detect effects of concern.

!

Evaluation of physiological and behavioral changes (i.e., functional
data) in animal studies also are limited in terms of the scope of
effects assessed and the measurements employed. As with human studies,
it is possible that one or more effects of concern may be occurring but
procedures for measuring these effects were not or could not be
incorporated in the study design, leaving the possibility of a false
negative.

! Because of the limited range of measures of behavioral and
physiological effects evaluated historically, functional data obtained
from human and animal studies should not be relied on solely, to the
exclusion of other kinds of pertinent information, when weighing the
evidence for selection of the critical effect(s) that will be used as
the basis of the RfD or RfC.

**3.1.2 RATIONALE.1.2 RATIONALE**

Many of the adverse acute and longer-term effects of anticholinesterase
organophosphorous pesticides that have been observed in humans were
described by Morgan (1989) and updated by Reigart and Roberts (1999):

> 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
> excessive contraction of the pupil of the eye (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.

As noted, many of the effects described above may be seen after acute
exposures of humans to anticholinesterase pesticides. There also are
case reports describing long(er)-term effects following acute high-level
exposures. Little information exists describing effects following
long(er)-term, low-level exposures to humans. Several cholinesterase
inhibiting chemicals from both the organophosphorous and carbamate
classes have been used and/or are being explored for usefulness in the
treatment of certain neuromuscular diseases and neurocognitive disorders
such as Alzheimer=s disease (Taylor, 1996a; Standaert and Young, 1996).
The literature describing the results of clinical trials and imposition
of treatment regimens for these drugs may be useful in providing
insights into the nature (both therapeutic and adverse) of human
responsiveness to these substances.

Increasing levels of exposure generally result in progressively more
serious effects, although the exact pattern of effects differs among
anticholinesterase chemicals and may be influenced by the age of the
patient, genetic differences, drug interactions, and other factors.
Different cholinesterase-inhibiting chemicals may, and generally do,
produce different spectra of clinical signs and behavioral effects. This
complexity, in part, may arise from differences between the absorbed
chemicals in distribution between the central and peripheral nervous
systems and differential binding in those nervous system compartments,
or differential interactions with the two major types of cholinergic
receptors (*i.e*., muscarinic and nicotinic receptors). The nature and
temporal pattern of effects also depends on the magnitude, duration, and
frequency of exposure, as well as whether metabolic activation is
needed. Perhaps one third of the effects caused by anticholinesterase
chemicals (*e.g.*, headache, confusion, tremor, and convulsions) can be
attributed primarily to effects on the central nervous system (Minton
and Murray, 1988). For many effects, however, it is difficult to
distinguish whether they are centrally or peripherally mediated or both.

For any pesticide, including cholinesterase inhibitors, OPP may require
different toxicology studies in laboratory animals, depending upon the
use and exposure patterns of the substance. Not all of these studies
include the requirement for measurement of cholinesterase activity or
the effects occurring as a consequence of its inhibition. Those that do
or could are noted with an asterisk (\*). The key studies required for a
food-use pesticide of conventional chemistry are:

! Acute oral, dermal, and inhalation lethality tests in mammals;

! Acute or subchronic (90-day) delayed neurotoxicity study in hens;

! Acute and subchronic (90-day) neurotoxicity screening battery in
rats\*, which includes:

>  Functional observational battery, which is a set of structured
> observations outside the home cage, including assessments of autonomic
> signs, pupillary response to light or pupil size, arousal, reactivity,
> posture and gait, grip strength, limb splay, and simple sensory
> reflexes (e.g., tail pinch and a sudden sound);
>
>  Automated motor activity;
>
>  Histopathology of neural tissue from animals prepared by *in situ*
> perfusion;
>
> 

Responses to visual or proprioceptive (i.e., sense of body position or
awareness of pressure) stimuli are optional, but not commonly done.

! 21-Day or subchronic (90-day) dermal toxicity study in mammals\*;

! Subchronic (90-day) inhalation study in mammals\* (if appropriate on
basis of anticipated human route of exposure);

! Two chronic toxicity studies\*, one in the rat and one in the dog;

! Two prenatal developmental toxicity studies\*, one in a rodent and one
in a non-rodent species; and

! Two-generation reproduction study in rodents\*; and

! Developmental neurotoxicity study in rats\*, which includes, in pups:

>  detailed observations, developmental landmarks, motor activity,
> auditory startle reflexes, learning and memory test, and
> neuropathology on postnatal days 11 and 60
>
>  detailed observations for neurological effects also are made in dams

While they never have been a part of EPA\'s data requirements and, thus,
there are no EPA testing guidelines for them, human hazard
identification studies on some pesticides have been submitted by the
chemical=s sponsor(s) and, in the past, prior to the passage of FQPA,
considered for use in risk assessments. These hazard identification
studies typically are designed to identify no-effect levels for
ChEI-associated enzyme activity and, sometimes, for some clinical
effects. Although many of these human hazard identification studies with
cholinesterase inhibitors are acute (i.e., single dose) in their
exposure duration, some have incorporated short-term (e.g., 4-10 day) or
longer (e.g., 21-28 day) repeat dosing. Measures of cholinesterase
inhibition in either whole blood (which is a mixture of plasma and
RBCs), or separately in RBCs and plasma are usually included. Sometimes,
reporting of some clinical symptoms and signs are included; in a few
cases, objective physiological measures, such as blood pressure, pulse
rate or temperature, have been reported.

Human hazard identification studies can be designed to detect more
effects in addition to blood enzyme inhibition (e.g., mild sweating and
nausea) compared to animal studies, due to self-reporting of complaints,
including sensory, cognitive, and psychological effects. Formal
evaluations (by interview or test), however, are very uncommon as are
measurements of physiological parameters like heart function (e.g.,
heart rate and blood pressure) and breathing rate. More sophisticated
neurobehavioral test batteries, such as intelligence tests or simple
memory tests, used in epidemiological studies (for example, Anger, et
al., 1996), are rarely, if ever, used in human hazard identification
studies of cholinesterase-inhibiting organophosphorous and carbamate
pesticides.

The reports of certain kinds of animal studies include detections of
overt clinical signs, including many of the autonomic signs, and motor
effects, such as tremors. In the rodent neurotoxicity screening battery
studies, the data are gathered systematically by observers unaware of
treatment. The measurements of effects are defined quantitatively,
albeit, usually on an ordinal scale (*e.g.*, +1, +2). Valid screening
studies also include automated and quantitative measures of motor
activity, grip strength, and limb splay, though changes in these
measures are not a distinguishing characteristic of cholinesterase
inhibition. EPA=s test guidelines for the neurotoxicity screening
batteries were published in 1991. OPP has received data from these
neurotoxicity screening studies on many of the anticholinesterase
organophosphorous and carbamate pesticides. Of the roughly 30 effects
that may occur following acute exposures as listed by Morgan (1989) and
updated by Reigart and Roberts (1999), perhaps one third would not be
seen in routine animal studies, or even in the neurotoxicity screening
battery, as they are currently designed, especially the sensory,
cognitive, and psychological effects. Thus, because of the limitations
in the study design and conduct of both human and animal studies, OPP
may not understand fully the profile of effects of concern that may
result from exposure to the cholinesterase-inhibiting pesticides.

**3.2 NERVOUS SYSTEM CHOLINESTERASE INHIBITION.2 NERVOUS SYSTEM
CHOLINESTERASE INHIBITION**

**3.2.1 CONCLUSIONS.2.1 CONCLUSIONS**

! Inhibition of acetylcholinesterase in the nervous system (both central
and peripheral) is generally accepted as a key component of the
mechanism of toxicity leading to adverse cholinergic effects. The
inhibition of this enzyme provides direct evidence of potential adverse
effects. Interference with the timely deactivation of neuronal or
neuroeffector acetylcholine results in the protraction of the actions of
acetylcholine at these sites, which in turn results in adverse
cholinergic effects. Because the inhibition of acetylcholinesterase is a
key event that can lead to adverse effects, data showing this response
provide valuable information in assessing potential hazards posed by
anticholinesterase pesticides.

! Measures of acetylcholinesterase activity in both central and
peripheral nervous tissues are important for a full assessment of the
potential for hazard because the enzyme and each chemical may have
different pharmacokinetic and pharmacodynamic properties in each
compartment of the nervous system. ! The relationships between the
functional effects and changes in acetylcholinesterase activity in both
nervous system compartments often are difficult to characterize with
existing data for a variety of reasons (e.g., development of tolerance,
heterogeneity of cholinergic pathways including the molecular form(s) of
AChE present at each location, limited data on the regional distribution
of acetylcholinesterase, the time course of inhibition in each region,
and limited evaluation of functional effects).

**3.2.2 RATIONALE.2.2 RATIONALE**

The inhibition of acetylcholinesterase is a key step in the mechanism of
toxicity of certain organophosphorous and carbamate pesticides (Mileson,
et al, 1998; Mileson, 1999a; US EPA, 1999a, 1999c), and, therefore,
measures of cholinesterase inhibition represent a critical biochemical
biomarker of potential adverse effects. Inhibition of
acetylcholinesterase in the central nervous system is considered to be
an indicator of an adverse effect. Nonetheless, reductions in neural
AChE activity may not always be accompanied by overt clinical signs or
symptoms because, for example, the critical functions of those specific
neurons may not be sufficiently evaluated to detect related changes or
tolerance may have developed. The time at which potential functional
effects are evaluated may also contribute to an apparent lack of
concordance between functional effects and the neurochemical effects
(i.e., cholinesterase inhibition). Based on these, and, perhaps, other
factors, it is difficult to determine, with accuracy or consistently,
the degree of cholinesterase inhibition that will cause specific
physiological or behavioral changes. Thus, OPP considers a
treatment-related decrease in brain or peripheral tissue AChE activity,
in itself, toxicologically important. Data showing such a decrease are
appropriate for use as a critical effect for the derivation of RfDs and
RfCs, as well as for characterizing potential human hazards.

Historically, data on central nervous system AChE inhibition have come
from single or repeated exposure animal studies, in which whole brain
homogenates are assayed at one or two time points. For the past several
years, more detailed measurements of brain AChE inhibition have been
required. These requirements, as part of the neurotoxicity screening
battery, or as separate studies, have sought to characterize the time
course of inhibition in plasma, RBCs, and brain, including in specific
brain regions, after acute and 90-day exposures. Even so, most of the
existing data sets will generally contain measures only of whole brain
AChE activity, but not usually regional brain measurements, or
time-course data, particularly following acute exposures. The lack of
regional brain measures appears to be a limitation, given that the
distribution of cholinergic pathways and the concentration and molecular
form of AChE in different brain regions is not homogenous. Thus, whole
brain measurements of AChE inhibition may reveal little or no change in
activity while masking significant changes in specific brain regions
associated with particular cholinergically-mediated functions (e.g*.*,
the hippocampus and memory).

Unfortunately, measures of AChE inhibition in peripheral neural tissues
or neuroeffector junctions are rare. AChE inhibition data from the
peripheral nervous system potentially have unique value because many of
the adverse signs and symptoms associated with exposure to
anticholinesterase pesticides (e.g*.*, diarrhea, excess salivation) are
a result of effects on the peripheral nervous system. Because of the
potential pharmacokinetic differences between the central and peripheral
nervous compartments, measures of AChE activity in both of these systems
are important for the full assessment of chemicals on the nervous
system. Certain chemicals may have equivalent access to a specific
compartment, in both degree and rate of interaction. On the other hand,
there are others for which the rate of access to, and concentration in,
peripheral tissues is far greater than in the central nervous system.
These patterns could shift with longer term exposures.

Although AChE inhibition data in peripheral nervous system tissues have
not been required in toxicological studies submitted to EPA and, at the
moment, no standard protocol exists for the generation of such data, OPP
indicated in 1997 that the collection of these measures potentially
could become an alternative to the use of blood cholinesterase
inhibition measures in animal studies in the hazard and risk assessment
process. As discussed earlier, the SAP (SAP, 1997) and an expert panel
of ILSI (Mileson, et al. 1999b) have stated that it is feasible to
measure AChE inhibition in peripheral nervous system tissues. The 1997
SAP report asserted, \"it is important that joint efforts be mounted to
evaluate AChE inhibition in the peripheral neural tissues *per se* and
in the neuroeffector junctions.\" The SAP expressed the view that it is
technically feasible to routinely conduct AChE assays on the peripheral
nervous system, while recognizing the difficulties involved. The SAP
further suggested that skeletal muscles, heart, lung, salivary glands,
diaphragm, and autonomic ganglia (*e.g.*, superior cervical ganglia) be
considered as appropriate tissues to examine. The SAP considered that
standardized and reproducible dissection and homogenization of tissue,
assays with minimal tissue dilution, selection of the most relevant
tissue targets, and standardization of tissue storage conditions were
the most important technical issues to resolve when measuring AChE
activity in the peripheral nervous system. Work is underway in EPA=s
National Health and Environmental Effects Laboratory (NHEERL) to develop
and standardize protocols for assaying enzyme activity in various
peripheral tissues (e.g., see Marshall, et al., 1999).

**3.3 BLOOD CHOLINESTERASE INHIBITION.3 BLOOD CHOLINESTERASE
INHIBITION**

**3.3.1 CONCLUSIONS.3.1 CONCLUSIONS**

! Inhibition of blood cholinesterases (i.e., plasma and red blood cell)
is not itself an adverse effect, but may indicate a potential for
adverse effects on the nervous system. As a matter of science policy,
blood cholinesterase data are considered appropriate surrogate measures
of potential effects on peripheral nervous system acetylcholinesterase
activity in animals, for CNS acetylcholinesterase activity in animals
when CNS data are lacking and for both peripheral and central nervous
system acetylcholinesterase in humans.

! As such, blood cholinesterase inhibition data are considered
appropriate endpoints for derivation of reference doses or
concentrations when considered in a weight-of-the-evidence analysis of
the entire database on a single pesticide or on two or more pesticides
assigned to a common mechanism of toxicity group, where
acetylcholinesterase inhibition is the common mechanism of toxicity.

! Red blood cell measures of acetylcholinesterase inhibition, if
reliable, generally are preferred over plasma data. Since the red cell
contains only acetylcholinesterase, the potential for exerting effects
on neural or neuroeffector acetylcholinesterase may be better reflected
by changes in red blood cell acetylcholinesterase than by changes in
plasma cholinesterases which contain both butyrylcholinesterase and
acetylcholinesterase in varying ratios depending upon the species. This
conclusion rests on data showing that chemicals may have significantly
different interactions with AChE and BuChE, including their affinities
for binding with the enzymes.

! Although RBC acetylcholinesterase data are generally preferred, in
some cases, reliance on measures of RBC may not be appropriate because
of methodological issues concerning blood measures of cholinesterase
activity. When making weight-of-the-evidence judgments concerning the
selection of RBC versus plasma measures of cholinesterase inhibition as
endpoints for derivation of reference doses or concentrations, it is
critical to consider all aspects of the information database, including
the adequacy of the study protocol, quality of the data, dose-dependency
of the responses, as well as available data on measures of brain
acetylcholinesterase inhibition and functional effects.

! Plasma contains both butyrylcholinesterase and acetylcholinesterase in
varying ratios depending upon the species. The separate characterization
of RBC and plasma measures of cholinesterase inhibition provides an
additional means of measuring effects. Additionally, having separate RBC
and plasma data allow for more informative animal-to-human comparisons.

**3.3.2 RATIONALE.3.2 RATIONALE**

As a biomarker of exposure, blood cholinesterase inhibition can be
correlated with the extent of exposure. As discussed earlier, there is
often a direct relationship between a greater magnitude of exposure and
an increase in incidence and severity of clinical signs and symptoms as
well as blood cholinesterase inhibition. In other words, the greater the
exposure, the greater the cholinesterase inhibition in the blood and the
greater the potential for an adverse effect to occur. Both plasma and
RBC measures of cholinesterase inhibition also provide:

 pharmacokinetic evidence of absorption of the pesticide and/or its
active metabolite(s) into the bloodstream and systemic circulation; and

 pharmacodynamic evidence of binding to AChE, the neural form of the
target enzyme, or to plasma BuChE, an enzyme similar in structure to
AChE

Because the interaction with AChE is widely accepted as a key event of
the mechanism of toxicity for anticholinesterase pesticides, inhibition
of this cholinesterase in the blood creates the presumption that a
chemical also is causing inhibition of neural AChE. Chemicals are
absorbed into the blood and transported to the peripheral nervous
system. Pharmacokinetically, the blood compartment and the peripheral
nervous system are \"outside of\" the central nervous system, i.e.,
separated from the CNS by the blood-brain barrier. Thus, blood measures
of cholinesterase activity are viewed as a better surrogate for the
effects on AChE in the peripheral nervous system than are enzyme changes
in the central nervous system. Because data on AChE inhibition in the
peripheral nervous system have rarely been gathered in animals, blood
cholinesterase inhibition measures are generally the only information
available to assess the potential of chemicals to inhibit AChE in the
peripheral nervous system. In human studies, blood cholinesterase
inhibition measures serve as surrogates for effects in both the central
and peripheral nervous systems because neither of these neural tissues
is available for evaluation directly. As discussed earlier, evaluations
of clinical signs and symptoms have limitations, and thus should not be
relied on solely, to the exclusion of other data. Therefore, blood
cholinesterase inhibition data are considered appropriate endpoints for
derivation of reference doses or concentrations when considered in a
weight-of-the-evidence analysis of the entire database on a single
pesticide or on two or more pesticides assigned to a common mechanism of
toxicity group, where acetyl- cholinesterase inhibition is the common
mechanism of toxicity.

The usefulness of collecting blood cholinesterase inhibition data (both
RBC and plasma) is illustrated further by its use in monitoring workers
for occupational exposures (even in the absence of signs, symptoms, or
other behavioral effects). Blood cholinesterase inhibition (RBC and/or
plasma) is considered as providing a sufficient basis for removing
workers from the exposure environment, given the assumption that if one
were to protect against enzyme inhibition, one would protect against
effects of graver concern. For example, the California Department of
Health Services (CDHS) requires monitoring of agricultural workers who
have contact with highly toxic organophosphorous or carbamate compounds
(EPA Toxicity Category I or II pesticides; LD~50~  500 mg/kg in
rats)(CDHS, 1988). CDHS removes workers from the workplace whose plasma
levels show 40% or greater cholinesterase inhibition from baseline, or
whose red blood cell cholinesterase levels show 30% or greater
inhibition. Workers may not return until their cholinesterase values
return to within 80% of baseline. The World Health Organization (WHO)
also has guidelines with the same RBC action levels (i.e., 30% or
greater inhibition), and considers plasma inhibition of 50% of baseline
to

indicate a \"toxic\" decrease (Fillmore and Lessinger, 1993). Fillmore
and Lessinger also reviewed the California program and found that \"The
relative risk of pesticide poisoning was increased in workers whose
initial baseline plasma levels were low, or if their levels had already
dropped to 60-80% of their baseline previously in the season.\"

Although a pesticide=s effect(s) on either RBC and plasma cholinesterase
activity is considered to provide information on its potential to
inhibit AChE in the nervous system, data from RBCs, which contain AChE
exclusively, may better reflect neuronal AChE inhibition than data from
the plasma, which is a variable mixture of butyrylcholinesterase and
acetylcholinesterase. As discussed earlier, acetylcholinesterase is the
enzyme involved in the mechanism of toxicity for the cholinergic effects
of anticholinesterase pesticides. Although BuChE is somewhat similar in
structure to AChE, BuChE is nevertheless sufficiently different in
important ways which often result in it having binding affinities to
anticholinesterase agents as well as other characteristics that are
quite different from those of acetylcholinesterase (Silver, 1974; Taylor
and Radic, 1994). The composition of plasma cholinesterases varies
widely among humans, dogs, and rats, the species for which these
measures are most typically made. Human plasma is overwhelmingly BuChE
with a ratio of BuChE to AChE of 1,000:1 (Edwards and Brimijoin, 1983).
In dogs, there is a little more than 10% acetylcholinesterase in plasma
with a ratio of BuChE to AChE of 7:1 (Scarsella *et al.*, 1979). In
rats, plasma contains approximately 50% or more of AChE with a BuChE to
AChE ratio of 1:3 in males and 2:1 in females (Edwards and Brimijoin,
1983). While it is technically possible to ascertain the contribution of
each ChE to the level of inhibition in plasma, this type of data is
rarely available. Thus, the relationship between blood measures of AChE
and BuChE or other factors is usually not known. For these reasons, a
treatment-related decrease in plasma cholinesterase activity, viewed in
isolation, provides less insight into the potential of a chemical to
cause neural AChE inhibition than do data on RBC AChE inhibition.

Historically, there have been technical difficulties with the
measurement of the inhibition of plasma and RBC cholinesterase(s),
particularly for the latter (see Wilson, et al., 1996). Although in
recent years there have been improvements in blood measures of
cholinesterase activity, it is important to consider carefully the
methodological issues that may affect the accuracy and variability of
the data when assessing the effects of pesticides on cholinesterase
activity in blood. There are many methods available for measuring blood
cholinesterase activity. The colorimetric method, based on the Ellman
reaction, is considered a reliable method when performed properly, and
is commonly used for measuring plasma and RBC cholinesterase activity
(Ellman, *et al.*, 1961; US EPA, 1992; ASCP, 1994). While well suited to
the measurement of cholinesterase inhibition induced by
organophosphorous pesticides, the Ellman method may underestimate
cholinesterase activity in both plasma and RBC following carbamate
exposure because of the relatively unstable binding of the carbamate
esters to the acetylcholinesterase. The radiometric method may be better
suited for measuring carbamate-inhibited cholinesterase (Johnson and
Russell, 1975; Wilson, et al., 1996). The refinement of measurement
methods continues in NHEERL. If, and when, the conduct of future animal
studies on pesticides includes the measurement of peripheral nervous
system acetylcholinesterase activity, in addition to central nervous
system measures, then less reliance can be placed on the use of blood
measures as critical effects for the derivation of RfDs/RfCs in the risk
assessment process. However, as noted above, blood measures will, in any
case, continue to provide important information in characterizing human
hazard because they still will provide a means of animal-to-human
comparison of cholinesterase inhibition. As noted above, work on
standardizing methods for measuring acetycholinesterase activity in the
peripheral nervous system is underway. OPP/EPA expects that the
standardization and use of these methods will result in a database that
improves the scientific understanding of the risks of
cholinesterase-inhibiting compounds.

**4. WEIGHT-OF-THE-EVIDENCE ANALYSIS FOR SELECTION OF CRITICAL EFFECTS4.
WEIGHT-OF-THE-EVIDENCE ANALYSIS FOR SELECTION OF CRITICAL EFFECTS**

The present science policy has been prepared considering the comments
received from the SAP and the public in 1997 and during the public
comment period in 1998. This revised policy continues to embrace the
weight-of-the-evidence approach of considering all relevant data in an
integrative manner that was described in the 1997 OPP document (US EPA,
1997). This revised policy expands the discussion of the approach and
clarifies the weight-of-the-evidence approach by describing more
explicitly under what conditions and how plasma and/or RBC
cholinesterase data would be considered. The policy also re-emphasizes
the potential usefulness of collection of peripheral neural data on AChE
inhibition to reduce reliance on the surrogate blood measures.

OPP is using the weight-of-the-evidence approach described here to
analyze individual studies as well as the complete database on a
pesticide when selecting critical effects for hazard assessment. The
primary objective of the weight-of-the-evidence analysis for
anticholinesterase pesticides is to select Points of Departure (PoDs)
(i.e., NOAELs, LOAELs, or benchmark doses) for critical effects to be
used in the calculation of RfDs, RfCs or margins of exposure (MOE) for
all of the routes and durations of exposure appropriate for a pesticide
given its use and exposure patterns when, after review of the entire
toxicological database, it is concluded that the cholinergic effect(s)
induced by the substance being evaluated do, in fact, represent the
critical effect(s). Briefly, the weight‑of‑the‑evidence approach
includes consideration of all available data on:

! clinical signs and other physiological and behavorial effects in
humans and animals;

! symptoms in humans;

! central nervous system acetylcholinesterase inhibition;

! peripheral nervous system acetylcholinesterase inhibition;

! red blood cell acetylcholinesterase inhibition; and

! plasma cholinesterase inhibition (BuChE in humans; mixed AChE/BuChE in
animals).

A comparison of the pattern of doses required to produce physiological
and behavioral effects and cholinesterase inhibition in different
compartments is conducted. In addition to these parallel analyses of the
dose‑response information, comparisons of the temporal aspects (e.g.,
time of onset and peak effects and duration of effects) of each relevant
endpoint are made. This analysis should be done for each relevant route
and duration of exposure (e.g., acute, intermediate and/or chronic
exposures) for each available species/strain/sex of animals.
Furthermore, the potential for differential sensitivity/susceptibility
of adult versus young animals (i.e*.*, effects following perinatal or
postnatal exposures) to anticholinesterase chemicals should be assessed.
These analyses should be conducted in the context of the adequacy of the
protocols used and the quality of the available data. Based on this
weight-of-the-evidence analysis for an anticholinesterase pesticide, OPP
may select as the critical effects any one or more of the behavioral and
physiological changes or enzyme measures listed above.

Although clinical signs/symptoms, physiological and behavioral changes
are considered very important for characterizing an adverse effect in
humans, these endpoints are not given disproportionate emphasis or
relied on solely, or even always necessarily preferred, in selecting
critical effects for risk assessment because the evaluations of such
endpoints have limitations. Comprehensive measures of AChE activity in
nervous system tissues, particularly as they may reflect age-related
differences, are considered very important and are given considerable
prominence in the weight-of-the-evidence analysis for selection of
critical effects because, as discussed earlier, acetylcholinesterase
inhibition is considered a key event in the mechanism of toxicity for
the cholinesterase-inhibiting organophosphorous and carbamate pesticides
and a substantial body of literature exists which links enzyme
inhibition with a broad range of adverse effects. Thus, data on
cholinesterase inhibition may be viewed as predictors of potential
adverse responses mediated via cholinergic pathways and may be used
instead of, or in the absence of, data on clinical signs and symptoms,
and other physiological and behavioral effects. Direct measures of AChE
inhibition in the neural target tissues, (i.e., central and peripheral
nervous systems) are preferred. However, when such data are missing or
inadequate, they would obviously receive less weight in the analysis. In
these circumstances, measures of cholinesterase inhibition in the blood
(plasma and/or RBC) are viewed as reasonable surrogates for the
peripheral nervous system given that the blood is the pharmacokinetic
compartment into which chemicals are absorbed and transported to the
peripheral nervous system. In animals, data on blood cholinesterase
inhibition also are considered important companion data for central
nervous system AChE inhibition data, even though the brain constitutes a
different pharmacokinetic compartment. As noted earlier, blood measures
(both plasma and RBC) of cholinesterase activity in human studies must
serve as surrogates for enzyme activity in both central and peripheral
nervous systems, in light of the lack of availability of data on these
parameters. As discussed in Section 3.3, within the blood compartment,
RBC AChE data, if reliable, are generally preferred over plasma data.
Even though plasma contains a mixture of AChE and BuChE, plasma
cholinesterase data should be evaluated and considered in the parallel
analyses as described below. Also, there may be certain situations where
plasma cholinesterase inhibition may be selected as the critical effect
for the risk assessment.

Evaluation of the statistical and toxicological significance of the
study results and application of uncertainty factors follows the
Agency\'s established procedures for derivation of an RfD or RfC and the
principles articulated in the FQPA 10X Safety Factor policy. A
description of the strengths, weaknesses, and limitations of the
database is included; this evaluation may lead to the identification of
data needed to refine the data base and the risk assessment. Any
residual concerns (i.e., significant uncertainties) are accommodated for
when making the FQPA 10X Safety Factor determination.

Practically, the weight-of-the-evidence analysis may be viewed as having
several steps: first, the individual studies are evaluated; second, all
studies in the database and their relationship to one another are
examined in an integrated manner; and lastly, the critical effects are
selected for risk assessment and additional data needs identified. Below
is a more detailed discussion of these steps.

**4.1 ANALYSIS OF INDIVIDUAL STUDIES.1 ANALYSIS OF INDIVIDUAL STUDIES**

For a full evaluation of an anticholinesterase pesticide, the important
elements of a study should include:

! Evaluations of physiological and behavioral effects;

! Measures of central nervous system acetylcholinesterase activity (in
animal studies) (often these will be whole brain measures rather than
measures in specific brain regions)

! Measures of peripheral nervous system acetylcholinesterase inhibition
(in animal studies) (rarely available at the present time);

! Measures of RBC and plasma cholinesterase inhibition.

First, each study is critically evaluated. Results should be assessed in
the context of both statistical *and* biological significance. No fixed
percentage of change (e.g. 20% for cholinesterase enzyme inhibition) is
predetermined to separate adverse from non-adverse effects. OPP\'s
experience with the review of toxicity studies with
cholinesterase-inhibiting substances shows that differences between pre-
and post-exposure of 20% or more in enzyme levels is nearly always
statistically significant and would generally be viewed as biologically
significant. The biological significance of statistically-significant
changes of less than 20% would have to be judged on a case-by-case
basis, noting, in particular, the pattern of changes in the enzyme
levels and the presence or absence of accompanying clinical signs and/or
symptoms. The study evaluation involves consideration of, among other
factors: the adequacy of study protocol and design (e.g., experimental
group size and characteristics (e.g., single gender or both), dose
spacing, methods used for neurochemical and functional evaluations);
whether pre-exposure data were obtained in the subsequently-exposed
individuals (i.e. measures are taken before and after treatment in the
same individual, then statistically analyzed as such) or a separate
control group was used, and the general conduct of the study. The
consistency of the findings within the study when repeated measures are
taken, the dose-dependency of the responses, as well as the temporal
aspects of effects (e.g., the time-of-onset, steady state, time-to-peak
effects and the time until complete recovery) and the statistical
significance of any differences measured between unexposed and exposed
groups are to be examined before reaching any conclusions regarding
biological significance. The relationship of the different effects seen
to one another also should be considered in interpreting the findings.
Following critical evaluation of the validity of the study, candidate
points of departure (i.e., NOAELs, LOAELs, or benchmark doses) are
identified or calculated.

**4.2 INTEGRATIVE ANALYSIS OF THE DATA BASE.2 INTEGRATIVE ANALYSIS OF
THE DATA BASE**

When evaluating the entire database and selecting an endpoint(s) as the
critical effect(s) to serve as the PoD in the derivation of a RfD or
RfC, parallel analyses of the dose-response (i.e., changes in magnitude
of enzyme inhibition or of a different effect with increasing dose) and
the temporal pattern of all relevant effects will be compared across all
of the different compartments affected (*e.g*., plasma, RBC, peripheral
nervous system, brain), and for the functional changes to the extent the
database permits. The overall adequacy of the test protocols and the
quality of the data also are important elements of the analysis. The
consistency (or, the lack thereof) of LOAELs, NOAELs, or BMDs for each
category of effects (e.g., clinical signs, cholinesterase inhibition in
the various compartments, etc.) for the test species/strains/sex
available and for each duration and route of exposure should be noted.
If scientifically valid, reliable, and ethically appropriate to use,
human data may be preferable to animal data because they preclude the
need for extrapolation of results across species, avoiding the
uncertainties attendant to this aspect of the risk assessment process.
Confidence in the selection of an endpoint(s) for derivation of an RfD
or RfC is enhanced by the factors described in Table 1. The findings for
anticholinesterase pesticides will span a broad continuum, and their
databases will range from those which are comprehensive and robust to
those which are limited and of poor quality. Thus, end point selection
and weight-of-the-evidence judgments must be made on a case-by-case
basis. For example, often cholinesterase inhibition data in a single
compartment may be inconsistent across studies involving the same
species or strain. In some cases, large differences may be noted in the
magnitude of cholinesterase inhibition in one compartment in comparison
to all the other compartments. In other cases, there is no dose-effect
relationship for cholinesterase inhibition in one or more compartments.
Time course data for cholinesterase inhibition also are often limited.
Brain measures of AChE activity are often limited to whole brain at
termination of an animal study. So, a typical database for an
anticholinesterase pesticide will likely contain a number of
inadequacies that can have a broad spectrum of influence from none to
substantial on the selection of critical effects. It should be
emphasized, however, that the lack of, or deficiency in, any one factor
listed in Table 1 would not necessarily discount the usefulness of a
study in selecting an endpoint for calculation of an RfD or RfC.
Functional evaluations are limited in both human and animal studies.
Therefore, as described earlier, measures of cholinesterase inhibition
are included in the weight-of-the-evidence evaluation; the reliance on
all relevant data is considered to be both scientifically sound and
public health protective. Cholinesterase inhibition in the blood may
occur at lower doses than other cholinergic effects (e.g., brain AChE
inhibition, functional effects). The NOAEL or equivalent benchmark dose
for RBC AChE inhibition and that for plasma and/or brain may not be the
same. This could be due to methodological problems or to the different
binding affinities of a pesticide to AChE compared to those for BuChE or
to a number of other factors. As explained in Section 3.3, if the
measurements of AChE inhibition in RBCs are considered methodologically
sound, these data generally are preferred over plasma cholinesterase
activity data as predictors of neural AChE activity, even if the plasma
NOAEL/BMD is lower. However, if the RBC data are unreliable (e.g.,
questions exist about the methodology or there is no dose-dependency) or
the dose response for inhibition of plasma cholinesterase more closely
approximates that for AChE inhibition in the nervous system than does
the dose response for RBC acetylcholinesterase inhibition, plasma
cholinesterase inhibition may be the more prudent endpoint to use to
represent the critical effect.

Occasionally, because of methodological difficulties or for other,
poorly-understood reasons, empirical correlations between the doses that
cause plasma and brain cholinesterase inhibition (in the same or other
studies) may be stronger than those between the doses for RBC and brain
enzyme inhibition.

The weight-of-the-evidence approach emphasizes the determination of the
quality of the cholinesterase data, especially the RBC measures.
Standard operating procedures for measuring cholinesterase activity have
continued to evolve over the last decade (Wilson, et. al.*,* 1996;
Hunter, et. al.*,* 1997); detailed information on the method(s) and
procedures used for measurements of cholinesterase activity following
treatment is important. The method used for carbamate pesticides is
particularly important because the reliability of data on cholinesterase
effects depends not only on the specific methodology used, but to a
great extent on sample processing (given the readily reversible nature
of the carbamylated AchE).

**4.3 COLLECTION OF PERIPHERAL NERVOUS SYSTEM CHOLINESTERASE INHIBITION
DATA.3 COLLECTION OF PERIPHERAL NERVOUS SYSTEM CHOLINESTERASE INHIBITION
DATA**

As discussed earlier, the 1997 FIFRA Scientific Advisory Panel endorsed
collection of peripheral nervous system AChE data as being technically
feasible and advised OPP that these data may be a better indicator of
cholinergic effects than are blood cholinesterase measures. The ILSI
Panel provided more technical guidance along with a number of
recommendations for further studies to improve the methodologies, while,
nonetheless concluding that such measures could be taken now (Mileson,
et al., 1999b). OPP agrees that peripheral nervous system measurements
from a suitable set of tissues could provide an alternative to blood
measures. OPP, with ongoing technical and research support from NHEERL,
will continue to support the development and validation of methodologies
for measuring peripheral neural AChE inhibition. This was a major aspect
of OPP=s policy in 1997 and was endorsed by the 1997 SAP. Once a
methodology is validated, data from such studies will be sought on a
regular basis and used to supplement or replace blood measures which now
serve as a surrogate for the peripheral nervous system. In the interim,
any data on peripheral tissues will be evaluated and incorporated into
the risk assessment on a case-by-case basis. OPP strongly encourages the
development of any data aimed at refining risk assessments based upon
blood measures to be focused on peripheral nervous system measures of
AChE. Additional data to differentiate between the acetylcholinesterase
and butyrylcholinesterase in plasma, a procedure recommended by the SAP
in 1997, would also be useful.

Additional studies to provide data on metabolism, pharmacokinetics and
pharmacodynamics also may be useful to aid in the characterization of
the cholinesterase inhibiting properties and potential hazard of
organophosphorous and carbamate pesticides. To lessen the uncertainties
inherent in route-to-route extrapolation, endpoint specific data could
be collected on exposure routes of interest, such as cholinesterase
inhibition following dermal exposure.

**5. CONCLUSION5. CONCLUSION**

The elements of the weight-of-the-evidence evaluations used for
selecting toxicity endpoint(s) are summarized in Table 2.
Weight-of-the-evidence judgments must be sound and supported by the data
on the individual pesticides. The risk assessor should provide a hazard
characterization that summarizes the endpoint data that are available
for consideration, discusses the strengths, limitations and
uncertainties of the data, and describes how well the data supports the
conclusions. The rationale for selection of the critical effect(s) must
be clearly articulated in this characterization.

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[^1]: Adverse effects include alterations from the baseline that
    diminish an organism\'s ability to survive, reproduce, or adapt to
    the environment. Neurotoxicity is defined as an adverse change in
    the structure or function of the central and/or peripheral nervous
    system following exposure to an agent (US EPA 1998a).

[^2]: ACritical effect@ is defined in EPA=s Integrative Risk Information
    System (IRIS) as Athe first adverse effect, or its known precursor,
    that occurs as the dose rate increases.@ Information on the
    derivation of reference doses or reference concentrations can be
    found at the IRIS web site http://www.epa.gov/ngispgm3/iris/rfd.htm.

[^3]: Draft benchmark software for hazard endpoints is currently
    available at http:\\www.epa.gov\\ncea\\bmds.

[^4]: Summary reviews of the cholinergic components of the nervous
    system and of the toxicity of anticholinesterase chemicals can be
    found in several chapters of two widely-available textbooks
    (Ecobichon (1996), Hoffman, et al*.*, (1996), and Taylor (1996a,
    1996b)) and in Dementi (1997) which served as a technical support
    document to the 1997 policy document.

[^5]:
    > 5A \"symptom\" is defined as a condition which is a departure from
    > normal function reported by the person experiencing and reporting
    > that condition (e.g., headache, nausea). A \"clinical sign\" is an
    > objectively-measured effect (e.g., heart rate, blood pressure)
    > indicative or suggestive of a condition for an individual (human
    > or animal) observed and recorded by another such as a physician.

[^6]:
    > 6 EPA is currently reviewing its policy concerning human studies
    > with respect to ethical and scientific standards for their
    > acceptability and use in risk assessments, particularly with
    > respect to decisions under FQPA. The Agency neither requires nor
    > encourages the conduct of human hazard identification studies to
    > detect potential adverse effects of pesticides. EPA has held two
    > meetings of a joint SAP/SAB (Science Advisory Board) panel
    > (December 1998 and November, 1999 (US EPA, 1998c,1999d)) on the
    > ethical elements of this issue. The Panel is expected to issue its
    > report in the Summer of 2000.