645R97101
1997
OFFICE OF RESEARCH AND DEVELOPMENT
' National Health and Environmental Effects Research Laboratory
PESTICIDES AND CHILDREN
Health Effects Research
Progress Report
December, 1997
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CONTENTS
S
^ Introduction 3
.J Research Program Summary 4
FY96-97 Program Highlights 6
Research Progress in Base Program on Pesticides and Children
Age-Related Differences in Sensitivity to Pesticides 7
Toxicity Testing Procedures for Immature Animals 10
Research Progress in Projects Supported through Internal RFP on Pesticides
and Children
The Effects of Pesticides on Learning and Memory 12
The Biochemical Effects of Pesticides on the Central Nervous System 13
The Effects of Pesticides on the Immune System and Allergic Response .... 14
Neurochemical Changes and Behavioral Effects Induced by Pesticides 15
The Effects of Pesticides on Reproductive Toxicity 16
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
U.S. Environmental Protection Agency
*igion 5, Library (PL-12J) O
11 West Jackson Boulevard, 12th Ftaar
Chicago, II 60604-3590
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INTRODUCTION
The purpose of this report is to communicate results from the Pesticides and Children
Research Program of the EPA's National Health and Environmental Effects Research
Laboratory (NHEERL).
CONTENT
The report contains
• a summary of our Pesticides and Children Research Program, including
an explanation of its regulatory and programmatic context, the overall
program goal, the rationale for the program, and the research strategy
• two sections that describe projects supported by our base program in
Pesticides and Children
• a detailed description of the projects supported by the 1995 Pesticides and
Children initiative, including a summary of research accomplishments and
anticipated progress for the near future
The format of this report is still evolving, and we welcome feedback. Readers with
comments or requests for further information are encouraged to contact:
Sue McMaster
National Health and Environmental Effects Research Laboratory (MD-51A)
U.S. EPA
Research Triangle Park, N.C. 27711
Phone: (919) 541-3844 or FAX: (919) 541-1440
E-mail: mcmaster.suzanne@epamail.epa.gov
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PESTICIDES AND CHILDREN
RESEARCH PROGRAM SUMMARY
Infants and children differ from adults in their
dietary consumption of--and susceptibility to --
pesticides. Consequently, they may be at
increased risk from exposure to pesticides.
Protecting children from the potentially
harmful effects of pesticides is a fundamental
responsibility mandated by several environ-
mental laws. These include the Federal
Insecticide, Fungicide, and Rodenticide Act
(FIFRA), which gives EPA the authority to
regulate the distribution and use of pesticides,
and the recently enacted Food Quality
Protection Act (FQPA), which revises and
strengthens FIFRA by requiring that special
consideration be given to infants and children.
Protection is provided under the FQPA by
considering the unique diets of children,
requiring safe pesticide tolerance levels for
children, and proposing an additional safety
factor to account for potential increased
exposure and sensitivities of children. These
statutes are implemented through EPA's
Office of Prevention, Pesticides and Toxic
Substances (OPPTS) and Regional Offices.
Scientific guidance for successful
implementation of these regulations is
provided by EPA's Office of Research and
Development (ORD) through its Pesticides
and Children Research Program. NHEERL,
which conducts effects-based research within
ORD, supports the Pesticides and Children
Research Program in two ways: through an
on-going, base program on pesticides and
through a program initiated in 1995 that
specifically targets the unique susceptibility of
the young to pesticide exposures.
PROQRAMiGOAg
To advance understanding of factors that may
make children more vulnerable than adults to
the health effects of pesticides.
EPA scientists have long been concerned that
infants and children, who are undergoing
crucial periods of development and matura-
tion, might be especially vulnerable to the
toxic effects of pollutants. Differences in
metabolism, diet, and activity and behavior
patterns have the potential to place this group
at increased risk. In 1993, these concerns
were underscored by the National Academy of
Sciences (NAS) in their report, Pesticides in
the Diets of Infants and Children. This report
highlighted the critical need for research on
infants and children exposed to pesticides.
The Academy concluded that the health
effects experienced by children are different
from those of adults and that the federal
government should do more to address their
unique risks. The report set forth several
recommendations intended to safeguard the
health of infants and children, such as
reducing the degree of uncertainty in
estimates of risk due to dietary pesticide
residues, developing standardized tests using
immature animals as part of the basic
evaluation of pesticides for toxicity, and
assessing age-related differences in
sensitivity to pesticides.
ORD's research programs are founded on
principles of risk assessment. In the area of
health effects, the programs are guided by the
human health risk assessment paradigm
formulated by the National Academy of
Sciences. The risk paradigm consists of four
fundamental steps that contribute to risk
management decisions: hazard identification,
dose-response assessment, exposure
assessment, and risk characterization.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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NHEERL supports the first two steps of this
process (hazard identification and dose-
response assessment) through the
development of test methods, predictive
models, and scientific data that strengthen
regulatory and policy decisions.
NHEERL's Pesticides and Children Research
Program, which is part of a larger effort aimed
at protecting the health of sensitive
subpopulations, emphasizes two areas of
study:
• evaluating age-related differences in
sensitivity to pesticides, and
developing toxicity testing procedures
for immature animals.
The program is multidisciplinary in scope,
consisting of projects in neurotoxicology,
reproductive toxicology, immunotoxicology,
and developmental toxicology. Specific
objectives include characterizing differences
in response to pesticides as a function of age,
developing data that will help OPPTS
determine whether current pesticide tolerance
levels are sufficiently protective of children,
and improving current testing guidelines for
predicting the long-term effects of pre- and
perinatal pesticide exposures.
During 1995, NHEERL expanded the scope of
its base pesticide program by initiating an
internal Request for Proposals (RFP) to
address research needs specifically outlined
in the NAS report mentioned above. Five new
projects were awarded funding. These
projects, described at the end of this report,
augment our base program by supplementing
the database on age-dependent toxicity to
pesticides and by explaining some of the
principles of developmental toxicity. Results
will be used to evaluate current testing
guidelines and to reduce uncertainty in the
risk assessment of pesticides.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PESTICIDES AND CHILDREN RESEARCH
FY96-97 PROGRAM HIGHLIGHTS
When studies by our Laboratory demonstrated that young rats were much more sensitive
than adults to the toxic effects of chlorpyrifos, a cholinesterase-inhibiting pesticide, we began
to explore possible reasons for this difference. We have found that young rats are more
sensitive not because the target enzyme (acetylcholinesterase) is more sensitive, but rather
because they are deficient in enzymes that detoxify the pesticide.
We found quantitative differences in both the dose-response and time-course of neurological
effects resulting from exposure to chlorpyrifos. Rat pups approximating the infant/toddler
stage were about five times more sensitive to this pesticide than adults, while pups
approximating pre-adolescents were about twice as sensitive. Moreover, the onset and
recovery of both behavioral and biochemical effects was age-dependent.
Using a well-established animal model for myopia, we showed that chlorpyrifos interferes
with the visual regulation of eye growth, which helps explain Japanese reports showing a
correlation between the use of organophosphates and the incidence of myopia in human
populations.
In collaboration with the National Institute of Environmental Health Sciences on the long-
term effects of perinatal exposure to pesticides, we have shown that the primary adult effects
of early exposure to methoxychlor (an endocrine disrupting pesticide) are reproductive.
We found that the developing rat was more sensitive than the developing mouse to the
immunosuppressive effects of certain contaminants, which led to the recommendation to
include the rat as a test species in immunotoxicity testing guidelines.
PROJECTS SUPPORTERfHRQU(^jrcrERNAL|lI=iyRg;l2^16) ^rr:
Our research using a rodent model of human asthma suggests that exposure to carbaryl
causes systemic immune suppression and exacerbates immune-mediated lung
inflammation. These results were awarded Best Presentation by a Postdoctoral Fellow by
the Immunotoxicology Specialty Section at the 1997 annual meeting of the Society of
Toxicology. Related studies in immature animals will enable us to investigate the
mechanism of pesticide-enhanced asthmatic symptoms and determine the effect of age at
the time of sensitization on response.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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AGE-RELATED DIFFERENCES
IN SENSITIVITY TO PESTICIDES
Evaluating age-related differences in pesticide
toxicity is an integral part of pesticide research
in NHEERL. Our approach is to expose
laboratory animals to pesticides during
different stages of development and examine
the effect of age on toxic response and
toxicokinetics (pesticide distribution and
detoxification by the body). Classes of
pesticides under investigation include
pyrethroids, endocrine disrupters (which exert
their toxicity by mimicking or interfering with
the actions of hormones during development),
and cholinesterase inhibitors. The studies are
multidisciplinary in scope, involving analyses
of developing organ systems, changes in
behavior, and biochemical responses.
Special emphasis is being placed on neural
growth and differentiation, neurobehavioral
changes, neurochemistry, allergic response,
immune function, and reproductive
competency and function. Results from these
studies are improving our understanding of
the principles of developmental toxicity and
the differences in response between adults
and children. When viewed in their entirety,
these studies provide valuable insights into a
number of issues of concern to risk
assessors, including the extrapolation of
animal data to humans, the adequacy of
current uncertainty factors used in calcula-
tions of risk assessment, the identification of
the most appropriate adverse effect for
calculations of the reference or benchmark
dose, and the determination of safe levels
(tolerances) of pesticide residues.
RESEARCH PROGRESS
During the early 1990s, our research
demonstrated that young animals can be up
to 21 times more sensitive than adults to the
neurotoxic effects of a pyrethroid insecticide.
We subsequently showed that this difference
in sensitivity was due to immature metabolic
processes which, upon maturation in adult
animals, led to the detoxification of the
pesticide. Similar age-dependent effects
were found with cholinesterase-inhibiting
pesticides: we showed that young rats were 2-
9 times more sensitive than adults to the
lethal effects of some organophosphates and
carbamates.
Because cholinesterase-inhibiting pesticides
share a common mode of toxicity (the
inhibition of acetylcholinesterase activity), it
was generally believed that their toxic effects
would likewise be similar. However, our more
recent research (from FY95 to the present,
discussed below) indicates that the effects of
this class of pesticide are not the same across
all compounds, and that differences in
response occur among age groups.
Neurobehavioral changes and neuro-
chemical responses. We are examining
age-related differences in the neurotoxicity of
pesticides by systematically measuring
behavioral changes and biochemical
responses in rats at various stages of
development (equivalent to infants and
toddlers, pre-adolescents, and young adults).
Effects on learning and memory, motor
activity, sensory function, cholinesterase
activity, and cholinergic receptor binding are
being investigated. Exposure parameters
include dosing duration (acute vs. repeated),
age at the time of exposure, and age at the
time of testing. The purpose of these studies
is to compare the dose-response and time-
course of behavioral and biochemical
changes in rats of different ages.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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Our results with chlorpyrifos, a cholinesterase-
inhibiting pesticide, indicate that there are
quantitative differences in both the dose-
response and time-course of neurological
effects, and that the magnitude of these
differences depends on the age of the animal
at the time of exposure. In dose-response
studies conducted in FY95, we confirmed that
very young animals are more sensitive to the
acute neurotoxic effects of chlorpyrifos: we
found that seventeen-day-old pups
(approximating the infant/toddler stage) are
about five times more sensitive than adults,
while 27-day-old pups (approximating pre-
adolescents) are about twice as sensitive as
adults. In FY96, we investigated the time-
course of these effects in young and adult
animals. We found that the onset and
recovery of both behavioral and biochemical
effects depends on age. Our studies of
chlorpyrifos are near completion, and
corresponding studies will be conducted using
aldicarb, a carbamate pesticide. Because
aldicarb has different kinetic properties than
chlorpyrifos, the contrast between the two
pesticides will contribute to our understanding
of the importance of kinetics to differences in
susceptibility. We also intend to evaluate the
effects of repeated (as opposed to acute)
exposure to chlorpyrifos in order to establish
age-related differences between exposure
scenarios. (Contacts: V. Moser, S. Padilla,
Neurotoxicology Division)
Toxicodynamic and toxicokinetic studies.
The above-described findings indicate that
young rats are more sensitive to the toxic
effects of cholinesterase-inhibiting pesticides,
but the mechanisms that account for the
enhanced sensitivity are unclear. For this
reason, we have begun to examine
toxicodynamic and toxicokinetic factors that
might help explain age-related differences in
sensitivity. We felt that if we could
understand the mechanism involved, it would
be easier to extrapolate our findings to the
human situation.
In toxicodynamic studies conducted during
FY96, we showed that acetylcholinesterase in
young rats is not more sensitive than adult
acetylcholinesterase to inhibition by anti-
cholinesterate pesticides (including chlor-
pyrifos, aldicarb, carbaryl, and malathion).
This suggests that the sensitivity of young
animals is not due to the obvious
toxicodynamic consideration. Therefore, we
turned our attention to toxicokinetic factors.
We concentrated our toxicokinetic studies on
two common enzymes that detoxify
organophosphate pesticides: A-esterase
(chlorpyrifos-oxonase) and carboxylesterase.
In studies conducted during FY96-97, we
examined the activity profiles of these two
enzymes in young and adult rats. Our
developmental profiles indicated that young
rats have low levels of these enzymes in their
liver and plasma relative to adults. As the
animals mature, their enzyme levels increase,
and the increase correlates with increased
resistance to chlorpyrifos toxicity. These
results suggest that kinetic differences play a
major role in age-related sensitivity to
chlorpyrifos. It appears that young rats are
more sensitive than adults to cholinesterase-
inhibiting pesticides not because the target
enzyme (acetylcholinesterase) is more
sensitive, but rather because they lack
enzymes necessary for detoxifying the
pesticide. While the carboxylesterase
detoxification pathway is probably not an
important issue in humans (humans have very
little carboxylesterase), A-esterase activity
levels may be meaningful. Young humans
(up to the age of two), like young rats, are
deficient in A-esterase activity, which implies
that infants and toddlers may be more
sensitive than adults to pesticides like
chlorpyrifos. (Contacts: S. Chanda, S.
Padilla, Neurotoxicology Division)
Ocular toxicity. On-going studies are testing
the hypothesis that exposure to organo-
phosphate pesticides during development
results in myopia and that the effect is age-
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
8
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dependent. These studies reflect an interest
in Japanese reports showing a correlation
between the use of organophosphates and
the incidence of myopia in human
populations. During FY96, we tested this
hypothesis by exposing chickens, a well-
established animal model for experimental
myopia, to chlorpyrifos during early post-
hatching development (days 2-9). Our results
showed that chlorpyrifos interferes with the
visual regulation of eye growth, which could
potentially lead to a greater incidence of
myopia in exposed populations. (Contact: W.
Boyes, Neurotoxicology Division)
Long-term effects on the nervous,
immune, and reproductive systems. In
collaboration with the National Institute of
Environmental Health Sciences (NIEHS), we
are evaluating the long-term effects of
perinatal exposure to pesticides. The
objective is to determine whether early
exposure to pesticides results in persistent
changes to major biological functions (i.e.,
functional effects manifested in adulthood).
We are exposing rats in utero (and up to 42
days after birth, depending upon the effect
under evaluation), after which changes are
assessed in the developing nervous, immune,
and reproductive systems.
Our first studies were conducted during FY95-
96 with methoxychlor, a known endocrine-
disrupting pesticide. Endocrine-disrupting
chemicals have received widespread attention
in recent years, and many of their reported
effects are related to reproductive dysfunction
and developmental abnormalities. As
expected, few neurological or immunological
alterations were detected. However, the
reproductive function of these rats was
severely compromised, and we have
concluded that the primary effects in adults of
early exposure to methoxychlor are
reproductive.
During FY97, we completed most of our
planned testing of carbaryl, a cholinesterase
inhibitor. Although data analyses are not yet
complete, preliminary results indicate that
substantial carbaryl exposure during
development has few adverse effects on adult
immune or reproductive endpoints. (The
results of neurobehavioral testing are
discussed on page 13 of this report.)
Studies of the long-term effects of chlorpyrifos
were begun late in FY96. Additional
pesticides to be studied include parathion,
atrazine, and trichlorfon. (Contacts: V. Moser,
S. Padilla, S. Barone, R. MacPhail,
Neurotoxicology Division; R. Smialowicz,
Experimental Toxicology Division)
Reproductive toxicity. Scientists in our
Experimental Toxicology Division are studying
the effects of an endocrine-disrupting
pesticide, dichlorodiphenylethylene (DDE, a
metabolite of DDT) on the disposition of
testosterone in adult and developing rats.
During FY96-97, we measured the disposition
of DDE in the tissues of adult rats and, as
expected, found that it sequestered in tissues
with high lipid content. We also studied the
anti-androgenic effects of DDE and observed
a dose-dependent decrease in seminal
vesicle weight. These and other of our data
are being used to develop a pharmacokinetic
model in adult rats. Presently, we are
conducting research on the developing rat.
We are designing a method in fetuses to
detect anti-androgenic activity following in
utero exposure to DDE. We plan to
extrapolate our pharmacokinetic model in
adult rats to simulate in utero disposition and
response. (Contact: T. Leavens,
Experimental Toxicology Division)
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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TOXICITY TESTING PROCEDURES
FOR IMMATURE ANIMALS
One of the cornerstones of the OPPTS
regulatory program is the compendium of
testing guidelines used to gather toxicological
information on pesticides. These guidelines
are required by FIFRA for pesticide regis-
tration arid reregistration. OPPTS relies upon
NHEERL and others in ORD to help develop,
validate, revise and review these guidelines.
The development of tests that specifically
address the toxicity of pesticides in immature
animals is clearly needed if infants and
children are to be adequately protected.
Studies are currently underway in NHEERL
that could lead to the revision of current
testing guidelines for developmental
toxicological endpoints, including teratogenic,
reproductive, neurological, and immunological
effects. We are studying different exposure
scenarios, biochemical. changes, and
behavioral endpoints in established rodent
models in an effort to improve existing test
procedures, and we are developing and
validating new test methods to augment the
current guidelines. Particular attention is
being paid to methods and models that
provide insight into potentially sensitive
subpopulations, including children. We also
are assisting in the revision of the multi-
generational testing protocol, which will lead
to internationally harmonized testing
guidelines. This research serves to enhance
EPA's ability to obtain relevant health effects
data from manufacturers of pesticides and
industrial chemicals and to improve the quality
of risk assessments.
RESEARCH PROGRESS
Neurotoxicity. Because the central nervous
system (CNS) continues to develop after birth,
it may be especially vulnerable to toxicant
exposure during developmental periods. The
objective of this research is to evaluate
diverse endpoints associated with growth and
differentiation of the CNS to determine
whether developing animals are uniquely
sensitive to the neurotoxicity of pesticides.
Prior to FY95, our Laboratory showed that
neurobehavioral tests used in rodent studies
were only appropriate for pups that were at
least 16 days old; before day 16, pups have a
limited behavioral repertoire. No attempt had
been made to develop a test in which a single
endpoint could be used to assess behavior
throughout the lifetime of the animal. We
therefore initiated a study to establish
baseline control levels using a modified
functional operational battery protocol at 17
days of age. In FY95 we reported our findings
from this study, which revealed informative
developmental profiles for each of the test
measures (e.g., sensory responses and
neuromuscular tests). For some of the
endpoints, we found significant differences
between males and females emerging at the
age of sexual maturity (from days 40 to 60).
(Contact: V. Moser, Neurotoxicology Division)
Currently, studies are underway to examine
the developmental expression of biochemical
markers of effect. Results from these studies
are being used to complement our projects
that have predominantly behavioral
components. We are studying the enzymes
acetylcholinesterase (AChE) and butyryl-
cholinesterase (both of which are
hypothesized to play critical roles as CNS
growth regulators) and the neurotransmitter,
acetylcholine. In FY96, we evaluated patterns
of enzyme activity in the brain and showed
that regional activity of the two enzymes is
qualitatively different in pups relative to adults.
We are now attempting to determine whether
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
10
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inhibition of these enzymes by cholinesterase-
inhibiting pesticides can lead to alterations in
neural development. Initial studies are being
conducted with chlorpyrifos. We are
examining developmental patterns of enzyme
activity to determine whether neurotrophic
mechanisms have been altered at the cellular
or molecular level. These measures will then
be compared to analyses of other molecular
milestones of development and to behavioral
and other developmental landmarks. Future
studies of the effects of developmental
exposure to chlorpyrifos on regional
acetylcholine levels, a putative developmental
morphogen, are in the planning stages.
(Contacts: S. Barone, S. Padilla,
Neurotoxicology Division)
We also are evaluating subtle neurotoxic
effects from pesticide exposures, such as
deficits in learning and memory. Eyeblink
conditioning is an example of a test method
used to study chemical-induced alterations in
learning. This learning procedure can be
assessed in animals and humans from
virtually any age group. Our rodent models of
cognitive development have made it possible
to examine this neurotoxicant effect and to
relate the effect to underlying neurological
mechanisms. Moreover, we can compare
effects directly across age. Studies
conducted during FY95 contributed to an
understanding of the underlying neural
circuitry controlling the eyeblink response.
We found that early disruption of the
developing cerebellum impairs eyeblink
conditioning. These data are important
because they help us determine the site of
action for pesticides having effects in this test.
(Contacts: M. Stanton, Neurotoxicology
Division)
Another test method that addresses
neurotoxicity is visual contrast sensitivity
(VCS). During FY96, our scientists showed
that using VCS scores in analyses of compu-
terized test results helps differentiate cognitive
and sensory effects in children. VCS is
already being used in medical diagnosis and
subclinical detection of neurotoxicity in adults,
and the possibility of using such a test in
children may be useful in future studies
related to the FQPA. Because of the
apparent sensitivity of the VCS test for
detecting neurotoxicity in humans, we plan to
develop a similar procedure in animals.
(Contact: K. Hudnell, Neurotoxicology
Division)
Immunotoxicity. NHEERL scientists played
a crucial role during FY95-96 in developing
guidelines for immunotoxicity testing. One of
the key recommendations for testing--the
inclusion of the rat as a test species in
addition to the mouse-would not have been
possible without the research conducted in
our Laboratory. Our studies had shown that
the developing rat was more sensitive than
the developing mouse to the immuno-
suppressive actions of certain contaminants.
(Contact: R. Smialowicz, Experimental
Toxicology Division)
Developmental toxicity. Standard testing
guidelines for developmental toxicity generally
involve dosing pregnant animals during the
period of major organogenesis. Concerns
have been raised about possible confounding
factors in study design, such as isolation of
pups from the mother during the exposure
period, which may adversely affect the growth
and development of the offspring. We
conducted a study during FY96 to examine
this potential confounder, and results showed
that maternal separation stress in itself did not
appear to affect reproductive development.
(Contacts: C. Lau, G. Klinefelter,
Reproductive Toxicology Division)
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PROJECTS SUPPORTED THROUGH INTERNAL RFP ON PESTICIDES AND CHILDREN
The Effects of Pesticides on Learning and Memory
Research is being conducted to assess age-related differences in subtle neurobehavioral effects
of pesticides. We are exposing rodents perinatally to cholinesterase-inhibiting pesticides shown to
affect learning (carbaryl and chlorpyrifos), and then evaluating the animals for learning deficits and
memory impairments once they reach adulthood. Complementary evaluations are being performed
in rodents exposed as adults. We plan to relate results from our learning tests with neurochemical
endpoints to determine whether changes in learning and memory can be correlated with changes
in neurochemical responses.
(RlSECfiCHTQUESTIONJ
Does early exposure to pesticides produce long-lasting effects on learning and memory?
Can better tests be developed to detect the effects of pesticides on learning and memory?
By addressing age-related differences in sensitivity to the developing nervous system, this proposal
responds to an area of concern specifically identified by the NRC report. Different hierarchical levels
of learning and memory will be studied, the results of which may be used to document differences
in risk based on age of exposure. Tests for cognitive function, if found useful for evaluating the
neurotoxic potential of pesticides, may lead to a revision of testing guidelines for new and existing
pesticides.
During FY96-97, the neurological effects of carbaryl were assessed in a perinatal dosing study. We
administered several learning tests, including 1) habitation of motor activity in a novel environment,
2) initial acquisition of an operant (bar-press) response, 3) acquisition of intermittently reinforced
operant behavior patterns, and 4) repeated acquisition of operant response sequences. Our
findings indicated no effect of perinatal exposure on the habituation of motor activity in adulthood.
Experiments with chlorpyrifos-also perinatally exposed-are now underway. Thus far, we have
observed no treatment-related effect on habituation, spontaneous (operant-level) bar pressing, or
initial acquisition of the operant bar-press response. Effects on the acquisition of intermittently
reinforced behavior patterns in perinatal chlorpyrifos-treated adult rats are currently being analyzed.
For more information contact: Robert C. MacPhail, Ph.D.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PROJECTS SUPPORTED THROUGH INTERNAL RFP ON PESTICIDES AND CHILDREN
The Biochemical Effects of Pesticides on the Central Nervous System
Research is being conducted to determine whether exposure to cholinesterase-inhibiting pesticides
(carbaryl and chlorpyrifos) causes toxic effects to the central nervous system (CNS) that are
expressed differently in developing and adult animals. Our specific aims are to develop an exposure
strategy, measure key neurochemical endpoints associated with growth and differentiation of the
CNS in young and adult animals, and determine mechanisms of action. The mechanisms we are
investigating include the unique trophic role of acety- and butyryl-cholinesterase during
development, the role of acetylcholine as a morphogen, and the role of cholinergic tone in
modulating neurotrophic factors (e.g., nerve growth factor and brain-derived growth factor).
vr •<•.•— ^^.~K^fry^
•>> :"' ';:,:•;:-, •*•;-•
Are the neurochemica, neuroanatomica, or functional effects of exposure to pesticides
different in the young and adult CNS?
Does exposure to cholinesterase-inhibiting pesticides result in qualitatively different effects
based on alternative mechanisms of action?
What is the effect of early exposure to pesticides on growth and differentiation of the CNS?
NRC RECOMMENJMTIO^^ ,:ft j - -^:::,;
Due to the broad range of biochemical assessments that will be made, this research will contribute
to the database on pesticide toxicity, which addresses the NRC's concern regarding paucity of data.
Qualitative (and possibly quantitative) differences in response between the mature and developing
CNS will likely be observed. The in-depth assessment of neurochemical markers of effect will be
useful for risk assessment and will complement similar projects with predominantly behavioral
components.
During FY96-97, we conducted studies to assess developmental outcome following late gestational
exposure to chlorpyrifos. We measured a variety of neurochemical endpoints (including cholin-
esterase activity, regional DNA and protein content, and serum thyroid hormone levels) in different
regions of the brain; we examined developmental landmarks (e.g., eye opening, vaginal opening,
estrus cyciicity, and testes weights); and we conducted long-term assessments using the Functional
Observational Battery, motor activity, and Morris water maze. We are continuing to follow the long-
term effects of gestational exposure to chlorpyrifos. Our analysis of the time-course and degree of
cholinesterase activity following exposure revealed that the fetus is not necessarily protected by the
placenta from pesticide exposure. Our data indicate that the dynamic increase in synthesis of
cholinesterase in the fetus may mask the real exposure of the fetal nervous system. Current work
continues to focus on neurotrophic mechanisms (quantitative and qualitative differences in acetyl-
and butyryl-cholinesterase, acetylcholine, and neurotrophins resulting from developmental exposure
to chlorpyrifos). This mechanistic approach includes both in vivo and in vitro examinations of
markers of proliferation and differentiation.
For more information contact: Stanley Barone, Ph.D.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PROJECTS SUPPORTED THROUGH INTERNAL RFP ON PESTICIDES AND CHILDREN
The Effects of Pesticides on the Immune System and Allergic Response
The impact of pesticide exposure on the development of allergy to house dust mites is being studied
in adult and young animals using dieldrin and carbaryl. We are using an established rodent model
for allergic sensitization to test two hypotheses: 1) that exposure to pesticides promotes the
development of allergic sensitization to house dust mites, and 2) that this effect is greater in young
animals than in mature animals. Investigators are focusing on a tightly drawn and clinically relevant
set of parameters to evaluate immune function, pulmonary hyperreactivity, and lung inflammation.
Does early exposure to pesticides impair the immune system?
Can pesticides help trigger asthmic responses?
Are there biochemical changes (biomarkers) that represent an early indication of exposure
to pesticides?
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Data from these studies should indicate whether the young represent a sensitive subpopulation for
immune response and whether pesticide exposures play a role in the development of allergic lung
disease. Dose-response relationships will be used to extrapolate information from animal toxicology
studies to pesticide-exposed human populations. Mechanistic information on immune responses
obtained from this study will prove useful to risk assessment.
During FY96, the impact of exposure to carbaryl on the development of an allergic response to
house dust mites (HDM) was studied in adult animals using a rodent model developed by our
Laboratory. This model exhibits key features of human asthma, such as airway hyperreactivity. Our
experimental design involves sensitizing rats with HDM either systemically (by injection) or locally
(intratracheally) and then exposing them orally to a pesticide for a two-week period. At the end of
the exposure period, the animals are challenged with HDM, and various immunological endpoints
are measured. Results suggest that low doses of carbaryl given to systemically sensitized rats
cause systemic immune suppression, while high doses up-regulate local specific immune response
to antigen and exacerbate immune-mediated lung inflammation. These results were presented at
the 1997 annual meeting of the Society of Toxicology and were awarded Best Presentation by a
Postdoctoral Fellow by the Immunotoxicology Specialty Section. More recently, results from locally
sensitized animals have shown that carbaryl plays an inhibitory role in HDM-induced asthma. In an
effort to understand the mechanism of pesticide-enhanced asthmatic symptoms, our future studies
will examine the influence of carbaryl on production of mediators of inflammatory response
(cytokines) in both adult and immature, 3-week-old rats. It is hoped that by examining young
animals we can determine the effect of age at the time of sensitization on response.
For more information contact: Mary Jane Belgrade, Ph.D.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PROJECTS SUPPORTED THROUGH INTERNAL RFP ON PESTICIDES AND CHILDREN
Neurochemical Changes and Behavioral Effects Induced by Pesticides
Research to test the behavioral and neurochemical effects of exposure to pesticides (chlorpyrifos
and methoxychlor) is being performed in young and adult rats to explore age-related differences in
neurotoxicity. We are evaluating the affect of acute and chronic pesticide exposures on behavioral
endpoints (learning and memory) using a T-maze and the eye blinking response. Both long-term
and immediate effects are being studied. Changes in behavior will be related to neurochemistry in
the brain and thyroid.
RESEARCH(QUESTIONS
What are the long-term and immediate effects of exposures to pesticides and do they differ
in mature and immature animals?
How do behavioral effects (changes in learning and memory) relate to effects on
neurochemical processes?
What are the effects of early exposure to pesticides on the endocrine system?
NRC RECOMMENDATIONS ADDRESSED!
This project is evaluating pesticide effects on the neural and endocrine systems, both of which were
identified as areas of concern by the NRC. Our objective is to produce empirical evidence regarding
qualitative and quantitative differences in the effects of pesticides across age, thereby helping to
fill gaps in data. By linking behavioral effects with effects on underlying neurochemical processes,
this research is contributing to the development of biologically-based approaches to risk
assessment. This research is also helping to determine the adequacy of current animal testing
guidelines by addressing the importance of age of exposure and by possibly developing more
sensitive biological or behavioral methods for use in these evaluations.
Postdoctoral fellows were recruited for this project during FY96 and joined the Laboratory in early
FY97. Studies are now underway comparing weanling and adult rats for effects of acute exposure
to chlorpyrifos on cognitive and neurochemical endpoints; these studies should be completed during
FY98. Effects of chronic exposure will be examined later in FY98.
For more information contact: Mark E. Stanton, Ph.D.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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PROJECTS SUPPORTED THROUGH INTERNAL RFP ON PESTICIDES AND CHILDREN
The Effects of Pesticides on Reproductive Toxicity
This research will determine how estrogenic and anti-androgenic pesticides administered at a young
age affect the development and function of the mature reproductive system. Rabbits will be used
in this research because its prolonged period of reproductive development closely approximates the
infancy-adolescence phase in humans. However, the rabbit model has not been used routinely in
studies of this type; therefore, a subordinate objective of this research is the establishment of the
rabbit as a relevant animal model for study of male reproductive competency. Animals will be
exposed to o,p-DDT and p,p'-DDE during development and evaluated for alterations in endocrine
markers, semen (sperm number, motility, and morphology), fertility, and reproductive organ
changes.
RESEARCH QUESTIONS
Is the reproductive system of the young more susceptible to pesticides than that of the
mature animal?
What exposure levels pose a risk?
Is the rabbit a good model for reproductive toxicity?
What are the effects of pesticides on endocrine markers, on semen (sperm motility,
morphology), on fertility, and on reproductive organ development?
This study should provide qualitative information on the susceptibility of the developing reproductive
system to pesticides, it should provide quantitative (dose-response) data that will enhance
biologically-based risk assessment models, and it should provide mechanistic explanations useful
for human risk assessment. Moreover, results from this study will be used to determine the
appropriateness of the rabbit model for reproductive toxicity testing; these methods could then be
applied to OPPTS testing guidelines.
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This project is in the initial design phase.
For more information contact: Gary R. Klinefelter, Ph.D.
PESTICIDES IN THE DIET OF INFANTS AND CHILDREN
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