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
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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.
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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).
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       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.
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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.
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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.
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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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U.S. Environmental Protection Acencrv
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