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Science Issue Paper:
Chlorpyrifos Hazard and Dose Response
Characterization
August 21, 2008
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Prepared by:
Health Effects Division
Office of Pesticide Programs
US Environmental Protection Agency
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TABLE OF CONTENTS
1.0 Introduction 4
2.0 Updated Points of Departure & Uncertainty Factors 8
2.1. Pathways of Exposure 8
2.2. Summary of 2000 Human Health Risk Assessment 9
2.3. Data Available for Consideration 10
2.3.1. Human Information 10
2.3.1.1. Deliberate Dosing Studies in Human Subjects 10
2.3.1.2. Epidemiology Studies 10
2.3.1.3. Studies on Metabolism and Toxicokinetics 10
2.3.2. Animal Studies 10
2.4. Dose Response Assessment 10
2.4.1. AChE Inhibition 10
2.4.2. Effects on the Developing Nervous System 10
2.5. Proposed Points of Departure (PoD) 10
2.5.1. Oral Route 10
2.5.2. Dermal & Inhalation Routes 10
2.6. Extrapolation/Uncertainty Factors 10
2.7. Issues for the FIFRA SAP 10
3.0. Summary of Key Data Used in PoD & UF Determination 10
3.1. Metabolism and Toxicokinetics 10
3.1.1. Metabolic Profile 10
3.1.1.1. Ontogeny of Metabolic Processes in the Young 10
3.1.1.2. Metabolic Changes During Pregnancy 10
3.1.2. Data on Tissue Dosimetry 10
3.1.3. Conclusions 10
3.2. Inhibition of Acetylcholinesterase (AChE) 10
3.2.1. Gestational exposure 10
3.2.2. Post-natal, acute exposures 10
3.2.3. Post-natal, repeated exposures 10
3.2.4. Method of administration 10
3.2.5. Preliminary conclusions 10
3.3. Effects on the Developing Nervous System 10
3.3.1. Behavioral Effects in Rats 10
3.3.2. Behavioral Effects in Mice 10
3.3.3. Discussion 10
3.4. Human Epidemiology: Observations in Children 10
3.5. Extrapolation Factors 10
4.0 Summary & Next Steps 10
5.0 References 10
Attachment 1.0.RBC and brain ChE activity in dams and fetuses from
comparative ChE studies following gestational exposure (From
USEPA, 2006, Section II.B.2) 10
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Table of Figures
Figure 1. Schematic of decision points in the updated chlorpyrifos hazard identification 5
Figure 2. Major metabolic pathways of chlorpyrifos metabolism (Reproduced from
Timchalk et al, 2005) 10
Figure 3. Plot of brain AChE inhibition in post-natal pups following a single dose of 1
mg/kg 10
Figure 4. Plot of brain AChE inhibition in post-natal pups following repeated dosing at
1.5 mg/kg 10
Figure 5. Multiple possible mechanisms of chlorpyrifos (From Slotkin 2006) 10
Figure 6. Mental Development Index (MDI) results from CHAMACOS, Mt. Sinai, and
Columbia University 10
Figure 7. Dose-response of chlorpyrifos oxon on inhibition of brain AChE activity.
Extracted from Figure 2.a in Cole et al (2005) 10
Table of Tables
Table 1. Toxicological endpoints and uncertainty factors selected in the 2000 human
health risk assessment for chlorpyrifos 10
Table 2. Summary of Benchmark Dose Analyses for Acute and Repeated AChE Studies
in Rat 10
Table 3. Summary of tests in Adults (at least 5 weeks of age) Following Gestational
and/or Early Postnatal Dosing of 1 mg/kg/day Chlorpyrifos 10
Table 4. Potential composite factors for chlorpyrifos 10
Table 5. Summary of repeated studies evaluating gestational exposure to maternal rats
and fetuses 10
Table 6. Summary of acute studies evaluating post-natal exposure to juvenile rats 10
Table 7. Summary of repeated studies evaluating post-natal exposure to juvenile rats. 10
Table 8. Effects following gestational or postnatal exposure to 1 or 5 mg/kg chlorpyrifos
administered subcutaneously in DMSO 10
Table 9. Preliminary Results of DDEF analysis for Intra-Species Extrapolation for TK
(UFAK) based on PON 1 Activity 10
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1.0 Introduction
Chlorpyrifos (0,0-diethyl-0-3,5,6-trichloro -2-pyridyl phosphorothioate) is a broad-
spectrum, chlorinated organophosphate (OP) insecticide. In 2000, nearly all residential
uses were voluntarily cancelled by Dow AgroSciences but agricultural use remains.
The 2000 human health risk assessment was largely based on adult laboratory animal
data (rat or dog) for cholinesterase inhibition and the application of default uncertainty
factors. Since 2000, there has been extensive research on various aspects of
chlorpyrifos including its neurological effects in animals and humans following
gestational and post-natal exposures, its pharmacokinetics, and mechanism of action.
Additional, there are currently several regulatory efforts on-going for chlorpyrifos which
have led EPA to update the hazard assessment and hazard characterization of
chlorpyrifos. These efforts include the review of chlorpyrifos under the Pesticide
Registration Improvement Act (PRIA) and registration review. In addition, In addition,
the Natural Resources Defense Council (NRDC) and the Pesticide Action Network
North America (PANNA) have petitioned the Agency to revoke all tolerances and cancel
all uses of chlorpyrifos1.
In recent years, U.S. and international efforts have made significant improvements in
the scientific basis for human health risk assessments by increasing the use of
mechanistic and kinetic data, and the Agency has emphasized the use of mode of
action information in characterizing potential health effects of exposure to environmental
agents (US EPA, 2005). International efforts including ILSI's (International Life
Sciences Institute) and the International Programme on Chemical Safety's (IPCS)
human relevance framework for evaluating the qualitative and quantitative relevance of
a particular animal model of action in humans discuss the use of chemical specific and
generic data when considering animal to human extrapolation (Seed et al, 2005; Meek
et al 2003, Boobis et al, 2008). The IPCS guidance on developing Chemical Specific
Adjustment Factors (CSAFs; WHO, 2005) describes the use of kinetic and mechanistic
data to derive inter- and intra-species extrapolation factors based on data instead of
reliance solely on default factors. Consistent with these efforts, this issue paper and the
associated appendices emphasize toxicokinetics (TK) and toxicodynamic (TD) data to
evaluate and apply the new chlorpyrifos research from animals and humans.
Figure 1 provides a schematic of the key steps involved in the update to the
chlorpyrifos hazard/dose-response characterization. The first step involves selecting a
point of departure (PoD). A PoD is a point on the dose-response curve which is at the
low end of the observable data and is used as the starting point for extrapolation. A key
component of hazard assessment is the selection and determination of the critical effect
for predicting and estimating human health risk. The Agency's goal is to select a critical
effect that will be protective for other toxicities. Depending on the compounds chemical
and toxicity profile/characteristics, different critical effects may (or may not) be
1 The Agency notes that the NRDC petition includes multiple science issues, many which are addressed
in this issue paper. Some other issues, particularly results of the Agricultural Health Study and exposures
related to volatilization of chlorpyrifos are not addressed here but are being addressed in other on-going
efforts.
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manifested for different durations of exposure, routes, or lifestages. Historically the
Agency has selected PoDs for chlorpyrifos and other OPs based on inhibition of
acetylcholinesterase (AChE). An important component of the current analysis is the
comparison of doses causing inhibition of AChE at various durations, routes, and
lifestages with doses resulting in other toxicities.
Figure 1. Schematic of decision points in the updated chlorpyrifos hazard
identification
Step 1
Establish Point
of Departure
(PoD)
Select the
toxicologic
al effect?
Determine
the value
Benchmark
dose modeling
Cholinestesterase
Inhibition in
blood, peripheral
tissues, or brain
NOAEL/LOAEL
Approach
Other toxicities
(e.g., behavior)
Step 2
Animal to
Human
Extrapolation
PBPK/PD Modeling
Determine level of
refinement based on
available data &
models
Data-Derived Extrapolation
Factors Using PK and/or PD
Default Approach, 10X
StepS
Within Human
Variability
Determine level of
refinement based on
available data &
models
PBPK/PD Modeling
Data-Derived Extrapolation
Factors Using PK and/or PD
Default Approach, 10X
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PoDs can be no-observed-effect-levels (NOAELs), lowest-observed-effect-levels
(LOAELs), or derived from benchmark dose (BMD) modeling. The use of BMD
modeling to derive PoDs is the preferred approach when sufficient data are available to
reliably support modeling. The NOAEL/LOAEL approach historically used by EPA does
not account for the variability in the experimental results, which are due to
characteristics of the study design, such as dose selection, dose spacing, and sample
size. This paper includes BMD modeling on selected AChE studies using a similar
dose-response model (i.e., decreasing exponential) used in the OP and A/-methyl
carbamate cumulative risk assessments and supported by the FIFRA SAP on multiple
occasions (FIFRA SAP 2001, 2002, 2005a, 2005b, 2008). BMD estimates are provided
for pregnant rats and young post-natal (PN) rat pups (post-natal day one or PND1).
The Agency plans to extend this analysis up to pups up to age PND 17. BMD modeling
has not been attempted by the Agency thus far on data from other toxicities due to lack
of dose-response data amenable to BMD modeling in the majority of such studies.
The next steps in the updated hazard/dose response characterization of chlorpyrifos
involve consideration of animal to human extrapolation and within human variability.
The preferred approach for extrapolating from the PoD to lower doses is to incorporate
mode of action information and use sophisticated models like physiologically-based
pharmacokinetic (PBPK)/ pharmacodynamic (PD) or biologically-based dose-response
(BBDR) models. When such models are not available, uncertainty or extrapolation
factors are used typically used to extrapolate from animal to humans (i.e., inter-species)
and among the human population to account for sensitive individuals (i.e., intra-
species). Historically, the Agency has used default 10-fold factors to account for inter-
and intra-species extrapolation. More recently, with increased emphasis on the use of
TK and TD data in risk assessment, the derivation of uncertaint factors (UFs) derived
from data, instead of default factors, has also increased. With the intent of improving
the scientific basis for the chlorpyrifos risk assessment, in this issue paper the Agency
has considered the availability of current models and mechanistic data for chlorpyrifos
to use in animal to human and within human extrapolation. Overall, the available PBPK
models, although well-developed and supported for non-pregnant adults, do not include
calculations for pregnancy (e.g., no placental compartment) and for young children less
than 5 years old and thus can not be used in a quantitative manner in extrapolation in
this analysis.
In the absence of PBPK models for use in extrapolation, the Agency proposes to
use chemical specific adjustment factors derived from data over the application of the
10X default factors. As such, the Agency has used the 2005 IPCS guidance on
Chemical-Specific Adjustment Factors to evaluate available TD and TK data in animals
and humans and to determine the extent to which such data support data-derived or
chemical-specific extrapolation factors. In short, the Agency has concluded that with
regard to TD characteristics, due to likelihood of different mechanisms of toxicity of
chlorpyrifos and lack of identifiable and measurable key events for modes of action not
related to AChE inhibition, the Agency proposes not to refine the TD component of the
animal to human and within human variability factors. The Agency further proposes that
the TK component of the animal to human factor can not be refined. This
determination was based in large part of key differences in rat and human development
with regard to the temporal maturation of detoxification enzymes and the related
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difficulty in matching appropriate TK parameters for rats and humans. Due to the
availability of extensive data on paraoxonase-1 (PON1) in many populations worldwide
and in different lifestages, the Agency has performed a series of calculations to assess
within human variability for TK with regard to PON1 activity.
This issue paper 1) summarizes data relevant to infants, children, and pregnant
women from many areas of research, 2) provides an interpretation of these data in the
context of human health risk assessment, and 3) offers options for updating the PoD(s)
and extrapolation factors (intra- and interspecies UFs) for chlorpyrifos. In this
document, proposed PoDs are UFs are discussed first followed by summaries of
information on metabolism, AChE inhibition, effects on the developing brain in
experimental animals, and epidemiology of children exposed pre- or post-natally.
These summaries provide an integrative synthesis of available information and
preliminary conclusions about specific aspects of the data that are most relevant to the
selection of PODs and extrapolation factors.
Seven appendices to this issue paper contain an extensive review of the scientific
literature and provide the background, scientific support, and analyses performed so far
by the Agency:
> Appendices A-D describe metabolism and pharmacokinetics,
acetylcholinesterase (AChE) inhibition data, other modes of action and toxicities
besides AChE inhibition, and epidemiological studies in mothers and children.
> Appendix E provides an analysis of data for chlorpyrifos which may be used to
inform data-derived inter- and/or intra-species extrapolation factors.
> Appendix F provides the results of benchmark dose modeling on selected studies
with sufficient dose-response information.
> Appendix G provides draft data evaluation records (DER) for deliberate dosing
studies with human subjects.
Appendices A-E primarily contain summary information and which represent varying
levels of detail and discussion. The literature reviews contained in the appendices are
not meant to be exhaustive review but instead are meant to describe key studies and
findings.
This document is not a full and complete risk assessment/characterization. This
paper does not address the FQPA 10X factor for infants and children. The Food
Quality Protection Act (FQPA, 1996) requires the Agency to add a presumptive
additional 10X safety factor for the protection of infants and children to take into
account, among other things, the "completeness of data with respect to exposure and
toxicity to infants and children (emphasis added)." EPA may assign a safety factor
different than 10X if reliable data show that factor to be safe. In making this
determination, EPA considers both toxicity and exposure issues. Since an exposure
assessment is not included here, a complete analysis for the FQPA 10X is also not
discussed here. It is important to note that the Agency has not developed any final
conclusions regarding updates to the chlorpyrifos hazard assessment. At this time, the
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Agency has progressed to a stage where feedback and peer review on the overall
direction of the assessment is warranted.
2.0 Updated Points of Departure & Uncertainty Factors
2.1. Pathways of Exposure
In any risk assessment, before the PoDs and UFs are determined, it is important
to first consider the relevant exposure scenarios or pathways (by route and duration),
what population groups are exposed, and how they are exposed. This information
guides the relevant toxicity data needed to evaluate the exposure scenarios of interest
from different routes, duration of exposure, and lifestages.
• What uses remain for chlorpyrifos?
The remaining uses of chlorpyrifos are primarily agricultural.
• What routes of exposure are relevant for chlorpyrifos?
The general population can be exposed to chlorpyrifos via the oral route in
food and water (i.e., from run-off or leaching). Exposure (via the inhalation
and/or dermal routes) may also occur through volatilization and spray drift for
those people living in close proximity to fields treated with chlorpyrifos. Workers
who apply or handle chlorpyrifos are exposed via the inhalation and dermal
routes. To the extent possible, it is preferred to use route specific data when
such data are available as TK properties (e.g., absorption) vary among the
dermal, inhalation, and oral routes and route specific data helps account for
these TK differences. Gestational and post-natal studies in pups are rarely
available via the dermal and inhalation routes, particularly at low doses. As such,
route-to-route extrapolation may be necessary for these routes.
• What age groups or lifestages need to be considered?
For the general population, all age groups could be exposed in the diet.
The following age groups are typically analyzed by OPP: infants (children <1); 1
to 2 years old; 3 through 5 years old; 6 through 12 years old; 13 through 19 years
old; 20 through 49 year olds; 50 years of age and greater; and females of
childbearing age (13-49 years old). These age groups were selected since they
provide a broad representation of potential exposures. For this effort, the
Agency has focused on children (<1-18 years old) and females of childbearing
age as these groups represent potentially susceptible populations.
For workers, adults are evaluated. OPP does not develop separate
exposure or risk estimates for female and male workers but instead assesses the
more sensitive sex. In the case of chlorpyrifos, females are the more appropriate
sex for consideration as they may be or could become pregnant when
chlorpyrifos application or post-application activities occur.
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• What duration(s) of exposure are appropriate?
For dietary risk assessments for single chemical risk assessments, OPP
typically evaluates acute (24 hour) and chronic (1 year) exposures.
Worker risk is evaluated by short- (1-30 days), intermediate- (30 days-6
months), and long-term (>6 months to a year) exposure durations. The
appropriate duration of exposure for worker risk is determined by the exposure
pattern of a particular chemical and the relevant worker activities for
mixing/loading/applying and/or post-application activities (e.g., thinning, picking).
It is not unusual for workers to be exposed to the same pesticide for repeated
days.
2.2. Summary of 2000 Human Health Risk Assessment
To provide context to the reader, the PoDs and UFs used in the 2000 risk
assessment are shown below. Some of the scenarios evaluated in 2000 are not
relevant due to the cancellation of many residential uses. For the 2000 human health
risk assessment, EPA evaluated the available registrant submitted studies and scientific
literature and identified NOAELs and LOAELs based on statistically significant plasma
and RBC ChE inhibition for use in risk assessment. Blood ChE inhibition was used as
the endpoint for all scenarios. Doses from adult animals were selected from oral,
dermal, and inhalation studies for use in route specific risk assessments. Table 1
represents a summary of the toxicological endpoints for acute oral, chronic oral, dermal
and inhalation scenarios from the 2000 risk assessment.
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Table 1. Toxicological endpoints and uncertainty factors selected in the 2000 human health risk assessment for chlorpyrifos
EXPOSURE
SCENARIO
DOSE
(mg/kg/day)
ENDPOINT
STUDY
Acute Dietary
(single day)
NOAEL=0.5
IF = 100
FQPA=10
1 mg/kg/day: 40% plasma cholinesterase inhibition at peak time of
inhibition (6 hours post exposure) in adult males (RBC not measured)
(Mendrala and Brzak 1998)
1.5 mg/kg/day: Significant 23.7% plasma and 29.7% RBC ChE inhibition
at 4 hours post exposure in adult males
Blood Time Course Study (Mendrala and Brzak 1998,
44648102) with support from Zheng et al. (2000)
Chronic Dietary
NOAEL=
0.03
UF= 100
FQPA=10
Significant Plasma and RBC cholinesterase inhibition at 0.2 to 0.33
mg/kg/day
0.22 mg/kg/day: Significant 33-67% J, plasma and 24-46% J, RBC ChE
activity (90 day dog, Barker 1989);
0.3 mg/kg/day: 43%J, plasma and 41%J,% RBC ChE activity relative to
controls (2-week DNT Hoberman et al. 1998a,b) and 52%J, plasma and 39%
| RBC ChE activity relative to controls (2-week DNT companion study,
Mattsson et al. 1998)
0.33 mg/kg/day: Significant 15-51% plasma ChE inhibition in both sexes,
19-31% RBC ChE inhibition at 104 weeks vs. controls (2-yr rat, Crown et al.
1990)
Weight of Evidence from 5 studies:
• 2 year dog: McCollister et al. 1971/ Kociba et al.
1985, MRIDs 00064933/00146519;
• 90 day dog: Barker 1989, MRID 42172801;
• 2 yr rat: Crown et al., 1990, MRID 42172802;
• 90 day rat: Crown et al. 1985, MRID 40436406;
rat DNT: Hoberman et al. 1998a,b, MRID 44556901; rat
DNT companion: Mattsson et al. 1998, MRID 44648101.
Short-Term
Dermal
(1-30 days)
Dermal
NOAEL =5
Plasma and RBC cholinesterase inhibition of 45 and 16%, respectively at 10
mg/kg/day following 4 days.
21-day dermal rat and 4 day probe study (Calhoun and
Johnson 1988, MRID#: 40972801)
Intermediate- and
Long-Term
Dermal
(1 month to
chronic)
Oral
NOAEL =0.03
Plasma and RBC cholinesterase inhibition.
3% dermal absorption factor is required due to the use of an oral NOAEL (b).
Weight of Evidence from 5 studies (see chronic dietary
above):2 year dog; 90 day dog; 2 yr rat; 90 day rat;
developmental neurotoxicity
Short-,and
Intermediate-
Term
Inhalation
(1 day to 6
months)
Inhalation
NOAEL=
0.1
Lack of effects in 2 rat inhalation studies at the highest dose tested.
Two 90 day rat inhalation studies (Corley et al. 1986a,b
MRID #: 40013901 & 40166501; Newton 1988, MRID #:
40908401
Makhteshim-Agan)
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EXPOSURE
SCENARIO
Long-Term
Inhalation
(> 6 months)
DOSE
(mg/kg/day)
Oral
NOAEL~
0.03
100%
absorption
(relative to oral
absorption)
ENDPOINT
Plasma and RBC cholinesterase inhibition
STUDY
Weight of Evidence from 5 studies (see chronic dietary
above):
2 year dog; 90 day dog; 2 yr rat; 90 day rat; developmental
neurotoxicity
UF = Uncertainty Factor; a) Use absorbed dermal NOAEL of 0.15 mg/kg/day (5 mg/kg/day * 0.03 dermal absorption factor) for comparison with absorbed biomonitoring exposure.
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2.3. Data Available for Consideration
The toxicity studies considered in the Agency's review are summarized in below
in Section 3.0 and in Appendices B, C, D, E, and G. The scientific literature on
chlorpyrifos is extensive and includes data from many sources, in animals and humans,
and includes guideline studies with standard protocols and literature studies with
atypical study designs. Sources of human information include deliberate dosing
studies, epidemiology studies, and metabolism studies (in vitro and in vivo). Like many
pesticide chemicals, there are a variety studies evaluating different durations of
exposure, different animal species, systemic toxicity, reproductive and developmental
toxicity, neurotoxicity, and developmental neurotoxicity (DNT). A significant component
of the current review includes numerous literature studies which have considered many
different effects in multiple species. Many of the animal studies from the literature
involve unusual study designs, measure unique biological parameters or outcomes, and
use novel techniques. Many of the studies considered in this review use only a single
dose for a particular age or lifestage and many others only use high doses at or near
lethality. Studies that use only a single dose do not provide any information about the
shape of the dose-response curve and those using only high doses may be less
relevant when extrapolating human risk at low environmental exposures. Overall, the
Agency has given more weight to studies and outcomes replicated by multiple
laboratories and/or with multiple techniques and to studies which utilized relevant
routes/durations of exposure and relatively low and/or multiple doses.
2.3.1. Human Information
2.3.1.1. Deliberate Dosing Studies in Human Subjects
Three deliberate dosing studies in adult (non-pregnant) humans are available which
measure AChE activity and urinary levels of chlorpyrifos and/or its metabolites (See
Appendix G). The Agency has determined that the deliberate dosing studies in adults
are not appropriate for use in PoD or UF derivation in the current proposal. These
studies provide valuable information on correlating oral or dermal exposure with levels
of chlorpyrifos and/or TCP in blood and urine. In addition, these studies also provide
information on time course of absorption, metabolism, and excretion. Kisicki et al
(1999) also includes PON1 genotype information. Due to the availability of quality TK
information, these studies have been used in the past by the Agency to aid in
interpreting biomonitoring data. Specifically, results from Nolan et al (1982) have been
used previously by the Agency in estimating (i.e., back-calculating) chlorpyrifos
exposure based on urinary levels of TCP. Nolan et al (1982) has also been used to
derive a dermal absorption factor in humans.
The blood ChE data from these studies have not been proposed for use in deriving
PoDs or UFs. The reason why the blood ChE data from these studies are not proposed
for use in deriving PoDs or UFs is based on several factors. First, there are
experimental laboratory data that indicate that the developing nervous system may be
susceptible to chlorpyrifos by mechanisms related to cholinergic and noncholinergic
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mechanisms. Findings in epidemiology studies in children support the animal studies.
These human studies do not include the potentially susceptible populations being
evaluated in the current effort, namely pregnant woman and children and thus do not
consider toxicity endpoints other than AChE inhibition (and related clinical signs). Nolan
et al (1982) and Griffin et al (1999) only include a single dose group for a particular
route. Griffin et al (1999) reports no changes in AChE inhibition and in Kisicki et al
(1999) changes were only seen in one person. Studies with only a single dose group
have been criticized in the past by the Human Studies Review Board (HSRB).
Moreover, the HSRB has not supported the use of NOAEL studies in risk assessment
since absence of an effect (LOAEL) raises questions about whether the investigators
and the study design were able to detect an effect.
The Agency will solicit comment from the Panel on the Agency's preliminary
decisions not to use the deliberate dosing studies with human subjects as a PoD or to
reduce the inter-species UF but instead to use the TK data from these studies to help
interpret biomonitoring and epidemiology studies. Depending on the comments by the
Panel on these issues, the Agency may, if appropriate, take these studies to the Human
Studies Review Board (HSRB) in the near future for the necessary scientific and ethical
review.
2.3.1.2. Epidemiology Studies
One unique aspect of the chlorpyrifos literature is the availability of epidemiology
studies in three cohorts of mothers and children. These studies are summarized in
Section 3.D with more details provided in Appendix D. The epidemiology studies
provide important information about potential human outcomes related to the potential
effects of OPs on the developing brain. Moreover, they provide data which supports the
human relevance of outcomes observed in the laboratory animal studies. They are not
for use in directly deriving the PoDs or UFs for several reasons. The Agency is aware
of an effort by Drs. Dale Hattis and Robin Whyatt to develop a PBPK model which
includes a placental compartment for assessing tissue dosimetry to the fetus and which
accounts for intra-species TK variability. The investigators then plan to use that model
to estimate a human PoD from the blood biomarker reported in Whyatt et al (2003).
This work has only just begun and will likely take several years. These epidemiology
studies are, however, informative is considering factors which contribute to population
variability and in evaluating the types of toxicities and their respective human relevance
observed in animal studies.
Similar to many other epidemiology studies, these studies have not measured
urinary or blood metabolites at or near the timing of pesticide applications. Each of the
cohorts has been exposed to chlorpyrifos to some extent. There is some limited dose-
response information that provides support for the contribution of chlorpyrifos to the
birth and neurodevelopmental outcomes reported. The Columbia University team has
correlated the timing of the voluntary cancellation of indoor uses of chlorpyrifos with
maternal and cord blood levels of chlorpyrifos (Whyatt et al. 2003, 2004). They have
further associated 'high' levels of chlorpyrifos with birth and neurodevelopmental
outcomes. Since the voluntary cancellation of the indoor uses, maternal and umbilical
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cord blood levels of chlorpyrifos in the Columbia University cohort have dropped
substantially.
In addition to chlorpyrifos, each cohort has been exposed to multiple pesticides,
including other OPs. This is particularly true for the study conducted at the University of
California at Berkley where the cohort has been exposed to many OPs (Eskenazi et al.
2004, 2007; Bradman et al. 2003, 2005, 2007). The potential neurodevelopmental
mechanisms, and the related dose responses, other than AChE inhibition are not well
understood. As such, given the lack of a reliable biomarker of effect for these toxicities,
determining the quantitative contribution of chlorpyrifos to the reported outcomes
separate from the other OPs is challenging. With improved understanding, the Agency
may, in the future, be able to better characterize the linkage between blood or urinary
levels of chlorpyrifos and/or its metabolites with health outcomes. At this time, the
Agency has used the reported levels of chlorpyrifos and its metabolites simply as
markers of exposure without an attempt to link and estimate dose-effects relationships.
2.3.1.3. Studies on Metabolism and Toxicokinetics
A key component of this issue paper is the use of the 2005 IPCS guidance on
Chemical Specific Adjustment Factors to evaluate available metabolic data for use in
informing inter- and intra-species extrapolation factors. The Agency prefers to use
extrapolation factors informed by data rather than applying default factors in order to
improve the scientific support for a risk assessment. The metabolic profile of
chlorpyrifos is well characterized in animals and humans, due, in part, to a large body of
in vivo and in vitro metabolism studies from rats and humans (See Section 2.A and
Appendix A). The Agency has evaluated the extent to which available data on
carboxylesterase, butryl cholinesterase (BuChE), PON1 (or A-esterase), and P450s are
sufficiently robust to develop data-derived inter- or intra-species factors. This analysis
is summarized in Section 2.E with more details provided in Appendix E.
In brief, there a variety of studies which inform human metabolism of chlorpyrifos.
Human and rat metabolism studies in adults provide the major metabolites and the time
course of absorption, metabolism and excretion. In vitro studies using human and rat
tissues on multiple enzymes (carboxylesterase, BuChE, A-esterase or PON1, P450s)
are available. Studies on metabolism and TK of chlorpyrifos provide characterization for
interpreting gestational and post-natal toxicity studies as these studies provide data on
tissue dosimetry and ontogeny of detoxification enzymes.
In general, the in vitro studies do not contain sufficient numbers of samples
across different age groups and lifestages for quantifying within human variability. Inter-
species extrapolation is challenging for gestational effects given differences in rat and
human pregnancy with regard to birth and maturation of metabolic processes. There
are uncertainties surrounding appropriate matching of rat and human information for
purposes of extrapolation. However, there are extensive data on the population
variability of PON1 which is being considered for use in addressing the TK component
of the intraspecies factor.
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2.3.2. Animal Studies
The Agency has focused its effort to derive PoDs on animal studies, particularly
those in rats as rat because this is the species most often used in both literature and
guideline studies. Sections 3.B and 3.C and Appendices B and C contain the Agency's
review of available animal studies. The animal database on chlorpyrifos is large and
includes evaluation a many different toxicities in multiple age groups and lifestages.
The focus of the Agency's review has been on studies in pregnant animals and/or post-
natal studies in juveniles as these groups represent potentially susceptible
subpopulations.
The Agency's analysis contains two major components. The first component
includes evaluation of AChE data from gestational and post-natal studies. This review
includes both qualitative and quantitative considerations. First, a qualitative evaluation
across many studies considered lifestage and age-related sensitivity and also a variety
of issues such as duration of exposure and method/route of administration. The AChE
review also includes a BMD analysis of selected studies (see below) which contain
sufficient dose-response data and represent a variety of lifestages and age groups.
The second component of the review includes evaluation of toxicities not directly
associated with AChE inhibition. Early in the review, the Agency had considered
performing a MOA analysis using the MOA Framework (see website:
http://www.who. int/ipcs/methods/harmonization/areas/non_cancer/en/index. htm I).
However, it became clear in the initial review that sufficient data, particularly with
respect to dose response and temporal concordance, are currently unavailable to
perform a thorough MOA analysis. In the formal risk assessment, the Agency may
consider doing a mode of action/human relevance framework analysis to identify key
data deficiencies and research. In the mean time, the Agency is proposing to simply
compare the dose levels used in these studies with those in AChE studies. This
comparison informs the PoD determination as discussed below. In addition, the Agency
has compared types of effects shown in animals with reported epidemiological
outcomes. This comparison of animal studies and human outcomes provides support
for the potential of chlorpyrifos at sufficiently high doses to result in effects on the
developing brain.
2.4. Dose Response Assessment
PoDs can be NOAELs, LOAELs, or derived from BMD modeling (Figure 1).
Numerous scientific peer review panels over the last decade have supported the
Agency's application of the BMD approach as an improvement over the historically
applied approach of using NOAELs or LOAELs and as a scientifically supportable
method for deriving PoDs in human health risk assessment. The NOAEL/LOAEL
approach does not account for the variability and uncertainty in the experimental results,
which are due to characteristics of the study design, such as dose selection, dose
spacing, and sample size (USEPA, 2000). With the BMD approach, all the dose
response data are used to derive a PoD. For multiple AChE studies, there are sufficient
dose response data to support a BMD analysis. This analysis is described below.
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The Agency has not performed BMD analysis on studies evaluating the effect of
chlorpyrifos on the developing brain as these do not provide dose response data
amenable to BMD modeling analysis. Specifically, these studies, in general, may
include only a single dose at a particular age, do not report graded responses (i.e., all or
nothing effect), and/or show non-monotonic dose response curves (e.g., response goes
up then down). For these studies, the Agency simply considered the doses used.
2.4.1. AChE Inhibition
As a preliminary analysis, the Agency has conducted BMD modeling on selected
AChE studies described in Appendix B. These studies were selected based on the
availability of at least two treatment groups. In addition, these studies were selected as
they represented a variety of ages and durations of exposure. Selected studies include
repeated gestational exposures to the dam and acute exposures to PND1 pups. The
Agency is extending this analysis to include additional acute studies in pups up to age
PND17 and to include repeated dosing studies in pups. More details of the BMD
modeling reported here can be found in Appendix F.
The Agency has used a decreasing exponential dose-response model similar to
that used for the OP and A/-methyl carbamate cumulative risk assessments and
previously reviewed and supported by the FIFRA SAP on several occasions (FIFRA
SAP 2001, 2002, 2005a, 2005b, 2008). Consistent with risk assessment on other OP
and NMCs compounds, the Agency has used a benchmark response level of 10% and
has thus calculated BMDi0s and BMDI_i0s. The BMDio is the estimated dose where
AChE is inhibited by 10% compared to background. The BMDLio is the lower
confidence bound on the BMDio. Extensive analyses conducted as part of the OP
cumulative risk assessment (USEPA, 2002) have demonstrated that 10% is a level that
can be reliably measured in the majority of rat toxicity studies, and is generally at or
near the limit of sensitivity for discerning a statistically significant decrease in AChE
activity across the brain compartment and is a response level close to the background
AChE level. The Agency uses the BMDL, not the BMD, for use as the PoD since the
BMDL accounts for variability of the data (USEPA, 2000). The BMDio provides a point
of comparison across studies. Table 2 provides the results of the preliminary BMD
analysis.
Typically, studies submitted for pesticide registration and most studies from the
public literature only measure brain and/or blood ChEs. It is rare for data from
peripheral tissues to be available for consideration. Chlorpyrifos is unique in that
multiple studies are available which provide such peripheral data. Table 2 does not
include BMD results for plasma ChE measures. Consistent with OPP's ChE policy,
plasma ChE data from animals are used for risk assessment when RBC AChE data are
not reliable and/or when peripheral AChE measures are not available. This is not the
case for chlorpyrifos; reliable RBC and peripheral data are both available.
As shown in Table 2, the preliminary BMD analysis has included brain, blood,
and peripheral (heart) ChE inhibition data. As of August, 2008, the Agency had
completed a preliminary analysis of the registrant submitted studies (Hoberman et al.
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1998a, b, Mattsson et al. 1998, MRID44556901, 44648101, Maurissen et al. 2000,
Mendrala and Brzak 1988), in addition to published literature studies by Betancourt and
Carr (2004), Zheng et al (2000), Moser et al (2006), and Timchalk et al (2006).
Data from Zheng et al (2000), Moser et al (2006), and Timchalk et al (2006) were
provided by the authors to OPP for analysis. Regarding the Timchalk et al (2006) data,
the Agency has not reported BMDio/BMDI_i0s for the PND5 chlorpyrifos treated groups
(1 and 10 mg/kg) because the values for some individual animals at this age group were
inconsistent with all other data in the study (reported to have 3X more activity than other
animals of same age). BMD estimates from Zheng et al (2000) are also not reported. A
preliminary analysis was conducted on Zheng et al (2000). The BMD estimates are
considered low confidence because the AChE data in this study at low doses at or near
10% inhibition are highly variable. Analysis of PND17 data from Moser et al (2006) and
Timchalk et al (2006) will be submitted to the Panel in a supplemental report. In
addition, BMD analysis of repeated dosing studies in post-natal pups will be provided to
the Panel in a supplemental report.
The Agency has only reported BMDio values for acute dosing from Betancourt
and Carr (2004). The published report for this study does not provide all the necessary
information needed for a complete BMD analysis, particularly the lower 95% confidence
limits (i.e., BMDLio). Specifically, the actual sample size and standard deviation for all
treatment groups were not reported in the publications (ranges were reported for
sample sizes; standard error were reported for some groups but not all). The Agency
has estimated the missing values based on the results from a variety of studies from
Russell Carr's laboratory at Mississippi State University in the analysis.
For acute post-natal exposures in PND1 and 12, the brain BMDi0s are 0.12 and
0.64 mg/kg, respectively. The BMD10for RBC AChE in PND12 pups 0.25 mg/kg. There
are no peripheral data available in pups amenable to BMD modeling. However, given
the remarkable similarity in response between peripheral (heart) and RBC AChE data in
dams, use of RBC AChE data from PND12 pups as a surrogate for peripheral data is
reasonable. Among the BMD10s estimated for brain AChE inhibition in repeated dosing
adult studies, there is little variability across the studies. Specifically, BMDi0s from
gestational studies in dams range between 0.5 and 1.2 mg/kg/day. For dams, BMDL10s
for RBC and heart AChE are approximately 10-fold lower than values for brain AChE
inhibition. Like the brain AChE results, little variability is also seen among the blood and
heart AChE data in dams where BMDi0s range from 0.06 to 0.16 mg/kg/day.
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Table 2. Summary of Benchmark Dose Analyses for Acute and Repeated AChE Studies in Rat
Reference
Age
Brain
(mg/kg/day)
BMD10
BMDL10
RBC
(mg/kg/day)
BMD10
BMDL,o
Heart
(mg/kg/day)
BMD10 BMDL10
Acute/Single Dose Studies
Betancourt and Carr (2004)
Timchalk et al. (2006)
PND1
PND 12
0.12
0.64
NR
0.54
NA
0.25 0.08 NA
Repeat Dose Studies
2006 Cumulative RA
Dow
(Hoberman et al. 1998a,b,
MRID44556901); Maurissen,
2000
Dow
(Mattsson et al. 1998
44648101); Mattsson, 2000
Dow
(Mattsson et al. 1998
44648101); Mattsson, 2000
Repeated >21
days,
Adult Female
(non-pregnant)
Dam, GD6-20
Dam, GD6-20
Dam, LD 1
1.48
0.65
Hindbrain
1.10
Forebrain
1.17
Hindbrain
0.96
Forebrain
1.11
1.26
0.54
Hindbrain
0.81
Forebrain
0.98
Hindbrain
0.55
Forebrain
0.77
NA
0.06
0.14
0.079
0.03
0.08
0.0498
NA
0.16 0.12
0.109 0.056
NA= Not applicable;
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2.4.2. Effects on the Developing Nervous System
While there are a number of studies demonstrating long-term effects of peri-natal
chlorpyrifos exposure, most of those studies were conducted with only one or two dose
levels, and often both doses showed similar effects. Thus, the data are not amenable to
benchmark dose modeling. As discussed in Section 3.3 and Appendix C, when
comparing across studies, these studies provide a basis for concern that a dose of 1
mg/kg/day, in rats and mice, administered for as few as four consecutive days will
produce a variety of neurobehavioral outcomes in the offspring. These effects at 1
mg/kg are summarized in Table 3.
Across all studies, the lowest tested dose tested was 0.3 mg/kg/day. This dose
was used as the lowest dose in the DNT study, which involved repeated oral exposures
to the dam through gestation and until postnatal day 10. No behavioral effects were
observed at any time in the offspring exposed to this dose level, although the Agency
notes that brain morphometric changes (seen at 1 mg/kg/day) are not available from
this dose group. This same dose was used by Jett et al (2001) dosed every fourth day
on PND 7, 11 and 15 in one group of rats and on PND 22 and 26 in a second group.
Jett et al (2001) used subcutaneous injection in peanut oil. Significant effects on
learning in a Morris water maze was observed in the 0.3 mg/kg/day dose group, but
only when they were receiving the treatment while testing was being conducted (PND
24-28). Thus, these results may have been influenced by the concurrent dosing and
reflect acute toxicity of chlorpyrifos. For these reasons, 0.3 mg/kg/day cannot be
confidently used as either a NOAEL or LOAEL.
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Table 3. Summary of tests in Adults (at least 5 weeks of age) Following
Gestational and/or Early Postnatal Dosing of 1 mg/kg/day Chlorpyrifos
(excluding effects of higher doses). All data are in rats except where indicated.
Behavioral
Domain
Locomotor
activity
Learning &
Memory
Neuromotor
response
Social
Interactions
(mice)
Depression
Neurotransmitter
system
involvement
Device/Task
Figure-8 maze
Open-field (mice)
T maze
Elevated plus maze
Radial arm maze
T maze
Radial arm maze
T maze
Passive avoidance (mice)
Acoustic startle
Conspecific behaviors
Recognition
Agonistic behaviors (male)
Induced maternal behaviors
(female)
Elevated plus maze
Chocolate milk preference
Cholinergic
Serotonergic
Outcomes at 1 mg/kg/day
Rate of habituation increased or
decreased, depending on exposure
window and gender
Increased
Decreased early in training
Increased with PND 1-4 dosing
Not altered
Not altered
Increased errors early in training;
decreased errors in females exposed
PND 1 -4; not altered with PND 1 1 -1 4
dosing
Not altered
Not altered
Not altered
Not altered
Not altered
Increased
Increased
Increased time in open arms (both
species but gender differences)
Decreased
Muscarinic subsensitivity; no change
in nicotinic
Abnormal sensitivity
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2.5. Proposed Points of Departure (PoD)
As discussed above, chlorpyrifos exposure is expected to occur through the oral,
dermal, and inhalation routes. PoDs are needed for each route.
2.5.1. Oral Route
The Agency has proposed three options for the oral PoDs. The Agency will
solicit comment on these options as well as alternative approaches.
Option 1: The first option proposes to use the PoDs which were based on rat RBC and
plasma ChE inhibition in the 2000 risk assessment for acute oral, dermal and inhalation
exposures, and rat and dog blood AChE for chronic oral exposures (Table 2). The 2000
risk assessment included a weight of the evidence discussion primarily on adult rat and
dog AChE guideline studies and adult data from Zheng et al (2000). This option would
involve application of the NOAELS for blood AChE inhibition from route specific studies
(oral, dermal, inhalation) in rats or dogs to all populations. The acute oral PoD would be
0.5 mg/kg/day and the repeated oral PoD would be 0.03 mg/kg/day. The dermal and
inhalation NOAELs would be 5 and 0.1 mg/kg/day, respectively.
Option 2: As mentioned above, some AChE studies provide sufficient dose response
data amenable for BMD modeling, the preferred approach to deriving PoDs. AChE
studies in repeated gestational studies in dams (heart) and acute post-natal pups (brain
and RBC) provide BMDio and BMDLio values in the same range—0.06-0.12 mg/kg/day.
In Option 2, the Agency proposes to use a PoD of 0.1 mg/kg/day for oral exposure of all
age groups and all durations (i.e., acute and chronic).
This value of 0.1 mg/kg/day is not derived from a single value. Instead, the
proposed value of 0.1 mg/kg/day is derived from a weight of the evidence using BMD10s
and BMDLioS from brain and RBC AChE in young pups (PND1 and 12) in acute studies
and peripheral (heart) AChE in repeated gestational studies with dams. As such,
multiple lifestages are considered in the proposed PoD: pregnant dams, PND1 pups,
and PND12 pups. With regards to the developing brain, PND 12 pups are similar to
newborns. It can be argued that direct dosing of PND1 pups does not directly match
human exposures since PND 1 pups are closer to third trimester human fetuses.
However, the PND 1 data add support that very young animals may be more sensitive
than adults following acute exposures. The proposed PoD of 0.1 mg/kg/day is also
supported by the PND 12 blood data. Furthermore, the proposed value is 3-10 fold
lower than causing effects on the developing brain reported in other laboratory animal
studies and thus is expected to be protective for those effects. The lowest dose used in
any study evaluating the potential for chlorpyrifos to affect learning/memory is 0.3 mg/kg
from Jett et al (2001). The proposed PoD of 0.1 mg/kg/day is 3-fold lower (i.e., more
health protective endpoints) than this dose of 0.3 mg/kg/day. The Agency further notes
that these BMDLio values are approximately 10-fold lower (more health protective) than
the lowest dose (1 mg/kg/day) used in many gestational and post-natal studies
evaluated toxicities other than AChE inhibition (See Appendix C).
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Thus, the Agency believes that the proposed PoD of 0.1 mg/kg/day provides a
scientifically supportable value because it was derived 1) BMD modeling which is
preferred over the use of NOAELs/LOAELs, 2) incorporates information from multiple
susceptible lifestages, 3) and is protective of toxicities other than AChE inhibition,
including behavioral effects remaining in adulthood.
Option 3: The third option is a blend of Options 1 and 2 and separates the PoD for
acute and repeated exposures. The Agency proposes to use the same PoD of 0.1
mg/kg/day discussed above for all populations but only for acute oral exposure and for
short-term dermal exposure to workers (1-30 days) as discussed in Option 1. For
chronic dietary exposure, a PoD of 0.03 mg/kg/day for all populations is proposed. This
is the same PoD used in the 2000 risk assessment. This value of 0.03 mg/kg/day is
based on the NOAELs and LOAELs for plasma and RBC ChE inhibition in five studies
(2-year study in dog, 90-day study in dog, 90-day study in rat, 2-year feeding in rat, the
DNT study). This value of 0.03 mg/kg/day is supported by the BMDLio of 0.03
mg/kg/day in pregnant dams for RBC AChE inhibition (Table 2).
2.5.2. Dermal & Inhalation Routes
Route specific data are preferred because such data accounts for potential
differences in absorption, distribution, or metabolism. In the case of chlorpyrifos, dermal
and inhalation studies are available which identify NOAELs for these routes in adult
rats. With respect to inhalation exposure, there are two nose only studies with vapor
chlorpyrifos which provides a NOAEL of 287 ug/m3 or 20 ppb (0.1 mg/kg/day).
Similarly, there are two dermal studies which together provide a dermal NOAEL in adult
rats of 5 mg/kg/day. These studies do not include pregnant dams, fetuses or post-natal
pups and therefore do not consider potentially susceptible populations. In the absence
of data in these groups, the Agency will continue to use route specific studies, as
appropriate. An alternative for dermal exposure is to use an oral PoD derived from
susceptible populations (as discussed above) with a dermal absorption factor.
Specifically, the Agency could use a dermal absorption of 3% from human subjects
(Nolan et al, 1982). The Agency will solicit comment from the Panel what toxicity and/or
TK studies (if any) in pregnant dams, fetuses, and/or post-natal pups could be
conducted to better inform the dermal and inhalation risk assessments.
2.6. Extrapolation/Uncertainty Factors
In previous risk assessments, the Agency has applied the default 10X factors for
both inter- and intra-species extrapolation in addition to the FQPA 10X. In 2005, the
WHO published its guidance for deriving chemical specific adjustment factors (CSAFs;
WHO, 2005). The guidance is based in large part on analyses by Renwick (1993) and
Renwick and Lazarus (1998) and describes the use of TK and TD data in lieu of the
default 10X safety factors for human sensitivity and experimental animal-to-human
extrapolation. EPA has an on-going effort to develop similar guidance and has used
these concepts in some risk assessments, including several pesticides.
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As discussed in detail in Appendix E, the Agency has applied the 2005 IPCS
Chemical Specific Adjustment Factors Guidance in the chlorpyrifos analysis.
Understanding MOA is an important component of deriving data derived extrapolation
factors (DDEFs) in that MOA provides the foundation for understanding which TK and
TD factors are critical for extrapolation. Although, inhibition of AChE is a well
established neurotoxic mode of action for OPs (including chlorpyrifos), chlorpyrifos may
have multiple modes of action resulting in its effects on the developing nervous system.
When deriving data-derived factors, it is necessary to consider each mode of toxicity,
critical effect, target organ, and lifestage because the magnitude of extrapolation factors
may differ among different toxicities and life stages.
The MOA involving inhibition of AChE leading to clinical signs of neurotoxicity
and changes in behavior has been well-documented for many OPs. If AChE inhibition
was the only mode of action affecting toxicity of chlorpyrifos, it may be possible to derive
extrapolation factors for the TD component of animal to human (UFAD) and possibly
within human variability (UFHD) because there are data which describe the molecular
structure and in vitro effects of the AChE in different species and data which evaluate
the in vitro effects of juvenile and adult AChE inhibition. However, other potential
modes/mechanisms that may affect the developing brain are less understood and none
are sufficiently robust to establish key events with dose-response and temporal
concordance. Given the remaining uncertainty regarding the modes(s) of action
affecting the developing brain—and specifically differences in animals and humans and
within human variability—the Agency has elected to not develop a DDEF for UFAD or
UFHD. As such, the Agency is proposing to apply the default 3X for inter- and intra-
species TD extrapolation (i.e., UFAD and UFHD).
As discussed in detail in Appendix E, the Agency evaluated the extent to which
data on carboxylesterases, P450s, and paraoxonase (PON1, or A-esterase) support
development of DDEFs of inter- and intra- TK extrapolation (i.e., UFAK and UFHK).
Based on differences in rat and human pregnancy with regard to birth and maturation of
metabolic processes, there are uncertainties surrounding appropriate metabolic
parameters for animal to human extrapolation of juveniles. This uncertainty in
combination with limited data precludes the development of a DDEF for inter-species
TK extrapolation (i.e., UFAK). Thus, the Agency proposes to apply the default 3X for
UFAK. Data on carboxylesterases are not sufficiently robust for intra-species TK
extrapolation (i.e., UFHK)- Data on P450s are complicated by multiple enzymes each
with its own maturation profile. Others have evaluated the P450 literature for use in
derivation of child specific UFs with poor success (Ginsberg et al, 2004a). There are,
however, extensive data on PON1 which allow for development of a factor for intra-
species TK extrapolation (i.e., UFHK)- The Agency has reviewed data on PON1 activity
and enzymes levels among different populations and age groups. These data show
that age-related maturation of PON1 is a larger source of variability compared with
different genetic polymorphisms among adults, particularly for the PON-192 Q/R
polymorphism.
The Agency is proposing two options for the intra-species TK extrapolation factor
(UFHK): one option involves using a UFHK derived from PON1 data and a second option
involves using a default factor. Specifically, the Agency is considering the use a 12X
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factor based on chlorpyrifos oxonase (CPOase) data in mothers and newborns (Holland
et al, 2006) as the basis of the UFHK. This factor was derived in a manner consistent
with IPSC (2005); the ratio of the 50th percentile in mothers and 5th percentile in
newborns was calculated.
There are uncertainties associated with use of PON1 data as the source of
information for UFHK- The Agency has provided an extensive discussion of this in
Section 3.0 and in Appendix E. The Agency will solicit comment from the SAP on
several science issues related to interpreting and using PON1 data.
Table 4. Potential composite factors for chlorpyrifos
Factor
Inter-species
Intra-species
Toxicokinetics
3X
3Xor12X
Toxicodynamics
3X
3X
Composite Factor
Combined
10X
10Xor36X
100Xor360X
2.7. Issues for the FIFRA SAP
The Agency has not developed any final conclusions regarding PoDs or UFs. At this
time, the Agency has progressed to a stage in the review where feedback and peer
review on the overall direction of the assessment is warranted. The Agency is soliciting
comment from the panel on the proposed PoDs and UFs (data and approach) and also
on aspects of the literature review which provides the foundation of the proposals.
The following sections summarize key information on the metabolic profile of
chlorpyrifos, AChE data, other toxicities besides AChE inhibition, and epidemiology
studies in children. More detailed descriptions of these data can be found in
Appendices A-E
3.0. Summary of Key Data Used in PoD & UF Determination
3.1. Metabolism and Toxicokinetics
3.1.1. Metabolic Profile
The metabolism and TK of chlorpyrifos have been extensively studied in animals
and humans as well as in vitro systems. Many of these studies are summarized in
Appendix A. Overall, rats and humans show similar patterns of metabolism for
chlorpyrifos in adults. Although less information is available to compare rats to humans
with regard to pregnancy and post-natal maturation, as described below, the patterns of
metabolism appear to be generally similar in rats and animals.
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Chlorpyrifos undergoes metabolic transformations mainly by the liver enzymes
residing in the microsomes. Although, chlorpyrifos is lipophillic, its extensive metabolism
into water soluble metabolites does not lead to any accumulation of the parent material
or its metabolites in the body tissues. The initial metabolic attack on the chlorpyrifos is
its desulfuration, resulting in its bioactivation to the more toxic and potent AChE
inhibitor, the oxon form (Figure 2). However, the oxon is unstable and is rapidly
deactivated through hydrolytic cleavage by a process called dearylation releasing the
3,5,6-trichloro-2-pyridinol (TCP). Simultaneously along the desulfuration process,
dearylation will be acting on both the parent chlorpyrifos as well as on the oxon
metabolite leading to the release of TCP. TCP is further conjugated to form glycine or
glucuronide conjugates and eliminated into the urine. TCP is the major excreted
metabolite and used as the biomarker in PK, biomonitoring, and epidemiology studies.
There are several enzymes that play a role in the metabolism and toxicity of
chlorpyrifos (Figure 2). In addition to inhibition of AChE, the oxon also binds
stoichometrically to butyrlcholinesterse (BuChE; abundant in blood and other tissues).
In this regard BuChE is viewed as a scavenger of the oxon formed. The cytochrome
P450 family of microsomal enzymes (CYPs) is responsible for its metabolic activation
and deactivation. Another group of important enzymes in the detoxification of
chlorpyrifos is the A-esterases; one such A-esterase is paraoxonase (i.e., PON1).
These are calcium activated enzymes and are distributed in various tissues including
the liver, brain and blood. These act on the oxon by hydrolyzing it before reaching its
target AChE enzyme. The oxon also binds irreversibly to carboxylesterases.
Carboxylesterases are distributed among different issues (liver, blood, brain) with
highest abundance in the liver. The glutathione dependent enzymes play an important
role in the secondary metabolism of chlorpyrifos producing water soluble metabolites
that are readily excreted into the urine.
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Figure 2. Major metabolic pathways of chlorpyrifos metabolism (Reproduced from
Timchalk et al, 2005)
AChE Inhibition
Toxicity
Cl
ChU
Diethylthio phosphate
Chlorpyrifos-oxon
'CH,
Diethylphosphate
3,5,6-trichloro-2-pyridinol
(TCP)
:xr
OH ^M^ ^»ri
Cl
Conjugates-O" ^N^ ^Cl
Sulfate or glucuronides of TCP
3.1.1.1. Ontogeny of Metabolic Processes in the Young
Differential inhibition of the AChE enzyme itself does not appear to account for
the observed age-related sensitivity found in young animals as suggested by in vitro
studies (Benke and Murphy, 1975; Chanda et a/., 1995; Mortensen et al., 1996;
Atterberry et al., 1997). Rat fetuses and juveniles and newborn humans, however, have
lower capacity to detoxify than adults. This decreased capacity to detoxify has been
associated with increased sensitivity in rats. Specifically, in rats, A-esterase activity is
virtually nonexistent in the fetus (Lassiter et al., 1998) and increases from birth to reach
adult levels around PND21 (Mortensen et al., 1996; Li et al., 1997). Mortenson et al
(1996) showed that in the plasma level of CPOase2 was 1/11 that of adult animals. The
animal data regarding the role of carboxylesterase in mediating OP toxicity are also
quite extensive (e.g., Clement, 1984; Fonnum et al., 1985; Maxwell, 1992 a, b). Fetal
rats possess very little carboxylesterase activity (Lassiter et al., 1998) with increasing
! CPOase is A-esterase (PON1) activity specific to chlorpyrifos oxon
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activity as the postnatal rat matures, reaching adult values after puberty (50 days-of-
age; Morgan et al., 1994; Moser et al., 1998; Karanth and Pope, 2000). There are,
however, very little data in human tissues which could evaluate age-related maturation
of carboxylesterase expression. The available data come from Pope et al (2005) and
Ecobichan and Stephens (1973). Ecobichan and Stephens (1973) showed a steady
increase in AChE and ChE levels of infants beginning at birth up to adult levels. Pope
et al (2005) evaluated maturational expression of liver carboxylesterases in human liver
tissues from infants (2-24 months) and adults (20-36 years). The authors report
relatively small (and not statistically significant) differences in activities between children
ages 2-24 months and adults (20-36 years). The Agency notes, however, that
youngest age evaluated in the study was 2 months old and this individual had the
lowest level of carboxylesterase.
The temporal pattern of A-esterase activity (and carboxylesterases) correlates
reasonably well with studies on OP sensitivity. Several studies have shown an
increased sensitivity of newborn rats to OP compounds which are detoxified via the A-
esterase and/or carboxylesterase pathways (Gagne and Brodeur, 1972; Benke and
Murphy, 1975; Pope et al., 1991; Chambers and Carr, 1993; Padilla et al., 2000; 2002;
Karanth and Pope, 2000).
There are only studies in the literature that have assessed A-esterase activity in
children. Based on these studies, it appears that serum A-esterase levels are very low
in human infants compared to adults (Augustinsson and Barr, 1962; Mueller et al., 1983;
Ecobichon and Stephens, 1973; Holland et al, 2006; Chen et al, 2003). After birth, there
is a steady increase of this activity (Augustinsson and Barr, 1963). In a related study of
the age-dependence of total serum arylesterase (ARase) activity (of which a large
component is A-esterase activity), adult levels were achieved by two years-of-age
(Burlina et al., 1977). The Agency is aware of yet unpublished data of PON1 levels (A-
esterase) in children up to age 5 from Drs. Nina Holland and Brenda Eskanazi with a
much larger sample size (>200) than previous studies. These data were presented at
the American Society of Human Genetics meeting (Huen et al, 2007) and suggest that
paraoxonase activity may be lower than adult levels up to 47 months. After completion
of the data analysis and ultimately publication, these data will substantially improve the
overall understanding of the human ontogeny of PON1. Recent studies by Holland et al
(2006) and Chen et al (2003) have provided ARase and/or CPOase activities in
newborns and their mothers. These studies have been analyzed by the Agency as part
of consideration of data derived intraspecies extrapolation factor for chlorpyrifos
(Holland et al, 2006; Chen et al, 2003; Appendix E).
Maturation of the cytochrome P450s to detoxify or activate chlorpyrifos to the
oxon may also play a role in age-related differences in the young and adults. The most
important P450s for chlorpyrifos metabolism in humans are 1A2, 2B6, 2C19, and 3A4
(Buratti et al, 2002; Tang et al, 2001). It is important to note that CYP3A4 is deficient in
neonates; the fetal form, CYP3A7, is active in utero and immediately after birth (LaCroix
et al, 1997). In addition, CYP1A2 is barely detectable at birth and CYP 2C19 and 3A4
are 3 to 10-fold lower in newborns than other children and adults (Sonnier et al, 1998;
Vieria et al, 1996). Less is known about the development of CYP2B6 as this one is not
as extensively studied as other CYPs.
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3.1.1.2. Metabolic Changes During Pregnancy
Metabolic activities can be altered during pregnancy (Anderson 2006; Anger and
Piquette-Miller2008, Bologa etal. 1991; Carpintero et a/. 1996; Czekaj etal. 2000 and
2005; Dickman eta/.2008; Ejiri etal. 2005; Ferre etal. 2006; Mines 2007; Homma etal.
2000; Howard and Sugden 1993; Tsutsumi etal. 2001 )3. For example, Chanda et al
(2002) showed that pregnant female rats had lower plasma, brain, and liver
carboxylesterase activity compared to non pregnant females. Regarding A-esterase
(PON1) activity, Ferre et al (2006) showed that the paraoxonase (POase) in serum
decreased from a nonpregnant background of 146 U/L to 111 U/L in late gestation,
indicating 76% of normal activity in late gestation pregnant women. Carpintero et al
(1996), however, found that phenyl acetate metabolism increased from 23.6 to 33.5
(ikat/g in the third trimester. Data in mice support the findings of Ferre et al (2006)
findings in humans suggesting a reduction in A-esterase activity during pregnancy. In
mice, Weitman et al. (1983) found that PON1 activity towards parathion was 50
nmol/min/ml in non-pregnant females, but it decreased as low as 14 nmol/min/ml during
gestation (Weitman et al., 1983).
With regard to plasma ChE levels, Howard et al (1978) have shown that in six
healthy pregnant women levels of plasma ChE dropped by approximately 30% during
the first trimester but returned to close to pre-pregnancy levels in the third trimester.
Similarly, Venkataraman et al (1990), Whittaker, et al (1988), and De Peyster et al
(1994) both reported decreases in plasma ChE in pregnant women. Evans et al (1988)
showed that cholinesterase levels in 39 of 44 pregnant women dropped after
conception; in 20 of those women, the decline in cholinesterase activity continued
throughout pregnancy.
As mentioned above, the most important P450s for chlorpyrifos metabolism in
humans are 1A2, 2B6, 2C19, and 3A4. Bologa et al (1991) found that production of a
marker substrate for 1A2 activity dropped to 41% of non-pregnant levels by the third
trimester in epileptic women. While 1A2 and 2B activities decrease in pregnancy, 2C
and 3A activities increase. In pregnant AIDS patients, endogenous cortisol metabolism
(marker for 3A4 activity) increased by more than 2-fold (Homma et al, 2000).
There is a consistent pattern for several key detoxification enzymes that
metabolic activity may decrease during pregnancy. The reductions are not large in
magnitude and the importance of these decreases is unknown at environmental
exposures. However, these studies suggest the potential for a reduced capacity to
detoxify during pregnancy. Toxicity studies in rats add further support that reduced
ability to detoxify chlorpyrifos and/or the oxon effects sensitivity during pregnancy.
Female rats, particularly pregnant rats, appear to be more sensitive than adult male rats
to cholinesterase inhibition (Moser et al. 1998, Hoberman 1998a,b, Mattsson et al.
3 The Agency notes that metabolic changes during human pregnancy are also described in a public
comment to the SAP docket by Drs. Torka Poet and Charles Timchalk of Battelle Laboratory.
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1998). Moser and Padilla (1998) found that inhibition of ChE in brain tissues had a
sooner onset, a later peak effect, and a slower recovery in adult (approximately PND
70) females administered a single oral gavage dose of 80 mg/kg, compared to males.
3.1.2. Data on Tissue Dosimetry
An important issue when evaluating gestational exposure is the extent to which
the compound crosses the placenta and is thus available to affect fetal tissues. Cross
placental tissue dosimetry data are rarely available in humans for pesticide chemicals.
Umbilical cord data from one research group (Columbia University) are available for
chlorpyrifos. Specifically, Whyatt et al (2003) have shown that levels of chlorpyrifos in
maternal blood are similar to the levels measured in umbilical cord blood (Whyatt et al,
2003; Section 2.2). In gestational studies with rats, similar or higher levels of TCP in
fetal brain and blood compared to dams suggests that chlorpyrifos and/or its
metabolites reach the target tissue (brain) in the fetus (Hunter al, 1999; Mattsson et al,
1998, 2000). Hunter et al, (1999) showed that the concentration of TCP in the fetal
brain was 2-3- fold higher than the TCP concentration in the maternal brain in time
course and dose-response studies. In a study by Mattsson et al (1998, 2000),
concentrations of chlorpyrifos, the oxon, and TCP were measured in the blood of
maternal and fetal tissues and in milk. Mattsson et al (1998, 2000) found that TCP
levels in maternal and fetal blood were similar and chlorpyrifos levels were
approximately 2-fold higher in maternal blood than fetal blood. The oxon is highly
unstable and was only detected at high doses in the fetus (and not dams). In another
gavage gestational exposure study, Akhtar et al (2006) exposed rats to chlorpyrifos
from GD 0-20 in fetal and maternal tissues on GD21. It is difficult to make conclusions
regarding dose relationships in this study due to a small range of dose. The
investigators saw some inconsistent trends. Total residues of chlorpyrifos were higher in
the fetuses than in the dams and brain concentrations of chlorpyrifos were greater or
similar to dam levels. When considered together, these studies (Whyatt et al, 2003;
Hunter al, 1999; Mattsson et al,1998, 2000; Akhtar et al, 2006) support the conclusion
that chlorpyrifos and/or its metabolites are likely available to the fetus at similar (and
possibly higher) levels compared with maternal tissues.
With regard to post-natal exposure to chlorpyrifos through breast milk, there is
some limited data that show that chlorpyrifos can be found in breast milk. Mattsson et
al (1998, 2000) provided data in rat milk which suggest that chlorpyrifos can reach milk
at lower doses (0.3 mg/kg/day). There are very little human breast milk data in the
literature except for persistence organic pollutants and none for chlorpyrifos from
current U.S. exposure levels
3.1.3. Conclusions
TK studies from humans and rats support a preliminary conclusion that
chlorpyrifos and/or its metabolites may be available to the fetus, likely at levels similar to
maternal tissues. The Agency further concludes that TK differences in young and
adults play an important role in the age-dependant sensitivity with chlorpyrifos
(described below in Sections 3.B-D). Additional information in pregnant animals and
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humans suggest that metabolic capacity to detoxify may be reduced during pregnancy,
although the relevance of these changes is not known at low environmental levels.
3.2. Inhibition of Acetylcholinesterase (AChE)
Chlorpyrifos, like other OPs, binds to and phosphorylates the enzyme, AChE, in
both the central (brain) and peripheral nervous systems leading to accumulation of
acetylcholine and, ultimately, to clinical signs of toxicity. This mode of action, in which
AChE inhibition leads to neurotoxicity, has been well described (Mileson et al, 1998). In
2000, the Agency concluded for chlorpyrifos that inhibition of ChE was the most
sensitive effect in all of the animal species evaluated and in humans, regardless of
exposure duration. For the current analysis, the Agency has reviewed the studies
submitted for registration as well as searched the public literature for studies in which
pregnant animals and/or juvenile animals were exposed to chlorpyrifos. ChE inhibition
is most commonly reported for the blood (plasma and RBC) and brain (whole or
subsections), although a few studies have evaluated inhibition in peripheral tissues such
as the heart, diaphragm, or lung. The following text provides a summary of key studies;
more details, including extensive tables, can be found in Appendix B.
The Agency has examined time course information. In the available studies, the
time to peak inhibition following exposure to chlorpyrifos was independent of age and
method of exposure and varied from 2 to 24 hours across different studies but was
typically between 3 and 6.5 hours. Recovery in young rats occurs faster than in adult
animals (Chakraborti et al., 1993; Moserand Padilla, 1998; Popeetal., 1991; Pope and
Liu, 1997). In the fetus, comparison of Lassiter et al. (1998a) and Ashry et al. (2002)
indicates that the time of peak brain ChE inhibition is the same (4-5 hours) following
repeated (7 mg/kg/day) or acute oral exposure (50 mg/kg/day) to the dam. In contrast,
peak brain ChE inhibition in the dam is later following lower repeat exposure (7
mg/kg/day peaks at 5 hours, Lassiter et al. 1998a) compared to a single dose in late
gestation (2 hours) at high doses (50 mg/kg/day, Ashry et al. 1992).
Tables 5-7 provide summary information from AChE studies in gestational and
post-natal studies in rats. More detailed versions of these tables can be found in
Appendix B. The information provided here focuses on effects at or near a dose of 1-
1.5 mg/kg. This dose has been used by numerous investigators evaluating both AChE
inhibition and other toxicities. As such this dose provides a comparison point for
comparing among studies, different toxicities, duration of exposure, ages, lifestages,
and methods of administration. Comparisons across different studies need to be made
with care as timing of sampling varies among studies which impacts results.
3.2.1. Gestational exposure
In gestational studies with chlorpyrifos, AChE activity is generally inhibited more
in dams than in the fetus (Table 5). A similar pattern has been seen for many other
OPs (USEPA, 2006, Attachment 1). As such, it would appear that the fetus may be
protected by the dam. However, rat fetal brain ChE activity increases 4.3 times from
GD14 to GD18 and that activity increases another 3 times from GD18 to PND1.
According to Lassiter et al (1998a), "new synthesis of uninhibited cholinesterase
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molecules may dilute the inhibited molecules such that the fetal brain cholinesterase
activity recovers more quickly than the maternal brain." Therefore, at a given time after
exposure, cholinesterase may appear less inhibited by chlorpyrifos in the fetus
compared to adults because the fetus recovers more quickly by rapidly synthesizing
new brain cholinesterase. Further support for Lassiter's comments are found in TK
studies mentioned above. Following gestational exposure to the dam, Hunter et al.
(1999) found that levels of TCP in the fetal brain were 2-3 times higher than in the
maternal brain. Additional data are found in Mattsson et al (1998, 2000) who showed
that chlorpyrifos levels were 2-fold higher in maternal blood than fetal blood but TCP
levels were similar. Thus, when the dam is exposed to chlorpyrifos, the fetus is as well-
-likely at similar levels. As such, although the AChE data consistently shows more
inhibition in the dam compared with the fetus, the fetus may not actually be protected by
the dam. Therefore, AChE data in fetuses from repeated dosing gestational studies
may not accurately reflect potential fetal toxicity at a particular dose.
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Table 5. Summary of repeated studies evaluating gestational exposure to
maternal rats and fetuses.
Study
Mattsson
etal.
(1998,
2000)
Hoberman
etal.
1998a,b,
Maurissen
etal
(2000)
Mattsson
etal.
1998,
(2000)
Qiao et al.
(2002)
Route
(vehicle)
oral
gavage
(corn oil)
oral
gavage
(corn oil)
oral
gavage
(corn oil)
s.c.
injection
(DMSO)
Time of
exposure
GD6-20
GD6-20
GD6-
PND1
GD 17-20
Time of
measurement
post-dosing
4hrs
4-5 hrs
2hrs
GD21
Dose
1
mg/kg/day
1
mg/kg/day
1
mg/kg/day
1
mg/kg/day
Fetal
inhibition
8% (NS)
0%
5% (NS)
4% (NS)
0%
N/A
N/A
N/A
5% (NS)
0%
0%
5% (NS)
2% (NS)
3% (NS)
6% (NS)
Maternal
inhibition
10%/7%
(p<0.02)
12%/7%
(NS)
87%/85%
(p<0.02)
77%/60%
(p<0.02)
49%/50%
(p<0.02)
68.9%
84.4%
17.9%
6% (NS)
6% (NS)
90%
(p<0.02)
80%
(p<0.02)
40%
(p<0.02)
brainstem
forebrain
Compartment
forebrain
hindbrain
RBC
plasma
heart
plasma
RBC
brain
forebrain
hindbrain
RBC
plasma
heart
N/A
N/A
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3.2.2. Post-natal, acute exposures
In post-natal studies where pups are directly exposed, the degree of ChE
inhibition is clearly age dependant following single exposures (Table 6, Figure 3). In
general, blood and peripheral measures are more inhibited at the same dose compared
with brain measures. As mentioned in the metabolism section, newborn and juvenile
rats are more sensitive to AChE inhibition caused by chlorpyrifos than adult rodents, not
because of a difference in the affinity of chlorpyrifos oxon to AChE, but because
maturation of detoxification enzymes (Iyer, 2001). This ontogeny (and resulting
reduced sensitivity) is evident in Figure 3 where the degree of brain AChE inhibition
decreases with the age of the post-natal pup.
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Table 6. Summary of acute studies evaluating post-natal exposure to juvenile
rats.
Study
Dam et al.
(2000)
Betancourt
and Carr
(2004)
Timchalk
etal.
(2006)
Zheng et
al, 2000
Timchalk
etal.
(2006)
Timchalk
etal.
(2006)
Moser et
al. 2006
Route
(vehicle)
s.c. injection
(DMSO)
oral gavage
(corn oil)
oral gavage
oral gavage
(peanut oil)
oral gavage
oral gavage
oral gavage
(corn oil)
Age
PND1
PND1
PND5
PND7
PND12
PND17
PND17
Time of
measurement
post-dosing3
2hrs
12hrs
3hrs
4hrs
6hrs
24hrs
4.5 hrs
Dose
1 mg/kg
1 .5 mg/kg
1 mg/kg
1 .5 mg/kg
1 mg/kg
1 mg/kg
0.5 mg/kg
2 mg/kg
0.5 mg/kg
2 mg/kg
Inhibition
70% (M); 25%
(F)
80% (M); 10%
(F)
60% (M); 35%
(F)
58%
22.1%
45.7%
62.1%
17%
32%
51%
5.2%
27.0%
33.4%
2.1%
15%
21 .9%
0%
10%
10%
40%
Compartment
brainstem
cerebellum
forebrain
forebrain
brain
RBC
plasma
frontal cortex
RBC
plasma
brain
RBC
plasma
brain
RBC
plasma
brain
whole blood
a. Reported time of peak effect
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Figure 3. Plot of brain AChE inhibition in post-natal pups following a single dose
of 1 mg/kg
c
o
o
c
o
_c
LLI
.E
O
E
00
100
90
80
70
60
50
40
30
20
10
0
6 8 10 12
Age of Post-Natal Rat Pup
14
16
18
3.2.3. Post-natal, repeated exposures
Table 7 and Figure 4 summarize information from repeated dosing studies in
post-natal pups using a dose of 1-1.5 mg/kg/day as a point of comparison. Repeated
dosing studies show similar degrees of brain AChE inhibition independent of duration of
exposure. For example, Guo-Ross et al (2007) and Richard and Chambers (2005)
each measured similar amounts of brain AChE inhibition but Guo-Ross et al (2007)
dosed pups with only 4 exposures whereas Richard and Chambers (2005) used 6
exposures. The pattern of similar degrees of AChE inhibition across repeated dosing
post-natal studies likely reflects the rapid nature of AChE recovery observed by multiple
investigators (Chakraborti etal., 1993; Moserand Padilla, 1998; Popeetal., 1991; Pope
and Liu, 1997). This pattern is less evident at higher doses where AChE inhibition has
reached >70-80% and/or where metabolic processes may be saturated.
One exception to this is the PND1-11 group in Betancourt and Carr (2004) where
no brain AChE inhibition was reported. When evaluating the results within the
Betancourt and Carr (2004) study, there is a decrease in inhibition following repeated
dosing studies from PND1-3, 1-6, and 1-11 suggesting that as the pups mature, they
become less sensitive. A similar but less pronounced trend was observed by Richards
and Chambers (2005) who showed that PND1-6 and 1-12 dosing resulted in similar
degrees of inhibition. In the PND1-21 group, a somewhat lower amount of brain AChE
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inhibition was observed (36%) compared with the PND1-6 and 1-12 groups. There are
potential explanations for this. First, the results of Richards and Chambers (2005) may
be explained based on the timing of measurement, PND1-6 animals were measured at
6 hours post-dosing but the PND 1-21 was measured 24 hours post-dosing.
Alternatively, the reduced brain AChE inhibition could have resulted from maturation of
detoxification pathways resulting in decreased inhibition.
It is notable that the trend shown in Figure 4 is distinctly different from results in
adult studies for most OPs. Typically in adult rats, AChE inhibition increases with
repeated exposures. In other words, at a common dose level, more inhibition is
observed after repeated exposures compared with a single exposure.
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Table 7. Summary of repeated studies evaluating post-natal exposure to juvenile
rats.
Study
Guo-Ross et
al. (2007)
Betancourt
and Carr
(2004)
Song et al.
(1997)
Richardson
and
Chambers
(2005)
Betancourt
and Carr
(2004)
Betancourt
and Carr
(2004)
Richardson
and
Chambers
(2005)
Richardson
and
Chambers
(2005)
Zheng et al
(2000)
Route
(vehicle)
oral
gavage
(corn oil)
oral
gavage
s.c.
injection
(DMSO)
oral
gavage
(corn oil)
oral
gavage
oral
gavage
oral
gavage
(corn oil)
oral
gavage
(corn oil)
oral
gavage
(peanut oil)
Time of
exposure
PND 1-4
PND 1-3
PND 1-4
PND 1-6
PND 1-6
PND1-11
PND1- 12
PND 1-21
PND7-20
Time of
measurement
post-dosing
4 hrs
24hrs
24 hrs
6 hrs
24 hrs
24 hrs
12 hrs
24 hrs
4 hrs
Dose
1
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
1
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
1.5
mg/kg/day
Inhibition
25%
45%
27%
24%
49%
28%
None
43%
36%
42%
57%
59%
Compartment
brain
forebrain
brainstem
brain (excluding
cerebellum and
medulla-pons)
forebrain
forebrain
brain (excluding
cerebellum and
medulla-pons)
brain (excluding
cerebellum and
medulla-pons)
frontal cortex
RBC
plasma
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Figure 4. Plot of brain AChE inhibition in post-natal pups following repeated
dosing at 1.5 mg/kg
o
"c
o
o
M-
0
^^
c
o
15
IE
HI
r-
O
5
m
1 VJVJ
90 -
80 -
70 -
60 -
50 -
40 -
30 -
20 -
10 -
0 -
t
• f
*
O
10 12 14
Number of doses
16
18
20
22
3.2.4. Method of administration
AChE studies available for chlorpyrifos use a variety of methods of
administration. The two most common are oral gavage and subcutaneous injection,
particularly with DMSO. Some have suggested that the TK properties of a particular
chemical may vary by method of administration, thereby impacting the amount of AChE
inhibition observed in a particular study. However, the Agency's analysis suggests that
the inhibition levels may be more similar than previously believed.
In general, study designs in gestational studies vary widely among laboratories
with regard to doses used, number of repeated doses, and gestational days of dosing
which makes comparing the results problematic. Chanda and Pope (1996) exposed
dams from GD 12-19 via subcutaneous injection with peanut oil and showed 75% brain
inhibition 24 hours after the last dose in the dams at a dose of 6.25 mg/kg/day. Hunter
et al (1999) and Lassiter et al (1998a) exposed dams using corn oil gavage from GD14-
18 at 7 mg/kg/day and observed 68% and 69% brain AChE inhibition 24 hours after the
last dose, respectively. The gestational days of dosing differs between the studies and
the number of doses differs between the studies-8 and 5 for Chanda and Pope (1996)
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and the EPA studies (Hunter et al, 1999; Lassiter et al, 1998a), respectively. Even
when considering the differences in study designs, there is notable similarity in the
amount of measured brain AChE inhibition at 24 hours after the last dose in the studies
using subcutaneous injection and oral gavage—75% and 68-69% (Chanda and Pope,
1996; Hunter et al, 1999; Lassiter et al, 1998a, respectively).
Comparison of post-natal studies show that brain AChE inhibition at similar dose
levels (e.g., Tables 5 and 6) yields remarkably similar results in young pups (ages
PND1 -5). For example following an acute dose of 1 or 1.5 mg/kg/day in PND1 pups,
60% and 58% forebrain AChE inhibition were noted from subcutaneous injection with
DMSO and corn oil gavage, respectively (Dam et al, 2000; Betacourt and Carr, 2004).
Following exposure at 1 mg/kg/day from PND1 to PND4, 24% and 25% brain AChE
inhibition were noted from subcutaneous injection with DMSO and corn oil gavage,
respectively (Song et al, 1997; Guo-Ross et al, 2007). The time measurements of these
studies were at 24 hour and 4 hours post-dosing for subcutaneous injection and
gavage, respectively. The Agency also notes that preliminary (not yet replicated or
published by the authors) data by Carr and Narr presented at SOT (2008) showed
striking similarity in the time course and amount of brain AChE inhibition in PND10 pups
exposed at 5 mg/kg from subcutaneous injection with DMSO and corn oil gavage.
Moreover, the amount of brain AChE (25-28%) in the Carr poster is similar to that
PND11 pups exposed at the same dose from Dam et al (2000) who used subcutaneous
injection (15-30% brain stem, but 15-35% for brain) at 4 hours post-dosing.
A recent study Marty et al (2007) provides TK data which supports findings of
AChE studies mentioned above. Specifically, Marty et al (2007) compared methods of
administration for PND5 pups exposed to 1 mg/kg/day chlorpyrifos via corn oil gavage,
subcutaneous injection with DMSO, and oral exposure in milk. Across the three
methods of administration, Marty et al (2007) showed relatively small (2-fold or less)
differences in: 1) AUC for chlorpyrifos and TCP; 2) % lives for TCP; and 3) similar time
to peak effect for chlorpyrifos and TCP. Based on the findings of Marty et al (2007),
there appear to be only small differences in TK characteristics in PND5 pups exposed
via corn oil gavage, subcutaneous injection with DMSO, and exposure in milk.
The Agency has concluded for young pups, at least up to post-natal day 5 in rat,
that administration via the oral route and subcutaneous injection provide remarkably
similar results and that post-natal studies up to PND 5 in either route are relevant for
risk assessment. Less data are available to compare routes/methods of administration
for older pups and no comparative TK data are available for gestational exposures. As
more studies are available in the future, the Agency may, if appropriate, extend this
conclusion to include older pups. The lack of comparative PK for oral gavage and
subcutaneous injection in pregnant dams and fetuses in considered an important data
gap in quantitatively evaluating dose response data in subcutaneous injection studies.
However, the Agency can not discount the findings of subcutaneous injection
gestational studies at this time.
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3.2.5. Preliminary conclusions
Numerous AChE studies are available in different lifestages and ages in rats.
These studies vary widely by the level and number of doses used, availability of time
course information, and method of administration. The Agency has preliminarily
concluded the following
• Repeated dosing gestational studies which show less fetal brain AChE inhibition
compared with the dam may not reflect actual toxicity to the pup. This conclusion
is based, in large part, on TK data comparing blood and brain levels of
chlorpyrifos and/or its metabolites in fetal and dam tissues.
• Following acute post-natal exposure studies, there is an age-dependant
sensitivity that decreases as the pups mature.
• When considering the repeated dosing post-natal studies across laboratories,
there is little variability with respect to degree of brain AChE inhibition across
different durations of exposure. Within a laboratory, however, decreases in
sensitivity have been observed with longer duration of exposure.
The Agency will solicit comment on each of these preliminary conclusions and the
science which does and does not support them.
3.3. Effects on the Developing Nervous System
There is a growing body of literature on the effects of chlorpyrifos in the developing
brain which indicate that gestational and early postnatal exposure can lead to
neurochemical and behavioral alterations into adulthood; these changes are observed
long after potential AChE inhibition has recovered. Indeed, some authors report finding
no or only marginal inhibition of fetal or neonatal brain AChE at the doses causing such
effects. Figure 5 provides a schematic of some of the mechanisms or toxic pathways
that have been proposed for chlorpyrifos (Slotkin, 2006). One such hypothesis involves
the morphogenic role of AChE, such that inhibition may adversely impact the
development of the central and peripheral nervous systems to cause permanent
damage (Brimijoin and Koenigsberger, 1999 and Bigbee et al, 1999). Although multiple
mechanisms have been proposed (described in Appendix C), a coherent mode of action
with supportable key events, particularly with regard to dose-response and temporal
concordance, has not yet been elucidated.
Many recent studies elaborate on the alterations induced by peri-natal chlorpyrifos
exposure (see Appendix C). The results of these studies contribute to the overall hazard
characterization of chlorpyrifos and are important in on-going efforts to define pathways
of toxicity for chlorpyrifos. In this section, however, the Agency has focused on studies
evaluating behavioral measures following gestational and/or postnatal exposure to
chlorpyrifos. Behavioral measures are available from multiple laboratories and in two
species. Although some differences among the studies have been observed, the
behavioral outcomes are generally reliable, and are particularly valuable as they show
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qualitative similarities to some neurodevelopmental findings reported in children
(described in Section 2.D below).
Figure 5. Multiple possible mechanisms of chlorpyrifos (From Slotkin 2006)
Oxidative
Stress
Direct Actions on
Cholinergic
Receptors
Interaction with
Signaling Intermediates
Signaling
Cascades
Transcription
Factor
Expression,
Function
AChE
Inhibition:
CPF Oxon
3.3.1. Behavioral Effects in Rats
A series of recent behavioral studies from a single laboratory (Drs. Slotkin, Levin,
and co-workers) have described a variety of effects in adult Sprague-Dawley rats that
were treated with chlorpyrifos during several different periods of development: early
gestation (GD 9-12; Icenogle et al 2004); late gestation (GD 17-20; Levin et al, 2002) or
after birth on PND 1-4 (Aldridge et al., 2005c; Levin et al., 2001) or PND 11-14 (Levin et
al, 2001). Chlorpyrifos, either 1 and/or 5 mg/kg, was administered subcutaneously in
DMSO. Exposures took place either during gestation (GD 9-12 or GD 17-20) or else
after birth (PND 1-4 or PND 11-14), and behavioral testing was initiated at
approximately 4 weeks of age, continuing for several months. These studies are
summarized in Table 8. AChE inhibition was not directly measured in any of these
studies.
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Table 8. Effects following gestational or postnatal exposure to 1 or 5 mg/kg
chlorpyrifos administered subcutaneously in DMSO
Reference
Window
Dose
Assessment PND
Chocolate Milk
Preference
Elevated Plus
maze
Motor Activity
Radial arm maze
(RAM) working &
reference memory
T-maze activity &
alternation
Startle reflex &
prepulse inhibition
Ketanserin
Challenge
RAM
Scopolamine
Challenge
RAM
Mecamylamine
Challenge
RAM
Icenogle
(2004)
GD9-12
1 or 5
mg/kg(a)
-4-17 weeks
.»
tcenter
crosses
t habituation
rate
t errors early
in training
-i- activity
early in
session
No effect
~
^
scopolamine
effect
No effect
Levin
(2002)
GD 17-20
1 or 5
mg/kg(b)
-4-17 weeks
~
~
^9
habituation
rate
9 t errors
early in
training
-i- activity
early in
session
~
~
?;
scopolamine
effect
No effect
Levin
(2001)
PND 1-4
1 mg/kg
-4-17
weeks
~
~
No effect
9 4- errors
throughout,
$ \ errors
early in
training
$ 4- activity
middle of
session
~
~
No effect
No effect
Aldridge
(2005)
PND 1-4
1 mg/kg
-7-17 weeks
6*/?^
preference
6"t time in
open arms &
tcenter
crosses
~
9 4- errors
throughout,
$ \ errors
early in
training
~
~
(57$ t errors
~
~
Levin
(2001)
PND 11-14
5 mg/kg
-4-17 weeks
~
~
6*/?^
habituation
rate
No effect
$ 4- activity
middle of
session
~
~
?;
scopolamine
effect
No effect
(a) No statistical interaction of treatment and sex, therefore sexes were combined for analyses. Data are
for the 5 mg/kg dose group which was most affected, 1 mg/kg dose group had little if any effect except in
the T maze; ® Effects significant only at 1 mg/kg dose for RAM, both doses for T maze and motor
activity;(c) - not tested.
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Cognitive function has been repeatedly tested in these studies, using both a T
maze to measure spontaneous alternation (a crude form of learning) and the radial arm
maze (RAM). The RAM evaluates both working (short-term) and reference (long-term)
memory throughout several weeks of training. As shown in Table 8, adult rats treated
with chlorpyrifos during gestation or early lactational periods showed altered learning on
both forms of memory; only when exposure occurred on PND 11-14 was there no effect
of treatment. In general, chlorpyrifos-treated rats displayed more errors early in the
training sessions, but were able to learn the task eventually. Gender differences in this
pattern of response emerged but were dependent on the period of exposure - the effect
was seen in GD 17-20 females, but PND 1-4 males. On the other hand, female rats
treated PND 1-4 displayed fewer errors than controls throughout the training session.
The authors have interpreted this counterintuitive result as indication of an attenuation
of normal gender differences, since the treated female rats showed the same error rate
as males, or else possibly an enhancement of cholinergic response (Levin et al., 2001;
Aldridge et al., 2005a,b,c). It is interesting to note that spontaneous alternation was not
altered in any of the studies.
The involvement of underlying neurotransmitter systems may be delineated by
using pharmacological challenges. After RAM training, rats were administered
scopolamine (a muscarinic antagonist which produces acute cognitive deficits),
mecamylamine (a nicotinic antagonist which by itself does not alter cognitive function),
and ketanserin (a serotonergic antagonist, which does not alter cognitive function). In
most cases, the normal amnesic effect of scopolamine was blocked in the rats treated
gestationally or in later lactation, but not in the PND 1-4 groups. Mecamylamine had no
effect in any groups in any study. On the other hand, chlorpyrifos-treated rats showed a
dose-related increase of errors when challenged with ketanserin, which had no effect in
controls. Taken together, these data indicate that the muscarinic cholinergic system
may be sub-functional as a result of treatment, and these effects are specific for gender
and window of exposure. In addition, the serotonergic system may be playing an
abnormal role to enable cognitive abilities. Thus, the authors' interpretations over these
studies are that there is a wide window of vulnerability to chlorpyrifos ranging from early
gestation (neurulation) to late lactation that can impair cholinergic circuits used in
learning and memory (Aldridge et al., 2005a,b,c).
Other chlorpyrifos effects on the serotonergic nervous system were evaluated
using other behavioral tests. Treated rats showed alterations in preference for a
preferred substance (chocolate milk) and increased time in the open arms of an
elevated plus maze (which evaluates anxiety). These effects, along with the abnormal
responsiveness to ketanserin in the RAM, suggest long-term differences in this system
following early postnatal exposures. These behavioral effects are concordant with
ongoing neurochemical studies from the same laboratory, as described in Appendix C.
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Gender-selective deficits in locomotor development are also reported in several
of these studies, where most evaluations were conducted within a couple of weeks of
exposure (starting at 4 weeks of age). Activity levels recorded as latencies in the T
maze were decreased in the early portions of the test sessions in gestationally exposed
rats. Marginal decreases were observed in PND 1-4 male rats, but only in the middle of
training. Overall activity measured in a figure-8 maze was never altered, but the rate of
habituation (the change in activity during the test session) was either increased or
decreased, depending on gender and window of exposure. An earlier study from the
same laboratory (Dam et al., 2000) also reported activity changes at 4 weeks of age,
but in this case, only males showed decreased open-field activity and rearing following
exposure to 1 mg/kg on PND 1-4. Using a different dosing paradigm, Carr et al. (2001)
did not find open-field activity changes in rats receiving 3 mg/kg/day from PND 1-21, but
when the doses were escalated (up to 6 mg/kg/day or 12 mg/kg/day) decreases were
observed 1-2 weeks after exposure ended. There is an indication of long-term changes
in activity in that the number of center crosses was increased in the elevated plus maze
in both studies where it was evaluated, and this test took place after RAM training. On
the other hand, however, latencies in the RAM were never altered by chlorpyrifos.
While it appears that there is little consistency in these findings, it is important to note
some differences in the procedures and apparati in which activity was measured.
There is only one other study of which we are aware which involved short-term
developmental dosing with chlorpyrifos, followed by a test of learning and memory. Jett
and coworkers (2001) used a Morris water maze, which evaluates spatial learning and
memory, to test adolescent (24-28 day old) Long-Evans rats. One group received
subcutaneous injections of chlorpyrifos (peanut oil vehicle) at 0.3 or 7 mg/kg on PND 7,
11, and 15, the other on PND 22 and 26. Only the high dose slowed learning in the
earlier postnatal exposure group, whereas both doses were effective in the postweaning
exposure group. There was no brain AChE inhibition in comparably treated rats
sacrificed on PNDs 7, 8, 16, or 28. This dosing regimen is unique to this study and not
comparable to the other studies described above. Since dosing occurred either shortly
before or during testing, the results may be confounded and reflect acute toxicity of
chlorpyrifos. These results have not been repeated, not has testing been conducted at
later times after exposure using this paradigm.
It is important to note that the guideline developmental neurotoxicity study
includes measures of locomotor activity as well as learning and memory, with
assessments occurring at weaning and again at 2 months of age. In the chlorpyrifos
DNT study conducted by the manufacturer (Maurissen et al., 2000), dams were dosed
from GD 6 to PND 11. There were changes in the startle response (decreased
amplitude and increased latency) in PND22 rats, as well as a suggestion (non-
significant) of increased activity levels in adult offspring at the high dose, 5 mg/kg/day;
however, this dose level was maternally and developmental^ toxic. No significant
changes were reported using a T maze delayed alternation task at either age.
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3.3.2. Behavioral Effects in Mice
Persistent effects of chlorpyrifos following gestational and/or early postnatal
exposure were also demonstrated in CD-1 mice in a series of studies from the
laboratory of Dr. Calamandrei. One study involved subcutaneous administration in
DMSO on either PND 1-4 or 11-14 (Ricceri et al., 2003), while the others included both
oral administration of the dams on (3D 15-18 followed by dosing of the pups on PND11 -
14 (Ricceri et al., 2006; Venerosi et al., 2006). The combination of pre- and postnatal
exposures renders interpretation more difficult, and less comparable to the studies in
rats.
Ricceri et al (2003) included assessment of brain AChE activity after exposure on
PND 1-4 or PND 11-14 and report inhibition only at 1 hr (but not later times) after dosing
on PND 4 (little difference between the dose groups). There was no effect in the mice
dosed on PND 11-14. The Agency notes that mice may be less sensitive than to rats to
chlorpyrifos (USEPA, 2000), yet the dose of 1 mg/kg/day was sufficient to produce
significant inhibition in the young pups. AChE activity measured in a later study (Ricceri
et al., 2006) also reported no brain inhibition in pups dosed PND 11-14, but serum
activity was lowered (to approximately 50% control levels) in both postnatal chlorpyrifos
groups.
Ricceri and colleagues (2003) measured a variety of social, motor, and cognitive
behaviors before weaning and up until PND 60. Notable persistent behavioral effects
were apparent and included increased open-field locomotor activity (on PND 25, in the
PND 11-14 group mice only, both doses), more activity in a novel environment (on PND
35, all treatment groups), and more aggressive responses by males in the social
interaction test (PND 45, all treatment groups). On PND 60, passive learning, however,
was not affected.
In later studies by the same group (Ricceri et al., 2006; Venerosi et al, 2006),
exposure occurred during GD 15-18 at 3 or 6 mg/kg chlorpyrifos (oral administration to
the dam), followed by exposure of the offspring on PND 11-14 at a dose of 1 or 3 mg/kg
chlorpyrifos (subcutaneous administration in peanut oil). Focusing on the mice that
received vehicle first, then chlorpyrifos, or vice versa, allows evaluation of treatment
effects pre- or postnatally. Ricceri et al. (2006) reported that, when tested as adults,
motor activity was increased in mice that received the high dose during GD 15-18.
Postnatal exposure increased male agonistic behaviors in the high-dose group
(supporting their finding from 2003), and induced-maternal behaviors in both dose
groups. Postnatally treated females also spent more time in the open arms of an
elevated plus maze. In another study of adult females (Venerosi et al., 2006), the
authors reported that while chlorpyrifos exposure during gestation altered social
behavior toward the same and new partners, these outcomes were not observed when
the mice also received chlorpyrifos postnatally. Such potential compensation of
gestational exposure requires additional study.
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3.3.3. Discussion
In general, studies in the literature have used doses of 1 to 6 mg/kg/day
chlorpyrifos, during gestation and/or lactation. Exceptions are the Jett study (Jett et al.,
2001) and the manufacturer's developmental neurotoxicity study (Maurissen et al.,
2000), both of which used 0.3 mg/kg/day as the lowest dose. While the latter detected
no changes at that lowest dose, the former reported changes in water maze learning
that took place shortly after or during dosing. The studies from the Slotkin/Levin and
Calamandrei laboratories provide evidence that adults may exhibit persistent behavioral
changes following peri-natal exposures. Since both laboratories included a dose of 1
mg/kg/day, some comparisons in response may be made - these are summarized in
Table 3 (Section 2.0). While the precise pattern and direction of changes are not
always consistent, this is not totally unexpected given the many experimental factors
that are different, including window of exposure, age at testing, specificity of behavior
measured, and many others. When evaluated as a whole, however, these studies
provide a basis for concern for susceptibility for persistent effects of chlorpyrifos as low
as 1 mg/kg/day on neurodevelopment.
In conclusion, there is a growing body of literature with laboratory animals
indicating that gestational and/or early postnatal exposure to chlorpyrifos may cause
persistent behavioral effects into adulthood. There are also concurrent changes in brain
neurochemistry based on both in vivo and in vitro studies that may underlie these
behavioral changes into adulthood. The cholinergic nervous system is a target for
chlorpyrifos, through AChE inhibition associated with acetylcholine destruction at the
synapse and the morphogenic role of this enzyme, as well as possible effects on
transporters and receptors. Although there are suggestions for targets for chlorpyrifos
other than on the cholinergic system, there is no conclusive evidence identifying them at
this time. Specifically, it is still difficult to definitely discern the initiation of the
neurochemical changes (i.e., serotonin, macromolecules, etc) reported from concurrent
catalytic functional AChE inhibition or an effect on a morphogenic function of AChE.
This inability to compare is usually due to study design issues and/or lack of time course
information and/or pharmacokinetic data on tissue dosimetry. The studies do, however,
provide qualitative descriptions of how the developing rodent brain has a susceptibility
to chlorpyrifos exposure with consequences lasting beyond the duration of any
observable AChE inhibition and into adulthood.
3.4. Human Epidemiology: Observations in Children
Three major prospective epidemiology cohort studies are looking at pre- and
post-natal pesticide exposure in minority mothers and infants, birth outcomes, genetic
susceptibility plus long-term childhood neurobehavioral and neurodevelopment
outcomes. Funded by multiple Federal Agencies, including US EPA, the study sites are:
(1) Columbia University, NYC, (2) Mt Sinai, School of Medicine, NYC, both with multi-
ethnic urban poor women and infants, and (3) University of California at Berkeley
(Center for Health Assessment of Mothers and Children of Salinas, CHAMACOS) with
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women and their children from farm worker populations. These studies and their
reported findings are described in detail in Appendix D.
The following text focuses on outcomes related to mental and cognitive function.
Birth outcomes (e.g., birth weight, birth length, head circumference) have been reported
by some studies (Whyatt et al. 2003, Berkowitz et al. 2004). However, the type of birth
outcome and direction of the change (i.e., increase vs. decrease), vary among the three
cohorts. For example, one group (Mt. Sinai) reported decreased head circumference
(albeit small) when maternal urinary TCP levels were above the limit of detection (>11
ug/L) and PON1 status was considered (Berkowitz et al. 2004). In contrast, increases
in head circumference were associated with increasing maternal urinary DAPs in the
CHAMACOS cohort (Eskenazi et al. 2004) and the Columbia team reported no changes
in head circumference (Whyatt et al. 2004). The Agency does not discount the reported
birth outcomes. Instead, the Agency has elected to emphasize those outcomes which
have been replicated by multiple cohorts. Furthermore, as described above, there are
multiple animal studies which support the concept that gestational exposure can result
in effects on behavior which persist beyond any initial AChE inhibition. Thus, when the
animal and epidemiology studies are considered in combination, there is strong
evidence from multiple human and animal studies regarding the potential for
neurodevelopmental effects of chlorpyrifos.
It is unusual for the Agency to have data from three large, prospective cohorts for
consideration in human health risk assessment. Each provides unique and somewhat
complementary information:
• The Columbia University NYC cohort includes predominately African American
and Dominican women and children. This team has reported indoor air, maternal
and cord blood measures of parent chlorpyrifos, and multiple birth and
neurodevelopmental outcomes. This cohort was exposed during pregnancy to
chlorpyrifos and other pesticides indoors and in food. One focus of the publications
from this group involves comparisons between pre- and post cancellation of indoor
uses of chlorpyrifos.
• Mount Sinai NYC cohort includes women and children who are Puerto Rican
Hispanic, African American, and Caucasian. This team has associated urinary
metabolites (TCP and/or DAPs) with some birth and neurodevelopmental outcomes.
This group has placed an emphasis on relating outcome information with PON1
status. The enrollment of the Mt. Sinai cohort overlapped with the cancellation of
residential uses of chlorpyrifos. However, the researchers have not evaluated the
impacts of the residential phase out on the health outcomes measured in their
publications.
• The CHAMACOS cohort includes mothers and children from farm families who
live in the Salinas Valley, California and who are predominately of Mexican descent.
This cohort is exposed to many pesticides, including multiple OPs (Table A-1 in
Appendix D), from multiple pathways such as occupational exposures and take-
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home exposures. This team has collected information on PON1 status but has not
yet published findings associating PON1 status with health outcomes. This team
has, however, associated urinary DAP metabolites with some birth and
neurodevelopmental outcomes.
These studies have been performed using prospective epidemiology methods
such that exposure measures come before the potential outcomes. Chemical measures
of blood and urine pesticide analytes are done by, or with, testing methods of the
Centers Disease Control and Prevention (CDC) in ways that can be compared to
reference ranges in the National Health and Nutrition Examination Survey (NHANES)
for DAPs and/or TCP in urine and chlorpyrifos in blood (Columbia only). All three
studies used well developed neurodevelopmental measures, which provide
comparability. Both the Columbia and CHAMACOS cohorts used the standardized
Bayley Scales of Infant Development II (BSID-II)I, a widely used, normative value-
referenced, developmental test for young children that is used frequently to diagnose
developmental delay and is known to be highly sensitive to low level intrauterine
exposures. All three also used the Child Behavior Checklist (CBCL) to assess behavior
problems such as attention problems, attention deficit hyperactivity disorder (ADHD),
and pervasive developmental disorder (PDD) problems. The Mt. Sinai results for
Bayley Mental Development Index (MDI) and the Bayley Psychomotor Development
Index (PDI) are still in preparation (Engel et al., in prep).
In an effort to characterize the exposures and outcomes in the epidemiology
studies, the Agency developed a series of detailed tables which compare blood levels of
chlorpyrifos and urine levels of TCP in humans and animals across studies and
compared these levels with AChE inhibition data where possible. These tables are
found in Appendix D. Due to differences in study design between epidemiology and
laboratory studies, the interpretation of these tables is challenging and problematic. In
animal or human laboratory studies, time course studies provide valuable information
such as the time of maximum effect and/or maximal blood or tissue levels and time to
recovery. In contrast, in epidemiology studies, the timing of exposure is unknown and
thus the timing of measurements in relation to when exposure occurred is also
unknown. Specifically, the urine and blood measures in these studies are not timed
with applications, so it is difficult to correlate these results with known exposures in the
home or agricultural field. The epidemiology studies have taken spot samples of urine
(as opposed to 24 hour samples). There is uncertainty associated with spot samples as
they may not capture all the pesticide exposures because of the short-half life of the OP
pesticides in the body.
Compared with epidemiology studies, laboratory studies, whether animal or
human, are highly controlled situations where the amount, timing, and route of exposure
are known. In laboratory experiments, important endpoints such as AChE inhibition and
behavioral measures or clinical signs can be observed with care. In addition, the
magnitude of exposure can be controlled for. Specifically, exposure pathways like food
or residential exposures could be controlled for during a laboratory study compared with
epidemiology studies where the magnitude of exposure may vary significantly among
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individuals and among different days. Similarly, the route of exposure is known for in
the laboratory study but is unknown in the epidemiology studies. For example, in the
human deliberate dosing studies, the subjects were exposed via the oral route. With
regard to the epidemiology studies, mothers were likely exposed through the diet (oral)
and from residential uses (dermal, inhalation).
Unlike the birth outcomes which show variable results across the cohorts, delays in
mental development were reported in all three cohorts (Columbia, Mt. Sinai and
CHAMACOS). Both Mt. Sinai and CHAMACOS cohorts report abnormal reflexes in
neonates associated with urinary maternal DAP levels. For each Iog10 unit increase in
total DAPs, these authors report a 32 percent (Engel et al., 2007) and 26 percent
(Young et al., 2005) increased risk of abnormal reflexes.
Figure 6. Mental Development Index (MDI) results from CHAMACOS, Mt. Sinai,
and Columbia University
Prenatal OPs and Bayley
Mental Development Index
Berkeley Mt. Sinai Columbia
(Log10DAPs) (Log10DAPs) (High v. Low CPF)
Adj b Adj b Adj b
6 Months
1 Year
2 Years
3 Years
-1.2
-1.3
-3.5**
-1.3
-1.9**
p<0.1 **p<0.05
Eskenazi et al. 2007; Engel et al. in preparation; Rauh et al. 2006
Source: Rauh 2008, presentation to EPA, April. Used with permission.
Increases in pervasive developmental disorder were reported in both the
Columbia and CHAMACOS cohorts. In the Columbia study (Rauh et al. 2006) these
effects in 3 year old children were associated with high (>6.17 pg/g) chlorpyrifos blood
umbilical cord levels, while the CHAMACOS cohort reported these effects in 2 year old
children to be associated with increases in total urinary maternal and child DAPs and
DMPs, and child DEPs for 12 month old children (Eskenazi et al. 2007). The Mount
Sinai team has not yet published findings for the 2 year old children but these results
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are currently under preparation for publication. Based on preliminary information
shared with the Agency by the investigators at Mt. Sinai, these results are expected to
show increasing urinary DAPs were associated with lower Mental Developmental Index
(Engel et al. in prep, Figure 6). Thus, prenatal OP exposure has been reported to be
associated with delays in mental development in 2 and 3 year old children, increased
odds of abnormal reflexes in neonates, and increased odds of pervasive developmental
disorder in children 2 and 3 years of age.
In the CHAMACOS study, however, TCP in maternal urine was not associated
with any Bailey or CBCL adverse outcomes in children, and there were no reported
associations between PDI or attentional deficits and urinary OP concentrations. It
should be recognized that the Columbia study reported effects for 3 yrs olds, while the
CHAMACOS study has only published data for 2 yr old children thus far. In addition,
the urinary levels of TCP in the CHAMACOS study were much lower (median 3.5 ug/L
compared to 7.5 ug/L for the Mt. Sinai cohort) than the Mt. Sinai cohort and this could
partially explain the differences in the study results.
The Agency believes that the Columbia University studies provide the most
relevant information for evaluating the human health effects of chlorpyrifos. These
studies specifically evaluated chlorpyrifos in maternal and umbilical cord blood levels
rather than the TCP and/or DAP urinary metabolites in maternal urine reported in the
Mount Sinai and CHAMACOS studies. Many OPs can contribute to total urinary DAP
concentrations. TCP is a common metabolite of multiple pesticides: chlorpyrifos,
chlorpyrifos-methyl and trichlorpyr. It is also the primary environmental degradate of
chlorpyrifos and is found on food treated with chlorpyrifos. As such, environmental
and/or dietary exposures to TCP can also contribute to urinary TCP levels and exposure
to multiple OPs complicates interpretation of DAP data.
In the Columbia University cohort, recruitment of study participants overlapped
with residential use cancellation; there was a sharp decline in use during this period.
Chlorpyrifos levels dropped substantially in maternal personal air and plasma and cord
blood plasma samples after cancellation. For children born before the cancellation,
'high' chlorpyrifos exposure in cord plasma was associated with decreased birth weight
and length. In contrast, this relationship was no longer significant for newborns born
after the cancellation because the blood levels dropped and only one child was in the
high group. Likewise, there was no association with chlorpyrifos and
neurodevelopmental outcomes after the cancellation, again because all but one of the
children had cord blood levels less than 6.17 pg/g.
There were multiple chemical exposures in the Columbia University cohort study,
including diazinon and propoxur that are cholinesterase inhibitors, and o-phenylphenol,
a disinfectant/fungicide, all of which were measured in 100% of air samples at higher
median concentrations than chlorpyrifos. The mean umbilical cord levels were lower
than chlorpyrifos (1.1, 3.1 and 4 pg/g for diazinon, 2-isopropoxyphenol and chlorpyrifos,
respectively). It is also likely that the diazinon and propoxur metabolites in blood were
possibly underestimated because of the relatively short half lives, and the lack of
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information to correlate the time of sample collection with pesticide application. The
study authors report that after controlling for both diazinon and 2-isopropxyphenol
(metabolite of propoxur) exposure in cord plasma, the associations between birth weight
and length and cord plasma (In)chlorpyrifos remained statistically significant (p< 0.02)
and the effect size remained similar to that seen without 2-isopropoxyphenol in the
model (Whyatt et al. 2004). However, a similar analysis was not reported for the
neurodevelopmental outcomes, so it is not clear if these chemicals have any influence
on the chlorpyrifos findings.
While neurodevelopment deficits may be multifactor in origins, it is not always
possible to identify the sources for each case. As discussed previously, these children
in cohort are from poor multi-ethnic populations and urban neighborhoods and may
experience other health disparities that compound pesticide exposure. Such disparities
are linked to health care access, low income and low education, as well as exposure to
urban air pollutants. Cicchetti (2007) published a critique of neurodevelopmental
outcomes reported by Rauh et al. (2006). Specifically, Dr. Cicchetti commented on the
study population socio-demographics like maternal education-and said that Rauh et al.
(2006) masked, but did not eliminate educational bias by dichotomizing maternal
education into high school graduate or non-graduate. Furthermore, that the "high" and
"low" exposure groups differed in their race/ethnicity characteristics which confounds
race/ethnicity with exposure, and the finding that more high exposure children had
ADHD is meaningless. Dr. Rauh responded to Cicchetti by saying the "high" and "low"
groups were clearly defined based on the previous report of reduced birth weight among
children with exposure levels above 6.17 pg/g (Whyatt et al. 2004). High school degree
was used to adjust for maternal education because the sample was uniformly low
income, and thus education was the preferred covariate for social class. Maternal
intelligence, although controlled was not significant in their analysis. Rauh et al. (2006)
controlled for race/ethnicity in all models and also used a stratified analysis showing a
significant chlorpyrifos effect within each ethnic group, independent of race. They used
Achenbach's Child Behavior Checklist (CBCL) to assess behavior problems rather than
make a diagnosis because ADHD is hard to diagnose in preschool-aged children.
Cicchetti (2007) also commented on the significance of differences in
neurodevelopmental measures and suggested the mean Mental Development Index
scores were clinically meaningless, and indicated that there were no standards defining
"high" and "low" chlorpyrifos exposure. Rauh et al. (2007) disagrees with Dr. Cicchetti
claim that the significant chlorpyrifos effect on Mental Development Index was
"clinically meaningless" and indicates that a Bayley developmental score < 85 prompts
referral to early intervention services, and exposures that produce small shifts in the
mean often result in more children who meet the diagnostic criteria.
The Agency can not rule out the potential for multiple AChE-inhibiting pesticides
impacting the health outcomes reported in the children. In the individual chemical risk
assessments for all three, indoor residential exposures provided risk estimates above
the Agency's level of concern. As a result, some indoor uses for all three pesticides
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have been voluntary cancelled by registrants4. This conclusion regarding muti-
chemical exposure does not preclude the potential contribution of chlorpyrifos in the
reported health outcomes. Given, that measured levels of chlorpyrifos have been
statistically associated with multiple birth and neurodevelopmental outcomes and these
blood levels have been correlated in time with the chlorpyrifos phase-out, the Agency
has preliminarily concluded that chlorpyrifos likely played a role in these outcomes. The
Agency will solicit comment from the panel on the preliminary conclusions with regard to
the usefulness of information from each cohort for the chlorpyrifos risk assessment, the
associations between chlorpyrifos exposure and health outcomes, and the qualitative
similarities noted between the epidemiology studies with animal studies.
3.5. Extrapolation Factors
The following text is a summary of more detailed information which can be found
in Appendix E on developing and using DDEFs and the specific evaluation of TK and
TD data for chlorpyrifos.
In previous risk assessments, the Agency has applied the default 10X factors for
both inter- and intra-species extrapolation in addition to the FQPA 10X safety factor.
U.S. and international efforts have made significant efforts to improve the scientific
basis for human health risk assessments by increasing the use of mechanistic and
kinetic data. One such area is the decreased reliance on default uncertainty factors
through the development of Chemical Specific Adjustment Factors (often called Data-
Derived Extrapolation Factors, DDEFs). In 2005, the WHO published its guidance for
deriving chemical specific adjustment factors (CSAFs; WHO, 2005). The guidance is
based in large part on analyses by Renwick (1993) and Renwick and Lazarus (1998)
and describes the use of TK and TD data as a means of replacing the traditional 10X
safety factors for human sensitivity and experimental animal-to-human extrapolation.
EPA has an on-going effort to develop similar guidance and has used these concepts in
some risk assessments including several pesticides.
A preferred approach to extrapolate from animals to humans and within humans
would be to use a PBPK or other sophisticated model. However, such a model is not
currently available for assessment of chlorpyrifos exposure during pregnancy or for
young children. In the absence of such a model, extrapolation factors to account for
inter- and intra-species variability are used. Such factors based on data are more
scientifically robust than use of default factors. The Agency has evaluated the extent to
which data are available to develop DDEFs for chlorpyrifos. Given the remaining
uncertainty regarding the modes(s) of action affecting the developing brain, the Agency
has elected to not develop a DDEF for UFAD or UFHD- As such, the Agency proposes to
apply the default 3X for inter- and intra-species TD extrapolation (i.e., UFAD and UFHD).
4 Diazinon residential uses were also phased out, with retail sales for indoor uses ceasing by December
2002, a year after chlorpyrifos.
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The Agency has evaluated data on P450s, carboxylesterases, BuChE, and
PON1 for inter-species TK extrapolation (i.e., UFAK). Due to limited data and based on
differences in rat and human pregnancy with regard the timing of maturation of
metabolic processes, there are uncertainties surrounding appropriate metabolic
parameters for animal to human extrapolation. This uncertainty precludes the
development of a DDEF for inter-species TK extrapolation (i.e., UFAK). Thus, the
Agency proposes to apply the default 3X for UFAK.
Regarding within human variability, the Agency again evaluated data on P450s,
carboxylesterases, BuChE, and PON1. Studies on carboxylesterases and BuChE are
limited by number of sample/subjects and/or by lack of data in juveniles. Data on P450s
are complicated by multiple enzymes each with its own maturation profile. Others have
evaluated the P450 literature for use in derivation of child specific UFs with poor
success (Ginsberg et al, 2004a). One of the key detoxification enzymes of chlorpyrifos,
paraoxonase 1 (PON1) is an A-esterase which can metabolize chlorpyrifos oxon without
inactivating the enzyme (Sultatos and Murphy, 1983). Extensive population variability
data from blood are available for PON1.
There are multiple PON1 polymorphisms reported in the literature including two
in the coding region, at least 13 in the noncoding region, and more than 150 single
nucleotide polymorphisms (snp; Jarvik et al, 2003). The amount of information on each
varies widely. The Q/R polymorphism at position 192 results from a Gln/Arg substitution
and affects catalytic efficiency (Humbert et al, 1993; Adkins, et al, 1993; Blatter Garin et
al, 1997; Mackness et al, 1998). Specific to activity on chlorpyrifos oxon, the R192
alloform has a higher catalytic efficiency of hydrolysis compared to the Q192 alloform
(Cole et al, 2005). This would suggest that individuals with the Q192 alloform may be
more sensitive. In the preliminary analysis, the Agency has focused on the PON1-192
polymorphism since it has been studied more extensively than any other, has been
linked to chlorpyrifos oxon sensitivity in animal studies, and has been evaluated in
studies attempting to associate PON1 status with health outcome following OP pesticide
exposure in adults and children.
The analysis summarized in Table 9 is preliminary. The Agency will be soliciting
comment from the SAP on several aspects of the current analysis. As described in
detail in Appendix E, the Agency has followed the 2005 IPCS guidance. In that
guidance, UFHK can be determined as the ratio of the dose metric at a lower percentile
(e.g., 10th, 5th, 2.5th, 1st percentile of the distribution) for those deemed sensitive and a
central tendency measure of the general population. The 50th and 5th percentiles were
calculated for each genotype and/or age group. Ratios of the 50th-percentile and the
5th-percentile were calculated. The Agency has performed calculations on the QQ, QR,
and RR genotypes but has only reported the results for the QQ and QR genotypes here
as these groups are potentially more sensitive to chlorpyrifos or its oxon (Holland et al,
2006; Cole et al, 2005; Furlong et al, 2005). Data on paraoxonase (POase), ARase, and
CPOase have been evaluated. There are two alternatives to performing the
calculations: 1) compare the 5th percentile of the QQ group to the 50th percentile of the
QQ group 2) or to compare the 5 percentile of the QQ group to the 50 percentile of
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the QR group. The QR would, theoretically, represent the intermediate-speed
metabolizers and potentially more representative of a central tendency estimate.
However, as shown below, in most studies and among substrates, the QQ-QQ and QQ-
QR ratios provide similar results thus suggesting that either comparison may be
suitable.
They represent values under consideration for intra-species TK extrapolation
(i.e., UFAK). For purposes of comparison, the default UFHKis 3X. Thus, values which
differ substantially from 3X are of particular interest. Ultimately, the UFAKwill be
combined with the 3X UFnofor the intra-species extrapolation factor.
For the majority of studies evaluated where only adults were included, the
resulting ratio of 50th/5th percentile ratios were 3X or less. This suggests that for a
the default 3X factor is a reasonable approximation of within human variability.
In the four scenarios which considered newborns and mothers, the values are
substantially greater than 3X—ranging from approximately 7X up to 31X. Based on this
finding, the Agency preliminarily concludes that age-related maturation is the major
contributor to population variability with respect to PON1 activity. CPOase data are the
most appropriate for assessing population variability with respect to detoxification of
chlorpyrifos oxon. CPOase data are limited in that only one study reports CPOase data
in newborns and mothers (Holland et al, 2006). For CPOase, the QQ-QQ and QQ-QR
ratios provide similar results, approximately 11-12X5.
5 The Agency notes that this factor of 12X differs from the population variation estimates reported by the study
authors. Holland et al, (2006) report population variation estimates of approximately 70-fold in mothers and
newborns for CPOase. These values are derived from a comparison of the lowest and highest values as thus
represent the minimum and maximum.
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Table 9. Preliminary Results of DDEF analysis for Intra-Species Extrapolation for
TK (UFAK) based on PON1 Activity.
Nationality/Ethnicity
American, adult female,
multiple ethnic groups7
American, adult male,
multiple ethnic groups7
African-American,
newborns5
African American, mothers5
African American, mothers &
newborns5
Caucasian;4
Caucasian newborns5
Caucasian mothers5
Caucasian mothers &
newborns5
Hispanic newborns5
Hispanic mothers5
Hispanic mothers &
newborns5
Iranians2
Latino newborns6 (n = 130)
Latino mothers6 (n = 130)
Latino mothers & newborns6
Peruvians1
Workers (high OP exp)3
Workers (low OP exp)3
POase
QQsoth/
QQsth
1.3
1.4
QRsOth/
QQsth
3.4
3.5
Not reported
1.7
2.2
1.6
9.9
1.9
1.1
1.1
4.6
8.2
5.1
312
3.1
2.3
2.3
A Rase
QQsoth/
QQsth
QRsOth/
QQsth
Not reported
Not reported
2.6
1.6
7.2
2
2.3
1.5
12.0
2.2
1.7
7.8
1.3
3.1
1.6
18.5
1.9
1.1
1.1
2.7
1.8
8.3
2
2.3
1.4
11.4
2.1
1.7
8.2
1.1
4
1.6
17.5
2.2
1.1
1.1
CPOase
QQsoth/ QRsOth/
QQsth QQsth
1.3 1.4
1.3 1.2
Not reported
2.3 3
1.7 1.8
10.8 11.6
1 Catano, et al (2006), 2 Sepahvand, et al (2007), 3 Sirivarasei, et al (2007), 4 Brophy, et al
(2001), 5 Chen, et al (2003), 6 Holland, et al (2006), 7Kisicki et al (1999)
PON1 activity is affected by many things including genetic status in the coding
region such as the PON1-192 and PON1-55 genotypes, but also in the regulatory
region (Brophy et al, 2001; Deakin et al, 2003). For example, Deakin et al (2003)
describe three polymorphisms in the PON1 promoter; each polymorphism leads to
differences in activity. Furthermore, PON1 activity can be affected by environmental
factors like smoking (Nishio and Watanabe, 1997; James et al, 2000; Jarvik et al, 2000),
fat content in the diet (Shih et al, 1998; Hedrick et al, 2000), consumption of
antioxidants (Aviram et al, 2000) or consumption of alcoholic beverages (Hayek et al,
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1997; van der Gaag et al, 1999). In pregnant women and umbilical cord blood, POase
activity can also be affected by duration of labor or the type of delivery (Viachos et al,
2006). It is interesting to note that the PON 1-192 R allele has been associated with pre-
term births (Lawlor et al, 2006; Chen et al, 2004). With regard to changes during
pregnancy, available studies show different results. Ferre et al (2006) showed a
decrease in paraoxon hydroxylation of approximately 25% in late gestation compared to
nonpregnant background levels. Carpintero, et al (1996), however, found that phenyl
acetate metabolism increased from approximately 40% in the third trimester.
Serum A-esterase levels are very low in human infants compared to adults
(Augustinsson and Barr, 1962; Mueller et al., 1983; Ecobichon and Stephens, 1973;
Holland et al, 2006; Chen et al, 2003). After birth, there is a steady increase of this
activity (Augustinsson and Barr, 1962). Similarly, Burlina et al (1977) evaluated age-
dependence of total serum arylesterase activity and showed that adult levels were
achieved by two years-of-age. The Agency is aware of yet unpublished data on POase,
ARase and CPOase in children up to age 5 from Drs. Nina Holland and Brenda
Eskanazi with a much larger sample size (>200) than previous studies. These data
were presented at the ASHG (Huen et al, 2007) and suggest that POase activity may be
lower than adult levels up to 47 months. After completion of the data analysis and
ultimately publication, these data will substantially improve the overall understanding of
the human ontogeny of POase, ARase and CPOase. The findings of the older literature
(Augustinsson and Barr, 1962; Mueller et al., 1983; Ecobichon and Stephens, 1973)
combined with more recent studies by Holland et al (2006) and Chen et al (2003)
support a similar conclusion that newborns and young children have lower levels of
PON1 than do adults. More specifically, newborns have lower levels of PON1 than do
other age groups. As such, these findings suggest that development and use of a
DDEF for intra-species TK extrapolation from newborn and maternal data would be
protective of other age groups since PON1 levels are expected to be lowest at birth.
Some have suggested that PON1 status is a key contributor in chlorpyrifos
sensitivity whereas others have suggested that a significant amount of OP must be
present in the blood or brain for PON1 activity to affect toxicity based on generally low
affinity (Km, 0.1-10 mM; Aldridge and Reiner, 1972; Fonnum and Sterri, 2006; Timchalk
et al, 2002b). This concept, namely relevance of PONIat environmentally relevant
concentrations, is key for determining its potential use in human health risk assessment.
In addition, a key uncertainty in the use of PON1 data in the risk assessment is the
extent to which reliance on population variability from a single enzyme (i.e., PON1)
reflects actual variability given that multiple detoxification pathways are functioning
which may modulate deficits. The Agency has considered data from multiple sources in
this evaluation: information from PBPK model simulations, in vitro studies, animal
studies, and human epidemiology.
PBPK Model Simulations: PBPK modeling is valuable tool as it provides a
computational approach to evaluate the relative importance of specific metabolic
parameters such as the relatively high Km of PON1. Moreover, a PBPK model involves
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consideration of multiple pathways simultaneously and thus can consider the extent to
which one or more metabolic pathways may modulate deficits in other pathways.
Timchalk et al (2002b) performed Monte Carlo analysis of PON1 levels from
adults for the QQ, QR, and RR genotypes using the chlorpyrifos PBPK model. In these
simulations, at lower doses (~5 ug/kg) CPOase was not a determinant in the outcome.
However, at higher doses (~0.5-5mg/kg), the authors suggest that CPOase may be a
determinant in toxicity. The authors further suggest that other esterase detoxification
pathways may adequately compensate for lower CPOase activity; hence an increased
sensitivity to low CPOase is not observable until other detoxification pathways or
esterases have been appreciably depleted or overwhelmed.
The same group of investigators has used PBPK modeling to evaluate changes
in PON1 consistent with newborn levels and changes during pregnancy (Public
comment to the FIFRA SAP by Drs. Poet and Timchalk). The PBPK simulations
reported in the public comment provide similar findings as Timchalk et al (2002b) in that
reductions in PON1 levels, including levels consistent with the 12-fold DDEF shown in
Table 3, did not have substantial impact on BuChE inhibition levels. The models
discussed in the public comment have not been through substantial peer review and
have not been published in the literature. Moreover, they do not provide information on
dose or effects to the fetus and on effects in children younger than 5 years old. They
do, however, provide information that suggests that at environmentally relevant
concentrations of chlorpyrifos, PON1 status may not be a determinant in toxicity in older
toddlers and adults.
In vitro data: Due to the relatively high Km of PON1, Mortenson et al (1996)
tested the ability of CPOase to hydrolyze the oxon at physiologically relevant
concentrations (e.g., nM to low uM). Mortenson et al (1996) reported that CPOase
activity in rats was indeed capable of hydrolyzing physiologically relevant concentrations
of chlorpyrifos oxon; thereby suggesting that CPOase may hydrolyze the oxon at low
environmental concentrations. In a recent study by Sogorb et al (2008), serum
albuminase activity was compared to POase, CPOase, and diazoxonase. At
concentrations of chlorpyrifos oxon up to 5 uM, CPOase was effective at preventing any
meaningful reductions in AChE activity. On the other hand, a clear dose-dependant
increase in AChE inhibition was noted for serum albuminase. As stated by the authors,
"the activity associated with PON1 was able to fully protect AChE in the case of
chlorpyrifos-oxon, where the contribution of albumin was barely significant."
In vivo animal data: To investigate the role of PON1 on chlorpyrifos sensitivity,
Cole et al (2005) used a transgenic mouse model which expresses human PON1Q192
or PON1R192 at equivalent levels in the absence of endogenous mouse PON1. The
investigators compared effects of chlorpyrifos and the oxon following dermal exposure
to mice. They showed that adult mice expressing PON1-Q192 were significantly more
sensitive to the oxon than were mice expressing PON1-R192. As shown in Figure 7,
this sensitivity was evident at all tested doses but was more pronounced at higher
doses.
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Figure 7. Dose-response of chlorpyrifos oxon on inhibition of brain AChE
activity. Extracted from Figure 2.a in Cole et al (2005).
PON1-R192
PON1-Q192
O PON1-;-
0%
0 1 2
CPO dosage (mg/kg, dermal)
Studies in agricultural workers: There are a couple studies available which have
evaluated effect of PON1 status in agricultural workers who handle OPs; these studies
provide inconsistent findings. It is important to note that all three studies are limited to
varying degrees and suffer from many of the same weakness as other case controlled
studies. For example, each is limited by small sample size, the amount of exposure
information collected, and recall bias. Both studies provide little exposure information,
including which OPs that the workers were exposed to. Povey et al (2005) evaluated
sheep dippers in the UK who reported chronic ill health and have handled OPs. In this
study, for self-reported symptoms consistent with OP poisoning, odds ratios for QR or
RR genotype were approximately 2-fold higher than those for the QQ genotype. Lee et
al (2003) reported an increased incidence of reported symptoms consistent with chronic
OP exposure in QQ or QR genotypes in 100 workers in South Africa (odds ratio of 2.9,
confidence interval 1.7-6.9).
Epidemiology studies in children & mothers: With regard to effects in children,
three publications by the same group at Mt. Sinai, New York report associations
between maternal PON1 activity and birth outcomes (Engel et al, 2007; Berkowitz et al,
2004; Wolff et al, 2007). PON1 activity measurements for the Mt. Sinai cohort are found
in Chen et al (2003).
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In Berkowitz et al (2004), maternal levels of TCP above the limit of detection in
combination with low maternal PON1 activity were associated with a significant (albeit
small) reduction in head circumference. Berkowitz et al (2004) further reported that
maternal PON1 levels, not PON1 genetic polymorphisms, were associated with reduced
head size. In a follow up study, Engel et al (2007) reported that abnormal reflexes were
associated with total dimethylphosphate (DMP) metabolites when ARase activity was
included in the analysis. Diethylphosphates6 (chlorpyrifos is a diethylphosphate; DEP)
and total di-alkylphosphate (DAPs) were associated with abnormal reflexes without
inclusion of PON1 status in the analysis. Wolff et al (2007) evaluated the association
between DAP levels and birth outcomes from the same cohort. With the lowest tertile
ARase activity, urinary DEPs were associated with lower birth weight and DMPs with
shorter birth length. Wolff et al (2007) also reported that birth length was shorter for RR
mothers compared with QQ mothers.
As of August, 2008, the Agency was not aware of any studies published in the
literature evaluating PON1 status and health outcome in children from the CHAMACOS
cohort. PON1 status has been measured in the CHAMACOS cohort and reported by
Holland et al (2006). The investigators have communicated plans to present data on
PON1 status and birth outcomes at the upcoming ISEE/ISEA meeting (2008). The
Columbia University investigators have not measured PON1 status in the mothers or
children in the other NY cohort.
In summary, animal studies using in vivo studies in transgenic animals and in
vitro techniques support that PON1 status effects sensitivity to chlorpyrifos oxon.
Studies in transgenic animals must be interpreted with care as they represent an
artificial model—human genes expressed in the mouse. Moreover, the Cole et al
(2005) evaluates primarily high doses. Human epidemiology data on agricultural
workers and in children are limited. Results of epidemiological studies in workers would
be more convincing with larger sample sizes and a prospective study design. The
reported associations reported by Engel et al (2007), Berkowitz et al (2004) and Wolff et
al (2007) in children would be more convincing if similar findings were available in
another cohort of children and mothers. Such data from the CHAMACOS cohort may
be available in late 2008 or early 2009.
Key preliminary conclusions in the chlorpyrifos hazard characterization are: 1)
juveniles are more sensitive than adults and 2) this sensitivity is derived, at least in part,
based on TK differences in young and adults, including PON1 (or A-esterase). There
are remaining uncertainties regarding the relevance of PON1 at environmentally
relevant concentrations and further uncertainties regarding the extent to which other
detoxification pathways modulate deficiencies in PON1 activity. However, on balance,
population variability with respect to PON1 status can not be ruled out as a determinant
in tissue dose, and ultimately toxicity, to the fetus or to very young children. As shown
below, the Agency is proposing two options for the intra-species TK extrapolation factor
(UFHK). The first option involves using a UFHK of 12X derived from PON1 data (from
CPOase activity in newborns and mothers (Holland et al, 2006; Table 4)) which would
6 Chlorpyrifos is a diethylphosphate OP.
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lead to an intra-species extrapolation factor of 36X when combined with the 3X for
UFHo. The other option involves using the default factor of 3X for both the UFHK and the
UFHD which would lead to an intra-species factor of 10X. The Agency will solicit
comment on the science which supports both of these options.
4.0 Summary & Next Steps
The Agency has performed a review of the scientific literature under the context
of its use in human health risk assessment. This review provides the foundation for
proposed PoDs and UFs. Both the preliminary conclusion from the literature review and
the proposed PoDs and UFs will be reviewed by the SAP in September, 2008.
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Attachment 1.0. RBC and brain ChE activity in dams and fetuses from comparative ChE studies following
gestational exposure (From USEPA, 2006, Section II.B.2)
OP
Ace p hate
MRID 46151805
Azinphos methyl
MRID 46291 101
Cholinesterase &
Group
Dose
GD 21 Dams
RBC
Brain
GD 21 Fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Fetuses
RBC
Brain
Dose (mg/kg/day)
0
1.6360±
0.7461
8.6009 ±
1.4779
1.7284±
0.5776
1.4688±
0.0871
0
1.43 ±0.31
11.1 ±0.5
1.36 ±0.28
2.2 ±0.1
0.5
1.9691 ±
0.7684
7.1673±
0.8621 (17)
1.9883±
0.7651
1.3613±
0.1320
1
2.3221 ±
0.5884
7.0441 ±
0.900(18)
1.4476±
0.2403
1.2915 ±
0.1313(12)
0.2
1.41 ±0.30(1)
10.8 ±0.7 (3)
1.30 ±0.07 (4)
2.3 ±0.1
2.5
1.4638 ±
0.7615
5.096
±0.933(41)
1.0662±
0.3121
1.2586±
0.1666(14)
0.9
1.39 ±0.43 (3)
10.0± 1.9(10)
1.31 ±0.15(4)
2.3 ±0.2
10
1.5202 ±
0.6202
3.3112 ±
0.5209 (62)
1.3385±
0.5334
0.8816 ±
0.1254(40)
1.2
1.05 ±0.20 (27)
10.7±0.7(4)
1.32 ±0.17 (3)
2.2 ±0.1 (0)
Section II.B.2 -Page 77 of 81
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OP
Chlorpyrifos
MRID 44648102
% activity compared to
control
Diazinon
MRID 45842602
Dicrotophos
MRID 46153201
Cholinesterase &
Group
Dose
GD 20 Dams
RBC
Hindbrain
GD 20 Fetuses
RBC
Hindbrain
Dose
GD 20 Dams
RBC
Brain
GD 20 Male fetuses
RBC
Brain
GD 20 Female fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Male fetuses
RBC
Brain
GD 20 Female fetuses
RBC
Brain
Dose (mg/kg/day)
0
0
1.1 06± 0.163
17.272± 1.041
1.1 88± 0.230
2.383 ±0.194
1.208 ±0.143
2.311± 0.198
0
2593 ±21 8
4.78 ±0.99
2546± 112
1.75± 0.34
2523 ± 455
1.57± 0.18
0.3
73.7**±14.5
101.1±7.2
102.2±20.3
107.0±5.0
0.084
1.183 ±0.165
16.925± 1.066
1.392 ±0.183
2.380± 0.262
1.325±0.172
2.360± 0.395
0.05
2342 ±79 (10)
4.26± 1.06(10)
2423± 351
1.51± 0.25(14)
2362± 50
1.36± 0.13(13)
1
17.6**±6.7
92.0*±2.2
106.4±16.7
99.7±5.6
0.825
0.71 9± 0.223 (35)
16.675± 0.617
1.319± 0.230
2. 194 ±0.161
1.363± 0.254
2.231± 0.234
0.2
1638± 120(37)
2.49± 0.51 (48)
1923± 190(24)
1.22± 0.28(30)
1825± 207(28)
1.22± 0.11 (24)
5
4.9**±2.8
24.0**±4.8
7.9**±4.3
46.1**±9.3
26.23
0.00± 0.00 (100)
3.228 ±0.229 (81. 3)
0.247 ±0.162 (79.2)
1.689± 0.348 (29.1)
0.217± 0.148 (82.0)
1.822±0.372(21.2)
1.0
1282 ±226 (51)
1.03 ±0.21 (78)
1311± 124(49)
0.77± 0.08(56)
1414± 142(44)
0.72± 0.02(54)
Section II.B.2 -Page 78 of 81
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OP
Dimethoate
MRID 45529702
Disulfoton
MRID 46635901
Fosthiazate
Not yet assigned
Methamidophos
MRID 46660901
Cholinesterase &
Group
Dose
GD 20 Dams
RBC
Brain
GD 20 Fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
Dose (mg/kg/day)
0.0
1669 ± 180
12,838 ± 1373
1213 ±79
1781 ± 175
0
2.02±0.34
11.97±0.53
1.27±0.16
1.81±0.30
0
3931± 1474.5
49446±2189.8
2644± 644.1
6612 ±679.5
0
1.64 ±0.286
10.82 ±0.271
0.1
1563 ±224 (6)
13,044 ±530 (-2)
1225±98(-1)
1569 ± 173(12)
0.042
1.66±0.31 (18)
11.35±0.50(5)
1.21±0.20
1.75±0.28
0.1
3831 ± 757.3
48974 ± 1364.5
3283 ± 992.4
6328 ± 476.3
0.10
1.68± 0.220
10.40± 1.711
0.5
1459 ±278 (13)
11, 563 ±300 (10)
1181 ± 172(3)
1600 ± 136(10)
0.168
1.13±0.37(44)
8.12±0.44(32)
1.02±0.19(20)
1.74±0.26
0.7
2193 ±712.2 (44)
47135 ± 1510(5)
2893± 738.3
6251 ± 649.5 (5)
1.03
0.84± 0.117(49)
4.86 ±0.416 (55)
3.0
709 ± 104(58)
5094 ± 1081 (60)
834 ± 183(31)
1188 ± 164(33)
0.694
0.20±0.13(90)
1.76±0.19(85)
0.22±0.11 (83)
1.18±0.21 (35)
5
20 ±0.0 (99)
5152± 1718.9(90)
1851 ±593.4(30)
51 82 ±684.5 (22)
3.12
0.45 ±0.1 18 (73)
2.32± 0.173(79)
Section II.B.2 -Page 79 of 81
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OP
Methyl parathion
MRID 45646501
Phorate
MRID 46241402
Terbufos
MRID 46240802
Cholinesterase &
Group
GD 20 Fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Male fetuses
RBC
Brain
GD 20 Female fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Male fetuses
RBC
Brain
GD 20 Female fetuses
RBC
Brain
Dose
GD 20 Dams
RBC
Brain
GD 20 Male fetuses
RBC
Brain
Dose (mg/kg/day)
1.29 ±0.196
1.56 ±0.157
0
1500.1 ±255.03
13.48 ±0.807
1041. 3± 145.79
2.10± 0.116
1090.4 ± 163.7
2.06 ±0.152
0
35.98 ± 1.12
2.95 ±0.54
7.05 ±0.83
0.57 ±0.01
6.80± 0.99
0.59± 0.04
0
42.30 ±5.00
3.00 ± 1.12
5.16 ± 1.48
0.59± 0.11
1.13±0.147
1.51± 0.089
0.03
1702.3 ±386.36
13.58 ±0.428
1082.2 ± 160.9
2.05± 0.095
1118. 0± 131.13
2.12±0.14
0.03
33.92 ±3.76
2.88 ±0.74
5.72 ±0.51 (19)
0.58 ±0.04
5.81± 0.91
0.57 ±0.04
0.03
40.68 ±4.00
3.00± 0.79
4.63 ± 1.86
0.53± 0.05
0.72 ±0.1 33 (44)
1.08 ±0.125 (31)
0.30
979.5± 283.80
(35)
12.26 ±0.527 (9)
1075.0 ± 135.32
2.04 ±0.173
1010. 2 ± 130.36
2.06 ±0.174
0.1
30.99 ±4.82 (14)
2.94± 0.70
5.69 ±0.66 (19)
0.56± 0.03
5.48± 0.89
0.58± 0.02
0.1
14.42± 4.04(66)
1.96± 0.68(35)
2.51± 0.86(51)
0.48±0.04(19)
0.38 ±0.075 (55)
0.77 ±0.061 (51)
0.60
632.9± 124.52(58)
9.35± 1.026(31)
808.9 ± 186.38(22)
1.97± 0.073
894.9± 215.77(18)
2.02 ±0.092
0.2
27.64 ±5.16 (23)
1.73 ±0.67 (41)
6.42 ±0.56
0.60 ±0.03 (6)
6.28 ±0.78
0.59 ±0.02
0.3/0.2
4.46± 1.64(89)
0.69± 0.19(77)
1.62 ±0.69 (69)
0.36 ± 0.09 (39)
Section II.B.2 -Page 80 of 81
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OP
Cholinesterase &
Group
GD 20 Female fetuses
RBC
Brain
Dose (mg/kg/day)
4.32 ±0.85
0.53 ±0.04
4.52 ±0.99
0.57 ±0.04
1.99± 1.09(54)
0.50± 0.05
1.76 ±0.75 (59)
0.36± 0.07(32)
Section II.B.2 - Page 81 of 81
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