EPA-HSRB-11-02
Paul Anastas, PhD
EPA Science Advisor
Office of the Science Advisor
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Subject: April 13-14, 2011 EPA Human Studies Review Board Meeting Report
Dear Dr. Anastas,
The United States Environmental Protection Agency (EPA or Agency) requested that the
Human Studies Review Board (HSRB) provide scientific and ethics reviews of several com-
pleted studies and scenarios involving intentional exposure of human subjects to pesticides or
other EPA regulated compounds.
The first of these scenarios, a set of three interrelated studies measuring dermal and inha-
lation exposure of professional agricultural workers who spray pesticides using open-cab airblast
equipment, was conducted by the Agricultural Handler Exposure Task Force, LLC (AHETF).
These studies (AHE62, AHE63 and AHE64) were conducted after publication of the EPA's ex-
panded final rule for protection of subjects in human research (40 CFR 26) on February 6, 2006
(71 Federal Register 24, 6137). These studies were conducted under a single scenario monograph
(MRID 48326701), which incorporates the data from these three studies into a single dataset. An
additional set of data, collected as part of a third-party sponsored protocol (AHE07) conducted
prior to publication of the EPA's expanded final rule for protection of subjects in human re-
search, will also be included in this data set. This data set will be posted to the Agricultural Han-
dlers Exposure Database (AHED®), and used generically to estimate daily dermal and inhalation
exposures of workers who treat agricultural crops with conventional pesticides using open-cab
airblast equipment.
In addition, the Board reviewed a completed study of dermal and inhalation exposure of
professional janitorial workers who clean indoor surfaces with an antimicrobial pesticide product
using ready-to-use wipes or a trigger spray bottle and wipe, conducted by the Antimicrobial Ex-
posure Assessment Task Force II (AEATF II). This study (AEA-02) was also conducted after
publication of the EPA's expanded final rule for protection of subjects in human research. The
data will be posted to the Biocide Handlers Exposure Database (BHED®), and used generically
to estimate daily dermal and inhalation exposures of those who wipe indoor surfaces with anti-
microbial pesticides.
The Board also reviewed a published study involving intentional human exposure to the
zinc oxide contained in sunscreens. The Agency proposes to rely on this study, conducted after
publication of the EPA's expanded final rule for protection of subjects in human research, for
regulatory actions.
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Finally, the Board considered additional sponsor-provided information regarding the Ki-
sicki et al. (1999) study on chlorpyrifos, which was originally discussed by the HSRB in June
2009 (EPA HSRB 2009). As the review and discussion of this additional information was con-
ducted independently rather than at the request of the Agency, the Board’s conclusions are at-
tached to this meeting report as Appendix 1.
The enclosed report provides the Board’s response to EPA charge questions presented at
the April 13-14, 2011 meeting.
Assessment of Comuleted AHETF Research Studies A11E62 . AHE63 and AHE64: Deter-
mination of Dermal and Inhalation ExDosure to Workers During Airblast A Iications of
Liquid Sprays Using Open Cab Equipment ( MRID 48289611. 48289612. 48289613.
48289614 48289615. 48289616 and 48326701).
Science
The Board concluded that the research reported in the completed monograph, associated
field study reports, and associated supplemental documents was conducted in a manner that was
reasonably faithful to the design and objectives of the protocol and governing documents of the
AHETF.
The Board also concluded that the Agency has adequately, but not completely, cons i-
dered the limitations on these data that should be considered when using the data in estimating
the dermal and inhalation exposure of those who apply conventional pesticides with open-cab
airblast equipment. Additional limitations and concerns have been identified by the Board, and
the conclusion as to the generalizability of these data requires further consideration and analysis.
Ethics
The Board concluded that the study was conducted in substantial compliance with sub-
partsKandL4OCFR26.
Assessment of Completed AEATF II Research Study AEAO2: Measurement of Potential
Dermal and Inhalation Exuosure during Application of a Liquid Antimicrobial Pesticide
Product Using Tri aer Snrav and Wipe or Ready-to-Use Wipes for Cleaning Indoor Sur-
faces (MRID 48375601).
Science
The Board concluded that the research reported in the completed monograph, associated
field study reports, and associated supplemental documents was conducted in a manner that was
reasonably faithful to the design and objectives of the protocol and governing documents of the
AEATF II.
The Board also concluded that the Agency has adequately, but not completely, cons
dered the limitations on these data that should be considered when using the data in estimating
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dermal and inhalation exposures of handlers who are applying antimicrobial pesticides using a
trigger spray or ready-to-use wipes.
Ethics
The Board concluded that the study was conducted in substantial compliance with sub-
parts K and L 40 CFR 26.
Assessment of Published Research Study MRID 48387301: Gulson et al. (2010) Small
Amounts of Zinc from Zinc Oxide Particles in Sunscreens Applied Outdoors Are Absorbed
throu2h Human Skin.
Science
The Board concurred with the Agency’s assessment that the Gulson et al. (2010) study
provides some potentially useflul data on the dermal absorption of zinc from zinc oxide nanopar-
tides in sunscreen applied to human skin, despite the multiple limitations identified by the
HSRB. However, the Board advised the Agency to proceed with caution when using these data
in risk assessment, as these data cannot be used as a stand-alone set to assess dermal absorption
of zinc oxide.
Ethics
The Board concluded that there was insufficient information available at the time of the
April meeting to determine whether the Gulson et a!. (2010) study was conducted in substantial
compliance with procedures at least as protective as those in subparts A - L of EPA’s regulation
at 40 CFR Part 26. The Board recommended that EPA seek additional information from Macqu-
arie University or the study investigator (including a copy of the research protocol and unsigned
informed consent form) and that information be provided to the HSRB for re-consideration.
Sincerely,
ç s
Sean Philpott, PhD, MSBioethics
Chair
EPA Human Studies Review Board
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NOTICE
This report has been written as part of the activities of the EPA Human Studies Review
Board, a Federal advisory committee providing advice, information and recommendations on
issues related to scientific and ethical aspects of human subjects research. This report has not
been reviewed for approval by the Agency and, hence, the contents of this report do not neces-
sarily represent the view and policies of the Environmental Protection Agency, nor of other
agencies in the Executive Branch of the Federal government, nor does the mention of trade
names or commercial products constitute a recommendation for use. You may obtain further
information about the EPA Human Studies Review Board from its website at
httD://www.eDa.gov/osalhsrb . You may also contact the HSRB Designated Federal Officer, via
e-mail at ord-osa-hsrb epa.gov
In preparing this document, the Board carefully considered all information provided and
presented by the Agency presenters, as well as information presented by public commenters.
This document addresses the information provided and presented within the structure of the
charge by the Agency.
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US ENVIRONMENTAL PROTECTION AGENCY
HUMAN STUDIES REVIEW BOARD
Chair
Sean Philpott, PhD, MSBioethics, Director for Research Ethics, The Bioethics Program of Union
Graduate College and the Mount Sinai School of Medicine, Schenectady, NY
Vice Chair
Janice Chambers, PhD, DABT, Fellow ATS, William L. Giles Distinguished Professor, Director,
Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State
University, Mississippi State, MS
Members
George Fernandez, PhD, Professor of Applied Statistics, Director of the University of Nevada-
Reno Center for Research Design and Analysis, University of Nevada-Reno, Reno, NV
Vanessa Northington Gamble, MD, PhD, University Professor of Medical Humanities, Professor
of Health Policy and American Studies, The George Washington University, Washington, DC
Sidney Green, Jr., PhD, Fellow of the ATS, Professor, Department of Pharmacology, Howard
University College of Medicine, Washington, DC
Dallas E. Johnson, PhD, Professor Emeritus, Department of Statistics, Kansas State University,
Manhattan, KS
Michael D. Lebowitz, PhD, FCCP, Professor (NTE) of Medicine-Research/Scholar, University
of Arizona, Tucson, AZ
Jose E. Manautou, PhD, Associate Professor of Toxicology, Department of Pharmaceutical
Science, University of Connecticut School of Pharmacy, Storrs, CT
Jerry A. Menikoff, MD, JD, Director, Office for Human Subjects Research, Office of the
Secretary, Department of Health and Human Services, Rockville, MD
*Rebecca Parkin, PhD, MPH, Professorial Lecturer (EOH) Human Risk Assessment, School of
Public Health and Human Services, The George Washington University, Washington, DC
William Popendorf, PhD, MPH, Professor Emeritus, Department of Biology, Utah State Univer-
sity, Logan, UT
Virginia Ashby Sharpe, PhD, Medical Ethicist, National Center for Ethics in Health Care, Veter-
ans Health Administration, Washington, DC
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Linda J. Young, PhD, Professor, Department of Statistics, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL
Human Studies Review Board Staff
Jim Downing, Executive Director, Human Studies Review Board Staff, Office of the Science
Advisor, United States Environmental Protection Agency, Washington, DC
* Not in attendance at the April 13-14, 2011 Public Meeting
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INTRODUCTION
On April 13-14, 2011, the United States Environmental Protection Agency’s (EPA or
Agency) Human Studies Review Board (HSRB) met to address scientific and ethical issues con-
cerning two completed study monographs. One study measured levels of dermal and inhalation
exposure received by janitorial workers who apply antimicrobial pesticides using trigger spray or
ready-to-use wipes. The second was comprised of three completed, interrelated studies measur-
ing levels of dermal and inhalation exposure received by pesticide applicators who spray pesti-
cides with open-cab airblast equipment. These studies, along with a fourth study conducted prior
to publication of the EPA’s expanded fmal rule for protection of subjects in human research,
were compiled into a single scenario monograph (MRID 48326701), which incorporates the data
from these studies into a single data set. in accordance with 40 CFR 26.1602, EPA sought HSRB
review of these two completed study monographs. The completed studies are discussed more ful-
ly below.
In addition, the Agency sought HSRB review of one published study of dermal absorp-
tion of zinc (Zn) from zinc oxide (ZnO) nanoparticles in sunscreen applied to human skin. This
study, conducted after publication of the EPA’s expanded fmal rule for protection of subjects in
human research, was identified by Agency scientists from the peer reviewed literature. This
study, which the Agency proposes to rely upon for regulatory actions, is discussed in detail be-
low.
Finally, the HSRB considered additional sponsor-provided information regarding a pre-
Rule intentional exposure study of chlorpyrifos, originally discussed by the Board in June 2009.
The Board’s review and discussion of this additional information was conducted independently
rather than at the request of the EPA. The Board’s discussion and conclusions are attached to this
meeting report as Appendix 1.
REVIEW PROCESS
On April 13-14, 2011, the Board conducted a public face-to-face meeting in Arlington,
Virginia. Advance notice of the meeting was published in the Federal Register as “Human Stu-
dies Review Board; Notice of Public Meeting” (76 Federal Register 59, 17122).
Following welcoming remarks from Agency officials, the Board heard presentations from
EPA on the following topics: three completed studies (compiled into a single monograph) mea-
suring dermal and inhalation exposure received by pesticide applicators who spray pesticides
with open-cab airblast equipment, one completed study measuring levels of exposure received by
janitorial workers when applying antimicrobial pesticides using a trigger spray or ready-to-use
wipes, and one published study measuring dermal absorption of zinc (Zn) from zinc oxide (ZnO)
nanoparticles in sunscreen applied to human skin.
The Board also asked clarifying questions of several study sponsors and/or research in-
vestigators, including:
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Ms. Megan Boatwright, Analytical Coordinator, Golden Pacific Laboratories
Dr. Victor Cafiez, Risk Assessment Solutions; Commenting on behalf of the Agricultural
Handler Exposure Task Force, LLC (AHETF).
Dr. Richard Collier, Administrative Committee Chair, AHETF.
Dr. Has Shah, Manager, Antimicrobial Exposure Assessment Task Force II (AEATF II).
Mr. Robert Testman, Vice-President, Golden Pacific Laboratories.
Public oral comments were provided by:
Dr. Victor Cai ez, Risk Assessment Solutions; Commenting on behalf of the AFIETF.
Dr. Richard Collier, Administrative Committee Chair, AHETF.
Dr. Has Shah, Manager, AEATF II.
One written public comment was submitted by Ms. Carol Dansereau, Executive Director
of the Farm Worker Pesticide Project, Seattle, WA.
For their deliberations, the Board considered the materials presented at the meeting, oral
comments, and Agency background documents (e.g., published literature, sponsor and investiga-
tor research reports, study protocols, data evaluation records, and Agency science and ethics re-
views of proposed protocols and completed studies). A comprehensive list of background docu-
ments is available online at httD://www.regulations.gov .
CHARGE TO THE BOARD AND BOARD RESPONSE
Assessment of Completed AHETF Research Studies A11E62. AHE63 and AHE64: Deter-
mination of Dermal and Inhalation Exposure to Workers During Airblast Anplications of
Liquid Sprays Using Open Cab Equipment .
Overview of the Study
Three separate field studies were conducted, each monitoring dermal and inhalation ex-
posure of workers to commercially available pesticides while spraying tree or trellis crops in
three different U.S. states where open-cab airblast equipment is commonly used in production
agriculture. A total of 13 professional agricultural handlers were monitored as they applied pesti-
cides using open-cab airblast equipment: three adult men applying pesticides to grape vines in
California (AHE62), five adult men applying pesticides to grape vines in New York (AHE63),
and five adult men applying pesticides to pecan trees in Oklahoma (AHE64). The scenario de-
sign, protocols for the three studies, SOPs and governing documents were reviewed favorably by
the HSRB at its October 21-22, 2008 (EPA HSRB 2008b).
Monitored on actual days of work, study participants handled from 7 to 90 lbs of active
ingredient (carbaryl or malathion), spraying 3 to 24 acres in 1.4 to 10.6 hours. Dermal exposure
was measured using hand washes, face/neck wipes, and whole body dosimeters (100% cotton
union suits) for the remainder of the body (torso, arms, and legs). Inhalation exposure was meas-
ured using personal air sampling pumps and OVS mounted on the shirt collar. Results represent
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dermal exposure with and without chemical-resistant hats while wearing a long-sleeved shirt,
pants, shoes/socks and chemical-resistant gloves, and inhalation exposure without respiratory
protection.
The Agency proposes to use data from these three studies, along with a previous open-
cab airbiast study involving 15 male participants applying pesticides to apple, orange, peach and
pear trees in Florida, Georgia and Idaho (AHEO7; Smith 2005), to estimate generically daily
dermal and inhalation exposures of workers who treat agricultural crops with conventional pest i-
cides using open-cab airbiast equipment.
Science
Charge(s) to the Board
1. Was the research reported in the AHETF completed monograph report and associated
field study reports faithful to the design and objectives of the protocol, SOPs and governing doc-
uments?
2. Has EPA adequately characterized, from a scientific perspective, the limitations on
these data that should be considered when using the data in estimating exposure of those who
apply pesticides with open-cab airblast equipment?
Board Response to the Charge(s)
HSRB Recommendation
The Board concluded that the research reported in the completed monograph, associated
field study reports, and associated supplemental documents was conducted in a manner that was
reasonably faithful to the design and objectives of the protocol and governing documents of the
AHETF.
The Board also concluded that the Agency has adequately, but not completely, consi-
dered the limitations on these data that should be considered when using the data in estimating
the dermal and inhalation exposure of those who apply conventional pesticides with open-cab
airblast equipment. Additional limitations and concerns have been identified by the Board, and
the conclusion as to the generalizability of these data requires further consideration and analysis.
HSRB Detailed Recommendations and Rationale
The HSRB concluded that the research reported in the completed monograph report and
the associated field study reports of Open Cab Airblast Applicator Exposures (Bruce 2010a;
Bruce 2010b; Bruce 2010c; Klonne and Holden 2010; Smith 2005) was faithful to the design and
objectives of the protocol, SOPs, and governing documents. Four limitations were suggested as
potential additions to those outlined in the Agency’s Reviews of the monograph and associated
field study reports (Crowley 2011; Crowley and Sarkar 2011). The magnitude of one of these
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limitations cannot be determined but could significantly impact the accuracy of the measured
exposures in handlers who are not wearing a chemical resistant (CR) hat.
The AHETF seems to have responded well to most of the concerns expressed by the
HSRB during their prior reviews of the protocol (EPA HSRB 2006; EPA HSRB 2008b). For
instance, the use of an informal survey/poll of local experts that identified the absence of the
“Kinkelder” airbiast sprayer, the use of a refmed and better defmed recruitment process, and the
inclusion of descriptive records of applicator behavior. In the end, the analytical chemistry me-
thods seemed appropriate and well chosen, the quality assurance (QA) considerations appeared
to be in place, and the desired study conditions were largely obtained.
Although no formal statistical test for differences among crops or clusters was provided,
the visual evidence in the Science Review (Figure 1; Crowley 2011, 12) is strong support for
their similarity. The Board supports the Agency’s plan to combine the datasets and to use a
mixed-model approach for statistical analysis to account for potential data clustering for both the
existing data and new monitoring. The Board also agrees with a similar approach and conclusion
regarding the negligible effect of canopies used by six of the 28 monitoring units (MUs).
The use of head patches inside and outside CR hats was innovative. Its use obviated the
need for an MEA for the portion of the head covered by the hat, probably increased the accuracy
of measuring exposure with a hat, and yielded potentially useful estimates of the protection from
exposure that the hat provided. At the same time, the use of this technique presents two limita-
tions in estimating exposures without such a hat.
First, it could be argued that when estimating exposure without such a CR hat that the
amount reaching the inner patch should not be counted. This argument would apply if the path-
way by which the active ingredient that was deposited onto the inner patch was via a micro-scale
form of drift under the hatband (“penetration”) and not directly through the hat’s material (“per-
meation”). The effect on the overall dermal exposure of this sort-of double counting the patch
data is insignificantly small (on the order of 0.1%) due to the very small deposition measured on
the inner patch.
Second, the use of this innovation may have reduced the accuracy of measuring exposure
without a hat because the presence of a wide-brimmed hat probably also reduced deposition onto
the exposed face and neck from what it would have been had the hat not been there at all. The
magnitude of this reduction is undetermined and seems undeterminable with the information
available. The brim is likely to have acted somewhat like a canopy, only much closer. Its effect
is likely to have been greater on the portions of the face and neck closest to the underside of the
hatband and to decrease on the portions further away, such as the tip of the nose. The effect of
the brim’s protection is probably clear but not overwhelming.
The Agency might reconsider applying a 1/2 limit of quantification (LOQ) factor when
no active ingredient is found in the backup portion of the OVS air samples. A proper sample
should have no airborne chemical in that portion. In fact, finding active ingredient there implies
some degree of overloading of the front portion of the sampling media. Consistently assigning a
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non-detect measurement in the backup portion of a value of 1/2 limit of detection (LOD) results
in a biased result.
Four additional limitations were suggested. The first is to include such qualitative data as
the absence of the “Kinkelder” airbiast sprayer from the array of equipment included within the
study. This sprayer is an older model noted by the local experts to be prone to spray drift. The
implications of higher drift on handler exposures depend on a combination of environmental
conditions and work practices. However, the broader point is that such qualitative information
could become a valuable resource to future users of the exposure database.
The second limitation is the potential underestimation of spray actually deposited onto
the wiped portion of the subjects’ head due to shielding by the brim of the CR hats that were al-
ways worn.
The third limitation is the possible exceptional status of one monitoring unit involved in
exposure monitoring for open-cab spraying of Oklahoma pecans (Study AHE64; Monitoring
Unit A5). This MU used nozzle pressure that was twice as high as that recommended for the
type of nozzle (and three to four times as high as reported by any other MU using this type of
nozzle). The Board recognized that increasing nozzle pressure will cause a decrease in droplet
size and will be one of the reasons for an increase in spray drift. This MU also changed from
spraying in both directions to spraying in only one direction for the last third of his application
time. This was the only MU for which such a comment was made. Two reasons were suggested
by the Board for why an applicator might make such a change in spraying direction: either to na-
vigate sloping terrain or/and to avoid windblown drift. No information is available regarding the
terrain, but the initial one way pattern resulted in this MU spraying only when driving upwind.
Both of these reasons could cause this MU’s exposures to be low. Whether these differences
would otherwise disqualify this MU is unknown.
The fourth limitation is the need to rely on model accuracy for estimates to be interpreta-
ble. The primary objective is to estimate the geometric mean, the arithmetic mean, and the 95 th
percentile of normalized exposures (exposure per pound of active ingredient handled [ AaiH])
within 3-fold with 95% confidence. The secondary objective is to assess proportionality
(ln(exposure) = ln(AaiIl)) [ CHANGE] against the alternative of independence in ln(exposure)
and ln(AaiH) with 80% power. It is critical to carefully evaluate the validity of the model to en-
sure that the mean of the normalized exposures on the log scale (and the geometric mean on the
data scale) are interpretable. Because clusters are part of the study’s design, a random cluster ef-
fect was included in the model. How the presence of a cluster effect impacts interpretation
should be considered carefully.
In the Agency’s Review (Crowley and Sarkar 2011), an alternative approach was taken in
which the normalized exposures were modeled using a lognormal distribution. The validity of
this approach depends on two assumptions. First, the expectation of ln(exposure/AaiH) must be
constant. It is important to assess the validity of the linear relationship between ln(exposure) and
ln(AaiH . Second, for the approach to be valid, the mean response of ln(exposure/AaiH) should
be the same for every cluster. Otherwise, a single lognormal distribution would not describe the
normalized exposures. Perhaps this is why some of the Q-Q plots for the lognormal distribution
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(Appendix B; Crowley and Sarkar 2011, 24-5) indicate some lack of fit, especially in the tails. (It
is not possible to be sure that these correspond to the inhalation data for which a significant clus-
ter effect was present because the graphs as presented to the Board were not labeled.)
The Agency would benefit from carefully considering all assumptions underlying a pro-
posed statistical approach and then checking the validity of each. Care should be taken to ensure
that the results can be meaningfully interpreted. The term “arithmetic mean” should be changed
to “mean,” especially when a model is used to estimate the population mean. It would be helpful
for the axes of all graphs to be labeled. Lastly, in the write-up of results, implying that the failure
to reject a null hypothesis indicates that the null hypothesis is true should be avoided.
The Agency should note that more detailed suggestions and analyses may be provided by
some Board members independent of this consensus Board report.
Ethics
Charge to the Board
Does the available information support a determination that the studies were conducted in
substantial compliance with subparts K and L of 40 CFR Part 26?
Board Response to the Charge
HSRB Recommendation
The Board concurred with the Agency’s assessment (Sherman 2011a) that the study was
conducted in substantial compliance with subparts K and L 40 CFR 26.
HSRB Detailed Recommendation and Rationale
The documents provided include reports of each of three field studies conducted on be-
half of the AHETF (Bruce 2010a; Bruce 20 lOb; Bruce 2010c). The submitted study documents
state that the study was conducted in substantial compliance with the ethical and regulatory stan-
dards of 40 CFR 26, Subparts K and L. The requirements of FIFRA §12(a)(2)(P) and Title 3, §
6710 of the California Code of Regulations also apply.
The three protocols were each reviewed and approved pursuant to the standards of the
Common Rule (45 CFR Part 46, Subpart A) by an independent human subjects review commit-
tee, Independent Institutional Review Board, Inc. (IIRB, Inc.) of Plantation, FL. Minutes of
IIRB, Inc. meetings and a copy of IIRB, Inc. policies and procedures were provided (Bruce
2010d; Bruce 2010e; Bruce 2010f; IIRB 2010a; IIRB 2010b; Sherman 201 la). IIRB, Inc. is reg-
istered with the US Office for Human Research Protections (OHRP; Reg. # 10RG0002954) and
is fully accredited by the Association for the Accreditation of Human Research Protection Pro-
grams, Inc. (AAHRPP).
1. The Board concurred with the conclusions and factual observations relating to the study, as
detailed in the EPA’s Ethics Review (Sherman 201 Ia) and summarized briefly below.
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a. Prior HSRB and Agency Review.
Because each of these three studies was initiated after 7 April 2006, prior submission of
the protocol and supporting materials to EPA was required by 40 CFR §26.1125. The re-
quirements of 40 CFR §26.1125 for prior submission of the protocol to EPA and of
§26.1601 for HSRB review of the protocol were satisfied. The scenario designs and study
protocols were initially approved by IIRB, Inc. in July 2008. The HSRB discussed these
three protocols at its October 2008 meeting, concurring with the Agency’s assessment
that these three proposed open cab airbiast field study protocols, if revised as suggested
by the Agency and the HSRB, would meet the applicable requirements of 40 CFR Part
26, subparts K and L (EPA HSRB 2008b).
b. Responsiveness to HSRB and Agency Recommendations.
The initial ethics reviews by the Agency (Carley and Evans 2008a; Carley and Evans
2008b; Carley and Evans 2008c) and by the HSRB (EPA HSRB 2008b) provided nine
recommendations with regard to these three protocols. All of those recommendations,
and the responses made with regard to them, are detailed in the submitted documents
(Sherman 2011, 25). The HSRB agreed with the Agency that the comments by the Agen-
cy and HSRB were addressed satisfactorily.
2. The Board concluded that this study, as conducted, met all applicable ethical requirements for
research involving human participants, in accordance with the following criteria:
a. Acceptable risk-benefit ratio.
The risks to study participants were minimized appropriately and were justified by the
potential societal benefits, particularly data on the dermal and inhalation exposure of pro-
fessional pesticide applicators to the liquid pesticides they apply to orchard and trellis
crops using an airbiast sprayer drawn by a vehicle with no cab. These data could be used
to develop mechanisms to protect future persons who apply these liquid pesticides.
Minors and pregnant or lactating women were excluded from participation, with preg-
nancy confirmed by over-the-counter pregnancy testing on the day of study or by opt-out.
The potential of stigma resulting from study exclusion was also appropriately minimized.
Clear stopping rules and medical management procedures were in place, and no adverse
events or other incidents of concern related to product exposure were reported.
The study was designed to minimize the risks of exposure to the test compounds, subject
to being able to accomplish the purposes of the study.
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b. Voluntary and informed consent of all participants.
The study protocol included several mechanisms designed to minimize coercive recruit-
ment and enrollment.
Monetary compensation was not so high as to unduly influence participation.
In addition, it is worth noting that twelve of the thirteen subjects were owner-operators,
and thus less likely to be as vulnerable to coercion or undue influence as an employee
might be.
3. Eight minor protocol deviations were reported. In addition, there was one unreported but mi-
nor protocol deviation. These are documented in detail in the EPA Ethics Review (Sherman
201 la, 8-10). The Board agrees with the Agency’s assessment that these deviations did not
compromise the informed consent process or put the study participants at increased risk.
4. The Board agrees with the observations in the EPA Ethics Review (Sherman 201 la) with re-
gard to heat index thresholds. In particular, in November 2009—after this research had been
conducted—the SOP for these types of studies was revised to lower the acceptable heat index
threshold from 120° F to 105° F. However, as noted in Table 4 of the EPA Ethics Review
(Sherman 2011 a, 15), the heat index values for the subjects enrolled in this study never ex-
ceeded 105° F, so this was not an issue for this research.
Assessment of Comnieted AEATF II Research Study AEAO2: Measurement of Potential
Dermal and Inhalation Exposure durina Application of a Liquid Antimicrobial Pesticide
Product Usina Tri er Snrav and Wipe or Ready-to-Use Wipes for Cleaning Indoor Sur-
faces .
Overview of the Study
The objective of this study was to measure individual exposures to a surrogate antimi-
crobial pesticide (didecyl dimethyl ammonium chloride; DDAC) while cleaning surfaces with a
trigger spray and wipe or with ready-to-use wipes. A total of 36 volunteers participated in the
study, 18 cleaning surfaces using a trigger spray and wipe and 18 cleaning surfaces using ready-
to-use wipes in one of three building types (an office building, a Rite Aid pharmacy building, or
a retired teacher’s memorial building in Fresno, CA). Participants cleaned surfaces for one of six
pre-determined wiping times (30-60, 60-90, 90-120, 120-150, 150-180 and 180-210 minutes to-
tal cleaning time, respectively). The study protocol, SOPs and governing documents were
reviewed favorably by the HSRB at its April 9-10, 2008 meeting (EPA HSRB 2008a).
Dermal and inhalation exposure monitoring was conducted while study participants
cleaned surfaces. Dermal exposure was measured by inner and outer body dosimeters. Airborne
concentrations of the surrogate compound were monitored in the participant’s breathing zone us-
ing an OVS tube sample collector connected to a personal sampling pump. Environmental cond i-
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tions were also recorded at the time of monitoring, and observers made notes, photographs and
videos of participant activity throughout the wiping period.
These exposure data will be used to populate a database representing inhalation and der-
ma! exposure during a number of antimicrobial handler scenarios. A scenario is defmed as a pes-
ticide handling task based on activity (e.g., application) and equipment type (e.g., mop & bucket,
ready-to-use wipes, pressure treatment of wood facilities, painting). These data will be used by
the Agency to estimate dermal and inhalation exposures of antimicrobial handlers who are apply-
ing pesticides using a trigger spray and wipe or ready-to-use wipe under a variety of scenarios.
Science
Charge(s) to the Board
1. Was the research reported in the AEATF II completed wipe study report faithful to the
design and objectives of the protocol and governing documents of the AEATF II?
2. Has EPA adequately characterized, from a scientific perspective, the limitations on
these data that should be considered when using the data in estimating exposure of those who
clean indoor surfaces with antimicrobial pesticides using a trigger-bottle and wipes or ready-to-
use wipes?
Board Response to the Charge(s)
HSRB Recommendation
The Board concluded that the research reported in the completed study reports and asso-
ciated supplemental documents was conducted in a manner that was reasonably faithful to the
design and objectives of the protocol and governing documents of the AEATF II.
The Board also concluded that the Agency has adequately, but not completely, characte-
rized the limitations on these data that should be considered when using the data in estimating
dermal and inhalation exposures of antimicrobial handlers who are applying pesticides using a
trigger spray or ready-to-use wipes.
HSRB Detailed Recommendations and Rationale
The Board concurred with the EPA’s assessment (Leighton 2011) that the AEATF II
made the appropriate changes to the protocol proposed by the EPA and HSRB and has executed
the study in a way that is reasonably faithful to the design and objectives of the protocol and go-
verning documents of the AEATF II. Although several protocol deviations occurred, they were
unlikely to have adversely affected the reliability of the data. The Agency’s analysis of the data,
especially their use of adjustments for the QAIQC field recovery samples, was an improvement
in estimating exposure based on actual monitoring.
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There was one potentially important shortcoming of the study. The calculated rates at
which the active ingredient was applied to the treated surfaces did not achieve the stated target
application rate of 7 mg of active ingredient per m 2 of treated surface. Only one of the partici-
pants using ready-to-use wipes and only two of the participants using a trigger spray achieved
the 7 mg/rn 2 target. Neither the origin nor the significance of not meeting this target-is known.
While the protocol failed to achieve this technical target, this target was not recognized hereto-
fore to potentially be in conflict with achieving the important goal of letting the subjects “wipe
surfaces as they would normally work” (Selim 2011, 43). By all appearances, the study did
achieve the latter goal.
The Board believes that the EPA has insufficiently characterized from a scientific pers-
pective the limitations on these data. There were limitations that were not satisfactorily ad-
dressed. As an example, there were too many non-detects in the whole body dosimeters, which
created the positive regression lines for the dermal exposures by anchoring at the low end. The
assumptions of their modeling should be tested. Then, the appropriate regression models should
be run on the results of the hand wipes (exposure by AaiH). If this is done, the effects of the non-
detects can be evaluated in a step-wise fashion.
The results of the non-parametric bootstrap had narrower confidence limits and should be
used instead of the parametric bootstrap results. Interpretations of the results should be re-
evaluated, as they are currently too strong given the lack of any statistical significance. The lack
of any relationship of inhalation exposure to AaiH should be accepted. The results of evaluating
the K-factor, especially for inhalation, show the study to be underpowered to evaluate this asso-
ciation. Conclusions that estimates at the high end of AaiH are overestimates are definitely incor-
rect in the threshold model, which shows values below the estimated line at high AaiH and high-
er values in the middle of the range of AaiH that are elevating the curve.
Two potential new limitations to the study were also identified. One is an outgrowth of a
search to explain the result that the dermal unit exposure value for the ready-to-use wipe applica-
tions is twice as large as that for the trigger spray bottle. The difference in results may be due to
the different ways in which the amount of active ingredient handled was calculated in the two
studies, and this explanation has implications to the validity of extrapolations based on AaiH.
The two methods treated about the same amount of surface area in the same amount of time and
resulted in about the same exposure, but the amount of biocide handled by the trigger spray bot-
tle applicators was about twice that handled by ready-to-use wipe applicators. The study was not
designed to tell how much active ingredient (ai) actually reached the target surface nor if the two
methods were equally effective in treating the surfaces. Wiping has no intrinsic means to control
the rate at which the active ingredient is applied. The rate depends completely on the applicator.
The methods used to calculate the amount of ai handled may not directly relate to the amount.
Neither method accounts for the amount of ai that stayed on the wipe or rag at the time it was
discarded, and this unknown may have affected the calculated amount of ai handled. The possi-
bility that the almost two-fold difference in the amounts handled (and in unit exposure values) is
attributable to the difference in the amount left on the wipes and rags suggests a limitation to the
data that should be considered when using the data. The need to extrapolate via unit exposures
may be elevated due to the fact that the test conditions were well less than the target application
rates of 7 mg aiim 2 .
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The second limitation stems from the predominance of hand exposures. Noting that the
hands account for 92% of the dermal exposure for the trigger spray bottle uses (Selim 2011, 107)
and 98% for the ready-to-use wipe users (Selim 2011, 98) brings attention to a potentially impor-
tant limitation of the recovery correction factor based on the DDAC dermal exposure and recov-
ery study conducted by Boatwright (2007). Although this study investigated the recovery at two
dermal application rates, it appears that it did not investigate the effect of retention time on re-
covery from the skin. Without data one can only conjecture that the behavior of DDAC might
mimic the behavior of the few insecticides in which similar tests have included time. In their
January 2007 report, the EPA’s Scientific Advisory Panel pointed out the importance of a chem-
ical’s residence time on the skin in the recovery efficiency (EPA SAP 2007). (This report was
also cited by the HSRB in their June 2007 policy on MEAs.) Parts of that discussion implied
that, in general, recoveries from skin are (or appear to be) higher soon after deposition and de-
crease as either the chemical resides on the skin for longer durations or continuous or repeated
applications saturate the ability of the skin to retain as much of the ai deposited later as it could
earlier. Both of these scenarios result in what appear to be decreases in the recovery efficiency
of those chemicals from skin as exposure time increases.
The same concerns about the use of a single lognormal distribution when there are cluster
effects, raised by the Board in its discussion of the AHETF open cab airbiast study, also apply
here.
The Agency should note that more detailed suggestions and analyses may be provided by
some Board members independent of this consensus Board report.
Ethics
Charge to the Board
Does the available information support a determination that the study was conducted in
substantial compliance with subparts K and L of 40 CFR Part 26?
Board Response to the Charge
HSRB Recommendation
The Board concurred with the Agency’s assessment (Sherman 201 Ib) that the study was
conducted in substantial compliance with subparts K and L 40 CFR 26.
HSRB Detailed Recommendation and Rationale
The documents provided include a report of a completed wipe study conducted on behalf
of the AEATF II (Selim 2011). The submitted study documents state that the study was con-
ducted in substantial compliance with the ethical and regulatory standards of 40 CFR 26, Sub-
parts K and L. The requirements of FIFRA §12(a)(2)(P) and Title 3, § 6710 of the California Code
of Regulations also apply.
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The protocol was reviewed and approved pursuant to the standards of the Common Rule
(45 CFR Part 46, Subpart A) by an independent human subjects review committee, IIRB, Inc. of
Plantation, FL. Minutes of IIRB, Inc. meetings and a copy of IIRB, Inc. policies and procedures
were provided (IIRB 2010a; IIRB 2010b; Selim 2011; Sherman 201 ib). As stated previously,
IIRB, Inc. is registered with OHRP and is fully accredited by AAHRPP.
1. The Board concurred with the conclusions and factual observations relating to the study, as
detailed in the EPA’s Ethics Review (Sherman 201 ib) and summarized briefly below.
a. Prior HSRB and Agency Review.
Because this study was initiated after April 7, 2006, prior submission of the protocol and
supporting materials to EPA was required by 40 CFR §26.1125. The requirements of 40
CFR §26.1125 for prior submission of the protocol to EPA and of 26.1601 for HSRB
review of the protocol were satisfied. The HSRB reviewed the protocol at its April 2008
meeting, concluding that “if the proposed mop and wipe scenario design, protocol, and
supporting documentation is revised as suggested in EPA’s review, the research would
meet the applicable requirements of 40 CFR part 26, subparts K and L” (EPA HSRB
2008a, 2).
b. Responsiveness to HSRB and Agency Recommendations.
The HSRB noted two instances where the sponsor did not address comments raised by
the HSRB in its April 2008 report. In the first instance, the HSRB recommended that the
consent form be revised to explain that the “underlying purpose of the study will be to
collect information that will be provided to the EPA, and that the EPA would use that in-
formation to determine the appropriate standards for allowable exposures to products
such as the test compound” (EPA HSRB 2008a, 25). In the second instance, the Board
recommended that the recruitment flyer should explain that the research would measure
inhalation as well as dermal exposure (EPA HSRB 2008a, 26). The sponsors could not
explain the lack of responsiveness to these two recommendations. The Board concluded
that the lack of responsiveness to these two HSRB recommendations did not compromise
the ethical conduct of the study, but recommended that the Agency follow up with the
sponsor to determine why these recommendations were not addressed.
2. The Board concluded that this study, as conducted, met all applicable ethical requirements for
research involving human participants, in accordance with the following criteria:
a. Acceptable risk-benefit ratio.
The risks to study participants were minimized appropriately and were justified by the
potential societal benefits, particularly data on the dermal and inhalation exposure of pro-
fessional janitorial workers to the liquid antimicrobials they apply. These data could be
used to develop mechanisms to protect future persons who apply these liquid antimicro-
bials.
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Minors and pregnant or lactating women were excluded from participation. All study par-
ticipants were at least 18 years old. All females self-administered an over-the-counter
pregnancy test on the day of monitoring, and all such tests were negative. No nursing
mothers were enrolled in the study.
The study was designed to minimize the risks of exposure to the test compounds, subject
to being able to accomplish the purposes of the study. The study involved the use of a
commonly used, commercially available cleaning product, in a matter consistent with its
labeling.
Clear stopping rules and medical management procedures were in place. There was one
report of a participant leaving early because he was feeling ill. Although the observation
log does not indicate whether the worker was feeling ill as a result of his participation in
the research or whether his illness was unrelated to the study, documentation provided to
the Agency and the HSRB subsequent to the April 2011 meeting suggests that this partic-
ipant was unwell prior to study initiation. However, there was no indication that there had
been any follow up of the participant. Although there is little reason to think that this par-
ticipant would develop a serious and unexpected medical problem related to his participa-
tion after leaving the study site, the Board recommended that the Agency work with
sponsors to develop a SOP for following up with participants who leave a study early be-
cause of illness.
b. Voluntary and informed consent of all participants.
The study protocol included several mechanisms designed to minimize coercive recruit-
ment and enrollment.
Monetary compensation was not so high as to unduly influence participation.
3. Two minor protocol deviations were reported. In addition, there were two unreported but mi-
nor protocol deviations. The reported deviations were that: 1) the protocol called for study
participants to take 10-minute rest breaks, but most participants declined or took breaks that
were less than 10 minutes in length; and 2) a photograph of a participant’s face was taken at
one monitoring site, although no images were included in the report.
The Agency review noted one unreported deviation (Sherman 201 ib). This deviation in-
volved the enrollment of three participants (one who was monitored and two who were alter-
nates) who self-reported that their health was only “fair,” despite the requirement that all partici-
pants be in “good health”. In response to a similar protocol deviation that occurred during the
related AEATF II mop study discussed at the October 2010 HSRB meeting, the Board rec-
ommended the AEATF II develop a SOP for clarif ’ its health status reporting and inclusion
criteria (EPA HSRB 2010). This study was conducted prior to the October 2010 HSRB meet-
ing.
The HSRB also discovered an additional unreported deviation, namely the use of an “LRB-
unapproved form to collect personal information about volunteers and to sign a ‘Worker
Page 19of30
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Health Statement” (Selim 2011, 3). These forms were stored separately from the field phase
data. The sponsors could not explain this deviation from the protocol.
The HSRB concluded that these protocol deviations did not compromise the informed con-
sent process or put the study participants at increased risk.
Assessment of Published Research Study by Gulson el aL (2010): Small Amounts of Zinc
from Zinc Oxide Particles in Sunscreens AnDlied Outdoors Are Absorbed throuEh Human
Skin .
Overview of the Study
In the Gulson et a!. (2010) study, 20 human volunteers were exposed to sunscreens con-
taining stable isotopes of zinc oxide (ZnO) for the purpose of measuring dermal absorption of
zinc (Zn) from ZnO nanoparticles of varying size. Such metal oxide nanoparticles are being used
more and more frequently in sunscreens because they reflect and absorb ultraviolet (UV) light as
transparent coatings, rather than as opaque coatings as with traditional metal oxide sunscreens.
The study was conducted in Australia in March 2009. Ten male and ten female subjects,
ranging from 19 to 66 years of age, were enrolled. Study participants wore UV protective upper
body clothing with a patch removed to expose a small section of skin on the back. Sunscreen
containing stable isotope 68 Zn oxide, formulated as “bulk” particles (110 ±46 nanometers [ nm]
in size) or as nanoparticles (19 ± 8 nm in size), was applied twice daily for a period of five days
to the exposed section of skin on the back. Eleven participants (5 males and 6 females) applied
68 Zn oxide formulated as nanoparticles and nine participants (5 males and 4 females) applied
68 Zn oxide formulated as bulk particles. Participants were encouraged to wear other non-zinc
types of sunscreen on the areas of skin not covered by the UV-resistant clothing. The test materi-
al was removed from each subject’s back at the end of each day with an alcohol-lanolin wipe.
Urine and blood samples were taken eight days before sunscreen application, immediate-
ly before the first application, at the end of each of the five days of application, and six days after
the last a 1 plication. Dermal absorption of Zn was calculated from the changes in the ratios of
68 Zn to 6 Zn in urine and blood samples during the course of the experiment; Zn was measured
using Multi-Collector Inductively Coupled Mass Spectrometry (MC-ICP-MS).
Science
Charge(s) to the Board
1. Is the Gulson et a!. (2010) study scientifically sound, providing reliable data?
2. If so, is the Gulson era!. (2010) study relevant for qualitative use in support of an as-
sessment of the absorption of zinc oxide through the skin?
Page 20 of 30
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Board Response to the Charge(s)
HSRB Recommendation
The Board concurred with the Agency’s assessment (Ryrnan and Dole 2011) that the
Gulson eta!. (2010) study provides some potentially useful data on the dermal absorption of zinc
from ZnO nanoparticles in sunscreen applied to human skin, despite the multiple limitations
identified by the HSRB. However, the Board advised the Agency to proceed with caution when
using these data in risk assessment, as these data cannot be used as a stand-alone set to assess
dermal absorption of zinc oxide.
HSRB Detailed Recommendations and Rationale
The Gulson et a!. (2010) study provides some potentially useful data on the dermal ab-
sorption of zinc from zinc oxide nanoparticles in sunscreen applied to human skin. The analytical
methods used in the study appeared to be sound. The use of the 68 Zn as a tracer for Zn from the
sunscreen was an innovative approach for assessing absorption and the presence of a chemical in
body fluids (e.g., blood or urine) when the same chemical is also present endogenously. The
Board also agreed with the comments of the Agency (Ryman and Dole 2011) that the distinction
between bulk and nanoparticles was not as absolute as indicated in the study since the lower
range of size for bulk particles is within the range of the size for nanoparticles. This overlap in
size between the groups of particles should not affect the outcome of the studies and the interpre-
tation of the results significantly. However, the Board raised a number of caveats, including lack
of a proper control group, small sample size, and sample contamination, which limits the utility
of this study in risk assessment of zinc oxide-based sunscreen.
While the analytical approaches used in the Gulson et a!. (2010) study to measure Zn in
blood and urine were solid, the study design itself was faulty. For example, there were no un-
treated controls to verify the consistency of Zn levels in untreated individuals. The presence of
increases in 68 Zn in the person who applied the sunscreen creates questions regarding the
changes that might occur with time independent of sunscreen application. The design was not a
carefully controlled experimental trial, and therefore was subject to a variety of behavioral dif-
ferences among the subjects as they pursued their beach recreation activities that are likely to
have influenced the results.
Despite the fact that the tracer analytical technique that measured Zn in blood and urine
did an excellent job in distinguishing endogenous Zn from Zn absorbed from the sunscreen, this
procedure did not distinguish between dermal penetration of soluble Zn from penetration of in-
tact nanoparticles, or from absorption via dermal versus oral routes. Therefore, this study has
many uncertainties associated with the interpretation of the data, and does not provide informa-
tion regarding the penetration of nanoparticles through intact, healthy skin. The study thus is of
limited value in judging the level of intact nanoparticle absorption through intact skin.
The sunscreen formulation contained isopropyl myristate, which is a known chemical en-
hancer of skin penetration, while the wash off solution used contained alcohol and lanolin. The
impact of these two variables on Zn absorption was not considered, nor did the report explain
Page 21 of3O
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how much Zn was removed by the daily alcohol and lanolin wash off procedure. Another source
of concern is the potential impact of ethylenediaminetetraacetic acid (EDTA), a commonly used
preservative and stabilizer, on the zinc oxide nanoparticle formulation. The authors did not ad-
dress the possibility that EDTA can chelate ionic Zn released from the nanoparticle formulation.
This is an important consideration since chelation of free zinc in the skin by EDTA can influence
the degree of dermal absorption. EDTA is routinely used in this type of sunscreen formulation,
but its impact on dermal Zn absorption was not addressed.
The final study design was based on a pilot study in which the zinc oxide active ingre-
dient was 51%, while the 68 Zn isotope in the final study was 99% enriched 68 Zn. The authors
provide insufficient information on the pilot study (e.g., age, gender, or skin type of subjects).
The Board concurs with the Agency’s assessment of lack of clarity on how the pilot study was
used in the design of the final study and subject selection.
Other variables not accounted for include the frequency and time of garment changes and
the type of activities the subjects engaged in after the first 30-minute exposure interval. There
was no mention either of the total length of the subjects’ sun exposure and the timing between
the first and second daily exposure. Although UV exposure was estimated to be the same for all
subjects, differences in length and intensity of physical activity likely contributed to the degree
of sweating and blood flow to the skin. No information was provided on the time of the second
application of the sunscreen in relationship to the first application. Description of dosing inter-
vals was missing.
The authors of the Gulson eta!. (2010) study provided no justification or criteria for se-
lecting subjects 4, 8, 9 and 13 used for complete analysis of all samples collected (Figure 8;
2010, 146). The use of retention rates to address the issue of urine sample contamination was a
very good approach; however, there was no mention of how these potential contaminations oc-
curred and the potential for hand contamination to have caused hand-to-mouth dosing of the sub-
jects. The investigators also did not consider the effect of the ZnO that was retained in the appli-
cator’s hand(s) on the quantities of the dosing rates reported in Table 1 of the manuscript (GuI-
son eta!. 2010,143).
The investigators speculated that the six urine specimens could have been contaminated
with 68 Zn from sunscreen on the participants’ hands during collection. In addition, it appears that
the authors did not attempt to minimize, control for, or account for the possibility of the conta-
minated hand-to-mouth route of exposure among their subjects. While the authors provided a
rather convincing post-hoc series of arguments to support a hypothesis of contaminated urine
samples (Gulson et a!. 2010, 146-7), they provided no description of a mechanism for how the
zinc would have gotten from a subject’s back into the urine sample. Since the collection con-
tainers were pre-cleaned and the beach towels provided to the subject were renewed daily in or-
der to eliminate any potential source of sample contamination, the reader is left to infer that six
of the ten female subjects somehow got a portion of the sunscreen from their backs onto their
hands and subsequently from their hands into the urine collection containers. If this transport of
sunscreen occurred with these subjects, then it is also plausible that 68 Zn from the sunscreen was
on the hands of all the subjects but that not all subjects contaminated their urine samples. If that
were the mechanism, then it also seems likely that sometime during the day these subjects had
Page 22 of 30
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one or more occasions to transfer sunscreen into their mouths via food, beverages, mouth wiping
and other hand-to mouth activities. Thus, it is plausible, if not likely, that the hands of many or
all subjects were contaminated with 68 Zn, and that either some (if not most) of the 68 Zn measured
in blood and urine was the result of the transfer from the subject’s back into their hands and then
to their mouths, leading to gastrointestinal absorption of 68 Zn. This alternative plausible me-
chanism could completely negate the validity of the authors’ conclusions regarding dermal ab-
sorption.
Gulson and his colleagues may have also made significant errors in the quantities of the
doses reported in Table I (Gulson et a!. 2010, 143) by not considering the amount of ZnO that
was retained on the applicator’s hands after dosing each subject. The report does not say wheth-
er or not the applicator was wearing gloves or using the same one-fmger application technique as
described for their pilot study. Gloves would have been important in the pilot study to avoid par-
ticulate contamination of the samples being generated for examination by scanning electron mi-
croscopy. It is reasonable to expect that a significant amount of sunscreen would have been re-
tained on the applicator’s glove or hand after each application. It is also reasonable to expect
that a larger fraction of the first application was retained on the applicator’s hand than the frac-
tion retained from subsequent applications. Thus, the effect that the retention of sunscreen on
the applicator’s gloves or hands would have had on individual subjects cannot be determined.
While the source of the j 68 Zn% in the blood from the applicator reported in Figure 4B
(Gulson eta!. 2010, 144) is unknown, the similarity of the A 68 Zn% in her blood to that reported
for the blood of the test subjects indicates that the applicator’s absorbed dose was similar to that
of the test subjects. Both of the following possible sources of her dose have serious implications
to the results. If the source were via the hand-to-mouth route, contamination due to field practic-
es would seem to be even more widespread than just intra-subject. If the source of her 68 Zn were
from dermal absorption through her hands but if the absorption rate through hands is less than
through the skin on the back (as has been reported in studies assessing anatomical variations in
dermal absorption of OP insecticides), the similarity of the blood and urinary excretion rates for
the applicator and the subjects would imply that the amount retained on the applicator’s hands
could have been several times larger than the dermal doses reported to be applied to the subject’s
backs in Table I (Gulson eta!. 2010, 143). Such dose values thus may be more inaccurate.
While the authors did not even mention isotope fractionation in their article, the effect of
isotope fractionation probably caused the authors to slightly over-estimate their calculated Zn
absorption factor. Isotope fractionation refers to the separation of one isotope in comparison to
either a lighter or heavier isotoge of the same element; in this case, the human body’s preferen-
tial absorption or retention of 6 Zn versus 64 Zn. This phenomenon was reported to occur in both
of the two “other studies of biological samples” cited by Gulson Ct al. (2010, 144). Isotopic
fractionation may have been the unrecognized cause of the fmding by Gulson et a!. that the mean
68 Znft 4 Zn ratio in their pre-exposure blood samples (0.4158) was 7% higher than the
18.8%/48.6% = 0.3868 natural background referred to in the manuscript (2010, 141). However,
the effect of isotope fractionation may only reduce the 68 Znt 4 Zn ratio by a few percent.
The researchers also failed to establish the temporal stability of an individual’s 68 Zn/ 64 Zn
ratio upon which their conclusion of an increase of that ratio in blood or urine is based (Gulson et
Page 23 of 30
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a!. 2010). The only example of time-series data that was located in the material presented to the
Board was the results by Ohno et a!. (2005), which measured the 68 Zn/ 64 Zn ratio in blood sam-
ples collected once per month from just one person in which the average month-to-month varia-
tion was reported to be ±6.6% (a coefficient of variation of ±6.2%). This natural variation is
considerably more than the 0.42% and 0.23% mean post-exposure increases reported by Gulson
eta!. (2010) over 13 and 19 days from day 0 to the end of the exposures and the six-day follow-
up, respectively. The magnitude of this natural variation and the lack of an untreated control
group suggest that the accuracy of the reported absorption factor could have been erroneous.
Regarding gender-related differences in dosing, the authors provide insufficient informa-
tion for readers to tell whether there was any interaction between the subject’s individual body
weights (not reported) and “a significant difference between doses for males and females with a
mean of 4.6 mg/cm 2 for males versus 3.7 mg/cm 2 for females” (Gulson eta!. 2010, 142). With-
out any implication of probability, it is possible that the fact that significant differences between
genders were found only when comparing their change in 68 Znft 4 Zn ratios without body weight
(reported in Figure 5A) but not when comparing their total zinc (i 68 Zn%) content in whole blood
using the subject’s weight and gender (reported in Figure SB) was a spurious association.
Regarding study sample selection, there are two major statistical concerns with the sam-
ple’s demographic constitution. The report indicates that the data came from 20 subjects split
between the nanoparticle group, with 6 females and 5 males and the bulk group with 4 females
and 5 males (Table 1; Gulson eta!. 2010, 143). This table, listing the subjects’ demographics,
showed greater similarities among the subjects due to family connections. Therefore, the ques-
tion arises whether the sample of individuals used in the study represents a random sample of the
reference population or whether is it a convenience sample. Because the experimental error or
within treatment variation could be underestimated in a non-random, convenient, and small sam-
ple, the statistical validity of the data used in this study is questionable. The second issue is re-
lated to the known outlier (subject 7, enrolled in the nanoparticle group). This subject’s ob-
served data point was relatively larger than all other data points (e.g., Figures 4 and 5; Gulson et
a!. 2010, 144). Also, an adverse or allergic reaction was mentioned in the report as the likely
reason for the large response observed in the case of subject 7. Therefore, this single observation
could be the main cause of the observed skewedness in the experimental data. The researchers
performed a log transformation to correct this high degree skewedness, which may be caused by
this single outlier. The statistical significance reported in the paper (gender x treatment) may
have disappeared if they had excluded this observation from the analysis or performed any robust
weighted linear model analysis to adjust for this influential data.
Based on these concerns the statistical validity of the data used and the conclusion de-
rived may be questionable. Thus, the results reported could be treated as exploratory in nature
and further studies are needed to confirm the conclusion.
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Ethics
Charge to the Board
Is there adequate information to determine that the Gulson eta!. (2010) study was con-
ducted in substantial compliance with procedures at least a protective as those in subparts A - L
of EPA’s regulation at 40 CFR Part 26?
Board Response to the Charge
HSRB Recommendation
The Board concluded that there was insufficient information available at the time of the
April meeting to determine whether the Gulson eta!. (2010) study was conducted in substantial
compliance with procedures at least as protective as those in subparts A - L of EPA’s regulation
at 40 CFR Part 26. The Board recommended that EPA seek additional information from Macqu-
arie University or the study investigator, including a copy of the research protocol and an un-
signed informed consent form, and that this information be provided to the HSRB for re-
consideration.
HSRB Detailed Recommendation and Rationale
This is the first time EPA has asked the HSRB to review a study from the published lite-
rature that was conducted after EPA’s amended Rule for the Protection of Human Subjects of
Research. The study was conducted in Sydney, Australia and received approval from the ethics
committee of Macquarie University, as required by the Australian Code for the Responsible
Conduct of Research (2007).
As noted in EPA’s ethics review (Parsons 2011), 40 CFR Part 26, subparts A through L
do not apply; the research was neither conducted or supported by EPA nor was it conducted by a
person with the intention to submit the results to EPA. FIFRA §12(a)(2)(P) does not apply to this
research since the compound tested is not a pesticide. Thus, the applicable acceptance standards
are as follows:
40 CFR §26.1703. Prohibition of reliance on research involving intentional exposure of
human subjects who are pregnant women (and therefore their fetuses), nursing women, or
children. Except as provided in §26.1706, in actions within the scope of 26.1701 EPA
shall not rely on data from any research involving intentional exposure of any human
subject who is a pregnant woman (and therefore her fetus), a nursing woman, or a child.
40 CFR §26.1705 Prohibition of reliance on unethical human research with non-pregnant,
non-nursing adults conducted after April 7, 2006. Except as provided in §26.1706, in ac-
tions within the scope of §26.1701, EPA shall not rely on data from any research initiated
after April 7, 2006, unless EPA has adequate information to determine that the research
was conducted in substantial compliance with subparts A through L of this part, or if
conducted in a foreign country, under procedures at least as protective as those in sub-
Page 25 of 30
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parts A through L of this part. This prohibition is in addition to the prohibition in
§26.1703.
The HSRB determined that most of the information that the Board has about the ethical
conduct of the Gulson eta!. (2010) study is without supporting documentation and thus any con-
clusions about the ethical conduct of the study would be inferential. Although the Board thought
that there is no reason to believe that the conduct of the reported research was fundamentally un-
ethical, in the absence of supporting documentation like the informed consent form and research
protocol, the Board concluded that it did not have adequate information to determine that partici-
pant selection was voluntary, that information about risks and benefits was sufficiently commu-
nicated, that study participants understood the nature of the research or their right to withdraw
from the study, or that the female participants were neither pregnant nor nursing. Thus, the Board
concluded that it lacked adequate information to determine that the research was conducted un-
der procedures at least as protective as those in subparts A through L of 40 CFR Part 26.
In the absence of the informed consent form, the Board had insufficient information to
determine how study risks and benefits may or may not have been characterized, whether partic-
ipants were informed of their right to withdraw from the study, whether participation involved
reasonable or unreasonable compensation or pressure to participate with family members. The
Board offers the following reasoning for its recommendation:
1. Acceptable risk-benefit ratio.
The Board agreed with the EPA review (Parsons 2011) that the main risks to participants
were in blood sampling and possible adverse reaction to the sunscreen formulations. Accord-
ing to Gulson eta!., the sunscreen formulations contained an enriched level of a stable iso-
tope of zinc, which does not pose a higher risk than naturally occurring zinc and “many”
commercially available sunscreens contain “nanoparticulate Ti02 and/or ZnO” (2010, 140).
Thus, although the risk-benefit balance was not discussed in the study report, the risks appear
to be low.
No minors were enrolled in the study. However, apart from an email from the study invest i-
gator to the Agency (Parsons 2011), the Board had no evidence that pregnant or lactating
women were excluded from participation.
The study reports that one participant (Subject 7) had an adverse reaction to the sunscreen.
The nature and severity of the adverse reaction was not described.
According to the published study, the risks were mitigated as follows: 1) risks of sun expo-
sure were mitigated with UV-protective clothing and advice to wear sunscreen; 2) Subject 7
ceased participation in the application portion of the study after an adverse reaction; and 3) a
trained phlebotomist drew participants’ blood.
The Board concluded that although the risks of the study were low, the “adequacy of infor-
mation” standard should not be strictly correlated with risk. That is, the threshold for adequa-
cy of information should not be understood in proportion to the amount of risk posed by the
Page 26 of 30
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study (if the risk is low, then the threshold for adequacy can be low). The Board determined
that such a standard would exclude other requirements of ethical research including voluntary
informed consent, respect for participants, and fair participant selection.
2. Voluntary and informed consent of all participants.
The Board concluded that it did not have adequate information to determine that participant
selection in the Gulson eta!. (2010) study was conducted in substantial compliance with pro-
cedures at least as protective as those in subparts A - L of 40 CFR Part 26.
Board members raised a concerns about the fairness and voluntariness of participant selec-
tion, including: 1) the investigator holding recruitment discussions with potential participants
in his home; 2) selecting participants who were “personal contacts” of the investigator; 3) in-
clusion of participants from the same family, suggesting that the study was conducted in a
context where subjects may have experienced undue pressure to participate; and 4) a lack of
information about the type and level of compensation that participants were offered. In addi-
tion, although the published paper states that “venous blood and urine samples were collected
8 days before exposure” (Gulson et a!. 2010, 140) an email from the study investigator to the
Agency (Parsons 2011) stated that “the subjects attended a meeting at my house 1 week be-
fore the trial started and I described the trial and any risks.” This apparent inconsistency in
the timing of the informed consent process and the collection of study-related samples raises
some concerns.
Although the Board recognizes that this research was reviewed and approved by the
Macquarie University Human Research Ethics Committee, which is registered with the Australi-
an National Health and Medical Research Council, it concluded that ethics committee approval
alone does not provide adequate information to determine that the Gulson et a!. (2010) study was
conducted in substantial compliance with procedures at least as protective as those in subparts A
- L of EPA’s regulation at 40 CFR Part 26. The Board thus recommended that EPA seek add i-
tional information from Macquarie University or the study investigator, including a copy of the
research protocol and unsigned informed consent form, and that this information be provided to
the HSRB for re-consideration.
Page 27 of 30
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REFERENCES
Australian National Health and Medical Research Council, Australian Research Council, Aus-
tralian Vice-Chancellor’s Committee. 2007. National Statement on Ethical Conduct in Human
Research. Canberra, AUS: Australian National Health and Medical Research Council. Available
online at http://www.nhmrc.gov.au/publications/synopses/el2syn.htm . Accessed April 14, 2011.
Boatwright, M. 2007. Determination of Removal Efficiency of Didecyl Dimethyl Ammonium
Chloride (DDAC) from Hand Surfaces Using Isopropyl Alcohol/Water Wash. Study Number
050211. Unpublished study prepared by Golden Pacific Laboratories. MRID 47214801.
Bruce, E.D., for the Agricultural Handler Exposure Task Force (AHETF). 2010a. Determination
of Dermal and Inhalation Exposure to Workers During Airblast Applications of Liquid Sprays
Using Open Cab Equipment in California Trellis Crops. Study Number AHE62. Dated Novem-
ber 3, 2010. Unpublished document prepared by the AHETF. MRID 48289611.
Bruce, E.D., for the AHETF. 20 lOb. Determination of Dermal and Inhalation Exposure to Work-
ers During Airblast Applications of Liquid Sprays Using Open Cab Equipment in New York
Trellis Crops. Study Number AHE63. Dated November 3, 2010. Unpublished document pre-
pared by the AHETF. MRID 48289612.
Bruce, E.D., for the AI-IETF. 2010c. Determination of Dermal and Inhalation Exposure to Work-
ers During Airblast Applications of Liquid Sprays Using Open Cab Equipment in Oklahoma
Tree Nuts. Study Number AHE64. Dated November 3, 2010. Unpublished document prepared
bytheAHETF. MRID 48289613.
Bruce, E.D., for the AHETF. 2010d. IIRB Correspondence Report for Cluster Report AHE62.
Dated November 4, 2010. Unpublished document prepared by the AHETF. MRID 48289614.
Bruce, E.D., for the AHETF. 2010e. IIRB Correspondence Report for Cluster Report AHE63.
Dated November 4, 2010. Unpublished document prepared by the AHETF. MRID 48289615.
Bruce, E.D., for the A1-IETF. 2010f. IIRB Correspondence Report for Cluster Report AHE64.
Dated November 4, 2010. Unpublished document prepared by the AHETF. MRID 48289616.
Carley, J., and J. Evans. 2008a. Science and Ethics Review of AHETF Scenario Design and Pro-
tocol A}1E62 for Exposure Monitoring in California Trellis Crops. Dated September 23, 2008.
Unpublished document prepared by the Health Effects Division, United States Environmental
Protection Agency.
Carley, J., and J. Evans. 2008b. Science and Ethics Review of AHETF Scenario Design and Pro-
tocol AHE63 for Exposure Monitoring in New York Grapes. Dated September 23, 2008. Unpub-
lished document prepared by the Health Effects Division, United States Environmental Protec-
tion Agency.
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Carley, J., and J. Evans. 2008c. Science and Ethics Review of AFIETF Scenario Design and Pro-
tocol AHE64 for Exposure Monitoring in Oklahoma Pecans. Dated September 23, 2008. Unpub-
lished document prepared by the Health Effects Division, United States Environmental Protec-
tion Agency.
Crowley, M. 2011. Review of Agricultural Handler Exposure Task Force (AHETF) Open Cab
Airbiast Applicator Exposure Monitoring Studies: A1-1E62, AHE63, AHE64. Dated March 1,
2011. Unpublished document prepared by the Health Effects Division, United States Environ-
mental Protection Agency.
Crowley, M., and B. Sarkar. 2011. Review of Agricultural Handler Exposure Task Force
(AHETF) Monograph: Open Cab Airblast Application of Liquid Sprays. Dated January 11,
2011. Unpublished document prepared by the Health Effects Division, United States Environ-
mental Protection Agency.
EPA Human Studies Review Board. 2006. June 27-30, 2006 Human Studies Review Board
Meeting Report.
EPA Human Studies Review Board. 2008a. April 9-10, 2008 Human Studies Review Board
Meeting Report.
EPA Human Studies Review Board. 2008b. October 21-22, 2008 Human Studies Review Board
Meeting Report.
EPA Human Studies Review Board. 2009. June 24-25, 2009 Human Studies Review Board
Meeting Report.
EPA Human Studies Review Board. 2010. October 27-28, 2010 Human Studies Review Board
Meeting Report.
EPA Scientific Advisory Panel. 2007. January 7-12, 2007 Scientific Advisory Panel Meeting
Report.
Gulson, B., M. McCall, M. Korsch, eta!. 2010. Small Amounts of Zinc from Zinc Oxide Particle
in Sunscreens Applied Outdoors are Absorbed Through Human Skin. Toxicological Sciences
118(1): 140-9. MRJD 48387301.
Independent Institutional Review Board, Inc. (IIRB, Inc.). 2010a. Human Research Protection
Plan. Unpublished materials prepared by IIRB, Inc.
IIRB, Inc. 20 lOb. Current Membership Roster. Unpublished materials prepared by IIRB, Inc.
Kisicki, J, C. Seip, and M. Combs. A Rising Dose Toxicological Study to Determine the No-
Observable-Effect-Levels (NOEL) For Erythrocyte Acetylcholinesterase (AChE) Inhibition and
Cholinergic Signs and Symptoms of Chlorpyrifos at Three Dose Levels. Dated April 15-19,
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1999. Unpublished study prepared by MDS Harris Laboratories under Project No. 21438 and
Dow AgroSciences Study No. DR K-0044793-284. MRID 44811002.
Klonne, D.R., and L.R. Holden for the AHETF. 2010. Agricultural Handler Exposure Scenario
Monograph: Open Cab Airbiast Application of Liquid Sprays. Dated December 15, 2010. Un-
published document prepared by the AHETF. MRID 48326701.
Leighton, T. 2011. Science Review of the AEATF II Wipe Human Exposure Monitoring Study
(Trigger Spray & Wipe and Ready-to-Use Wipes Scenarios). Dated March 17, 2011. Unpub-
lished document prepared by the Office of Pesticide Programs, United States Environmental Pro-
tection Agency.
Macquarie University Ethics Review Committee. 2011. Research Ethics Correspondence and
Approval Letters. Unpublished document submitted to the United States Environmental Protec-
tion Agency.
Ohno, T., A. Shinohara, M. Chiba, and T. Hirata. 2005. Precise Zn Isotopic Ration Measure-
ments of Human Red Blood Cell and Hair Samples by Multiple Collector-ICP-Mass Spectrome-
try.
Analytical Sciences 2 1(4): 425-8.
Parsons, L. 2011. Ethics Review of Intentional Exposure Human Toxicity Study. Dated March
16, 2011. Unpublished document prepared by the Office of Pesticide Programs, United States
Environmental Protection Agency.
Ryman, J., and T. Dole. 2011. Data Evaluation Record: 68 Zinc Oxide Nanoparticles/888502.
Dated March 15, 2011. Unpublished document prepared by the Health Effects Division, United
States Environmental Protection Agency.
Selim, S. 2011. Project No. AEAO2, Report No. 070264: A Study for Measurement of Potential
Dermal and Inhalation Exposure During Application of a Liquid Antimicrobial Pesticide Product
Using Trigger Spray and Wipe or Ready- to- Use Wipes for Cleaning Indoor Surfaces. Dated
January 26, 2011. Unpublished document prepared by Golden Pacific Laboratories, LLC. MRID
48375601.
Sherman, K. 201 la. Ethics Review of Completed AHETF Open Cab Airbiast Scenario Worker
Exposure Monitoring Study. Dated March 8, 2011. Unpublished document prepared by the Of-
fice of Pesticide Programs, United States Environmental Protection Agency.
Sherman, K. 201 lb. Ethics Review of Completed AEATF II Wipe Scenario Worker Exposure
Monitoring Study. Dated March 17, 2011. Unpublished document prepared by the Office of Pes-
ticide Programs, United States Environmental Protection Agency.
Smith, L.D. 2005. Determination of Dermal and Inhalation Exposure to Workers During Appli-
cation of a Liquid Pesticide Product by Open Cab Airblast Application to Orchard Crops. Study
Number AHEO7. Amended August 23, 2005. Unpublished document prepared by the AHETF.
MRID 46448201.
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April 13-14, 2011 EPA Human Studies Review Board Meeting Report
ADpendix 1
Reconsideration of Two Concerns Previously Raised by the HSRB in its June 2009 Review
of a Pre-Rule Intentional Human Dosing Study Involving Chiorpyrifos (Kisicki et al. 1999)
In addition to the other topics considered at the April 2011 meeting, the Board considered
additional information regarding the Kisicki et al. (1999) study on chlorpyrifos, originally
reviewed by the HSRB in June 2009 (EPA HSRB 2009). Additional information was supplied
by Dow AgroSciences about two concerns raised by the HSRB at the June 2009 meeting, but this
information was not supplied to the HSRB until after the report of that meeting had been
finalized.
In its June 2009 report, the Board raised four concerns about the Kisicki et a!. (1999)
study. Two of these concerns were addressed in the additional information provided by Dow
AgroSciences.
The first of these concerns was about the substantially lower level of oral absorption of
chlorpyrifos calculated in the Kisicki et a!. (1999) study as compared with the level of absorption
calculated in an earlier study conducted Nolan eta!. (1982). The Kisicki eta!. study reported
35% absorption as compared with the 70% absorption reported by Nolan et a!. for the same
dosing level (0.5 mg/kg). The Board was skeptical as to whether the reason presented for the
differences between the level of oral absorption between the two studies, namely that the gelatin
capsule used in the Kisicki et a!. study would have taken longer to dissolve and would therefore
have resulted in lower absorption, were valid.
The second of these concerns was the lack of documentation that urine samples were
subjected to acid hydrolysis. Acid hydrolysis would be necessary to liberate the chlorpyrifos
metabolite trichloropyridinol (TCP) from any conjugates in the sample. Had acid hydrolysis not
been performed, the subsequent quantitation of TCP in the urine samples would have
underestimated the levels of TCP. In its June 2009 report (EPA HSRB 2009) the Board
concluded that, had the hydrolysis step not been performed, the lower oral absorption reported in
Kisicki et a!. (1999) might have been the result of inaccurate measurement of urinary TCP
levels.
The information provided to the Agency by Dow AgroSciences addressed both of these
concerns. This supplementary information indicated that the urine was indeed subjected to acid
hydrolysis using methods that had been previously shown to free TCP from any conjugates. The
information provided by Dow AgroSciences also indicated that the chlorpyrifos used in the
Kisicki et a!. (1999) study was placed in a gelatin capsule in a crystalline form, with the
remainder of the capsule filled with lactose. By contrast, in the Nolan eta!. (1982) study the
chlorpyrifos was dissolved in methylene chloride and applied as a solution to the lactose tablets.
The particle size of the chlorpyrifos used in the Kisicki et a!. study thus was larger than that used
in the Nolan et a!. study. It is possible that these larger particles could have been absorbed more
slowly than the smaller particles.
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After considering this addition information, the Board concluded that its original
recomiiendations about the Kisicki et al. (1999) study should be amended as follows:
1. It is logical that larger particles of a material such as chlorpyrifos would be absorbed
more slowly than smaller particles. The differences in absorption between the Nolan
et al. (1982) and Kisicki et al. (1999) studies may have resulted, at least in part, from
the different sizes of chlorpyrifos particles in the two formulations.
2. The quantitation of urinary TCP was accurate because the urine was subjected to acid
hydrolysis and heat to liberate conjugated TCP.
The Board also concluded, however, that two other issues discussed by the HSRB at the June
2009 meeting remain unaddressed. These issues still raise concern about the reliability and utility
of the blood and urine measurements of chlorpyrifos and/or TCP from Kisicki et al. (1999) for
risk assessment purposes.
REFERENCES
EPA Human Studies Review Board. 2009. June 24-25, 2009 Human Studies Review Board
Meeting Report.
Kisicki, J, C. Seip, and M. Combs. A Rising Dose Toxicological Study to Determine the No-
Observable-Effect-Levels (NOEL) For Erythrocyte Acetylcholinesterase (AChE) Inhibition and
Cholinergic Signs and Symptoms of Chiorpyrifos at Three Dose Levels. Dated April 15-19,
1999. Unpublished study prepared by MDS Harris Laboratories under Project No. 21438 and
Dow AgroSciences Study No. DR K-0044793-284. MIRID 44811002.
Nolan, R.J., D.L. Rick, R.L. Freshour, et a!. 1982. Chlorpyrifos: Pharmacokinetics in Human
Volunteers Following Single Oral and Dermal Doses. Dated August 1982. Unpublished study
prepared by the Dow Chemical Company under Protocol HEB-DR-0043-4946-4. MRID 124144.
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