lack of standardization between studies, the paucity of research
specifically directed towards evaluation of the above-mentioned factors
and the complexity of the test system, elaboration of anything more than
a very basic general model is not possible.

HSRB Consensus and Rationale

	The Board noted that the choice of intermittent vs. continuous exposure
designs depends upon the goal of the study (e.g. relative or complete
protection) as well as other factors. The Board concluded that more
research is needed to determine the relative benefits of these designs
and concurred with EPA that future guidance on this matter would be
helpful.

Issue 3: Complete Protection Time

	Dr. Matt Kramer, a USDA statistician, has suggested that the precision
of CPT estimates in repellent testing could be significantly increased
by defining failure of efficacy as the mean time from treatment to a
series of several [e.g., five] landings or bites.  The Board consultants
were asked to determine whether this approach would markedly increase
the precision of CPT estimates without requiring additional subjects
and, if so, whether the increased precision would justify
the incremental risk to the subjects resulting from their exposure to
mosquitoes.  The consultants also were asked to consider the
practicalities involved in testing long-lasting repellents to the point
of five landings.

The complete protection time for a subject is clearly defined as the
time of repellent application to the first bites/LIBe’s with or
without the confirmation of a second bites/LIBe’s.  It is unclear what
the mean of the times to the first 5 bites/LIBe’s is measuring.  A
discussion on the precision seems irrelevant when one is not so sure
what is being measured.  Besides, these 5 times are all correlated
within each subject, and so the efficiency gain is not so obvious due to
lack of independence among measurements.  With the censoring of efficacy
failure seen in a number of mosquito repellency studies, it is
unrealistic to be able to observe, say first 5 bites or LIBe’s, and
probably impractical to test to the point of 5 bites/LIBe’s.

Perhaps more critical is the artifact of taking average of times to the
first five bites/LIBe’s, which is to overestimate the time to efficacy
failure.  This seems to make sense if one is interested in understanding
the average mosquito biting/LIBing behavior.  However, it seems
unsuitable if one is interested in characterizing the “efficacy” of
repellent products.

The rationale for this idea of measuring times to five bites/LIBe’s
became very clear during the consultant’s presentation.  If the
population for statistical inference is the population of mosquitoes and
the statistical inference is on the mosquito biting behavior, it makes
sense.  However, that is not the case in these mosquito repellency
efficacy studies.  The population for statistical inference is the
population of repellent users, not mosquitoes, and the statistical
inference is on the time from application of repellent to failure of its
efficacy.

Currently there is no consensus on whether continuous or intermittent
methods are most desirable. Prior to the new regulations, continuous and
intermittent methods were used. A preference for the intermittent method
became dominant after the new regulations were in place because it is
more protective of individual subjects since they are exposed to less
bites. However the use of continuous methods might also call for less
subjects.

HSRB Consensus and Rationale

Based upon information provided by the consultants, the Board remains
unclear of what the mean of the times to the first 5 bites/(landings
with intent to bite) would measure of relevance to EPA determinations of
efficacy.  Therefore The Board concluded that precision of CPT estimates
in repellent testing would not be significantly increased by defining
failure of efficacy as the mean time from treatment to a series of
several [e.g., five] landings or bites.

	The Board concluded it would be helpful to their deliberations if
protocols submitted to the HSRB included rationales for sample size,
outcome measures, number of treatment groups and controls, why a field
study is preferable, why a specific environment was selected, how
different environments differ, and how controls for environmental shifts
in temperature or time of day are determined.

	The Board understands that the need for smaller sample sizes and the
accompanying lack of power must be balanced with subject protection, but
it is also important to understand which variables can be controlled.
The expertise of control and treated subjects with respect to detecting
mosquito landings must be balanced and the activity of subjects also
should be controlled.

D.  Completed Insect Repellent Efficacy Study (SCI-001) of DEET
Formulations

Charge to the Board

Is this study sufficiently sound, from a scientific perspective, to be
used to assess the repellent efficacy of the formulations tested against
mosquitoes?  Please comment specifically on:

Whether participation in field testing by several subjects on the day
after they had been treated with a different test repellent is likely to
have affected the validity of the results for those subjects on those
days.

Board Response

The active ingredient DEET formulated in three forms was tested for its
ability to repel mosquitoes from the legs of volunteers by the protocol
presented and modified by Carroll-Loye.  The protocol had been modified
based on the suggestions and input of EPA and the HSRB.  The results
were reported in SCI.001.1, LipoDEET 302; SCI.001.2, LipoDEET 3434; and
SCI.001.3 Coulston’s Duranon, all compared to Ultrathon (approved by
the military). All experiments were conducted using Good Laboratory
Practices.

The dosimetry for the three products was done in the laboratory on July
3-5, 2007.  The field tests were conducted at Site 1 in Butte County, a
grassland habitat, on July 7, 8 and 15, and at Site 2 in Glenn County, a
forest habitat, on July 12, 13 and 14.  Different mosquito species
composition occurred at the two sites. Ten subjects were used for the
dosimetry tests.  Ten subjects were used for each of the three products.
Some of the subjects participated for all three of these test articles
as well as for the Ultrathon. The subjects were required to be above 18
years of age and no more than 55 years of age, active in rural outdoor
settings, and having used no repellent on the day before the test. Only
legs were tested in this study because of greater biting pressure on
legs than arms. There were two experienced persons serving as negative
controls (i.e., without any repellent product) to confirm mosquito
biting pressure (and biting pressure was maintained throughout the
period of the study, defined as at least one Landing with Intent to
Bite, LIBe, per min at Site 1 and the second site selected to be Site
2). Experimental subjects, in pairs, monitored LIBe’s during a one
minute interval each 15 minutes, until the First Confirmed LIBe (FCLIBe)
could be determined. Stopping rules were employed.  No evidence of West
Nile Virus (WNV)  was present in either test site. Mosquitoes landing
were taken to the laboratory for later identification, and for screening
for WNV, Western Equine Encephalitis, and St. Louis Encephalitis Virus,
and all mosquitoes were negative.  All subjects wore Tyvek coverall,
head nets and surgical gloves, and they worked in pairs.  There was a
one minute observation period during each 15 minute interval, starting
150-210 minutes post application.  Toward the end of the day, controls
were reduced from 2 to 1 subject to limit exposure to mosquitoes.
Complete protection time (CPT) was measured, defined as the time to the
First Confirmed Landing with Intent to Bite (FCLIBe). Adequate LIBing
pressure was maintained throughout the test period.

LipoDEET 302 is 30% DEET on lipid spheres designed to improve the
durability and to improve the cosmetic properties. Six females and four
males were tested for dosimetry, 7 females and 3 males at Site 1, 3
females and 7 males at Site 2.  The MOE was 522.  It yielded a CPT of
10.3 ± 1.3 hr in Site 1 and 9.5 ± 1.8 hr in Site 2.

LipoDEET 3434 is DEET at 34.34%; this protocol SCI.001.2 was amended to
test a different test article than the original protocol described  Five
females and five males were tested at Site 1, 4 females and 6 males at
Site 2.  The MOE was 492.  It yielded a CPT of 10.6 ± 1.3 hr in Site 1
and 10.4 ± 1.9 hr in Site 2.

Coulson’s Duranon is 20% DEET in microscopic protein spheres to
reduced skin absorption of DEET, improve cosmetic properties and inhibit
evaporation.  Five females and five males were tested at Site 1, 3
females and 7 males at Site 2.  The MOE was 856.  It yielded a CPT of
8.4 ± 1.9 hr in Site 1 and 9.5 ± 1.3 hr in Site 2.

Data were presented for the comparison article, Ultrathon, CPT of 10.1
± 2.3 hr for Site 1 and 10.0 ± 2.2 hr for Site 2.  While this
comparison may have been of interest to the sponsor, it is unclear why
this information was provided in this report.

The report was clearly written and a greater attention to statistical
analysis was provided than has been provided in the past by Carroll-Loye
Biological Research.  The study was justified in that additional insect
repellents that are more efficacious and/or more acceptable cosmetically
to the public would be an advantage from both the standpoint of health
(to reduce the chances of contracting a mosquito-borne disease) and of
comfort.  The information should be generalizable to the public,
although the exclusions, which were highly appropriate, excluded some
subpopulations that would likely use insect repellents.  The experiment
was necessary to determine the field efficacy of these test
formulations, and the experiments were set up to meet the study
objective.  Measurements taken were appropriate for the objective and
quality assurance considerations were in place.

The experiment was conducted according to the approved protocol with
some deviations. One of the most serious deviations, which was not
acknowledged in the description, was the fact that some of the subjects
were tested on the day following a test with another product, despite
the fact that the protocol stated “Use of insect repellent within one
day of preceding the study” was an exclusion criterion.  Additionally,
the tests on any given repellent (including the repellent used in study
WPC-001) were not conducted on a single day and the statistical
treatment of the data did not account for different days of testing.

The deviation of not allowing a day of non-repellent use before testing
was of concern to the Board because of potential persistence of the
repellent of one day’s test into the test of the following day.
However, information provided by the Carroll-Loye Biological Research
via public comments indicated that the repellent was carefully washed
off at the end of the experiment both with soap and water and with
alcohol.  The public comments also indicated that the reason for the one
day wait in the protocol was not related to concerns about the
persistence of the repellent.  Therefore this deviation did not affect
the validity of the results obtained.  In addition, the public comments
indicated that the reason for the compression of the tests on these
several test materials into a short span of time was because of the
reports that West Nile virus-contaminated mosquitoes were moving close
to the test area and that this test region would have been unsafe in a
matter of just a few more days. This explanation was adequate to explain
why the testing days were set up as they were.  It would have been
advisable to have had this deviation and the rationale for it presented
in the report.

Charge to Board

b.	The effects of changes to the experimental design resulting in
evaluation of repellents using fewer than ten subjects per treatment per
day, followed by pooling of results by site for statistical analysis.

Board Response

Strengths

One of the strengths of the study is the inclusion of the positive
control treatment with 3M’s Ultrathon.

Weaknesses

SCI-001 studies are very troubling from a statistical design point of
view.  There is some indication that there was no desire to compare the
various repellents to one another, and yet each of reports compared a
repellent to a positive control, the Ultrathon repellent.  The
statistical analysis assumes that there are 20 different subjects on
each repellent over two sites, but many of the subjects wore both
repellents and a few subjects were used in both sites.  Subjects that
were used at both sites were subjects:  8, 13, 14, 15, 37, 40, 46, 52,
53, 60, 61, 62, 63, 67, 71, 72, and two control subjects.  Almost all
subjects wore more than one repellent.  This is not an inherent flaw in
the design, although if subjects are going to be used more than once,
there should have been some counterbalancing of the repellents to the
subjects.  The major flaw is in the statistical analysis, particularly
when repellents are being compared to one another as is done in the
reports by comparing each repellent to Ultrathon.  The analysis assumes
that there are 20 different subjects involved in each of the three
write-ups.  That is, the analysis assumes that the experimental design
is a parallel subjects design and it is clearly not.

In the combined  experiments, there were only 33 unique subjects,
representing 80 data points, excluding the negative controls.  Subjects
do not appear to have been randomly allocated to four test materials,
between Sites 1 and 2, and over five days.  This lack of statistical
independence of data between different test materials, Sites 1 and 2,
and different dates of experiment renders the analysis incorrect.  For
example, due to overlap of subjects between Sites 1 and 2, one cannot
pool the results by site for statistical analysis.  Also there is
substantial overlap of subjects among and between test materials, the
comparison among and between test materials cannot be made.  This
renders the analysis scientifically invalid.

In SCI-001, there is 0% censoring.  This differs to previous studies
conducted and reported by Carroll-Loye Biological Research, Inc and
reviewed by the Board.  As an example, there was 40% censoring in
EMD-004.1, 10-30% censoring in EMD-004.2, and 90-100% censoring in
EMD-004.3.  The Board supports the EPA’s evaluation that “Further
clarification is needed to verify the accuracy of the data generated.”

The data analysis (pairwise comparison between three test materials and
the positive control Ultrathon) is inadequate and is inconsistent with
the experimental design and conduct of the study in which subjects were
allocated to four treatment groups.  As was suggested by the HSRB in its
January 2007 meeting, the appropriate analysis would have been an
analysis of variance type.  Wilcoxon rank sum test in the presence of
censored data is known as Gehan’s test, and is known to be inefficient
for comparison of Kaplan-Meier estimates.  A more appropriate test
comparison of censored time to event data is the log rank test or better
yet a regression analysis for censored time to event data based on Cox
proportional hazards regression models.   The analysis of the number of
LIBe’s is inappropriate as it ignores the duration of follow-up during
which LIBe’s are recorded.  An appropriate analysis would have been
Poisson analysis.

Having ten subjects spread over  five days and combining the results
into the current analysis has a potential to confound the effect of
repellent with the different mosquito pressures on different days. This
would have been better if it were planned in advance.  The whole idea is
that the experimenter has  control over the experiment by using random
mechanism for subject selection and for allocation of subjects to
repellents, days and sites. One solution would be to perform the
analysis of variance as was done when the results for two sites were
pooled, i.e., by including the day as the main effect and the two-way
repellent by day interaction (repellent effect modification by day
effect) in the analysis of variance, in Table 5, on page 17 of 217, of
the report for SCI-001.3.  Of course, this requires making the
assumption that the experimenter had adequate control of the experiment
as described above and that observations are statistically independent,
which is not the case.  With the proper experimental design using site
and day of experiment as blocking factors, the analysis of variance
would allow evaluation of the three main effects of repellent, day and
site, along with the two-way and three-way interactions.  There is no
inherent problem with pooling across studies.  Study site can be
considered as a blocking factor, and as such it would generally be
appropriate to pool across the two blocks.  This assumes independence of
data from sites, namely, no overlap in subjects between the sites.  This
was not the case for these experiments, and as a consequence the data
cannot be pooled across the two sites.

Pooling of results by site for statistical analysis is generally
preferable if the other experimental design aspects are the same, which
appears to be the case with the reported studies.  The main advantage of
a  pooled analysis versus   marginal analyses  for each site is
two-fold: 1) by doubling the sample size for the analysis, you increase
the statistical efficiency by a factor of 1.41 in the point estimation
of the CPT exactly or in the analysis of variance approximately (as you
lose one degree of freedom for site effect) and 2) you can assess the
effect of repellent more accurately by accounting for the potential
effect of site difference in mosquito pressure.  However, this assumes
that the data from two sites are independent, which is again obviously
not the case.  There are three subjects (40, 18 and 62) used in both
sites with LipoDEET 302, two subjects (53 and 46) used in both sites
with Ultrathon, and three subjects (40, 8 and 63) used in both sites
with Duranon.

HSRB Consensus and Rationale

While the Board concluded that the participation of several subjects on
the day after they had been treated with a different test repellant was
not ideal and there were errors (i.e. choice of test limb) in the study,
this did not affect the validity of the results.

However with only 33 subjects for 80 data points (excluding the negative
controls), the overlap  of some of the same subjects for different test
materials, for Sites 1 and 2, and for different dates of the experiment
without proper experimental design and control, the Board concluded that
it is impossible to interpret the reported data adequately thus
rendering scientific validity of the results into question.  In
addition, the study may not have been sufficiently sound to estimate
within  population variances.

Thus, the Board concluded that the study was not sufficiently sound,
from a scientific perspective, to be used to assess the repellent
efficacy of the formulations tested against mosquitoes.

Charge to Board

2.  Does available information support a determination that this study
was conducted in substantial compliance with subparts K and L of EPA
regulations at 40 CFR part 26?  Please comment specifically on:

a.	The decision to use a different test formulation in place of one of
the test materials described in the protocol reviewed by the IRB, EPA
and the HSRB.

b.	How to assess the ethical conduct of an insect repellency study
involving multiple test formulations when there is an ethical deficiency
in the conduct of the study with respect to one of the test
formulations.  If the ethical deficiency warrants not relying on the
results of the testing with regard to one test formulation, under what
circumstances (if any) does the ethical deficiency affect the
acceptability of the results from testing the other formulations?

Board Response

Brief Overview of the Study

The basic protocol for these studies (SCI-001) was initially reviewed at
the January 2007 meeting of the HSRB, at which time the Board concluded
that the study would meet the requirements established in the
Environmental Protection Agency’s final human studies rule (40 CFR
Part 26) pending minor revision.  In particular, the Board was concerned
about several matters relevant to subject recruitment and the overall
conduct of the study.  For example, the protocol did not describe how
untreated controls would be recruited, but instead implied that controls
will be recruited in the same manner as subjects in the “exposure”
arm—via “word-of-mouth” and a Volunteer Data Base maintained by
the Principal Investigator.  The Board recommended that the protocol
clarify how untreated controls will be recruited, and that the IRB of
record (Independent Investigational Review Board [IIRB], Inc. of
Plantation, FL) review any materials used for recruiting purposes,
including any telephone scripts fliers, emails, letters, or local ads.
The Board also felt that the risks associated with DEET exposure during
the course of the study are mischaracterized, and that the
informed-consent document was also structured in a manner that did not
apply to unexposed control subjects.  In light of these deficiencies,
the Board recommended that the informed-consent document be redesigned
and re-reviewed by IIRB (EPA HSRB 2007a).

Subsequent to the aforementioned meeting of the HSRB, the informed
consent documents were revised in accordance with Board and EPA
recommendations, submitted to IIRB for re-review, and approved (Carley,
2007). It is unclear, however, whether the protocol was also revised in
accordance with the Board and EPA recommendations, and submitted to the
IIRB for review.

The documents provided by Carroll-Loye (Carroll, 2007a; Carroll, 2007b;
Carroll, 2007c) specifically state that each study was conducted in
compliance with the requirements of the U.S.  EPA Good Laboratory
Practice Regulations for Pesticide Programs (40 CFR 160); 40 CFR 26
subparts K and L; FIFRA § 12(a)(2)(P); and the California State EPA
Department of Pesticide Regulations for study monitoring (California
Code of Regulations Title 3, Section 6710).  Each study was also
reviewed and approved by a commercial human subjects review committee,
IIRB.  Documentation provided to the EPA by IIRB indicated that it
reviewed these studies pursuant to the standards of the Common Rule (45
C.F.R. Part 46, Subpart A) and determined them to be in compliance with
that Rule.

As submitted to the Agency, each completed study consists of two
interdependent analyses: 1) a dosimetry study designed to determine the
amount of an insect-repelling compound (30% or 34% DEET in liposomal
capsules, or 20% DEET in protein capsules) that typical users would
typically apply when provided with a lotion formulations; and 2) an
efficacy study designed to measure the effectiveness of each compound as
a mosquito repellent.  For each dosimetry and efficacy studies,
commercially available and obtained 3M Ultrathon (34.34% polymerized
DEET) was used as a comparator.  Ultrathon is the principal insect
repellent used by American military forces and is considered to be one
of the most effective insect repellents available.

Dosimetry and efficacy studies for personal insect repellents containing
LipoDEET 302 and 3434 (30% and 34% DEET in liposomal capsules,
respectively) and Duranon (20% DEET in protein capsules) were conducted
from July 3 through August 2, 2007 (Carroll 2007a; Carroll 2007b;
Carroll 2007c).  All studies (one using each test compound) were
performed simultaneously at a laboratory site in Davis, California, and
at field sites in Butte and Glenn Counties, California, by researchers
at Carroll-Loye Biological Research.  The study sponsor was Scientific
Coordination, Inc., of Rockville, Maryland.  The studies were conducted
using products from two manufacturers:  LipoDEET 302 and 3434 was
manufactured and supplied by DermAegis, Inc. of Rockford, Illinois;
Duranon was manufactured and supplied by Sawyer Products of Safety
Harbor, Florida.  It is important to note, however, that the protocol
originally reviewed and approved at the January 2007 HSRB meeting listed
a different set of test compounds; at the sponsor’s request
Carroll-Loye Biological Research changed one of the test compounds from
Insect Guard II (EPA Reg. No. 54287-8) to LipoDEET 3434 (an unregistered
compound) at the sponsor’s request.

Dosimetry was determined by direct measurement of compound application.
The efficacy of each as a mosquito repellent was determined by measuring
the ability of the formulations to prevent mosquito landings (defined as
“Lite with Intent to Bite”; LIBe) under field conditions.
Mosquitoes were aspirated mechanically prior to biting; prior to
initiation of the efficacy study, all volunteers will be trained both to
recognize a mosquito landing with the intent to bite and to remove such
mosquitoes with an aspirator using laboratory-raised, pathogen-free
mosquitoes in a controlled laboratory setting.  During the field
studies, participants worked in pairs to facilitate identification and
aspiration of LIBing mosquitoes during brief exposure periods.  The
strengths and weaknesses of each study design are described above.

The dosimetry study enrolled a total of 10 individuals, each of who
tested all four formulations (the three test compounds and the
comparator).  Each efficacy study enrolled 10 subjects for each
formulation at each of the two field sites.  Many volunteers
participated in multiple analytic phases.  For example, six of the 10
participants in the dosimetry study also participated in one or more of
the field tests.  In total, 39 volunteers participated in at least one
analytic phase of SCI-001.1, SCI-001.2 or SCI-001.3.  Fifteen of these
volunteers also participated in the dosimetry and/or efficacy phase of
Carroll-Loye study WPC-001 described below.  In addition to these 41
volunteers, two controls (described as “experienced personnel” and
who were untreated with repellent) also participated to determine
ambient LIBe pressure at field site. On any given day at a field site,
the same pair of participants served as controls for SCI-001.1,
SCI-001.2, SCI-001.3 and the concurrently run WPC-001 study.  Five
individuals served (repeatedly) as controls during each of the six field
tests; two also participated in the repellent efficacy tests, to give a
cumulative total of 45 volunteers.  In addition, three alternate
participants were enrolled to: 1) replace any individual who withdrew;
and 2) protect the confidentiality of any participant excluded from the
study as a result of pregnancy or other potentially stigmatizing
condition, as described below.

Critique

The Board concurred with the factual observations of the ethical
strengths and weaknesses of the study, as detailed in the EPA’s
Science and Ethics Review (Carley, 2007).  The risks to study
participants, in general, were minimal and were justified by the likely
societal benefits, including data on the efficacy of these new
formulations (30% or 34% DEET in liposomal capsules, or 20% DEET in
protein capsules) as personal insect repellents.  Higher concentrations
of DEET are commercially available and have been used as repellents for
years; the subjects enrolled in this study were thus unlikely to be at
increased risk of experiencing adverse side effects upon exposure.
Reactions to mosquito bites are usually mild and easily treated with
over-the-counter steroidal creams.  The study also excluded individuals
who have a history of such severe skin reactions to further minimize the
risk of a participant experiencing a severe physical reaction to a
mosquito bite.  In addition, the study protocol was designed
specifically to minimize the likelihood that a mosquito will bite,
through the use of clear stopping rules, limited exposure periods, and
paired observation; no side effects or adverse events were reported.  To
minimize the risk that study participants will be exposed to illnesses
like WNV, the study protocol called for field tests of repellent
efficacy to be conducted only in areas where known vector-borne diseases
have not been detected by county and state health or vector/mosquito
control agencies for at least one month.  Mosquitoes collected during
the field studies also were subjected to serologic or molecular analyses
to confirm that they were free of known pathogens.  Finally, the study
protocol also included several mechanisms designed to minimize coercive
recruitment and enrollment, compensation was not considered to be so
high as to unduly influence participation, and minors and pregnant or
lactating women were explicitly excluded from volunteering (pregnancy
being confirmed by requiring all female volunteers to undergo a
self-administered over-the-counter pregnancy test on the day of the
study).  The potential stigmatization resulting from study exclusion was
minimized by the use of so-called “alternate” participants, allowing
for volunteers to withdraw or be excluded from participating without
unduly compromising their confidentiality.

Regrettably, several serious protocol deviations occurred during the
conduct of these studies. Most notably, due to the study
investigator’s failure to obtain IRB approval for fundamental changes
to one of the study protocols, the research described in SCI-001.1,
SCI-001.2 and SCI-001.3 does not comport with the applicable
requirements of 40 CFR Part 26, subparts K and L.

First, as mentioned previously, many study volunteers participated in
multiple analytic phases of SCI-001.1, SCI-001.2 and SCI-001.3, testing
the field efficacy of different insect repellents on different days.
Many volunteers participated in field trials conducted on consecutive
days, and were re-randomized to receive the same or a different test
compound each time.  The approved protocol, however, includes clear
criteria excluding any participant who “[used an] insect repellent
within one day preceding the study” (c.f., Carroll, 2007a; Carroll,
2007b; Carroll, 2007c).  This change is unlikely to have increased the
risks to study participants, but several Board members nevertheless
raised the concern that compromising these exclusion criteria may
adversely affect the scientific validity of the study data (as described
above).  The decision to disregard previously established exclusion
criteria was also made without submitting the protocol for approval or
informing study participants of this change, and may represent a
departure from accepted review and approval practices.  There was some
disagreement among Board members, however, as to whether the phrase
“one day preceding the study” specifically excluded participants who
had used or been exposed to an insect repellent on the day preceding a
field trial, or simply prior to consent and enrollment in the study
overall.  Dr. Carroll submitted a response to the EPA that implied the
former (Carroll, 2007d), but the Board nevertheless recognized the
uncertainty surrounding this issue.

Second, to minimize the risk that study participants will be exposed to
arthropod-borne illnesses like WNV, for example, during its review of
another Carroll-Loye protocol at the April 2007 HSRB meeting (EPA HSRB,
2007b) the Board recommended conducting serologic or molecular analyses
to confirm that mosquitoes collected during the field studies were free
of known pathogens.  Study investigators did conduct such analyses in
the context of these studies, but did so without submitting the protocol
for approval or informing study participants of these analyses in the
informed consent document.  Although this change is likely to have
actually reduced the risks faced by study participants, it is
nevertheless a substantial departure from accepted review and approval
practices.

Finally, and most seriously, at the sponsor’s request Carroll-Loye
Biological Research changed one of the test compounds from Insect Guard
II (EPA Reg. No. 54287-8) to LipoDEET 3434 (an unregistered compound)
without IRB or HSRB review or approval.  Dr. Carroll has justified the
decision to substitute a test material by stating that, in conversations
that he has previously had with the IRB and the California EPA, that IRB
review and approval is needed only for “proposed changes … that are
likely to increase risk to participants” (Carroll 2007d, 3).  Because
the substituted material (LipoDEET 3434) was nearly identical to another
compound approved for testing (LipoDEET 302), and contained a
concentration of DEET considerably less than that in already approved
and marketed personal insect repellents, Dr. Carroll felt that the
substitution posed no increase in risk to study participants and fell
within the “latitude” given to study investigators with respect to
minor protocol changes.  The Board disagreed, feeling that a change of
test materials was not simply a minor protocol change; although it is
unlikely that study participants were subjected to greater risk, the
substitution of LipoDEET 3434 for Insect Guard II was a protocol change
of sufficient magnitude to warrant IRB notification and review.  More
importantly, the failure to seek IRB review and approval is a
significant and serious departure from accepted review and approval
practices, as well as a violation of the letter and intent of the
Agency’s Final Human Studies Rule.

Federal guidelines from the Office of Human Research Protections (OHRP)
clearly state that all proposed protocol changes must be reviewed by the
IRB of record at convened meetings, in accordance with HHS regulations
at 45 CFR 46.108(b), although institutions may adopt policies describing
the types of minor changes in previously approved research that can be
approved under an expedited review procedure in accordance with HHS
regulations at 45 CFR 46.110(b)(2).  Except in cases when necessary to
prevent imminent harm to study participants, an investigator should
never institute a protocol change without IRB review.  Federal
regulations regarding review and approval of human subjects research,
for example, explicitly prohibit investigators from implementing any
protocol changes without prior IRB approval unless such changes are
necessary to prevent immediate, serious harm to study participants.  It
is never an investigator’s prerogative to determine which protocol
changes warrant IRB review and which do not; only the IRB (or some
authority other than the investigator) has that authority.

The failure of Carroll-Loye Biological Research to 1) obtain IRB
approval of the revised protocol; and 2) report these protocol
deviations to the Independent Investigational Review Board in a timely
manner are serious regulatory breaches.  The failure of Dr. Carroll to
notify the EPA and the HSRB of protocol deviations such as the
substitution of LipoDEET 3434 for Insect Guard II in submitted study
documents for studies SCI-001.1 and SCI-001.3 is also troubling.  The
Board recommended the Carroll-Loye Biological Research report these
deviations to the IIRB as soon as possible and work with that
organization to develop and implement a corrective course of action.

Because of the serious nature of these deviations, the research
described in SCI-001.2 using the unapproved pesticide formulation failed
to comport with the applicable requirements of 40 CFR Part 26, subparts
K and L, and the Board recommended that the Agency not accept for
regulatory decision-making purposes any of the data obtained during the
conduct of that particular study.  Furthermore, this particular ethical
deficiency also affects the acceptability of the results from testing
the other two formulations. SCI-001.1, SCI-001.2 and SCI-001.3 were run
concurrently, and study participants enrolled in all three studies ran
the risk of being randomized to receive treatment with the unapproved
investigational compound, LipoDEET 3434.  It is thus impossible to
separate the data collected in SCI-001.1 and SCI-001.3 from that
collected in SCI-001.2, and the Board recommended that the Agency not
utilize any the data obtained during the conduct of these three studies.

HSRB Consensus and Rationale

Based on a careful review of the study documents provided, the Board
concluded that the research was conducted in a manner that failed to
meet the applicable requirements of §40 CFR 26, subparts K and L.  The
study investigator failed to obtain IRB approval for fundamental changes
to one of the study protocols and, as a result, the data collected from
these three concurrently run studies cannot be used by the Agency for
regulatory decision-making purposes.

E.  Completed Insect Repellent Efficacy Study with Oil of Lemon
Eucalyptus (WPC-001)

Charge to the Board

1.  Is the research conducted under WPC-001 sufficiently sound, from a
scientific perspective, to be used to assess the repellent efficacy of
the formulation tested against mosquitoes?  Please comment specifically
on whether participation in field testing by several subjects on the day
after they had been treated with a different test repellent is likely to
have affected the validity of the results for those subjects on those
days.

Board Response

The active ingredient Oil of Lemon Eucalyptus (OLE) formulated as a pump
spray was tested for its ability to repel mosquitoes from the legs of
volunteers by a study presented by Carroll-Loye.  All experiments were
conducted using GLP. This study was conducted similarly to and in
conjunction with the DEET efficacy studies of SCI-001, and the
descriptions of the experimental conditions and design of SCI-001,
above, are appropriate for this study and will not be repeated here.

The test product had 30% OLE in a pump spray. Seven females and three
males were tested at Site 1, 3 females and 7 males at Site 2.  It
yielded a CPT of 6.1 ± 1.5 hr in Site 1 and 4.2 ± 0.8 hr in Site 2.

As was discussed for the SCI-001 study, the experiment was conducted
according to the approved protocol with some deviations, with the most
serious being the lack of at least a one day wait for some of the
subjects before they were tested with another product.  The Board’s
analysis of this deviation and design of this experiment was discussed
above for SCI-001, and is pertinent to this experiment also and will not
be repeated here.

	The science assessment of WPC-001 was the same as that for SCI-001.
However, unlike SCI-001 the residual effects of successive days of
testing were unlikely to have had an effect (CPTs were shorter) and the
data therefore are useable.  Treatment of subjects on successive days is
not intellectually satisfying, but it was unlikely to have a significant
effect.

HSRB Consensus and Rationale

The Board concluded that despite problems estimating variability and
some of the same design problems found in study SCI-001, the
Carroll-Loye study WPC-001 assessing the repellent efficacy of the
formulation tested was sufficiently sound for the purposes for which it
was intended.

Charge to Board

2.  Does available information support a determination that the research
covered by WPC-001 was conducted in substantial compliance with subparts
K and L of EPA regulations at 40 CFR part 26?  If the conduct of any
part of SCI-001 is deemed not to substantially comply with the
requirements of subparts K and L, please comment specifically on how to
assess the ethical conduct of research conducted under WPC-001 in light
of the fact that it was conducted at the same times and at the same
places as the research covered under protocol SCI-001.

Brief Overview of the Study

The protocol for this study was initially reviewed at the April 2007
meeting of the Human Studies Review Board, at which time the Board
concluded that the study would meet the requirements established in the
Environmental Protection Agency’s final human studies rule (40 CFR
Part 26) pending minor revision.  In particular, the Board was concerned
about several matters relevant to subject recruitment and the overall
conduct of the study.  To minimize the risk that study participants
would be exposed to arthropod-borne illness, for example, the Board
recommended that investigators trap landing mosquitoes or other vectors
for pooled serologic or nucleic acid-based testing and alert research
participants if they had been inadvertently exposed to vector-borne
pathogens.  Secondly, the Board expressed concerns about plans to
recruit research subjects in Florida, as these recruitment procedures
were not described adequately in the protocol and supporting materials.
Finally, the Board raised questions about the informed consent
procedures for control subjects, suggesting that the informed consent
procedures should be modified to more clearly explain the risks to
control (untreated) research subjects.  In light of these deficiencies,
the Board recommended that the protocol and informed-consent document be
revised and submitted to re-review by IIRB (EPA HSRB, 2007b).  The
protocol and informed consent documents subsequently were revised in
accordance with Board and EPA recommendations, submitted to IIRB for
re-review, and approved (Carley, 2007).

The documents provided by Carroll-Loye (Carroll, 2007e) specifically
state that each study was conducted in compliance with the requirements
of the U.S. EPA Good Laboratory Practice Regulations for Pesticide
Programs (40 CFR 160); 40 CFR 26 subparts K and L; FIFRA § 12(a)(2)(P);
and the California State EPA Department of Pesticide Regulations for
study monitoring (California Code of Regulations Title 3, Section 6710).
 Each study was also reviewed and approved by a commercial human
subjects review committee, IIRB.  Documentation provided to the EPA by
IIRB indicated that it reviewed these studies pursuant to the standards
of the Common Rule (45 CFR Part 46, Subpart A) and determined them to be
in compliance with that Rule.

As submitted to the EPA, the completed study consists of two
interdependent analyses: 1) a dosimetry study designed to determine the
amount of oil of lemon eucalyptus-based personal insect repellent that
typical users would typically apply when provided with a pump spray
formulation; and 2) an efficacy study designed to measure the
effectiveness of the pump spray formulation as a mosquito repellent.
The dosimetry and efficacy studies were conducted from July 10 through
August 1, 2007 (Carroll, 2007e).  The studies were performed
simultaneously at a laboratory site in Davis, California, and at field
sites in Butte and Glenn Counties, California, by researchers at
Carroll-Loye Biological Research.  The study sponsor was WPC Brands,
Inc. of Bridgeton, Missouri.  The studies were conducted using product
manufactured and supplied by ChemRite CoPac, Inc. of Lannon, Wisconsin.

Dosimetry was determined by direct measurement of compound application.
The efficacy of each as a mosquito repellent was determined by measuring
the ability of the formulations to prevent mosquito landings (defined as
“Lite with Intent to Bite”; LIBe) under field conditions.
Mosquitoes were aspirated mechanically prior to biting; prior to
initiation of the efficacy study, all volunteers were  trained both to
recognize a mosquito landing with the intent to bite and to remove such
mosquitoes with an aspirator using laboratory-raised, pathogen-free
mosquitoes in a controlled laboratory setting.  During the field
studies, participants worked in pairs to facilitate identification and
aspiration of LIBing mosquitoes during brief exposure periods.  The
strengths and weaknesses of each study design are described above.

The dosimetry study enrolled a total of 10 individuals.  Each efficacy
study enrolled 10 subjects at each of the two field sites.  Many
volunteers participated in multiple analytic phases. For example, six of
the 10 participants in the dosimetry study also participated in one of
the field tests.  In total, 23 volunteers participated in at least one
analytic phase of WPC-001. Fifteen of these volunteers also participated
in the dosimetry and/or efficacy phase of Carroll-Loye studies
SCI-001.1, SCI-001.2 or SCI-001.3 described previously (Carroll, 2007a;
Carroll, 2007b; Carroll, 2007c).  In addition to these 23 volunteers,
four controls (described as “experienced personnel” and who were
untreated with repellent) also participated to determine ambient LIBe
pressure at field sites.  On any given day at a field site, the same
pair of participants served as controls for WPC-001 and the
concurrently-run SCI-001.1, SCI-001.2, and SCI-001.3 studies.  Three
individuals served (repeatedly) as controls during each of the three
field tests; all three also participated in the repellent efficacy
tests, giving a cumulative total of 27 volunteers. In addition, three
alternate participants were enrolled to: 1) replace any individual who
withdrew; and 2) protect the confidentiality of any participant excluded
from the study as a result of pregnancy or other potentially
stigmatizing condition, as described below.

Critique

The Board concurred with the factual observations of the ethical
strengths and weaknesses of the study, as detailed in the EPA’s
Science and Ethics Review (Carley, 2007). In general, the research
described in WPC-001 comports with the applicable requirements of 40 CFR
Part 26, subparts K and L.  The risks to study participants were minimal
and were justified by the likely societal benefits, including data on
the efficacy of this formulation as a personal insect repellent.  Based
on toxicological data currently available, the subjects enrolled in this
study were unlikely to be at increased risk of experiencing adverse side
effects upon exposure.  Reactions to mosquito bites are usually mild and
easily treated with over-the-counter steroidal creams.  The study also
excluded individuals who have a history of such severe skin reactions to
further minimize the risk of a participant experiencing a severe
physical reaction to a mosquito bite.  In addition, the study protocol
was designed specifically to minimize the likelihood that a mosquito
would bite, through the use of clear stopping rules, limited exposure
periods, and paired observation; no side effects or adverse events were
reported.  To minimize the risk that study participants will be exposed
to illnesses like WNV, the study protocol called for field tests of
repellent efficacy to be conducted only in areas where known
vector-borne diseases have not been detected by county and state health
or vector/mosquito control agencies for at least one month.  Mosquitoes
collected during the field studies also were subjected to serologic or
molecular analyses to confirm that they were free of known pathogens.
Finally, the study protocol also included several mechanisms designed to
minimize coercive recruitment and enrollment, compensation was not
considered to be so high as to unduly influence participation, and
minors and pregnant or lactating women were explicitly excluded from
volunteering (pregnancy being confirmed by requiring all female
volunteers to undergo a self-administered over-the-counter pregnancy
test on the day of the study).  The potential stigmatization resulting
from study exclusion was minimized by the use of so-called
“alternate” participants, allowing for volunteers to withdraw or be
excluded from participating without unduly compromising their
confidentiality.

Although a majority of the Board concluded that research described in
WCP-001 comports with the applicable requirements of 40 CFR Part 26,
subparts K and L, and that there was no clear and convincing evidence
that the conduct of the research was fundamentally unethical, further
comments are warranted on certain events that took place during the
conduct of the study.  In particular, as noted in the EPA’s review of
these studies, several significant protocol deviations occurred.

As discussed in greater detail in the Board’s review of Carroll-Loye
studies SCI-001.1, SCI-001.2 and SCI-001.3, for example, some members
were concerned that the study investigator failed to obtain IRB approval
for significant changes to the study protocol.  Many of the study
volunteers participated in multiple analytic phases of WPC-001 (as well
as phases of SCI-001.1, SCI-001.2 and SCI-001.3), participating in field
trials conducted on consecutive days and being re-randomized to receive
the same or a different test compound each time.  The approved protocol,
however, includes clear criteria excluding any participant who “[used
an] insect repellent within one day preceding the study” (c.f.,
Carroll, 2007e).  As mentioned previously, however, there was
substantial disagreement among Board members as to whether this
constitutes a deviation from accepted review and approval practices.

The failure of Carroll-Loye Biological Research to: 1) obtain IRB
approval of the revised protocol and consent forms; and 2) report these
protocol deviations to IIRB in a timely manner are serious regulatory
breaches.  The Board recommends the Carroll-Loye Biological Research
report these deviations to the IIRB as soon as possible and work with
that organization to develop and implement a corrective course of
action.

Although the research conducted under WPC-001 was conducted at the same
times and at the same places as the research covered under protocol
SCI-001, there is no evidence that study participants enrolled in this
study ran the same risk of being randomized to receive treatment with an
unapproved investigational compound as participants in SCI-001.1,
SCI-001.2 and SCI-001.3.  A majority of the Board thus concluded that
there was no clear and convincing evidence that the conduct of study
WPC-001 was fundamentally unethical, and concluded that the research
described therein meets the applicable requirements of §40 CFR 26,
subparts K and L.  A significant minority of the Board, however, argued
that the Agency should not utilize any data obtained during the conduct
of this study; these Board members concluded that the study
investigator’s repeated failure in this and other studies to obtain
IRB approval for significant protocol changes or to report study changes
and protocol deviations to the IRB, created an unprecedented pattern of
departure from accepted review and approval practices that could have
study participants at increased risk or significantly impaired the
informed consent process.

HSRB Consensus and Rationale

The Board agreed that the investigator’s failure to report changes to
the IIRB were ethical violations. However while a majority of the Board
agreed that these violations did not rise to the level of substantial
noncompliance, some Board members believed it did meet that level.  Thus
a majority of the Board concurred with the initial assessment of the
Agency that the study submitted for review by the Board meets the
applicable requirements of §40 CFR 26, subparts K and L.

F(1).  Proposed Carroll-Loye Picaridin Insect Repellent Efficacy Studies
(SPC-001)

Charge to the Board

1.  If the proposed research described in Protocol SPC-001 from
Carroll-Loye Biological Research is revised as suggested in EPA’s
review, does the research appear likely to generate scientifically
reliable data, useful for assessing the efficacy of the test substances
for repelling mosquitoes?

Board Response

The active ingredient Picaridin in three formulations will be tested in
the field for its ability to repel mosquitoes by the Carroll-Loye
company.  The active ingredient will be formulated into 1) a 7% pump
spray that will also be used for a 10% pump spray, an aerosol and a
5.75% towelette; 2) a 15% pump spray that will also be used for a 15%
aerosol and a 12% towelette; and 3) a 15% lotion that will also contain
a sunscreen.  All experiments will be conducted using GLP.  A dosimetry
experiment with 10 individuals will be performed to determine the amount
of product that would be utilized by people using the product as
directed.  The experiment will be a field study.  Two locations in
California would be used.

Legs and/or arms will be tested.  There will be two experienced persons
serving as negative controls (i.e., without any repellent product) to
confirm mosquito biting/landing pressure. Experimental subjects, in
pairs, will monitor LIBe’s during a one minute interval each 15
minute, until the First Confirmed LIBe (FCLIBe) can be determined.
Stopping rules will be employed. The Complete Protection Time (CPT) will
be determined.

With respect to the pertinent science criteria established earlier by
the HSRB for completed studies:

The scientific question was stated (i.e., to test the efficacy of
Picaridin in several formulations to repel mosquitoes).

Because existing data were not adequate to answer the question of
efficacy, new studies involving human subjects are necessary.

The potential benefits of the study are clear, i.e., that an effective
repellent would be available that would have either greater efficacy
and/or fewer drawbacks than what was currently approved.

The risks are minimal because the active ingredient is of very low
toxicity, the other formulation ingredients are of very low toxicity,
the mosquitoes will be aspirated before they have an opportunity to
bite, and the regions selected will not have evidence of West Nile or
encephalitis viruses.

Deficiencies related to information about the lotion formulation have
been addressed.

Study Design Criteria

The purpose of the study is clearly defined (i.e., efficacy testing).

There are specific objectives (i.e., to determine the CPT that Picaridin
in three formulations displays as a mosquito repellent).

The sample size will be 10 individuals per product along with 2
experienced individuals to confirm mosquito biting pressure.  A
dosimetry experiment prior to the field experiment will quantify the
amount of repellent being used.

The Board had some concern that with respect to data interpretation, a
stronger solution would be used to estimate CPT for the lower
concentration towelettes.  However, it was explained during the meeting
that the concentration quoted for the towelette also included the weight
of the fabric, and the picaridin solution was identical to the spray.

There was also insufficient information regarding relevant
environmental, mosquito and subject factors that might contribute to
variability.

Participation Criteria:

The participants will be representative of a subset  of the population
of concern; however, there are others in the population unlike these
participants who are likely to use these products, but it would either
be unethical to test them or would be less appropriate to test them.
The participating population is considered appropriate and reasonable.

Measurement Criteria

There was concern about using data from the tested products to
extrapolate for other products.  Extrapolating information from the
7-percent pump spray to a 10-percent pump spray formulation is
acceptable, however there are problems with extrapolating from the
7-percent pump spray to the 5.75-percent towelette because the towelette
may not administer the same dose. On the other hand, if the dosimetry
phase demonstrates the towelette administers approximately the same
dose, extrapolation would be acceptable. Quality assurance will be a
part of the experimental plan.  If the products will not be compared,
randomization is less of an issue if the experiments can be performed on
separate days, however randomization and allocation of subjects to
treatments needs to be fully specified.

There were also statistical concerns that data generated would not be
appropriately analyzed, especially as it related to the assignment of
subjects to conditions.

The measurements will be accurate and reliable.

The measurements will be appropriate to the question being asked.

Quality assurance will be a part of the experimental plan.

Laboratory and Field Conditions

Laboratory experiments are not proposed, except for the dosimetry

Field experiments will be appropriate.

The study will include a stop rule plan, medical management plan, and a
safety monitor.

	HSRB Consensus and Rationale

While the Board agreed that the study rationale, formulations to be
tested and data collection procedures were scientifically sound, the
protocol did not adequately explain the relationship between the study
design and analytic plan nor did it include an appropriate statistical
analysis plan (including estimation of variability) that could be
evaluated for its validity or utility.  There was also insufficient
information regarding relevant environmental, mosquito and subject
factors that might contribute to variability. Thus, the Board concluded
that the proposed research does not appear likely to generate
scientifically reliable data, useful for assessing the efficacy of the
test substance for repelling mosquitoes.

Charge to the Board

b. If the proposed research described in Protocol SPC-001 from
Carroll-Loye Biological Research is revised as suggested in EPA’s
review, does the research appear to meet the applicable requirements of
40 CFR part 26, subparts K and L?

Board Response

Brief Overview of the Study

The research is to be conducted by Carroll-Loye Biological Research, a
private laboratory in Davis, California by using healthy volunteers and
a controlled environment. The study sponsor is Spectrum Brands, Inc. of
Bridgeton, Missouri, a division of United Industries Corporation.  The
study protocol was reviewed and approved by a commercial human subjects
review committee, IIRB of Plantation, Florida. Minutes of IIRB meetings
were provided to the EPA as a separate document, and documentation
previously provided to the EPA by IIRB indicates that it reviewed this
protocol pursuant to the standards of the Common Rule (45 CFR Part 46,
Subpart A).

The research protocol submitted consisted of two interdependent studies:
1) a dosimetry study designed to determine the amount of an
insect-repelling compound, known as picaridin, that normal subjects
would typically apply when provided with one of three compound
formulations (7% Picardin Pump Spray [data will be bridged to 10% Pump
Spray and Aerosol and 5.75% Towelette],15% Picaridin Pump Spray [data
will be bridged to 15% Aerosol and 12% Towelette], and 15% Picaridin
Lotion formulated with sunscreen); and 2) an efficacy study designed to
measure the effectiveness of picaridin as a mosquito repellent.
Dosimetry will be determined either by passive patch dosimetry (spray
formulations) or by direct measurement of compound application (lotion
formulation).  The efficacy of each formulation as a mosquito repellent
will be determined by measuring the ability of the formulations to
prevent mosquito landings (defined as “Lite with Intent to Bite”;
LIBe) under field conditions. Mosquitoes will be aspirated mechanically
prior to biting; prior to initiation of the efficacy study, all
volunteers will be trained both to recognize a mosquito landing with the
intent to bite and to remove such mosquitoes with an aspirator using
laboratory-raised, pathogen-free mosquitoes in a controlled laboratory
setting.  During the field studies, participants will work in pairs to
facilitate identification and aspiration of LIBing mosquitoes during
brief exposure periods.  The strengths and weaknesses of each study
design are described above.

The dosimetry study will enroll a total of 10 subjects, each of whom
will test all three formulations.  The efficacy study will enroll 10
subjects per test formulation per field site, for a total of 30
subjects. Efficacy will be tested independently at two different sites,
representing two different environments.  Two controls (described as
“experienced personnel” and who were untreated with repellent) also
participated to determine ambient LIBe pressure at each field site,
giving a cumulative total of 28 volunteers.  Subjects may participate in
either or both studies, making the total number of volunteers enrolled
no less than 32 but no greater than 74.  In addition, three alternate
subjects will be enrolled to: 1) replace any subject who withdraws from
participating; and 2) protect the confidentiality of any subject
excluded from the study as a result of pregnancy or other potentially
stigmatizing condition, as described below.

Critique

The Board concurred with the factual observations of the strengths and
weaknesses of the study, as detailed in the EPA’s Science and Ethics
Review (Carley and Sweeney, 2007a). The proposed research described in
Protocol SPC-001 comports with the applicable requirements of 40 CFR
Part 26, subparts K and L.

The risks to study participants are minimal and justified by the likely
societal benefits, including data on the efficacy of picaridin as a
mosquito repellent. The risks to study participants are three-fold: 1)
reaction to test materials themselves; 2) exposure to biting arthropods;
and 3) possible exposure to arthropod-borne diseases.

The active ingredient of these three repellent formulations is
commercially available and is present at similar concentrations in other
EPA-registered products; specifically, picaridin is registered and
marketed as an insect repellent in the United States under the
registered trade name BayrepelTM and the brand name Autan.  As
volunteers with known allergic reactions to insect repellents and common
cosmetics are excluded from participating in this study, enrolled
participants are unlikely to be at increased risk of experiencing
adverse side effects upon exposure.  Clear stopping rules also have been
developed, as have plans for the medical management of any side effects
or adverse events associated with product exposure.

Reactions to mosquito bites are usually mild and easily treated with
over-the-counter steroidal creams.  The study also excluded individuals
who have a history of such severe skin reactions to further minimize the
risk of a participant experiencing a severe physical reaction to a
mosquito bite.  In addition, the study protocol was designed
specifically to minimize the likelihood that a mosquito will bite.  All
volunteers will be trained both to recognize LIBe’s and to remove such
mosquitoes with an aspirator prior to biting. Risk of bites is further
minimized through the use of clear stopping rules, limited exposure
periods, and paired observation.

To minimize the risk that study participants will be exposed to
illnesses like WNV, the study protocol calls for field tests of
repellent efficacy to be conducted only in areas where known
vector-borne diseases have not been detected by county and state health
or vector/mosquito control agencies for at least one month.  Mosquitoes
will be collected during the field studies and subjected to serologic or
molecular analyses to confirm that they were free of known pathogens.

Finally, the study protocol also includes several mechanisms designed to
minimize coercive recruitment and enrollment, compensation does not
appear to be so high as to unduly influence participation, and minors
and pregnant or lactating women are explicitly excluded from
volunteering (pregnancy being confirmed by requiring all female
volunteers to undergo a self-administered over-the-counter pregnancy
test on the day of the study).  In reviewing similar protocols submitted
by Carroll-Loye Biological Research at previous HSRB meetings, for
example, the Board has expressed concern about the potentially coercive
nature of study subject recruitment.  Although the study is to be
conducted by Carroll-Loye Biological Research, a private research
laboratory in Davis, California, the Principal Investigator of the study
and Co-Owner of the research laboratory, Dr. Scott P. Carroll, also is
an adjunct faculty member of the Department of Entomology at the
University of California, Davis.  As the majority of research
participants will be recruited from the University’s student
population, including from Dr. Carroll’s own department, the Board
previously recommended that the protocol and consent documents be
altered to define clearly the mechanisms in place to prevent coercion.
The current protocol includes several mechanisms, including the
exclusion of any student or employee the Study Director, a substantial
waiting period between recruitment and study enrollment, and an
interview by Dr. Carroll, designed to minimize coercive subject
recruitment and enrollment.

In accordance with the EPA’s final human studies rule, 40 CFR §§
26.1701-1704,  minors and pregnant women are explicitly excluded from
participation, the latter being confirmed by requiring all female
volunteers to undergo a self-administered over-the-counter pregnancy
test on the day of the study.  In reviewing previous protocols, the
Board has previously raised concerns about the potentially stigmatizing
nature of a positive test, and recommended that investigators develop
additional protections to ensure that the results of over-the-counter
pregnancy tests would be kept private.  The use of so-called
“alternate” subjects is one such safeguard; that study participants
may be designated as alternate subjects and automatically excluded from
participation allows for potentially pregnant volunteers to withdraw
without compromising their confidentiality.

HSRB Consensus and Rationale

The Board concurred with the initial assessment of the Agency that the
revised protocol submitted for review by the Board meets the applicable
requirements of §40 CFR 26, subparts K and L.

F(2).  Proposed Carroll-Loye Picaridin Insect Repellent Efficacy Studies
(SPC-002)

Charge to Board

1.  If the proposed research described in Protocol SPC-002 from
Carroll-Loye Biological Research is revised as suggested in EPA’s
review, does the research appear likely to generate scientifically
reliable data, useful for assessing the efficacy of the test substances
for repelling ticks?

Board Response

The active ingredient Picaridin in three formulations will be tested in
the laboratory for its ability to repel ticks by the Carroll-Loye
company. The active ingredient will be formulated into 1) a 7% pump
spray that will also be used for a 10% pump spray, an aerosol and a
5.75% towelette; 2) a 15% pump spray that will also be used for a 15%
aerosol and a 12% towelette; and 3) a 15% lotion that will also contain
a sunscreen. These are the same formulations as would be tested in
protocol SPC-001 (to test repellency against mosquitoes). All
experiments will be conducted using GLP..  A dosimetry experiment with
10 individuals will be performed to determine the amount of product that
would be utilized by people using the product as directed; this amount
will be applied by technicians to the subjects in the experiment.
Subjects will be trained in the procedures.  One of each subject’s
arms will serve as a negative control to validate each experimental tick
for its questing behavior.

Ticks will be laboratory reared American dog ticks (Dermacentor
variabilis) and deer ticks (Ixodes scapularis).  Because these are
laboratory reared, the ticks are anticipated to be disease-free.

This protocol is very similar to protocols on tick repellency submitted
by this investigator in the past, using the metric of repellency from
the treated area on the arms of the subjects during a 3 minute
observation period during each 15 minutes of the test.  The Complete
Protection Time from the First Confirmed Crossing will be calculated.

With respect to the pertinent science criteria established earlier by
the HSRB for completed studies:

The scientific question was stated (i.e., to test the efficacy of
Picaridin in several formulations to repel ticks).

Because existing data were not adequate to answer the question of
efficacy, new studies involving human subjects are necessary.

The potential benefits of the study are clear, i.e., that an effective
repellent would be available that would have either greater efficacy
and/or fewer drawbacks than what was currently approved.

It is likely that the benefits would be realized because repellent
efficacy will be determined in carefully designed laboratory
experiments.

The risks are minimal because the active ingredient is of very low
toxicity, the other formulation ingredients are of very low toxicity,
the ticks should be removed before they have an opportunity to bite, and
the laboratory reared ticks will not possess any diseases.

Study Design Criteria

The purpose of the study is clearly defined (i.e., efficacy testing for
tick repellency).

There are specific objectives (i.e., to determine the Complete
Protection Time that Picaridin in three formulations displays as a tick
repellent).

The sample size will be 10 individuals per product. A dosimetry
experiment prior to the field experiment will quantify the amount of
repellent being used.

It is anticipated that the findings from this study can be generalized
beyond the study sample.

Participation Criteria

The participants will be representative of some of the population of
concern, but are not representative of the entire population, as
acknowledged by the Investigator; however, since it would either be
unethical to test them or would be less appropriate to test them, the
participating population is considered appropriate and reasonable.

Measurement Criteria

The measurements of tick crossing will be accurate and reliable.

The measurements will be appropriate to the question being asked.

Quality assurance will be a part of the experimental plan.

Laboratory and Field Conditions

Laboratory experiments are proposed.

Field experiments are not proposed.

The EPA science review did not identify any questions or concerns that
needed to be addressed by the HSRB. As was true for protocol SPC-001,
the Board had some concern that, with respect to data interpretation, a
more concentrated product appeared to be proposed to estimate CPT for
the lower concentration towelettes.  However, it was explained during
the meeting that the concentration quoted for the towelette also
included the weight of the fabric, and the Picaridin solution was
identical to the spray.

There were a number of concerns from a statistical perspective. The
protocol states in section 8.3.1 that “[s]ubjects will be assigned to
the treatment groups on the basis of a randomly assigned subject
number,” 1-10 to Lotion, 11-20 to 7% Pump, and 21-30 15% Pump.
However, it also states in section 8.3.2 that “individual subjects may
test more than one repellent, on separate days.”  These two statements
are inconsistent as the former statement implies 30 distinct and unique
subjects, whereas the latter implies an overlap, which may render the
statistical interpretation of the data very difficult, depending on how
the experiment is carried out.  If there is going to an overlap of
subjects among test groups, an appropriate experimental design needs be
employed to allow proper statistical inference.  It is also unclear what
is meant by the sentence “The experiment will be partially randomized
by subjects” in section 8.2 on experimental design.

As for the method of analysis, the statement that “Kaplan-Meier
analyses provide median estimates with substantially reduced error
estimates” is incorrect as it can go either way.  One cannot make a
direct comparison between the median and its 95% confidence interval
based on the Kaplan-Meier estimate of survival function and the mean and
the 95% confidence interval based on the normal theory.  Also the
statement that the median based on Kaplan-Meier method is “less
sensitive to data censoring” is incorrect.

Complete Protection Time defined as the mean time across all treated
subjects from application of the repellent to the First Confirmed
Crossing cannot be estimated due to censoring; neither can the standard
deviation or the confidence interval be accurately calculated.  The more
appropriate statistic in the presence of censoring is the median and the
confidence interval based on the Kaplan-Meier estimate of the survival
distribution for the time to efficacy failure.

The Board disagreed with the EPA’s assessment on compliance with
applicable scientific standards on the following items:

Quantification of efficacy of the test materials is inappropriate as the
normality assumption is inadequate and due to potential censoring.

Discussion of the statistical power is irrelevant as there is no
statistical test of hypotheses is involved.

Justification of the sample size for the repellency phase is not
scientific and thus inadequate.

The Carroll-Loye Protocol SPC-002 7/10/07 document contained the Site
Questionnaire (pp 63-66 of 70) and the Study Specific Instructions (p 67
of 70) for SPC-001, which must be in error, as they refer to mosquitoes,
instead of ticks. There appears to be discrepancies in the Material
Safety Data Sheets (MSDS) in the above document: On pp 59 and 61 the EPA
Registration Number is identical (121-92), but Product Item Numbers are
different (53667 on p 59 and 53661 on p 61) for the identical
formulation. There did not seem to be a MSDS included for the
formulation with sunscreen.

The IRB is universally understood as an acronym for “Institutional
Review Board.”  Therefore, reference to “Independent Investigational
Review Board” as “Independent IRB” is inappropriate and
misleading.

HSRB Consensus and Rationale

The Board concluded that the research appears likely to generate
scientifically reliable data, useful for assessing the efficacy of the
test substances for repelling ticks, provided that the revisions
suggested by EPA are incorporated and that the experimental design is
made more specific to the allocation of the test substances into three
groups of subjects and that there is no overlap of subjects from one
test group to the other, if the data are intended to be compared among
test substances.

The Board urges EPA to consider the design of newer studies and the
designs already used for existing products to make certain that labels
reflect information of comparative value to consumers.  If there are
currently inconsistencies in the information used to authorize label
information on efficacy, the Board urges EPA to develop new guidelines
or revisions to the current guidelines that will balance accuracy of
data collected, safety to human subjects and consistency in the ultimate
labels produced.

Charge to Board

2.  If the proposed research described in Protocol SPC-002 from
Carroll-Loye Biological Research is revised as suggested in EPA’s
review, does the research appear to meet the applicable requirements of
40 CFR part 26, subparts K and L?

Board Response

Brief Overview of the Study

The research is to be conducted by Carroll-Loye Biological Research, a
private laboratory in Davis, California by using healthy volunteers and
a controlled environment.  The study sponsor is Spectrum Brands, Inc. of
Bridgeton, Missouri, a division of United Industries Corporation.  The
study protocol was reviewed and approved by a commercial human subjects
review committee, Independent Investigational Review Board, Inc. (IIRB)
of Plantation, Florida. Minutes of IIRB meetings were provided to the
EPA as a separate document, and documentation previously provided to the
EPA by IIRB indicates that it reviewed this protocol pursuant to the
standards of the Common Rule (45 CFR Part 46, Subpart A).

The research protocol submitted consists of two interdependent studies:
1) a dosimetry study designed to determine the amount of an
insect-repelling compound, known as picaridin, that normal subjects
would typically apply when provided with one of three compound
formulations (7% Picardin Pump Spray [data will be bridged to 10% Pump
Spray and Aerosol and 5.75% Towelette],15% Picaridin Pump Spray [data
will be bridged to 15% Aerosol and 12% Towelette], and 15% Picaridin
Lotion formulated with sunscreen); and 2) an efficacy study designed to
measure the effectiveness of IR3535 as a tick repellent.  Dosimetry will
be determined either by passive patch dosimetry (spray formulations) or
by direct measurement of compound application (lotion formulation).  The
efficacy of IR3535 as a tick repellent will be determined by placing
Western black-legged ticks (Ixodes pacificus) on picaridin-treated and
untreated forearms and measuring the speed and distance that moving
insects would migrate  into the treated area.  The strengths and
weaknesses of each study design are described above.

The dosimetry study will enroll a total of 10 subjects, each of whom
will test all three formulations. The efficacy study will enroll 10
subjects per test formulation.  Each subject will serve as his/her own
control.  Participants in the dosimetry study may or may not participate
in the efficacy study, and participants in the efficacy study may also
test different formulations on different days; this makes the total
number of volunteers enrolled in both the dosimetry and efficacy studies
no less than 10 but no greater than 40. In addition, three alternate
subjects will be enrolled to: 1) replace any subject who withdraws from
participating; and 2) protect the confidentiality of any subject
excluded from the study as a result of pregnancy or other potentially
stigmatizing condition, as described below.

Critique

The Board concurred with the factual observations of the strengths and
weaknesses of the study, as detailed in the EPA’s Science and Ethics
Review (Carley and Sweeney, 2007b). Once the recommended changes
outlined therein are incorporated into the protocol, the proposed
research described in Protocol SPC-002 should comport with the
applicable requirements of 40 CFR Part 26, subparts K and L.

The risks to study participants are minimal and justified by the likely
societal benefits, including data on the efficacy of these
picaridin-based formulations as a tick repellent. The risks to study
participants are three-fold: 1) reaction to test materials themselves;
2) exposure to biting arthropods; and 3) possible exposure to
arthropod-borne diseases.

The active ingredient of these three repellent formulations is
commercially available and is present at similar concentrations in other
EPA-registered products; specifically, picaridin is registered and
marketed as an insect repellent in the United States under the
registered trade name BayrepelTM and the brand name Autan.  As
volunteers with known allergic reactions to insect repellents and common
cosmetics are excluded from participating in this study, enrolled
participants are unlikely to be at increased risk of experiencing
adverse side effects upon exposure.  Clear stopping rules also have been
developed, as have plans for the medical management of any side effects
or adverse events associated with product exposure.

The risks of bites are negligible and minimized by the study design;
tick questing and biting behavior is slow, and study participants are
trained to remove ticks from their forearms prior to biting.

The ticks used for the study are bred and raised in a laboratory
environment and are considered to be pathogen-free, minimizing the risk
of vector-borne disease.  The Agency noted in their review (2007b) that
the tick-borne rickettsial illness Rocky Mountain Spotted Fever (RMSF)
has been passed within tick colonies through a transovarian transmission
mechanism, so the laboratory colonies in question should either be
tested for RMSF prior to initiation of SPC-002, or the consent form and
protocol altered accordingly to reflect the low but not negligible risk
posed to trial participants.

The study protocol also includes several mechanisms, similar to those
described above in the Board’s review of Study Protocol SPC-001,
designed to minimize coercive recruitment and enrollment, compensation
does not appear to be so high as to unduly influence participation,
minors and pregnant or lactating women are explicitly excluded from
volunteering (pregnancy being confirmed by requiring all female
volunteers to undergo a self-administered over-the-counter pregnancy
test on the day of the study), and the use of so-called “alternate”
subjects allows for volunteers to withdraw or be excluded without
compromising their confidentiality. The Board’s only concern about
subject recruitment was the unjustified exclusion of study participants
older than 55 years of age; most Board members felt that a clear
rationale for excluding older volunteers should be provided, or the
exclusion criteria changed.

HSRB Consensus and Rationale

The Board concurred with the initial assessment of the Agency that the
revised protocol submitted for review by the Board meets the applicable
requirements of §40CFR26, subparts K and L.

G.  Proposed ICR Picaridin Insect Repellent Efficacy Study (A 117)

Charge to the Board

1.  If the proposed research described in ICR’s proposed picaridin
protocol is revised as suggested in EPA’s review, does the research
appear likely to generate scientifically reliable data, useful for
assessing the efficacy of the test substances for repelling mosquitoes
of the genus Culex?

Board Response

This protocol presented by ICR was designed to determine in laboratory
studies whether two Picaridin-containing products will repel mosquitoes
of the genus Culex. This product is already registered and the study, if
showing efficacy of the product in repelling Culex, would allow
repellency of this genus to be added to the label.  This is important
because Culex is one of the major carriers of West Nile virus, and
efficacy against this genus would be of interest to consumers.  The
particular products to be tested are Avon Skin-So-Soft SSS Bug Guard
Plus Picaridin Insect Repellent and Avon Skin-So-Soft SSS Bug Guard Plus
Picaridin Insect Repellent spray.  The hypothesis was that 8 hours of
protection against Culex quinquefasciatus, a species known to be a
vector for West Nile virus, would be demonstrated by the two products.

The protocol was clearly written.  Laboratory reared insects that were
disease free would be used. The protocol proposed the use of the
guideline amount of repellent, not an amount determined by a dosimetry
study, and would monitor bites, not landings, in order to be consistent
with the methods used to obtain the label currently borne by these
products.  The negative control would use only landings, avoiding bites
as much as possible, and would be used to guarantee that a landing rate
of at least 5 in 60 seconds was maintained; if the landing rate dropped
below this, more mosquitoes would be added to the cages.

This protocol appeared to follow guideline procedures, and data from a
study such as this would be necessary to modify label claims to include
efficacy against Culex, particularly if Culex was not represented or was
not represented well in the original field studies, which presumably was
the case. The procedures seem to be straightforward.  Pre-testing the
subjects for attractiveness to Culex should yield more consistent
results than if such pretesting was not done.

There were a few points of confusion.  The reference to dose range
finding (pg. 27) was not clear, particularly when the standard guideline
dose level was proposed. It is unclear whether confounding from the
subject’s other arm containing a different product, or the presence of
the arms of the second subject in the cage would occur.  However both
subjects would use the same two products and both products would contain
the same active ingredient, i.e., Picaridin, so there is probably
little, if any, confounding likely. There was a question as to whether
the laboratory temperature and humidity would be similar to that
occurring in the field.  There was also a question of how, with an 8
hour limit to the test, an average of 8 hours efficacy with a 2 hour
deviation might be obtained.  It was suggested that a Q test was not the
most appropriate statistical method, and that the Kaplan Meier test
might be the better approach.

Two of the aspects of this protocol are at variance with what the Board
had previously concluded regarding other insect repellency protocols,
and these deserve specific discussion.  One is the use of the standard
guideline dosage instead of a dosimetry test to determine the dosage to
be used.  While in a totally new study, the dosimetry test would yield a
dosage more representative of the amount of product that the consumer
would use, in this situation, this study is supposed to match a
previously conducted field study with the same products.  It is
therefore appropriate to use the same dosage as was used in the earlier
field study.  Likewise, the previous field study used bites. While the
Board has been impressed with the added safety to participants imparted
by the landing endpoint over the bite endpoint, these data would need to
match the data obtained in the previous field study, which used bites as
the endpoint.  In addition, the consultants indicated that landings did
not necessarily predict bites, leading to even more support for the use
of bites in order to match the earlier study with bites.

	HSRB Consensus and Rationale

The Board concluded that this study would be appropriate to confirm
efficacy against Culex, was designed to meet the current EPA guidelines,
and was generally clear and adequately designed, with the exception of
the appropriate statistics.  If revised consistent with EPA’s
recommendations and the Board’s suggestions, the study should yield
valid data regarding the efficacy of these products in repelling Culex.

Charge to Board

2.  If the proposed research described in ICR’s proposed picaridin
protocol is revised as suggested in EPA’s review, does the research
appear to meet the applicable requirements of 40 CFR part 26, subparts K
and L?

Board Response

Brief Overview of the Study

The proposed study (Spero, 2007) would evaluate the efficacy of two
different skin-applied lotion formulations of picaridin-based insect
repellents under laboratory conditions; these two formulations are Avon
Skin-So-Soft SSS Bug Guard Plus Picaridin Insect Repellent (EPA Reg. No.
806-29) and Avon Skin-So-Soft SSS Bug Guard Plus Picaridin Insect
Repellent Spray (EPA Reg. No. 806-31.

The research is to be conducted by ICR, a commercial organization based
in Baltimore, Maryland; ICR provides testing and regulatory consulting
services for companies developing and marketing pesticides and
insecticides in the United States and Canada.  The study is managed by
toXcel, LLC of Gainesville, Virginia.  The sponsor of this study is Avon
Products, Inc. of New York, New York.  The submitted documents assert
that the study will be conducted in accordance with the ethical and
regulatory standards of 40 CFR 26, Subparts K and L, as well as the
requirements of FIFRA §12(a)(2)(P), and the U.S. EPA’s GLP Standards
described at 40 CFR 160. The protocol was reviewed and approved by an
independent human subjects review committee, Essex Investigational
Review Board (EIRB), Inc., of Lebanon, New Jersey prior to submission to
the Agency.

Efficacy of the two picaridin-based formulations will be evaluated by
using healthy volunteers. The study will be performed at ICR’s
laboratory in Baltimore, Maryland, and test the effectiveness of the two
compounds as mosquito repellents by measuring the ability of each
formulation to prevent mosquito bites under laboratory conditions.  The
strengths and weaknesses of the study design are described above.

The effectiveness study will enroll a total of 13 subjects.  Of the 13
participants, twelve will be treated and test the effectiveness of the
two picaridin-based repellent formulations.  The compounds will be
applied to 250 cm2 patches of skin on the forearms of each study
participant; one compound will be applied to the right forearm and one
to the left forearm, with the effectiveness of each formulation
simultaneously evaluated.  Treated skin will be exposed to mosquitoes
for five minutes at half-hour intervals by having study participants
insert their forearms into 8 ft.3 test “cages” containing 100
laboratory-reared, pathogen-free female C. quinquefasciatus.  The
efficacy of each repellent formulation will be ascertained by measuring
the time from application to "breakdown" of repellency, with
“breakdown" defined in the protocol as either two confirmed bites in a
single five-minute exposure period, or one bite in each of two
consecutive exposure periods.  Treated study participants will work in
pairs, observing mosquito landings and alerting attendant ICR staff of
potential bites; ICR staff will determine whether or not these are
confirmed bites. Probes (i.e., "bites" where the mosquito punctures the
skin but does not collect blood) and bites from mosquitoes that do not
fully alight (i.e., all six legs on the surface of the exposed skin)
will not be considered as confirmed bites.  Once breakdown has occurred
for a particular repellent formulation, no further exposure of the
subject's treated skin will occur.  The study protocol justifies the
enrollment of twelve treated participants by stating that ten volunteers
are needed to obtain statistical validity; an additional two
participants will be enrolled as alternates to “allow for drop outs”
(Spero and Gaynor, 2007).

One study participant, chosen by lottery (a “coin toss”), will
remain untreated and will be monitored to determine mosquito-biting
pressure under laboratory conditions.  A 250-cm2 patch of untreated skin
will be exposed for five minutes at half-hour intervals, with the
ambient biting pressure determined by measuring the number of mosquitoes
landing on the skin.  A minimum rate of 5 landings per minute is
necessary for the laboratory trial to be conducted or continued.
Landing mosquitoes will be “shaken” away by the study participant
while ICR staff counts the number of landings.

Critique

The supporting documentation provided by the study investigators,
sponsor and EIRB, as submitted to the Agency, appear to meet the
regulatory requirements of 40 CFR 26.1115a and 40 CFR 26.1125. A
description of EIRB procedures was provided to the EPA with a claim of
confidentiality, so were not available for review by the HSRB. Agency
staff, however, reviewed the documentation provided by EIRB and
determined these procedures and policies to be in compliance with the
applicable standards of the Common Rule (45 CFR 46, Subpart A). The
protocol as submitted to the Agency thus is substantially compliant with
the regulatory requirements of review and documentation, minor
deficiencies not withstanding.

The Board concurred with the factual observations of the strengths and
weaknesses of the study, as detailed in the EPA’s Science and Ethics
Review (Carley and Sweeney, 2007c). Specifically, the Board agreed with
the Agency’s recommendations that ICR: 1) revise the current protocol
discussion of risks and benefits; 2) provide more information about
ICR’s subject recruitment and enrollment processes; and, 3) alter the
data collection form to remove all identifiable information.  Most Board
members disagreed, however, with the EPA’s recommendation that study
investigators provide a clearer justification for relying on time to FCB
as a measure of repellent efficacy. Previous field studies using time to
FCB as an endpoint for these particular repellents efficacy have already
be submitted to and approved by the Agency.  These additional laboratory
studies are to be conducted solely for the purpose of justifying
additional label claims as per Agency requirements.  For consistency,
use of time to FCB, using pathogen-free colonies of mosquitoes under
controlled conditions, is justified.

Once these changes are incorporated into the protocol, the proposed
research described in ICR A117 should comport with the applicable
requirements of 40 CFR 26, subparts K and L. In brief, the risks to
study participants are minimal and justified by the likely societal
benefits, including data on the efficacy of these picaridin-based
formulations as a repellent for one of the key mosquito genera known to
transmit WNV in the United States.  At first glance, the potential risks
to study participants are three-fold: 1) reaction to test materials
themselves; 2) exposure to biting arthropods; and, 3) exposure to
arthropod-borne diseases.

These two picaridin-based repellent formulations are commercially
available in the United States, and have been used widely as cosmetic
and personal care products with little evidence of toxic effects.
Volunteers with known allergic reactions to insect repellents and common
cosmetics are excluded from participating in this study, and the amount
of skin treated with picaridin is limited, so enrolled participants are
unlikely to be at increased risk of experiencing adverse side effects
upon exposure to the test materials.  Clear stopping rules also have
been developed, as have plans for the medical management of any side
effects or adverse events associated with product exposure.  The Board
did recommend, however, that the informed consent documents be modified
to more accurately represent the known toxic risks associated with acute
picaridin exposure, particularly via ocular, oral and respiratory routes
of exposure.

The endpoints of the study protocol require two confirmed mosquito bites
to document breakdown of repellent effectiveness.  Reactions to mosquito
bites are usually mild and easily treated with over-the-counter
steroidal creams; such a cream, in addition to CalomineTM and rubbing
alcohol, will be provided to study participants to alleviate minor
symptoms associated mosquito bites.  The study excludes individuals who
have a history of such severe skin reactions to further minimize the
risk of a participant experiencing a severe physical reaction to a
mosquito bite.

The mosquitoes used for the study are bred and raised in a laboratory
environment and are considered to be pathogen-free, minimizing the risk
of vector-borne disease.  In the Agency’s Scientific and Ethics review
of the protocol (Carley and Sweeney, 2007b), a concern was raised that
the risk of arthropod-borne illnesses is not adequately discussed in the
informed consent document; the Agency recommended that a discussion of
these risks be added.  The Board agreed.

The differential risks to untreated control subjects (chosen by lottery
rather than via separate enrollment of more experienced study
participants) are listed in both the protocol and the informed consent
document.  The Board did note, however, a misleading statement in the
consent document, in which mosquitoes will be “brushed [off] by ICR
staff” from untreated control subjects arms prior to biting.  The
protocol stated that untreated controls will “shake” landing
mosquitoes off.  This inconsistency must be corrected.

Furthermore, it is clear from the protocol that all study volunteers, be
they untreated controls or treated volunteers, will be asked to expose
an untreated arm to the mosquito colony before the efficacy study
begins, in order to test “attractiveness.”  The attractiveness
component of the study is not listed in the informed consent document,
and must be added.  The informed consent document also lists one of the
societal benefits of the study as bringing a “new repellent to
market.”  The study as described, however, is designed to simply allow
labeling change.  The repellent formulations under evaluation are
already on the market.

A more detailed explanation of study recruitment is also needed; the
exact procedures for recruiting study participants are unclear.  As
currently written, however, compensation for study participation is not
so high as to unduly influence enrollment, and employees and contractors
of ICR, toXcel and the sponsor (as well as family members) are excluded
from participation.  In accordance with the newly promulgated provisions
in the EPA’s final human studies rule (40 CFR §§ 26.1701-1704),
minors and pregnant women are explicitly excluded from participation,
the latter being confirmed by requiring all female volunteers to undergo
a self-administered over-the-counter pregnancy test on the day of the
field study.  The use of two potential “alternate” subjects allows
for volunteers to withdraw or be excluded without compromising their
confidentiality.

Finally, the Board was concerned that an overlooked risk of study
participation might be the strain associated with the physical
requirements imposed upon volunteers.  Each study participant will be
asked to spend a full day in a warm, humidified laboratory environment.
Although the likelihood of an adverse event precipitated by the physical
strains of study participation is small, these risks nevertheless should
be incorporated into the informed consent document, and a clear plan for
medical monitoring and treatment be articulated.

HSRB Consensus and Rationale

The Board concurred with the assessment of the Agency that the protocol
ICR A117 submitted for review by the Board, if revised as suggested in
both EPA’s review and by the Board, would meet the applicable
requirements of 40 CFR 26, subparts K and L.

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Epidemiology (STROBE) Statement: Guidelines for Reporting Observational
Studies. PLoS Med 4(10): e296   HYPERLINK
"http://dx.doi.org/10.1371/journal.pmed.0040296"
doi:10.1371/journal.pmed.0040296

The Human Studies Review Board (HSRB or Board) has discussed and
provided advice to EPA on scientific and ethical issues related to the
conduct of field studies to evaluate the efficacy of mosquito repellent
products.  The HSRB has reviewed both proposals for new field studies
and the results of completed studies.  The HSRB has noted that, although
there are many similarities across studies, not all studies employ the
same study design.  The HSRB has identified several methodological
issues for which additional background information would assist the
Board in its evaluation of such studies.

BACKGROUND

Currently, EPA requires all pesticide products that claim to repel
mosquitoes to provide data on the duration of efficacy under field
conditions at two biologically distinct sites.  These data are derived
from human research with subjects who have been treated with the
repellent formulations in the field.  The Agency evaluates the duration
of repellent efficacy for a subject by calculating the time from
application of the repellent to the occurrence of an event indicating an
efficacy failure.  Historically, for field studies of mosquito
repellency, EPA has used the “first confirmed bite” as an indication
of efficacy failure on a test subject.  Several recent studies have
shifted to the “first confirmed landing with intent to bite;” EPA
has accepted this alternative endpoint.  A “confirmed landing” on a
test subject is a mosquito landing followed by a second landing on the
same subject within a specified period of time (usually 30 minutes)
after the initial landing.

Field studies typically involve 6 – 10 subjects who have been treated
with a defined amount of the test material.  Each subject is then
regularly and repeatedly exposed to ambient mosquito populations for a
fixed interval of time until the subject experiences an efficacy failure
followed by a confirmation with the specified period of time. Mosquito
landing pressure (representing intent to bite) at a site is monitored by
concurrently exposing untreated subjects to mosquito landings.  A study
is considered valid only if there are at least a specified minimum
number of mosquito landings on untreated subjects during each exposure
interval.

On October 25, 2007, the HSRB will discuss scientific aspects of the
design of field studies to assess the efficacy of mosquito repellents.
For this meeting the Board has requested consultants to provide
specialized information or assistance to the Board.   The Board is
particularly interested in the frequency, duration and timing of
exposure of subjects to potential mosquito landings.  The Board requests
each consultant to respond briefly to the series of questions below.
Please send the responses to the HSRB Chair and Designated Federal
Official (DFO) at least one week before the meeting—i.e., by no later
than October 18.  All responses will subsequently be provided to the
other consultants, the HSRB members, and EPA staff for their review, and
will be posted on   HYPERLINK "http://www.regulations.gov"
www.regulations.gov  under docket ID number, EPA-HQ-ORD-2007-0942.  HSRB
consultants will be available at the meeting to
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	What do data show about the variability of the time intervals
between first and subsequent landings in mosquito repellent field
trials?

	What is the current scientific understanding of how factors other
than repellent efficacy could affect the likelihood that an initial
event—a mosquito landing or mosquito bite—would be “confirmed”
by another similar event within 30 minutes?  Please address at least
these factors:

o	Characteristics of mosquito populations

o	Characteristics of test sites

o
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	Can the impact of such factors on the likelihood or timing of an
initial and confirming event be predicted?  Can it be quantified?

At its June 27 - 29, 2007 meeting the Board learned that different
designs with different “length-biased” sampling for mosquito
repellent field studies are in use.  One design exposes subjects to
potential mosquito landings for one minute of every 15 minutes; another
design exposes subjects to potential mosquito landings for five minutes
of every 30 minutes.  The DFO is separately providing a CD containing
the background materials for the June 27 – 29, 2007 HSRB meeting.  The
protocols are loaded on the CD.  These designs have different
“length-biased” sampling.

	What is the methodological rationale for the two different designs?

	Which design is used more widely in the field? Why?

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direction or magnitude of the effects be predicted?  How might these
influences be analyzed and accounted for in collecting, reporting and
analyzing repellent efficacy data?

Dr. Matt Kramer, a USDA statistician who has served as a consultant, has
suggested that the precision of estimates of Complete Protection Time
(CPT) in repellent testing could be significantly increased by defining
a failure of efficacy as the mean time from treatment to a series of
several landings or bites.  He has stated:

The precision of CPT increases when it is estimated beyond time to
[First Confirmed Bite] FCB or FCLanding.  How well CPT can be estimated
depends on the distribution of so many bites beyond FCB.  The number of
mosquitoes that will bite (n) will determine results of the test.  Each
person in the field should be his/her own control; that way it is
possible to know n per person, and reduce person-to-person variability.

If using the mean time to the first 5 bites, the SE will decrease
proportionally as n increases (n = 5 in this case).  That is equivalent
to an increase in the power of the test of 5 times.  This method allows
for detecting formulation differences near the CPT.

	Does this approach, indeed, increase the precision of estimates of
CPT markedly without requiring additional subjects?

	If so, would this increased precision justify the incremental risk
to the subjects resulting from their exposure to a great?

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Proposed Final Draft v.1 Dated January 10, 2008 Do Not Cite or Quote

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