January 20, 2015
EPA-HSRB-14-03
Thomas Burke, Ph.D.
EPA Science Advisor
Office of the Science Advisor
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Subject: November 5, 2014 EPA Human Studies Review Board Meeting Report
Dear Dr. Burke,
The United States Environmental Protection Agency (EPA or Agency) requested that the
Human Studies Review Board (HSRB) provide scientific and ethics reviews of two items: a new
open pour protocol proposed by the Agricultural Handler Exposure Task Force, LLC (AHETF)
and a pre-Rule publication by Frampton et al. (2002) concerning human exposure to nitrogen
dioxide. The Board's key responses to the charge questions are summarized in this letter and are
detailed in the enclosed final meeting report.
AHETF Protocol (AHE 170) - Open Pour Loading of Granules
Science
• The Board concludes that given the clearly defined boundaries of the protocol, if
performed as described and as recommended by the HSRB, the AHE170 protocol is
likely to generate scientifically reliable data that will be useful for assessing the exposure
of those who perform open pour loading of granular pesticide products.
Ethics
• If the research is performed as described, it is likely to meet the applicable requirements
of 40 CFR part 26, subparts K and L.
A Published Report by Frampton et al. (2002) on Nitrogen Dioxide Effects on Airway and
Blood Cells
Science
• The sample collection and laboratory analysis parts of this study are scientifically sound.
However, the chamber exposure data are not reliable without more information regarding
the actual exposure levels to nitrogen dioxide.
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• The statistical analysis is adequate to justify the significant differences that the authors
identified, but is not adequate to detect all of the differences that may have occurred. This
study may be used in a quantitative way as part of a weight-of-evidence analysis to
support effects that might occur at the exposure levels reported (0.6 and 1.5 ppm), but
this study (as published) is not relevant to support the existence of no effects at the
claimed levels of exposure.
Ethics
The Board concluded that the published report by Frampton et al. (2002) submitted for
review meets the applicable requirements of 40 CFR part 26 subpart Q, and that the data
within this article may be considered acceptable for EPA's reliance, contingent upon the
determination of their scientific validity.
Sincerely,
Rebecca T. Parkin, PhD, MPH
Chair
EPA Human Studies Review Board
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NOTICE
This report has been written as part of the activities of the EPA Human Studies Review Board, a
Federal advisory committee providing advice, information and recommendations on issues
related to scientific and ethical aspects of human subjects research. This report has not been
reviewed for approval by the Agency and, hence, the contents of this report do not necessarily
represent the view and policies of the Environmental Protection Agency, nor of other agencies in
the Executive Branch of the Federal government, nor does the mention of trade names or
commercial products constitute a recommendation for use. You may obtain further information
about the EPA Human Studies Review Board from its website at http://www.epa.gov/osa/hsrb.
You may also contact the HSRB Designated Federal Officer, via e-mail at ord-osa-
hsrb@epa.gov
In preparing this document, the Board carefully considered all information provided and
presented by the Agency presenters, as well as information presented by public commenters.
This document addresses the information provided and presented within the structure of the
charge by the Agency.
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US ENVIRONMENTAL PROTECTION AGENCY
HUMAN STUDIES REVIEW BOARD
Chair
Rebecca Parkin, Ph.D., M.P.H., Professorial Lecturer, Environmental and Occupational Health
and Epidemiology & Biostatistics, Milken Institute School of Public Health, The George
Washington University, Washington, DC
Vice Chair
Jewell H. Halanych, M.D., Assistant Professor, Internal Medicine Residency Program,
Montgomery Regional Campus, University of Alabama at Birmingham, Montgomery, AL
Members
Gary L. Chadwick, Pharm.D., M.P.H., C.I.P., Senior Consultant, HRP Consulting Group, Inc.
Fairport, NY
Liza Dawson, Ph.D., Research Ethics Team Leader, Division of AIDS National Institutes of
Health (NIH), National Institute of Allergy and Infectious Disease (NIAID), Bethesda, MD
George Fernandez, Ph.D., Statistical Training Specialist, SAS Institute, Statistical Training and
Technical Services, Sparks, NV
Kyle L. Galbraith, Ph.D., Manager, Human Subjects Protection, Carle Foundation Hospital,
Urbana, IL
Edward Gbur, Jr., Ph.D., Professor, Agricultural Statistics Laboratory, University of Arkansas,
Fayetteville, AR
Elizabeth Heitman, Ph.D., Associate Professor of Medical Ethics, Center for Biomedical Ethics
and Society, Vanderbilt University Medical Center, Nashville, TN
John C. Kissel, Ph.D., Professor, Department of Environmental and Occupational Health, School
of Public Health, University of Washington, Seattle, WA
Randy Maddalena, Ph.D., Physical Research Scientist, Indoor Environment, Lawrence Berkeley
National Laboratory, Berkeley, CA
William Popendorf, Ph.D., M.P.H., Professor Emeritus, Department of Biology, Utah State
University, Logan, UT
Kenneth Ramos, M.D., Ph.D., Pharm.B., Associate Vice President, Precision Health Sciences,
Professor of Medicine, Arizona Health Sciences Center, Tucson, AZ
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Suzanne M. Rivera, Ph.D., M.S.W., Vice President for Research and Technology Management,
Case Western University, Cleveland, OH
Jun Zhu, Ph.D., Professor of Statistics and of Entomology, Department of Statistics, University
of Wisconsin - Madison, Madison, WI
Human Studies Review Board Staff
Jim Downing, Executive Director, Human Studies Review Board Staff, Office of the Science
Advisor, United States Environmental Protection Agency, Washington, DC
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INTRODUCTION
On November 5, 2014, the United States Environmental Protection Agency's (EPA or Agency)
Human Studies Review Board (HSRB or Board) met to address the scientific and ethical charge
questions related to two agenda items: a new protocol proposed by the Agricultural Handler
Exposure Task Force, LLC on open pouring of granules (AHE170), and a published article by
Frampton et al. (2002) regarding airway and blood cell responses to nitrogen dioxide exposures.
REVIEW PROCESS
The Board conducted a public meeting using Adobe Connect1, on November 5, 2014. Advance
notice of the meeting was published in the Federal Register as "Human Studies Review Board;
Notification of a Public Meeting" (EPA, 2014, pp. 62437-62439).
Following welcoming remarks from Agency officials, the Board heard presentations from EPA
for the two agenda items in sequence. This Final Report of the meeting describes the HSRB's
discussion, recommendations, rationale and consensus in response to each charge question.
For each agenda item, Agency staff first presented their review of the science and the Board
asked the Agency presenters clarifying questions. The staff then described their review of the
ethical aspects and the Board asked clarifying questions about those. The Board solicited public
comments and next asked Agency staff to read the Charge Questions for the document under
consideration. The Board discussed the science questions first and then the ethics question. The
Chair then called for a vote to confirm concurrence on a summary statement in response to each
charge question.
For their evaluation and discussion, the Board considered materials presented at the meeting, oral
comments, the original protocol and published report, related materials and published articles,
and the Agency's science and ethics reviews. There were no public comments presented at this
meeting. A comprehensive list of background documents is available online at
http://www.epa.gov/hsrb/.
CHARGE TO THE BOARD AND BOARD RESPONSE
AHETF Protocol (AHE 170) - Open Pour Loading of Granular Pesticide Products
Overview of the Study
The Agricultural Handler Exposure Task Force, LLC (AHETF) designs and implements study
protocols to assess workers' exposures in a variety of workplace scenarios. In this proposal,
AHETF plans to evaluate experienced handlers' dermal and inhalation exposures to conventional
pesticide formulations in granular form (e.g., passable through 4-79 mesh sieves). The study will
1 Accessed at http://epa. connectsolutions. com/hsrb.
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focus on open pour loading of pesticides from non-bulk containers into typical agricultural
application equipment; application of these pesticides will not be evaluated in this study. EPA
plans to use the results to estimate exposures to a variety of pesticides loaded in comparable
conditions.
A 7x3 study design has been selected. Seven monitoring areas (MA) across the U.S. have been
identified to represent the various climatic, crop and geographic conditions under which the test
substances are applied. Within each area, three monitoring units (MU) will be determined; one
handler will comprise each MU. The sample of handlers will be identified using a two-step
process: screening for appropriate and willing commercial growers, who have appropriately
experienced and interested workers; potential participants will be recruited through notices
and/or meetings. Similarity restrictions will be used to ensure purposive diversity of the study.
Eligible participants will be provided with written informed consent materials and time to review
them before deciding whether they will take part in the study. Participants will be given an
opportunity in the consent process to request their individual research results.
Growers will select one pesticide from a list of 10, known to be stable and having well-known
analytical methods, for use on their property. During regular work days, participants will conduct
typical but partially scripted tasks; e.g., transferring 50-lb. bags into spreaders, stacking bags,
loading and unloading bags, and/or cleaning up equipment. One of the three ranges of the
amount of the active ingredient handled (AaiH), from 5 to 400 Ibs. AaiH, will be randomly
assigned to the 4+ hour monitoring period for each MU. At least three loads will be poured per
monitoring period. The workers will wear two layers of clothing: full-body work clothing plus a
long-sleeved, long-legged whole body dosimeter (WBD) underneath. The under layer will be
used to assess whole-body dermal exposures. Face/neck wipe and hand wash samples will be
collected before exposure, before breaks and at the end of the monitoring period. Breathing zone
air samples will be collected using standard personal air monitoring equipment (either fiber
filters in cartridges or Occupational Safety and Health Agency (OSHA) Versatile Sampler (OVS)
tubes with personal air sampling pumps). Fortified field samples will be used in triplicate to
assess the stability of the active ingredient (AI) in the field, during transport and storage, and on
sampling materials, such as face/neck wipes and the dosimeter. The study team will observe
participants during their monitoring periods and note environmental conditions, personal
protective equipment used, and participant activities.
The primary statistical objective is to estimate exposures within three-fold of the actual
population values. The secondary objective is to ensure that the study has at least 80% power to
distinguish proportionality from independence between exposure and AaiH.
Science
Charge to the Board
• Is this research likely to generate scientifically reliable data, useful for assessing the
exposure of those who perform open pour loading of granular pesticide products?
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Board Response to the Charge
HSRB Recommendations
• The Board concludes that given the clearly defined boundaries of the protocol, if
performed as described and as recommended by the HSRB, the AHE170 protocol is
likely to generate scientifically reliable data that will be useful for assessing the exposure
of those who perform open pour loading of granular pesticide products.
HSRB Detailed Recommendations and Rationale
Overall, the Board agreed with the Agency's scientific assessment that the AHE170 protocol
"addresses the technical aspects of applicable exposure monitoring guidelines and is likely to
produce scientifically valid and useful data" (Evans and Sherman, 2014, p. 2) for assessing the
exposure of those who perform open pour loading of granular pesticide products. Some aspects
of the study are commented on below; these provide opportunities to improve the design or
identify points that should be considered by the Agency when interpreting and using the data.
1. Surrogate Related Issues
The AHETF has identified a range of pesticides to use as surrogates for all active ingredients in
granular formulations. The use of multiple pesticides as surrogates increases the chances of
successfully identifying representative MUs for each AaiH level in each MA. The protocol states
that "any of the AHETF surrogates can be used for generating exposure data for this scenario"
(Collier, 2014, p. 35). However, the validity of this assertion for a given surrogate pesticide
depends on the persistence of that pesticide in the granular formulation over the period of time
between the start of exposure and the collection of the dosimeter or skin washes. If a given
surrogate pesticide is lost from the formulation by volatilization, degradation, or by irrecoverable
sorption into the skin during the exposure period, then the resulting exposure measurement for
that specific pesticide will be biased low and result in an underestimate of exposure.
Differences in volatilization for each surrogate pesticide can be evaluated in part by comparing
results of the measured vapor-phase inhalation exposure (measured with the OVS tubes) relative
to the particle bound formulation (measured on the OVS filter). Differences in the degradation
rate on the dosimeter and air samplers for the surrogates can be determined in part by the
recovery of spiked pesticide from the fortified samplers if volatilization is found to be negligible.
The data analysis for the protocol should include, at a minimum, a qualitative assessment of the
relative persistence of the selected surrogate pesticides over the duration of the exposure. For
those surrogates that are not completely conserved in/on the granular formulation and/or on the
WBD, the data analysis should include a quantitative assessment of chemical loss and how that
loss impacts the measured exposure for the open pour loading of granules.
Quantifying the loss of surrogate pesticide due to irrecoverable dermal sorption is more difficult
in the absence of absorbed dose (AD) measurements, which are beyond the scope of the
protocol. Clearly the loss of surrogate pesticide during the monitoring period by sorption into the
dermal layer will bias the exposure measurements low, but it also has implications for the safety
of the study and the calculation of margins of exposure (MOEs) as discussed below. The
archived literature is rich with information about the performance of WBD (cotton long
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underwear) as dosimeters for dermal exposure, particularly as a primary outer garment (i.e., as
used in the Jazzercise™ exposure assessment scenario2). The efficiency of WBDs is affected by
the efficiency of the fabric in collecting and retaining particles for the duration of the scenario-
based activity, and retaining AI in the light of volatilization and loss via transfer to the skin. It is
not clear whether there is good information on the relationship between the WBD-based
exposure data and absorbed dose during field studies. For example, the protocol did not include
information about how factors such as temperature and sweat saturation of the WBD garment
would influence the estimation of absorbed dose from a whole body dosimeter.
2. Non-conservatism of the protocol
Non-conservatism of study results can be produced from uncertainty in the relationship between
1) exposure estimates based on face/neck wipe and hand wash and 2) the sorption of active
ingredients from granules contacting the skin. For example, hands and exposed skin are to be
washed to assess exposure. It is well-established that wash recovery efficiency declines with
delay time (e.g., Fenske & Lu, 1994; Fenske etal., 1996). Granular formulations are typically
about 10% AI (i.e., 100,000 ppm); this may be greater than saturation for most or all
compound/carrier combinations. Therefore, some fraction of the AI on the outer surfaces of the
granules will be readily available for transfer to skin and may behave, upon contact with skin, as
pure compound. Recovery of this material would likely be incomplete even with rapid washing,
so results will not be conservative. In addition, delay until washing will be variable and will
introduce more variability in recovery efficiency. Particles are going to be very large so they
will likely fall from the skin prior to washing, and some AI will be absorbed into the skin beyond
the point of recovery; therefore, using either process as a basis would underestimate the
exposure. Furthermore, the protocol provides for use of new gloves for each worker. This avoids
exposure to residues in gloves from activities unrelated to the study but is non-conservative in
that gloves can be contaminated under normal working conditions and workers are very unlikely
to use fresh gloves every day. The Board recommends that these issues be considered and
addressed in interpretation of study results.
3. Non-conservatism of the estimate of MOEs
The uncertainties in absorbed dose relative to the measured exposure can also lead to non-
conservatism of stated margins of exposure and consequently of the study's safety. For example,
four of the proposed surrogates (chlorpyrifos, imidacloprid, pendamethalin, and tefluthrin) have
MOEs that reflect assumed dermal bioavailabilities that are less than 100%. Those assumed
dermal bioavailabilities are probably taken from (non-specified) studies that are or may be very
dissimilar to conditions relevant to granular pour operations (i.e., dry WBDs versus sweat soaked
WBDs). Dermal bioavailability is not independent of loading conditions. Therefore, the assumed
bioavailabilities cannot be presumed to be transferable. The Agency should review and
document the loading conditions used to develop the dermal absorption factors for these four
2 Around 1999, EPA's Office of Pesticide Programs decided to use transfer coefficients derived from Jazzercise™ to
represent one hour of extreme exercise or four hours of typical activities. For more information on this, see
"Overview of Issues Related to the Standard Operating Procedures for Residential Exposure Assessment" presented
at the FIFRA Scientific Advisory Panel meeting on September 21, 1999 (pp. 2, 41, and 45-50). For technical
information, see Ross, J. et al. (1990). Measuring potential dermal transfer of surface pesticide residue generated
from indoor fogger use. Chemosphere. 20:349-360.
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surrogates for their relevance to the field conditions to be tested within this protocol to assure
that the MOEs are indeed adequate.
4. Statistical and sample design issues
The chosen sampling design that involves stratifying a large portion of the country into
monitoring areas and selecting monitoring units within each area is an appropriate sampling
design based on the nature of this study. The justification of the selection of monitoring areas is
reasonable, given practical and cost constraints.
The limited randomness in the selection of monitoring units that would likely be achieved by the
recruitment process as described in the documents was discussed at some length. Although the
Board felt the proposed approach was acceptable given practical constraints, it recommends that,
if feasible, some form of classification system be developed that would identify the mechanism
by which each MU was recruited and in particular would identify those, if any, that might need
to be recruited by "traditional recruitment" in order to provide an after-the-fact (future) indicator
of the randomness of the MU selection process.
The objective of creating a database of exposures over a range of exposure scenarios may be
achieved with the proposed sampling design for three types of agricultural pesticides, viz.:
herbicides, insecticides, and fungicides. The design attempts to take into account
climate/environment (7 MAs), usage (11 crops), pesticides (10), and amount of AI handled (3
AaiH ranges). In addition, the documents indicate that the best case scenario would be to have
each MU within an MA be classified in a different AI range. Having at least one MU in every
one of these combinations clearly is not possible with the chosen sample size.
It has been stated that no data currently exist for this particular exposure issue (Collier, 2014, p.
11; Evans and Sherman, 2014, p. 3). Each MU in the study is expected to perform a variety of
tasks, but it is not clear if all or some or only one the 10 pesticides will be handled during the
monitoring. Since the goal is to have each MU in an area have a different AaiH level, with three
MUs and three AaiH levels, it appears that there are no replications of AaiH levels within an
area. As a result, the variability among humans who would be exposed to a particular scenario
cannot be addressed using only the information from this study.
Implementation of this protocol will provide quantitative information on a variety of exposure
situations related to open pour or granule formulations. While statistical analyses are not the goal
of this study, the information will surely be used in some way by someone for that purpose
eventually. Lack of replication of the human variability would make it difficult or likely
impossible for any user of the database to conduct further statistical analyses.
Ethics
Charge to the Board
• Is the research likely to meet the applicable requirements of 40 CFR part 26, subparts
K and L?
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Board Response to the Charge
HSRB Recommendation
• If the research is performed as described, it is likely to meet the applicable requirements
of 40 CFR part 26, subparts K and L.
HSRB's Detailed Recommendation and Rationale
Overall, the Board agreed with the agency's assessment that this study is ethically acceptable.
The Board's detailed reasoning is given below.
1. Risks to subjects
The risks to study participants are outlined in section 2.3 of the protocol (Collier, 2014, pp. 62-
67), as follows: a) risk of heat-related illness, b) risk associated with scripting of field activities;
c) psychological risks; d) risk of exposure to surfactants and e) risk of exposure to surrogate
chemicals. Pregnant and nursing women will be excluded.
a. Regarding heat-related illness, the protocol states that this risk may be increased due to the
fact that the subjects must wear an extra layer of clothing (long underwear (or WBD) for
exposure monitoring during the manual pouring procedure). Likely, the more significant
factors for heat-related illness would be temperature, humidity and level of activity of the
subject. In other words, the addition of a single layer of clothing may not be significant in
the overall risk of heat-related illness. Therefore, the statement that the use of the extra
clothing constitutes greater than minimal risk in this study might be an overstatement.
To minimize risk of heat-related illness, the protocol appropriately outlines steps for
monitoring temperature and if necessary, using cycles of work and rest, moving the worker to
a cooler environment, or conducting the testing during early morning or night when
temperatures are lower. Given that the subjects will be individuals who are familiar with the
manual pouring procedures and will be in good health, the risks are reasonable.
b. The risk of scripted activities is likely to be negligible, since it involves reducing the load
size of the material to achieve three load/apply cycles per MU.
c. Likewise, the psychological risks appear to be minimal; they relate to changing clothes in the
presence of a researcher, and for women, taking a pregnancy test, which is done in a private
room without disclosure of test results to anyone outside the research team.
d. The protocol describes the risk from use of a surfactant (0.01% v/v Aerosol® OT in
water). The surfactant is used as a very dilute solution and risks of skin irritation are likely to
be negligible. An eye wash is available in case of accidental exposure to the eyes (Collier,
2014, pp. 64-65).
e. However, the handling of surrogate materials itself could, in theory, constitute greater than
minimal risk. The standard of minimal risk relates to the probability and magnitude of harm
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experienced in daily life3 and this is generally interpreted as the daily life of healthy
individuals—which could reasonably be taken to mean individuals in the general (not
occupational) population who are not exposed to pesticides on a daily basis. In other words,
the fact that agricultural workers are exposed to pesticides in their daily work does not
necessarily mean this exposure would a priori be considered minimal risk using a general
healthy person standard.
The risks of exposure to surrogate materials are described in section 2.3.5 (Collier, 2014, pp.
65-67). Since the study involves one day of exposure, the risks are those associated with
acute toxicity, rather than chronic exposure. Table 1 of the protocol shows the dermal MOE
for each surrogate (Collier, 2014, p. 66). The calculated MOEs meet or exceed the required
minimum MOE in each case. Therefore, the risks are considered acceptable according to
EPA standards for exposure to organophosphate pesticides (Collier, 2014, pp. 65-66).
Whether or not the overall study constitutes minimal risk, or greater than minimal risk, is
difficult to determine, but the risks are reasonably low and are well managed as described in
the protocol.
Workers engaged in the pouring procedures for the study will be experienced pesticides
handlers, will be using appropriate personal protective equipment (PPE) and will be
reminded of safe handling practices.
2. Selection of subjects
The subjects will be offered participation in the study after their employer has agreed to allow
recruitment of his/her workers (Collier, 2014, p. 32). Workers will be informed of the study and
have an opportunity to learn about the study without their supervisors being present. Employers
will sign consent documents stating that they will not influence or coerce their employees to
participate in the study. It is not clear whether employees might be prohibited or discouraged
from joining the study if employers did not feel it was in their (the employer's) interest to allow
participation. Pregnant and nursing women will be excluded. Some workers in agricultural
settings have low levels of literacy; these individuals will not be excluded from participation.
3. Informed consent
Informed consent will be conducted in individual sessions using prepared materials in English or
in Spanish. For workers who cannot read or who are not comfortable reading consent materials,
the materials will be read aloud by the study staff in the presence of a witness, per SOP AHETF
11.1.3 (Collier, date, pp. 261-266). These informed consent arrangements are acceptable.
3 Minimal risk means that the probability and magnitude of harm or discomfort anticipated in the research are not
greater in and of themselves than those ordinarily encountered in daily life or during the performance of routine
physical or psychological examinations or tests. The definition is in the Code of Federal Regulations at 40 CFR
26.102(1); it is available at http://www.gpo.gov/fdsys/granule/CFR-1999-title40-voll/CFR-1999-title40-voll-sec26-
102/content-detail. html
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4. Privacy and confidentiality
The main privacy issues relate to possible photographs or videos during the study activities,
changing of clothing in the presence of researchers, and pregnancy testing. These risks are
appropriately managed as described in section 2.3.3 of the protocol (Collier, 2014, pp. 64).
5. Monitoring of data
The protocol describes monitoring for heat-related illness in Section 2.3.1 (Collier, 2014, p. 62).
Workers will be observed and reminded to use appropriate PPE during handling of the product.
Nearby medical facilities will be identified and transportation and costs for any needed medical
care will be provided, per section 2.3.5 (Collier, 2014, pp. 65-67).
6. Vulnerable or disadvantaged subjects
Given that some agricultural workers are economically disadvantaged, or may have low literacy
levels, these individuals are potentially vulnerable. It is acceptable to enroll these subjects, given
that the study involves low levels of risk, participants are appropriately compensated for their
time, and consent procedures require adequate oral explanation of the study for those who cannot
read. It is important that language issues are adequately addressed—for example, that bilingual
or Spanish speaking witnesses are available if needed. For other workers who are not fluent in
English, or who do not read English and are not Spanish-speakers, they would need to be
excluded from the study due to the lack of translated forms and materials.
In summary, the study has appropriate procedures and protections in place for human subjects.
The level of risk is low and the procedures to minimize risk are reasonable. The social value of
conducting the research is significant and the overall risk/benefit ratio is acceptable.
A published report by Frampton et al. (2002) of intentional human exposures to nitrogen
dioxide and their effects on airway and blood cells4 5
Overview of the Study
This standard, three-period cross-over inhalation toxicity study was conducted to assess the
"effects of nitrogen dioxide (NCh) on airway inflammation, blood lymphocyte recruitment to the
lung, and susceptibility of airway cells to infection with influenza virus and RSV [respiratory
syncytial virus]" (Frampton et al., 2002, p. L161). Twenty-one (12 male, 9 female) adult subjects
participated in this study; they were recruited, from the population of students and community
members around the University of Rochester. Eligible participants were evaluated individually
in an environmental chamber during 1995-1996. The participants averaged 27 years old, were
non-smokers, had no history of cardiac or respiratory diseases, and were free of respiratory
infections for at least six weeks. They were exposed three times (to air and to 0.6 ppm and 0.15
ppm NCh) for three hours each, during which they exercised intermittently several times. The
4 Chair's note: Dr. Gary Chadwick recused himself from Board decisions and votes related to this study.
5 Following this meeting, the EPA decided to not rely on this study.
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exposure levels typified those found in indoor environments with unvented combustion sources
(Frampton etal., 2002, p. LI55). The three exposure days were separated by at least three weeks.
Airway exposures were measured using standard spirometry methods, fiberoptic bronchoscopy,
and airway brush biopsies. Blood cell counts were performed on the bronchoscopy and brush
biopsy samples. Flow cytometry was used to assess cell differential counts, blood lymphocytes
and other metrics. Cell cultures were used to determine the viability of respiratory and epithelial
cells in the presence of influenza and RSV strains.
Analysis of variance (ANOVA) methods were used to evaluate the effects related to NCh
exposures. Period, carryover, treatment effect and gender differences were examined. Residuals
were assessed to determine whether the assumption of normality had been violated.
Science
Charge to the Board
• Is this study scientifically sound, providing reliable data?
• If so, is this study adequate for quantitative use in support of an inhalation risk
assessment for the use of nitrogen dioxide as a medical equipment sterilant?
Board Response to the Charge
HSRB Recommendations
• The sample collection and laboratory analysis parts of this study are scientifically sound.
However, the chamber exposure data are not reliable without more information regarding
the actual exposure levels to nitrogen dioxide.
• The statistical analysis is adequate to justify the significant differences that the authors
identified, but is not adequate to detect all of the differences that may have occurred. This
study may be used in a quantitative way as part of a weight-of-evidence analysis to
support effects that might occur at the exposure levels reported (0.6 and 1.5 ppm), but
this study (as published) is not relevant to support the existence of no effects at the
claimed levels of exposure.
HSRB Detailed Recommendations and Rationale
At its core, this paper describes some very detailed and scientifically sound biological sample
collection and analytical methods. However, the Board noted issues that add to the Agency's
review (Leshin, 2014). Specifically, the HSRB has four concerns related to the operation of the
exposure chamber that greatly weaken the Board's confidence in the reliability of the study's
data and two concerns about statistical methods (two concerns) that weaken the study's
reliability and constrain its potential power.
1. Exposure chamber
a. In regard to the chamber, on p. LI56 Frampton et al. (2002) state that "For comfort,
temperature and relative humidity were maintained at 37.1 ± 3.0°C and 21.2 ± 0.92%
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(mean ± SD), respectively." This temperature (corresponding to 98.8°F) is outside of the
normal comfort range and comprises a potential heat stress setting, especially when
combined with an exercise regime "sufficient to increase the minute ventilation to 40
L/min" for 10 of each 30 minutes for three hours (Frampton et al., 2002, p. L156). No
recognition of this risk is mentioned within the paper (or its associated consent
information).
The authors state "The capabilities for generating and maintaining pollutant levels and
constant temperature and humidity have been described previously (48)" (Frampton et
al., 2002, p. LI56). This citation (ref 48) is probably in error and should be ref. 49 (and
likely reference 49 should be reference 48).6 Reference 49 is a three-page extended
conference abstract that describes the designed operating capabilities of this chamber:
"This system [(S), (DX), (R) and (H)] was designed to vary the chamber temperature
from about 10°C to 31.5°C and relative humidities [sic] from about 25 to 85%" (Utell et
al., 1984, p. 220); these limits are repeated on page 221 in Table 1. Thus, the reported
research by Frampton et al. (2002) seems to have been conducted outside of both the
temperature and humidity ranges for which the chamber was designed. No comment
regarding exceeding these limits was found within the paper.
Two of the Frampton et al. (2002) claims regarding NCh on page LI 56 are without any
supporting methods, data, or references but are virtually word-for-word from the aerosols
reported in reference 49 (Utell et al., 1984, p. 220). The first claim, "This [referring to
-0.3 atmospheric changes/min] enabled NCh levels to reach >90% of target levels within
4 min" (Frampton et al., 2002, p. L156), is technically incorrect. Comments elsewhere in
reference 49 read, "A volumetric control is incorporated into the chamber air supply to
stabilize the air flow at 10 mVmin" (the same flow rate as in Frampton et al., 2002) and
"the effective chamber volume (less instrumentation furniture, etc.) is approximately 40
m3" (both quotes are from Utell et al., 1984, p. 220). Thus, the ideal (best case) air
exchange time is 40 m3 divided by 10 mVmin or the four minutes referred to by the
authors.7 However, 2.3 air changes (at least 9 minutes) are required in order for any
injected air component (like NCh) to reach >90% of target levels (derived from: -ln(l -
C/Ctarget) = -ln(l - 0.9) = -ln(0.1) = 2.3). Less than ideal mixing would have extended the
room's actual air exchange rate by 10% to 50% of its ideal value, and thus the time to
reach 90% of its target level could have been 10 to 15 minutes. Because the authors do
not describe the startup sequence of the exposure chamber, the Board could not determine
how long in advance NCh might have been injected into the chamber and the magnitude
of the effect of this delay on the exposure conditions experienced by the test subjects.
6 Their reference 48 is "Utell MJ, Frampton MW, Roberts NJ Jr, Finkelstein JN, Cox C, and Morrow PE.
Mechanisms of nitrogen dioxide toxicity in humans. Health Effects Institute Research Report 43:1-44, 1991"
(Frampton et al., 2002, p. L165). It appears that this reference to 48 should be to reference 49 that is "Utell MJ,
Morrow PE, Hyde RW, and Schreck RM. Exposure chamber for studies of pollutant gases and aerosols in human
subjects: design considerations. JAerosol Sci 15: 219-221, 1984" (Frampton et al., 2002, p. L165). Reference 49
was probably also mistakenly cited on p. L155 of the Frampton et al. (2002) paper in reference to toxicity
mechanisms "Because of its oxidative potential and limited solubility, NCh is a deep lung irritant, and accidental
exposures to high concentrations can cause acute lung injury and death (13, 49)."
7 Less than ideal mixing will extend this time to more than four minutes. On p. 221 of reference 49, the authors
mention "a wall-mounted air circulation unit which is normally kept in operation to assist in chamber mixing" (Utell
et al., 1984). Without more specifics, such a unit would probably keep the room's actual air exchange rate within
10% to 50% of its ideal value.
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However, even a 15-minute transient is a small fraction of subjects' three-hour exposure
periods.
d. Another claim is potentially of much greater concern. In their Methods section, the
authors state that "The concentrations of NCh at the 3- and 6-ft levels within the chamber
varied by no more than 5% of the mean" (Frampton et al., p. LI 56). Furthermore, in the
Results, the authors claim that "Actual achieved NCh concentrations were 0.61 ± 0.02
and 1.50 ± 0.02 (SD) ppm" (Frampton etal, 2002, p. LI57). However, no method of
measuring the Mh was mentioned; measuring NCh concentrations is not a trivial matter,
and its method is worthy of a description. While these oversights might just reflect the
authors' focus on biologic responses and these results may be a repeatable measure of
chamber performance, it is also possible that this claim of uniformity was simply copied
from reference 49 that states "Sampling of test aerosol concentrations at 3' and 6' levels
(3x3 matrix) within the chamber has revealed that the aerosol concentrations vary by no
more than ± 5% of the mean" (Utell et al, 1984, p. 220). In contrast to those test aerosol
concentrations, Figure 1 of reference 49 (Utell et al., 1984, p. 220) indicates only one
location for environmental monitoring ["EM"]. In fact, the authors state later that
"Chamber monitoring is based on environmental factors, e.g. temperature, air flow,
relative humidity, and the pollutant per se" (Utell et al., 1984, p. 220). This statement
referenced by Frampton et al. to that citation "for generating and maintaining pollutant
levels," and the lack of a method to measure Mh imply that the authors of Frampton et
al. may have based their exposure values on the rate at which the 5000 ppm NCh
compressed gas was emitted into the venturi mixer and diluted into the 10 mVmin air
supplied to the exposure chamber; i.e., the reported concentrations of NCh may be merely
calculated but unverified assumptions. Neither the length nor the material of the ducting
that carried the air from the control and mixing equipment located in an adjacent
engineering space to the exposure chamber was described within either paper. Since NCh
is fairly reactive, it is at least possible - if not likely - that some of the supplied NCh was
lost en route to (or within) the exposure chamber. If the test concentrations were
calculated rather than measured, they were probably not as high as expected and reported.
This uncertainty makes the exposure side of this study seem unreliable.
Although there is one author (Utell) common to both Frampton et al. (2002) and
reference 49 (Utell et al, 1984), the four concerns noted above suggest that the authors of
the former paper may have had a limited understanding of the exposure chamber itself.
2. Statistical issues
The statistical design and analysis procedures are clearly stated on page LI 57 of Frampton etal.
(2002). The study's three-period cross-over design with washout periods was commonly used in
this type of situation. Based on a document containing background material, an undescribed pre-
study power analysis apparently indicated that four subjects for each of the six treatment
sequences would provide adequate power (Sherman, 2014, p. 45). Additional subjects were
recruited to allow for dropouts but data from only 21 subjects are reported in the paper; this
number of actual participants does have an effect on the study's power. The analyses of variance
included period and cross-over effects. Checks on model assumptions were carried out. These
analysis procedures are standard and strengthen the acceptability of the numerical results.
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a. The first statistical concern relates to the treatment of outliers. Frampton et al. (2002)
state that "Data means shown in RESULTS include all study subjects, even though
statistical outliers were excluded for the ANOVA" (p. LI 57). Deletion of outliers is an
acceptable practice as long as it is reported as part of the methods. However, no
indication was given as to the criteria by which an observation was identified as an
outlier. More importantly, there was neither mention of which variables were affected
nor the severity of the problem. As a consequence, in the Results, parts or all of Figures
1 through 6 do not accurately reflect the numerical results of the statistical analysis (pp.
L159-L161). Moreover, the extent of the inaccuracies cannot be determined; Figure 4A
(p. L160) is a good example of this issue. The authors indicate that the only significant
effect is the NCh main effect. However, the figure shows a very large difference between
male and female responses at 1.5 ppm which would be indicative of a potential gender x
NCh level interaction. At best, this leads to potential confusion as to which effects may
actually be significant.
b. The level of the second statistical concern depends upon the intended use of the Frampton
et al. (2002) data. The ANOVA tests the authors used were adequate to support their
conclusions that pulmonary responses were observed. However, in some cases the paper
could have used more powerful statistical tests; these might have detected other
significant responses that were not recognized. A good example is the respiratory
ventilation data that comprise the first two data columns of Table 2 (at rest and while
exercising) (p. L158). In both columns, the mean respiratory rates progress with
increasing dose of NCh, but the only test mentioned was Student's t-test for total NCh
intake. The differences in ventilation at the two doses subjectively look rather large (i.e.,
the increase in rates for all but the females while exercising are in the range of+4 to
+9%); these could have been significant if the results had been analyzed using paired t-
tests. Another statistical option might have been some form of regression analysis, but
the authors only provided data for 0.6 to 1.5 ppm exposures, instead for the three sets of
data (including 0 ppm) as was done for all other comparisons. The existence of such
increases in ventilation rate would be consistent with the effects of such a respiratory
irritant and with the decreases in hematocrit that were observed and presented in Figure 1
(p. L159) for all three test levels (0, 0.6, and 1.5 ppm).
In summary, the statistical methods used in the study do not detract from the significant
responses that the authors identified nor from their conclusions, but the limitations to these tests
imply that the authors may not have identified all of the statistically significant responses to
these exposures that may be of interest to the Agency.
Ethics
Charge to the Board
• Does the study meet the applicable requirements of 40 CFR part 26 subpart Q?
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Board Response to the Charge
HSRB Recommendation
• The Board concluded that the published report by Frampton et al. (2002) submitted for
review meets the applicable requirements of 40 CFR part 26 subpart Q, and that the data
within this article may be considered acceptable for EPA's reliance, contingent upon the
determination of their scientific validity.
HSRB's Detailed Recommendation and Rationale
The Board agreed overall with the Agency's assessment of the ethics of the study (Sherman,
2014). The Frampton et al. (2002) article, published in the American Journal of Physiology of
Lung Cellular and Molecular Physiology, is the report of a medical study intended to test the
effects of nitrogen dioxide (NCh) on airway inflammation, blood cells, and resistance to
respiratory viral infection in 21 normal, non-smoking adult male and female volunteers. The
investigation was conducted at the University of Rochester, School of Medicine and was funded
by the National Institutes of Health and the Center for Indoor Air Research. It was designed as a
study of intentional exposure to NCh as an environmental pollutant, but EPA identified the work
as a potential source of data for evaluating NCh as a sterilant of medical equipment.
In the Methods and Study Design section of the article, the investigators report that the study was
approved by the University of Rochester's Research Subjects Review Board, the university's
institutional review board (IRB) for protection of human subjects in research (Frampton et al,
2002, pp. LI56). At that time the IRB would have been operating under regulatory standards of
the 1996 version of 45 CFR 468 and the ethical guidance of the Belmont Report9.
The article states that investigators enrolled 21 subjects (9 females, 12 males) ages 18-40 and
that "Informed consent was obtained" (Frampton et al., 2002, p. LI56). When interviewed by
Agency staff in August 2014 and later contacted by email, the article's first author, Dr. Mark
Frampton, reported that none of the participants was pregnant or nursing, and that subjects were
tested for pregnancy before each exposure (Sherman, 2014, p.30).
In response to a request from EPA staff, the University of Rochester's IRB provided electronic
copies of available records on this study, which included the study's protocol and consent
document, and the IRB's approval letter. It may be assumed that the IRB approved the protocol
following the above regulatory requirements in place at the time the study was reviewed.
The Board concurred with the conclusions of the OPP's Ethics Review (Sherman, 2014) that the
reported research does not rely on data from intentional exposure of any human subject who was
a pregnant or nursing woman or a child. There is no evidence that the conduct of the research
was fundamentally unethical. There is no evidence that 1) the research was conducted in a way
that placed participants at increased risk of harm (based on the knowledge available at the time
the study was conducted) or 2) vulnerable populations were targeted. However, because
8 Protection of Human Subjects in the U.S. Code of Federal Regulations, 45 CFR part 46. For a timeline of these
federal regulations, go to http://histonf.nih.gov/about/timelines_laws_human.html
9 Available at httpj//wwwJihs,goy/oto
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recruitment processes focused on the University community where the study was conducted, and
the protocol does not address additional protections for students or employees, there is a
possibility that some participants' ability to give informed consent may have been impaired by
coercion.
1. Assessment of risks and benefits
The article reports that many Americans are exposed to levels of NCh as an indoor air pollutant
resulting from combustion, such as when natural gas-fueled ice resurfacing machines are used in
enclosed ice rinks (Frampton etal., 202, p. L155). The primary benefit of the study was to
society, by generating knowledge of NCh's health effects. The highest concentration studied was
a level stated in the consent form to be a level observed in homes where gas stoves are used.
Risks of exposure to NCh are not clearly spelled out in the consent document, although the short-
term risks of the bronchoscopy are identified. Neither the article nor the consent form addresses
any potential benefit to participants or the balance of risks and benefits. The University of
Rochester IRB's approval of this study can be interpreted as its assessment that the risks to
participants did not outweigh the study's anticipated benefits under ethical and regulatory
standards in place at the time of its review.
2. Equitable selection of study participants
Although vulnerable populations were not targeted, it is not possible to determine whether
selection of study participants was equitable or whether individual members of vulnerable
populations were enrolled in the study. According to the protocol and consent document,
participants were compensated a total of $550 for taking part in all three phases of the study,
with progressively increased exposure to NCh, a blood draw, and bronchoscopy at each stage.
Participants received $50 for completion of the first phase, an additional $50 for completion of
the second phase, and additional $450 for completion of the third phase. Although the
compensation schedule was structured to promote participants' completion of all three phases,
the amount and schedule appear do not appear to have been disproportionate or coercive.
3. Voluntary and informed consent of all participants
Participants were recruited from among members of the University of Rochester community in
Rochester, New York, including students and employees of the University. Although vulnerable
populations were not targeted for the study, it is not possible to tell whether participants from
potentially vulnerable populations were recruited. Similarly, there is no indication that the study
protocol included mechanisms designed to minimize coercive recruitment and enrollment of
students and employees who may have been in a subordinate position to the researchers.
To participate in the study, subjects were required to be healthy, non-pregnant adults with no
history of smoking. No children were enrolled. After an initial conversation with the study
coordinator by phone, potential participants met with one of the investigators to discuss the
study. Potential participants received a copy of the consent document to take home for further
review before deciding whether to enroll. In his communication with EPA staff, Dr. Frampton
reported that participants signed consent documents, and documents from the IRB call for
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signatures from the participant, an investigator, and a witness. Participants were informed in the
consent document that they were able to withdraw from the study at any time.
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References
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Ethical Principles and Guidelines for the Protection of Human Subjects of Research.
Washington, DC: U.S. DHEW, The National Commission for the Protection of Human
Subjects of Biomedical and Behavioral Research. Retrieved from
http://www.hhs.gov/ohrp/humansubjects/guidance/belmont.html
U.S. Department of Health and Human Services (DHHS) (1996). 45 CFR part 46. Code of
Federal Regulations. Washington, DC: U.S. Department of Health and Human Services
(DHHS). Retrieved December 8, 2014, from
http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html (2009 version)
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Evans, J., & Sherman, K. (2014). Science and Ethics Review of AHETF Scenario Design and
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Frampton, M. W., Boscia, J., Roberts, M. J., et al. (2002, January). Nitrogen dioxide exposure:
effects on airway and blood cells. American Journal of Physiology of Lung Cellular and
Molecular Physiology, 282, L155-L165.
Leshin, J. (2014). Nitrogen Dioxide: Evaluation of inhalation study, Frampton et al 2002.
Washington, DC: U.S. Environmental Protection Agency, Office of Chemical Safety and
Pollution Prevention.
Sherman, K. (2014). Ethics Review of Human Toxicity Study with Nitrogen Dioxide.
Washington, DC: U.S. Environmental Protection Agency, Office of Pesticide Programs.
U.S. Environmental Protection Agency (EPA). (2010, July 1). 40 CFR part 26. Code of Federal
Regulations. Washington, DC: U.S. Government Printing Office. Retrieved December 9,
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2014, from http://www.gpo.gov/fdsys/pkg/CFR-2010-title40-vol l/xml/CFR-2010-title40-
voll-part26.xml
U.S. Environmental Protection Agency (EPA). (2014, October 17). Human Studies Review
Board; Notification of a Public Meeting. 79 FR [FederalRegister]', pp. 62437-62439.
Retrieved November 2, 2014, from
https://www.federalregi ster.gov/articles/2014/10/17/2014-24757/human-studies-revi ew-
board-notification-of-a-public-meeting
Utell, M., Frampton, M., Jr, R. N., JN, F., Cox, C., & Morrow, P. (1991). Mechanisms of
Nitrogen Dioxide Toxicity in Humans. Boston, MA: Health Effects Institute.
Utell, M., Morrow, P., Hyde, R., & Schreck, R. (1984). Exposure chamber for studies of
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Science, 75,219-221.
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