1 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 St udies Review Board Meeting Report

Dear Dr. Burke,

The United States Environmental Protection Ag ency (EPA or Agency) requested that the
Human Studies Review Bo ard (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 summ arized 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 recommende d 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 load ing 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 laborat ory analysis parts of this study are scientifically sound. However, the chamber exposure data are not re liable without more information regarding the actual exposure leve ls to nitrogen dioxide.2  The statistical analysis is adequate to jus tify 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 expos ure 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 th e 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 Board3 NOTICE

This report has been written as part of the ac tivities of the EPA Human Studies Review Board, a Federal advisory committee providing advice, information and recommendations on issues
related to scientific and ethica l aspects of human subjects rese arch. 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 po licies of the Environmenta l Protection Agency, nor of other agencies in the Executive Branch of the Federal governme nt, 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-osahsrb@epa.gov

In preparing this document, the Board carefu lly considered all information provided and presented by the Agency presenters, as well as information presented by public commenters. This document addresses the information provide d and presented within the structure of the charge by the Agency.4 US ENVIRONMENTAL PROTECTION AGENCY
HUMAN STUDIES REVIEW BOARD

Chair

Rebecca Parkin, Ph.D., M.P.H., Professorial Lect urer, Environmental and Occupational Health and Epidemiology & Biostatistics, Milken Inst itute School of Public Health, The George Washington University, Washington, DC

Vice Chair

Jewell H. Halanych, M.D., Assistant Professo r, 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 a nd Infectious Disease (NIAID), Bethesda, MD

George Fernandez, Ph.D., Statisti cal Training Specialist, SAS Inst itute, Statistical Training and Technical Services, Sparks, NV

Kyle L. Galbraith, Ph.D., Manager, Human Subjects Protection, Carl e Foundation Hospital, Urbana, IL

Edward Gbur, Jr., Ph.D., Professor, Agricultural St atistics Laboratory, University of Arkansas, Fayetteville, AR

Elizabeth Heitman, Ph.D., Associat e 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 Scientis t, Indoor Environment, Lawrence Berkeley National Laboratory, Berkeley, CA

William Popendorf, Ph.D., M.P.H., Professor Em eritus, Department of Biology, Utah State University, Logan, UT

Kenneth Ramos, M.D., Ph.D., Pharm.B., Associat e Vice President, Precision Health Sciences, Professor of Medicine, Arizona He alth Sciences Center, Tucson, AZ5 Suzanne M. Rivera, Ph.D., M.S. W., Vice President for Resear ch and Technology Management, Case Western University, Cleveland, OH

Jun Zhu, Ph.D., Professor of Sta tistics and of Entomology, Departme nt of Statistics, University of Wisconsin – Madison, Madison, WI

Human Studies Revi ew Board Staff

Jim Downing, Executive Director, Human Studies Review Board St aff, Office of the Science Advisor, United States Environmenta l Protection Agency, Washington, DC6 INTRODUCTION

On November 5, 2014, the United States Environm ental Protection Agency’s (EPA or Agency)
Human Studies Review Boar d (HSRB or Board) met to address the scientific and ethical charge questions related to two agenda items: a new protocol propos ed 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 res ponses to nitrogen di oxide exposures.

REVIEW PROCESS

The Board conducted a public meeting using Adobe Connect 1 , on November 5, 2014. Advance notice of the meeting was published in the Federal Register as “Human Studies Review Board;
Notification of a Public M eeting” (EPA, 2014, pp. 62437-62439).

Following welcoming remarks from Agency offici als, the Board heard presentations from EPA for the two agenda items in sequence. This Fi nal Report of the meeti ng describes the HSRB’s discussion, recommendations, rationale and consen sus in response to ea ch charge question.

For each agenda item, Agency staff first presen ted their review of th e 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 qu estions about those. The Board solicited public comments and next asked Agency staff to read the Charge Questions for the document under
consideration. The Board discusse d the science questions first a nd then the ethics question. The
Chair then called for a vote to confirm concurre nce on a summary statement in response to each charge question.

For their evaluation and discussion, the Board consid ered materials presente d at the meeting, oral comments, the original protocol and published report, related ma terials and published articles, and the Agency’s science and ethics reviews. There were no public comments presented at this meeting. A comprehensive list of backgr ound documents is available online at http://www.epa.gov/hsrb/

.

CHARGE TO THE BOARD AND BOARD RESPONSE

AHETF Protocol (AHE 170) – Open Pour Lo ading of Granular Pesticide Products

Overview of the Study

The Agricultural Handler Exposure Task For ce, LLC (AHETF) designs and implements study protocols to assess workers’ exposures in a vari ety of workplace scenarios. In this proposal, AHETF plans to evaluate experi enced handlers’ dermal and inha lation exposures to conventional pesticide formulations in granul ar form (e.g., passable through 4-79 mesh sieves). The study will

1 Accessed at http://epa.connectsolutions.com/hsrb.7 focus on open pour loading of pesticides from no n-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 monito ring 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, thr ee 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 writte n informed consent materials and time to review them before deciding whether they will take pa rt in the study. Participants will be given an opportunity in the consent process to re quest their individua l research results.

Growers will select one pestic ide from a list of 10, known to be stable and having well-known analytical methods, for use on their property. Duri ng regular work days, participants will conduct typical but partially scripted tasks; e.g., transf erring 50-lb. bags into sp readers, 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 lbs. 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 monito ring period. Breathing zone air samples will be collected using standard personal air monitoring equipment (either fiber filters in cartridges or Occupational Safety and Health Ag ency (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 th e 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 mon itoring periods and note enviro nmental conditions, personal protective equipment used, a nd participant activities.

The primary statistical objective is to estimat e exposures within three-fold of the actual population values. The secondary objective is to en sure that the study has at least 80% power to distinguish proportionality from inde pendence between exposure and AaiH.

Science

Charge to the Board

 Is this research likely to generate scientif ically reliable data, us eful for assessing the exposure of those who perform open pour lo ading of granular pesticide products?8 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 recommende d 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 load ing 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 ex posure monitoring guidelines and is likely to produce scientifically valid a nd useful data” (Evans and Sh erman, 2014, p. 2) for assessing the exposure of those who perform ope n pour loading of granular pe sticide products. Some aspects of the study are commented on below; these pr ovide opportunities to improve the design or identify points that should be considered by th e Agency when interpretin g 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 pest icides 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 th is assertion for a given surrogate pesticide depends on the persistence of that pesticide in the granular form ulation over the period of time between the start of exposure a nd the collection of the dosimeter or skin washes. If a given surrogate pesticide is lost from the formulati on by volatilization, degradation, or by irrecoverable sorption into the skin during the exposure pe riod, 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 pe sticide 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 th e OVS filter). Differences in the degradation rate on the dosimeter and air samplers for the surrogates can be determ ined in part by the recovery of spiked pesticide from the fortified samp lers if volatiliz ation is found to be negligible. The data analysis for the protocol should include, at a minimum, a qualitative assessment of the relative persistence of th e selected surrogate pesticides ove r the duration of the exposure. For those surrogates that are not co mpletely conserved in/on the gra nular formulation and/or on the WBD, the data analysis should include a quantita tive assessment of chemi cal loss and how that loss impacts the measured exposure for the open pour loading of granules.

Quantifying the loss of surrogate pe sticide due to irrec overable dermal sorption is more difficult in the absence of absorbed dose (AD) meas urements, which are beyond the scope of the protocol. Clearly the loss of su rrogate pesticide during the mon itoring period by sorption into the dermal layer will bias the exposure measurements lo w, but it also has imp lications for the safety of the study and the calculati on of margins of exposure (MOE s) as discussed below. The
archived literature is rich with informati on about the performance of WBD (cotton long9 underwear) as dosimeters for dermal exposure, part icularly as a primary outer garment (i.e., as used in the JazzerciseTM exposure assessment scenario 2 ). The efficiency of WBDs is affected by the efficiency of the fabric in collecting and re taining particles for the duration of the scenariobased activity, and retaining AI in the light of volatiliza tion 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 studie s. For example, the protocol did not include information about how factors such as temper ature and sweat saturati on of the WBD garment
would influence the estimation of abso rbed 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. Fo r 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 et al ., 1996). Granular formul ations 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 fr action of the AI on the outer surfaces of the granules will be readily availabl e for transfer to skin and may be have, 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 additi on, 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, an d some AI will be absorbed into the skin beyond the point of recovery; therefor e, using either process as a basis would underestimate the exposure. Furthermore, the protoc ol provides for use of new gloves for each worker. This avoids exposure to residues in gloves from activities unr elated to the study but is non-conservative in that gloves can be contaminated under normal work ing conditions and workers are very unlikely to use fresh gloves every day. The Board recomm ends that these issues be considered and addressed in interpre tation 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 nonconservatism of stated margins of exposure and c onsequently of the study’s safety. For example, four of the proposed surrogates (chlorpyrifos, im idacloprid, pendamethalin, and tefluthrin) have
MOEs that reflect assumed dermal bioavailabil ities that are less than 100%. Those assumed dermal bioavailabilities are probab ly taken from (non-specified) st udies 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 Pesticid e Programs decided to use transfer co efficients derived from JazzerciseTM 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 Pr ocedures for Residential Expo sure 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 . 20:349-360.10 surrogates for their relevance to th e field conditions to be tested w ithin this protocol to assure that the MOEs are indeed adequate.

4. Statistical and sample design issues The chosen sampling design that involves st ratifying 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 justif ication of the selection of monitoring areas is reasonable, given practical and cost constraints.

The limited randomness in the selection of monito ring units that would likely be achieved by the recruitment process as described in the documen ts was discussed at some length. Although the Board felt the proposed approach was acceptable gi ven 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 an d in particular would identify t hose, if any, that might need to be recruited by “traditional recr uitment” in order to provide an after-the-fact (future) indicator
of the randomness of the MU selection process.

The objective of creating a databa se of exposures over a range of exposure scenarios may be achieved with the proposed sampling design for th ree types of agricultu ral pesticides, viz.: herbicides, insecticides, and fungicides. The design attempts to take into account climate/environment (7 MAs), usage (11 crops), pe sticides (10), and amo unt 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 diffe rent AI range. Having at least one MU in every one of these combinations clearly is no t possible with the chosen sample size.

It has been stated that no data currently exist for this partic ular exposure issue (Collier, 2014, p. 11; Evans and Sherman, 2014, p. 3). Each MU in th e study is expected to perform a variety of tasks, but it is not clear if all or some or onl y one the 10 pesticides will be handled during the monitoring. Since the goal is to ha ve each MU in an area have a di fferent 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 w ho would be exposed to a particular scenario cannot be addressed using only th e information from this study.

Implementation of this protocol will provide qua ntitative information on a variety of exposure situations related to open pour or granule formulations. While stat istical 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 applicab le requirements of 40 CFR part 26, subparts
K and L?11 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 a ssessment 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) ri sk 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, th e protocol states that this ri sk may be increased due to the fact that the subjects must wear an extra layer of clothi ng (long underwear (or WBD) for exposure monitoring during the manual pouring procedure). Likely, the more significant
factors for heat-related illne ss would be temperature, humidity and level of activity of the subject. In other words, the addition of a si ngle layer of clothing may not be significant in
the overall risk of heat-related illness. Therefore, the statem ent that the use of the extra clothing constitutes greater than minimal risk in this study might be an overstatement.

To minimize risk of heat-rela ted illness, the protocol a ppropriately outlines steps for monitoring temperature and if n ecessary, using cycles of work and rest, moving the worker to a cooler environment, or conducting the te sting during early morn ing or night when temperatures are lower. Given that the subjec ts will be individuals w ho are familiar with the manual pouring procedures and will be in good health, the risk s are reasonable.

b. The risk of scripted activities is likely to be negligible, since it i nvolves reducing the load
size of the material to achieve th ree 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 out side 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 it self could, in theory, constitute greater than
minimal risk. The standard of minimal risk re lates to the probability and magnitude of harm12 experienced in daily life 3 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 da ily basis. In other words, the fact that agricultu ral workers are exposed to pesticid es 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 risk s are those associated with acute toxicity, rather than chr onic exposure. Table 1 of the protocol shows the dermal MOE for each surrogate (Collier, 2014, p. 66). The cal culated MOEs meet or exceed the required minimum MOE in each case. Therefore, the ri sks are considered acceptable according to EPA standards for exposure to organophospha te pesticides (Collier, 2014, pp. 65-66). Whether or not the overall study constitutes minima l risk, or greater than minimal risk, is difficult to determine, but the risks are reason ably low and are well managed as described in the protocol.

Workers engaged in the pouring procedures fo r 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 par ticipation in the study after thei r 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 wit hout their supervisors bei ng present. Employers will sign consent documents stating that they wi ll not influence or coerce their employees to participate in the study. It is not clear whethe r employees might be prohibited or discouraged from joining the study if employers did not feel it was in their (the employe r’s) interest to allow participation. Pregnant and nursing women will be excluded. Some workers in agricultural settings have low levels of lite racy; these individuals will not be excluded from participation.

3. Informed consent

Informed consent will be conducte d 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.I.3 (Collier, date, pp. 261-266). These inform ed 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(i); it is available at http://www.gpo.gov/fdsys/granule/CFR-1999 -title40-vol1/CFR-1999-title40-vol1-sec26-

102/content-detail.html13 4. Privacy and confidentiality

The main privacy issues relate to possible pho tographs or videos dur ing the study activities, changing of clothing in the presence of research ers, and pregnancy testing. These risks are appropriately managed as described in secti on 2.3.3 of the protocol (Collier, 2014, pp. 64).

5. Monitoring of data

The protocol describes monitoring for heat-relate d illness in Section 2.3. 1 (Collier, 2014, p. 62).
Workers will be observed and reminded to use a ppropriate 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 secti on 2.3.5 (Collier, 2014, pp. 65-67).

6. Vulnerable or disadvantaged subjects

Given that some agricultural workers are economi cally disadvantaged, or may have low literacy levels, these individuals are potenti ally vulnerable. It is acceptabl e to enroll these subjects, given that the study involves low levels of risk, participants are appr opriately compensated for their time, and consent procedures requ ire 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 n eeded. For other workers who are not fluent in English, or who do not read English and are no t 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 mi nimize risk are reasonable. The social value of conducting the research is signi ficant and the overall risk/ben efit ratio is acceptable.

A published report by Frampton et al. (2002) of intentional hum an exposures to nitrogen dioxide and their effects on airway and blood cells 4 5

Overview of the Study

This standard, three-period cross-over inhalati on toxicity study was conducted to assess the “effects of nitrogen dioxide (NO 2 ) on airway inflammation, blood lymphocyte recruitment to the lung, and susceptibility of airway cells to infection with influe nza 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 popula tion of students and community members around the University of Rochester. Elig ible 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 respirat ory diseases, and were free of respiratory
infections for at least six weeks. They were exposed thre e times (to air and to 0.6 ppm and 0.15 ppm NO 2 ) for three hours each, during which they ex ercised intermittently several times. The

4 Chair’s note: Dr. Gary Chadwick recused himself fro m Board decisions and votes related to this study.

5 Following this meeting, the EPA decided to not rely on this study.14 exposure levels typified those found in indoor environments with unvented combustion sources (Frampton et al., 2002, p. L155). The three exposure days were separated by at le ast 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 de termine the viability of respiratory and epithelial cells in the presence of influenza and RSV strains.

Analysis of variance (ANOVA) methods were us ed to evaluate the effects related to NO 2 exposures. Period, carryover, treatment effect an d gender differences were examined. Residuals
were assessed to determine whether the a ssumption of normality had been violated.

Science

Charge to the Board

 Is this study scientifically s ound, providing reliable data?

 If so, is this study adequate for quantita tive 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 laborat ory analysis parts of this study are scientifically sound. However, the chamber exposure data are not re liable without more information regarding the actual exposure leve ls to nitrogen dioxide.

 The statistical analysis is adequate to jus tify 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 expos ure 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 de tailed 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 f our concerns related to the operation of the exposure chamber that greatly weaken the Board’ s confidence in the re liability of the study’s
data and two concerns about st atistical methods (two concer ns) that weaken the study’s reliability and constrain its potential power.

1. Exposure chamber

a. In regard to the chamber, on p. L156 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%15 (mean ± SD), respectively.” This temperatur e (corresponding to 98.8°F) is outside of the normal comfort range and comprises a potenti al heat stress setti ng, especially when combined with an exercise regime “suffici ent 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 with in the paper (or its associated consent information).
b. The authors state “The capabilities for gene rating and maintaining pollutant levels and constant temperature and humidity have b een described previously (48)” (Frampton et al. , 2002, p. L156). This citation (ref. 48) is pr obably 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 designe d 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 hum idities [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 wa s found within the paper.

c. Two of the Frampton et al. (2002) claims regarding NO 2 on page L156 are without any supporting methods, data, or refe rences but are virtually word-for-word from the aerosols reported in reference 49 (Utell et al ., 1984, p. 220). The first clai m, “This [referring to ~0.3 atmospheric changes/min] enabled NO 2 levels to reach >90% of target levels within 4 min” (Frampton et al ., 2002, p. L156), is technically inco rrect. Comments elsewhere in reference 49 read, “A volumetric control is incorporated into the chamber air supply to
stabilize the air flow at 10 m 3 /min” (the same flow rate as in Frampton et al ., 2002) and “the effective chamber volume (less instrume ntation furniture, etc.) is approximately 40

m 3 ” (both quotes are from Utell et al ., 1984, p. 220). Thus, the ideal (best case) air exchange time is 40 m 3 divided by 10 m 3 /min 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 NO 2 ) to reach >90% of target le vels (derived from: -ln(1 –
C/C target ) = -ln(1 – 0.9) = -ln(0.1) = 2.3). Less th an 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 b een 10 to 15 minutes. Because the authors do not describe the startup seque nce of the exposure chamber, the Board could not determine how long in advance NO 2 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, Robe rts 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. J Aerosol 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 oxidativ e potential and lim ited solubility, NO 2 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 th an 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.16 However, even a 15-minute transient is a sm all fraction of subject s’ three-hour exposure periods.
d. Another claim is potentially of much greater concern. In their Methods section, the
authors state that "The concentrations of NO 2 at the 3- and 6-ft levels within the chamber varied by no more than 5% of the mean" (Frampton et al ., p. L156). Furthermore, in the Results, the authors claim that “Actual achieved NO 2 concentrations were 0.61 ± 0.02 and 1.50 ± 0.02 (SD) ppm” (Frampton et al ., 2002, p. L157). However, no method of measuring the NO 2 was mentioned; measuring NO 2 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 thes e 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 (3 x 3 matrix) within the chamber has revealed that the aerosol conc entrations 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 base d 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 genera ting and maintaining pollutant levels,” and the lack of a method to measure NO 2 imply that the authors of Frampton et al. may have based their exposure values on the rate at which the 5000 ppm NO 2 compressed gas was emitted into the ve nturi mixer and diluted into the 10 m 3 /min air supplied to the exposure chamber; i.e. , the reported concentrations of NO 2 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 NO 2 is fairly reactive, it is at least possible - if not likely - that some of the supplied NO 2 was lost en route to (or within) the exposure chamber. If the test concentrations were calculated rather than measured, they were pr obably not as high as expected and reported. This uncertainty makes the exposure si de of this study seem unreliable.

Although there is one author (U tell) common to both Frampton et al . (2002) and reference 49 (Utell et al ., 1984), the four concerns noted a bove suggest that the authors of the former paper may have had a limited understanding of the expos ure chamber itself.

2. Statistical issues The statistical design and analysis procedures are clearly stated on page L157 of Frampton et al. (2002). The study’s three-period cr oss-over design with washout periods was commonly used in this type of situation. Based on a document containing background materi al, an undescribed prestudy power analysis apparently indicated that four subjects for each of the six treatment sequences would provide adequate power (S herman, 2014, p. 45). Additional subjects were recruited to allow for dropouts but data from only 21 subjects ar e 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 strength en the acceptability of the numerical results.17 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 ANO VA” (p. L157). Deleti on 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 wh ich 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 conseque nce, in the Results, parts or all of Figures 1 through 6 do not accurately reflect the numerical results of the stat istical analysis (pp. L159-L161). Moreover, the extent of the in accuracies cannot be de termined; Figure 4A (p. L160) is a good example of this issue. Th e authors indicate that the only significant
effect is the NO 2 main effect. However, the figure shows a very large difference between male and female responses at 1.5 ppm which wo uld be indicative of a potential gender ×
NO 2 level interaction. At best, th is leads to potential confus ion 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 test s the authors used were adequate to support their conclusions that pulmonary responses were obs erved. However, in some cases the paper could have used more powerful statistical tests; these might have detected other significant responses that were not rec ognized. 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 NO 2 , but the only test mentioned wa s Student’s t-test for total NO 2 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 si gnificant if the results had been analyzed using paired ttests. Another statistical op tion 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 identifi ed nor from their conclusions, but the limitations to these tests imply that the authors may not have identified a ll of the statistically si gnificant responses to these exposures that may be of interest to the Agency.

Ethics

Charge to the Board

 Does the study meet the applicable requi rements of 40 CFR part 26 subpart Q?18 Board Response to the Charge

HSRB Recommendation

 The Board concluded that th e 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 as sessment 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 medica l study intended to test the effects of nitrogen dioxide (NO 2 ) 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 NO 2 as an environmental pollutant, but EPA identified the work as a potential source of data for evaluating NO 2 as a sterilant of medical equipment.

In the Methods and Study Design s ection of the article, the inves tigators report that the study was approved by the University of Rochester’s Rese arch Subjects Review Board, the university’s institutional review board (IRB) for protecti on of human subjects in research (Frampton et al., 2002, pp. L156). At that time the IRB would have been operating under regulatory standards of
the 1996 version of 45 CFR 46 8 and the ethical guidance of the Belmont Report 9

. 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. L156). 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 Un iversity of Rochester’s IRB provided electronic copies of available records on this study, wh ich included the study’s protocol and consent document, and the IRB’s approval letter. It may be assumed that the I RB 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 co nduct of the research was fundamentally unethical. Ther e is no evidence that 1) the research was conducted in a way that placed participants at increased risk of ha rm (based on the knowledge available at the time the study was conducted) or 2) vulnerable popul ations were targeted. However, because

8 Protection of Human Subjects in the U.S. Code of Fede ral Regulations, 45 CFR part 46. For a timeline of these federal regulations, go to http://history.nih.gov/about/timelines_laws_human.html

9 Available at http://www.hhs.gov/ohrp/humansubjects/guidance/belmont.html19 recruitment processes focused on the Universi ty community where the study was conducted, and the protocol does not address ad ditional protections for stude nts or employees, there is a possibility that some participants’ ability to gi ve informed consent may have been impaired by
coercion.

1. Assessment of risks and benefits

The article reports that many Amer icans are exposed to levels of NO 2 as an indoor air pollutant resulting from combustion, such as when natural gas-fueled ice resurfacing machines are used in enclosed ice rinks (Frampton et al ., 202, p. L155). The primary benefit of the study was to society, by generating knowledge of NO 2 ’s health effects. The highest concentration studied was
a level stated in the consent form to be a le vel observed in homes wh ere gas stoves are used. Risks of exposure to NO 2 are not clearly spelled out in the consent document, although the shortterm risks of the bronchoscopy are identified. Neither the article nor the consent form addresses any potential benefit to particip ants or the balance of risks and benefits. The University of Rochester IRB’s approval of this study can be in terpreted as its assessment that the risks to participants did not outweigh th e 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 target ed, it is not possible to determine whether selection of study participants was equitable or whether i ndividual members of vulnerable populations were enrolled in th e 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 in creased exposure to NO 2 , 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 ha ve been disproportionate or coercive.

3. Voluntary and informed cons ent of all participants

Participants were recruited from among members of the Universi ty of Rochester community in Rochester, New York, including students and em ployees of the University. Although vulnerable populations were not targeted fo r the study, it is not possible to tell whether participants from potentially vulnerable populati ons were recruited. Similarly, there is no indication that the study protocol included mechanisms designed to mini mize coercive recruitment and enrollment of students and employees who may have been in a subordinate position to the researchers.

To participate in the study, subj ects were required to be heal thy, non-pregnant adults with no history of smoking. No children were enrolled. After an initi al conversation with the study coordinator by phone, potential participants met w ith one of the investigators to discuss the study. Potential particip ants 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 doc uments, and documents from the IRB call for20 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.21 References

U.S. Department of Health, Educ ation and Welfare (DHEW) (1979). The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research. Washington, DC: U.S. DHEW, The Nationa l Commission for the Protection of Human Subjects of Biomedical and Behavi oral Research. Retrieved from http://www.hhs.gov/ohrp/humansubj ects/guidance/belmont.html

U.S. Department of Health and Huma n 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/gui dance/45cfr46.html (2009 version)

Collier, R. H. (2014, July 25). Determination of Dermal and I nhalation Exposures to Workers During Open Pour Loading of Granules, AHETF Study No. AHE170. Agricultural Handler Exposure Task Force, LLC.

Evans, J., & Sherman, K. (2014). Science and Ethics Review of AHETF Scenario Design and Protocol (AHE170) for Exposure Monitoring of Workers during Open Pour Loading of

Granules. Washington, DC: U.S. Environmental Protection Agency, Office of Pesticide Programs.

Fenske, R., & Lu, C. (1994). Determination of handwash removal effi ciency: incomplete removal of the pesticide chlorpyrifos fr om skin by standard handwash techniques. American Industrial Hygiene Association Journal, 55 (5), 425-432.

Fenske, R., Schulter, C., Lu, C., & Allen, E. (199 8). Incomplete removal of the pesticide captan from skin by standard handwash exposure assessment procedures. Bulletin of Environmental Contamination and Toxicolocy, 61 (2), 194-201.

Frampton, M. W., Boscia, J., Roberts, M. J., et al. (2002, January). Nitr ogen 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 Protecti on Agency, Office of Chemical Safety and

Pollution Prevention.

Sherman, K. (2014). Ethics Review of Human Toxici ty Study with Nitrogen Dioxide. Washington, DC: U.S. Environmental Protec tion 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 Pr inting Office. Retrieved December 9,22 2014, from http://www.gpo.gov/fdsys/pkg/CFR- 2010-title40-vol1/xml/CFR-2010-title40- vol1-part26.xml

U.S. Environmental Protection Agency (EPA ). (2014, October 17). Human Studies Review Board; Notification of a Public Meeting. 79 FR [Federal Register] , pp. 62437-62439.
Retrieved November 2, 2014, from https://www.federalregiste r.gov/articles/2014/10/17/2014- 24757/human-studies-reviewboard-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 pollutant gases and aerosols in hum an subjects: design considerations. Journal of Aerosol
Science, 15 , 219-221.