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Page 1 of 30 EPA

- HSRB

- 11

- 02 Paul Anastas, PhD EPA Science Advisor Office of the Science Advisor 1200 Pennsylvania Avenue, NW Washington, DC 20460 Subject: April 13

- 14, 2011 EPA Human Studies Review Board Meeting Report Dear Dr. Anastas, The United States Environm ental Protection Agency (EPA or Agency) requested that the Human Studies Review Board (HSRB) provide scientific and ethics reviews of several co mpleted studies and scenarios involving intentional exposure of human subjects to pesticides or other EPA regulated compounds. The first of these scenarios, a set of three interrelated studies measuring dermal and inh alation exposure of professional agricultural workers who spray pesticides using open

- cab airblast equipment, was conducted by the Agricultural Handl er Exposure Task Force, LLC (AHETF). These studies (AHE62, AHE63 and AHE64) were conducted after publication of the EPA’s e xpanded final rule for protection of subjects in human research (40 CFR 26) on February 6, 2006 (71 Federal Register 24, 6137). These studies were conducted under a single scenario monograph (MRID 48326701), which incorporates the data from these three studies into a single dataset. An additional set of data, collected as part of a third

- party sponsored protocol (AHE07) conducted prior to publication of the EPA’s expanded final rule for protection of subjects in human research, will also be included in this data set. This data set will be posted to the Agricultural Handlers Exposure Database (AHED®), and used genericall y to estimate dail y dermal and inhalation exposures of workers who treat agricultural crops with conventional pesticides using open

- cab airblast equipment. In addition, the Board reviewed a completed study of dermal and inhalation exposure of professional janitorial worke rs who clean indoor surfaces with an antimicrobial pesticide product using read y - to
- use wipes or a trigger spray bottle and wipe, conducted by the Antimicrobial E xposure Assessment Ta sk Force II (AEA TF II). This study

(AEA

- 02) was also conducted after pub lication of the EP

A’ s expanded final rule for protection of subjects in human research. The data will be posted to the Biocide Handlers Exposure Database (BHED®), and used genericall y to estimate daily

dermal and inhalation exposures of those who wipe indo or surfaces with ant imicrobial pesticides. The Board also reviewed a published study involving intentional human exposure to the zinc oxide contained in sunscreens. The Agency proposes to rel y on this study, conducted after to publication of the EPA’s e xpanded final rule for protection of subjects in human research, for regulatory

actions.Final Draft Document Dated May 20, 201 1

Page 2 of 30 Finally , the Board considered additional sponsor

- provided information regarding the Kisicki et al . (1999) study

on chlorpy

rifos, which was originall y discussed b y th e HSRB in June 2009 (EPA HSRB 2009). As the review and discussion of this additional information was co nducted independently

rather than at the request of the Agency , the Board’s conclusions are attached to this meeting report as Appendix 1. The enclose d report provides the Board’s response to EPA charge questions presented at the April 13

- 14, 2011 meeting. Assessment of Completed AHETF Research Studies AHE62, AHE63 and AHE64: Dete rmination of Dermal and Inhalation Exposure to Workers During Airblast A pplications of Liquid Sprays Using Open Cab Equipment (MRID 48289611, 48289612, 48289613, 48289614, 48289615, 48289616 and 48326701) . Science The Board concluded that the research reported in the completed monograph, associated field study

reports, and associated supplemental documents was conducted in a manner that was reasonabl y faithful to the design and objectives of the protocol and governing documents of the AHETF. The Board also concluded that the Agency has adequatel y, but not completely, cons idered the limitations on these data that should be considered when using the data in estimating the dermal and inhalation exposure of those who apply conventional pesticides with open

- cab airblast equipment. Additional limitations and concerns have been i dentified by the Board, and the conclusion as to the generalizability of these data requires further consideration and anal ys is. Ethics The Board concluded that the study was conducted in substantial compliance with subparts K and L 40 CFR 26. Assessmen t of Completed AEATF II Research Study AEA02: Measurement of Potential Dermal and Inhalation Exposure during Application of a Liquid Antimicrobial Pesticide Product Using Trigger Spray and Wipe or Ready - to - Use Wipes for Cleaning Indoor Su rfaces (MRID 48375 601). Science The Board concluded that the research reported in the completed monograph, associated field study

reports, and associated supplemental documents was conducted in a manner that was reasonabl y faithful to the design and objectives of the pro tocol and governing documents of the AEATF II.

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Page 3 of 30 The Board also concluded that the Agency has adequatel y, but not completely, cons idered the limitations on these data that should be considered when using the data in estimating dermal and inhalation exposures of handlers who are appl yi ng antimicrobial pesticides using a trigger spray

or ready

- to
- use wipes. Ethics The Board concluded that the study was conducted in substantial compliance with subparts K and L 40 CFR 26.
Assessment of Published Research S tudy MRID 48387301: Gulson et al. (2010) Small Am ounts of Zinc from Zinc Oxide Particles in Sunscreens Applied Outdoors Are Absorbed through Human Skin. Science The Board concurred with the Agency ’s assessment that the Gulson et al. (2010) study provide s some potentially useful data on the dermal absorption of zinc from zinc oxide nanoparticles in sunscreen applied to human skin, despite the multiple limitations identified by the HSRB. However, the Board advised the Agency to proceed with caution when us ing these data in risk assessment, as these data cannot be used as a stand

- alone set to assess dermal absorption of zinc oxide. Ethics The Board concluded that there was insufficient information available at the time of the April meeting to determine wh ether the Gulson et al . (2010) study was conducted in substantial compliance with procedures at least as protective as those in subparts A

- L of EPA’s regulation at 40 CFR Part 26. The Board recommended that EPA seek additional information from Macquarie University

or the study

investigator (including a copy of the research protocol and unsigned informed consent form) and that information be provided to the HSRB for re

- consideration. Sincerel y,

Sean Philpott, PhD, MSBioethic s Chair EPA Human Studies Review BoardFinal Draft Document Dated May 20, 201 1

Page 4 of 30 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 nece ssarily represent the view and policies of the Environmental Protection Agency , nor of other age ncies 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 carefull y considered all information provided and presented b y the Agency presenters, as well as information presented b y public commenters. This document addresses the information provided and presented within the structure of the charge b y the Agency

.

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Page 5 of 30 US ENVI RONMENTAL PROTECTION AGENCY HUMAN STUDIES REVIEW BOARD Chair Sean Philpott, PhD, MSBioethics, Director for Research Ethics, The Bioethics Program of Union Graduate College and the Mount Sinai School of Medicine, Schenectady, NY Vice Chair Janice Chambe rs, PhD, DABT, Fellow ATS, William L . Giles Distinguished Professor, Director, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University , Mississippi State, MS Members George Fernandez, PhD, Professor of Appli ed Statistics, Director of the University of Nevada

Reno Center for Research Design and Anal ys is, University of Nevada

- Reno, Reno, NV Vanessa Northington Gamble, MD, PhD, University Professor of Medical Humanities, Professor of Health Policy

and American Studies, The George Washington University , Washington, DC Sidney Green, Jr., PhD, Fellow of the ATS, Professor, Department of Pharmacology , Howard University

College of Medicine, Washington, DC Dallas E. Johnson, PhD, Professor Emeritus, Department of St atistics, Kansas State University , Manhattan, KS Michael D. Lebowitz, PhD, FCCP, Professor (NTE) of Medicine

- Research/Scholar , University of Arizona, Tucson, AZ Jose E. Manautou, PhD, Associate Professor of Toxicology , Department of Pharmaceutical Scien ce, University

of Connecticut School of Pharmacy , Storrs, CT Jerry A. Menikoff, MD, JD, Director, Office for Human Subjects Research, Office of the Secretary , Department of Health and Human Services, Rockville, MD *Rebecca Parkin, PhD, MPH, Professorial Lecturer (EOH) Human Risk Assessment, School of Public Health and Human Services, The George Washington University , Washington, DC William Popendorf, PhD, MPH, Professor Emeritus, Department of Biology, Utah State Unive rsity , L ogan, UT Virginia Ashby

S harpe, PhD, Medical Ethicist, National Center for Ethics in Health Care, Vete rans Health Administration, Washington, DCFinal Draft Document Dated May 20, 201 1

Page 6 of 30 Linda J. Young, PhD, Professor, Department of Statistics, Institute of Food and Agricultural Sciences, University

of Florida, Gainesvil le, FL Human Studies Review Board Staff Jim Downing, Executive Director, Human Studies Review Board Staff, Office of the Science Advisor, United States Environmental Protection Agency , Washington, DC * Not in attendance at the April 13

- 14, 2011 Public Meeting

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Page 7 of 30 INTRODUCTION On April 13

- 14, 2011, the United States Environmental Protection Agency ’s (EPA or Agency) Human Studies Review Board (HSRB) met to address scientific and ethical issues co ncerning two completed study monographs. One study

measure d levels of dermal and inhalation exposure received b y janitorial workers who apply antimicrobial pesticides using trigger spray or read y - to
- use wipes. The second was comprised of three completed, interrelated studies measuring levels of dermal and inhalat ion exposure received by pesticide applicators who spray pest icides with open

- cab airblast equipment. These studies, along with a fourth study conducted prior to publication of the EPA’s expanded final rule for protection of subjects in human research, wer e compiled into a single scenario monograph (MRID 48326701), which incorporates the data from these studies into a single data set. In accordance with 40 CFR 26.1602, EPA sought HSRB review of these two completed study monographs. The completed studies are discussed more fu lly below. In addition, the Agency

sought HSRB review of one published study of dermal absor

ption of zinc (Zn) from zinc oxide (ZnO) nanoparticles in sunscreen applied to human skin. This study , conducted after publication of the EPA’s expanded final rule for protection of subjects in human research, was identified by Agency scientists from the peer reviewed literature. This study , which the Agency

proposes to rel y upon for regulatory actions, is discussed in detail b elow. Finally , the HSRB considered additional sponsor

- provided information regarding a pre

Rule intentional exposure study of chlorpy

rifos, originall y discussed b y the Board in June 2009. The Board’s review and discussion of this additional information was conducted indepen dently rather than at the request of the EPA. The Board’s discussion and conclusions are attached to this meeting report as Appendix 1. REVIEW PROCESS On April 13

- 14, 2011, the Board conducted a public face

- to
- face meeting in Arlington, Virginia. Advance notice of the meeting was published in the Federal Register as “Human Studies Review Board; Notice of Public Meeting” (76 Federal Register 59, 17122). Following welcoming remarks from Agency officials, the Board heard presentations from EPA on the follo wing topics: three completed studies (compiled into a single monograph) me asuring dermal and inhalation exposure received b y pesticide applicators who spray pesticides with open

- cab airblast equipment, one completed study measuring levels of exposure recei ved b y janitorial workers when applying antimicrobial pesticides using a trigger spray or ready

- to
- use wipes, and one published study measuring dermal absorption of zinc (Zn) from zinc oxide (ZnO) nanoparticles in sunscreen applied to human skin. The Board also asked clarifying questions of several study sponsors and/or research i nvestigators, including:Final Draft Document Dated May 20, 201 1

Page 8 of 30 Ms. Megan Boatwright, Analy tical Coordinator, Golden Pacific Laboratories Dr. Victor Cañez, Technical Chair, Agricultural Handler Exposure Task Force, L LC (AHETF). Dr. Richard Collier, Administrative Committee Chair, AHETF. Dr. Has Shah, Manager, Antimicrobial Exposure Assessment T ask Force II (AEA

TF II). Mr. Robert Testman, V

ice

- President, Golden Pacific Laboratories. Public oral comments were provided by : Dr. Victor Cañez, Technical Chair, AHETF. Dr. Richard Collier, Administrative Committee Chair, AHETF. Dr. Has Shah, Manager, AEA TF II. One written public comment was submitted by Ms. Carol Dansereau, Executive Director of the Farm Worker Pesticid e Project, Seattle, WA. For their deliberations, the Board considered the materials presented at the meeting, oral comments, and Agency

background documents (e.g., published literature, sponsor and investig ator research reports, study protocols, data eva luation records, and Agency science and ethics r eviews of proposed protocols and completed studies). A comprehensive list of background doc uments is available online at http://www.regulations.gov

. CHARGE TO TH E BOARD AND BOARD RESPONSE Assessment of Completed AHETF Research Studies AHE62, AHE63 and AHE64: Dete rmination of Dermal and Inhalation Exposure to Workers During Airblast Applications of Liquid Sprays Using Open Cab Equipment . Overview of the Study T hree separate field studies were conducted, each monitoring dermal and inhalation e xposure of workers to comme r cially available pesticides while spraying tree or trellis crops in three different U.S. states where open

- cab airblast equipment is commonly use d in production agriculture. A total of 13 professional agricultural handlers were monitored as they applied pest icides using open

- cab airblast equipment: three adult men applying pesticides to grape vines in California (AHE62), five adult men applying pes ticides to grape vines in New York (AHE63), and five adult men appl yi ng pesticides to pecan trees in Oklahoma (AHE64). The scenario d esign, protocols for the three studies, SOPs and governing documents were reviewed favorably b y the HSRB at its October 21

- 22, 2008 (EPA HSRB 2008b). Monitored on actual day s of work, study participants handled from 7 to 90 lbs of active ingredient (carbaryl or malathion), spraying 3 to 24 acres in 1.4 to 10.6 hours. Dermal exposure was measured using hand washes, face/neck wipes, and whole bod y dosimeters (100% cotton union suits) for the remainder of the body (torso, arms, and legs). Inhalation exposure was mea sured using personal air sampling pumps and OVS mounted on the shirt collar. Results represent

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Page 9 of 30 dermal exposure with and without chemical

- resistant hats while wearing a long

- sleeved shirt, pants, shoes/socks and chemical

- resistant gloves, and inhalation exposure without respiratory protection. The Agency

proposes to use data from these three studies, along with a prev ious open

cab airblast stud y involving 15 male participants applying pesticides to apple, orange, peach and pear trees in Florida, Georgia and Idaho (AHE07; Smith 2005), to estimate generically daily dermal and inhalation exposures of workers who treat agricultural crops with conventional pest icides using open

- cab airblast equipment. Science Charge(s) to the Board 1. Was the research reported in the AHETF completed monograph report and associated field study

reports faithful to the design and objective s of the protocol, SOPs and governing do cuments? 2. Has EPA adequatel y characterized, from a scientific perspective, the limitations on these data that should be considered when using the data in estimating exposure of those who apply pesticides with op en - cab airblast equipment? Board Response to the Charge(s) HSRB Recommendation The Board concluded that the research reported in the completed monograph, associated field study

reports, and associated supplemental documents was conducted in a manner that was reasonabl y faithful to the design and objectives of the protocol and governing documents of the AHETF. The Board also concluded that the Agency has adequatel y, but not completely, cons idered the limitations on these data that should be considere d when using the data in estimating the dermal and inhalation exposure of those who apply conventional pesticides with open

- cab airblast equipment. Additional limitations and concerns have been identified by the Board, and the conclusion as to the generali zability of these data requires further consideration and anal ys is. HSRB Detailed Recommendations and Rationale The HSRB concluded that the research reported in the completed monograph report and the associated field study reports of Open Cab Airblast Applicator Exposures (Bruce 2010a; Bruce 2010b; Bruce 2010c; Klonne and Holden 2010, Smith 2005) was faithful to the design and objectives of the protocol, SOPs, and governing documents. Four limitations were suggested as potential additions to those outli ned in the Agency’s Reviews of the monograph and associated field study

reports (Crowley

2011; Crowley

and Sarkar 2011). The magnitude of one of theseFinal Draft Document Dated May 20, 201 1

Page 10 of 30 limitations cannot be determined but could significantly impact the accuracy

of the measured exposures i n handlers who are not wearing a chemical resistant (CR) hat. The AHETF seems to have responded well to most of the concerns expressed by the HSRB during their prior reviews of the protocol (EPA HSRB 2006; EPA HSRB 2008b). For instance, the use of an in formal survey /poll of local experts that identified the absence of the “Kinkelder” airblast spray er, the use of a refined and better defined recruitment process, and the inclusion of descriptive records of applicator behavior. In the end, the analy tical ch emistry

m ethods seemed appropriate and well chosen, the quality assurance (QA) considerations appeared to be in place, and the desired study conditions were largely

obtained. Although no formal statistical test for differences among crops or clusters was provided, the visual evidence in the Science Review (Figure 1; Crowley 2011, 12) is strong support for their similarity . The Board supports the Agency ’s plan to combine the datasets and to use a mixed

- model approach for statistical anal ys is to account fo r potential data clustering for both the existing data and new monitoring. The Board also agrees with a similar approach and conclusion regarding the negligible effect of canopies used by six of the 28 monitoring units (MUs). The use of head patches ins ide and outside CR hats was innovative. Its use obviated the need for an MEA for the portion of the head covered by the hat, probabl y increased the accuracy of measuring exposure with a hat, and yi elded potentially useful estimates of the protection from exposure that the hat provided. At the same time, the use of this technique presents two limit ations in estimating exposures without such a hat. First, it could be argued that when estimating exposure without such a CR hat that the amount reaching the i nner patch should not be counted. This argument would apply if the pathway by which the active ingredient that was deposited onto the inner patch was via a micro

- scale form of drift under the hatband (“penetration”) and not directly through the hat’s mate rial (“pe rmeation”). The effect on the overall dermal exposure of this sort

- of double counting the patch data is insignificantl y small (on the order of 0.1%) due to the very small deposition measured on the inner patch. Second, the use of this innovatio n may have reduced the accuracy of measuring exposure without a hat because the presence of a wide

- brimmed hat probabl y also reduced deposition onto the exposed face and neck from what it would have been had the hat not been there at all. The magnitude of this reduction is undetermined and seems undeterminable with the information available. The brim is likely to have acted somewhat like a canopy , only much closer. Its effect is likely to have been greater on the portions of the face and neck closest to the underside of the hatband and to decrease on the portions further away , such as the tip of the nose. The effect of the brim’s protection is probably clear but not overwhelming. The Agency

might reconsider appl yi ng a 1/2 limit of quantitation (LOQ) fac tor when no active ingredient is found in the backup portion of the OVS air samples. A proper sample should have no airborne chemical in that portion. In fact, finding active ingredient there implies some degree of overloading of the front portion of the sampling media. Consistently assigning a non

-

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Page 11 of 30 detect measurement in the backup portion of a value of 1/2 limit of detection (LOD) results in a biased result. Four additional limitations were suggested. The first is to include such qualitative data as the absence of the “Kinkelder” airblast spray er from the array of equipment included within the study . This spray er is an older model noted by the local experts to be prone to spray drift. The implications of higher drift on handler exposures depend on a combination of environmental conditions and work practices. However, the broader point is that such qualitative information could become a valuable resource to future users of the exposure database. The second limitation is the potential underestimati on of spray actually deposited onto the wiped portion of the subjects’ head due to shielding b y the brim of the CR hats that were a lway s worn. The third limitation is the possible exceptional status of one monitoring unit involved in exposure monitorin g for open

- cab spraying of Oklahoma pecans (Study AHE64; Monitoring Unit A5). This MU used nozzle pressure that was twice as high as that recommended for the ty pe of nozzle (and three to four times as high as reported by an y other MU using this ty pe of no zzle). The Board recognizes that i ncreasing nozzle pressure will cause a decrease in droplet size and an increase in spray drift. This MU also changed from spraying in both directions to spra ying in onl y one direction for the last third of his application time. This was the onl y MU for which such a comment was made. Two reasons were suggested b y the Board for wh y an appl icator might make such a change in spraying direction: either to navigate sloping terrain or/and to avoid windblown drift. No information is available regarding the terrain, but the initial one way pattern resulted in this MU spraying only when driving upwind. Both of these reasons could cause this MU’s exp o sures to be low. Whether these differences would otherwise disqualify this MU is unknown. The fourth limitation is the need to rely on model accuracy

for estimates to be interpret able. The primary

objective is to estimate the geometric mean, the arithmetic mean, and the 95 th percentile of normalized exposures (exposure per pound of a ctive ingredient handled [AaiH]) within 3

- fold with 95% confidence. The secondary objective is to assess proportionality (ln( exposure ) = ln(

AaiH ) ) against the alternative of independence in ln( exposure ) and ln( AaiH ) with 80% power. It is critical to car efully evaluate the validity

of the model to ensure that the mean of the normalized exposures on the log scale (and the geometric mean on the data scale) are interpretable. Because clusters are part of the study ’s design, a random cluster effect should be included in the model. How the presence of a cluster effect impacts interpretation should be considered carefull y. In the Agency ’s Review (Crowley and Sarkar 2011), an alternative approach was taken in which the normalized exposures were modeled using a lognormal distribution. The validity of this approach depends on two assumptions. First, the expectation of ln( exposure/AaiH ) must be linear. It is important to assess the validity of the linear relationship between ln( exposure ) and ln( AaiH ). Second, for the approach to be valid, there cannot be a cluster effect. Otherwise, the mean of the distribution would change with cluster so that a single lognormal distribution would not describe the normalized exposures. Perhaps this is why some of the Q

- Q plots fo r the lo g-Final Draft Document Dated May 20, 201 1

Page 12 of 30 normal distribution (Appendix B; Crowley and Sarkar 2011, 24

- 5) indicate some lack of fit, e speciall y in the tails. (It is not possible to be sure that these correspond to the inhalation data for which a significant cluster effect was present because the graphs are not labeled.) The Agency

would benefit from carefully considering all assumptions underlying a pr oposed statistical approach and then checking the validity of each. Care should be taken to ensure that the results can be meaningfull y i nterpreted. The term “arithmetic mean” should be changed to “mean,” especiall y when a model is used to estimate the population mean. It would be helpful for the axes of all graphs to be labeled. Lastl y, in the write

- up of results, implying that the failure to reject a null h ypothesis indicates that the null hypothesis is true, should be avoided. The Agency

should note that more detailed suggestions and analy ses may be provided by some Board members independent of this consensus Board report. Ethics Cha rge to the Board Does the available information support a determination that the studies were conducted in substantial compliance with subparts K and L of 40 CFR Part 26? Board Response to the Charge HSRB Recommendation The Board concurred with the Agency ’s assessment (Sherman 2011a) that the study was conducted in substantial compliance with subparts K and L 40 CFR 26. HSRB Detailed Recommendation and Rationale The documents provided include reports of each of three field studies conducted on b eha lf of the AHETF (Bruce 2010a; Bruce 2010b; Bruce 2010c). The submitted study documents state that the study

was conducted in substantial compliance with the ethical and regulatory standards of 40 CFR 26, Subparts K and L. The requirements of FIFRA §12(a)(2 )(P) and Title 3, § 6710 of the California Code of Regulations also apply. The three protocols were each reviewed and approved pursuant to the standards of the Common Rule (45 CFR Part 46, Subpart A) b y an independent human subjects review commi ttee, Ind ependent Institutional Review Board, Inc. (IIRB, Inc.) of Plantation, FL. Minutes of IIRB, Inc. meetings and a cop y of IIRB, Inc. policies and procedures were provided (Bruce 2010d; Bruce 2010e; Bruce 2010f; IIRB 2010a; IIRB 2010b; Sherman 2011a). IIRB, In c. is re gistered with the US Office for Human Research Protections (OHRP; Reg. # IORG0002954) and is fully accredited b y the Association for the Accreditation of Human Research Protection Programs, Inc. (AAHRPP). 1. The Board concurred with the conclusion s and factual observations relating to the study , as detailed in the EPA’s Ethics Review (Sherman 2011a) and summarized briefly below.

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Page 13 of 30 a. Prior HSRB and Agency Review. Because each of these three studies was initiated after April 7, 2006, prior submi ssion of the protocol and supporting materials to EPA was required b y 40 CFR §26.1125. The r equirements of 40 CFR §26.1125 for prior submission of the protocol to EPA and of §26.1601 for HSRB review of the protocol were satisfied. The scenario designs and study protocols were initially

approved b y IIRB, Inc. in July 2008. The HSRB discussed these three protocols at its October 2008 meeting, concurring with the Agency ’s assessment that these three proposed open cab airblast field study protocols, if revised as suggested by the Agency

and the HSRB, would meet the applicable requirements of 40 CFR Part 26, subparts K and L (EPA HSRB 2008b). b. Responsiveness to HSRB and Agency Recommendations. The initial ethics reviews by the Agency

(Carley and Evans 2008a ; Carley and Evans 2008b; Carley

and Evans 2008c) and by the HSRB (EPA HSRB 2008b) provided nine recommendations with regard to these three protocols. All of those recommendations, and the responses made with regard to them, are detailed in the submitted d ocuments (Sherman 2011, 25). The HSRB agreed with the Agency that the comments by

the Age ncy and HSRB were addressed satisfactoril y. 2. The Board concluded that this study , as conducted, met all applicable ethical requirements for research involving hum an participants, in accordance with the following criteria:
a. Acceptable risk

- benefit ratio. The risks to study

participants were minimized appropriatel y and were justified by the potential societal benefits, particularl y data on the dermal and inhalation exposure of pr ofessional pesticide applicators to the liquid pesticides they appl y to orchard and trellis crops using an airblast spray er drawn b y a vehicle with no cab. These data could be used to develop mechanisms to protect future persons who appl y these liquid pesticides. Minors and pregnant or lactating women were excluded from participation, with pre gnancy confirmed by

over - the

- counter pregnancy testing on the day

of study or by opt
- out. The potential of stigma resulting from study exclusion w as also appropriately minimized. Clear stopping rules and medical management procedures were in place, and no adverse events or other incidents of concern related to product exposure were reported. The study was designed to minimize the risks of exposure to the test compounds, subject to being able to accomplish the purposes of the study

. b. Voluntary

and informed consent of all participants.Final Draft Document Dated May 20, 201 1

Page 14 of 30 The study protocol included several mechanisms designed to minimize coercive recrui tment and enrollment. Mo netary compensation was not so high as to unduly influence participation. In addition, it is worth noting that twelve of the thirteen subjects were owner

- operators, and thus less likely

to be as vulnerable to coercion or undue influence as an employee mi ght be. 3. Eight minor protocol deviations were reported. In addition, there was one unreported but m inor protocol deviation. These are documented in detail in the EPA Ethics Review (Sherman 2011a, 8

- 10). The Board agrees with the Agency ’s assessment that these deviations did not compromise the informed consent process or put the study participants at increased risk. 4. The Board agrees with the observations in the EPA Ethics Review (Sherman 2011a) with r egard to heat index thresholds. In particular, in N ovember 2009―after this research had been conducted―the SOP for these ty pes of studies was revised to lower the acceptable heat index threshold from 120o F to 105o F. However, as noted in Table 4 of the EPA Ethics Review (Sherman 2011a, 15), the heat index values for the subjects enrolled in this study never e xceeded 105o F, so this was not an issue for this research. Assessment of Completed AEATF II Research Study AEA02: Measurement of Potential Dermal and Inhalation Exposure during Application of a Liqu id Antimicrobial Pesticide Product Using Trigger Spray and Wipe or Ready - to - Use Wipes for Cleaning Indoor Su rfaces. Overview of the Study The o bjective of this study was to measure individual exposures to a surrogate antim icrobial pesticide (didecyl dim ethyl ammonium chloride; DDAC) while cleaning surfaces with a trigger spray

and wipe or with ready

- to
- use wipes. A total of 36 volunteers participated in the study , 18 cleaning surfaces using a trigger spray and wipe and 18 clearing surfaces using ready to
- use wipes in one of three building t ypes (an office building, a Rite Aid pharmacy building, or a retired teacher ’s memorial building in Fresno, CA). Participants cleaned surfaces for one of six pre - determined wiping times (30

- 60, 60

- 90, 90

- 120, 120

- 150, 1 50

- 180 and 180

- 210 minutes t otal cleaning time, respectively). The study protocol, SOPs and governing documents were reviewed favorabl y b y the HSRB at its April 9

- 10, 2008 meeting (EPA HSRB 2008a). Dermal and inhalation exposure monitoring was conducted while study participants cleaned surfaces

. Dermal exposure was measured by inner and outer bod y dosimeters. Airborne concentrations of the surrogate compound were monitored in the participant’ s breathing zone u sing an OVS tube sample collector connected t o a personal sampling pump. Environmental conditions were also recorded at the time of monitoring, and observers made notes, photographs and videos of participant activity throughout the wiping period.

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Page 15 of 30 These exposure data will be used to populate a datab ase representing inhalation and de rmal exposure during a number of antimicrobial handler scenarios. A scenario is defined as a pe sticide handling task based on activity (e.g., application) and equipment t yp e (e.g., mop & bucket, read y - to
- use wipes, pressur e treatment of wood facilities, painting). These data will be used b y the Agency

to estimate dermal and inhalation exposures of antimicrobial handlers who are appl ying pesticides using a trigger spray and wipe or ready

- to
- use wipe under a variet y of scenarios. Science Charge(s) to the Board 1. Was the research reported in the AEATF II completed wipe study report faithful to the design and objectives of the protocol and governing documents of the AEATF II ? 2. Has EPA adequatel y characterized, from a scientific perspective, the limitations on these data that should be considered when using the data in estimating exposure of those who clean indoor surfaces with antimicrobial pesticides using a trigger

- bottle and wipes or ready

- to
use wipes? Board Respo nse to the Charge(s) HSRB Recommendation The Board concluded that the research reported in the completed monograph, associated field study

reports, and associated supplemental documents was conducted in a manner that was reasonabl y faithful to the desi gn and objectives of the protocol and governing documents of the AEATF II. The Board also concluded that the Agency has adequatel y, but not completely, cons idered the limitations on these data that should be considered when using the data in estimating dermal and inhalation exposures of antimicrobial handlers who are applying pesticides using a trigger spray

or ready

- to
- use wipes. HSRB Detailed Recommendations and Rationale The Board concurred with the EPA’s assessment (Leighton 2011) that the AEATF II made the appropriate changes to the protocol proposed by the EPA and HSRB and has executed the study in a way that is reasonabl y faithful to the design and objectives of the protocol and g overning documents of the AEATF II. Although several protocol dev iations occurred, they were unlikely to have adversely

affected the reliability

of the data. The Agency ’s analy sis of the data, especiall y their use of adjustments for non

- detects in the fortified samples (of which there were more than a few) was an improv ement. There was one potentially important shortcoming of the study . The calculated rates at which the active ingredient was applied to the treated surfaces did not achieve the stated target application rate of 7 mg of active ingredient per m2 of treate d surface. Only one of the partic i-Final Draft Document Dated May 20, 201 1

Page 16 of 30 pants using read y - to
- use wipes and onl y two of the participants using a trigger spray achieved the 7 mg/m 2 target. Neither the origin nor the significance of not meeting this target is known. While the protocol failed to achieve this technical target, this target was not recognized heret ofore to potentiall y be in conflict with achieving the important goal of letting the subjects “wipe surfaces as they

would normally

work” (Selim 2011, 43). By all appearances, the study did achieve the latter goal. The Board believes that the EPA has insufficiently characterized from a scientific per spective the limitations on these data. There were limitations that were not satisfactoril y a ddressed. As an example, there were too man y non - detects in the whole bod y dosimeters, which created the positive regression lines for the dermal exposures by anchoring at the low end. The assumptions of their modeling should be tested. Then, the appropriate regression models should be run on the results of the hand wipes (exposure by AaiH). If this is done, the effects of the non

detects can be evaluated in a step

- wise fashion. The results of the non

- parametric bootstrap had narrower confidence limits and should be used instead of the parametric boots trap results. Interpretations of the results should be re

evaluated, as they

are currently

too strong given the lack of any statistical significance. The lack of an y relationship of inhalation exposure to AaiH should be accepted. The results of evaluating the K

- factor, especiall y for inhalation, show the study to be underpowered to evaluate this ass ociation. Conclusions that estimates at the high end of AaiH are overestimates are definitely inco rrect in the threshold model, which shows values below the esti mated line at high AaiH and higher values in the middle of the range of AaiH that are elevating the curve. Two potential new limitations to the study were also identified. One is an outgrowth of a search to explain the result that the dermal unit exposure value for the ready

- to
- use wipe applic ations is twice as large as that for the trigger spray bottle. The difference in results may be due to the different way s in which the amount of active ingredient handled was calculated in the two studies, and this explanation has implications to the validity of extrapolations based on AaiH. The two methods treated about the same amount of surface area in the same amount of time and resulted in about the same exposure, but the amount of biocide handled b y the trigger spray bo ttle applicators was about twice that handled b y ready

- to
- use wipe applicators. The study was not designed to tell how much active ingredient (ai) actually reached the target surface nor if the two methods were equall y effective in treating the surfaces. Wiping has no intrinsic means to control the rate at which the active ingredient is applied. The rate depends completely on the applicator. The methods used to calculate the amount of ai handled may not directl y relate to the amount. Neither method accounts for the amount of ai that stay ed on the wipe or rag at the time it was discarded, and this unknown may have affected the calculated amount of ai handled. The poss ibility that the almost two

- fold difference in the amounts handled (and in unit e xposure values) is attributable to the difference in the amount left on the wipes and rags suggests a limitation to the data that should be considered when using the data. The need to extrapolate via unit exposures may be elevated due to the fact that the test conditions were well less than the target application rates of 7 mg ai/m 2

. The second limitation stems from the predominance of hand exposures. Noting that the hands account for 92% of the dermal exposure for the trigger spray bottle uses (Selim 20 11, 107)

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Page 17 of 30 and 98% for the read y - to
- use wipe users (Selim 2011, 98) brings attention to a potentially impo rtant limitation of the recovery correction factor based on the DDAC dermal exposure and reco very study conducted b y Boatwright (2007). Although this s tudy investigated the recovery at two dermal application rates, it appears that it did not investigate the effect of retention time on recovery from the skin. Without data one can onl y conjecture that the behavior of DDAC might mimic the behavior of the f ew insecticides in which similar tests have included time. In their January 2007 report, the EPA’s Scientific Advisory Panel pointed out the importance of a che mical’s residence time on the skin in the recovery efficiency

(EPA SAP 2007). (This report was also cited by

the HSRB in their June 2007 policy on MEAs.) Parts of that discussion implied that, in general, recoveries from skin are (or appear to be) higher soon after deposition and to d ecrease as either the chemical resides on the skin for longer durations or continuous or repeated applications saturate the ability of the skin to retain as much of the ai deposited later as it could earlier. Both of these scenarios result in what appear to be decreases in the recovery efficiency of those chemicals fr om skin as exposure time increases. The Agency

should note that more detailed suggestions and analy s e s may be provided by some Board members independent of this consensus Board report. Ethics Charge to the Board Does the available information support a determination that the study was conducted in substantial compliance with subparts K and L of 40 CFR Part 26? Board Response to the Charge HSRB Recommendation The Board concurred with the Agency ’s assessment (Sherman 2011b) that the study was conduc ted in substantial compliance with subparts K and L 40 CFR 26. HSRB Detailed Recommendation and Rationale The documents provided include a report of a completed wipe study conducted on behalf of the AEATF II (Selim 2011). The submitted study documents st ate that the study was co nducted in substantial compliance with the ethical and regulatory standards of 40 CFR 26, Subparts K and L. The requirements of FIFRA §12(a)(2)(P) and Title 3, § 6710 of the California Code of Regulations also apply. The protocol was reviewed and approved pursuant to the standards of the Common Rule (45 CFR Part 46, Subpart A) by an independent human subjects review committee, IIRB, Inc. of Plantation, FL . Minutes of IIRB, Inc. meetings and a cop y of IIRB, Inc. policies and proced ures were provided (IIRB 2010a; IIRB 2010b; Selim 2011; Sherman 2011b). As stated previously , IIRB, Inc. is registered with OHRP and is fully accredited by

AAHRPP.Final Draft Document Dated May 20, 201 1

Page 18 of 30 1. The Board concurred with the conclusions and factual observations relating to the stu dy , as detailed in the EPA’s Ethics Review (Sherman 2011b) and summarized briefly below.
a. Prior HSRB and Agency Review. Because this study

was initiated after 7 April 2006, prior submission of the protocol and supporting materials to EPA was requir ed b y 40 CFR §26.1125. The requirements of 40 CFR §26.1125 for prior submission of the protocol to EPA and of §26.1601 for HSRB review of the protocol were satisfied. The HSRB reviewed the protocol at its April 2008 meeting, concluding that “if the propo sed mop and wipe scenario design, protocol, and supporting documentation is revised as suggested in EPA’s review, the research would meet the applicable requirements of 40 CFR part 26, subparts K and L” (EPA HSRB 2008a, 2). b. Responsiveness to HSRB and Agency Recommendations. The HSRB noted two instances where the sponsor did not address comments raised by the HSRB in its April 2008 report. In the first instance, the HSRB recommended that the consent form be revised to explain that the “underlying purp ose of the study will be to collect information that will be provided to the EPA, and that the EPA would use that i nformation to determine the appropriate standards for allowable exposures to products such as the test compound” (EPA HSRB 2008a, 25). In the second instance, the Board recommended that the recruitment flyer should explain that the research would measure inhalation as well as dermal exposure (EPA HSRB 2008a, 26). The sponsors could not explain the lack of responsiveness to these two recommendations. The Board concluded that the lack of responsiveness to these two HSRB recommendations did not compromise the ethical conduct of the study , but recommended that the Agency follow up with the sponsor to determine why these recommendations were not addressed. 2. The Board concluded that this study , as conducted, met all applicable ethical requirements for research involving human participants, in accordance with the following criteria:
a. Acceptable risk

- benefit ratio. The risks to study

participants were minimized appropriatel y and were justified by the potential societal benefits, particularl y data on the dermal and inhalation exposure of professional janitorial workers to the liquid antimicrobials they appl y. These data could be used to develop mechanisms to protect future persons who apply these liquid antimicr obials. Minors and pregnant or lactating women were excluded from participation. All study participants were at least 18 y ears old. All females self

- administered an over

- the

- counter pre gnancy

test on the day of monitoring, and all such tests were negative. No nursing mothers were enrolled in the study

.

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Page 19 of 30 The study was designed to minimize the risks of exposure to the test compounds, subject to being able to accomplish the purposes of the study . The stud y involved the use of a commonly

used, commercially

available cleaning product, in a matter consistent with its labeling. Clear stopping rules and medical management procedures were in place. There was one report of a participant leaving earl y because he was feeling ill. Although the observation log does not indicate whether the worker was feeling ill as a result of his participation in the research or whether his illness was unrelated to the study , documentation provided to the Agency

an d the HSRB subsequent to the April 2011 meeting suggests that this parti cipant was unwell prior to study initiation. However, there was no indication that there had b een an y follow up of the participant . Although there is little reason to think that this p articipant would develop a serious and unexpected medical problem related to his particip ation after leaving the study site, the Board recommended that the Agency work with sponsors to develop a SOP for following up with participants who leave a study earl y b ecause of illness. b. Voluntary

and informed consent of all participants. The study protocol included several mechanisms designed to minimize coercive recrui tment and enrollment. Monetary compensation was not so high as to unduly influence particip ation. 3. Two minor protocol deviations were reported. In addition, there were two unreported but m inor protocol deviations. The reported deviations were that: 1) the protocol called for study participants to take 10

- minute rest breaks, but most participants declined or took breaks that were less than 10 minutes in length; and 2) a photograph of a participant’s face was taken at one monitoring site, although no images were included in the report. The Agency

review noted one unreported deviation (Sherman 2011b). This deviation i nvolved the enrollment of three participants (one who was monitored and two who were alternates) who self

- reported that their health was only “fair,” despite the requirement that all partic ipants be in “good health” . In response to a similar protocol deviation that occurred during the related AEATF II mop study discussed at the October 2010 HSRB meeting, the Board re commended the AEATF II develop a SOP to clarify its health status reporting and inclusion criteria (EPA HSRB 2010). T h e Board noted that this study was conducted prior to the Oct ober 2010 HSRB meeting. The HSRB also discovered an additional unreported deviation, namel y the use of an “IRB
unapproved form to collect personal information about volunteers and to sign a ‘Worker Health Statement’” (Selim 2011, 3). These forms were stored separatel y from the field phase data. The sponsors could not explain this deviation from the protocol. The HSRB concluded that these protocol deviations did not compromise the informed co nsent process or put the study participants at increased risk.Final Draft Document Dated May 20, 201 1

Page 20 of 30 Assessment of Published Research Study by Gulson et al. (2010): Small Amounts of Zinc from Zinc Oxide Particles in Sunscreens Applied Outdoors Are Absorbed through Human Skin. Overview of the Study In the Gulson et al. (2010) study , 20 human volunteers were exposed to sunscreens co ntaining stable isotopes of zinc oxide (ZnO) for the purpose of measuring dermal absorption of zinc (Zn) from particles of varying size. Such metal oxide nanoparti cles are being used more and more frequently

in sunscreens because they reflect and absorb ultraviolet (UV) light as transp arent coatings, rather than as opaque coatings as with traditional metal oxide sunscreens. The study was conducted in Australia in March 2009. Ten male and ten female subjects, ranging from 19 to 66 ye ars of age, were enrolled. Study participants wore UV protective upper body clothing with a patch removed to expose a small section of skin on the back. Sunscreen containing stable isot ope 68 Zn oxide, formulated as “bulk” particles (110 ± 46 nanometers [nm] in size) or as nanoparticles (19 ± 8 nm in size), was applied twice daily for a period of five day s to the exposed section of skin on the back. Eleven participants (5 males and 6 females) applied 68 Zn oxide formulated as nanoparticles and nine participants (5 males and 4 females) applied 68 Zn oxide formulated as bulk particles. Participants were encouraged to wear other non

- zinc ty pes of sunscreen on the areas of skin not covered by th e UV
- resistant clothing. The test material was removed from each subject’s back at the end of each day with an alcohol

- lanolin wipe. Urine and blood samples were taken eight day s before sunscreen application, immediat ely before the first application, at the end of each of the five day s of application, and six day s after the last application. Dermal absorption of Zn was calculated from the changes in the ratios of 68 Zn to 64 Zn in urine and blood samples during the course of the experiment; Zn was measured using Multi

- Collector Inductively Coupled Mass Spectrometry (MC

- ICP

- MS). Science Charge(s) to the Board 1. I s the Gulson et al . (2010) study scientificall y sound, providing reliable data? 2. If so, is the Gulson et al . (2010) study relevant for qual itative use in support of an a ssessment of the absorption of zinc oxide through the skin? Board Response to the Charge(s) HSRB Recommendation The Board concurred with the Agency ’s assessment (Ry man and Dole 2011) that the Gulson et al. (2010) study pr ovides some potentially useful data on the dermal absorption of zinc

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Page 21 of 30 from ZnO nanoparticles in sunscreen applied to human skin, despite the multiple limitations identified by

the HSRB. However, the Board advised the Agency to proceed with caution when usin g these data in risk assessment, as these data cannot be used as a stand

- alone set to assess dermal absor

p tion of zinc oxide. HSRB Detailed Recommendations and Rationale The Gulson et al. (2010) study provides some potentially useful data on the dermal absorption of zinc from zinc oxide nanoparticles in sunscreen applied to human skin. The anal yt ical methods used in the study appeared to be sound. The use of the 68 Zn as a tracer for Zn from the sunscreen was an innovative approach for assessing absorptio n and the presence of a chemical in body fluids (e.g., blood or urine) when the same chemical is also present endogenousl y. The Board also agreed with the comments of the Agency (Ry man and Dole 2011) that the distinction between bulk and nanoparticles was not as absolute as indicated in the study since the lower range of size for bulk particles is within the range of the size for nanoparticles. This overlap in size between the groups of particles should not affect the outcome of the studies and the interp r etation of the results significantly . However, the Board raised a number of caveats, including lack of a proper control group, small sample size, and sample contamination, which limits the utility of this study

in risk assessment of zinc oxide

- based sunsc reen. While the analy

tical approaches used in the Gulson et al. (2010) study to measure Zn in blood and urine were solid, the study design itself was fault y. For example, there were no untreated controls to verify

the consistency

of Zn levels in untreat ed individuals. The presence of increases in 68 Zn in the individual who applied the sunscreen (hereafter referred to as “the appl icator”) creates questions regarding the changes that might occur with time independent of sunscreen application. The design was not a carefully controlled experimental trial, and therefore was subject to a variet y of behavioral differences among the subjects as they pursued their beach recreation activities that are likel y to have influenced the results. Despite the fact that the tracer analy tical technique that measured Zn in blood and urine did an excellent job in distinguishing endogenous Zn from Zn absorbed from the sunscreen, this procedure did not distinguish between dermal penetration of soluble Zn from penetration of i ntact nanoparticles, or from absorption via dermal versus oral routes. Therefore, this study has many uncertainties associated with the interpretation of the data, and does not provide inform ation regarding the penetration of nanoparticles through intact, healthy skin. The study

thus is of limited value in judging the level of intact nanoparticle absorption through intact skin. The sunscreen formulation contained isoprop yl myristate, which is a known chemical e nhancer of skin penetration, while the was h off solution used contained alcohol and lanolin. The impact of these two variables on Zn absorption was not considered, nor did the study report e xplain how much Zn was removed b y the dail y alcohol and lanolin wash off procedure. Another source of conce rn is the potential impact of eth yl enediaminetetraacetic acid ( EDTA), a commo nly used preservative and stabilizer, on the zinc oxide nanoparticle formulation. The authors did not address the possibility that EDTA can chelate ionic Zn released from the nan oparticle formulation. This is an important consideration since chelation of free zinc in the skin by EDTA canFinal Draft Document Dated May 20, 201 1

Page 22 of 30 influence the degree of dermal absorption. EDTA is routinely used in this ty

pe of sunscreen formulation, but its impact on dermal Zn absorption was not addressed. The final study

design was based on a pilot study in which the zinc oxide active ingr edient was 51%, while the 68 Zn isotope in the final study was 99% enriched 68 Zn. The authors provide insufficient information on the pilot study ( e.g., age , gender, or skin ty pe of subjects). The Board concurs with the Agency ’s assessment of lack of clarit y on how the pilot study was used in the design of the final study and subject selection. Other variables not accounted for include the frequ ency and time of garment changes and the ty pe of activities the subjects engaged in after the first 30

- minute exposure interval. There was no mention either of the total length of the subjects’ sun exposure and the timing between the first and second dail y exposure. Although UV exposure was estimated to be the same for all subjects, differences in length and intensit y of ph ysical activity likel y contributed to the degree of sweating and blood flow to the skin. No information was provided on the time of the second application of the sunscreen in relationship to the first application. Description of dosing intervals was missing. The authors of the Gulson et al. (2010) study provided no justification or criteria for s electing subjects 4, 8, 9 and 13 used for complete analy sis of all samples collected (Figure 8; 2010, 146). The use of retention rates to address the issue of urine sample contamination was a very good approach; however, there was no mention of how these potential contaminations o ccurred and the potential for hand contamination to have caused hand

- to
- mouth dosing of the subjects. The investigators also did not consider the effect of the ZnO that was retained in the appl icator’s hand(s) on the quantities of the dosing rates reported in Table 1 of the manuscript (Gu lson et al. 2010, 143). The investigators speculated that contamination of urine samples could result from subject’s hands during urine collection. Six urine samples were allegedl y contaminated with 68 Zn from sunscreen. In add ition, it appears that the authors did not attempt to minimize, control for, or a c count for the possibility of the contaminated hand

- to
- mouth route of exposure among their sub jects. While the authors provided a rather convincing post

- hoc series of arguments to support a hy pothesis of contaminated urine samples (Gulson et al. 2010, 146

- 7), they provided no d escription of a mechanism for how the zinc would have gotten from a subject’s back into the urine sample. Since the collection containers were pre

- cleaned and the beach towels provided to the subject were renewed daily in order to eliminate any potential source of sample contamination, the reader is left to infer that six of the ten female subjects somehow got a portion of the sunscreen from their backs onto their hands and subsequently from their hands into the urine colle ction containers. If this transport of sunscreen occurred with these subjects, then it is also plaus ible that 68 Zn from the sunscreen was on the hands of all the subjects but that not all subjects co ntaminated their urine samples. If that were the mechanism, then it also seems likely that som etime during the day

these subjects had one or more occasions to transfer sunscreen into their mouths via food, beverages, mouth wiping and other hand

- to mouth activities. Thus, it is plaus ible, if not likely , that the hands of many or all subjects were contaminated with 68 Zn, and that either some (if not most) of the 68 Zn measured in blood and urine was the result of the transfer from the subject’ s back into their hands and then to their mouths, leading to gastrointestinal a b-

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Page 23 of 30 sorption of 68 Zn. This alternative plausible mechanism could completely negate the validity of the authors’ conclusions regarding dermal absorption. Gulson and his colleagu es may have also made significant errors in the quantities of the doses reported in Table 1 (Gulson et al. 2010, 143) by not considering the amount of ZnO that was retained on the applicator’s hands after dosing each subject. The report does not say whether or not the applicator was wearing gloves or using the same one

- finger application technique as described for their pilot study . Gloves would have been important in the pilot study to avoid particulate contamination of the samples being generated for ex amination by scanning electron m icroscop y. It is reasonable to expect that a significant amount of sunscreen would have been retained on the applicator’s glove or hand after each application. It is also reasonable to expect that a larger fraction of the first application was retained on the applicator’s hand than the fra ction retained from subsequent applications. Thus, the effect that the retention of sunscreen on the applicator’s gloves or hands would have had on individual subjects cannot be determine d. While the source of the Δ 68 Zn% in the blood from the applicator reported in Figure 4B (Gulson et al. 2010, 144) is unknown, the similarity of the Δ 68 Zn% in her blood to that reported for the blood of the test subjects indicates that the applicator’s abso rbed dose was similar to that of the test subjects. Both of the following possible sources of the applicator’s dose have serious i mplications to the results. If the source were via the hand

- to
- mouth route, contamination due to field practi c es would seem to be even more widespread than just intra

- subject. T he source of her 68 Zn could be from dermal absorption through her hands , but if the absorption rate through hands is less than through the skin on the back (as has been reported in studies assessing anatomical variations in dermal absorption of OP insecticides), the similarity of the blood and urinary

excr etion rates for the applicator and the subjects would imply that the amount retained on the applic ator’s hands could have been several times larger than the dermal doses reported to be applied to the subject’s backs in Table 1 (Gulson et al. 2010, 143). Thus, s uch dose values may be inacc urate. While the authors did not even mention isotope fractionation in their article, the effect of isotope fractio nation probably caused the authors to slightly over - estimate their calculated Zn absorption factor. Isotope fractionation refers to the separation of one isotope in comparison to either a lighter or heavier isotope of the same element; in this case, the h uman body ’s prefere ntial absorption or retention of 68 Zn versus 64 Zn. This phenomenon was reported to occur in both of the two “other studies of biological samples” cited by Gulson et al. (2010, 144 ). Isotopic fra ctionation may

have been the unrecognized cause of the finding b y Gulson et al . that the mean 68 Zn/ 64 Zn ratio in their pre

- exposure blood samples (0.4158) was 7% higher than the 18.8%/48.6% = 0.3868 natural background referred to in the manuscript (2010, 141). However , the effect of isotope fracti onation may onl y reduce the 68 Zn/ 64 Zn ratio by a few percent. The researchers also failed to establish the temporal stability of an individual’s 68 Zn/ 64 Zn ratio upon which their conclusion of an increase of that ratio in blood or urine is based (Gulson e t al. 2010). The only

example of time

- series data that was located in the material presented to the Board was the results by

Ohno et al. (2005), which measured the 68 Zn/ 64 Znratio in blood samples collected once per month from just one person in which the average month

- to
- month vari ation was reported to be ±6.6% (a coefficient of variation of ±6.2%). This natural variation isFinal Draft Document Dated May 20, 201 1

Page 24 of 30 considerabl y more than the 0.42% and 0.23% mean post

- exposure increases reported b y Gulson et al . (2010) over 13 and 19 day s from d ay 0 to the end of the exposures and the six

- day follow

up, respectivel y. The magnitude of this natural variation and the lack of an untreated control group suggest that the accuracy of the reported absorption factor could have been erroneous. Regardin g gender

- related differences in dosing, the authors provide insufficient inform ation for the reader to tell whether there was an y interaction between the subject’s individual body weights (not reported) and “a significant difference between doses for males and females with a mean of 4.6 mg/cm 2 for males versus 3.7 mg/cm 2 for females” (Gulson et al. 2010, 142). Without any implication of probability , it is possible that the fact that significant differences b e tween genders were found only

when comparing the ir change in 68 Zn/ 64 Zn ratios without body weight (reported in Figure 5A) but not when comparing their total Zn content in whole blood u s ing the subject’s weight (reported in Figure 5B) was a spurious association. However, the author’s calculations for Δ 68 Zn% did take into account differences in gender and body weight b y adjus ting for fat

- free body

mass via BMI. Regarding study

sample selection, there are two major statistical concerns with the sample’s demographic constitution. The report indicates that the data came from 20 subjects split between the nanoparticle group, with 6 females and 5 males and the bulk group with 4 females and 5 males (Table 1; Gulson et al. 2010, 143). This table, listing the subjects’ demographics, showed greater similarit ies among the subjects due to family connections. Therefore, the que stion arises whether the sample of individuals used in the study represents a random sample of the reference population or whether is it a convenience sample. Because the experimental er ror or within treatment variation could be underestimated in a non

- random, convenient, and small sample, the statistical validity of the data used in this study is questionable. The second issue is r elated to the known outlier (subject 7, enrolled in the nanoparticle group). This subject’s o bserved data point was relatively larger than all other data points (e.g., Figures 4 and 5; Gulson et al. 2010, 144). Also, an adverse or allergic reaction was mentioned in the report as the likel y reason for the larg e response observed in the case of subject 7. Therefore, this single observation could be the main cause of the observed skewedness in the experimental data. The researchers performed a log transformation to correct this high degree skewedness, which may be caused by this single outlier. The statistical significance reported in the paper (gender x treatment) may have disappeared if they

had excluded this observation from the anal ys is or performed any robust weighted linear model analy sis to adjust for thi s influential data. Based on these concerns , the statistical validity of the data used and the conclusion d erived may

be questionable. Thus, the results reported could be treated as exploratory in nature and further studies are needed to confirm the co nclusion. Ethics Charge to the Board Is there adequate information to determine that the Gulson et al . (2010) study was co nducted in substantial compliance with procedures at least a protective as those in subparts A

- L of EPA’s regulation at 40 CFR Part 26?

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Page 25 of 30 Board Response to the Charge HSRB Recommendation The Board concluded that there was insufficient information available at the time of the April meeting to determine whether the Gulson et al . (2010) study was conducted in substantial complian ce with procedures at least as protective as those in subparts A

- L of EPA’s regulation at 40 CFR Part 26. The Board recommended that EPA seek additional information from Macquarie University

or the study

investigator, including a copy of the research pro tocol and an unsigned informed consent form, and that this information be provided to the HSRB for re

consideration. HSRB Detailed Recommendation and Rationale This is the first time EPA has asked the HSRB to review a study from the published lit erature that was conducted after EPA’s amended Rule for the Protection of Human Subjects of Research. The stud y was conducted in Sydney, Australia and received approval from the ethics committee of Macquarie University , as required by the Australian Code for the Responsible Conduct of Research (2007). As noted in EPA’s ethics review (Parsons 2011), 40 CFR Part 26, subparts A through L do not apply ; the research was neither conducted or supported by EPA nor was it conducted b y a person with the intention to submi t the results to EPA. FIFRA §12(a)(2)(P) does not apply to this research since the compound tested is not a pesticide. Thus, the applicable acceptance standards are as follows: 40 CFR §26.1703. Prohibition of reliance on research involving intentional exp osure of human subjects who are pregnant women (and therefore their fetuses), nursing women, or children. Except as provided in §26.1706, in actions within the scope of §26.1701 EPA shall not rely

on data from any

research involving intentional exposure of any human sub ject who is a pregnant woman (and therefore her fetus), a nursing woman, or a child. 40 CFR §26.1705 Prohibition of reliance on unethical human research with non

- pregnant, non - nursing adults conducted after April 7, 2006. Except as provided in §26.1706, in a ctions within the scope of §26.1701, EPA shall not rely on data from any

research initiated after April 7, 2006, unless EPA has adequate information to determine that the research was conducted in substantial compliance with subparts A thr ough L of this part, or if conducted in a foreign country , under procedures at least as protective as those in subparts A through L of this part. This prohibition is in addition to the prohibition in §26.1703. The HSRB determined that most of the information that the Board has about the ethical conduct of the Gulson et al . (2010) study is without supporting documentation and is thus inferential. Although the Board thought that there is no reason to believe that the conduct of the reported research was fun damentally unethical, in the absence of supporting documentation likeFinal Draft Document Dated May 20, 201 1

Page 26 of 30 the informed consent form and research protocol, the Board concluded that it did not have ad equate information to determine that participant selection was voluntary , that information abo ut risks and benefits was sufficientl y communicated, that study participants understood the nature of the research or their right to withdraw from the study , or that the female participants were ne ither pregnant nor nursing. Thus, the Board concluded that it lacked adequate information to d etermine that the research was conducted under procedures at least as protective as those in subparts A through L of 40 CFR Part 26. In the absence of the informed consent form, the Board had insufficient information t o determine how stud y risks and benefits may or may not have been characterized, whether parti cipants were informed of their right to withdraw from the study , whether participation involved reasonable or unreasonable compensation or pressure to participate with family members. The Board offers the following reasoning for its recommendation: 1. Acceptable risk

- benefit ratio. The Board agreed with the EPA review (Parsons 2011) that the main risks to participants were in blood sampling and possible adverse r eaction to the sunscreen formulations. According to Gulson et al ., the sunscreen formulations contained an enriched level of a stable is otope of zinc, which does not pose a higher risk than naturally occurring zinc and “many” commerciall y available sunscr eens contain “nanoparticulate TiO2 and/or ZnO” (2010, 140). Thus, although the risk

- benefit balance was not discussed in the study report, the risks appear to be low. No minors were enrolled in the study . However, apart from an email from the study invest igator to the Agency

(Parsons 2011), the Board had no evidence that pregnant or lactating women were excluded from participation. The study reports that one participant (Subject 7) had an adverse reaction to the sunscreen. The nature and severity

of the adverse reaction was not described. According to the published study , the risks were mitigated as follows: 1) risks of sun exp osure were mitigated with UV

- protective clothing and advice to wear sunscreen; 2) Subject 7 ceased participation in the applicati on portion of the study after an adverse reaction; and 3) a trained phlebotomist drew participants’ blood. The Board concluded that although the risks of the study were low, the “adequacy of info rmation” standard should not be strictly correlated with ris k. That is, the threshold for adequ acy of information should not be understood in proportion to the amount of risk posed by the study (if the risk is low, then the threshold for adequacy can be low). The Board determined that such a standard would exclude other requirements of ethical research including voluntary informed consent, respect for participants, and fair participant selection.

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Page 27 of 30 2. Voluntary

and informed consent of all participants. The Board concluded that it did not have adequate information t o determine that participant selection in the Gulson et al . (2010) study was conducted in substantial compliance with pr ocedures at least as protective as those in subparts A

- L of 40 CFR Part 26. Board members raised a concerns about the fairness and vo luntariness of participant sele ction, including: 1) the investigator holding recruitment discussions with potential participants in his home; 2) selecting participants who were “personal contacts” of the investigator; 3) i nclusion of participants from the same famil y , suggesting that the stud y was conducted in a context where subjects may have experienced undue pressure to participate; and 4) a lack of information about the ty

pe and level of compensation that participants were offered. In add ition, a l though the published paper states that “venous blood and urine samples were collected 8 day s before exposure” (Gulson et al . 2010, 140) an email from the study investigator to the Agency (Parsons 2011) stated that “the subjects attended a meeting at my house 1 w eek b efore the trial started and I described the trial and any risks.” This apparent inconsistency in the timing of the informed consent process and the collection of study

- related samples raises some co n cerns. Although the Board recognizes that this res earch was reviewed and approved by the Macquarie Universit y Human Research Ethics Committee, which is registered with the Austral ian National Health and Medical Research Council, it concluded that ethics committee approval alone does not provide adequate i nformation to determine that the Gulson et al . (2010) study was conducted in substantial compliance with procedures at least as protective as those in subparts A

- L of EPA’s regulation at 40 CFR Part 26. The Board thus recommended that EPA seek add itional information from Macquarie Universit y or the study investigator, including a cop y of the research protocol and unsigned informed consent form, and that this information be provided to the HSRB for re

- consideration.Final Draft Document Dated May 20, 201 1

Page 28 of 30 REFERENCES Australian National Health and Medical Research Council, Australian Research Council, Au stralian Vice

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.

# DRAFT

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