Human Biological Responses to Low Level Ozone
Phase: NA
Conditions: Environmental Exposure, Nasal Inflammation
Sponsor: University of North Carolina, Chapel Hill

Human biological responses to low level ozone 
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Human biological responses to low level ozone 
David B. Peden, MD 
Version Number:  1.2 
11 May 2016 
NCT02857283 
 

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STATEMENT OF COMPLIANCE 
The study will be conducted in accordance with the International Conference on 
Harmonisation guidelines for Good Clinical Practice (ICH E6), the Code of Federal 
Regulations on the Protection of Human Subjects (45 CFR Part 46), and the NIDCR 
Clinical Terms of Award. All personnel involved in the conduct of this study have 
completed human subjects protection training. 
 
 
 
 
 
 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and 
according to local legal and regulatory requirements and applicable US federal 
regulations and ICH guidelines. 
Principal Investigator or Clinical Site Investigator: 
Signed: 
 
Date:  
 
Name: David B. Peden, MD 
 
Title: Professor of Medicine 
 

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TABLE OF CONTENTS 
 
 
 
PAGE 
STATEMENT OF COMPLIANCE ................................................................................................. I 
SIGNATURE PAGE ..................................................................................................................... I 
TABLE OF CONTENTS .............................................................................................................. II 
LIST OF ABBREVIATIONS ....................................................................................................... IV 
PROTOCOL SUMMARY ........................................................................................................... VI 
1 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ......... 9 
1.1 
Background Information .......................................................................................... 9 
1.2 
Rationale ................................................................................................................ 9 
1.3 
Potential Risks and Benefits ................................................................................... 9 
1.3.1 
Potential Risks ............................................................................................ 9 
1.3.2 
Potential Benefits ...................................................................................... 11 
2 
OBJECTIVES ................................................................................................................... 12 
2.1 
Study Outcome Measures .................................................................................... 12 
2.1.1 
Primary ...................................................................................................... 12 
2.1.2 
Secondary ................................................................................................. 12 
3 
STUDY DESIGN .............................................................................................................. 13 
3.1 
Subject Inclusion Criteria ...................................................................................... 13 
3.2 
Subject Exclusion Criteria ..................................................................................... 13 
3.3 
Strategies for Recruitment and Retention ............................................................. 14 
3.4 
Treatment Assignment Procedures ....................................................................... 14 
3.5 
Subject Withdrawal ............................................................................................... 14 
3.6 
Study Product Description .................................................................................... 14 
3.7 
Accountability Procedures for the Study Product .................................................. 14 
3.8 
Assessment of Subject Compliance with Study Product Administration ................ 14 
3.9 
Concomitant Medications/Treatments ................................................................... 14 
4 
STUDY SCHEDULE ......................................................................................................... 15 
4.1 
Withdrawal Visit .................................................................................................... 18 
4.2 
Unscheduled Visit ................................................................................................. 18 
4.3 
Laboratory Procedures/Evaluations ...................................................................... 18 
4.3.1 
Clinical Laboratory Evaluations ................................................................. 18 
4.3.2 
Special Assays or Procedures ................................................................... 18 
4.3.3 
Reporting of SAEs and AEs to IRB ............................................................ 19 
5 
ETHICS/PROTECTION OF HUMAN SUBJECTS ............................................................. 20 
5.1 
Informed Consent Process ................................................................................... 20 
5.2 
Subject Confidentiality .......................................................................................... 20 
6 
DATA HANDLING AND RECORD KEEPING ................................................................... 21 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
ATS/ERS 
American Thoracic Society/European Respiratory Society 
BAU 
Branchial Artery Ultrasound 
BP 
Blood Pressure 
CEMALB 
Center for Environmental Medicine, Asthma and Lung Biology 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
ECG 
Electrocardiogram 
ELF 
Epithelial Lining Fluid Collection 
EPA 
Environmental Protection Agency 
FEV1 
Forced Vital Capacity in 1 second 
FA 
Filtered Air 
FMD 
Flow Mediated Dilation 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HRV 
Heart Rate Variability 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IRB 
Institutional Review Board 
LV 
Left Ventricular 
MedDRA 
Medical Dictionary for Regulatory Activities 
N 
Number (typically refers to subjects) 
NL 
Nasal Lavage 
NEC 
Nasal Epithelial Cell Biopsy 
NSAID 
Non-Steroidal Anti-inflammatory Medications 
O3 
Ozone 
OHRP 
Office for Human Research Protections 
PHI 
Protected Health Information 
PI 
Principal Investigator 
QA 
Quality Assurance 

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QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
 

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PROTOCOL SUMMARY 
Title: 
Human biological responses to low level ozone 
Précis: 
Subjects will be exposed to low levels of ozone approximating 
a typical, metropolitan, summer day, ranging from 0.06ppm to 
0.08ppm.  Subjects will be sedentary, to approximate a typical 
day.  Nasal, pulmonary and cardiovascular endpoints will be 
measured. 
Objectives: 
Primary:  To explore whether low level ozone exposure, 
reflective of a typical metropolitan summer day, will cause 
measurable inflammation in nasal cells or affect cardiovascular 
endpoints in a sedentary human. 
Population: 
15 healthy control subjects 
Description of  
Intervention: 
Subjects will be exposed to low level ozone, ranging from 
0.06ppm to 0.08ppm during the course of the exposure 
session.  Subjects will be sedentary.  Nasal, pulmonary and 
cardiovascular endpoints will be measured. 
Study Duration: 
60 months. 
Subject 
Participation 
Duration: 
No more than 7 weeks. 
Estimated Time to 
Complete 
Enrollment: 
24 months. 
 
 

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Schematic of Study Design 
 
 
 
 
Prior to  
Enrollment 
 
 
 
 
 
 
Visit 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit 2 
Within 6 weeks 
of visit 1; >3 
days after visit 1 
 
 
 
 
 
 
 
 
 
 
Total N 15:  All subjects will have completed either CEMALB 98-0799 or EPA 95-
0518 prior to invitation into this study.  Obtain informed consent. Screen potential 
subjects by inclusion and exclusion criteria; obtain history, document. 
Screening Visit: 
Update medical history including medications, collect vital signs including height 
and weight, urine pregnancy test if indicated, 12 lead ECG, spirometry, 
venipuncture, physical exam, induced sputum, NEC biopsy.  Introduce the subject 
to the chamber area.  Discharge the subject with instructions for the psychological 
surveys, and with contact information. 
 
Baseline Visit: 
Update medical history and medication use, urine pregnancy test if indicated, vital 
signs.  The following samples will be collected / tests performed: ELF collection, 
NL, NEC biopsy, spirometry, BAU, FMD, LV ultrasound. 
Arm 2 
O3 
Arm 1 
Filtered air 

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Visit 3 
Within 2 weeks 
of visit 2 
 
 
 
 
 
 
 
 
Visit 4 
24 hours later 
 
 
 
 
 
 
 
 
 
 
Visit 5, 6 and 7 
6 weeks later 
Repeat intervals 
 
Follow up visit:  
Subjects will return 24 hours later.   Vital signs and AE’s will be collected.  Then 
symptom questionnaire, HRV, venipuncture, ELF, NL, Spirometry and sputum 
induction will be collected. 
Exposure visit: 
Vital signs will be collected, and subject will be queried for AEs.  HRV will be 
obtained, spirometry performed, O3 monitor placed, telemetry placed, and 
symptom questionnaire obtained.  A 6.5 hour exposure at O3 levels varying from 
0.06 to 0.08 with the subject resting or with mild treadmill walking will take place.  
At the end of exposure the following samples will be collected:  Symptom 
questionnaire, HRV, FMD, LV ultrasound, ELF and NL, spirometry. 
Crossover: 
Repeat visit 2 as visit 5, visit 3 
as visit 6 and visit 4 as visit 7 

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1 INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE  
1.1 
Background Information 
Air pollutants including ozone have been implicated in affecting health outcomes.  In 
particular, high level ozone exposure has been shown to affect pulmonary function and 
cause pulmonary inflammation (Silverman, 1976; Hazucha 1987; Devlin, 
1991).   Troubling community-based work has implicated high ozone levels as being 
correlated with increased pediatric asthma ED visits (Strickland, 2010).  Because of 
adverse health effects, EPA standards for safe ozone levels have been set, currently at 
0.07 ppm (2008).  Still, it is estimated that 100 million Americans live in areas where 
ozone levels periodically remain above the EPA standard.  And while this EPA standard 
had been set based on available data, it remained unclear at the time whether low-level 
ozone exposure, such as 0.06 ppm, might affect health as well. 
Our group previously examined lung function and inflammatory response in adults 
exposed to low-level ozone, 0.06 ppm exposure for 6.6 hours, while undergoing 
intermittent moderate exercise (Kim, 2011).  We found that in response to low-level 
ozone exposure (0.06 ppm) with exercise, lung function declines and neutrophilic airway 
inflammation is observed.  What remains unclear, is whether low-level ozone alone – 
without exercise – will cause similar health effects.  If low-level ozone exposure causes 
measurable decrements in lung function in patients at rest, in a typical, sedentary day, 
this would be important for human health. 
1.2 
Rationale 
The purpose of this study is to assess the response in healthy adults to low-level ozone 
exposure (0.06-0.08 ppm) during a 6.6 hour sedentary day.  Specifically, we will assess 
effect of low-level ozone exposure on the following outcomes: changes in profile of 
nasal inflammatory cells, lower respiratory function, and systemic cardiovascular 
measures such as heart rate variability, blood pressure, fibrinogen, CRP, and lipid 
profiles. 
1.3 
Potential Risks and Benefits 
1.3.1 
Potential Risks 
Ozone exposure:  
Exposure to ozone has been studied at our institution and by other investigators and 
has shown minor effects in lung function in some individuals. Subjects may experience 
some degree of airway irritation or cough with exposure. These symptoms typically 
disappear 2 to 4 hours after exposure, but may last longer for particularly sensitive 
people. In the unlikely event that a subject develops medically significant symptoms 

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during the exposure period, the exposure will be terminated and the appropriate medical 
intervention will be provided by the physician on site. Exposure to ozone below 0.08 
ppm tends not to cause noticeable effects in most people, and 0.12 ppm is a level many 
people in the United States may be frequently exposed to, especially in the summer 
months.  Our subjects’ maximum exposure will be 0.08 ppm. 
  
Spirometry:  
Lung function testing may cause lightheadedness with repeated efforts, but this is 
unusual.  Subjects will be seated in a non-rolling chair, and will be instructed to let us 
know if they feel at all lightheaded during the breathing test.  We would then wait longer 
between each test in order to prevent lightheadedness 
  
Sputum collection (induction): 
Collection of sputum may cause wheezing in some individuals, and coughing repeatedly 
may make a subject’s chest hurt.  A physician is immediately available and rescue 
bronchodilator albuterol is on hand.  Subjects will be carefully monitored with pulmonary 
function testing to ensure a return to baseline before leaving the research lab.  Most 
people can do this test without any problem. 
  
Brachial artery ultrasound: 
There are no significant risks associated with ultrasound imaging of the brachial artery, 
or with the 5 minutes of reduced blood flow that is part of the test. Cutting off blood flow 
to the arm may result in mild discomfort or temporary sensations of tingling or 
numbness in the hand until the blood pressure cuff is released. About 1 in 200 patients 
develop a painless rash on the arm where the blood pressure cuff is placed; this 
disappears over several days.  
  
Ultrasound of the heart:  There is no risk to this procedure.   
  
Blood sampling from vein: 
Risks with having blood drawn include mild discomfort or bruising at the site.  A small 
risk of infection also exists.  Blood will be drawn by well-trained staff. 
 
Ambulatory ECG monitoring (Holter):  
Subjects may have minor skin irritation in the area where the electrodes are attached to 
the skin. Prior to electrode application, the skin will be treated with alcohol and a 
cleansing solution. If male, the skin will be shaved in the area where the electrodes are 
attached.  Subjects should not participate if skin is highly sensitive to electrode adhesive 
or gel.  Subjects will not be able to do activities which would result in the monitor getting 
wet, such as showering or swimming while the monitor is on. 
Nasal lavage and ELF procedure: There are no risks associated with the nasal lavage.  
The ELF can sometime make individuals sneeze. 

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Nasal epithelial cell collection: This procedure can cause mild temporary bleeding from 
the nose.  It will be treated if it occurs by squeezing the tip of the nose for 5-10 minutes. 
Nasal biopsy also is transiently painful.  
 Genetic sampling: 
There is risk of discrimination based on results.  A Federal law, the Genetic Information 
Nondiscrimination Act (GINA), generally makes it illegal for health insurance companies, 
group health plans, and most employers to discriminate against subjects based on 
genetic information.  GINA does not protect subjects against genetic discrimination by 
companies that sell life insurance, disability insurance, or long-term care 
insurance.  GINA does not protect subjects against discrimination based on an already-
diagnosed genetic condition or disease. 
  
Confidentiality: There is risk of breach of confidentiality of medical information and/or 
data.  All individuals who have been granted access to the data to perform their 
research-related duties will be bound by an agreement of confidentiality. Blood and 
sputum samples will be stored until all measurements are made for this study.  Any 
samples that are left over will continue to be stored.  We will give subjects a consent 
form which discusses sample storage, titled “Center for Environmental Medicine, 
Asthma, and Lung Biology Repository for storage of coded samples” (05-
2528).  Subjects are not required to provide permission for us to store these samples 
after this study is completed in order to participate in this study. 
Psychological questionnaires: It is possible that subjects might find the questions asked 
in the surveys to be upsetting. These questions are not meant to be intentionally 
upsetting, but personal questions have a small potential to cause psychological stress. 
Subjects are not required to answer any questions with which make you uncomfortable. 
 
There may be uncommon or previously unknown risks. You should report any problems 
to the researcher. 
 
1.3.2 
Potential Benefits 
There is no direct benefit to the subject.  Results from this study may help to determine 
the effects of ozone on the human airways and cardiovascular system. 

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2 OBJECTIVES 
2.1 
Study Outcome Measures 
2.1.1 
Primary 
The primary endpoint is the change in nasal PMN counts, measured in nasal lavage 
fluid, between FA and O3. 
2.1.2 
Secondary 
Secondary endpoints to be measured include NEC and ELF for inflammatory endpoints, 
spirometry for change from baseline, change in cardiovascular endpoints FMD, HRV 
and LV ultrasound in FA vs O3.  
Exploratory endpoints correlation of ambient ozone level using a novel, wearable 
device. 
 
 

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3 STUDY DESIGN 
This is a double blind, randomized, crossover study of healthy individuals, who are 
exposed to relevant levels of ozone vs filtered air, for 6.5 hours and while at rest.  This 
is intended to mimic exposure to ozone levels of typical summer day, on a typical 
sedentary day. 
Multiple samples will be collected, for both nasal and cardiovascular endpoints.  
Additional information will be collected in for phenotype comparisons. 
3.1 
Subject Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the 
following criteria: 
• Provide signed and dated informed consent form. 
• Willing to comply with all study procedures and be available for the duration of 
the study. 
• Aged 18 to 50. 
• In good general health as evidenced by medical history with Vital signs within 
normal limits on admission to the study: SpO2 > 94%, systolic blood pressure 
between 150-90 mm Hg, diastolic blood pressure between 100-60 mm Hg, 
afebrile. 
• FEV1 of at least 80% of predicted. 
3.2 
Subject Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation 
in this study: 
• Medical condition, laboratory finding, or physical exam finding including any 
chronic medical condition considered by the PI as a contraindication to the 
exposure study, including significant cardiovascular disease, diabetes requiring 
medication, chronic renal disease, chronic thyroid disease, or kidney disease, 
which precludes participation. 
• Use of disallowed concomitant medications including systemic or inhaled 
steroids, NSAIDS or aspirin within 72 hours of dosing, and inability to withhold 
these medications prior to each session of the study.  
• Pregnancy or nursing. 

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• History of current tobacco use, or more than a lifetime 5 pack year history of 
tobacco use.   
3.3 
Strategies for Recruitment and Retention 
Subjects will complete either UNC CEMALB screening and database protocol #98-0799 
or the Recruitment and Screening of Potential Participants for EPA studies #95-0518 
prior to enrollment into this study.  
3.4 
Treatment Assignment Procedures 
A randomization table will be provided by Haibo Zhou, Ph.D, who provides statistical 
assistance to the CEMALB.  TRC maintains the blinding list, and will release it to the 
study team at the end of the study, or earlier if an interim analysis is done.    
3.5 
Subject Withdrawal 
Subjects may withdraw voluntarily from the study at any time.  The investigator may 
terminate a subject’s participation if there is any concern for the health or well-being of 
the subject, or if the subject fails to follow the study requirements. 
3.6 
Study Product Description 
Ozone is generated by TRC, a contractor for the US EPA.  They have procedures in 
place both for generating and monitoring the O3 concentrations, and they will provide a 
separate protocol operations plan for this study. 
3.7 
Accountability Procedures for the Study Product 
At the completion of the study TRC will provide a written report for all chamber 
exposures.  The study may be unblinded at the request of the PI or other study medical 
doctor in the event a subject has an unexpected event and unblinding is necessary for 
subject treatment or for study continuation. 
3.8 
Assessment of Subject Compliance with Study Product Administration 
Subjects will be queried regarding adherence to medication restrictions.  
3.9 
Concomitant Medications/Treatments 
Subjects will be queried regarding concomitant medication use at each visit.  Any new 
medications will be documented and the PI or covering medical doctor will be notified, 
and that individual will decide if the subject may proceed with the study. 
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4 STUDY SCHEDULE 
Subjects will arrive for a consent visit and screening for the study.  After informed 
consent is obtained, we will: 
1. Collect/update medical history, including any medications 
2. Collect vital signs, including pulse, RR, BP, temp, SpO2 and breath sounds. 
3. Measure height and weight. 
4. Pregnancy test will be done for all women 
5. Obtain standard 12-lead ECG for baseline measurement. 
6. Perform spirometry according to ATS/ERS recommendations. 
7. Perform venipuncture for 50ml of blood for clinical labs, including CBC Diff, 
chemistries as well as baseline research labs. 
8. Perform physical examination (by a study doctor) to evaluate the subject for 
eligibility. 
9. Collect sputum for baseline measurements.  Induced sputum is a method for 
obtaining lower airway secretions.  Subjects will be asked not to eat for 2 hours 
prior to this test as food residue in the mouth may contaminate the samples.  Any 
subject who comes to this study with a diagnosis of mild asthma will be pre-
treated with 2-4 puffs of albuterol prior to sputum induction.  Subjects will breathe 
saline from an ultrasonic nebulizer through a mouthpiece for 7 minutes of each 
saline concentration (3%, 4%, and 5%) while seated.  After the first inhalation 
period subjects will be asked to rinse their mouths out with water, spit into the 
sink, and then gargle.  Then they will clear the throat, and blow their 
nose.  Subjects are then encouraged to expectorate samples from deep in the 
chest and spit them into a separate sample cup.  Spirometry will be assessed 
after each saline level to ensure no lung function decrements.  If the FEV1 
decreases 10% or more from baseline, the subject will not be advanced to the 
next level of saline; however the same level may be repeated once.  If the FEV1 
drops 20% or more the procedure is stopped and, if necessary, 2 puffs of 
albuterol will be given.  The physician may prescribe a second albuterol MDI 
treatment at his/her discretion.   
10. Perform nasal epithelial cell biopsy for baseline:  a non-invasive biopsy 
procedure will be performed to retrieve a small cluster of cells from each of the 
nasal cavities.  For the biopsy procedure, the subject will be seated comfortably 
in a straight-backed chair or reclining on an exam table with the head titled as far 
back as possible while remaining comfortable. A short, sterile plastic sampling 
device called a curette will be inserted into one of the nasal cavities and the 
surface of the nasal cavity will be stroked several times for approximately 5 
seconds in order to obtain a small cluster of cells.  Only one side will be collected 
at this visit.  The investigator, study physicians or other trained staff will collect 
these samples. 
11. Introduce the subject to the chamber area by walking them through. 
12. Subjects will be discharged with contact information for a study physician on call. 

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13. Psychological surveys: Once subjects have been qualified, they will be emailed a 
link to complete psychological surveys.  These will be performed at home, and at 
the convenience of the subject, prior to the first exposure session. 
Once the subject is deemed eligible, they will return for a baseline day to collect pre-
exposure measurements.  This visit must occur no less than 3 days and no more than 6 
weeks after the screening visit.  Subjects will be asked to withhold NSAIDs and 
antioxidant vitamins for 7 days prior, and to arrive fasting (water is acceptable). We 
will update any changes in medical history, including medication use, collect vital signs, 
and perform spirometry.   
The following samples will be collected: 
1. Urine pregnancy test: For all women, if more than 7 days have passed since 
the prior test. 
2. Epithelial lining fluid (ELF) collection.  This is done by having the subject 
spray normal saline into the nose, and then insert a small filter paper into each 
nostril.  Nose clips are applied for 2 minutes, then the filter paper is 
removed.  These samples are collected by study staff. 
3. Nasal lavage (NL): This is done by repetitive spraying of sterile normal saline 
irrigation solution into the nostril, followed by voluntary expelling of the fluid by 
the subject into a specimen collection cup. Both nostrils are sampled, these 
samples will be collected by study staff, 
4. Spirometry:  Standard spirometry to obtain FEV1 and FVC measurements will 
be done prior to sputum induction.  This is done by having the subject exhale 
forcefully and completely into a mouth piece attached to a pneumotach.  This will 
be repeated several times, and the procedure will be done following ATS/ERS 
standards.  
5.  Flow-mediated brachial artery dilation (FMD): This measurement is made by 
imaging the brachial artery with an ultrasound device.  The blood flow to the 
artery is occluded for 5 minutes, and the artery is again imaged when blood flow 
is resumed.  
6. Left ventricular strain: Left ventricular contractility will be assessed by 
measuring global longitudinal strain using 2-dimensional speckle tracking 
echocardiography.  Apical 4-chamber, 2-chamber, and long axis 2-dimensional 
ultrasound images of the heart will be acquired using a Philips EPIQ 7 ultrasound 
machine and stored for subsequent off-line analysis.  Global longitudinal strain 
will be quantified using Philips QLAB advanced quantification software. 
The subject will be discharged to home with contact information for a study physician 
who is on-call if the subject has any health concerns.  The subject will return the next 
morning, and after health history is updated and vital signs collected, the following will 
take place: 

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1. Heart rate variability (HRV):  Standard ECG leads will be placed on the 
subject’s chest, and connected to a monitor that records the heart rate and 
rhythm.  The subject will be asked to lie supine in a quiet, darkened room for up 
to 30 minutes.  The subject will do this before the exposure and immediately after 
the exposure.  The analysis for this measurement is computer based. 
2. Spirometry:  Standard spirometry will be measured prior to the subject entering 
the exposure chamber.  
3. Ozone monitor:  Subjects will wear an experimental ozone monitor, which is on 
a bracelet.  This is to validate the wearable monitor in an environment with 
known ozone concentration.  
4. Telemetry: Standard 3 lead ECG monitoring will be done while the subject is in 
the exposure chamber. 
5. Symptom questionnaire: Subjects will be asked to complete a symptom 
questionnaire prior to entering the chamber. 
7. The subject will enter the chamber.  The atmosphere will be determined 
randomly and will be either filtered clean air or ozone.  The ozone concentration 
will be varied from 0.06 to 0.08 through the course of the exposure, and the 
exposure will last for 6.5 hours.  The subjects will rest during the exposure with 
intermittent mild walking on the treadmill.  Subjects will be provided with a low fat 
lunch to eat while they are in the chamber. HR will be monitored continually while 
the subject is in the chamber, and blood pressure will be measured hourly.   A 
symptom questionnaire will be completed at the end of the exposure 
period.  Immediately upon exiting the chamber, the subjects will undergo HRV for 
up to 30 minutes, followed by FMD and LV strain measurement.  ELF and NL 
samples will be collected.  Nasal epithelial cells (NEC) will be obtained from the 
opposing nostril from screening or exposure session 1. 
Spirometry will be obtained prior to discharge, and at any point during the study if the 
subject experiences any distress or shortness of breath. Subjects will be discharged 
with contact information for a study physician on call.  
Subjects will return 24 hours later.  After updating the health history and collecting vital 
signs, the following measurements/samples will be collected: 
1. Symptom questionnaire 
2. HRV for up to 30 minutes 
3. Venipuncture as collected at screening day 
4. ELF 
5. NL 
6. Spirometry 
7. Sputum induction 
The subjects will be discharged with contact information for a study physician.   They 
will be scheduled to return for a second session, with a minimum of 2 weeks and a 

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maximum of 6 weeks between exposures.  The sessions will be identical except for the 
atmosphere in the chamber, which is randomized. 
  
  
Baseline measurements provide safety information prior to each exposure.  Given the 
level of the O3 and the procedures that subjects are asked to undergo, we do not 
anticipate any health concerns, but if they do arise, they will be immediate, therefore 
there are no scheduled visits beyond day 3 of the 2nd session.  However, subjects will 
be encouraged to contact the study coordinator or physician at any time up to a month 
later if they have any concerns regarding their health or effects from the study. 
4.1 
Withdrawal Visit 
If a subject withdraws from the study, any safety measures deemed necessary by the 
covering physician will be performed.  This includes physical examination with vital 
signs, ECG measurement, or spirometry. 
4.2 
Unscheduled Visit 
If a subject has an unexpected response to any study procedure, any safety measures 
deemed necessary by the covering physician will be performed.  This includes physical 
examination with vital signs, ECG measurement, or spirometry. 
4.3 
Laboratory Procedures/Evaluations 
4.3.1 
Clinical Laboratory Evaluations 
Blood will be sent to Labcorp, INC, for a complete blood count and chemistry values at 
screening. 
4.3.2 
Special Assays or Procedures 
NLF will be collected and processed to obtain a cellular and cell-free fraction as 
described by us before, which will be analyzed for markers of inflammation and changes 
in immune responses. Cell-free nasal lavage fluid will be used to assess changes in 
markers of inflammation, host defense responses, and overall immune status. NLF cells 
will be used to assess changes in PMN influx as well as gene expression profiles 
associated with the exposure. 
 
Superficial scrape biopsies will be obtained as described by us before and processed to 
yield both RNA and DNA. Total RNA will be analyzed for gene expression profiles 
associated with the exposures, while DNA samples will be used to determine epigenetic 
changes associated with the exposure. 

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Epithelial lining fluid (ELF) will be obtained by placing a strip of synthetic absorbent 
material (SAM) into the nostril, securing it with a nose clip, and removing it after about 2 
minutes (you may want to copy language from our TCORS SOP here). ELF strips will 
be stored at -80oC until elution using PBS+BSA. ELF fluid will be used to assess 
changes in immune and inflammation mediators. 
 
Sputum will be processed as previously published –in brief: plug material from sputum 
will be weighted and homogenized with DPBS (8x wt of selected plug material), 
centrifuged and supernatants recovered for proteomic assessment; sputum will be 
further homogenized with DTT (0.1%), filtered, centrifuged and the cell pellet recovered. 
The cell pellet will be re-suspended in an appropriate volume to a desired cell 
concentration (1x106 cells/ml) for cytospin slide generation; slides will be fixed and 
stained and read for differential leukocyte analysis. The remaining cells will be 
resuspended in Trizol reagent for RNA extraction and future mRNA/miRNA analysis 
using Nanostring technology.   
 
Sputum: MesoScale Discovery to assay fluid phase proteins (cytokines); 
hemacytometry to assay total cell counts; NanoString technology to assay genes of 
interest; Flow cytometry: to assay functional cellular endpoints such as phagocytosis, 
intracellular oxidative burst, cell surface markers 
 
In addition, we will genotype for SNPs of interest such as GSTM1 and other oxidant related 
genotypes; we will analyze mRNA expression for inflammation, immune and oxidant associated 
genes. 
4.3.3 
Reporting of SAEs and AEs to IRB 
AEs and SAEs will be reported per UNC standard reporting procedures. 
 

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5 ETHICS/PROTECTION OF HUMAN SUBJECTS 
5.1 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual agreeing to 
participate in the study and continues throughout study participation.  Extensive 
discussion of risks and possible benefits of study participation will be provided to 
subjects and their families, if applicable.  A consent form describing in detail the study 
procedures and risks will be given to the subject.  Consent forms will be IRB-approved, 
and the subject is required to read and review the document or have the document read 
to him or her.  The investigator or designee will explain the research study to the subject 
and answer any questions that may arise.  The subject will sign the informed consent 
document prior to any study-related assessments or procedures.  Subjects will be given 
the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate.  They may withdraw consent at any time throughout the course 
of the study.  A copy of the signed informed consent document will be given to subjects 
for their records.  The rights and welfare of the subjects will be protected by 
emphasizing to them that the quality of their clinical care will not be adversely affected if 
they decline to participate in this study. 
The consent process will be documented in the clinical or research record.   
5.2 
Subject Confidentiality 
Subject confidentiality is strictly held in trust by the investigators, study staff, and their 
agents.  This confidentiality is extended to cover testing of biological samples and 
genetic tests in addition to any study information relating to subjects. 
The study protocol, documentation, data, and all other information generated will be 
held in strict confidence.  No information concerning the study or the data will be 
released to any unauthorized third party, however the subject may request copies of all 
clinical values, including lab results, ECG and spirometry. 

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6 DATA HANDLING AND RECORD KEEPING 
Paper documents will be maintained by the PI or designee.  All data will be entered into 
a REDCap database. 
This study will comply with the NIH Public Access Policy, which ensures that the public 
has access to the published results of NIH funded research. It requires scientists to 
submit final peer-reviewed journal manuscripts that arise from NIH funds to the digital 
archive PubMed Central upon acceptance for publication. 
The International Committee of Medical Journal Editors (ICMJE) member journals have 
adopted a clinical trials registration policy as a condition for publication.  The ICMJE 
defines a clinical trial as any research project that prospectively assigns human subjects 
to intervention or concurrent comparison or control groups to study the cause-and-effect 
relationship between a medical intervention and a health outcome.  Medical 
interventions include drugs, surgical procedures, devices, behavioral treatments, 
process-of-care changes, and the like.  Health outcomes include any biomedical or 
health-related measures obtained in patients or participants, including pharmacokinetic 
measures and adverse events. The ICMJE policy requires that all clinical trials be 
registered in a public trials registry such as ClinicalTrials.gov, which is sponsored by the 
National Library of Medicine.  Other biomedical journals are considering adopting similar 
policies. For interventional clinical trials performed under NIDCR grants and cooperative 
agreements, it is the grantee’s responsibility to register the trial in an acceptable 
registry, so the research results may be considered for publication in ICMJE member 
journals.  The ICMJE does not review specific studies to determine whether registration 
is necessary; instead, the committee recommends that researchers who have questions 
about the need to register err on the side of registration or consult the editorial office of 
the journal in which they wish to publish. 
 

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7 DATA ANALYSIS 
 
Aim 1. Characterize ozone induced changes in nasal PMN counts. 
This is a randomized, double blind, crossover pilot study, studying the changes in cells of nasal 
lavage (neutrophilla) after low-level ozone exposure. First, descriptive analysis will be 
conducted to examine potential ozone exposure effect and period effect. Second, two sample 
tests such as t-test and Mann Whitney test will be used to compare changes in response in clean 
air and ozone exposure groups. In general, carry-over effect is first tested. In the absence of 
significant carry-over effect, pooled data from both periods can be used to estimate the ozone 
exposure effect. Otherwise, only period 1 data can be used. We will report effect estimates, 
standard deviation as well as confidence intervals for ozone exposure effect and potential period 
effect. Finally, a random effects model will be fitted to assess the ozone exposure effect. 
Confidence intervals will be reported.  
 
Aim 2. Characterize ozone induced changes in inflammatory cytokines in nasal epithelian cell 
(NEC) biopsy and nasal epithelial lining fluid ELF 
The analysis plan is the same as in Aim 1. 
 
Aim 3. Characterize ozone induced changes in FMD, HRV, LVS, Brachial artery ultrasound, 
ambulatory ECG measures, & other cardiovascular measures. 
The analysis plan is the same as in Aim 1. 
 
Aim 4. Characterize ozone induced changes in spirometric measures 
The analysis plan is the same as in Aim 1. 
 
Aim 5. Investigate the performance of a wearable monitor for ambient ozone measurement. 
There are 2 current devices which will be used (descriptions and photos attached.  The subject 
may wear one or both of these devices.  The purpose of these devices at this time is strictly to 
assess the measured values obtained by the devices compared to the known O3 
atmosphere.  However, as these devices are prototypes there are two important questions.   The 

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first is will the sensors technically perform appropriately thought the exposure while being worn 
by volunteers.  In essence, the goal of this assessment is to simply see fi the devices work at all, 
and to identify specific technical modifications which may need to be made.  The second goal is 
to determine if the readings of functioning wearable sensors correlate well with the readings of 
the standard ozone monitors present in the exposure chamber (that is to demonstrate that they are 
accurate). All of the ozone sensors are non-invasive. These are exploratory endpoints 
 
In case of potential missing data, we will first carefully investigate the pattern of missing data 
and determine if it is informative missing. In case the missing is heavy, we will explore statistical 
methods such as multiple imputation to handle the missing data issue (Little and Rubin, 2014). 
The pilot design and sample size is motivated by our earlier similar study (Peden et al, 1995). 
The goal of this pilot study is to determine whether low-level ozone affects a) nasal epithelial 
inflammation, and b) exploratory cardiovascular endpoints such as heart rate variability.  If we 
find that cardiovascular endpoints are affected by low-level ozone in this small group, further 
studies will explore these, and perhaps other, cardiovascular markers and outcomes.   The aim 
of these future studies will be to further characterize the cardiovascular effects of low-level 
ozone exposure. 
The sample size for this study is determined on our primary endpoint outlined in Aim 1. The 
other aims are all secondary analysis using the same sample collected in Aim 1. We will not 
collect additional subjects for Aims 2-5. In previous literature, it is found that higher-level 
shorter duration ozone exposure caused a change in neutrophilia of 67 with a standard deviation 
of 31, while the change is 31 with a standard deviation of 17 in the clean-air group (Peden et al, 
1995). The aimed effect size is 36. If we assume the significance level is at 0.05, a sample size of 
13 subjects is adequate to ensure 90% power of detecting the effect. Approximately half of the 
subjects will receive air-ozone sequence, and another half will receive ozone-air sequence. Using 
a significance level of 0.05, the table below shows the sample size under various potential 
correlation coefficient. The calculation is based on a two-sided paired t test. 
 
Effect sizeCorrelation coefficientPower N 
36 
0 
0.8 10
0 
0.9 13
0.3 
0.8 8 
0.3 
0.9 10
0.5 
0.8 7 
0.5 
0.9 9 
Note: N means that N/2 subjects receive ozone-air sequence, and another N/2 subjects receive 
air-ozone sequence.  

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We anticipate the SD for the proposed study should behave similarly to the previous study. 
Nevertheless, we inflated our sample size to account for the situation that the true SD is larger 
than we assumed - our proposed sample size n=15 is larger than the real sample sizes for 
achieving 80% power, which is n=8 for correlation coefficient 0.3, and n=10 for corr. coeff at 0, 
respectively. 
The statistical analyses will be conducted under the supervision of Dr. Haibo Zhou (Professor of 
Biostatistics and Director of the CEMALB Biostatistics Core). 
 
  
 
Reference:  
Little, R. J. and Rubin, D. B. (2014). Statistical analysis with missing data. John Wiley & Sons. 
Peden, David B., R. Woodrow Setzer Jr, and Robert B. Devlin. "Ozone exposure has both a 
priming effect on allergen-induced responses and an intrinsic inflammatory action in the nasal 
airways of perennially allergic asthmatics." American journal of respiratory and critical care 
medicine 151.5 (1995): 1336-1345.