Phase II Randomized, Double-Blind, Placebo-Controlled Trial of Broccoli Seed and Sprout Extract (BSSE) to Evaluate Detoxification of Carcinogens in Firefighters
Phase: PHASE2
Conditions: Hematopoietic and Lymphatic System Neoplasm, Malignant Solid Neoplasm, Occupational Malignant Neoplasm
Sponsor: National Cancer Institute (NCI)

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DCP Protocol Template Version 18 
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VERSION NUMBER 
VERSION DATE 
REASON FOR CHANGE 
(high-level) 
V1 
October 3, 2022 
Initial Submission 
V2 
November 16, 2022 
Requested revisions; updated 
to template v12 
V3 
December 22, 2022 
Requested revisions per DCP 
concurrence review 12/5/22 
V4 
February 5, 2023 
Request revisions per DCP 
concurrence review 1/9/23 
V5 
March 21, 2023 
Requested revisions per DCP 
concurrence review 2/21/23; 
added option of remote 
consent 
V6 
April 28, 2023 
Requested revisions per DCP 
concurrence review 4/14/23 
incl. removing Appendix E/F 
to be stand alone documents. 
V7 
May 31, 2023 
Requested revisions per DCP 
concurrence review 5/15/23 
incl revision of table of 
contents, clarification of 24-
hr urine collection on the day 
of flashover training, and 
addition 24-hr urine 
questionnaire to Appendix E. 
V8 
August 14, 2023 
Updates per CIRB review 
7/27/23. General 
cleanup/formatting 
improvements. 
V8.1 
 
 
 
 
 
 
 
 
 
 
 
 
 
January 11, 2024 
The entry and end-of-study 
brief physical exams have 
been removed. Firefighters 
undergo an extensive annual 
physical exam as part of their 
occupational duties, which 
includes fire training. 
Consequently, the removal of 
the brief physical exams does 
not present any issues 
regarding eligibility 
evaluation or participant; 
safety for an intervention 
lasting 7-10 days with 

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broccoli seed and sprout 
extract. Moreover, 
eliminating the physical exam 
will ease the scheduling 
challenges encountered 
during the enrollment of the 
initial cohort of participants; 
minor updates as requested 
per DCP consensus review 
dated 9/5/23; general 
cleanup;Added information 
re: new gear rollout at TFD; 
update 
V8.2 
October 7, 2024 
The first cohort of six 
participants completed the 
study under protocol versions 
8.0 and 8.1. The urine 
samples were analyzed for 
levels of the benzene 
metabolite, SPMA (the 
primary study endpoint), at 
baseline and after flashover 
training. Under a different 
grant, we assessed SPMA 
levels after a different fire 
training setting (burn room 
training). The collective 
information suggested that 
measuring the benzene 
metabolite after either 
training fire is not a viable 
endpoint to evaluate the 
effects of BSSE on the 
detoxification of carcinogens 
due to the low and variable 
benzene exposure during the 
training fires. Additionally, 
we unblinded treatment 
assignment and found that 
the intervention received 
prior to the flashover training 
could not explain the low and 

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variable benzene exposure 
after the flashover training. 
 
Based on the available 
information, we propose an 
alternative design to assess 
changes in urinary 
acetaminophen mercapturate 
after acetaminophen 
administration as a surrogate 
biomarker for the 
upregulation of glutathione 
conjugation, the primary 
pathway responsible for the 
formation of SPMA from 
benzene. Using 
acetaminophen metabolism 
as a surrogate for assessing 
upregulation of Phase II 
detoxification pathways has 
been studied previously. 
 
We also propose a longer, 12-
week intervention period to 
allow for the evaluation of 
the effects of BSSE on 
epigenetic modifications, 
such as miRNA and DNA 
methylation, observed in 
incumbent firefighters. In 
addition, extending the 
intervention period provides 
the unique opportunity to 
explore the effects of BSSE on 
the detoxification of 
carcinogens, including 
benzene and polycyclic 
aromatic hydrocarbons 
(PAHs), to which firefighters 
are exposed during real world 
firefighting activities, such as 
actual fire and/or fuel leaks. 

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V8.3 
January 22, 2025 
Requested revisions per DCP 
concurrence review 11/5/24 
for greater clarity for urine 
collection and questionnaires, 
change in LAO PI, and 
addition of Appendix F; 
updated template from v12 
to v17. 
V8.4 
February 28, 2025 
Updates per DCP consensus 
review. 
V8.4 
May 6, 2025 
Updates per CIRB review; 
updated version date per 
CIRB review.  
V8.5 
November 12, 2025 
• 
The protocol was updated 
to DCP Version 18 (April 10, 
2025)  
• 
Due to the stringency of fire 
fighter schedule we needed 
to adjust visit schedules.  
This includes expanding 
allowable visit windows 
and timing of urine 
collections to be considered 
evaluable for the study 
endpoints. 
• 
Clarified need to obtain end 
of study measures while 
participant on study agent.  
• 
Clarified medical history to 
require month and year 
and allow use of 15th of the 
month for data collection if 
participant cannot recall 
the exact day of their 
annual physical. 
• 
Replaced post fire/fuel 
questionnaire with new 
abbreviated participant 
facing questionnaire 
(appendix F) 
• 
Added non-participant 
facing incident report for 
data collection (appendix 
H) 
• 
General protocol cleanup 
 

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COVER PAGE 
 
DCP Protocol #: UAZ22-11-01 
Local Protocol #: STUDY00003544 
 
 
 
 
 
 
Phase II Randomized, Double-Blind, Placebo-Controlled Trial of Broccoli Seed and Sprout Extract (BSSE) to 
Evaluate Detoxification of Carcinogens in Firefighters    
 
 
Lead Academic Organization (LAO) Name: University of Arizona Cancer Prevention Clinical Trials 
Network (CP-CTNet) 
 
 
 
 
 
 
 
Name of LAO Principal  
 
     Patricia Thompson, PhD 
Investigator:  
 
 
     Professor of Physiology 
 
 
 
 
 
     University of Arizona 
     1515 N Campbell Ave. 
     Tucson, AZ 85724 
     Tel: 520-626-3138 
     Fax: 520-626-5348 
     pcarino@arizona.edu 
 
Organization Name:  
     University of Arizona (AZ163) 
Protocol Principal Investigator:     Malvi Savani, MD, MS  
 
 
 
 
           Assistant Professor of Medicine  
 
 
 
 
 
     1515 N Campbell Ave 
 
 
 
 
 
     Tucson, AZ 85724  
 
 
 
 
 
     Tel: 520-626-8096 
 
 
 
 
 
     mbsavani@arizona.edu  
 
Organization:  
 
 
    George Washington University Cancer Center (DC014); not an accruing 
site 
Co-Principal Investigator:  
    Julie E. Bauman, MD, MPH 
Professor and Director of the GW Cancer Center  
 
 
 
 
 
    22nd & I Street NW 
 
 
 
 
 
    Washington, DC 20037 
 
 
 
 
 
    Tel: 202-741-2210 
 
 
 
 
 
    jebauman@email.gwu.edu  
 
Organization:  
 
 
   University of Arizona (AZ163) 
Co-Investigator: 
 
   Jefferey L. Burgess, MD, MS, MPH 
 
 
   Professor of Public Health  
1295 N. Martin Avenue 
 
   Tucson AZ 85724 
 
   Tel: 520-626-4918 
 
   jburgess@arizona.edu 

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Organization: 
 
 
   University of Arizona (AZ163) 
Statistician:  
 
 
   Chiu-Hsieh (Paul) Hsu, PhD 
 
 
 
 
 
   1515 N Campbell Ave.  
 
   Tucson, AZ 85724 
 
   Tel: 520-626-5054 
         pchhsu@arizona.edu  
 
Organization Name: 
   NCI/Division of Cancer Prevention 
Medical Monitor: 
   Malgorzata Wojtowicz, MD 
 
 
 
   NIH National Cancer Institute  
 
 
 
   Division of Cancer Prevention  
 
 
 
   9609 Medical Center Drive 
 
   Room 5E-104, MSC 9781  
 
   Bethesda, MD 20892  
 
   Tel: 240-276-7012 
 
   Fax: 240-276-7848 
 
   Malgorzata.wojtowicz@nih.gov  
 
Scientific Lead: 
   Malgorzata Wojtowicz, MD 
 
 
 
   NIH National Cancer Institute  
 
 
 
   Division of Cancer Prevention  
 
 
 
   9609 Medical Center Drive 
 
   Room 5E-104, MSC 9781 
 
   Bethesda, MD 20892 
 
   Tel: 240-276-7012 
 
   Fax: 240-276-7848 
 
   Malgorzata.wojtowicz@nih.gov  
 
Nurse Consultant: 
   Lisa Bengtson, MSN, RN, OCN 
 
 
 
   NIH National Cancer Institute  
 
 
 
   Bethesda, MD 20892  
 
 
 
   Lisa.bengtson@nih.gov  
 
IND Sponsor: 
NCI/Division of Cancer Prevention  
 
IND#:  
 
 
 
Agent(s)/Supplier: 
NCI/DCP  
 
 
Protocol Version Date: Nov 12, 2025 
 
Protocol Revision or   
Amendment #: 
Version 8.5  
 

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SCHEMA 
 
Phase II Randomized, Double-Blind, Placebo-Controlled Trial of Broccoli Seed and Sprout Extract (BSSE) to 
Evaluate Detoxification of Carcinogens in Firefighters    
 
Study Population: Healthy, non-smoking incumbent firefighters  
↓ 
Visit 1/Screening: Informed consent, clinical evaluation (medical history including date (month and year required, if date 
cannot be recalled 15th of month will be used) of last physical exam, height/weight, vital signs), baseline symptoms, blood 
draw for clinical labs and for research tests, concomitant medications (conmeds) and supplement use, Karnofsky 
performance status, baseline tobacco/alcohol and COVID assessment, pregnancy test (if applicable). Participants using 
any supplements containing active compounds in cruciferous vegetables must cease taking these supplements at least 14 
days prior to Baseline Visit. Participants must refrain from using non-study acetaminophen (or acetaminophen containing 
products) for 72 hours before the Baseline Visit. 
↓ 
Randomization: 66 participants will be randomized 1:1 to the following intervention: 
BSSE OR matched placebo  
 ↓ 
Visit 2/Baseline (can occur up to 12 weeks from screening visit): Vital signs, conmeds, pregnancy test (if applicable), any 
change in health status since visit 1 screen (if change reported, participant will be re-evaluated for eligibility as clinically 
indicated), a single dose of acetaminophen administration (2 x 500 mg), urine collection before acetaminophen dosing and 
for 8 (-1 and + 2) hours post acetaminophen dosing. Pre-Acetaminophen Spot Urine Questionnaire will be completed. 
Staff will provide urine collection supplies for the 8-hr urine collection, an Adverse Event (Symptom)/New Medicaton 
diary and Intake Calendar including instructions for use of study agent (BSSE). The 8-hr urine will be collected by the 
Tucson Fire Department (TFD) research liaisons or our study staff within 24 hours of completion. Staff will dispense 
study agent, and urine collection supplies for Post-Fire Spot Urine Questionnaire if fire/fuel leak activity occurs during the 
intervention period. 
↓ 
Study Agent Intervention Period: Participants take BSSE or matched placebo 4 tablets once daily for 12 weeks based 
on randomization assignment. The intervention period allows a window of −14 days to +7 days. Participants to collect a 
spot urine within 8 ± 2 hours post each fire/fuel leak activity and complete Post-Fire/Fuel Leak Spot Urine Questionnaire. 
The Spot Urine and Post-Fire/Fuel Leak Spot Urine Questionnaires will be collected by the TFD research liaisons or 
study staff within 24 hours of completion. Interim phone/email contact at week 1 (+7 days), 4±7 days, 8±7 days to assess 
adverse events (AEs), study agent compliance, and adequate completion of Adverse Event (Symptom)/New Medication 
Diary and Intake Calendar 
↓  
Visit 3/End-of-Intervention:  Occurs at 12 weeks with an allowable window of −14 to +7 days while the participant 
remains active on intervention.): Weight, vital signs, assess conmeds, collect study agent, review adverse events (AEs), 
collect Intake Calendar and Adverse Event (Symptom)/New Medication Diary, assess agent compliance, blood draw for 
clinical labs and research tests, Karnofsky Performance Status, follow-up tobacco/alcohol and COVID assessments, a 
single dose of acetaminophen administration (2 x 500 mg), spot urine collection before acetaminophen dosing. Pre-
acetaminophen Spot Urine Questionnaire will be completed.  Staff will provide urine collection supplies for the 8 (-1 and 
+2) hours post-acetaminophen urine collection. The 8-hr cumulative urine will be collected by the TFD research liaisons 
or our study staff within 24 hours of completion. 
↓ 
Follow-up: Telephone or email assessment of AEs 2 weeks ±7 days post intervention 
↓ 
Endpoints: 
Primary Endpoint: Change in urinary excretion of acetaminophen mercapturate after 12 weeks of study agent 
intervention,  assessed within the allowable window of 14 days before to 7 days after the 12-week time point.  

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Secondary Endpoints: Change in urinary excretion of acetaminophen glucuronide after 12 weeks of study agent 
intervention within the allowable window of 14 days before to 7 days after the 12-week time point, and assessment of the safety and 
tolerability of BSSE. 
Exploratory Endpoints: Glutathione S-Transferase Mu 1 (GSTM1) and Glutathione S-Transferase Theta 1 (GSTT1) 
genotypes and interaction with detoxification; urinary metabolomics; urinary excretion of mercapturic acid of benzene 
after fire/fuel leak activities; urinary excretion of metabolites of polycyclic aromatic hydrocarbons (PAHs) after fire/fuel 
leak activities; epigenetic modifications (microRNA and DNA methylation)  
 
  
 

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TABLE OF CONTENTS 
 
COVER PAGE ................................................................................................................................ 5 
SCHEMA ........................................................................................................................................ 7 
1. 
OBJECTIVES ...................................................................................................................... 12 
2. 
BACKGROUND .................................................................................................................. 12 
2.1 
Cancer Risk Among Firefighters ............................................................................................................... 12 
2.2 
Broccoli Seed and Sprout Products ........................................................................................................... 14 
2.3 
Rationale .................................................................................................................................................... 15 
3. 
SUMMARY OF STUDY PLAN ......................................................................................... 18 
4. 
Participant SELECTION ...................................................................................................... 19 
4.1 
Inclusion Criteria ....................................................................................................................................... 19 
4.2 
Exclusion Criteria ...................................................................................................................................... 20 
4.3 
Participant Consideration Regarding Alcohol Consumption ..................................................................... 21 
4.4 
Inclusion of Women and Minorities .......................................................................................................... 21 
4.5  
Recruitment ............................................................................................................................................... 21 
4.6  
Planned Accrual ........................................................................................................................................ 22 
5. 
REGISTRATION PROCEDURES ...................................................................................... 22 
5.1   Investigator and Research Associate Registration with CTEP .................................................................. 22 
6. 
NCI CENTRAL INSTITUTIONAL REVIEW BOARD..................................................... 23 
7. 
AGENT ADMINISTRATION ............................................................................................. 24 
7.1 
Dose Regimen and Dose Groups ............................................................................................................... 24 
7.2 
BSSE Administration ................................................................................................................................ 24 
7.3 
Run-in Procedures ..................................................................................................................................... 24 
7.4 
Contraindications....................................................................................................................................... 24 
7.5 
Concomitant Medications .......................................................................................................................... 25 
7.6 
Dose Modification ..................................................................................................................................... 25 
7.7 
Adherence/Compliance ............................................................................................................................. 25 
8. 
PHARMACEUTICAL INFORMATION ............................................................................ 26 
8.1 
BSSE (IND # 
 IND NCI, Division of Cancer Prevention)............................................................ 26 
8.2  
Acetaminophen .......................................................................................................................................... 27 
8.3 
Reported Adverse Events and Potential Risks ........................................................................................... 27 
8.4  
Availability ................................................................................................................................................ 28 
8.5 
Agent Distribution ..................................................................................................................................... 29 
8.6  
Agent Accountability ................................................................................................................................ 29 
8.7 
Packaging and Labeling ............................................................................................................................ 29 
8.8 
Storage ....................................................................................................................................................... 30 
8.9 
Registration/Randomization ...................................................................................................................... 30 
8.10 Blinding and Unblinding Methods ............................................................................................................ 30 
8.11 Agent Destruction/Disposal ....................................................................................................................... 31 
9.  
CLINICAL EVALUATIONS AND PROCEDURES ......................................................... 31 
9.1 
Schedule of Events .................................................................................................................................... 31 
9.2 
Schedule of Urine Samples and Spot Urine Questionnaire Collection ...................................................... 34 
9.3 
Baseline Testing/Pre-Study Evaluation ..................................................................................................... 35 
9.4 
Evaluation During Study Intervention ....................................................................................................... 35 
9.5 
Evaluation at Completion of Study Intervention ....................................................................................... 36 
9.6 
Post-intervention Follow-up Period ........................................................................................................... 36 
9.7 
Methods for Clinical Procedures ............................................................................................................... 36 
10.  CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION ................................. 36 
10.1 Primary Endpoint ...................................................................................................................................... 37 
10.2 Secondary Endpoints ................................................................................................................................. 37 
10.3 Off-Agent Criteria ..................................................................................................................................... 37 

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10.4 Off-Study Criteria ...................................................................................................................................... 37 
10.5 Study Termination ..................................................................................................................................... 37 
11. CORRELATIVE/SPECIAL STUDIES ............................................................................... 37 
11.1 Rationale for Methodology Selection ........................................................................................................ 37 
11.2 Comparable Methods ................................................................................................................................ 38 
12. SPECIMEN MANAGEMENT ............................................................................................ 39 
12.1 Laboratories ............................................................................................................................................... 39 
12.2 Collection and Handling Procedures ......................................................................................................... 39 
12.3 Shipping Instructions ................................................................................................................................. 40 
12.4 Tissue Banking .......................................................................................................................................... 41 
13. REPORTING ADVERSE EVENTS .................................................................................... 41 
13.1 Adverse Events .......................................................................................................................................... 42 
13.2 Serious Adverse Events ............................................................................................................................. 43 
14. STUDY MONITORING ...................................................................................................... 45 
14.1 Data Management ...................................................................................................................................... 45 
14.2   Electronic Case Report Forms ................................................................................................................... 45 
14.3 Source Documents ..................................................................................................................................... 46 
14.4 Data and Safety Monitoring Plan .............................................................................................................. 46 
14.5 Sponsor or FDA Monitoring ..................................................................................................................... 46 
14.6 Record Retention ....................................................................................................................................... 46 
14.7 Cooperative Research and Development Agreement (CRADA)/Clinical Trials Agreement (CTA) ........ 46 
15. STATISTICAL CONSIDERATIONS ................................................................................. 48 
15.1 Study Design/Description .......................................................................................................................... 48 
15.2 Randomization/Stratification ..................................................................................................................... 48 
15.3 Sample Size ............................................................................................................................................... 48 
15.4 Primary Objective, Endpoint(s), Analysis Plan ......................................................................................... 49 
15.5 Secondary Objectives, Endpoints, Analysis Plans..................................................................................... 49 
15.6 Reporting and Exclusions .......................................................................................................................... 50 
15.7 Evaluation of Toxicity ............................................................................................................................... 50 
15.8 Evaluation of Response ............................................................................................................................. 50 
15.9 Interim Analysis ........................................................................................................................................ 50 
15.10 Ancillary Studies ....................................................................................................................................... 51 
16. REGULATORY And ETHICAL CONSIDERATIONS ..................................................... 51 
16.1 Required Documents ................................................................................................................................. 51 
16.2 Informed Consent ...................................................................................................................................... 51 
16.3 Collection of Regulatory Documents ........................................................................................................ 51 
16.4 Other .......................................................................................................................................................... 52 
17.  ROSTER MANAGEMENT ................................................................................................. 52 
18. FINANCING, EXPENSES, AND/OR INSURANCE ......................................................... 52 
REFERENCES .............................................................................................................................. 52 
APPENDIX A ............................................................................................................................... 56 
APPENDIX B ............................................................................................................................... 57 
ALCOHOL AND TOBACCO QUESTIONNAIRE INSTRUCTIONS .............................................................. 57 
ALCOHOL ASSESSMENT-- BASELINE ......................................................................................................... 58 
ALCOHOL ASSESSMENT - FOLLOW-UP ...................................................................................................... 61 
TOBACCO ASSESSMENT – BASELINE ......................................................................................................... 63 
TOBACCO ASSESSMENT - FOLLOW-UP ...................................................................................................... 67 
NATIONAL AND LOCAL RESOURCES TO HELP WITH ALCOHOL ABUSE AND ALCOHOLISM ...... 70 
NATIONAL AND LOCAL RESOURCES TO HELP WITH QUITTING SMOKING ..................................... 71 
Appendix C  Participant clinical trial wallet card ......................................................................... 72 
APPENDIX D ............................................................................................................................... 73 
COVID 19 ASSESSMENT INSTRUCTIONS .................................................................................................... 73 
CP-CTNET COVID-19 BASELINE ASSESSMENT ......................................................................................... 74 

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CP-CTNET COVID-19 FOLLOW-UP ASSESSMENT ..................................................................................... 75 
APPENDIX E ................................................................................................................................ 77 
PRE-ACETAMINOPHEN SPOT URINE QUESTIONNAIRE ................................................... 77 
APPENDIX F ................................................................................................................................ 79 
POST-FIRE/FUEL LEAK SPOT URINE Collection QUESTIONNAIRE ................................. 79 
APPENDIX G ............................................................................................................................... 80 
REMOTE CONSENT INSTRUCTIONS ..................................................................................... 80 
APPENDIX H ............................................................................................................................... 81 
FIRE/FUEL INCIDENT REPORT ............................................................................................... 81 
 
 
 

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1. 
OBJECTIVES 
 
This is a phase II, randomized, double-blind, placebo-controlled trial to determine if BSSE intervention can 
upregulate Phase II detoxification pathways in firefighters.  
 
1.1 
Primary Objective  
• To determine whether BSSE increases the urinary excretion of mercapturic acid of acetaminophen, a 
surrogate for detoxification of the carcinogen benzene in healthy, incumbent firefighters. 
 
1.2 
Secondary Objectives  
• To evaluate the effects of BSSE on urinary excretion of acetaminophen glucuronide, a surrogate for 
detoxification of polycyclic aromatic hydrocarbons (PAHs). 
• To evaluate the safety and tolerability of BSSE.  
 
1.3 
Exploratory Objectives 
• To determine whether the GSTM1 and GSTT1 genotypes are important genetic modulators of BSSE-
induced detoxification of carcinogens.  
• To evaluate the effects of BSSE on urinary metabolomics.  
• To determine the effects of BSSE on the urinary excretion of the mercapturic acid of benzene after 
fire/fuel leak activities. 
• To determine the effects of BSSE on the urinary excretion of metabolites of PAHs after fire/fuel leak 
activities. 
• To determine the effects of BSSE on epigenetic modifications (blood microRNA and DNA 
methylation). 
 
2. 
BACKGROUND 
 
2.1 
Cancer Risk Among Firefighters 
There are approximately 1.06 million active firefighters in the United States1. Firefighters are routinely exposed 
to carcinogens during the course of their daily duties particularly from smoke exposure arising from active fire 
rescue, structural or incidental firefighting or combustion2. There is an expansive number of toxic components 
prevalent in smoke. Various elements including soot, polycyclic aromatic hydrocarbons (PAH), gases, and 
volatile organic compounds (VOCs) among others are exposures that firefighters encounter after close proximity 
to flames and after changing their protective equipment3. All fires release toxic and carcinogenic substances 
including benzene, 1,3-butadiene, and formaldehyde4. Additionally, newer building materials and furnishings 
that combust and pyrolyze result in significantly more toxicants from smoke than wildland fires or fires from 
buildings predating newer construction materials. Burgess et al. estimated the time firefighters spent in structural 
fires in two cities in Arizona including Tucson and Phoenix; total firefighter activity was 47.6 hours/year and 50 
hours/year in Phoenix and Tucson, respectively, displaying significant exposure to potential carcinogens5. Wood-
fueled fires remains a leading hazard that firefighters encounter globally. Wood smoke emits numerous 
compounds including PAHs and methylphenols (MP)6. These compounds, many of which are known 
carcinogens, increase the risk of cancer in firefighters.  
 
Several studies to date have demonstrated that firefighters are at an increased risk of developing various 
malignancies including melanoma, multiple myeloma, acute myeloid leukemia, prostate, kidney, brain, and 
respiratory tract cancers among others2, 7, 8. An extensive pooled cohort study of approximately 30,000 United 

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States firefighters from San Francisco, Chicago, and Philadelphia from 1950-2009 showed excess cancer 
mortality and incidence predominantly in the digestive (including colon) and respiratory tracts as well as the 
kidneys in male firefighters, and increased bladder cancer among women firefighters. This was the first study to 
also report an elevated mortality and incidence of malignant mesothelioma among US firefighters9. A study of 
34,796 male and 2,017 female career firefighters in Florida from 1981 to 1999 demonstrated an increased risk of 
bladder, testicular, and thyroid cancers in male firefighters and cervical and thyroid cancers in female firefighters, 
as well as Hodgkin disease10. The Florida Firefighter Cancer Registry evaluated 109,009 career firefighters from 
1981-2014 and reported an increased risk of thyroid, prostate, testicular, and melanoma cancers in male 
firefighters younger than age 50 while female firefighters were noted to have an increased risk of thyroid and 
brain cancer11.    
 
While several studies have established carcinogen exposure with an increased risk of malignancy among 
firefighters, there remains limited data pertaining to the cellular mechanistic drive. Epigenetic modifications 
including DNA methylation (DNAm), histone modification, and dysregulated microRNA (miRNA) expression 
are established in carcinogenesis with regulation of oncogenes and tumor suppressor genes. Studies of Tucson 
firefighters have demonstrated epigenetic modification associated with occupational exposures12. Jeong et al. 
examined blood from 52 incumbent and 45 new recruit nonsmoking firefighters for miRNA expression and found 
at least a 1.5-fold significant difference between groups with 9 microRNA identified13. Among incumbent 
firefighters, two of the three microRNAs with increased expression had activities in line with cancer promotion, 
and six of the miRNA with decreased expression had activities in line with tumor suppression. Similarly, Jung et 
al performed a longitudinal evaluation of whole blood miRNA expression in 52 new recruits prior to live-fire 
training and 20-37 months later taking into account cumulative fire-hours, fire-runs and time since most recent 
fire14. The study identified an increase in oncogenic miRNA and decrease in tumor suppressive miRNA with 
exposure. DNAm has also been shown as a cellular mechanism by which cancer risk is increased among 
firefighters. Zhou, et al. obtained peripheral blood samples from 45 incumbent and 41 new recruits non-smoking 
male firefighters which were subsequently analyzed for DNAm15. Genome-wide methylation was able to identify 
accurately the incumbent from new recruits as well as by the years of service among incumbents. The Sirtuin 
signaling pathway, p53 signaling, and 5’ AMP-activated protein kinase (AMPK) signaling were identified as 
potential mechanisms associated with increased cancer risk among firefighters. Beyond epigenetic changes, 
genotoxicity has been reported in Danish firefighters16. 
 
Several studies have utilized the training fire setting to evaluate the potential health hazard from smoke exposure 
in firefighters. An Australian training facility evaluated human resources records and self-reported data in a 
retrospective cohort and showed that individuals in the high probability of exposure group had an increased risk 
of overall cancers, testicular cancer, and melanoma relative to the general population17. Fire departments in 
Ontario where firefighter training exercises took place in burn houses examined air samples and skin and urine 
specimens of 28 firefighters prior to and after training fire exposure6. With wood as the primary fuel source 
employed in the training exercise, MP and PAH metabolites were measured. The study noted that air samples 
demonstrated 5 times greater concentration of MP than PAH whereas skin wipe samples from multiple body sites 
showed evidence of whole-body smoke exposure. Urine samples displayed several MPs and deconjugated PAH 
metabolites including hydroxynaphthalene, hydroxyfluorene, and hydroxyphenanathrene, among others. Despite 
large between-subject variations in operational roles, the creatinine-normalized levels of these markers remained 
significantly elevated. Recently, Rosting et al measured urine and air benzene and toluene concentrations pre- 
and post- fire drill in 9 firefighters. The study detected the benzene metabolite, s-phenylmercapturic acid (SPMA) 
and toluene metabolite s-benzylmercapturic acid (SBMA) in all urine samples 3 hours after the drill while 
benzene and toluene were also detected in air samples18. Structural fire training has been shown to contribute to 

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firefighters’ select chemical exposure as shown by urine analyzed for PAH metabolites and breath analyzed for 
VOCs including benzene8. The study found an increase in nearly all urinary PAH metabolites from pre- to 3-hour 
post training. Instructors that supervised three trainings per day also had an increased concentration at each 
collection with a 30-fold increase in 1-hydroxypyrene. Moreover, breath concentrations of benzene also increased 
2- to 7-fold immediately following training.  
 
With increasing awareness of the occupational exposure to hazardous chemicals during firefighting, advances in 
personal protective equipment (PPE) with the addition of particulate-blocking hoods as well as fire department 
protocols to include routine laundering of PPE after fire exposure are being developed19. Mayer et al. studied 
three PPE ensembles including a new knit hood, turnout jacket and pants, new particulate-blocking hood, turnout 
jacket and pants, and laundered particulate-blocking hood, with laundered turnout jacket and pants. Pre- and post-
fire training exposure exhaled breath samples and personal air sampling outside and inside the turnout jacket was 
collected in 24 firefighters to measure exposure to VOCs and naphthalene. The study found that benzene, toluene, 
and naphthalene concentrations inside the turnout jackets were similar to that of outside the turnout jacket 
concentrations. Additionally, benzene concentrations in breath samples were significantly elevated from pre- to 
post-fire training irrespective of PPE ensemble. This highlights the importance of developing alternative 
strategies for risk reduction.  
 
The commonly exposed chemicals such as benzene and PAHs are activated by Phase I metabolism to reactive 
metabolites and are detoxified through Phase II metabolism20. Cytochrome P450-dependent oxidation of benzene 
produces benzene oxide which is further either excreted as spontaneously rearranged phenol or further 
metabolized to hydroquinone and other metabolites21. On a cellular level, benzene oxide disrupts cellular function 
by reacting with peptides and proteins21, 22 Benzene oxide can be detoxified by glutathione S transferases (GST) 
to produce (SPMA). Similarly, the carcinogenic effect of PAH is dependent on oxidative metabolism with 
production of dihydrodiol epoxide metabolites and PAH quinones resulting in DNA damage leading to mutations 
in oncogenes and tumor suppressor genes23. PAH quinones and hydroxylated PAHs also modulate cell 
proliferation, DNA damage response via mitogen-activated protein kinases, Akt kinase, and calcium-dependent 
signaling pathways24. Glucuronidation of these active PAH metabolites represents a detoxification pathway of 
PAHs. Enhancing the detoxification of the commonly exposed chemicals is an arena that warrants further 
investigation.  
 
2.2 
Broccoli Seed and Sprout Products 
 
Broccoli extract potently induces phase II detoxification enzymes25 including GST, aldehyde dehydrogenase 
(ALDH), and NAD(P)H:quinone oxidoreductase (NQ01) which mitigate the effects of environmental 
carcinogens including benzene, aldehydes, and PAHs by enhancing their detoxification 26. More than 80% of 
inducer activity is driven by the phytochemical sulforaphane (SF) which is produced when its precursor 
glucoraphanin (GR) is hydrolyzed by myrosinase during food preparation or chewing 27. Mechanistically, SF 
disrupts the polyubiquitination of the NRF2 transcription factor mediated by its inhibitory protein, KEAP1. This 
frees NRF2 to translocate to the nucleus and bind to antioxidant response elements (AREs) in the promoter 
regions of target genes, inducing their transcription 28, 29. As GR is 20-50 times more concentrated in broccoli 
seeds relative to mature plants 27, 30, broccoli seed products (BSPs) were developed as a chemopreventive agent 
against carcinogen-induced cancers, and the safety and pharmacokinetics (PK) of various BSPs in humans has 
been established 29, 31, 32.  
 

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Proof-of-concept clinical trials in healthy volunteers have shown that BSPs rich in GR and/or SF are well-
tolerated and promote rapid, sustained detoxification of the airborne pollutants acrolein and benzene 29, 31, 33. In 
preclinical studies, SF has exhibited chemopreventive activity against carcinogen-induced stomach, skin, and 
breast cancers 34, 35. Studies in Nrf2-/- mice have shown that the chemopreventive effect of SF against 
benzo[a]pyrene-induced gastric cancer and 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer 
depends on the NRF2 signaling pathway 34. The relevance of NRF2 to oral cancer chemoprevention is highlighted 
by the enhanced susceptibility of Nrf2-/- mice to oral cancer induced by the carcinogen 4-nitroquinoline-1-oxide 
(4NQO), and the reduced susceptibility of Keap1-/- mice36. We have shown that SF reduces both the incidence 
and total burden of oral tumors in 4NQO-treated mice32.   
 
Avmacol (Broccoli Seed and Sprout Extract): The regular and extra strength Avmacol products (Avmacol and 
Avmacol ES, manufactured under GMP standards by Nutramax Laboratories, Inc.) are commercially available 
dietary supplements that contain GR plus the fully active enzyme myrosinase, thus yielding a higher and more 
consistent dose of SF upon ingestion. The regular Avmacol tablets contained GR-rich broccoli seed extract, 
freeze-dried broccoli sprouts for the myrosinase source, and the inert excipients required to form a tablet. 
Avmacol ES is the current, commercially available BSSE. Each tablet contains double the quantity of GR and 
myrosinase activity as the regular formulation. In addition, Avmacol ES contains extract of maitake mushroom, 
hypothesized to augment innate immunity37.  
 
2.3 
Rationale 
 
We have completed a series of healthy volunteer studies to confirm the bioavailability of SF when delivered as 
Avmacol, hereafter referred to as broccoli seed and sprout extract (BSSE) with results summarized below. 
 
• NCT02800265. In this pilot study, 10 healthy volunteers were treated with the regular strength BSSE, 8 
tablets daily for 3 days. This represented an exposure of 100 mg GR per day (SF equivalent of 140 µmol 
per day if 100% converted). Overnight urine (12 hours) was collected at baseline and after the third dose 
then evaluated by liquid chromatography tandem mass spectrometry for SF and its glutathione conjugates, 
SF-cysteine, and SF-N-acetylcysteine. Urinary excretion of SF and its metabolites was negligible for all 
participants at baseline. Relative to multiple historical studies administering GR, where mean 
bioavailability as SF metabolites was 5% over 24 hours, mean bioavailability as SF metabolites for BSSE 
was 20% over 12 hours (Bauman, unpublished data). In prior studies of GR, 60% of the bioavailable dose 
of GR is excreted in the urine as SF metabolites during the first 12 hours, and 40% in the subsequent 12 
hours. The estimated total mean bioavailability of BSSE was therefore estimated at 35%. This hypothesis 
was generally confirmed in a pure bioavailability study conducted by collaborators at Johns Hopkins, 
where urine was collected for 24 hours following a single dose of BSSE. In this study, total excretion of 
SF metabolites was 34.3% of dose (SD 9.7%) in the pilot phase and 32.8% of dose in the proton pump 
inhibitor phase38.  
• NCT03182959. In a recently completed pilot study, 11 subjects who had completed curative-intent 
treatment for tobacco-related head and neck squamous cell cancer were treated with two doses of BSSE 
in a randomized, crossover design: 4 tablets and 8 tablets for 4 weeks each, separated by a 4-week 
washout. The primary endpoint is NRF2 upregulation in buccal cells as measured by upregulation of 
mRNA of the target gene NQO1 and secondary endpoints include bioavailability of BSSE as SF 
metabolites and modulation of mucosal signatures of inflammation and immunity (NanoString nCounter® 
PanCancer panel). Analyses of bioavailability and mucosal bioactivity are ongoing. 

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• NCT03402230. We recently published the results of this randomized, crossover trial evaluating the 
detoxification of benzene and other tobacco carcinogens by BSSE in otherwise healthy current smokers 
(≥ 20 pack-years) 33, 39. Each participant was treated for 2 weeks with both low and higher-dose BSSE 
(148 µmol vs. 296 µmol of GR daily), separated by a 2-week washout, with randomization to low-high 
vs. high-low sequence.  
 
 
 
The primary endpoint of NCT03402230 was detoxification of benzene, measured by urinary excretion of its 
mercapturic acid, SPMA. Secondary endpoints included bioavailability, detoxification of acrolein and 
crotonaldehyde, modulation by GST genotype, and toxicity. Forty-nine participants enrolled, including 26 (53%) 
females with median use of 20 cigarettes/day. Low and higher-dose BSSE showed a mean bioavailability of 11% 
and 10%, respectively. As shown in Table 1 above, higher-dose BSSE significantly upregulated urinary excretion 
of the mercapturic acids of benzene (p = 0.04), acrolein (p < 0.01), and crotonaldehyde (p = 0.02), independent 
of GST genotype. Low dose BSSE also significantly upregulated urinary excretion of SPMA (p = 0.05). 
 
We have completed pilot studies to assess the metabolomic changes in firefighters pre- and post- fire. Several 
urinary metabolites that are associated with cancer risk or are known carcinogens were identified in the post-fire 
urine58. We plan to explore the modulatory effects of BSSE on metabolomic changes. To facilitate the 
metabolomic analysis, we are collecting additional information on beverage consumption history, over-the-
counter and prescription medication use, consumption of supplements/vitamins, pre-workouts and protein 
powders/shakes, blood and/or plasma donation history and turnout gear fit, which can impact the abundance of 
various metabolites.  
 
Our overall hypothesis is that BSSE intervention can lead to enhanced detoxification of the commonly exposed 
carcinogens in firefighters, which could ultimately reduce their cancer risk. We propose a phase II, randomized, 
Table 1. Urinary excretion of carcinogen metabolites in NCT03402230 
Carcinogen 
Metabolite 
BSSE Dose 
Level 
Pre (95% CI) 
pmol/mgCr 
Post (95% CI) 
pmol/mgCr 
Post/Pre 
(95% CI) 
p-
Value 
Benzene 
(SPMA) 
Low 
7.8 (6.3, 9.6) 
9.0 (7.2, 11.3) 
1.2 (1.0, 1.3) 
0.05 
High 
7.6 (6.2, 9.4) 
9.1 (7.3, 11.3) 
1.2 (1.0, 1.4) 
0.04 
Acrolein 
(3-HPMA) 
Low 
9934.5 
(8008.0, 
12,324.4) 
11,032.1 
(9161.1, 
13,285.2) 
1.1 (1.0, 1.3) 
0.11 
High 
9750.0 
(7903.7, 
12,027.6) 
12,450.1 
(10,658.8, 
14,542.5) 
1.3 (1.1, 1.5) 
<0.01 
Croton-aldehyde 
(3-HMPMA) 
Low  
10,962.6 
(9026.8, 
13,313.6) 
11,482.0 
(9677.5, 
13,622.9) 
1.1 (0.9, 1.2) 
0.56 
High 
10,808.6 
(8947.3, 
13,057.0) 
12,795.1 
(10,922.9, 
14,988.1) 
1.2 
(1.02,1.37) 
0.02 

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double-blind, placebo-controlled trial to evaluate if BSSE intervention can enhance Phase II detoxification 
pathways in firefighters. The trial was initially designed to determine the effects of BSSE on detoxification of 
carcinogens in the flashover training setting because this training activity provides a controlled exposure 
environment. Commonly exposed chemicals such as benzene and PAHs were consistently detected in firefighters’ 
urine collected following the training fire9,27. 
 
The first cohort of six participants completed the study under protocol versions 8.0 and 8.1. The urine samples 
were analyzed for levels of the benzene metabolite, SPMA (the primary study endpoint). Contrary to expectations, 
SPMA levels were not consistently elevated in post-flashover samples compared to baseline levels (see Table 2). 
It is likely that exposure to benzene during flashover is minimal. 
 
Table 2. Total amount of SPMA excreted over 24 hours (pmol). 
Participant ID – intervention prior to flashover 
training 
Baseline Post flashover training Post burn room training1 
315 - placebo 
848.25 
1660.27 
385.33 
316 – BSSE 
776.54 
1089.17 
2374.18 
317- BSSE 
1082.33 
650.58 
767.52 
318 - placebo 
342.52 
605.96 
310.36 
319 - BSSE 
755.12 
650.83 
503.92 
320 - placebo 
794.85 
724.70 
Missed 
collection 
1 no intervention prior to burn room training 
Based on these preliminary data, the study was temporarily closed to accrual to determine if any changes were 
needed in the study design and to optimize the study design if necessary. During the temporary closure, we 
unblinded the treatment assignment of this first cohort of six participants and found that the inconsistent elevation 
of benzene metabolite levels post-flashover could not be explained by the intervention (BSSE vs. placebo) 
received prior to flashover (see Table 2). 
 
We also worked in parallel through research collaboration to assess benzene exposure during another type of 
firefighter training (burn room training) and found that the SPMA levels were also not consistently elevated after 
the burn room training fire compared to baseline levels (see Table 2). The collective information suggests that 
exposure to benzene during the current fire training protocols is minimal, possibly due to procedures implemented 
by the fire department to minimize carcinogen exposure during training fires. These preliminary data suggest that 
measuring the benzene metabolite after the training fire is not a viable endpoint to evaluate the effects of BSSE 
on the detoxification of carcinogens due to the low and variable benzene exposure during the training fire. 
 
Based on the available information, we selected to assess the intervention effects on Phase II detoxification 
pathways by assessing changes in acetaminophen metabolism as a surrogate for detoxification of benzene and 
PAHs. Metabolism of acetaminophen occurs through three detoxification pathways: glucuronidation, sulfation, 
and glutathione conjugation. This makes acetaminophen metabolism a good model to study the detoxification 
pathways involved in protection against exposure to a wide range of exogenous and endogenous toxins. Assessing 
acetaminophen metabolism has been used previously as a relatively non-invasive approach to determine the 
effects of Spanish black radish, a cruciferous vegetable, on Phase II detoxification pathways40.  Changes in urinary 
acetaminophen mercapturate after acetaminophen administration will be used as a surrogate biomarker for the 
upregulation of glutathione conjugation, the primary pathway responsible for the detoxification of benzene to 

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SPMA. Changes in urinary acetaminophen glucuronide after acetaminophen administration will be used as a 
surrogate biomarker for the upregulation of glucuronidation, the primary pathway responsible for the 
detoxification of PAHs. 
 
We also propose a longer, 10-week intervention period to assess the modulatory effects of BSSE on epigenetic 
modifications, including dysregulated miRNA and DNA methylation, observed in incumbent firefighters.  The 
longer intervention period provides the unique opportunity to explore the effects of BSSE on the detoxification 
of carcinogens, including benzene and PAHs, which firefighters are exposed to during real world firefighting 
activities, such as actual fire and/or fuel leaks. Because of the unpredictability of occurrence of the real world fire 
and/or fuel leaks and uncontrolled exposure in actual fire and fuel leaks, we have moved the assessment of the 
detoxification of benzene and PAHs to exploratory objectives. For these exploratory endpoints, we plan to collect 
documentation of fire/fuel leak activities throughout the intervention period and adjust for these potential 
confounders.   
  
Increasing evidence suggests that tobacco and alcohol use are risk factors in the development of intraepithelial 
neoplasia and cancer. In addition, tobacco and alcohol use may adversely affect agent intervention, for example 
by altering the safety profile or metabolism of a drug. Standardized assessments of tobacco and alcohol use during 
clinical trials will aid in understanding the potential relationship between the use of these products and clinical 
endpoints or cancer prevention biomarkers. Therefore, NCI, DCP is including assessment of tobacco and alcohol 
use at baseline and end of treatment, to determine the potential impact of tobacco and alcohol use on 1) treatment 
toxicity and symptom burden, and 2) the efficacy of treatment intervention.  
 
The NCI, DCP Assessment of COVID-19 exposure and vaccine status at baseline and end of study will be used 
to determine the potential impact on 1) treatment toxicity and symptom burden, and 2) the efficacy of treatment 
intervention.  
 
3. 
SUMMARY OF STUDY PLAN 
 
Study Design: This is a phase II, randomized, double-blinded, placebo-controlled study to evaluate whether BSSE 
can upregulate Phase II detoxification pathways in firefighters. We plan to randomize a total of 66 participants 
(1:1 randomization) to receive BSSE or matched placebo for 12-weeks intervention, with an allowable window of 14 
days before or 7 days after the scheduled 12-week time point. Based on an estimated attrition rate of < 10%, we expect 
to have a minimum of 60 evaluable participants (a minimum of 30 participants per intervention assignment).   
 
Treatment Plan and Primary Assessments: After consent and confirmation of eligibility, participants will be 
randomized to BSSE or placebo in a double-blinded fashion. During their baseline visit, all participants will be 
asked to provide a spot urine sample and to participate in a short single-dose acetaminophen study with urine 
collection to assess their baseline metabolism of acetaminophen. Prior to starting, participants will be asked to 
complete a Pre-Acetaminophen Spot Urine Questionnaire (Appendix E) and to provide a spot urine.  Afterward, 
they will be asked to take two 500 mg acetaminophen tablets (i.e., a single extra strength dose of Tylenol) and 
collect all urine over the next 8 (-1 and + 2) hours (i.e., cumulative urine collection). After completing the pre 
and post-acetaminophen urine study, participants will take four tablets BSSE (Avmacol ES) or placebo daily for 
a 12-week intervention period, with an allowable window of 14 days before to 7 days after the scheduled 12-
week time point.   
 

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To assess the activity of BSSE to detoxify real-world exposures to carcinogens benzene and PAHs, during the intervention 
period, all participants will be asked to collect a spot urine within 8 ± 2 hours of their post-fire/fuel leak activity 
and to complete a Post-Fire/Fuel Leak Spot Urine Questionnaire (Appendix F). This post-fire/fuel leak spot urine 
and the Post-Fire/Fuel Leak Spot Urine Questionnaire (Appendix F) will be collected by the Tucson Fire 
Department (TFD) research liaisons or study staff within 24 hours of completion.  
 
At the end of intervention, participants will repeat the acetaminophen metabolism study. Participants must 
complete the end-of-intervention assessment prior to stopping the study agent to ensure that the acute effects of 
BSSE on acetaminophen metabolism can be accurately evaluated. During their end-of-study visit, all participants 
will again be asked to provide a spot urine sample and complete the Pre-Acetaminophen Spot Urine Questionnaire 
(Appendix E) before being instructed to take two 500 mg acetaminophen tablets and collect all urine over the 
next 8 (-1 and + 2) hours. With the baseline data, the end-of-intervention acetaminophen study will be used to 
assess the change in the metabolism of acetaminophen with intervention. Participants will discontinue the study 
agent only after completing their end-of-intervention pre- and post-acetaminophen urine collections. 
 
Additional Study Assessments: Blood collection will occur at screening and on the last visit for safety labs and 
assessment of epigenetic modifications. Blood collected at screening will be used for GST genotyping. Since up 
to 12 weeks may occur between Visit 1 (screening) and Visit 2 (baseline), participants will be asked at Visit 2 
about any changes in their health status since screening. If a change is reported, the participant will be re-evaluated 
for eligibility as clinically indicated, including repeat clinical laboratory testing as needed. Participants will be 
contacted by telephone or email (per their telecommunications preference) at 1 week + 7 days, 4 weeks ± 7 days 
and 8 weeks ± 7 days for adverse events (AEs), adherence to the study agent, and assessment of adequate 
completion of Adverse Event (Symptom)/New Medication Diary and Intake Calendar. Intake Calendar and 
Adverse Event (Symptom)/New Medication Diary will be collected during the end of intervention visit. Participants 
will be contacted by telephone or email (per their telecommunications preference) 2 weeks ±  7 days after the end 
of the treatment period for AE assessment. 
 
4. 
PARTICIPANT SELECTION 
 
The Protocol Risk for Auditing  for this study is low. 
 
High Risk studies require 100% review and confirmation of eligibility of all participants by the LAO prior 
to enrollment 
 
Low and Intermediate risk studies require confirmation of eligibility of the first two participants enrolled at 
each accruing LAO or AO. This review should take place as soon as possible but not more than two weeks 
following enrollment/randomization.  Continuing eligibility review can be determined based on the LAO 
assessment of AO performance and protocol complexity. 
 
For those international sites where data sharing is prohibited, this policy does not apply. 
 
4.1 
Inclusion Criteria  
 
4.1.1 Male or female incumbent firefighters who are current non-smokers.  
 
4.1.2 Age ≥18 years. 

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4.1.3 Karnofsky performance scale ≥ 70% (see Appendix A). 
 
4.1.4 Participants must have adequate and stable end organ and bone marrow function as defined below: 
Absolute neutrophil count 
≥ 1,000/microliter 
Platelets 
 
≥ 100,000/microliter 
Total bilirubin  
≤ 2 × institutional upper limit of normal (ULN)  
AST (SGOT)/ALT (SGPT) ≤ 2 × ULN 
 
Creatinine 
 
≤ 1.5 × ULN 
 
4.1.5 Participants on chronic suppressive antiviral therapy for herpes simplex virus (HSV) are eligible. 
 
4.1.6 The effects of BSSE on the developing human fetus at the recommended therapeutic dose are unknown. 
For this reason, women of child-bearing potential and men must agree to use adequate contraception 
(hormonal or barrier method of birth control; abstinence) prior to study entry and for the duration of study 
participation. Should a woman become pregnant or suspect she is pregnant while participating in this 
study, she should inform her study physician immediately. 
 
4.1.7  Ability to understand and the willingness to sign a written informed consent document. 
 
4.2 
Exclusion Criteria  
 
4.2.1 History of invasive cancer within the past 2 years, except for excised and cured non-melanoma skin cancer 
or carcinoma in situ of the cervix. Participants who continue adjuvant treatment for an index cancer 
occurring > 2 years ago, such as adjuvant hormonal therapy for breast cancer, are excluded. Participants 
who are on anti-neoplastic treatment for a chronic malignancy, such as multiple myeloma or chronic 
myelogenous leukemia, are excluded. 
 
4.2.2 Chronic, current or recent (within the past 2 weeks) use of systemic steroid doses equivalent to prednisone 
> 5 mg daily for continued use > 14 days. Use of inhaled steroids, nasal sprays, and topical creams for 
small body areas (< 10% body surface area) is allowed. 
 
4.2.3 Participants may not be receiving any other investigational agents. 
 
4.2.4 History of allergic reactions attributed to compounds of similar chemical or biologic composition to 
Avmacol ES (BSSE). 
 
4.2.5 Uncontrolled intercurrent illness including, but not limited to, serious ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, or cardiac arrhythmia, or psychiatric 
illness/social situations that would limit compliance with study requirements.  
 
4.2.6 Pregnant or lactating women. Pregnant women are excluded from this study because the effects of BSSE 
on the developing human fetus are unknown. Because there is an unknown but potential risk for AEs in 
nursing infants secondary to treatment of the mother with BSSE, breastfeeding should be discontinued if 
the mother is treated with BSSE. 
 

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4.2.7.   Participants with known human immunodeficiency virus (HIV), chronic hepatitis B virus (HBV), or 
hepatitis C virus (HCV) infection. Participants with HIV, HBV and HCV are excluded from this study 
because there is no information regarding the impact of anti-viral drugs on the bioavailability of Avmacol 
ES. SF is known to modulate certain phase 1 and phase 2 enzymes involved in drug metabolism. The 
potential for SF to alter the metabolism (either by increasing or decreasing) of antiviral therapy could have 
an effect on the efficacy of the pharmaceuticals to keep viral titers low and the disease under control. 
Since many of the drugs used in therapies of these viral infections have extensive CYP450 enzymatic 
impact and BSSE has its own enzymatic properties, there is concern for drug-to-drug interactions. 
 
4.2.8.   Ongoing use of any supplements containing active compounds in cruciferous vegetables such as SF and 
GR. The use of supplements related to the study agent may confound the study endpoints. Participant will 
be eligible if they agree to stop the  SF or GR product at least 14 days prior to the Baseline Visit.  
 
4.2.9. History of allergic reactions to acetaminophen or the formulation ingredients or any other contraindication 
to acetaminophen use. 
 
4.2.10 Unwilling or unable to refrain from the use of non-study acetaminophen (or acetmainphen containing 
products) for 72 hours prior to the baseline evaluation of acetaminophen metabolism and for 72 hours 
prior to the end-of-intervention evaluation of acetaminophen metabolism.   
 
4.3 
Participant Consideration Regarding Alcohol Consumption 
 
There is no safety concern associated with moderate or even heavy alcohol consumption in this study, provided 
participants do not exhibit underlying liver dysfunction. All participants will undergo screening for liver function, 
and those with clinically significant abnormalities—defined as AST or ALT >2× the upper limit of normal 
(ULN)—will be excluded to ensure participant safety. Regular alcohol use will neither limit eligibility nor require 
restriction during the study, with the exception of a 24-hour abstinence period immediately before and after 
administration of the acetaminophen probe. This temporary restriction is necessary to minimize any short-term 
impact of alcohol on the metabolism readouts. Outside of this window, participants will be encouraged to 
maintain their usual alcohol consumption habits in order to evaluate the intervention under real-world conditions. 
 
There is no concern that alcohol use will compromise the safety of participants or the integrity of the metabolic 
data collected to assess individual response to the intervention. While regular alcohol intake may affect steady-
state metabolism and detoxification pathways, this variability is not considered a risk. Alcohol use will be 
systematically recorded and treated as a covariate in statistical analyses to assess whether it modifies the effect 
of the intervention. 
 
4.4 
Inclusion of Women and Minorities 
 
Both men and women (as applicable) and members of all races and ethnic groups are eligible for this trial.  
 
4.5  
Recruitment  
Study participants will be recruited from Tucson and surrounding areas. Dr. Burgess has worked with the Tucson 
firefighters for 30 years, and on cancer risks in firefighters since 2015. This has included a study of exposures 
and cancer risk in Tucson firefighters funded by Federal Emergency Management Agency which enrolled 525 
participants, including 28 women and 55 Hispanic firefighters. The study team has been meeting with the long-

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term partners of Dr. Burgess, including Health and Safety Captain, Mr. John Gulotta from the TFD to discuss the 
current proposal concept and trial design. Mr. Gulotta has been instrumental to the recruitment of firefighters to 
Dr. Burgess’s prior study and has provided valuable input on the design of the proposed trial and is committed to 
support the recruitment of the proposed trial.  
 
 
4.6  
Planned Accrual 
 
The planned enrollment tables below match the sex, race and ethnicity demographic data for firefighters within 
southern Arizona. 
 
DOMESTIC PLANNED ENROLLMENT REPORT 
Racial Categories 
Not Hispanic 
or Latino: 
Female 
Not Hispanic 
or Latino: 
Male 
Hispanic or 
Latino: 
Female 
Hispanic or 
Latino: 
Male 
 
Total 
American Indian/Alaska Native 
 
2 
 
 
2 
Asian 
 
3 
 
 
3 
Native Hawaiian or Other 
Pacific Islander 
 
 
 
 
 
Black or African American 
1 
5 
 
 
6 
White  
5 
37 
1 
10 
53 
More Than One Race 
 
 
 
2 
2 
Total 
6 
47 
1 
12 
66 
 
 
5.       REGISTRATION PROCEDURES 
 
5.1   
Investigator and Research Associate Registration with CTEP 
 
Food and Drug Administration (FDA) regulations require IND sponsors to select qualified investigators.  NCI 
policy requires all persons participating in any NCI-sponsored clinical trial to register and renew their registration 
annually which is done via the Registration and Credential Repository (RCR). 
 
To register, all individuals must obtain a Cancer Therapy Evaluation Program (CTEP) Identity and Access 
Management (IAM) account (https://ctepcore.nci.nih.gov/iam). In addition, persons with a registration type of 
Investigator (IVR), Non-Physician Investigator (NPIVR), or Associate Plus (AP) (i.e., clinical site staff requiring 
write access to Rave or acting as a primary site contact) must complete their annual registration using CTEP’s 
web-based Registration and Credential Repository (RCR) (https://ctepcore.nci.nih.gov/rcr). Documentation 
requirements per registration type are outlined in the table below. 
 
Documentation Required 
IVR 
(Investigator) 
NPIVR 
(Non-
physician 
Investigator) 
AP 
(Associate 
Plus) 
A 
(Associate) 
AB 
(Associate 
Basic) 
FDA Form 1572 
 
 
 
 

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Financial Disclosure Form  
 
 
 
NCI Biosketch (education, 
training, employment, 
license, and certification) 
 
 
 
 
GCP training 
 
 
 
 
CV (optional) 
 
 
 
 
 
An active CTEP-IAM user account and appropriate RCR registration is required to participate in all CP-CTNet 
clinical trials.   
 
All personnel directly involved in the performance of procedures required by the protocol and the collection of 
data should be registered in the RCR.  
 
Personnel associated with the five registration types include, but is not limited to, the following: 
 
• Investigator (IVR) — MD, DO, or international equivalent 
• Non-Physician Investigator (NPIVR) — advanced practice providers (e.g., NP or PA) or graduate level 
researchers (e.g., PhD) 
• Associate Plus (AP) — clinical site staff (e.g., RN or CRA) with data entry access to RAVE. Also includes 
site administrator, data administrator, and consenting person. Individuals with an auditing role should register 
as an AP. 
• Associate (A) — other clinical site staff involved in the conduct of NCI-sponsored trials.  
• Associate Basic (AB) — individuals (e.g., pharmaceutical company employees) with limited access to NCI-
supported systems 
In addition, the site-protocol Principal Investigator (PI) must meet the following criterion: 
• Active registration status 
• The IRB number of the CIRB (IRB of record) listed on their Form FDA 1572 
 
Additional 
information 
can 
be 
found 
on 
the 
CTEP 
website 
at 
https://ctep.cancer.gov/investigatorResources/default.htm. For questions, please contact the RCR Help Desk by 
email at RCRHelpDesk@nih.gov. 
 
6.       NCI CENTRAL INSTITUTIONAL REVIEW BOARD 
  
The NIH policy on the Use of a Single Institutional Review Board for Multi-Site Research 
https://grants.nih.gov/grants/guide/notice-files/not-od-16-094.html became effective on January 25, 2018. In 
compliance with this policy, NCI Central IRB (NCI CIRB) is the sole IRB of record for all accruing sites 
conducting clinical trials through the CP-CTNet, all CP-CTNet  U.S.-based sites must be members of the NCI 
CIRB, and utilize the Cancer Prevention and Control  CIRB as their IRB of record. International sites should 
submit Research Ethics Board (REB) approval to the DCP Regulatory contractor following country-specific 
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Signatory Institutions must submit a Study Specific Worksheet (SSW) to the CIRB via IRBManager to indicate 
their intent to open the study locally. The CIRB’s approval of the SSW is then communicated to the PIs at the 
Signatory Institution and the Regulatory Contractor. In order for the SSW approval to be processed, the Signatory 
Institution must inform which CIRB-approved institutions aligned with the Signatory Institution are participating 
in the study. 
 
Should changes to the study become necessary, protocol amendments will be submitted to the DCP PIO according 
to DCP Amendment Guidelines. The DCP-approved amended protocol must be approved by the CIRB prior to 
implementation. 
 
7. 
AGENT ADMINISTRATION 
 
Intervention will be administered on an outpatient basis. Reported AEs and potential risks are described in 
Section 8.2.  
 
7.1 
Dose Regimen and Dose Groups 
 
The study agent is BSSE (administered as Avmacol ES) vs. matched placebo, 4 tablets once daily for 12 weeks.  
 
7.2 
BSSE Administration 
 
• Study agent will be dispensed at Baseline Visit. Each bottle will contain 182 tablets. Participants will 
receive two bottles, sufficient for 12 week intervention period with an allowable window of 14 days before 
to 7 days after the scheduled 12-week time point. 
• The participant will self-administer the study agent.  
• Participants will be instructed to take 4 tablets of the assigned study agent once daily with food around 
the same time each day until the day before the end of intervention visit. 
 
7.3 
Run-in Procedures  
 
Not applicable. 
 
7.4 
Contraindications 
 
Participants will be instructed to avoid alcohol consumption for 24 hours prior to, and 24 hours after, study-
specified acetaminophen administration. 
 
• 
Rationale for Alcohol Restriction Window: 
The 24-hour window before and after acetaminophen administration is a precautionary measure to 
minimize potential confounding effects of recent alcohol consumption on hepatic metabolism, 
specifically cytochrome P450 2E1 (CYP2E1) activity. Acute alcohol intake can transiently induce 
CYP2E1 or competitively inhibit it, depending on timing and dose, which may unpredictably alter the 
formation of N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of acetaminophen. 
While this risk is primarily of concern at supratherapeutic doses or with chronic alcohol use, we 

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included this conservative restriction to reduce variability in metabolic response that could compromise 
the study’s ability to detect treatment-related effects on detoxification pathways. 
 
 
 
 
7.5 
Concomitant Medications 
 
• Use of supraphysiologic, systemic steroid doses equivalent to prednisone > 5 mg daily for more than 14 
consecutive days is to be avoided, if medically possible, while on intervention. Should a participant 
require supraphysiologic, systemic steroid doses for > 14 days, they will be taken off study.  
• Participants are to avoid any herbs, herbal supplements, or nutraceuticals containing active compounds 
in cruciferous vegetables such as SF and GR while on study. Should a participant initiate any herbs, 
herbal supplements, or nutraceuticals containing SF and GR while on study, they will be taken off study. 
A daily multivitamin and/or mineral supplements are acceptable. 
• Participants will refrain from taking non-study acetaminophen (or any products containing 
acetaminophen) for 72 hours prior to and for 24 hours after study-specified acetaminophen 
administration. 
 
Participants will be asked about all medications (prescription and over-the-counter), vitamin and mineral 
supplements, and/or herbs taken  and documented by study staff during each study visit and during phone contacts 
1 week (+ 7 days), 4 weeks ± 7 days and 8 weeks ± 7 days after initiating study agent.  Staff will record start and 
stop date, dose and route of administration, and indication. Medications taken for a procedure (e.g., biopsy) should 
also be included. 
 
7.6 
Dose Modification 
 
If a participant misses a dose of BSSE or placebo, the dose is considered skipped and should not be replaced. If 
a participant vomits following a dose, the dose should not be retaken. Missed or vomited doses reported during 
phone contacts will be documented, along with symptoms.  
 
No dose modification will be made for Grade 1 AE. If a participant experiences a related Grade 2 AE (possibly, 
probably, or definitely attributable to study agent), no dose holding or modification of BSSE or placebo is 
required. If the related Grade 2 AE is unacceptable to the physician or participant, study agent may be held for 
up to 7 days at the discretion of the investigator. When the AE has resolved to acceptable Grade 2 or better, study 
agent will be restarted at the same dose. If the AE has not resolved to acceptable Grade 2 or better within 7 days, 
study agent will be permanently withdrawn, and the participant followed for resolution of AEs.  
 
If a participant experiences a related Grade 3 AE (possibly, probably, or definitely attributable to study agent) or 
any Grade 4 AE (regardless of relationship to study agent), study agent will be permanently withdrawn. 
Participants will be followed at least weekly for resolution of AEs.  
 
When possible and appropriate, participants withdrawn early will be encouraged to return for sample collection 
and clinical labs for safety. 
 
7.7 
Adherence/Compliance 

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7.7.1 Participants will be considered compliant for statistical analysis if they have taken ≥80% of their 
assigned study doses based on pill count. 
 
7.7.2 The primary measure of medication compliance is based on pill count and supplemented with the 
Participant Diary.  
 
8. 
PHARMACEUTICAL INFORMATION 
 
8.1 
BSSE (IND # 
 IND NCI, Division of Cancer Prevention)   
 
BSSE will be administered as Avmacol ES, provided by Nutramax Laboratories under IND # 
 and 
sponsored by NCI, DCP.  
 
The regular and extra strength (ES) Avmacol products (Avmacol and Avmacol ES, respectively, manufactured 
in tablet form under GMP standards by Nutramax Laboratories, Inc.) are commercially available dietary 
supplements that contain GR and the fully active enzyme myrosinase (MYROSIMAX). The combination of 
myrosinase and GR results in a higher and more consistent dose of SF upon ingestion of Avmacol or Avmacol 
ES. SF has been shown to mitigate the effects of environmental carcinogens including benzene, aldehydes, and 
PAHs found in tobacco smoke by enhancing their detoxification. Mechanistically, SF disrupts the 
polyubiquitination of the NRF2 transcription factor mediated by its inhibitory protein, KEAP1. This frees NRF2 
to translocate to the nucleus and bind to AREs in the promoter regions of target genes. Avmacol ES tablets, which 
contain approximately 2 times the amount of GR and myrosinase as the regular strength Avmacol tablets, also 
contain maitake mushroom extract. The maitake mushroom extract contains beta-glucans which are hypothesized 
to enhance the activity of SF and to augment innate immunity, providing an additional potential mechanism 
through which Avmacol ES can help support the body’s natural detoxification process and prevent cancer 
progression. A previous proof-of-concept study (NCT03402230) in which otherwise healthy current smokers 
(≥20 pack-years) were administered a low or high daily dose of  regular strength Avmacol for 2 weeks in a 
crossover design with a 2-week washout period between Avmacol treatment periods, showed a dose-dependent 
increase in the urinary excretion of the detoxification metabolites of benzene and acrolein, 2 carcinogens in 
cigarette smoke, indicating that Avmacol treatment could enhance the detoxification of at least some carcinogens 
in cigarette smoke. In the current study, the investigators propose to evaluate whether a dose of Avmacol ES at 
least equivalent to the higher daily dose of Avmacol from the previous study can upregulate Phase II 
detoxification pathways in firefighters. Demonstration of a sustained effect in a rigorous, placebo-controlled 
study would justify moving Avmacol forward in a primary or secondary chemoprevention setting in this at-risk 
cohort.  
 
The bioavailability of SF was determined to be 34.2% in healthy volunteers administered Avmacol based on 
quantitation of SF metabolites in the urine of individuals. The pharmacodynamic effects of Avmacol dosing was 
evaluated in peripheral blood mononuclear cells (PBMCs) isolated from study participants at baseline and 24 
hours following Avmacol administration. When compared with samples obtained prior to dosing, a significant 
increase in the expression of genes associated with cytoprotective, detoxification, and antioxidative functions 
(HO-1, HSP27, HSP70) and a decrease in the expression of genes associated with inflammation was observed in 
the samples obtained 24 hours after Avmacol administration38 
 

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Avmacol ES manufactured under cGMP by Nutramax Laboratories, Inc., is the current, commercially available 
BSSE dietary supplement for use in this study. Each Avmacol ES tablet contains 490 mg of active GR and 
myrosinase blend. It also contains maitake mushroom extract; the manufacturing specifications requires that each 
Avmacol ES tablet contains at least 30 mg of GR per tablet, double the quantity of GR activity relative to the 
Avmacol regular strength formulation. In addition to these active ingredients, Avmacol ES contains inactive 
excipients, i.e., microcrystalline cellulose, maltodextrin, hydroxypropyl cellulose, starch, and < 2% of 
croscarmellose sodium, silicon dioxide, and magnesium stearate. The GR content is 48 mg/tablet and myrosinase 
activity (assessed by conversion of GR to SF) is 13 mg/tablet for the specific Avmacol ES lot utilized in the study. 
Stability testing will be performed on the lot of Avmacol ES utilized for the study, and all clinical supplies will 
be used within expiry. Matching placebo tablets manufactured under cGMP by Nutramax Laboratories, Inc. will 
also be used in this study.  
 
8.2  
Acetaminophen 
Acetaminophen (500 mg tablets) for the study will be locally sourced by the University of Arizona study 
staff and dispensed by study personnel. Storage and handling of acetaminophen will be conducted in 
accordance with the manufacturer’s instructions. 
 
8.3 
Reported Adverse Events and Potential Risks 
 
Broccoli seed and sprout products including Avmacol have been studied extensively in more than 500 subjects 
in multiple phase 1 and phase 2 clinical studies. In most of these studies the BSP was administered as beverages 
prepared from broccoli sprouts or crude broccoli seed extracts normalized for GR or SF content to identify dose. 
No grade 3 or higher AE were reported. Reported grade 1 or 2 AEs have included poor taste of the study agent, 
most common during ingestion of SF-rich broccoli sprout extract beverages, eructation, flatulence, loose stool 
not meeting CTCAE criteria for diarrhea, diarrhea, abdominal pain or cramping associated with loose stool or 
diarrhea. During the first healthy volunteer study with Avmacol, the taste of the study agent was not a source of 
toxicity – likely because the taste is masked by encapsulation.  
 
The toxicities from NCT03402230, our completed, randomized, crossover trial evaluating the detoxification of 
benzene and other tobacco carcinogens by BSSE in otherwise healthy current smokers39, are summarized in Table 
3. Twenty-nine of 49 (59%) of participants reported at least one AE attributable to BSSE; all 29 (100%) were 
gastrointestinal AE. The most common AEs were loose stool not meeting CTCAE v.4 criteria for diarrhea (24%) 
or diarrhea (22%), all Grade 1. Ten of 49 (20%) participants experienced mild abdominal pain typically associated 
with loose stool or diarrhea. Despite the prevalence of mild gastrointestinal AEs, compliance with treatment was 
98%. These symptoms are consistent with other studies and expected due to the high fiber content of BSSE. The 
dose of BSSE in the current trial is identical to the high dose arm of NCT03402230. 
 
Table 3. Treatment-Emergent Adverse Events in Clinical Study of Avmacol for Detoxification of Tobacco 
Carcinogens in Heavy Smokers (NCT03402230)  
 
Toxicity 
Low Dose  
(4 Avmacol Regular Strength 
Tablets)a 
N=49 
High Dose  
(8 Avmacol Regular Strength 
Tablets)b 
N=49 
Both 
Arms 
N=49c 
Abdominal Pain 
1 (2%) 
10 (20%) 
10 (20%) 

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Bloating 
0 (0%) 
1 (2%) 
1 (2%) 
Diarrhea 
3 (6%) 
10 (20%) 
11 (22%) 
Loose Stool 
7 (14%) 
9 (18%) 
12 (24%) 
Flatulence  
3 (6%) 
7 (14%) 
10 (20%) 
Nausea 
1 (2%) 
0 (0%) 
1 (2%) 
Vomiting 
0 (0%) 
1 (2%) 
1 (2%) 
Weight Loss 
0 (0%) 
1 (2%) 
1 (2%) 
aLow dose: Subjects were given 4 Avmacol regular strength tablets per day. 
bHigh dose: Subjects were given 8 Avmacol regular strength tablets per day. 
cParticipants who had AE in either or both treatment arms. 
 
Acetaminophen is an FDA-approved medication that is available over the counter without a prescription. It is 
commonly used to reduce fever and relieve pain. The dosage used in this study is well within the FDA’s 
recommended daily limits. While side effects from taking study-specified acetaminophen in this study are 
unlikely, it is important to note that no medication, including FDA-approved over-the-counter drugs, is entirely 
free from risk. Possible side effects of acetaminophen may include: 
 
Liver warning: Severe liver damage may occur if: 
• 
adult takes more than 4,000 mg of acetaminophen in 24 hours 
• 
acetaminophen is taken with other drugs containing acetaminophen  
• 
adult has 3 or more alcoholic drinks every day while using acetaminophen   
Allergy alert: acetaminophen may cause severe skin reactions. Symptoms may include: 
• 
skin reddening 
• 
blisters 
• 
rash 
 
Acetaminophen (500 mg tablets) for the study will be locally sourced by the University of Arizona study staff 
and dispensed by study personnel. Storage and handling of acetaminophen will be conducted in accordance with 
the manufacturer’s instructions. 
 
8.4  
Availability 
 
Avmacol ES (490 mg of proprietary GR and myrosinase blend per tablet) is a commercially available dietary 
supplement manufactured and marketed by Nutramax Laboratories, Inc. Matching placebo tablets will also be 
manufactured by Nutramax Laboratories, Inc. Avmacol ES and matching placebo tablets will be supplied to 
investigators by the Division of Cancer Prevention (DCP), NCI. 
 
Avmacol ES and matched placebo are provided to the NCI under a Clinical Trials Agreement (CTA) between 
Nutramax, Inc. and the DCP, NCI (see §14.7). 
 
Acetaminophen is a commercially available antipyretic manufactured by several pharmaceutical companies. 
Acetaminophen will be purchased locally over the counter for the study by the University of Arizona study staff 
and dispensed by study personnel. Storage and handling of acetaminophen will be conducted in accordance with 
the manufacturer’s instructions.and will be provided to participants by study personnel. 
 

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8.5 
Agent Distribution 
 
Agents will only be released by NCI, DCP after documentation of CIRB approval of the DCP-approved protocol 
and consent is provided to DCP and the collection of all Essential Documents is complete (see DCP website for 
description of Essential Documents).  
 
NCI, DCP-supplied agents may be requested by the Investigator (or his/her authorized designees) at each 
Organization. DCP guidelines require that the agent be shipped directly to the institution or site where the agent 
will be prepared and administered. DCP does not permit the transfer of agents between institutions (unless prior 
approval from DCP is obtained). DCP does not automatically ship agents; the site must make a request. Agents 
are requested by completing the DCP Clinical Drug Request form: 
https://prevention.cancer.gov/sites/default/files/uploads/clinical_trial/Investigational-Agent-Request.docx (to 
include complete shipping contact information) and e-mailing the form to the DCP agent repository contractor:  
 
John Cookinham 
MRIGlobal 
DCP Repository 
1222 Ozark Street 
North Kansas City, MO 64116 
Phone: (816) 360-3805 
E-mail: NCI.DCP@mriglobal.org 
 
Acetaminophen (500 mg tablets) for the study will be locally sourced by the University of Arizona study staff 
and dispensed by study personnel. Storage and handling of acetaminophen will be conducted in accordance with 
the manufacturer’s instructions. 
 
8.6  
Agent Accountability 
 
The Investigator, or a responsible party designated by the Investigator, must maintain a careful record of the 
inventory and disposition of all agents received from DCP. The Investigator is required to maintain adequate 
records of receipt, dispensing and final disposition of study agent. This responsibility has been delegated to study 
site PI and the institution’s Research Pharmacy or study coordinator. Include on receipt record from whom the 
agent was received and to whom study agent was shipped, date, quantity and batch or lot number. On dispensing 
record, note quantities and dates study agent was dispensed to and returned by each participant.  
 
Acetaminophen (500 mg tablets) for the study will be locally sourced by the University of Arizona study staff 
and dispensed by study personnel. Storage and handling of acetaminophen will be conducted in accordance with 
the manufacturer’s instructions. 
 
8.7 
Packaging and Labeling 
 
BSSE tablets and matching placebo will be manufactured and packaged by Nutramax. Nutramax will ship the 
packaged BSSE (Avmacol ES) and matched placebo bottles to the NCI DCP Drug Repository, MRIGlobal, in 
Kansas City, MO. MRIGlobal will label bottles according to the blinded randomization schema, and ship to study 
site.  
 

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Each bottle will be labeled with a three-part label identifying study specific information including study title, 
DCP protocol number, dosing instructions, recommended storage conditions, the name and address of the 
distributor, and treatment ID.  
 
 
 
8.8 
Storage 
 
BSSE and placebo tablets should be stored at room temperature in a secure, locked area of the research facility, 
with temperature excursions permitted between 15–30°C (59–86°F). The CP-CTNet Lead Academic 
Organization, the DCP agent repository contractor, DCP Medical Monitor and Nurse Consultant should be 
notified in the event of a Temperature Excursion. 
 
Storage and handling of acetaminophen will be conducted in accordance with the manufacturer’s instructions. 
 
8.9 
Registration/Randomization 
 
This trial will use a web-based Registration/Randomization System, developed and maintained by the CP-CTNet 
Data Management, Auditing, and Coordinating Center (DMACC). The Help Desk includes technical personnel 
and administrators of the registration programs at the Data Management Center in Amherst, NY, USA. The Help 
Desk is available round the clock 7 days per week, except for New Year's Eve, Memorial Day, Independence 
Day, Thanksgiving Day, and Christmas Day. 
Frontier Science Randomization Help Desk 
4033 Maple Rd, Amherst, NY 14226 USA 
Phone: +1 716 834 0900 Extension 7301 
Email:  UserSupport_CP-CTNet@frontierscience.org 
 
Participants will be considered registered on the date they sign the approved informed consent document with a 
member of the study staff. Following registration, eligibility will be confirmed. After determination of eligibility 
and completion of informed consent document, eligible participants will be randomized (1:1) to either BSSE or 
placebo in double-blind fashion.   
 
Note: The Registration and Randomization process is documented in the "Stars User Guide". 
 
8.10 
Blinding and Unblinding Methods 
 
This is a randomized, double-blind (participants and investigators), placebo-controlled trial. Blinding and 
unblinding methods are specified as follows:  
• Investigators and clinical research coordinators will avoid speculating with the participant regarding the 
participant’s treatment group. 
• The individual authorized to break the blind, if conditions are met, is the Study PI in consultation with 
the DCP medical monitor. 
• The blind may be broken in the following two circumstances:  
o Medical necessity: The participant is experiencing a serious adverse event (SAE) AND 
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OR 
o Safety and informed consent for current and future participants: The participant is experiencing 
an SAE AND knowledge of treatment assignment would change understanding of the toxicity 
profile of BSSE, therefore altering safety and informed consent for current and future 
participants 
• Procedure for breaking the blind: 
o The SAE is reported according to procedures described in Section 13.0.  
o The treating investigator consults with the Study PI regarding the specific circumstances, and 
whether knowledge of treatment assignment would alter the clinical management of the SAE OR 
would change the understanding of the toxicity profile of BSSE such that safety and informed 
consent would be impacted for current and future participants. 
o If the SAE meets criteria for unblinding, the Study PI then authorizes DMACC to unblind 
treatment assignment for that participant. 
o DMACC unblinds the participant and informs the Study PI and the treating investigator: 1) 
unblinding has occurred; and 2) the treatment assignment of the unblinded participant.  
• The CIRB will be notified of the unblinding event. 
 
The NCI DCP Medical Monitor must be notified that the blind has been broken: 
 
Malgorzata Wojtowicz, MD, Medical Monitor 
NCI/Division of Cancer Prevention 
 
NIH National Cancer Institute 
 
9609 Medical Center Drive 
 
Room 5E-104, MSC 9781 
 
Bethesda, MD 20892 
 
PHONE: 240-276-7012 
 
FAX: (240) 276-7848 
malgorzata.wojtowicz@nih.gov 
8.11 
Agent Destruction/Disposal 
 
DCP-supplied agents: at the completion of investigation, all unused study agent will be returned to NCI, DCP 
Repository according to the DCP “Guidelines for AGENT RETURNS” and using he DCP form “DCP Returned 
Agents List”. 
 
9.  
CLINICAL EVALUATIONS AND PROCEDURES 
 
9.1 
Schedule of Events 
 
Evaluation/ 
Procedure 
Visit 1 
Consenting/ 
Screening 
 
Randomization 
Visit 2/ 
Baseline2 
Intervention 
Period11 
Visit 3/ 
End of 
Intervention11 
Follow-
up 
Informed Consent 
X 
 
 
 
 
 
Assess Eligibility 
X 
 
 
 
 
 
Medical History1 
X 
 
 
 
 
 

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Baseline 
Signs/Symptoms2 
X 
 
X 
 
 
 
Review for any 
change in health 
status since Visit 
1/screening2a 
 
 
X 
 
 
 
Tobacco and 
Alcohol Use 
Assessment (DCP 
Baseline) 
X 
 
 
 
 
 
Concomitant 
Medications3 
X 
 
X 
X 
X 
 
Baseline COVID 
Assessment 
X 
 
 
 
 
 
Vital Signs 
X 
 
X 
 
X 
 
Height, and 
Weight4 
X 
 
 
 
X4 
 
Karnofsky 
Performance 
Status 
X 
 
 
 
X 
 
Blood Draw: 
Clinical Labs 
(CBC/Diff, CMP) 
and Research 
Tests5  
X 
 
 
 
 
X 
 
Urine Pregnancy 
Test6 
X 
 
X 
  
  
 
Randomization7 
 
X 
 
 
 
 
Pre-
Acetaminophen: 
Collect Spot Urine 
and 
questionairre8, 9   
 
 
X 
 
X 
 
Administer 
Acetaminophen 
 
 
X 
 
X 
 
Post- 
acetaminophen 
cumulative 8-hr 
Urine  
Collection10 
 
 
X 
 
X 
 
Provide Intake 
Calendar and 
Adverse Event 
(Symptom)/New 
Medication Diary 
 
 
X 
 
 
 

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Dispense Study 
Agent11 
 
 
X 
  
 
 
Spot Urine 
Collection after 
Each Fire/Fuel 
Leak Activity12  
 
 
 
X 
 
 
Post-Fire/Fuel 
Leak Spot Urine 
Questionnaire12 
and Fire/Fuel 
Incident Report 
Appendix H12 
 
 
 
X 
 
 
Participant 
Contact and 
review of Adverse 
Events, 
new/change in  
medication and 
pill intake13  
 
 
  
X 
X 
X 
Collect Study 
Agent/Pill Count 
 
 
 
 
X 
 
Review Intake 
Calendar and 
Adverse Event 
(Symptom)/New 
Medication 
Diary14 
 
 
 
 
X 
 
Tobacco and 
Alcohol Use 
Assessment (DCP 
Follow-Up) 
 
 
 
 
X 
 
Follow-up COVID-
19 Assessment 
 
 
 
 
X 
 
1 Participants will be asked the day of the last physical exam (month and year required).  For those who do not recall the 
actual day of the month, the 15th of the month will be substituted. 
2 Baseline Visit should occur within 12 weeks of screening/consenting. Vital signs, conmeds, pregnancy test (if 
applicable), and any change in health status will be re-assessed at visit 2.   
2aIf a change in health status is reported at Visit 2, participant will be re-evaluated for eligibility as clinically indicated.  
3 Participants using any supplements containing active compounds in cruciferous vegetables such as SF and GR may be 
considered eligible if they agree to stop the SF or GR product at least 2 weeks prior to the Baseline Visit. 
4 Weight only to be collected at post screening visit. 
5 Research tests during Visit 1 include screening for GST genotypes and assessment of epigenetic modification (miRNA 
and DNA methylation). Research tests during Visit 3 include assessment of epigenetic modifications (miRNA and DNA 
methylation). 
6Urine pregnancy test is required only in women of child-bearing potential. 
7 Randomization will be completed after the subject is registered and confirmed eligible, after the Screening Visit and 
prior to Visit 2. 

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8 Participants will be instructed to avoid alcohol consumption for 24 hours prior to and 24 hours after study-specified 
acetaminophen administration. Participants will also be instructed to refrain from taking non-study acetaminophen (or any 
products containing acetaminophen) for 72 hours prior to and for 24 hours after study-specified acetaminophen 
administration.   
9 Over-the-counter and prescription medications, supplements use including pre-workout and protein beverages, other 
beverage consumption aside from water during the 24 hours prior to pre-acetaminophen dose spot urine collection, and 
blood and/or plasma donation history will be collected as part of the Pre-Acetaminophen Spot Urine Questionnaire.  
10The 8-hr (-1 and + 2 hr)  post acetaminophen urine will be collected by the TFD research liaisons or study staff within 
24 hours after completion. 
11 Participants will be instructed to begin taking the study agent within two weeks of the Baseline Visit. The intervention 
period will last a minimum of 12 weeks, with an allowable window of 14 days before to 7 days after the scheduled 12-
week time point. Participants will stop taking the study agent only after completing the End-of-Intervention Visit (Visit 
3), which includes the end-of-study acetaminophen dose and the associated 8-hour post-dose urine collection conducted 
within this same window. This design ensures that the effects of the study intervention on acetaminophen metabolism are 
assessed while participants are actively taking the study agent. 
12   The spot urine and the Post-Fire/Fuel Leak Spot Urine Questionnaire will be collected by the TFD research liaisons or 
study staff within 24 hours of completion. TFD research liaison will complete Fire/Fuel Incident Report Appendix H. 
13 Participants will be contacted by phone or email per their preferred method of telecommunication at week 1 + (7 days), 
week 4 ± 7 days and week 8 ± 7 days for evaluation and documentation of AEs, compliance, and proper completion of 
Adverse Event (Symptom)/New Medication Diary and Intake Calendar. 
14 Intake Calendar and Adverse Event (Symptom)/New Medication Diary will be collected during Visit 3/End of 
Intervention. 
 
9.2 
Schedule of Urine Samples and Spot Urine Questionnaire Collection 
Evaluation/Procedure 
 Visit 1 
Consenting & 
Screening 
Visit 2 
Baseline 
Intervention 
12 weeks  
Visit 3 
End of Intervention 
 
Pre-acetaminophen Spot Urine 
Collection 
 
X 
 
X 
Pre-Acetaminophen Spot 
Urine Questionnaire1 
 
X 
 
X 
Post-acetaminophen 8-hour 
Cumulative Urine Collection2 
 
X 
 
X 
Post-fire/fuel Leak Spot Urine 
Collection 3 
 
 
X 
 
Post-Fire/Fuel Leak Spot Urine 
Questionnaire4 
 
 
X 
 
1 Over the counter and prescription medications, supplements use including pre-workout and protein beverages, 
other beverage consumption aside from water during the 24 hours prior to pre-acetaminophen dose spot urine 
collection, and blood and/or plasma donation history will be collected as part of the Pre-Acetaminophen Spot 
Urine Questionnaire.  
2 The End-of-Intervention Visit (Visit 3), when participants stop taking the study agent, occurs at 12 weeks with 
an allowable window of 14 days before to 7 days after the scheduled time point. Participants must complete the 
end-of-study acetaminophen dose and the associated 8-hour post-dose urine collection while remaining on the 
study agent. Study agent is discontinued only after these assessments are completed. Remaining on the agent 
during the end-of-study acetaminophen collection ensures that the acute effects of the intervention on 
acetaminophen metabolism are accurately evaluated..The post acetaminophen 8 (-1 and + 2)  hour urine will be 
collected by the TFD research liaisons or study staff within 24 hours after completion. 

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3The post-fire/fuel leak 8 (± 2) hour spot urine will be collected by the TFD research liaisons or study staff within 
24 hours after completion. 
4Time of urine collection will be recorded along with a brief questionnaire on exposure, including type of fire and 
firefighter activity, along with post-fire washdown activity, will be collected as part of the Post Fire Urine Collection 
Questionnaire completed by the participant electronically and supplemented with a Fire Incident report to be completed 
by the TFD research liaison. The post-fire/fuel leak spot urine will be collected by the TFD research liaisons or study staff 
within 24 hours of completion. 
 
9.3 
Baseline Testing/Pre-Study Evaluation 
 
Visit 1 (Screening Evaluation): Participants will undergo a screening evaluation in which the informed consent 
form will be signed. To minimize participant burden, remote consent may be implemented in compliance to the 
instructions in Appendix G.  Participants will be assessed for study eligibility. Detailed inclusion and exclusion 
criteria are listed in sections 4.1 and 4.2. All participants will be evaluated for medical history including date 
(month and year required) of last physical exam, baseline signs/symptoms, tobacco/alcohol use (DCP baseline 
forms), COVID baseline assessment (DCP baseline form), and concurrent medication (conmed) and supplement 
use. In the event, the participant cannot recall the actual day of the month of the last physical exam, the 15th of 
the month will be collected as the date. All participants will undergo evaluation to obtain height, weight, vital 
signs (blood pressure, pulse, temperature), and Karnofsky performance status assessment. Blood will be collected 
for laboratory analysis with complete blood count with differential (CBC-diff) and comprehensive metabolic 
panel (CMP) as well as research testsincluding GST genotypes and assessment of epigenetic modification 
(miRNA and DNA methylation). Women of childbearing capacity will complete a urine pregnancy test. The 
participant’s preferred telecommunications contact method will be determined (telephone  or email). 
 
If a participant is using any supplements containing active compounds in cruciferous vegetables such as SF and 
GR and agrees to stop the SF or GR product at least 2 weeks prior to the Baseline Visit, he/she will undergo 
screening for eligibility to participate in this study.  
 
Randomization: Once determined eligible, participants will be randomized (1:1) to BSSE or matched placebo. 
 
9.4 
Evaluation During Study Intervention 
 
Visit 2 (Baseline): During this visit, participants will undergo assessment for vital signs and concomitant 
medications/supplements. They will be asked about any change in health status since Visit 1 (screening). If change 
in health status is reported, participant will be re-evaluated for eligibility as clinically indicated, including repeat 
clinical laboratory testing if needed.  Urine pregnancy test will be repeated, if applicable.  Two tablets of 500 mg 
acetaminophen will be administered to the participant and the participant will be instructed to collect a spot urine 
prior to acetaminophen dosing and to collect all urine for 8 (-1 and + 2) hours following the acetaminophen 
administration. Coordinators will administer the questions from the Pre-Acetaminophen Spot Urine 
Questionnaire (Appendix E) at the time of the pre-acetaminophen dose spot urine collection. Participants will be 
instructed to keep the post-acetaminophen dose urine in a cooler with ice packs, provided by the study staff, 
during the 8-hr. post-dose urine collection. Upon completion of the 8-hr post-dose urine collection, the sample 
will be picked up by the study staff or TFD research liaisons and processed within 24 hours of collection.  
Participants will be provided with BSSE or matched placebo and will be instructed to take 4 tablets of the assigned 
study agent each day with food until the day before the end of intervention visit. Participants will be instructed to 
start taking the study agent within 2 weeks of the Baseline Visit and notify the study staff by telephone or e-mail 
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for recording study agent intake and Adverse Event (Symptom)/New Medication Diary for new medications and 
side effects.  
 
Participants will also be instructed to collect a spot urine within 8 (± 2) hours post each of their fire/fuel leak 
activity, when feasible. Participants will be provided with the post-fire urine collection supplies.  Participants will 
be instructed to complete the Post-Fire/Fuel Leak Spot Urine Questionnaire (Appendix F)  when post-fire urine 
is collected.  The collected urine will be kept in a cooler with ice packs and picked up by the study staff or TFD 
research liaisons and processed within 24 hours of collection, when feasible. TFD research liaison will complete 
Fire/Fuel Incident Report (Appendix H) to document exposures. 
 
Participants will be contacted by study staff 1 week (+ 7 days),  4 weeks ± 7 days and 8 weeks ± 7 days after 
starting agent intervention to assess and document any AEs, compliance with study intervention including any 
missed pills, and appropriate completion of Adverse Event (Symptom)/New Medication Diary and Intake 
Calendar.   
 
9.5 
Evaluation at Completion of Study Intervention 
 
Visit 3 (End-of-Intervention): For the end-of-intervention evaluation, participants will be assessed for AEs, 
conmeds, and agent compliance. Intake Calendar and Adverse Event (Symptom)/New Medication Diary and any 
remaining study agent will be collected during the visit. Blood will be collected for laboratory analysis with CBC-
diff and CMP and for research tests including assessment of epigenetic modification (miRNA and DNA 
methylation). All participants will undergo evaluation to obtain weight, blood pressure, pulse, and temperature. 
Tobacco/alcohol use (DCP follow-up form), Karnofsky Performance Status, and COVID follow-up assessment 
will be performed during this visit. Two tablets of 500 mg acetaminophen will be administered to the participant 
and the participant will be instructed to collect a spot urine prior to acetaminophen dosing and to collect all urine 
for 8 (-1 and + 2) hours following the acetaminophen administration. Participants will be instructed to complete 
the Pre-Acetaminophen Spot Urine Questionnaire (Appendix E) for the pre-acetaminophen dose spot urine 
collection. Participants will be instructed to keep the post-acetaminophen dose urine in a cooler with ice packs, 
provided by the study staff, during the 8-hr post-dose urine collection.   Upon completion of the 8-hr post-dose 
urine collection, the sample will be picked up by the study staff or TFD research liaisons and processed within 
24 hours of collection.    
 
9.6 
Post-intervention Follow-up Period 
 
End of Study Assessment: Participants will be contacted by telephone or email 2 weeks ± 7 days after completing 
study treatment per their preference to assess AEs. 
 
Participant’s documentation of fire/fuel leak activities that occurred during the study agent intervention will be 
collected from the records of the TFD via the TFD research liaison to minimize participant burden. 
 
9.7 
Methods for Clinical Procedures 
 
Not Applicable. 
 
10.  
CRITERIA FOR EVALUATION AND ENDPOINT DEFINITION 
 

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10.1 
Primary Endpoint 
 
Primary endpoint. Compare the change (from baseline to end-of-intervention) in total amount of acetaminophen 
mercapturate excreted in the urine after acetaminophen dosing between BSSE and placebo arms.  
 
10.2 
Secondary Endpoints 
 
The secondary endpoints are: 
 
1) Compare the change (from baseline to end-of-intervention) in total amount of acetaminophen 
glucuronide excreted in the urine after acetaminophen dosing between BSSE and placebo arms.   
2) The safety and tolerability of BSSE, as measured by NCI CTCAE version 5. 
 
Exploratory objectives include the following analysis: 1) to assess whether the GSTM1 and GSTT1 genotypes are 
important genetic modulators of BSSE-induced detoxification of carcinogens in the setting of exposure to BSSE; 
2) to explore the effects of BSSE on urinary metabolome; 3) to evaluate the effects of BSSE on the urinary 
excretion of the mercapturic acid of benzene after fire/fuel leak activities, 4) to assess the effects of BSSE on 
urinary excretion of the metabolites of PAHs after fire/fuel leak activities, 5) to evaluate the effects of BSSE on 
epigenetic modifications (blood microRNA and DNA methylation). 
 
10.3 
Off-Agent Criteria  
 
Participants may stop taking study agent for the following reasons: completed the protocol-prescribed 
intervention, AE or SAE, inadequate agent supply, noncompliance, concomitant medications, medical 
contraindication. Participants will continue to be followed, if possible, for safety reasons and in order to collect 
endpoint data according to the schedule of events.  
 
10.4 
Off-Study Criteria 
 
Participants may go ‘off-study’ for the following reasons: the protocol intervention and any protocol-required 
follow-up period is completed, AE/SAE, any AE occurring in association with first administration of 
acetaminophen at Visit 2, lost to follow-up, non-compliance, concomitant medication, medical contraindication, 
withdraw consent, death, determination of ineligibility (including screen failure), pregnancy. 
 
10.5 
Study Termination 
 
NCI, DCP as the study sponsor has the right to discontinue the study at any time.  
 
11. 
CORRELATIVE/SPECIAL STUDIES 
 
11.1 
Rationale for Methodology Selection 
 
Integral Biomarkers 
• 
Urinary Excretion of Acetaminophen Metabolites: Urinary levels of acetaminophen and its metabolites 
(acetaminophen mercapture, acetaminophen glucuronide, and acetaminophen sulfate) will be quantified by 
liquid chromatography tandem mass spectrometry, as previously described 41.    

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Exploratory Biomarkers 
• Urinary Excretion of the Mercapturic Acid of the Carcinogen, Benzene: Urinary levels of the mercapturic 
acid of benzene will be quantified by liquid chromatography tandem mass spectrometry, as previously 
described33, 42. Specific analytes to be quantified include S-phenyl mercapturic acid (SPMA), a metabolite of 
benzene. 
 
• Urinary Excretion of PAH metabolites: Urinary excretion of PAH metabolites, including 1-hydroxypyrene, 
will be evaluated to assess detoxification of PAHs. PAH metabolites will be measured by liquid 
chromatography tandem mass spectrometry, as previously described43.   
 
• GSTM1 and GSTT1 Genotypes: Genomic DNA will be isolated from blood collected at the baseline clinic 
visit (week 1). GSTM1 and GSTT1 will be genotyped using standard methods as previously described 44. 
Briefly, two separate PCR reactions will be used to determine GSTM1 and GSTT1 homozygous deletion. Each 
amplification will include an internal PCA control (CYP1A1 fragment) to ensure null results are attributable 
to GST gene deletion but not due to PCR failure. PCR products will be analyzed by electrophoresis (1.5% 
agarose) and ethidium bromide staining. The presence or absence of GSTM1 and GSTT1 genes will be 
detected by the presence or absence of a band at 480 base-pairs (corresponding to GSTT1) and a band at 215 
base-pairs (corresponding to GSTM1). 
 
• 
Untargeted Urinary Metabolomics: Untargeted metabolomics will be performed as described previously 45. 
Briefly, pre-acetaminophen spot urine collected at both baseline and at the end of intervention as well as spot 
urine collected after each fire/fuel leak activity will be analyzed using an Exploris Orbitrap 480 for ultra-
high performance liquid chromatography in two modes: ESI C18 (reverse phase) positive and HILIC 
(hydrophilic interaction liquid chromatography) negative, with standard quality control, filtering, and 
imputation procedures. We plan to use Compound Discoverer for peak identification and annotation. Mass 
spectra will be matched against our in-house library, and against external libraries including ChemSpider 
and Metabolika. 
 
• 
miRNA: RNA will be isolated from blood and miRNA expression will be measured using the nCounter 
Human v3 miRNA expression panel (NanoString Technology Inc., Seattle, Washington) with 800 miRNAs 
from miRBase v21 as well as 5 housekeeping genes and 20 assay controls (six positive, eight negative, and 
six ligation controls)12-14. The panel includes greater than 95% of human miRBase reads.  
 
• DNA methylation: DNA will be isolated from blood and analyzed using Infinium MethylationEPIC array as 
described previously64. This is a genome-wide methylation analysis tool targeting ~930K unique methylation 
sites in the most biologically significant regions of the human methylome.  
 
 11.2 Comparable Methods 
 
The methods proposed for integral and exploratory biomarkers are standard methodologies used in other research 
studies, including our recently completed, randomized crossover study of low and high dose BSSE in otherwise 
healthy smokers (NCT03402230)33, 39 and our studies in firefighters12-15, 46. The resulting data will be able to be 
compared to existing data.  
 

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The methods proposed for untargeted urinary metabolomics are also standard methodologies used in our prior 
research studies of untargeted metabolomics in municipal and wildland-urban interface firefighters45. The results 
should be comparable to our prior studies, after correcting for batch effects. The quality control and extraction 
methods were also developed in line with other environmental studies in the United States using untargeted 
metabolomic approaches. 
 
12. 
SPECIMEN MANAGEMENT  
 
12.1 
Laboratories 
 
Clinical chemistry and hematology panels will be outsourced to a contracted commercial diagnostic laboratory 
service (i.e., Sonora Quest).  
 
The urinary biomarker analyses, including the acetaminophen metabolites and carcinogen metabolites, and 
untargeted metabolomics will be conducted in the University of Arizona Cancer Center Analytical Chemistry 
Shared Resource.   
 
miRNA analysis will be conducted in the University of Arizona Genetics Core facility. 
 
DNA methylation analysis will be performed in the University of Michigan Advanced Genomics Core. 
 
12.2 
Collection and Handling Procedures 
 
 
Blood for clinical chemistry and hematology panels 
Twelve and a half milliliters of blood (1 x 8.5 mL SST tube; 1 x 4 mL lavender-EDTA tube) will be collected at 
Visits 1 and 3 for CBC with diff and CMP. The SST tube will be held at room temperature for 30 min and then 
centrifuged. The lavender EDTA tube will be gently inverted to mix for anticoagulation.  Blood tubes will be 
prepped, labeled, and packaged according to the recommendation from the diagnostic laboratory.  All samples 
will be refrigerated prior to transfer to the commercial laboratory and sent for immediate analysis. 
 
Blood for genotyping and epigenetic modifications 
 
Visit 1, Screening:  
For genotying and DNA methylation - 1 x 6 mL Lavender EDTA tube will be collected. The lavender-EDTA 
tube will be inverted gently 5 times and the whole blood will be aliquoted evenly into 2 x 5 mL cryovials.  
 
For miRNA: 1 x 9 mL TempusTM Blood RNA tube will be collected.  Immediately after collection, the tube will 
be vigorously shaken for 10 seconds and aliquoted into 2 x 5mL cryogenic tubes.  
 
The cryo-vials/tubes will be labeled with the study ID, participant ID, and visit number/date and stored at -70°C 
or below until analysis.  
 
Visit 3, End of Intervention: 
For DNA methylation - 1 x  6 mL Lavender EDTA tube will be collected. The lavender-EDTA tube will be 
inverted gently 5 times and the whole blood will be aliquoted evenly into  2 x 5 mL cryovials.  
 

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For miRNA: 1 x 9 mL TempusTM Blood RNA tube will be collected.  Immediately after collection, the tube will 
be vigorously shaken for 10 seconds and aliquoted into 2 x 5mL cryogenic tubes.  
 
The cryo-vials/tubes will be labeled with the study ID, participant ID, and visit number/date and stored at -70°C 
or below until analysis.    
 
Urine for pregnancy test 
A single void urine will be collected and tested for pregnancy in the clinic according to package instructions. 
 
Urine collection before and after acetaminophen dosing 
A spot urine will be collected before acetaminophen administration and all urine will be collected for 8 (-1 and + 
2) hours after acetaminophen administration to assess acetaminophen metabolism. The urine volume will be 
recorded by study staff to the nearest 10 mL. Five 2 mL aliquots of urine will be retained for testing and the 
remaining urine discarded. The sample tubes will be labeled with the study ID, participant ID, visit number/date, 
sample type (pre-dose or post-dose) and stored at -70°C or below until analysis.  
 
Post Acetaminophen Urine Collection Window and Deviation Definition 
For the 8-hour post-acetaminophen urine collection, all efforts should be made to complete the cumulative 
collection within the designated 7–10-hour target window following acetaminophen dosing. 
 
A protocol deviation occurs when the cumulative urine collection is completed outside this window. Collections 
performed between 4 and 12 hours after acetaminophen dosing will be considered acceptable for analysis if the 
total urine volume is ≥200 mL, but will be documented as out-of-window. 
 
This allowance provides operational flexibility in field settings while maintaining adequacy for quantitative 
analysis of acetaminophen metabolites and preserving sample integrity.47 
 
Spot urine collection post fire/fuel leak activity 
Participants will collect a spot urine within 8 ± 2 hours post each of their fire/fuel leak activity, when feasible. 
Five 2 mL aliquots of urine will be retained for testing and the remaining urine discarded. The sample tubes will 
be labeled with the study ID, participant ID, date and time of collection, and sample type (post-fire) and stored at 
-70°C or below until analysis. 
 
Post-Fire/Fuel Leak Urine Collection Window and Deviation Definition 
 
For post-fire/fuel leak urine collections, participants should collect a single (spot) urine sample within 8 ± 2 hours 
of completing the fire or fuel leak activity. 
 
A protocol deviation occurs when the sample is collected outside this 6–10-hour window. Collections performed 
between 4 and 12 hours post-activity will be considered acceptable for analysis if the total urine volume is ≥30 
mL, but will be documented as out-of-window. 
 
This range provides operational flexibility in real-world field settings while ensuring adequate sample volume 
for quantitation of exposure-related metabolites and normalization to creatinine.48  
 
12.3 
Shipping Instructions 

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Collected frozen specimens (blood, urine) will be placed on ice packs and hand delivered to UA CP-CTNet 
research laboratory (University of Arizona Cancer Center, 1515 N Campbell Ave, Room 4971, Tucson, AZ) for 
storage until analysis.  
 
Frozen blood specimens will be shipped to the University of Michigan Advanced Genomics Core for DNA 
Methylation testing. The sample manifest and shipping dates will be provided to the University of Michigan 
Genomics Core and dates will be agreed upon prior to samples being shipped. All samples will be shipped using 
FedEx priority overnight, with 25-30 lbs. of dry ice per shipping container and shipping comply with IATA 
guidelines. Once samples are received by the Genomics Core, confirmation will be provided to the University of 
Arizona.  
 
The shipping address of the University of Michigan Advanced Genomics Core is listed below:  
The University of Michigan Advanced Genomics Core 
Room 122 
NCRC Building 14 
University of Michigan 
2800 Plymouth Rd. 
Ann Arbor, MI 48109-2800 
 
Contact person: 
Susan Dagenais 
sdagenai@umich.edu 
 
12.4 
Tissue Banking 
 
The NCI reserves the right to require the transfer of biologic specimens and data, or true copies of such data, 
acquired from research supported under this award to an eligible third party. This transfer can occur in order to 
preserve the specimens and data and/or to continue the research. Third parties supported under this award must 
be informed of this right.   
 
13. 
REPORTING ADVERSE EVENTS  
 
DEFINITION: An adverse event (AE) means any untoward medical occurrence associated with the use of a drug 
in humans, whether or not the untoward occurrence is considered drug related. Thus, an AE can include any 
unfavorable sign (e.g., an abnormal laboratory finding), symptom, or clinical outcome temporally associated with 
the use of a test drug, active control, or placebo, regardless of whether the event is thought to be related to the 
drug. An AE can arise with the use of a drug or biologic (e.g., use for a purpose other than FDA-approved 
indication or in combination with another drug) and with any route of administration, formulation, or dose, 
including an overdose. 
 
Please note that all abnormal clinical laboratory values that are determined to be of clinical significance based on 
a physician’s assessment are to be reported as AEs. A clinically significant lab value is one that indicates a new 
disease process, an exacerbation or worsening of an existing condition, or requires further action(s) to be taken. 
Those labs determined to be of no clinical significance or of unknown clinical significance (per the physician’s 
assessment) should not be reported as AEs. Any lab value of unknown clinical significance should continue to be 

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investigated/followed-up further for a final determination, if possible. (See the DCP Baseline and Adverse Event 
Reporting 
Guidelines 
[https://prevention.cancer.gov/clinical-trials/clinical-trials-management/cp-ctnet-
instructions-forms] for more detail on reporting abnormal clinical laboratory values.) 
 
A list of AEs that have occurred or might occur can be found in §8.2 Reported Adverse Events and Potential 
Risks, as well as the Investigator’s Brochure or package insert.  
 
13.1 
Adverse Events 
 
13.1.1 Reportable AEs 
 
All AEs that occur after the informed consent is signed and baseline assessments are collected must be recorded 
on the AE CRF whether or not related to study agent. 
 
13.1.2 AE Data Elements: 
 
The following data elements are required for AE reporting. 
• AE verbatim term 
• NCI Common Terminology Criteria for Adverse Events version 5.0 (CTCAE v5.0) AE term (MedDRA 
lowest level term) 
• CTCAE (MedDRA) System Organ Class (SOC) 
• Event onset date and event ended date 
• Treatment assignment code (TAC) at time of AE onset 
• Severity grade 
• Attribution to study agent (relatedness) 
• Whether or not the event was reported as a SAE 
• Whether or not the participant dropped due to the event 
• Outcome of the event 
 
13.1.3  Severity of AEs 
 
 
 
13.1.3.1 Identify the AE using CTCAE v5.0. The CTCAE provides descriptive terminology (MedDRA lowest 
level term) and a severity grading scale for each AE listed. A copy of the CTCAE can be found at: 
http://ctep.cancer.gov/protocolDevelopment/electronic applications/ctc.htm 
 
AE severity will be assessed according to the grade associated with the CTCAE term. AEs that do not have a 
corresponding CTCAE term will be assessed according to the general guidelines for grading used in the CTCAE 
v5.0. as stated below.  
 
CTCAE v5.0 general severity guidelines:  
 
Grade 
Severity 
Description  
1 
Mild 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 

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2 
Moderate 
Moderate; minimal, local or noninvasive intervention 
indicated; limiting age-appropriate instrumental activities of 
daily living (ADL)*. 
3 
Severe 
Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization 
indicated; disabling; limiting self-care ADL**. 
4 
Life-threatening 
Life-threatening consequences; urgent intervention indicated. 
5 
Fatal 
Death related to AE. 
   
*Instrumental ADL refers to preparing meals, shopping for groceries or clothes, using the telephone, managing 
money, etc. 
**Self-care ADL refers to bathing, dressing and undressing, feeding self, using the toilet, taking medications, 
and not bedridden. 
 
13.1.4 Assessment of relationship of AE to treatment 
 
 
The possibility that the AE is related to study agent will be classified as one of the following: unrelated, unlikely, 
possible, probable, definite. Criteria for these classifications are provided in DCP’s Serious Adverse Event Report 
Form: Instructions for Completion and Submission (https://prevention.cancer.gov/clinical-trials/clinical-
trials-management/cp-ctnet-instructions-forms). 
  
13.1.5 Follow-up of AEs 
 
All AEs, including lab abnormalities that in the opinion of the investigator are clinically significant, will be 
followed according to good medical practices and documented as such. Clinically significant AEs will be 
followed until resolution to ≤ Grade 1 or baseline, or until 4 weeks after completion of the intervention period, 
whichever is earlier. 
 
13.1.6 Collection of AEs 
 
AEs will be collected for each participant until the post-intervention follow-up assessment.   
 
13.2 
Serious Adverse Events  
 
13.2.1 DEFINITION: Regulations at 21 CFR §312.32 define an SAE as any untoward medical occurrence that 
at any dose has one or more of the following outcomes: 
 
• Death 
• A life-threatening AE  
(According to FDA safety guidance, an AE is considered life-threatening if, in the view of either the 
investigator or sponsor, its occurrence places the subject at immediate risk of death. It does not 
include an event that, had it occurred in a more severe form, might have caused death. Example: An 
allergic reaction resulting in angioedema of the larynx, allergic bronchospasm or anaphylaxis is 
considered life-threatening; however, an allergic reaction resulting only in a localized rash is not life-
threatening.)   
     • In patient hospitalization or prolongation of existing hospitalization  

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(NCI, DCP uses admission or stay (including emergency room) equal to or greater than 24 hours as the 
definition of hospitalization. Exceptions are hospitalization for treatment of a pre-existing condition 
[unless the condition increased in severity on study], outpatient surgery, planned/elective procedures, 
and procedures described in the protocol [e.g., pharmacokinetic sampling, surgery] even if the hospital 
stay is of the described length; however, it does include events resulting from any of these that fulfill 
other serious outcome criteria, e.g., prolongation of hospitalization or life-threatening.) 
• A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions 
• A congenital anomaly or birth defect 
• Important medical events that may not be immediately life-threatening or result in death or 
hospitalization should also be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the participant and may require intervention to prevent one of 
the other outcomes listed above. 
 
13.2.2 Reporting SAEs to DCP 
 
13.2.2.1 The accruing LAO and all Affiliated Organizations (AOs) will report SAEs on the DCP SAE Report 
Form as described in DMACC’s CP-CTNet SOP 02-01, found at  CP-CTNet SOP 02-01 Reporting Serious 
Adverse Events (cp-ctnet-dmacc.org) and DCP’s Serious Adverse Event Report Form: Instructions for 
Completion and Submission found at https://prevention.cancer.gov/clinical-trials/clinical-trials-management/cp-
ctnet-instructions-forms. 
 
13.2.2.2 Contact the DCP Medical Monitor, Protocol PI, and DCP Regulatory Contractor’s Safety Department 
within 24 hours of knowledge of the event. Contact via email is preferred, but phone contact is acceptable.  
 
Malgorzata Wojtowicz, MD 
NIH National Cancer Institute 
Division of Cancer Prevention 
9609 Medical Center Drive, Room 5E-104, MSC 9781 
Bethesda, MD 20892 
PHONE: 240-276-7012 
FAX: (240) 276-7848 
malgorzata.wojtowicz@nih.gov  
 
The contact information for the DCP Regulatory Contractor’s Safety Department is: phone: 650-691-4400 
x133; email: safety@ccsainc.com). 
 
Include the following information when contacting the DCP Medical Monitor, Protocol PI, and DCP 
Regulatory Contractor’s Safety Department: 
• Participant ID 
• Date and time of SAE onset 
• Date and time the accruing LAO or AO was notified about the SAE by the study participant or other 
person(s) 
• Name of person reporting the SAE 
• Call back phone number and email address 
• Accruing LAO or AO at which the subject is enrolled DCP protocol number 

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• Title of protocol 
• Suspected drugs (if any) 
• Description of the SAE, including attribution to the Investigational Agent 
 
13.2.2.3  The accruing LAO and AOs will email written SAE reports to the DCP Medical Monitor, Protocol PI, 
LAO Coordinator, and DCP Regulatory Contractor’s Safety Department within 48 hours of learning of the 
event using the Word SAE Report Form.    
  
13.2.2.4  The DCP Medical Monitor and the DCP Regulatory Contractor will determine which SAEs require 
submission to FDA or manufacturer as expedited safety reports. 
 
13.2.2.5  The accruing LAO and AOs will comply with applicable regulatory requirements related to reporting 
SAEs to the CIRB and local IRB/IEC as applicable. Specifically, if an SAE meets the definition of an 
unanticipated problem (UP; i.e., requires expedited reporting to FDA or the manufacturer as a safety report 
[serious, unexpected, and related to a study agent]), then it needs to be reported to the CIRB by the Signatory 
Institution PI at the accruing LAO or AO where the SAE occurred (see SOP 02-02 Reporting Protocol 
Deviations for more information). In addition to CIRB requirements, UPs must be reported to the accruing 
LAO’s or AO’s local IRB per local requirements. 
 
13.2.3 Follow-up of SAE 
 
Accruing LAO or AO staff should send follow-up reports as requested when additional information is available. 
Additional information should be entered on the DCP SAE Report Form in the appropriate format. Follow-up 
information should be sent to the DCP Medical Monitor, Protocol PI, LAO Coordinator, and DCP Regulatory 
Contractor’s Safety Department as soon as available. SAEs determined to be related to study drug will be 
followed for 30 days or until resolution of SAE or stabilization, whichever comes sooner. SAEs determined to be 
unrelated to study drug will be followed for 30 days or until resolution, whichever comes sooner.  
 
14. 
STUDY MONITORING 
            
14.1 
Data Management 
 
This study will report clinical data using Medidata RAVE, a cloud–based clinical trials data management system 
managed by the DMACC. RAVE will be the database of record for the protocol and subject to NCI and FDA 
audit. All RAVE users will be trained to use the system and will comply with the instructions in the guidelines 
provided to the LAO by the DMACC as well as applicable regulatory requirements such as 21 CFR; Part 11.  
 
14.2   Electronic Case Report Forms 
 
The System Variable and Attribute Report (SVAR) template will be used to create the study-specific eCRFs or 
SVAR workbook. DMACC will contact the LAO to determine if a meeting is needed to discuss the trial and 
eCRFs before the DMACC creates the draft SVAR. The SVAR template contains NCI Common Data Elements 
(CDEs) to facilitate data collection and analysis across studies. The SVAR template may require modification to 
capture the unique data elements (i.e., biomarkers) of each protocol and prepare a protocol-specific SVAR 
workbook. NCI CDEs, where available, shall be used for the initial SVAR workbooks and all subsequent 
workbook modifications.  

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More detailed information about the SVAR development process is available at: Program Resources | CP-CTNet 
DMACC website (cp-ctnet-dmacc.org) 
 
14.3 
Source Documents 
 
Source documentation for this trial will consist of protocol-specific source documents as well as selected clinical 
records pertinent for eligibility, medical history and physical findings, adverse events and study endpoints. Only 
those specific records will be copied for the source chart to preserve subject confidentiality. 
  
14.4 
Data and Safety Monitoring Plan 
 
The University of Arizona Cancer Center (UACC) Data and Safety Monitoring Board (DSMB) will provide 
oversight for subject safety for all UA CP-CTNet clinical trials consistent with the National Institutes of Health 
Policy for Data and Safety Monitoring dated June 10, 1998; further guidance statement issued by the NIH on 
June 5, 2000, and the policy for Data and Safety Monitoring by Data and Safety Monitoring Boards.  The UACC 
DSMB meets quarterly. 
 
Regular study-specific meetings will be used as a forum to review accrual rates, problematic issues relating to 
accrual and protocol implementation, adverse events occurrence, follow-up, and reporting; submission of all 
required study reports; and progress and outcomes of laboratory analyses.  
 
14.5 
Sponsor or FDA Monitoring  
 
The NCI, DCP (or their designee), pharmaceutical collaborator (or their designee), or FDA may monitor/audit 
various aspects of the study. These monitors will be given access to facilities, databases, supplies and records to 
review and verify data pertinent to the study.  
 
14.6 
Record Retention 
 
Clinical records for all participants, including eCRFs, all source documentation (containing evidence of study 
eligibility, history and physical findings, laboratory data, results of consultations, etc.), as well as CIRB records 
and other regulatory documentation will be retained by the Investigator in a secure storage facility in compliance 
with Health Insurance Portability and Accountability Act (HIPAA), Office of Human Research Protections 
(OHRP), Food and Drug Administration (FDA) regulations and guidances, and NCI/DCP requirements, unless 
the standard at the site is more stringent. The records for all studies performed under an IND will be maintained, 
at a minimum, for two years after the approval of a New Drug Application (NDA). For NCI/DCP, records will 
be retained for at least three years after the completion of the research. NCI will be notified prior to the planned 
destruction of any materials. The records should be accessible for inspection and copying by authorized persons 
of the Food and Drug Administration. If the study is done outside of the United States, applicable regulatory 
requirements for the specific country participating in the study also apply. 
 
14.7 
Cooperative Research and Development Agreement (CRADA)/Clinical Trials Agreement (CTA) 
 
The agent(s) supplied by DCP, NCI used in this protocol, is/are provided to the NCI under a Collaborative 
Agreement (CTA) between the Nutraceutical Company, Nutramax, Inc (hereinafter referred to as Collaborator(s)) 

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and the NCI Division of Cancer Prevention. Therefore, the following obligations/guidelines, in addition to the 
provisions in the “Intellectual Property Option to Collaborator” contained within the terms of award, apply to the 
use of Agent(s) in this study:  
 
14.7.1 Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be 
transferred or licensed to any party not participating in the clinical study. Collaborator(s) data for Agent(s) are 
confidential and proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol 
documents for studies utilizing investigational agents contain confidential information and should not be shared 
or distributed without the permission of the NCI. If a Participant participating on the study or participant’s family 
member requests a copy of this protocol, the individual should sign a confidentiality agreement. A suitable model 
agreement can be downloaded from the DCP website. 
 
14.7.2 For a clinical protocol where there is an Investigational Agent used in combination with (an) other 
Investigational Agent(s), each the subject of different collaborative agreements, the access to and use of data by 
each Collaborator shall be as follows (data pertaining to such combination use shall hereinafter be referred to as 
"Multi-party Data”). 
 
14.7.3 NCI must provide all Collaborators with prior written notice regarding the existence and nature of any 
agreements governing their collaboration with NIH, the design of the proposed combination protocol, and the 
existence of any obligations that would tend to restrict NCI's participation in the proposed combination protocol. 
 
14.7.4 Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by any other 
Collaborator solely to the extent necessary to allow said other Collaborator to develop, obtain regulatory approval, 
or commercialize its own investigational agent. 
 
14.7.5 Any Collaborator having the right to use the Multi-Party Data from these trials must agree in writing prior 
to the commencement of the trials that it will use the Multi-Party Data solely for development, regulatory 
approval, and commercialization of its own investigational agent. 
14.7.6 Clinical Trial Data and Results and Raw Data developed under a collaborative agreement will be made 
available exclusively to Collaborator(s), the NCI, and the FDA, as appropriate. All data made available will 
comply with HIPAA regulations. 
 
14.7.7 When a Collaborator wishes to initiate a data request, the request should first be sent to the NCI, who will 
then notify the appropriate investigators of Collaborator's wish to contact them. 
 
14.7.8 Any manuscripts reporting the results of this clinical trial must be provided to DCP for immediate delivery 
to Collaborator(s) for advisory review and comment prior to submission for publication. Collaborator(s) will have 
30 days (or as specified in the CTA) from the date of receipt for review. Collaborator shall have the right to 
request that publication be delayed for up to an additional 30 days in order to ensure that Collaborator’s 
confidential and proprietary data, in addition to Collaborator(s)’s intellectual property rights, are protected. 
Copies of abstracts must be provided to DCP for forwarding to Collaborator(s) for courtesy review as soon as 
possible and preferably at least three days prior to submission, but in any case, prior to presentation at the meeting 
or publication in the proceedings. Press releases and other media presentations must also be forwarded to DCP 
prior to release. Copies of any manuscript, abstract, and/or press release/ media presentation should be sent to the 
Protocol Information Office at NCI_DCP_PIO@mail.nih.gov.  
 
 
 
 

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The Protocol Information Office will forward manuscripts to the DCP Project Officer for distribution to the 
Collaborator(s). No publication, manuscript or other form of public disclosure shall contain any of Collaborator’s 
confidential/proprietary information. 
 
15. 
STATISTICAL CONSIDERATIONS 
 
15.1 
Study Design/Description 
 
This is a phase II, randomized, double-blinded, placebo-controlled study to evaluate whether BSSE can 
upregulate Phase II detoxification pathways in firefighters. 
  
 
15.2 
Randomization/Stratification  
 
Each participant will be randomly assigned to BSSE or matched placebo. Block randomization will be used to 
achieve balance in treatment group allocation. 
 
15.3 
Sample Size 
 
We are planning to randomize 33 participants to each group (i.e., BSSE and matched placebo) to achieve an 
evaluable sample size of 30 participants in each group (assuming loss to follow-up of <10%), where evaluable is 
defined as the initiation of treatment and the availability of pre-intervention and end-of-intervention urine samples 
for urinary acetaminophen mercapturate, a surrogate for detoxification of the carcinogen benzene to SPMA.  A 
sample size of 30 per group produces a one-sided 95% CI with a width no wider than 0.432 pooled standard 
deviations for the difference in change of log-transformed total amount of acetaminophen mercapturate excreted 
in the urine at the end of intervention from baseline between the BSSE and placebo groups. This sample size (i.e. 
30/group) is large enough to reasonably estimate the parameters for use in a future definitive trial and also 
consistent with the recommended sample size for a future trial designed with 90% power for detecting a small 
effect size of 0.20 Cohen’s d at a two-sided significance level of 5%65. In addition, a sample size of 30/group will 
also help us assess the potential efficacy of BSSE, by giving 80% power to detect an effect size of 0.65 Cohen’s 
d for change in log-transformed total amount of acetaminophen mercapturate based on a two-sample t-test at a 
one-sided significance level of 5%. The anticipated effect size for changes in acetaminophen mercapturate—a 
surrogate marker for detoxification of electrophilic environmental toxicants such as benzene, acrolein, and 
crotonaldehyde—is informed by our prior study of BSSE48  which showed a consistent and significant increase 
in the level of conjugated detoxification products of benzene, acrolein and crotonaldhyde in most subjects 
evaluated.  
 
While metabolism of drugs and toxicants varies widely across individuals, smokers typically exhibit elevated 
baseline detoxification activity due to chronic exposure-induced enzyme upregulation. As a result, smokers tend 
to be rapid metabolizers67.  As such, we anticipate an even greater change in acetaminophen detoxification 
products among healthy nonsmokers, who lack this background stimulation. However, to ensure a conservative 
and rigorous design, the current study is powered based on the observed effect size in smokers. A substantial 
body of literature supports the existence of both low and rapid metabolizers, reinforcing the premise that 
susceptibility to cancer from environmental exposures is closely tied to an individual’s detoxification capacity—
an inherently variable and modifiable trait even in healthy populations.PASS 2022 was used to perform sample 
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15.4 
Primary Objective, Endpoint(s), Analysis Plan 
 
The primary objective of this study is to evaluate the effects of BSSE on the urinary excretion of mercapturic acid 
of acetaminophen following acetaminophen dosing, a surrogate for detoxification of the carcinogen benzene. The 
change (from baseline to end-of-intervention) in total amount of acetaminophen mercapturate excreted in the 
urine after acetaminophen dosing will be compared between BSSE and placebo arms. Log-transformation will 
be applied to total amount of acetaminophen mercapturate before deriving the changes at the end of intervention 
from baseline since the total amount of acetaminophen mercapturate may be highly right skewed. Even if the 
total amount of acetaminophen mercapturate  is not highly right skewed, log-transformation will also allow us to 
compare the relative (%) change in total amount of acetaminophen mercapturate at the end of intervention from 
baseline between the BSSE and placebo groups through use of the geometric mean of total amount of 
acetaminophen mercapturate. The relative (%) change and the associated 95% CI by group, as well as the 
difference in the relative (%) change between BSSE and placebo groups and the associated 95% CI, will be 
reported. A two-sample t test will be performed to compare changes in the log-transformed total amounts of 
acetaminophen mercapturate between BSSE and placebo.   
 
15.5 
Secondary Objectives, Endpoints, Analysis Plans 
 
The secondary objectives and their respective endpoints are: 
1) To evaluate the effects of BSSE on urinary excretion of acetaminophen glucuronide, a surrogate for 
detoxification of PAHs. Similar to the total amount of acetaminophen mercapturate, log-transformation 
will be applied to the total amount of acetaminophen glucuronide excreted in the urine before deriving 
the changes at the end of intervention from baseline. A two-sample t test will be performed to compare 
changes in the log-transformed total amounts of acetaminophen glucuronide between BSSE and placebo.   
2) To evaluate the safety and tolerability of BSSE, as measured by NCI CTCAE version 5. To evaluate the 
safety of BSSE, toxicity will be measured by adverse events (AE) classified in accordance with NCI 
CTCAE version 5. The frequency and associated percentage of each specific AE will be tabulated by 
treatment group and then compared via Fisher’s exact test. Tolerability will be measured by adherence 
rate and will be compared between the BSSE and placebo via Fisher’s exact test. 
 
The exploratory objectives and their respective endpoints and analysis plans are:  
1) To evaluate whether the GSTM1 and GSTT1 genotypes are important genetic modulators of BSSE-
induced detoxification of carcinogens in the setting of exposure to BSSE. The correlation of GSTM1 and 
GSTT1 genotypes with detoxication of acetaminophen mercapturate/acetaminophen glucuronide will be 
assessed by linear regression for changes from baseline in the log-transformed acetaminophen 
mercapturate/acetaminophen glucuronide, in which BSSE and genotype indicators and interactions of 
BSSE, and genotype indicators are the covariates in the model. This will allow us to study whether the 
BSSE effect is significantly modulated by the genotypes. 
2) To explore the effects of BSSE on the urinary metabolome after fire/fuel leak activities. Urinary 
metabolome will be measured at baseline, post fire exposure, and post study agent intervention. A linear 
mixed effects model with BSSE and post-intervention indicators and their interaction term as the 
covariates will be fitted to changes from baseline in each of the log-transformed metabolite levels. This 
model will allow us to compare the change between BSSE and placebo at post fire exposure and post-
intervention, respectively, as well as comparing the changes at post-intervention from post fire exposure 
between BSSE and placebo.   

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3) To assess the effects of BSSE on the  urinary excretion of mercapturic acid of benzene after fire/fuel leak 
activities. Urine excretion of mercapturic acid of benzene will be measured at baseline, post fire exposure, 
and post study agent intervention. A linear mixed effects model with BSSE and post-intervention 
indicators and their interaction term as the covariates will be fitted to changes from baseline in the log-
transformed levels of mercapturic acid of benzene. This model will allow us to compare the change 
between BSSE and placebo at post fire exposure and post-intervention, respectively, as well as comparing 
the changes at post-intervention from post fire exposure between BSSE and placebo.   
4) To evaluate the effects of BSSE on the urinary excretion of metabolites of PAHs after fire/fuel leak 
activities. PAH metabolites will be measured at baseline, post fire exposure, and post study agent 
intervention. A linear mixed effects model with BSSE and post-intervention indicators and their 
interaction term as the covariates will be fitted to changes from baseline  in each of the log-transformed 
PAH metabolite levels. This model will allow us to compare the change between BSSE and placebo at 
post fire exposure and post-intervention, respectively, as well as comparing the changes at post-
intervention from post fire exposure between BSSE and placebo.   
5) To assess the effects of BSSE on pre- to post-intervention modifications of epigenetics (blood microRNA 
and DNA methylation). Two-sample t tests will be performed to compare changes in log-transformed 
expression levels between BSSE and placebo. 
 
Due to the exploratory nature of the secondary and exploratory endpoints and multiple comparisons associated 
with the evaluations, the  false discovery rate (FDR)  will be controlled at 10% using the Benjamin-Hochberg 
procedure and the results and findings will be interpreted cautiously. In addition,  the total number of comparisons 
will be reported to allow one to control for FDR at a specific rate, say, ≤10% via the adjusted p-value method. 
For exploratory endpoints, linear regression may be used to control for variables related to fire/fuel leak exposure 
when evaluating the effects of BSSE on those endpoints.    
 
15.6 
Reporting and Exclusions 
 
The definition of “evaluable” for the primary endpoint analysis is a participant who initiated protocol treatment 
and provided both pre-study agent intervention and post-study agent intervention urine samples for urinary 
acetaminophen mercapturate. All of the evaluable participants will be included in the primary analysis. 
 
Participants are considered compliant for secondary “per protocol” statistical analysis if they have taken ≥ 80% 
of their assigned study agent doses, as assessed by Study Diary and pill count. 
 
15.7 
Evaluation of Toxicity 
 
All participants will be evaluable for toxicity from the time of their first dose of BSSE vs. placebo. Descriptive 
statistics of the type and frequency of all AEs will be generated, including 95% confidence intervals. 
 
15.8 
Evaluation of Response 
 
All participants with endpoint data will be assessed for response to intervention, based on the endpoints described 
above in sections 15.4 and 15.5. 
 
15.9 
Interim Analysis 
 

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None. 
 
15.10 Ancillary Studies 
 
None. 
 
16. 
REGULATORY AND ETHICAL CONSIDERATIONS 
 
16.1 
Required Documents 
 
Besides the regulatory information that will be entered into the Registration and Credential Repository (see 
Section 5.1), the following documents are also required: 
 
16.1.1 Documentation of Federalwide Assurance (FWA) number for the LAO and all AOs. 
 
16.1.2 Signed Investigator’s Brochure/Package Insert acknowledgement form 
 
16.1.3 Delegation of Tasks Log form for the Lead Accruing Organization and all Accruing Sites signed by the 
PI for each site and initialed by all study personnel listed on the form.   
 
16.2 
Informed Consent 
 
All potential study participants will be given a copy of the CIRB-approved Informed Consent to review. The 
investigator will explain all aspects of the study in lay language and answer all questions regarding the study. If 
the participant decides to participate in the study, he/she will be asked to sign and date the Informed Consent 
document. The study agent(s) will not be released to a participant who has not signed the Informed Consent 
document. Individuals who refuse to participate or who withdraw from the study will be treated without prejudice. 
 
Participants must be provided the option to allow the use of blood samples, other body fluids, and tissues obtained 
during testing, operative procedures, or other standard medical practices for further research purposes. If 
applicable, statement of this option should be included within the informed consent document.  
 
Prior to study initiation, the informed consent document must be reviewed and approved by NCI, DCP, and the 
NCI CIRB. The NCI CIRB approves a model consent for each protocol. Each Signatory Institution inserts their 
CIRB-approved institutional boilerplate language into the model consent to create the CIRB-approved consent. 
If the model informed consent document is amended, Signatory Institutions must use the revised model informed 
consent document and insert their CIRB-approved institutional boilerplate language at the time the change 
becomes active.  
 
The NCI CIRB is the IRB of record and is the only IRB authorized to approve changes to the protocol or informed 
consent document. Institutions may require additional oversight that involves the local IRB, but the local IRB is 
not responsible for any regulatorily-required IRB actions.  
 
16.3 
Collection of Regulatory Documents 
 

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Regulatory documents will be collected by the DCP regulatory contractor and reviewed for completeness and 
accuracy.  
 
16.4 
Other 
This trial will be conducted in compliance with the protocol, the International Conference on Harmonisation’s 
(ICH) Good Clinical Practice (GCP) guidelines, and the applicable regulatory requirements. 
17.  
ROSTER MANAGEMENT 
 
The LAO is responsible for establishing, maintaining, and monitoring all its members that participate in CP-
CTNet studies. The LAO must have a “real-time,” comprehensive, consolidated roster of all its members with 
their relevant Cancer Therapy Evaluation Program (CTEP) institution codes, associated investigators, and 
research staff. This roster information is used for determining compliance with monitoring requirements. 
 
The LAO’s organizational rosters will be managed by the CP-CTNet Roster Management System website 
(https://applications.prevention.cancer.gov/cp-ctnet). Requests to add memberships to a roster will be done via 
this website. All requests require that the following documents be uploaded: 
• Consortium Letter of Commitment 
• Site Letter of Commitment 
• CV/NIH Biosketch 
 
18. 
FINANCING, EXPENSES, AND/OR INSURANCE 
 
Study procedures performed during study visits will be covered by the study budget.  Research tests, including 
serum and tissue biomarker evaluations, will not be billed to the subject.  Subjects may incur minimal out-of-
pocket expenses such as transportation but will not be charged for study agent or any study-related activities.  
Subjects that complete all required visits and procedures will receive monetary compensation of $350 U.S. dollars 
which they may use at their discretion for out-of-pocket cost such as transportation. If a subject is unable to 
complete the entire study, the amount of compensation will be based on how long the subject is in the study.   If 
injury occurs, medical care will be provided and charged to the subject’s insurer. 
 
 
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Modes of Action. Curr Drug Metab 2021, 22 (8), 584-595. DOI: 10.2174/1389200221999201125205725. Baird, W. M.; Hooven, L. 
A.; Mahadevan, B. Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ Mol Mutagen 
2005, 45 (2-3), 106-114. DOI: 10.1002/em.20095. 
(24) Hernandez, L. G.; van Steeg, H.; Luijten, M.; van Benthem, J. Mechanisms of non-genotoxic carcinogens and importance of a 
weight of evidence approach. Mutat Res 2009, 682 (2-3), 94-109. DOI: 10.1016/j.mrrev.2009.07.002. Nohmi, T. Thresholds of 
Genotoxic and Non-Genotoxic Carcinogens. Toxicol Res 2018, 34 (4), 281-290. DOI: 10.5487/TR.2018.34.4.281. 
(25) Prochaska, H. J.; Santamaria, A. B.; Talalay, P. Rapid detection of inducers of enzymes that protect against carcinogens. 
Proceedings of the National Academy of Sciences of the United States of America 1992, 89 (6), 2394-2398. 
(26) Kensler, T. W. Chemoprevention by inducers of carcinogen detoxication enzymes. Environmental health perspectives 1997, 105 
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I.; et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response 
elements. Biochemical and biophysical research communications 1997, 236 (2), 313-322. 
(27) Zhang, Y.; Tang, L. Discovery and development of sulforaphane as a cancer chemopreventive phytochemical. Acta 
pharmacologica Sinica 2007, 28 (9), 1343-1354. DOI: 10.1111/j.1745-7254.2007.00679.x. 
(28) Hong, F.; Freeman, M. L.; Liebler, D. C. Identification of sensor cysteines in human Keap1 modified by the cancer 
chemopreventive agent sulforaphane. Chemical research in toxicology 2005, 18 (12), 1917-1926. DOI: 10.1021/tx0502138. 
(29) Kensler, T. W.; Egner, P. A.; Agyeman, A. S.; Visvanathan, K.; Groopman, J. D.; Chen, J. G.; Chen, T. Y.; Fahey, J. W.; 
Talalay, P. Keap1-nrf2 signaling: a target for cancer prevention by sulforaphane. Topics in current chemistry 2013, 329, 163-177. 
DOI: 10.1007/128_2012_339. 
(30) Fahey, J. W.; Zhang, Y.; Talalay, P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against 
chemical carcinogens. Proceedings of the National Academy of Sciences of the United States of America 1997, 94 (19), 10367-10372. 
(31) Kensler, T. W.; Chen, J. G.; Egner, P. A.; Fahey, J. W.; Jacobson, L. P.; Stephenson, K. K.; Ye, L.; Coady, J. L.; Wang, J. B.; 
Wu, Y.; et al. Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a 
randomized clinical trial in He Zuo township, Qidong, People's Republic of China. Cancer epidemiology, biomarkers & prevention : a 
publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2005, 14 
(11 Pt 1), 2605-2613. DOI: 10.1158/1055-9965.EPI-05-0368. Egner, P. A.; Chen, J. G.; Wang, J. B.; Wu, Y.; Sun, Y.; Lu, J. H.; Zhu, 
J.; Zhang, Y. H.; Chen, Y. S.; Friesen, M. D.; et al. Bioavailability of Sulforaphane from two broccoli sprout beverages: results of a 
short-term, cross-over clinical trial in Qidong, China. Cancer prevention research 2011, 4 (3), 384-395. DOI: 10.1158/1940-
6207.CAPR-10-0296. Kensler, T. W.; Ng, D.; Carmella, S. G.; Chen, M.; Jacobson, L. P.; Munoz, A.; Egner, P. A.; Chen, J. G.; Qian, 
G. S.; Chen, T. Y.; et al. Modulation of the metabolism of airborne pollutants by glucoraphanin-rich and sulforaphane-rich broccoli 
sprout beverages in Qidong, China. Carcinogenesis 2012, 33 (1), 101-107. DOI: 10.1093/carcin/bgr229. Egner, P. A.; Chen, J. G.; 
Zarth, A. T.; Ng, D. K.; Wang, J. B.; Kensler, K. H.; Jacobson, L. P.; Munoz, A.; Johnson, J. L.; Groopman, J. D.; et al. Rapid and 
sustainable detoxication of airborne pollutants by broccoli sprout beverage: results of a randomized clinical trial in China. Cancer 
prevention research 2014, 7 (8), 813-823. DOI: 10.1158/1940-6207.CAPR-14-0103. 
(32) Bauman, J. E.; Zang, Y.; Sen, M.; Li, C.; Wang, L.; Egner, P. A.; Fahey, J. W.; Normolle, D. P.; Grandis, J. R.; Kensler, T. W.; 
et al. Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane. Cancer prevention research 2016, 9 (7), 547-557. DOI: 
10.1158/1940-6207.CAPR-15-0290. 
(33) Bauman, J. E.; Hsu, C. H.; Centuori, S.; Guillen-Rodriguez, J.; Garland, L. L.; Ho, E.; Padi, M.; Bageerathan, V.; Bengtson, L.; 
Wojtowicz, M.; et al. Randomized Crossover Trial Evaluating Detoxification of Tobacco Carcinogens by Broccoli Seed and Sprout 
Extract in Current Smokers. Cancers (Basel) 2022, 14 (9). DOI: 10.3390/cancers14092129. 
(34) Fahey, J. W.; Haristoy, X.; Dolan, P. M.; Kensler, T. W.; Scholtus, I.; Stephenson, K. K.; Talalay, P.; Lozniewski, A. 
Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-
induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (11), 7610-7615. 
DOI: 10.1073/pnas.112203099. Xu, C.; Huang, M. T.; Shen, G.; Yuan, X.; Lin, W.; Khor, T. O.; Conney, A. H.; Kong, A. N. 
Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin tumorigenesis in C57BL/6 mice by sulforaphane is mediated by nuclear 
factor E2-related factor 2. Cancer research 2006, 66 (16), 8293-8296. DOI: 10.1158/0008-5472.CAN-06-0300. Ramos-Gomez, M.; 
Kwak, M. K.; Dolan, P. M.; Itoh, K.; Yamamoto, M.; Talalay, P.; Kensler, T. W. Sensitivity to carcinogenesis is increased and 
chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proceedings of the National Academy 
of Sciences of the United States of America 2001, 98 (6), 3410-3415. DOI: 10.1073/pnas.051618798. 
(35) Cornblatt, B. S.; Ye, L.; Dinkova-Kostova, A. T.; Erb, M.; Fahey, J. W.; Singh, N. K.; Chen, M. S.; Stierer, T.; Garrett-Mayer, 
E.; Argani, P.; et al. Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. Carcinogenesis 2007, 28 
(7), 1485-1490. DOI: 10.1093/carcin/bgm049. Zhang, Y.; Kensler, T. W.; Cho, C. G.; Posner, G. H.; Talalay, P. Anticarcinogenic 
activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proceedings of the National Academy of 
Sciences of the United States of America 1994, 91 (8), 3147-3150. 

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(36) Ohkoshi, A.; Suzuki, T.; Ono, M.; Kobayashi, T.; Yamamoto, M. Roles of Keap1-Nrf2 system in upper aerodigestive tract 
carcinogenesis. Cancer prevention research 2013, 6 (2), 149-159. DOI: 10.1158/1940-6207.CAPR-12-0401-T. 
(37) Vetvicka, V.; Vetvickova, J. A Novel Glucan-Sulforaphane Combination Stimulates Immune Response to Influenza in Mouse 
Model. American Journal of Immunology 2016, 12 (2), 20.28. 
(38) Fahey, J. W.; Wade, K. L.; Stephenson, K. K.; Panjwani, A. A.; Liu, H.; Cornblatt, G.; Cornblatt, B. S.; Ownby, S. L.; Fuchs, E.; 
Holtzclaw, W. D.; et al. Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed 
Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration. Nutrients 2019, 11 (7). DOI: 
10.3390/nu11071489. 
(39) Bauman, J. E.; Hsu, C.-H.; Centuori, S.; Guillen-Rodriguez, J.; Garland, L.; Ho, E.; Bengston, L.; Wojtowicz, M.; Szabo, E.; 
Chow, H.-H. S. Randomized crossover trial evaluating detoxication of tobacco carcinogens by broccoli seed and sprout extract in 
heavy smokers. Cancer research 2021. 
(40) Evans, M.; Paterson, E.; Barnes, D. M. An open label pilot study to evaluate the efficacy of Spanish black radish on the induction 
of phase I and phase II enzymes in healthy male subjects. BMC Complement Altern Med 2014, 14, 475. DOI: 10.1186/1472-6882-14-
475. 
(41) Cook, S. F.; King, A. D.; van den Anker, J. N.; Wilkins, D. G. Simultaneous quantification of acetaminophen and five 
acetaminophen metabolites in human plasma and urine by high-performance liquid chromatography-electrospray ionization-tandem 
mass spectrometry: Method validation and application to a neonatal pharmacokinetic study. J Chromatogr B Analyt Technol Biomed 
Life Sci 2015, 1007, 30-42. DOI: 10.1016/j.jchromb.2015.10.013. 
(42) Haiman, C. A.; Patel, Y. M.; Stram, D. O.; Carmella, S. G.; Chen, M.; Wilkens, L. R.; Le Marchand, L.; Hecht, S. S. Benzene 
Uptake and Glutathione S-transferase T1 Status as Determinants of S-Phenylmercapturic Acid in Cigarette Smokers in the Multiethnic 
Cohort. PloS one 2016, 11 (3), e0150641. DOI: 10.1371/journal.pone.0150641. 
(43) Wang, Y.; Meng, L.; Pittman, E. N.; Etheredge, A.; Hubbard, K.; Trinidad, D. A.; Kato, K.; Ye, X.; Calafat, A. M. Quantification 
of urinary mono-hydroxylated metabolites of polycyclic aromatic hydrocarbons by on-line solid phase extraction-high performance 
liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2017, 409 (4), 931-937. DOI: 10.1007/s00216-016-9933-x. 
(44) Kelsey, K. T.; Nelson, H. H.; Wiencke, J. K.; Smith, C. M.; Levin, S. The glutathione S-transferase theta and mu deletion 
polymorphisms in asbestosis. Am J Ind Med 1997, 31 (3), 274-279. DOI: 10.1002/(sici)1097-0274(199703)31:3<274::aid-
ajim2>3.0.co;2-y. 
(45) Furlong, M. A.; Liu, T.; Snider, J. M.; Tfaily, M. M.; Itson, C.; Beitel, S.; Parsawar, K.; Keck, K.; Galligan, J.; Walker, D. I.; et 
al. Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach. Sci Rep 2023, 
13 (1), 20872. DOI: 10.1038/s41598-023-47799-x  From NLM Medline. 
(46) Goodrich, J. M.; Furlong, M. A.; Caban-Martinez, A. J.; Jung, A. M.; Batai, K.; Jenkins, T.; Beitel, S.; Littau, S.; Gulotta, J.; 
Wallentine, D.; et al. Differential DNA Methylation by Hispanic Ethnicity Among Firefighters in the United States. Epigenet Insights 
2021, 14, 25168657211006159. DOI: 10.1177/25168657211006159  From NLM PubMed-not-MEDLINE. Goodrich, J. M.; Jung, A. 
M.; Furlong, M. A.; Beitel, S.; Littau, S.; Gulotta, J.; Wallentine, D.; Burgess, J. L. Repeat measures of DNA methylation in an 
inception cohort of firefighters. Occup Environ Med 2022, 79 (10), 656-663. DOI: 10.1136/oemed-2021-108153  From NLM 
Medline. 
(47) Forrest, J. A.; Clements, J. A.; Prescott, L. F. Clinical pharmacokinetics of paracetamol. Clin Pharmacokinet 1982, 7 (2), 93-107. 
DOI: 10.2165/00003088-198207020-00001  From NLM. 
(48) Barr, D. B.; Wilder, L. C.; Caudill, S. P.; Gonzalez, A. J.; Needham, L. L.; Pirkle, J. L. Urinary Creatinine Concentrations in the 
U.S. Population: Implications for Urinary Biologic Monitoring Measurements. Environmental Health Perspectives 2005, 113 (2), 
192-200. DOI: doi:10.1289/ehp.7337. 

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APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
 
Karnofsky Performance Scale 
Percent 
Description 
100 
Normal, no complaints, no evidence of disease. 
90 
Able to carry on normal activity; minor signs or symptoms of disease. 
80 
Normal activity with effort; some signs or symptoms of disease. 
70 
Cares for self, unable to carry on normal activity or to do active work. 
60 
Requires occasional assistance but is able to care for most of his/her needs. 
50 
Requires considerable assistance and frequent medical care. 
40 
Disabled, requires special care and assistance. 
30 
Severely disabled, hospitalization indicated. Death not imminent. 
20 
Very sick, hospitalization indicated. Death not imminent. 
10 
Moribund, fatal processes progressing rapidly. 
0 
Dead. 
 
 
 

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APPENDIX B 
 
ALCOHOL AND TOBACCO QUESTIONNAIRE INSTRUCTIONS 
. 
• Data collection will be required for all CP-CTNet studies. 
o Data will be collected at baseline and end of every study. Data may also be collected at follow-up 
visits as determined by each protocol. If you wish to collect additional information beyond these 
core elements, you may certainly do so. However, all studies need to collect the basic elements in 
the attached eCRFs.  
o The eCRFs will be completed by the Site Staff or participant at the time of the designated visit. 
• Data will be submitted as part of the final clinical data set. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ALCOHOL ASSESSMENT-- BASELINE 
 
REGISTERING 
INSTITUTION  
 
_________________________________ 
PARTICIPANT 
ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions: 
For the following questions about drinking alcoholic beverages, a drink means a 12 oz. beer, a 5 oz. glass 
of wine, or one and a half ounces of liquor.  
When a number is requested in the response, please enter a whole number (i.e. “4”) and not a range or 
fraction of a number.  
 
1. In your entire life, have you had at least 12 drinks of any kind of alcoholic beverage?  
 
 Yes 
 No (End) 
 Refuse to answer (End) 
 Don’t know/Not sure 
 
2. In the past 12 months, on average, how often did you drink any type of alcoholic beverage?  
 
________ (Enter the number of days you drank based on the timeframe checked below.  Enter 0 if you 
never drank and skip to Question 6.)  
 Week  
 Month 
 Year 
 Refuse to answer 
 Don’t know/Not sure 
 
3. In the past 12 months, on those days that you drank alcoholic beverages, on average, how many drinks did 
you have per day?  
 
________ (Enter the average number of drinks per day)  
 
 Refuse to answer 
 Don’t know/Not sure 
 
4. In the past 12 months, on how many days did you have 5 or more drinks of any alcoholic beverage?  
 
_________ (Enter the number of days you had 5 or more drinks or enter 0 if none.) 
 
 Refuse to answer 
 Don’t know/Not sure 
 

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5. Was there ever a time or times in your life when you drank 5 or more drinks of any kind of alcoholic 
beverage almost every day?  
 
 Yes 
 No  
 Refuse to answer 
 Don’t know/Not sure 
 
6. If you do not currently drink alcoholic beverages, but did in the past, how long has it been since you last 
drank regularly?  
 
 Within the past month (0 to 1 month ago) 
 Between 1 and 3 months (1 to 3 months ago) 
 Between 3 and 6 months (3 to 6 months ago) 
 Between 6 and 12 months (6 to 12 months ago) 
 Between 1 and 5 years (1 to 5 years ago) 
 Between 5 and 15 years (5 to 15 years ago) 
 More than 15 years ago  
 Don’t know/Not sure 
 Never drank regularly 
 
7. At the heaviest point, either now or in the past, on the days when you drank, about how many drinks did you 
drink a day on the average?  
 
__________ (Enter the number of drinks a day) 
  
 Refuse to answer 
 Don’t know/Not sure 
 
8. How many years have you been drinking (or did drink) regularly?  
 
______ years 
 
 Refuse to answer 
 Don’t know/Not sure  
 
9. At what age did you begin drinking regularly?  
 
______ years of age 
 
 
 Refuse to answer 
 Don’t know/Not sure  
 

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10. What type(s) of alcohol do you drink? (Mark ALL that apply) 
 
 Wine 
 Liquor 
 Beer 
 Wine cooler 
 Other _______ (enter other type(s) of alcohol you drink) 
 
 
CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ALCOHOL ASSESSMENT - FOLLOW-UP 
 
 
REGISTERING 
INSTITUTION  
 
_________________________________ 
PARTICIPANT 
ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions: 
 
For the following questions about drinking alcoholic beverages, a drink means a 12 oz. beer, a 5 oz. glass 
of wine, or one and a half ounces of liquor.  
When a number is requested in the response, please enter a whole number (i.e. “4”) and not a range or 
fraction of a number. 
 
1. During the past 30 days, did you drink any alcoholic beverages?  
 
 Yes 
 No (End) 
 Refuse to answer (End) 
 Don’t know/Not sure 
 
2. During the past 30 days, how many days per week or per month did you drink any alcoholic beverages, on 
the average?  
 
________ (Enter number of days you drank based on the timeframe checked below.  Enter 0 if you did not 
drink.)  
 
 Week  
 Month 
 Refuse to answer 
 Don’t know/Not sure 
 
3. On the days when you drank, on average, about how many drinks did you have?  
 
________ (Enter the average number of drinks you had per day.) 
 
 Refuse to answer 
 Don’t know/Not sure 
 
4. In the past 30 days, on how many days did you have 5 or more drinks per day?  
 
_____________ (Enter the number of days you had 5 or more drinks or enter 0 if none.)  
 

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 Refuse to answer 
 Do not know/Not sure 
 
 
CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TOBACCO ASSESSMENT – BASELINE 
 
REGISTERING 
INSTITUTION  
 
_________________________________ 
PARTICIPANT ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions:  
When a number is requested in the response, please enter a whole number (i.e. “4”) and not a range or 
fraction of a number.  
 
Section A. Basic Cigarette Use Information 
 
1. Have you smoked at least 100 cigarettes (5 packs = 100 cigarettes) in your entire life? 
 
Yes  
 
 No → Skip to Section B 
 Don’t know/Not sure → Skip to Section B 
 
2. How old were you when you first smoked a cigarette (even one or two puffs)? 
 
_______ Years old 
 
 
3. How old were you when you first began smoking cigarettes regularly?   
 
_______ Years old 
 
  Check here if you have never smoked cigarettes regularly. 
 
4. How many total years have you smoked (or did you smoke) cigarettes? Do not count any time you may have 
stayed off cigarettes. 
 
______ Years (If you smoked less than one year, write “1.”) 
 
 
5. On average when you have smoked, about how many cigarettes do you (or did you) smoke a day? (A pack 
usually has 20 cigarettes in it).  
 
_____ Number of cigarettes per day 
 
6. Do you NOW smoke cigarettes?  
 
 Everyday 
 Some days 
 Not at all → Skip to question 8 
 
 

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7.  How soon after you wake up do you smoke your first cigarette?  
 
  Within 30 minutes 
  After 30 minutes 
 
8. How long has it been since you last smoked a cigarette (even one or two puffs)?  
 
First check which one of the following choices applies to you. Then, if applicable, write a number on the line 
for how many days, weeks, months, or years it has been since your last cigarette. 
 
 I smoked a cigarette today (at least one puff) 
 1-7 days → Number of days since last cigarette ________ 
 Less than 1 month → Number of weeks since last cigarette _________ 
 Less than 1 year → Number of months since last cigarette __________ 
 More than 1 year → Number of years since last cigarette ___________ 
 Don’t know/Don’t remember 
 
Section B. Use of Other Forms of Tobacco 
 
9.  Have you ever used other forms of tobacco, not including cigarettes? 
  
 Yes  
 No → Skip to Section C 
 
10. How often do you/did you use other forms of tobacco?  
 
 Every day → Number of times per day ________ 
 Some days → Number of days _______ per 
 Week 
 Month 
Year 
 
11. Which of the following products have you ever used regularly? 
 Check all that apply  
 
 Cigarettes 
 E-cigarettes or other electronic nicotine delivery system 
 Traditional cigars, cigarillos or filtered cigars 
 Pipes 
 Waterpipe 
 Hookah 
 Clove cigarettes or kreteks 
 Bidis  
 Smokeless tobacco, like dip, chew, or snuff 
 Snus 
 Paan with tobacco, gutka, zarda, khaini 

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 Other, Please specify: __________________________ 
 
12.  If you do not currently use other forms of tobacco, but did in the past, how long has it been since you last 
used other forms of tobacco regularly?  
 
 Within the past month (0 to 1 month ago) 
 Between 1 and 3 months (1 to 3 months ago) 
 Between 3 and 6 months (3 to 6 months ago) 
 Between 6 and 12 months (6 to 12 months ago) 
 Between 1 and 5 years (1 to 5 years ago) 
 Between 5 and 15 years (5 to 15 years ago) 
 More than 15 years ago 
 Don’t know/Not sure 
 Never used other forms of tobacco regularly 
 
Section C. Second-Hand Smoke Exposure 
 
13. Are you currently living with a smoker?   
 
Yes 
 
 No  
 
14. In the past 30 days, have you lived in a place where other people smoked cigarettes indoors?  
 
Yes 
 
 No  
 
15. In the past 30 days, have you worked in a place where other people smoked cigarettes indoors? 
 
Yes 
 
 No  
 
16. Thinking of all your childhood and adult years, have you ever lived in a place where other people smoked 
cigarettes indoors? 
 
 
 Yes 
In total, for about how many years? _______ If less than 1, write “1.”   
 No  
 
17. Thinking of all the years you have worked, have you ever worked in a place where other people smoked 
cigarettes indoors?   
 

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Yes 
→ In total, for about how many years? _______ If less than 1, write “1.”    
 No 
 
 
CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TOBACCO ASSESSMENT - FOLLOW-UP 
 
REGISTERING 
INSTITUTION 
 
_________________________________ 
PARTICIPANT 
ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions:  
When a number is requested in the response, please enter a whole number (i.e. “4”) and not a range or 
fraction of a number.  
 
1. Do you NOW smoke cigarettes?  
 
 Everyday 
 Some days 
 Not at all → Skip to Question 3. 
 Never smoked → Skip to Question 4 
 
2. On average, when you smoked, about how many cigarettes do you (or did you) smoke a day? (A pack usually 
has 20 cigarettes in it).     
_____ Number of cigarettes per day 
 
3. How long has it been since you last smoked a cigarette (even one or two puffs)?    
 
First check which one of the following choices applies to you. Then, if applicable, write a whole number on 
the line for how many days, weeks, months, or years it has been since your last cigarette. 
 
 I smoked a cigarette today (at least one puff) 
 1-7 days → Number of days since last cigarette ________ 
 Less than 1 month → Number of weeks since last cigarette _________ 
 Less than 1 year → Number of months since last cigarette __________ 
 More than 1 year → Number of years since last cigarette ___________ 
 Don’t know/Don’t remember 
 
4. Since your last visit, have you used other forms of tobacco, not including cigarettes?  
 
 Yes 
  No (End) 
 
5. How often do you/did you use other forms of tobacco?  
 
 Every day → Number of times per day ________ 
 Some days → Number of days _______ per 
 Week 
 Month 
Year 

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6. Since your last visit, which of the following products have you used?  Check all that apply 
 
 Cigarettes 
 E-cigarettes or other electronic nicotine delivery system 
 Traditional cigars, cigarillos or filtered cigars 
 Pipes 
 Waterpipe 
 Hookah 
 Clove cigarettes or kreteks 
 Bidis  
 Smokeless tobacco, like dip, chew, or snuff 
 Snus 
 Paan with tobacco, gutka, zarda, khaini 
 Other, Specify____________________________________________________________________ 
 
7. If you do not currently use other forms of tobacco, but did in the past, how long has it been since you last 
used other forms of tobacco regularly?  
 
 Within the past month (0 to 1 month ago) 
 Between 1 and 3 months (1 to 3 months ago) 
 Between 3 and 6 months (3 to 6 months ago) 
 Between 6 and 12 months (6 to 12 months ago) 
 Between 1 and 5 years (1 to 5 years ago) 
 Between 5 and 15 years (5 to 15 years ago) 
 More than 15 years ago 
 Don’t know/Not sure 
 Never used other forms of tobacco regularly 
 
The following instructions pertain to questions 8 - 10.  During each of the following time frames, please 
indicate whether you smoked cigarettes every day, some days, or not at all. 
 
8.  During study treatment 
 
 Smoked every day 
 Smoked some days 
 Did not smoke at all 
 Don’t know/not sure 
 Not applicable  
 
 
 

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9.  After the end of study treatment 
 
 
 Smoked every day 
 Smoked some days 
 Did not smoke at all 
 Don’t know/not sure 
 Not applicable (I have not completed the study treatment)  
 
10. Since your last visit to this clinic 
 
 
 Smoked every day 
 Smoked some days 
 Did not smoke at all 
 Don’t know/not sure 
 
 
CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 
 

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NATIONAL AND LOCAL RESOURCES TO HELP WITH ALCOHOL ABUSE AND ALCOHOLISM 
 
NIAAA’s online guide Treatment for Alcohol Problems: Finding and Getting Help is written for individuals, 
and their family and friends, who are looking for options to address alcohol problems. It is intended as a 
resource to understand what treatment choices are available and what to consider when selecting among them. 
https://pubs.niaaa.nih.gov/publications/treatment/treatment.htm 
Other resources: 
National Institute on Alcohol Abuse and Alcoholism www.niaaa.nih.gov  
301–443–3860  
National Institute on Drug Abuse www.nida.nih.gov  
301–443–1124  
National Clearinghouse for Alcohol and Drug Information www.samhsa.gov  
1–800–729–6686  
Substance Abuse Treatment Facility Locator www.findtreatment.samhsa.gov  
1–800–662–HELP  
Alcoholics Anonymous (AA) www.aa.org  
212–870–3400 or check your local phone directory under “Alcoholism”  
Moderation Management www.moderation.org 
212–871–0974  
Secular Organizations for Sobriety www.sossobriety.org  
323–666–4295  
SMART Recovery www.smartrecovery.org  
440–951–5357  
Women for Sobriety www.womenforsobriety.org  
215–536–8026  
Al-Anon Family Groups www.al-anon.alateen.org  
1–888–425–2666 for meetings  
Adult Children of Alcoholics  www.adultchildren.org 
310–534–1815  
 

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NATIONAL AND LOCAL RESOURCES TO HELP WITH QUITTING SMOKING 
NCI’s Smokefree.gov offers science-driven tools, information, and support that has helped smokers quit. You 
will find state and national resources, free materials, and quitting advice from NCI. 
Smokefree.gov was established by the Tobacco Control Research Branch of NCI, a component of the National 
Institutes of Health, in collaboration with the Centers for Disease Control and Prevention and other 
organizations. 
Publications available from the Smokefree.gov website include the following: 
• 
Clearing the Air: Quit Smoking Today for smokers interested in quitting. 
• 
Clear Horizons for smokers over age 50. 
• 
Staying Smoke-Free for Good for smokers who have recently quit. 
• 
Smoke-free for women, including pregnant women. 
• 
Smoke-free information in Spanish 
• 
Pathways to Freedom: Winning the Fight Against Tobacco for African American smokers. 
NCI’s Smoking Quitline at 1–877–44U–QUIT (1–877–448–7848) offers a wide range of services, including 
individualized counseling, printed information, referrals to other resources, and recorded messages. Smoking 
cessation counselors are available to answer smoking-related questions in English or Spanish, Monday through 
Friday, 8:00 a.m. to 8:00 p.m., Eastern time. Smoking cessation counselors are also available through LiveHelp, 
an online instant messaging service. LiveHelp is available Monday through Friday, 8:00 a.m. to 11:00 p.m., 
Eastern time. 
Your state has a toll-free telephone quitline. Call 1–800–QUIT–NOW (1–800–784–8669) to get one-on-one 
help with quitting, support and coping strategies, and referrals to resources and local cessation programs. The 
toll-free number routes callers to state-run quitlines, which provide free cessation assistance and resource 
information to all tobacco users in the United States. This initiative was created by the Department of Health 
and Human Services. For more information about quitlines, speak to an expert on the Smokefree.gov website. 
 

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APPENDIX D 
 
COVID 19 ASSESSMENT INSTRUCTIONS 
. 
• Data collection will be required for all CP-CTNet studies. 
o Data will be collected at baseline and end of every study. All studies need to collect the elements in 
the attached eCRFs.  
o The eCRFs will be completed by the Site Staff or participant at the time of the designated visit. 
• Data will be submitted as part of the final clinical data set. 
 
 

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CP-CTNET COVID-19 BASELINE ASSESSMENT 
 
REGISTERING 
INSTITUTION  
 
_________________________________ 
PARTICIPANT 
ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions: 
The following information is being collected for all Cancer Prevention Clinical Trials Network (CP-
CTNet) studies. Only information from before study entry should be reported on this form. 
 
Have you ever had a positive COVID-19 test in the last 3 months?  
 Yes 
 No  
 
 
Have you received a COVID-19 vaccine in the last 3 months? 
 
 Yes 
 No  
 Prefer not to answer 
 
 
 
Comments: 
_________________________________________________________________________________________
___________________________________________________________________ 
 
 
CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 

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CP-CTNET COVID-19 FOLLOW-UP ASSESSMENT 
  
REGISTERING 
INSTITUTION  
 
_________________________________ 
PARTICIPANT 
ID 
 
______________________ 
VISIT TYPE 
 
__________________________ 
VISIT DATE 
(MM/DD/YYYY) 
___ ___ / ___ ___ / ___ 
___ ___ ___ 
 
Instructions: 
The following information is being collected for all Cancer Prevention Clinical Trials Network (CP-
CTNet) studies. Only use this form to report information that has become available since the last 
completion of a CP-CTNet COVID-19 Assessment. 
 
Since your initial study visit, have you had a positive COVID-19 test?  
 Yes 
 No  
 
If Yes, was it in the last 3 months? 
 
 Yes 
 No  
 Prefer not to answer 
 
 
Since your initial study visit, have you received a COVID-19 vaccine? (Please Omit this question if study 
duration is less than 3 months) 
 
 Yes 
 No  
 Prefer not to answer 
 
If Yes, did was it in the last 3 months? (Please Omit this question if study duration is less than 3 months) 
 
 Yes 
 No  
 Prefer not to answer 
 
Comments: 
_________________________________________________________________________________________
___________________________________________________________________ 
 
 
 
 

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CRF completed by (check one):  
Study participant _____    
Study site staff      _____     Staff name (optional) ____________________________ 
Date __ __ / __ __ / __ __ __ __ 
                  (MM/DD/YYYY) 
 
 
 

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APPENDIX E 
PRE-ACETAMINOPHEN SPOT URINE QUESTIONNAIRE 
 
1. Have you taken any over-the-counter medications during the 24 hours before the urine collection? 
(Check all that apply) 
 
Aspirin or generic aspirin (acetylsalicylic acid) 
Motrin, Advil, or Ibuprofen 
Other over-the-counter medications (please list)  
 
No over the counter medications in the past 24 hours 
Prefer not to answer 
 
2. Have you taken any prescription medications during the 24 hours before urine collection? 
 
Yes, list other prescription medications:  
 
No 
Prefer not to answer 
 
3. Please select if you have taken any of the following during the 24 hours before urine collection (Check 
all that apply) 
 
Supplements/Vitamins  
Pre-workout 
Protein powder/shake 
None of the above  
Prefer not to answer 
 
4. [Question 3 = “Yes”] Please list all supplements/vitamins you have taken during the 24 hours before 
urine collection: 
 
 
5. Have you had anything other than water to drink during the 24 hours before urine collection? Check all 
that apply. 
 
Energy drink (for example Red Bull, Monster, other)  
Coffee and/or tea 
Regular soda 
Diet soda 
Alcoholic beverage 
Other, list other:  
 
None of the above 
Prefer not to answer 
 
 

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6. Have you ever donated blood and/or plasma? 
 
Yes 
No 
Prefer not to answer (End of survey) 
 
7. [Question 6 = “Yes”] When was the last time you donated blood or plasma? (checkbox) 
 
In the last 7 days  
In the last month 
In the last 6 months  
More than 6 months ago  
Prefer not to answer 

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APPENDIX F 
 POST-FIRE/FUEL LEAK SPOT URINE COLLECTION QUESTIONNAIRE 
(To be completed by Individual Firefighter) 
#1 Name: ___________________________________________________________ 
#2 Time of Urine Collection: ___________________________________________ 
#3 Fireground Role Information 
• 
Primary Role: ☐ Fire Attack ☐ Ventilation ☐ Search & Rescue ☐ Water Supply 
• 
Position During Incident: ☐ Offensive ☐ Defensive 
#4 Post-Fire Wash Down Performed On Scene: ☐ Yes ☐ No 
#5 Shower Taken Prior to Urine Collection: ☐ Yes ☐ No 
QR Code Scanned: ☐ Yes ☐ No (if applicable) 
 
 
 

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APPENDIX G 
REMOTE CONSENT INSTRUCTIONS 
1. The participant or their legally-authorized representative (LAR) receives a copy of the informed 
consent document (e.g., via mail, fax, email, or a web link) in advance of discussion regarding 
the study. If mailed, two copies must be mailed so the participant or LAR is able to retain a copy 
for reference when their signed document is returned to the site and they are waiting to receive 
the final copy with all necessary signatures back from the site. If an electronic-consent (e-
consent) document is provided, the content in the document must be the same as the paper-based 
consent document. 
2. The investigator or designee discusses the study with the potential participant either via telephone 
or video conferencing. The investigator/designee must have the same consent discussion via 
telephone/video conferencing that they would have had with the participant or LAR during an in-
person meeting. The investigator/designee must also implement a method to ensure the identity of 
the participant or LAR (e.g., verification of state identification or other identifying documents or 
use of personal questions or visual methods). 
3. If the potential participant or LAR agrees to participation, they sign the consent form and return it 
to the investigator (e.g., via mail, fax, email, or by signing the e-consent document). If postal mail 
is used, a pre-paid, self-addressed envelope should be provided to the participant or LAR to mail 
the signed consent form back to the investigator. The inclusion of a witness in the Remote 
Consent Procedures is dictated by local institutional policy and must follow FDA and OHRP 
requirements. When a witness is required, the research record must document the witness’ name 
and that they were present for the informed consent process. The inclusion of the witness’ 
signature on the consent form is dictated by local institutional policy. 
4. Once the research team receives the signed informed consent document from the participant or 
LAR, the investigator/designee who conducted the consent process must sign and date the 
document using the current date. Under the signature line, the investigator/designee must 
document whether consent was obtained over the telephone or video conferencing, the date of the 
telephone/video conference, and the date the signed consent was received. For 
example, “Discussed with [participant or LAR name] via [telephone or videoconferencing] 
on [insert date] and received signed consent form on [insert date].” Include a brief reason for 
performing the informed consent discussion over the telephone/videoconferencing. 
5. If the site has an informed consent policy that requires the witness to sign the consent document, 
the witness signs the informed consent. If the site does not have an informed consent policy that 
requires the signature of the witness on the consent document, then the name of the witness along 
with the date of the original consenting phone call is recorded in the research records to document 
the participation of the witness.   
6. The date the investigator/designee signs the informed consent document, not the date the consent 
discussion with the participant or LAR took place, is the official date of informed consent for the 
participant on the trial.   
7. The final informed consent document must be filed in the designated investigator/site regulatory 
file location. A copy of the final informed consent document, signed by the participant or LAR, 
the investigator, and the witness (if applicable), must be sent back to the participant via 
email/scan, fax, or postal mail. 
No research activities related to the study can begin until all steps of the informed consent process 
are complete. 

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APPENDIX H 
FIRE/FUEL INCIDENT REPORT 
 
To be completed by Liaison 
#1 Incident Details 
• 
Date: __________________________ 
• 
Time of Call: ___________________ 
• 
Time on Scene: _________________ 
• 
Run Number: ___________________ 
• 
Unit: ___________________________ 
• 
Incident Address: ____________________________________________________ 
#2 Type of Fire (check all that apply) 
• 
Structure: ☐ Residential ☐ Commercial ☐ Mobile Home 
• 
Vehicle: ☐ Car ☐ Truck ☐ RV/Trailer 
• 
Fuel Type: ☐ Gasoline ☐ Diesel ☐ Hybrid ☐ Electric (EV) 
#3 Firefighter Activity 
• 
Primary Role: ☐ Fire Attack ☐ Ventilation ☐ Search & Rescue ☐ Water Supply 
• 
Position During Incident: ☐ Offensive ☐ Defensive 
#4 On-Scene Photo Attached: ☐ Yes ☐ No 
#5 Additional Notes: