Clinical trial NCT06110494
Conditions: Apical Periodontitis Pulp Disease, Dental
Keywords: Pulp infection Nanotechnology antibacterial disinfection iron oxide nanoparticles
Sponsor: University of Pennsylvania
Start date: 2020-07-13; Completion date: 2022-03-10
Enrollment: 44 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The goal of this observational is study is to develop a protocol for root canal biofilms disinfection using a clinically approved and commercially available iron oxide nanoparticle formulation Ferumoxytol/H2O2 treatments. This protocol will be testing local single topical application of Ferumoxytol within the root canal system in patients going through routine root canal treatment, evaluate its potential as anti-biofilm treatment and compare it to the clinical gold standard disinfecting solution sodium hypochlorite (positive control) and saline (negative control).

Detailed description:
Patients presenting to the Department of Endodontics, School of Dental Medicine, University of Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with chronic apical periodontitis will be asked to take part in the study if they meet the inclusion criteria and volunteer to participate. After eligibility of the patient is confirmed, the patient will be assigned to treatments through the process of drawing lots from a box that was maintained in a locked cabinet. Before treatment, patients will be thoroughly informed about the nature, potential risks and alternatives of the study as well as the root canal treatment. Patients will be presented with a written consent form regarding the above mentioned study characteristics as well as the regular consent forms for the root canal therapy, including the consent form for endodontic treatement, acknowledgement of privacy practices and a patient understanding and informed consent form. Briefly, the patient will be anesthetized and the tooth isolated with rubber dam. 30% H2O2 followed by 3% NaOCl will be used to disinfect the tooth and the rubber dam. The removal of caries and the endodontic access will be carried out by sterile high-speed carbide burs.After access preparation with sterile burs and sterile saline irrigation, thermoplastic gutta percha was placed to temporarily block the orifice. The field, including the pulp chamber, is cleaned and disinfected as described previously. NaOCl is neutralized with 10% sodium thiosulfate. Contamination control sample (S0) will be taken from the internal cavosurface angle where the paper points will accidentally touch during sampling.After initial access to the root canal orifices, working length will be measured and a bacteriological sample will be taken from the targeted canals (S1). Sterile paper points will be placed into the canal, allowed to saturate and then transferred to a vial containing liquid dental transport media (LDT). For NaOCl group (Positive control), canals will be instrumented up to size 25/0.04 taper using 2mL of 3% NaOCl in between files. For Ferumoxytol/H2O2 group, canals will be instrumented up to size 25/0.04 taper using 2mL of a mixture of 6 mg/ml of Ferumoxytol with 3% H2O2. For saline only group, canals will be instrumented up to size 25/0.04 taper using 2mL of saline. When the final 25/0.04 taper apical size is reached, a second bacterial sample will be taken (S2). Before all samplings, sodium hypochlorite, Feramehe/ H2O2 and Saline. Canal contents will be deactivated with sodium thiosulphate for NaOCl,and saline wash will be used for Fer/H2O2 and saline treatments. A wash step with 1 mL saline was done to wash the deactivating solution, and paper points were used to dry the canals. A second bacterial sample was taken (S2) by placing LDT inside the canal, agitating it with 25/0.02 Hedstrom hand file, followed by absorbing the content with 2 paper points placed in the canal for 30 seconds each. The paper points will be placed inside a tube containing LDT. An additional step was included for all the test groups to evaluate if further irrigation after (S2) will lead to more reduction of bacterial counts inside the root canal system. This could inform future experiments evaluating the possibility of synergistic antimicrobial effects for the sequential Fer/H2O2 and NaOCl treatment. All canals in all groups were irrigated with 2 mL of 3% NaOCl (1 min), followed by ultrasonic irrigant activation for 30 seconds. This step was repeated once again making the total activation time 1 min and the total contact time of the irrigant 3 min. Upon completion of irrigation, a third bacterial sample (S3) was taken following the deactivation, washing, drying, and sampling steps as described previously. The remaining treatment sequences of the routine root canal therapy will be carried out after these procedures including further root-end enlargement and final routine irrigation protocol. The root canals will be dried with paper points, a medication (calcium hydroxide) will be placed and the teeth sealed with a temporary restoration. Patients will return after one to four weeks for completion of the root filling. For the any of the groups, the treatment procedures carried out during this investigation do not differ from the standard root canal treatment protocol with the exception of additional irrigation step with the experimental solution and the bacteriologic sampling procedures. The paper points used to take the bacteriological sampling will be transferred to the microbiology laboratory using a vial containing 1 ml of LDT. The laboratory procedures will be performed at the University of Pennsylvania Leon Levy Oral Health Sciences Building of the School of Dental Medicine in the Microbiology Laboratory Vial labels will contain information on tooth number, sample number (S0-S1-S2-S3) and the experimental group. The samples will be diluted and plated in culture plates. The culture plates will be incubated at 37°C in an anaerobic glove box containing 5% hydrogen, 5% CO2 and balance N2 for 5 days. After incubation the number of colony forming units will be determined by using a stereoscope. ANOVA and Students t-test will be used for statistical analysis.

Arms: arm 1: After access preparation with sterile burs and sterile saline irrigation and working length determination as part of routine root canal treatment, and after obtaining a pre-treatment bacteriological samples, 2 mL of a mixture of Ferumoxytol (6mg/mL)/H2O2(3%) was introduced into the canal. Canals were instrumented with 15/0.04, 20/0.04, and 25/0.04 rotary files using 2 mL of treatment solution after each file with a total of 8 mL of solution used and a total contact time of 10 minutes. arm 2: After access preparation with sterile burs and sterile saline irrigation and working length determination as part of routine root canal treatment, and after obtaining a pre-treatment bacteriological samples, 2 mL of 3% NaOCl was introduced into the canal. Canals were instrumented with 15/0.04, 20/0.04, and 25/0.04 rotary files using 2 mL of solution after each file with a total of 8 mL of treatment solution used and a total contact time of 10 minutes. arm 3: After access preparation with sterile burs and sterile saline irrigation and working length determination as part of routine root canal treatment, and after obtaining a pre-treatment bacteriological samples, 2 mL of 0.89% NaCl was introduced into the canal. Canals were instrumented with 15/0.04, 20/0.04, and 25/0.04 rotary files using 2 mL of treatment solution after each file with a total of 8 mL of solution used and a total contact time of 10 minutes.
Interventions: intervention 1: Iron oxide nanoparticles treatment Ferumoxytol/H2O2 intervention 2: NaOCl intervention 3: NaCl intervention 4: supplementary irrigation
Intervention descriptions: intervention 1: Participants receive a topical treatment through a root canal irrigation needle containing A mixture of 6mg/mL Ferumoxytol nanoparticles solution mixed with 3% H2O2 for 10 minutes contact time intervention 2: Participants receive a topical treatment through a root canal irrigation needle containing 3% NaOCl for 10 minutes contact time intervention 3: Participants receive a topical treatment through a root canal irrigation needle containing 0.89% NaCl for 10 minutes contact time intervention 4: All canals in all groups were irrigated with 2 mL of 3% NaOCl, followed by ultrasonic irrigant activation for 30s twice.

seconds. This step was repeated once again making the total activation time 1 min and the total contact time of the irrigant 3 min. Upon completion of irrigation, a third bacterial sample (S3) was taken following the deactivation, washing, drying, and sampling steps as described previously

Protocol documents:
Prot_SAP_003.pdf: 
Page 1 of 21 
 
 
 
 
 
 
 
 
 
 
 
 
 
A new clinical use of Ferumoxytol nanoparticles: 
an anti-biofilm treatment 
NCT06110494 
17-Dec-2021 
 
 
 
 
 
 
 
 
 
 
 


Page 2 of 21 
 
Protocol Details 
 
 
Resubmission* 
Yes 
 
 
 
Study Personnel 
Principal Investigator 
 
Study Contacts 
None 
Basic Info 
Confirmation Number: 
ceheehgd 
Protocol Number: 
828211 
Created By: 
 
Principal Investigator: 
 
Protocol Title: 
A new clinical use of Feraheme nanoparticles: an anti-biofilm treatment 
Short Title: 
Feraheme nanoparticles in Endodontics 
Protocol Description:  The purpose of this study is to to develop a protocol for biofilms disinfection with a clinically approved 
and commercially available iron oxide nanoparticle formulation Feraheme/H2O2 treatments. This 
protocol will be testing local single topical application of Feraheme within the canal system in 
patients going through routine endodontic treatment, evaluate its potential as 
anti-biofilm 
treatment and compare it to other currently used antibacterial protocols. 
Submission Type: 
Biomedical Research 
Application Type: 
FULL 
Name: 
Dept / School / Div: 
Campus Address 
Mail Code 
Address: 
5112 - Endodontics 
6003 
City State Zip: 
Phone: 
Fax: 
Pager: 
Email: 
HS Training Completed: 
Training Expiration Date: 
Name of course completed : 
EVANS BUILDING 
4001 SPRUCE ST 
PHILADELPHIA PA 19104-6003 
215-898-4927 
 
Yes 
03/01/2018 
CITI Protection of Human Subjects Research Training - ORA 


Page 3 of 21 
 
Other Investigator 
 
Responsible Org (Department/School/Division): 
 
5112 - Endodontics 
 
Key Study Personnel 
 
Disclosure of Significant Financial Interests* 
Does any person who is responsible for the design, conduct, or reporting of this research protocol have 
a FINANCIAL INTEREST? 
No 
 
Penn Intellectual Property* 
To the best of the Principal Investigator's knowledge, does this protocol involve the testing, development 
or evaluation of a drug, device, product, or other type of intellectual property (IP) that is owned by or 
assigned to the University of Pennsylvania? 
Yes 
 
Certification 
I have reviewed the Financial Disclosure and Presumptively Prohibited Conflicts for Faculty 
Participating in Clinical Trials and the Financial Disclosure Policy for Research and Sponsored Projects 
with all persons who are responsible for the design, conduct, or reporting of this research; and all required 
Disclosures have been attached to this application. 
Yes 
Name: 
Dept / School / Div: 
Campus Address 
Mail Code 
Address: 
5126 - SDM Deans Office 
City State Zip: 
Phone: 
Fax: 
Pager: 
Email: 
HS Training Completed: 
Training Expiration Date: 
Name of course completed : 
3737 CHSTNUT STREET 
APT 1410 
PHILADELPHIA PA 19104-0000 
Yes 
02/06/2020 
CITI Protection of Human Subjects Research Training - ORA 
Name: 
Department/School/Division: 
HS Training Completed: 
Training Expiration Date: 
Name of course completed: 
Orthodontics 
Yes 
06/15/2018 
CITI Protection of Human Subjects Research Training - ORA 


Page 4 of 21 
 
Biomedical Research 
Investigator Initiated Trial 
Is this an investigator-initiated trial? 
Yes 
 
Drugs or Devices* 
Does this research study involve Drugs or Devices? 
Yes: Drugs, products or devices prepared or used in a manner not in the FDA-approved labeling. 
 
IND Exemption 
 
Obtained 
 
For studies that fall under an IND exemption, please provide the number below 
For studies including IND or IDE's, please provide the number(s) below 
IDE Review* 
NOTE: For research involving investigational devices, you are required to review the guidance on 
Managing Research Device Inventory. Consult the Penn Manual for Clinical Research: https:// 
somapps.med.upenn.edu/pennmanual/secure/pm/investigational-product-management Please check the 
box Yes if you have reviewed the guidance. 
Yes 
 
Research Device Management* 
Please indicate how research device(s) will be managed. 
Not Applicable (no investigational devices) 
 
Drug, Herbal Product or Other Chemical Element Management * 
Please indicate how drugs, herbal products or other chemical entities will be managed. 
The drug, herbal product or other chemical entity will be received, stored and dispensed by the research 
team (please provide information in the protocol summary as to how this will be conducted) 
 
Radiation Exposure* 
Are research subjects receiving any radiation exposure (e.g. X-rays, CT, Fluoroscopy, DEXA, pQCT, 
FDG, Tc-99m, etc.) that they would not receive if they were not enrolled in this protocol? 
No 
 
Gene Transfer* 
Does this research involve gene transfer (including all vectors) to human subjects? 
No 
 
Human Source Material* 
Does this research include collection or use of human source material (i.e., human blood, blood products, 
tissues or body fluids)? 
No 
 
CACTIS and CT Studies* 
Does the research involve Center for Advanced Computed Tomography Imaging Services (CACTIS) and 
CT studies that research subjects would not receive if they were not part of this protocol? 
No 
 
CAMRIS and MRI Studies* 
Does the research involve Center for Advanced Magnetic Resonance Imaging and Spectroscopy 
(CAMRIS) and MRI studies that research subjects would not receive if they were not part of this protocol? 
No 
 
Investigational Agent or Device within the Operating Room* 
Does the research project involve the use of an investigational agent or device within the Operating 


Page 5 of 21 
 
Room? 
No 
 
Cancer Related research not being conducted by an NCI cooperative group* 
Does this protocol involve cancer-related studies in any of the following categories? 
No 
 
Processing of Materials* 
Will the research involve processing (such as over encapsulating, or compounding)? 
No 
 
In-House Manufacturing of Materials* 
Will the research involve processing (such as over encapsulating, or compounding)? 
No 
 
Medical Information Disclosure* 
Does the research proposal involve the use and disclosure of research subject's medical information for 
research purposes? 
Yes 
 
If the answer is YES, indicate which items is is provided with this submission: 
 
Modified research informed consent document that incorporates HIPAA requirements 
 
CTRC Resources* 
Does the research involve CTRC resources? 
No 
 
Pathology and Laboratory Medicine Resources* 
Will samples be collected by hospital phlebotomy and/or processed or analyzed by any of the clinical 
laboratories of the University of Pennsylvania Health System? 
No 
 
Research Involves Apheresis, Cell Collection, and/or Blood Product Collection* 
Does this research involve collection of blood products in the Penn Donor Center and/or the use of 
apheresis for treatment or collection of cells or other blood components? 
No 
 
Research involving blood transfusion or drug infusions* 
Will your research involve blood transfusion or infusion of study drug in 3 Ravdin Apheresis Unit for 
research purposes? 
No 
 
Trial in Radiation Oncology 
Is this research a prospective trial being done in Radiation Oncology, and if so, has this protocol been 
approved by the Radiation Oncology Protocol committee? 
N/A 
 
Study in Radiation Oncology 
Is this research a retrospective study being done in Radiation Oncology, and if so, has this project been 
reviewed by the Radiation Oncology Clinical Research Group? 
N/A 
 
Use of UPHS services* 
Does your study require the use of University of Pennsylvania Health System (UPHS) services, tests or 
procedures*, whether considered routine care or strictly for research purposes? 
No 
 
Primary Focus* 
Clinical Trial (prospectively assigning subjects to health-related interventions to evaluate outcomes) 


Page 6 of 21 
 
Protocol Interventions 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
 
Sponsors 
Business Administrator 
 
none 
 
Department budget code 
 
000 - 000 - 0 - 000000 - 0000 - 0000 - 0000 
 
Funding Sponsors 
Funding sponsors billing address 
If you have selected a commercial or industry sponsor, please provide the appropriate address and contact 
information for the Sponsor for the purposes of billing for IRB review fees (initial review, continuing 
review and convened modification fees apply here). If the Sponsor is not industry/ commercial, this 
information is not necessary to provide with your application. 
 
Funding sponsors gift 
Is this research being funded by a philanthropic gift? 
 
Regulatory Sponsor 
IND Sponsor 
 
none 
 
 
000 - 000 - 0 - 000000 - 0000 - 0000 - 0000 
 
Industry Sponsor 
 
None 
 
Project Funding* 
Is this project funded by or associated with a grant or contract? 
No 
 
Sponsor Funding 
Is this study funded by an industry sponsor? 
Sociobehavioral (i.e. cognitive or behavioral therapy) 
x Drug 
Device - therapeutic 
Device - diagnostic (assessing a device for sensitivity or specificity in disease diagnosis) 
Surgical 
Diagnostic test/procedure (research-related diagnostic test or procedure) 
Obtaining human tissue for basic research or biospecimen bank 
Survey instrument 
None of the above 


Page 7 of 21 
 
Status of contract 
 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
 
Multi-Site Research 
Other Sites 
No other sites 
 
Management of Information for Multi-Center Research 
N/A 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
 
Protocol 
Abstract 
Biofilms are aggregates of microorganisms that are attached to a surface and enmeshed in a protective 
extracellular polymeric matrix, causing many infectious diseases. Biofilm-associated infections are 
notoriously difficult to treat. Bacterial infections in the root canal lead to high rates of failure of endodontic 
treatments. Conventional drugs often fail to kill the microbes embedded within biofilms and are incapable 
of destroying the matrix. New approach using catalytic nanoparticles (CAT-NP) that are biocompatible 
and have potent anti-biofilm action has been proposed. Studies on CAT-NP have shown that CAT-NP 
rapidly catalyze low concentrations of H2O2 to generate free radicals in situ to degrade the biofilm matrix 
and simultaneously kill embedded bacteria with exceptional efficacy. Moreover, the treatment suppresses 
virulent biofilm formation without cytotoxicity in an animal biofilm model. Recently, a clinically 
approved and commercially available iron oxide nanoparticle formulation with similar catalytic activity 
and anti-biofilm properties of CAT-NP became available. The purpose of this study is to develop a 
protocol for biofilms disinfection with a clinically approved and commercially available iron oxide 
nanoparticle formulation Feraheme. This protocol will be testing a local single topical application of 
Feraheme within the canal system in patients going through routine endodontic treatment, will evaluate 
its potential as anti-biofilm treatment and compare it to other currently used antibacterial protocols 
 
Objectives 
Overall objectives 
The purpose of this study is to develop a better, more effective disinfection protocol against biofilms within 
the root canal system, subsequently, improve the success of root canal treatment in teeth with apical 
periodontitis. This protocol will be testing local single topical application of a very low dose of Feraheme 
within the canal system in patients going through routine endodontic treatment, will evaluate its potential 
as a novel antibacterial, anti-biofilm treatment by intra-canal bacterial sampling and compare it to other 
currently used antibacterial protocols 
 
Primary outcome variable(s) 
Studies has shown that the bacteria within tooth root canal system is the primary etiology for the root- end 
disease process. The reduction or elimination of microorganisms from the root canal system of infected 
teeth has been shown to correlate with the long-term success of the endodontic treatment. The main 
objective of root canal treatment is to disinfect root canal systems entirely and seal to prevent further 
contamination. However, with current techniques, it is impossible to eradicate bacteria and biofilm 
completely from the complex root canal system. This study is designed to evaluate the antibacterial 
efficacy of Feraheme/H2O2 and to compare it to the routinely used protocol. The outcome 


Page 8 of 21 
 
will be evaluated by taking bacterial samples from the root canals during routine root canal treatment after 
applying different disinfection protocols. Bacteria from each sample will be quantified. The primary 
outcome parameter will be the difference in bacterial reduction between the experimental group 
(Feraheme) and a comparison group (routinely used protocol). 
 
Secondary outcome variable(s) 
Complex anatomy limit the efficacy of disinfection solutions and protocols. By evaluating bacterial 
reduction in different canal configurations, we will have a better understanding on the efficacy of tested 
solutions. Furthermore, proof-of-concept experiments in endodontic treatment could open the door for 
Feraheme to be used in numerous clinical anti-biofilm applications. 
 
Background 
Endodontic research has established the importance of microorganisms in the pathogenesis of apical 
periodontitis (1-3). The reduction or elimination of microorganisms is a logical aim for successful 
endodontic treatment. Sjörgen et al (4) evaluated the long-term outcome after one step endodontic 
treatment of teeth with negative or positive cultures after instrumentation. 94% of the teeth filled after a 
negative culture succeeded, whereas only 68% of the canals filled after a positive culture had been detected 
were successful. Based on this study the optimal clinical success will be reached if a tooth is filled when 
the bacterial load of the canal is undetectable with culturing methods. Because culturing is as yet only 
effective as a research tool, the clinician should expect optimal success when treating an infected tooth 
with a technique or protocol that has been shown experimentally to result in consistent negative cultures. 
Byström et al (5-7) published a series of studies to evaluate the antibacteriological effects of the individual 
steps in the endodontic procedure. Only after instrumentation using endodontic instruments with NaOCl 
and EDTA irrigation and a dressing of calcium hydroxide it was possible to obtain a predictable negative 
culture (5-7). However, Histo-bacteriological studies have shown that even after meticulous chemo-
mechanical preparation using endodontic instruments with NaOCl and EDTA and obturation of the root 
canal system, a significant amount of bacterial biofilm is often present in the anatomical complexities of 
the root canal system (isthmuses, apical ramification, accessory canals, and dentinal tubules) and on the 
walls of the main root canal that was not engaged by the root canal instruments (8-10). Endodontic biofilms 
may play a key role in infections persisting after primary or secondary endodontic treatment, as 
microorganisms growing in biofilms are better protected from adverse environmental changes and other 
antimicrobial agents. Ricucci et al, (11) evaluated the prevalence of bacterial biofilms in untreated root 
canals of teeth showing apical periodontitis in 80% of the cases. Nevertheless, the persistence of 
endodontic infections is also attributed to the presence of the aforementioned anatomical complexities, 
which can harbor biofilm and protect it from the effects of the current disinfection solutions (12). Based 
on these facts, advanced disinfection strategies with the ability to disrupt biofilm are needed to achieve 
the goal of endodontic treatment and to overcome the persistence of endodontic infections, if possible 
using less harsh chemicals as currently used (e.g. NaOCl). Hydrogen peroxide (H2O2) is commonly used 
as a safe green chemical (13) for disinfection purposes or as tooth-whitening agent (at concentrations as 
high as 10%) because it generates free radicals that exhibit antibacterial activity or stain removal (through 
degradation of polymeric substances). However, the process of free radical generation is slow and H2O2 
by itself has a modest anti-biofilm or endodontic disinfection effects when used alone. It has been shown 
that catalytic iron oxide nanoparticles (CAT-NP) have enhanced peroxidase-like activity that catalyzes 
hydrogen peroxide (H2O2) to create a potent anti-biofilm action in vivo (14). Very low concentrations of 
CAT-NP (at 0.5 mg/ml) rapidly catalyze H2O2 to generate free radicals in situ that simultaneously degrade 
the biofilm matrix and kill embedded bacteria with exceptional efficacy within 5 minutes (5,000-fold more 
effective than H2O2 alone), without cytotoxic effects on oral tissues in vivo (14). Recently, in a 
preliminary in- vitro study we tested the antibacterial activity of CAT-NP/ H2O2 in comparison to 3% 
H2O2, 3% NaOCl, and 2% CHX, the latter two solutions are current disinfectants used in endodontic 
treatment. The solutions were topically placed on dentine blocks that were infected with E. faecalis, a 
commonly isolated microorganism from root canal treated teeth with persistent apical periodontitis. Our 
results show that the antibacterial activity of CAT-NP/H2O2 was significantly better than the conventional 
irrigants. CAT-NP/H2O2 was up to 30-fold more effective in bacterial killing compared to NaOCl and 
CHX. These results reveal the potential to exploit CAT-NP/H2O2 as a potent alternative approach for 
treatment of endodontic infections. Recently, an FDA-approved iron oxide nanoparticle formulation 
known as Feraheme became available commercially. Our in-vitro studies have shown that the peroxidase-
like activity of Feraheme is similar to CAT-NP and this nanoparticle with H2O2 exhibited similar catalytic 
activity and potent anti-biofilm effects against endodontic biofilms. Feraheme is approved as an 
intravenous treatment for iron deficiency with repeated dosing at 510 mg. For 


Page 9 of 21 
 
endodontic applications, only a single topical treatment of up to 6 mg (which is 85 times less than the 
FDA-approved dose) would be used for a transient exposure (for less than 10 minutes) of infected tooth 
canal in the oral cavity, supporting the potential clinical translation of a Feraheme/H2O2 anti- biofilm 
treatment. Proof-of-concept experiments in endodontic treatment could open the door for Feraheme to be 
used in numerous clinical anti-biofilm applications. The assessment of bacterial reduction during 
endodontic therapy by bacterial sampling and culturing is an established technique and has been used to 
evaluate treatment protocols or variations thereof (15-18). There is a current need for a better disinfecting 
agent that is capable of overcoming the limitations of the currently used irrigation solutions, and based on 
our preliminary findings, CAT-NP/H2O2 has a potential as a novel anti-biofilm clinical therapy with its 
potent antibacterial and anti-biofilm activities. The purpose of this study is to develop an irrigation 
disinfection protocol for eradicating biofilms with a clinically approved and commercially available iron 
oxide nanoparticle formulation Feraheme. This protocol will be testing local application of Feraheme 
within the canal system in patients going through routine endodontic treatment, will evaluate its potential 
as a novel anti-biofilm disinfection solution and compare it to other currently used antibacterial protocols.  
References: 1. Kakehashi S, Stanley H, Fitzgerald R. The effect of surgical exposures of dental pulps in 
germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965; 20:340-9. 
2. 
Bergenholtz G. Micro-organisms from necrotic pulp of traumatized teeth. Odontol Revy 1974; 25:347-
58. 3. Kerekes K, Tronstad L. Long-term results of endodontic treatment performed with a standardized 
technique. J Endodon 1990; 16:498-504. 4. Sjörgen U, Figdor D, Persson S, Sundqvist G. Influence of 
infection at the time of root filling on the outcome of endodontic treatment of teeth with apical 
periodontitis. International Endodontic Journal 1997; 30:297-306. 5. Byström A, Sundqvist G. 
Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. 
Scand J Dent Res. 1981;89:321-8. 6. Byström A, Sundqvist G. Bacteriologic evaluation of the effect of 
0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol. 1983;55:307-12. 
7. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of 
endodontic therapy. Int Endod J. 1985;18:35-40. 8. Nair, P.N., et al., Microbial status of apical root canal 
system of human mandibular first molars with primary apical periodontitis after "one-visit" endodontic 
treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2005. 99(2): p. 231-52. 9. Ricucci, D., et 
al., Histologic investigation of root canal-treated teeth with apical periodontitis: a retrospective study from 
twenty-four patients. J Endod, 2009. 35(4): p. 493-502. 10. Vera, J., et al., One- versus two-visit 
endodontic treatment of teeth with apical periodontitis: a histobacteriologic study. J Endod, 2012. 
38(8): p. 1040-52. 11. Ricucci D, Siqueira JF Jr. Biofilms and apical periodontitis: study of prevalence 
and association with clinical and histopathologic findings. J Endod. 2010;36:1277-88. 12. Kishen, A., 
Advanced therapeutic options for endodontic biofilms. Endodontic topics, 2010. 22: p. 99-123. 13. Andrea, 
G. F., C., Hydrogen peroxide in green oxidation reactions: recent catalytic processes. In Green Chemical 
Reactions, Tundo, P.; Esposito, V., Eds. Springer: The Netherlands, 2008; pp 191-212. 14. Gao, L.; Liu, 
Y.; Kim, D.; Li, Y.; Hwang, G.; Naha, P. C.; Cormode, D. P.; Koo, H., Nanocatalysts promote 
Streptococcus mutans biofilm matrix degradation and enhance bacterial killing to suppress dental caries 
in vivo. Biomaterials 2016, 101, 272-284. 15. Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M. 
Bacterial reduction with nickel-titanium rotary instrumentation. J Endod. 1998;24:763-7. 
16. Shuping GB, Orstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using nickel- 
titanium rotary instrumentation and various medications. J Endod. 2000;26:751-5. 17. McGurkin-Smith 
R, Trope M, Caplan D, Sigurdsson A. Reduction of intracanal bacteria using GT rotary instrumentation, 
5.25% NaOCl, EDTA, and Ca(OH)2. J Endod. 2005;31:359-63. 18. Wang CS, Arnold RR, Trope M, 
Teixeira FB. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod. 2007 
;33:1283-9. 
 
Study Design 
Phase* 
Not applicable 
 
Design 
Patients presenting to the Department of Endodontics, School of Dental Medicine, University of 
Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with chronic 
apical periodontitis will be asked to take part in the study if they meet the inclusion criteria specified below 
and volunteer to participate. Prior to patient recruitment a randomized chart was drafted that assigns teeth 
to either NaOCL or Feraheme/H2O2 (amended). After eligibility of the patient is confirmed, the randomly 
assigned protocol for the tooth to be treated will be looked up by the principal 


Page 10 of 21 
 
investigator. Before treatment, patients will be thoroughly informed about the nature, potential risks and 
alternatives of the study as well as the root canal treatment. Patients will be presented with a written 
consent form regarding the above mentioned study characteristics (amended to this IRB proposal) as well 
as the regular consent forms for the root canal therapy, including the consent form for endodontic 
treatement, acknowledgement of privacy practices and a patient understanding and informed consent form. 
Patients will be eligible to participate in the study for NaOCL or Feraheme/H2O2 if the following inclusion 
criteria are met: 1. Patients to welling to participate in the study. 2. Patients are 18 years or above. 3. Non-
contributory medical history (Patient can be seen for regular dental appointment in PDM; ASA classes I 
and II). 4. Tooth requiring root canal treatment with radiographic presence of periapical radiolucency and 
responding to thermal sensitivity testing negatively (difluordichlormethane at 50 °C) (Endo-Ice, 
Coltène/Whaledent Inc., Cuyahoga Falls, Ohio) and Negatively to EPT testing. 5. Tooth with adequate 
remaining tooth structure for proper isolation with rubber dam. 6. No history of previous endodontic 
treatment on the tooth. 7. Teeth with single canal and single and roots with single canals in multirooted 
teeth. Patients will not be eligible to participate in the study if any of the following exclusion criteria 
applies: 1. Self-reported Pregnancy. 2. Patients requiring antibiotic premedication prior to dental treatment. 
3. Patients with multiple drug allergies. 4. Patients with known hypersensitivity to Ferumoxytol 
nanoparticles or any iron products. 5. Patients who are scheduled for MRI for the head region within three 
months after Fer nanoparticles application. 6. Periodontal changes (pockets 3 mm, mobility Grade I or 
gingival edema). 7. Radiographic presence of resorptive processes. 8. Cracked and fractured teeth. 9. if 
one of the inclusion criteria is not met.The treatment and sampling procedures will largely follow the 
protocol described by Shuping et al. (19). Briefly, Patients presenting to the Department of Endodontics 
for evaluation and treatment of infected, necrotic teeth with chronic apical periodontitis will be asked if 
they want to participate in the study. Informed consent will be obtained by the principal investigator. The 
patient will be anesthetized, and the tooth will be isolated with rubber dam and opal dam will be placed 
around the tooth. The tooth, the clamp, and the rubber dam will be cleansed using 30% hydrogen peroxide 
and disinfected with 3% NaOCl. After access preparation with sterile burs and sterile saline irrigation, 
thermoplastic gutta percha will be placed to temporarily block the orifice. The field, including the pulp 
chamber, will be cleaned and disinfected as described previously. NaOCl will be neutralized with 10% 
sodium thiosulfate. After removal of the gutta percha from the root canal orifices and working length 
determination, Liquid Dental Transport Media (LDT) will be placed in the canal with a sterile 31g needle, 
and a #15 Hedstrom file will be used in a filing motion to scrape the canal walls and agitate the canal 
content. (S1) sample will be taken by placing a sterile paper point in the canal, allow it to saturate for 30 
seconds, and then transfer to a tube containing LDT. This step will be once more with a second paper 
point. The treatment solution (saline, NaOCl, or Fer/H2O2) 2 mL will be introduced into the canal. Canals 
will be instrumented with 15/0.04, 20/0.04, and 25/0.04 using 2 mL of treatment solution after each file 
with a total of 8 mL and a total contact time of 10 minutes. Canal contents will be deactivated with sodium 
thiosulphate for NaOCl, and saline wash will be used for Fer/H2O2 and saline treatments. A wash step 
with 1 mL saline will be done to wash the deactivating solution, and paper points will be used to dry the 
canals. A second bacterial sample will be taken (S2) by placing LDT inside the canal, agitating it with 
25/0.02 Hedstrom hand file, followed by absorbing the content with 2 paper points placed in the canal for 
30 seconds each. The paper points will be placed inside a tube containing LDT. An additional step was 
included for all the test groups to evaluate if further irrigation after (S2) will lead to more reduction of 
bacterial counts inside the root canal system. This could inform future experiments evaluating the 
possibility of synergistic antimicrobial effects for the sequential Fer/H2O2 and NaOCl treatment. All 
canals in all groups were irrigated with 2 mL of 3% NaOCl (1 min), followed by ultrasonic irrigant 
activation for 30 seconds. This step was repeated once again making the total activation time 1 min and 
the total contact time of the irrigant 3 min. Upon completion of irrigation, a third bacterial sample (S3) 
was taken following the deactivation, washing, drying, and sampling steps as described above.The 
remaining treatment sequences of the routine root canal therapy will be carried out after these procedures 
including further root-end enlargement and final routine irrigation protocol. The root canals will be dried 
with paper points, a medication (calcium hydroxide) will be placed and the teeth sealed with a temporary 
restoration. Patients will return after one to four weeks for completion of the root filling. For the any of 
the groups, the treatment procedures carried out during this investigation do not differ from the standard 
root canal treatment protocol with the exception of additional irrigation step with the experimental solution 
and the bacteriologic sampling procedures. The paper points used to take the bacteriological sampling will 
be transferred to the microbiology laboratory using a vial containing 1 ml of reduced transport fluid (LDT). 
The laboratory procedures will be performed at the University of Pennsylvanias Leon Levy Oral Health 
Sciences Building of the School of Dental Medicine in the Microbiology Laboratory (Dr. M. Koo's 
laboratory). Vial labels will contain information on tooth number, sample number (S0-S1-S2-S3) and the 
experimental group. Patient information will not be used in any case. LDT vials with samples will be 
vortexed before preparing aliquots. Samples with dilutions of 10, 100, and 1000-fold will be prepared 


Page 11 of 21 
 
under anaerobic conditions using sterile glassware. Cell culture dishes with anaerobic sheep blood agar 
will be inoculated with 0.25 ml of undiluted sample, as well as each of the three dilutions. The culture 
plates will be incubated at 37°C in an anaerobic glove box containing 5% hydrogen, 10% nitrogen, and 
85% CO2 for 7 days. After incubation the number of colony forming units will be determined by using 
a stereoscope. ANOVA and Students t-test will be used for statistical analysis. 
 
Study duration 
The projected time frame for this research project is from May 2017 to May 2018. The recruitment and 


Page 12 of 21 
 
treatment of patients with 60 teeth matching the inclusion criteria mentioned above can be reasonably 
achieved within these 12 months. Included patients will not participate in this proposed study longer than 
the time necessary for the routine root canal therapy. 
 
Resources necessary for human research protection 
Describe research staff and justify that the staff are adequate in number and qualifications to conduct 
the research. Describe how you will ensure that all staff assisting with the research are adequately informed 
about the protocol and their research related duties. Please allow adequate time for the researchers to 
conduct and complete the research. Please confirm that there are adequate facilities for the research. 
Patients will be treated in the Endodontic Clinic of the University of Pennsylvania's Department of 
Endodontics of the School of Dental Medicine (PDM). The Endodontic Clinic exclusively handles all root 
canal procedures carried out at the PDM with the exception of Dental Care Network facilities. By 
postgraduate residents alone, more than 1500 root canal procedures are done routinely per year. Patients 
are PDM patients and come from the Restorative Department of the PDM, emergency admissions or 
referred by outside dental clinics directly to the clinic. The recruitment and treatment of patients with 
60 teeth matching the inclusion criteria mentioned above can be achieved within a reasonable time. 16 
residents are enrolled at any time for postgraduate studies at the Endodontic Department. The instructions 
regarding the technical aspects and the human research protection issues will be given to the residents 
during specific research meetings that are held regularly in the Department for updates on progress and 
conduct of research activities within the Department. The primary investigator will inform all residents 
seeing patients about all aspects concerning the protection of human research participants and will review 
the research protocol and all associated consent forms to ensure calibration, proper conduct and 
participants protection at all times. Before treatment of an individual patient potentially suitable for 
treatment under the conditions of this investigation, the research consent form, the general root canal 
treatment consent form, OSHA, HIPPA, privacy notice etc. will be reviewed with the patient. Besides the 
principal investigator (BK), who is a full-time member of the Endodontic Department, and the co-
investigator (AB), who is one of the endodontic residents, endodontic residents are under supervision of 
full-time and/or part-time faculty members during patient treatments at any time. The principal investigator 
and co-investigator received training, conducted research in cell culture techniques and analysis. Needs 
for medical or psychological services are not a possible consequence of the bacterial sampling procedures 
to be undertaken during the routine root canal therapy for this research project. Any medical needs that 
should arise during the routine endodontic therapy are covered by the faculty and staff of the PDM or 
the University of Pennsylvania’s medical emergency services (511) as for any other dental procedure 
routinely carried out at the PDM. The laboratory part of the investigation will be performed in the 
Microbiology Laboratory (Dr. M. Koo's laboratory) of the Leon Levy Oral Health Sciences Building of 
the School of Dental Medicine. This laboratory is well equipped for cell culture and the handling of 
anaerobic bacteria, including an anaerobic glove box as well as all standard equipment for laboratory cell 
culture. 
 
 
 
Characteristics of the Study Population 
Target population 
Patients presenting to the Department of Endodontics, School of Dental Medicine, University of 
Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with chronic 
apical periodontitis. 
 
Subjects enrolled by Penn Researchers 
60 
 
Subjects enrolled by Collaborating Researchers 
0 
 
Accrual 
Patients will be treated in the Endodontic Clinic of the University of Pennsylvania's Department of 
Endodontics of the School of Dental Medicine (PDM). The Endodontic Clinic exclusively handles all root 
canal procedures carried out at the PDM, with the exception of Dental Care Network facilities. By 


Page 13 of 21 
 
postgraduate residents alone, more than 1500 root canal procedures are done routinely per year. Patients 
are PDM patients coming from the Restorative Department of the PDM, emergency admissions or referred 
from outside private clinics directly to the clinic. The recruitment and treatment of patients with 60 
teeth matching the inclusion criteria mentioned above can be achieved within a reasonable time. The 
clinical significance of this investigation will not be evaluated by statistical analysis based on overall 
sample size. A target negative culture threshold is well established in the literature for an acceptable 
success rate (refer to Background, reference 4) and the experimental and comparison groups are evaluated 
as successful (or unsuccessful) for their ability to reach this threshold (or not). Comparable studies 
investigating the reduction of intra-canal bacteria after changes in treatment protocol have been conducted 
with similar sample sizes (refer to Background, references 15-18). 
 
Key inclusion criteria 
Gender is not an inclusion or exclusion criteria. Patients will be eligible to participate in the study if the 
following inclusion criteria are met: 1. Patients to welling to participate in the study. 2. Patients are 18 
years or above. 3. Non-contributory medical history (Patient can be seen for regular dental appointment in 
PDM; ASA classes I and II). 4. Tooth requiring root canal treatment with radiographic presence of 
periapical radiolucency and responding to thermal sensitivity testing negatively (difluordichlormethane at 
50 °C) (Endo-Ice, Coltène/Whaledent Inc., Cuyahoga Falls, Ohio) and Negatively to EPT testing. 5. Tooth 
with adequate remaining tooth structure for proper isolation with rubber dam. 6. No history of previous 
endodontic treatment on the tooth. 7. Teeth with single canal and single and roots with single canals in 
multirooted teeth. 
Key exclusion criteria 
Economic status, gender, race or ethnicity are not of concern for the proposed investigation and therefore 
for study exclusion. The cut-off age of 18 years was only chosen so as to limit the need for parental consent. 
Patients will not be eligible to participate in the study if any of the following exclusion criteria applies: 1. 
Self-reported Pregnancy. 2. Patients requiring antibiotic premedication prior to dental treatment. 3. 
Patients with multiple drug allergies. 4. Patients with known hypersensitivity to Ferumoxytol nanoparticles 
or any iron products. 5. Patients who are scheduled for MRI for the head region within three months after 
Fer nanoparticles application. 6. Periodontal changes (pockets 3 mm, mobility Grade I or gingival edema). 
7. Radiographic presence of resorptive processes. 8. Cracked and fractured teeth. 9. if one of the inclusion 
criteria is not met. 
Vulnerable Populations 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
Populations vulnerable to undue influence or coercion 
Given the nature of the suggested study and the non-invasive procedures (irrigation within the closed 
environment of root canal, procedure of taking bacterial samples from a root canal) none of the 
potential participants appear to be likely to be vulnerable to coercion or undue influence or influence. 
Potential participants will receive the elected root canal treatment if they are participating in the study 
or if they are not participating in the study. In the event that Penn students or employees present for 
screening they will be informed that their decision regarding whether or not to participate will in no 
way impact their standing at the university. Given the number of patients seen on a regular basis in the 
Department of Endodontics and the common nature of the diseases pulp necrosis with apical periodontitis 
undue influence or coercion is not likely. 
Children Form 
Pregnant women (if the study procedures may affect the condition of the pregnant woman or 
fetus) Form 
Fetuses and/or Neonates Form 
Prisoners Form 
Other 
x None of the above populations are included in the research study 


Page 14 of 21 
 
Subject recruitment 
Patients presenting to the Department of Endodontics, School of Dental Medicine (PDM), University of 
Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with chronic 
apical periodontitis will be asked to take part in the study if they meet the inclusion criteria specified above. 
Patients will be asked if they were interested to participate in this investigation after a clinical diagnosis 
matching is confirmed and the patient/ tooth meets the inclusion criteria specified above. Initial clinical 
diagnosis will be made by a postgraduate resident and verified by the supervising faculty. The PI, a full-
time faculty, and the co-investigator, endodontic resident, will be informed about a potential study 
participant, verify the diagnosis and ask for participation (including consent and alternatives). No 
additional recruitment materials, such as radio/video scripts, flyers, internet postings, etc. will be used. No 
non-registered PDM patients will be asked to participate in the study. 
 
 
Will the recruitment plan propose to use any Penn media services (communications, marketing, etc.) for 
outreach via social media avenues (examples include: Facebook, Twitter, blogging, texting, etc.) or does 
the study team plan to directly use social media to recruit for the research? 
No 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
Subject compensation* 
Will subjects be financially compensated for their participation? 
No 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
If there is subject compensation, provide the schedule for compensation per study visit or session 
and total amount for entire participation, either as text or separate document 
 
N/A 
 
 
 
Study Procedures 
Suicidal Ideation and Behavior 
Does this research qualify as a clinical investigation that will utilize a test article (ie- drug or biological) 
which may carry a potential for central nervous system (CNS) effect(s)? 
No 
 
Procedures 
Patients presenting to the Department of Endodontics, School of Dental Medicine, University of 
Pennsylvania for evaluation and routine endodontic treatment of infected, necrotic teeth with chronic 
apical periodontitis will be asked to take part in the study if they meet the inclusion criteria specified below 
and volunteer to participate. Prior to patient recruitment a randomized chart was drafted that assigns teeth 
to either NaOCL, saline or Feraheme/H2O2 (amended). After eligibility of the patient is confirmed, the 
randomly assigned protocol for the tooth to be treated will be looked up by the principal investigator. 
Before treatment, patients will be thoroughly informed about the nature, potential risks and alternatives of 
the study as well as the root canal treatment. Patients will be presented with a written consent form 
regarding the above mentioned study characteristics (amended to this IRB proposal) as well as the regular 
consent forms for the root canal therapy, including the consent form for endodontic treatement, 
acknowledgement of privacy practices and a patient understanding and informed consent form. Patients 
will be eligible to participate in the study for NaOCL (positive control), saline (negative control or 
Feraheme/H2O2 if the following inclusion criteria are met: 1. Patients to welling to participate in the study. 
2. Patients are 18 years or above. 3. Non-contributory medical history (Patient can be seen for regular 
dental appointment in PDM; ASA classes I and II). 4. Tooth requiring root canal treatment with 
radiographic presence of periapical radiolucency and responding to thermal sensitivity testing negatively 


Page 15 of 21 
 
(difluordichlormethane at 50 °C) (Endo-Ice, Coltène/Whaledent Inc., Cuyahoga Falls, Ohio) and 
Negatively to EPT testing. 5. Tooth with adequate remaining tooth structure for proper isolation with 
rubber dam. 6. No history of previous endodontic treatment on the tooth. 7. Teeth with single canal and 
single and roots with single canals in multirooted teeth. If upon access this canal configuration can not be 
verified, the routine endodontic treatment will continue without bacterial sampling and the tooth not 
included in the investigation. Patients will not be eligible to participate in the study if any of the following 
exclusion criteria applies: 1. Self-reported Pregnancy. 2. Patients requiring antibiotic premedication prior 
to dental treatment. 3. Patients with multiple drug allergies. 4. Patients with known hypersensitivity to 
Ferumoxytol nanoparticles or any iron products. 5. Patients who are scheduled for MRI for the head region 
within three months after Fer nanoparticles application. 6. Periodontal changes (pockets 3 mm, mobility 
Grade I or gingival edema). 7. Radiographic presence of resorptive processes. 8. Cracked and fractured 
teeth. 9. if one of the inclusion criteria is not met. The treatment and sampling procedures will largely 
follow the protocol described by Shuping et al. (19). Briefly, Patients presenting to the Department of 
Endodontics for evaluation and treatment of infected, necrotic teeth with chronic apical periodontitis will 
be asked if they want to participate in the study. Informed consent will be obtained by the principal 
investigator. The patient will be anesthetized, and the tooth will be isolated with rubber dam and opal dam 
will be placed around the tooth. The tooth, the clamp, and the rubber dam will be cleansed using 30% 
hydrogen peroxide and disinfected with 3% NaOCl. After access preparation with sterile burs and sterile 
saline irrigation, thermoplastic gutta percha will be placed to temporarily block the orifice. The field, 
including the pulp chamber, will be cleaned and disinfected as described previously. NaOCl will be 
neutralized with 10% sodium thiosulfate. After removal of the gutta percha from the root canal orifices 
and working length determination, Liquid Dental Transport Media (LDT) will be placed in the canal with 
a sterile 31g needle, and a #15 Hedstrom file will be used in a filing motion to scrape the canal walls and 
agitate the canal content. (S1) sample will be taken by placing a sterile paper point in the canal, allow it to 
saturate for 30 seconds, and then transfer to a tube containing LDT. This step will be once more with a 
second paper point. The treatment solution (saline, NaOCl, or Fer/H2O2) 2 mL will be introduced into the 
canal. Canals will be instrumented with 15/0.04, 20/0.04, and 25/0.04 using 2 mL of treatment solution 
after each file with a total of 8 mL and a total contact time of 10 minutes. Canal contents will be deactivated 
with sodium thiosulphate for NaOCl, and saline wash will be used for Fer/H2O2 and saline treatments. A 
wash step with 1 mL saline will be done to wash the deactivating solution, and paper points will be used 
to dry the canals. A second bacterial sample will be taken (S2) by placing LDT inside the canal, agitating 
it with 25/0.02 Hedstrom hand file, followed by absorbing the content with 2 paper points placed in the 
canal for 30 seconds each. The paper points will be placed inside a tube containing LDT. An additional 
step was included for all the test groups to evaluate if further irrigation after (S2) will lead to more 
reduction of bacterial counts inside the root canal system. This could inform future experiments evaluating 
the possibility of synergistic antimicrobial effects for the sequential Fer/H2O2 and NaOCl treatment. All 
canals in all groups were irrigated with 2 mL of 3% NaOCl (1 min), followed by ultrasonic irrigant 
activation for 30 seconds. This step was repeated once again making the total activation time 1 min and 
the total contact time of the irrigant 3 min. Upon completion of irrigation, a third bacterial sample (S3) 
was taken following the deactivation, washing, drying, and sampling steps as described previously. The 
remaining treatment sequences of the routine root canal therapy will be carried out after these procedures 
further root-end enlargement and final routine irrigation protocol. The root canals will be dried with paper 
points, a medication (calcium hydroxide) will be placed and the teeth sealed with a temporary restoration. 
Patients will return after one to four weeks for completion of the root filling. For the any of the groups, 
the treatment procedures carried out during this investigation do not differ from the standard root canal 
treatment protocol with the exception of additional irrigation step with the experimental solution and the 
bacteriologic sampling procedures. The paper points used to take the bacteriological sampling will be 
transferred to the microbiology laboratory using a vial containing 1 ml of reduced transport fluid (LDT). 
The laboratory procedures will be performed at the University of Pennsylvania’s Leon Levy Oral Health 
Sciences Building of the School of Dental Medicine in the Microbiology Laboratory (Dr. M. Koos 
laboratory). Vial labels will contain information on tooth number, sample number (S0-S1-S2-S3) and the 
experimental group. Patient information will not be used in any case. LDT vials with samples will be 
vortexed before preparing aliquots. Samples with dilutions of 10, 100, and 1000-fold will be prepared 
under anaerobic conditions using sterile glassware. Cell culture dishes with anaerobic sheep blood agar 
will be inoculated with 0.25 ml of undiluted sample, as well as each of the three dilutions. The culture 
plates will be incubated at 37°C in an anaerobic glove box containing 5% hydrogen, 10% nitrogen, and 
85% CO2 for 7 days. After incubation the number of colony forming units will be determined by using a 
stereoscope. ANOVA and Students t- test will be used for statistical analysis. 
 
The following documents are currently attached to this item: 
 


Page 16 of 21 
 
There are no documents attached for this item. 
 
Deception 
Does your project use deception? 
No 


Page 17 of 21 
 
Analysis Plan 
After incubation the number of colony forming units will be determined by using a stereoscope. 
ANOVA and Students t-test will be used for statistical analysis. 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
 
Are you conducting research outside of the United States? 
No 
 
Data confidentiality 
 
Subject Confidentiality 
Patients will be presented with a written consent form regarding the above mentioned study characteristics 
(research consent form, amended to this IRB proposal) as well as the regular consent forms for the root 
canal therapy, including the consent form for endodontic treatement, acknowledgement of privacy 
practices and a patient understanding and informed consent form. Patients signed consent forms will 
collected and stored by the PI in a secure, restricted area. Study data will be stored on an institutionally 
secured and managed device or server. The information collected and used for research purposes will be 
the School of Dental School Medicine chart number, diagnosis and tooth number. No protected health 
information such as name, address, telephone number, date of birth, social security number, personal and 
family medical history or results from physical examinations, tests or procedures will be used or collected. 
The chart number will only be recorded for a possible future verification of the research data such as 
tooth number and diagnosis. Patients chart number is secured by a login and password only accessible 
to students, residents and faculty of the dental school. Diagnosis and tooth number will be used in this 
investigation for scientific publication or presentation at scientific meetings. 
 
Sensitive Research Information* 
Does this research involve collection of sensitive information about the subjects that should be excluded 
from the electronic medical record? 
No 
 
Subject Privacy 
Privacy refers to the person's desire to control access of others to themselves. Privacy concerns people, 
whereas confidentiality concerns data. Describe the strategies to protect privacy giving consideration to 
the following: The degree to which privacy can be expected in the proposed research and the safeguards 
that will be put into place to respect those boundaries. The methods used to identify and contact 
x Paper-based records will be kept in a secure location and only be accessible to personnel involved in 
the study. 
x Computer-based files will only be made available to personnel involved in the study through the use 
of access privileges and passwords. 
Prior to access to any study-related information, personnel will be required to sign statements 
agreeing to protect the security and confidentiality of identifiable information. 
x Wherever feasible, identifiers will be removed from study-related information. 
A Certificate of Confidentiality will be obtained, because the research could place the subject at risk 
of criminal or civil liability or cause damage to the subject's financial standing, employability, 
or liability. 
A waiver of documentation of consent is being requested, because the only link between the subject 
and the study would be the consent document and the primary risk is a breach of confidentiality. 
(This is not an option for FDA-regulated research.) 
Precautions are in place to ensure the data is secure by using passwords and encryption, because 
the research involves web-based surveys. 
Audio and/or video recordings will be transcribed and then destroyed to eliminate audible 
identification of subjects. 


Page 18 of 21 
 
potential participants. The settings in which an individual will be interacting with an investigator. The 
privacy guidelines developed by relevant professions, professional associations and scholarly 
disciplines (e.g., psychiatry, genetic counseling, oral history, anthropology, psychology). 
Patients signed consent forms will collected and stored by the PI in a secure, restricted area. The 
information collected and used for research purposes will be the School of Dental School Medicine chart 
number, diagnosis and tooth number. No protected health information such as name, address, telephone 
number, date of birth, social security number, personal and family medical history or results from physical 
examinations, tests or procedures will be used or collected. The chart number will only be recorded for 
a possible future verification of the research data such as tooth number and diagnosis. Patients chart 
number is secured by a login and password only accessible to students, residents and faculty of the dental 
school. 
Treatments will be done at the University of Pennsylvania School of 
Dental Medicine 
Endoodontic clinic. All root canal procedures within the Dental School is done in the Endodontic clinic 
where work stations are designed to accommodate patient's privacy. Endodontic clinic contains a 
separate private room in cases where research instructions or patient's concerns can be addressed. 
 
Data Disclosure 
Will the data be disclosed to anyone who is not listed under Personnel? 
The data will not be disclosed to anybody who is not listed under Personnel. 
 
Data Protection* 
 
Does your research request both a waiver of HIPAA authorization for collection of patient information 
and involve providing Protected Health Information ("PHI") that is classified as a "limited data set" 
(city/town/state/zip code, dates except year, ages less than 90 or aggregate report for over 90) to a recipient 
outside of the University of Pennsylvania covered entity? 
No 
 
Tissue Specimens Obtained as Part of Research* 
Are Tissue Specimens being obtained for research? 
No 
Name 
Street address, city, county, precinct, zip code, and equivalent geocodes 
All elements of dates (except year) for dates directly related to an individual and all ages over 89 
Telephone and fax number 
Electronic mail addresses 
Social security numbers 
Medical record numbers 
Health plan ID numbers 
Account numbers 
Certificate/license numbers 
Vehicle identifiers and serial numbers, including license plate numbers 
Device identifiers/serial numbers 
Web addresses (URLs) 
Internet IP addresses 
Biometric identifiers, incl. finger and voice prints 
Full face photographic images and any comparable images 
x Any other unique identifying number, characteristic, or code 
None 


Page 19 of 21 
 
Tissue Specimens - Collected during regular care* 
Will tissue specimens be collected during regulator clinical care (for treatment or diagnosis)? 
No 
 
Tissue Specimens - otherwise discarded* 
Would specimens otherwise be discarded? 
No 
 
Tissue Specimens - publicly available* 
Will tissue specimens be publicly available? 
No 
 
Tissue Specimens - Collected as part of research protocol* 
Will tissue specimens be collected as part of the research protocol? 
No 
 
Tissue Specimens - Banking of blood, tissue etc. for future use* 
Does research involve banking of blood, tissue, etc. for future use? 
No 
 
Genetic testing 
If genetic testing is involved, describe the nature of the tests, including if the testing is predicative or 
exploratory in nature. If predictive, please describe plan for disclosing results to subjects and provision of 
genetic counseling. Describe how subject confidentiality will be protected Note: If no genetic testing is to 
be obtained, write: "Not applicable." 
Not applicable 
 
 
 
Consent 
1. Consent Process 
Overview 
Patients routinely sign a consent form before being seen in the endodontic clinic. This form reviews the 
routine root canal procedure and any risks associated with it. We review routinely the consent form and 
health history with the patient and answer any questions that they may have on root canal procedures. The 
language used in the consent form is meant for lay-people and is not scientific in nature. If a patient chooses 
to participate in our study, they will sign an additional consent form for participation prior to participating. 
They will be made aware that if they do not wish to participate, they will not receive any treatment that is 
subpar as opposed to if they did agree to participate. The examiners will review the consent form with the 
patient and answer any questions they may have. If at any point, a patient decides they no longer want to 
participate, they will be excused from the study and will continue treatment as needed. 
 
Children and Adolescents 
N/A 
 
Adult Subjects Not Competent to Give Consent 
N/A 
 
2. Waiver of Consent 
Waiver or Alteration of Informed Consent* 
No Waiver Requested 
 
Minimal Risk* 
 
Impact on Subject Rights and Welfare* 


Page 20 of 21 
 
Waiver Essential to Research* 
Additional Information to Subjects 
Written Statement of Research* 
No 
 
If no written statement will be provided, please provide justificiation 
 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
 
Risk / Benefit 
Potential Study Risks 
Only the risks or discomforts that can arise during or after any routine root canal treatment procedure 
apply. Bacterial sampling will not have any adverse effect on the treatment or on treatment outcome. There 
are no potential risks expected from the noninvasive bacteriological sampling, including any potential 
physical, psychological, social, economic, monetary, legal or other risks. Identifying the antibacterial 
benefits of Feraheme/H2O2 irrigation is the goal of our experiment. Feraheme with much higher 
concentrations has been used in medicine as IV to improve and heal patients iron deficiency. Feraheme 
used in this experiment is limited within the root canal system and applied on dentine surfaces 
only. It will not be used systemically. Solution will be kept within the root canal system for a couple of 
minutes proposed in this protocol and will be evacuated by sterile saline, dental suction and paper points. 
Experiment was designed that the final root canal disinfection is not relying on experimental solutions 
antibacterial effect only. After bacterial sampling, antibacterial medication (CaOH2) will placed as done 
routinely to treat cases with pulp necrosis and apical periodontitis. Besides tooth number, diagnosis 
and the PDM chart number (for potential verification of study correctness) no patient data will be collected 
by the PI. All treatment procedures at the PDM, including the routine root canal treatment of the 
participants, during which the samples will be taken, are subject to strict HIPPA (privacy) and OSHA 
(hygiene) regulations. 
 
Potential Study Benefits 
The participant is not expected to get any personal benefit from taking part in this proposed research study. 
Microbial elimination is a key to the successful treatment of infected, necrotic teeth. Finding a better 
protocol to reduce intracanal infection will increase the successful outcome of endodontically treated 
necrotic, infected teeth and will result in an increased retention of natural teeth for the general population. 
The proposed study will verify by the means of microbial sampling and culturing, whether 
Feraheme/H2O2 irrigation protocol can provide better, effective disinfection on root canal surfaces and a 
significant reduction of intra-canal infection in-vivo. If this can be demonstrated by the proposed 
investigation it will have clinical significance as an improvement of root canal therapy. 
 
Alternatives to Participation (optional) 
If patient does not want to participate in the study, elected root canal treatment will be completed. 
 
Data and Safety Monitoring 
Investigators listed on the proposal will monitor the progress of the experiment. Routine meetings will be 
conducted to assess the number of samples collected, results, patient comments and complains, 
investigators' comments and findings. 
 
The following documents are currently attached to this item: 
 
There are no documents attached for this item. 
 
Risk / Benefit Assessment 
The risks or discomforts can arise during or after any routine root canal treatment procedure. There are no 
potential risks expected in this experiment. Our consent forms (for routine root canal procedures and for 
the experiment) contain clinic and investigator contact information. However, if occurs, patients' 


Page 21 of 21 
 
concerns or complaints will be reviewed routinely. 
 
 
 
General Attachments 
The following documents are currently attached to this item: 
HIPAA Authorization or Waiver (babeer.feahemeinendodontics.hipaa.consent.doc) 
Additional forms (feraheme-fda.pdf) 
Informed consent form (babeer.feahemeinendodontics.hipaa.consent.doc) 

ICF_002.pdf: 
 
 
 
 
 
 
 
 
 
 
 
A new clinical use of Ferumoxytol nanoparticles:  
an anti-biofilm treatment 
NCT06110494 
17-Dec-2021 
 


 
DATE:
17-Dec-2021
Institutional Review Board
TO:
Bekir Karabucak
3600 Civic Center Blvd., 9th Floor
CC:
Philadelphia, PA 19104
Phone: 215-573-2540
(Federalwide Assurance # 00004028)
RE:
IRB PROTOCOL#:
828211
PROTOCOL TITLE: A new clinical use of Feraheme nanoparticles: an anti-biofilm treatment
 
SPONSOR:
NO SPONSOR NUMBER
REVIEW BOARD:
IRB #6
IRB CONTINUING REVIEW: NOTICE OF APPROVAL
Dear Dr. Karabucak,
The above referenced protocol was reviewed and re-approved by the
Institutional Review Board using the expedited procedure set forth in 45 CFR
46.110 on 16-Dec-2021. This study has been determined to be eligible for
expedited review category(ies) 5, 1, 3.
This approval is for the period 16-Dec-2021 to 15-Dec-2022.
The documents included with the application noted below are approved:
-HSERA Continuing Review, confirmation code: dfcejbgh, submitted
12/15/2021
ONGOING REQUIREMENTS:
You must obtain IRB review and approval under 45 CFR 46 if you make
any changes to the protocol, consent form, or any other study documents
subject to IRB review requirements. Implementation of any changes
cannot occur until IRB approval has been given.
Reportable event, such as serious adverse events, deviations, potential
unanticipated problems, and reports of non-compliance must be reported
to the IRB in accordance with Penn IRB SOP RR 404.
When enrolling subjects at a site covered by the University of
Pennsylvania's IRB, a copy of the IRB approved informed consent form
with the IRB approved from/to stamp must be used unless a waiver of
written documentation of consent has been granted.


COMMITTEE APPROVALS: You are responsible for assuring and
maintaining other relevant committee approvals. This human subjects research
protocol should not commence until all relevant committee approvals have
been obtained.
If your study is funded by an external agency, please retain this letter as
documentation of the IRB's determination regarding your proposal.
If you have any questions about the information in this letter, please contact the
IRB administrative staff. A full listing of staff members and contact
information can be found on our website: http://www.irb.upenn.edu
***This letter constitutes official University of Pennsylvania IRB
correspondence. ***


 
1 
UNIVERSITY OF PENNSYLVANIA  
RESEARCH SUBJECT INFORMED CONSENT AND HIPAA AUTHORIZATION FORM 
Protocol Title:  
 
A new clinical use of Feraheme nanoparticles: an anti-biofilm 
treatment  
Principal Investigator:  
 
Dr. Bekir Karabucak 
Department of Endodontics School of Dental Medicine University of 
Pennsylvania 
240 S. 40th Street Philadelphia, PA 19104-6033  
215-898-4617 (office) 
215-573-2148 (fax) 
Endodontic Clinic 215-898-6062 (main) 
Emergency Contact 1: Dr. Alaa Babeer  
(610) 674-8970 (cell)                  
 
Why am I being asked to volunteer? 
You are being invited to participate in a research study. Your participation is voluntary which 
means you can choose whether or not you want to participate. If you choose not to participate, you 
will still receive treatment as needed. Please ask the study doctor and/or the research team about 
this form [[for the further assistance]] if you do not understand any of the medical terms.  
What is the purpose of this research study?  
The purpose of this study is to develop a better, more effective disinfection protocol against 
infections within the root canal system, subsequently, improve the success of root canal treatment. 
Identifying the antibacterial benefits of Feraheme/H2O2 irrigation is the goal of our experiment. 
Feraheme is an iron replacement product that have been used with much higher concentrations in 
medicine as injections to improve and heal patients with iron deficiency. Feraheme used in this 
experiment is limited within the root canal system and applied on the tooth surfaces only. It will not 
be used systemically. this agent is not approved for the study’s indications. 
 
How long will I be in the study? How many other people will be in the study?  
Your participation in this study will not require any more time than actually needed for the routine 
root canal treatment. We will be collecting information related to the diagnosis, chart number and 
tooth number. No protected health information such as name, address, telephone number, date of 
birth, social security number, personal and family medical history or results from physical 
examinations, tests or procedures will be used or collected  
There are no additional procedures, questionnaires, or surveys required. We will have about 60 
people participating in our study.  
What are the possible risks or discomforts?  
Only the risks or discomforts that can arise during or after any routine root canal treatment 
procedure apply. There are no potential risks expected from the noninvasive washing and 
sampling procedures, including any potential physical, psychological, social, economic, monetary, 
legal or other risks. Identifying the antibacterial benefits of Feraheme/H2O2 irrigation is the goal of 
our experiment. Feraheme with much higher concentrations have been used in medicine as 
IRB Approval From: 12-16-2021 To: 12-15-2022


 
2 
injections in the veins to improve and heal patients with iron deficiency. Feraheme systemic 
injections has been associated with increased amounts of iron in the blood, Hypersensitivity 
reactions in patients with multiple drug allergies, in only 2% of the cases it has been associated 
with adverse reactions of diarrhea, nausea, dizziness, hypotension (lowering of blood pressure), 
constipation, and peripheral edema (accumulation of fluid in upper and lower limbs). Feraheme 
used in this experiment is going to be 340 times less than the dose used systemically and it is 
limited within the root canal system and applied on tooth surfaces only. It will not be used 
systemically. Solution will be kept within the root canal system for a couple of minutes proposed in 
this protocol and will be evacuated by sterile saline, dental suction and paper points. Experiment 
was designed that the final root canal disinfection is not relying on experimental solutions 
antibacterial effect only. After bacterial sampling, the routine root canal treatment will be continued 
as needed. Besides tooth number, diagnosis and the PDM chart number (for potential verification 
of study correctness) no patient data will be collected by the Principle investigator. All treatment 
procedures at The University of Pennsylvania School of Dental Medicine, including the routine root 
canal treatment of the participants, during which the samples will be taken, are subject to strict 
HIPPA (privacy) and OSHA (hygiene) regulations. Your participation in this study will not require 
any more time than actually needed for the routine root canal treatment  
What are the possible benefits of the study?  
You are not expected to gain any benefit from taking part in this research study. The proposed 
study will verify by the means of microbial sampling and culturing, whether Feraheme/H2O2 
irrigation protocol can provide better, effective disinfection on root canal surfaces and a significant 
reduction of infection inside the root canal. If this can be demonstrated by the proposed 
investigation it will have clinical significance as an improvement of root canal therapy  
  
 
What other choices do I have if I do not participate?  
If they do not wish to participate, you will not receive any treatment that is subpar as opposed to if 
they did agree to participate. If at any point, you decide you no longer want to participate, you will 
be excused from the study and will continue treatment as needed.   
Will I be paid for being in this study?  
There will be no compensation provided for taking part in this study.  
Will I have to pay for anything?  
There is no additional cost for you or your insurance company associated with our study. 
When is the study over? Can I leave the Study before it ends?  
This study is expected to end after a sufficient number of people enroll in the project. Your 
involvement with the study ends when the root canal treatment appointment has ended. If you 
decide not to participate, you are free to leave the study at anytime. Withdrawal will not interfere 
with your future care.  
This study may also be stopped at any time by the Primary Investigator without your consent.  
Who can I call with questions, complaints or if I’m concerned about my rights as a research 
subject?  
If you have questions, concerns or complaints regarding your participation in this research study or 
if you have any questions about your rights as a research subject, you should speak with the 
IRB Approval From: 12-16-2021 To: 12-15-2022


 
3 
principal investigator listed in this form. If a member of the research team cannot be reached or 
you want to talk to someone other than those working on the study, you may contact the Office of 
Regulatory Affairs with any question, concerns or complaints at the University of Pennsylvania by 
calling (215) 898-2614.  
What information about me may be collected, used or shared with others?   
We will be collecting information related to the diagnosis, chart number and tooth number. No 
other protected health information such as name, address, telephone number, date of birth, social 
security number, personal and family medical history or results from physical examinations, tests 
or procedures will be used or collected. 
The U.S Office of Human Research Protections may receive your information. All human research 
is subject to their oversight.   
Once your personal health information is disclosed to others outside the School of Medicine, it may 
no longer be covered by federal privacy protection regulations  
Why is my information being used? 
The chart number will only be recorded for a possible future verification of the research data such 
as tooth number and diagnosis. your chart number is secured by a login and password only 
accessible to students, residents and faculty of the dental school  
. Your information and results of tests and procedures are used to: 
• do the research 
• oversee the research 
• to see if the research was done right 
• to evaluate and manage research functions.   
Who may use and share information about me?   
Additional to your clinical doctor who is performing the root canal, 
The following individuals may use or share your information for this research: 
• Dr. Bekir Karabucak (principal investigator listed above) 
• Dr. Alaa Babeer (emergency contact listed above) 
Who, outside of the School of Dental Medicine, might receive my information?  
The U.S Office of Human Research Protections may receive your information. All human research 
is subject to their oversight.   
The Principal Investigator or study staff will inform you if there are any additions to the list above 
during your active participation in the study. Any additions will be subject to University of 
Pennsylvania procedures developed to protect your privacy. 
 
How long may the School of Medicine use or disclose my personal health information?   
Your authorization for use of your personal health information for this specific study does not 
expire.  
Your information may be held in a research database.  However, the School of Medicine may not 
re-use or re-disclose information collected in this study for a purpose other than this study unless: 
• You have given written authorization  
• The University of Pennsylvania’s Institutional Review Board grants permission 
IRB Approval From: 12-16-2021 To: 12-15-2022


 
4 
• As permitted by law  
 
Your Personal Health Information will not be used or disclosed during your participation in this 
study. Besides tooth number, diagnosis and the Penn Dental Medicine chart number (for potential 
verification of study correctness) no patient data will be collected by the Principle Investigator. All 
treatment procedures at the University of Pennsylvania School of Dental Medicine, including the 
routine root canal treatment of the participants, during which the samples will be taken, are subject 
to strict HIPPA (privacy) and OSHA (hygiene) regulations  
Can I change my mind about giving permission for use of my information? 
Yes. You may withdraw or take away your permission to use and disclose your health information 
at any time. You do this by sending written notice to the investigator for the study.  If you withdraw 
your permission, you will not be able to stay in this study. 
 
What if I decide not to give permission to use and give out my health information? 
If you decide not to participate, you are free to leave the study at any time. Withdrawal will not 
interfere with your future care.  
You will be given a copy of this Research Subject consent form & HIPAA Authorization describing 
your confidentiality and privacy rights for this study.  
By signing this document, you are agreeing to participate in this research study, and you are 
authorizing us to use your chart number tooth number and diagnosis as described above. 
 
 
_________________________________    ____________________________   ______________ 
Name of Subject (Print)                                 Signature of Subject                          Date 
 
_________________________________    ____________________________   ______________ 
Name of Person Obtaining Consent             Signature                                            Date 
 
IRB Approval From: 12-16-2021 To: 12-15-2022