Antimicrobial Effect of Tropolone Containing Versus Tropolone Free Mouthwash - Randomized Clinical Trail
Phase: EARLY_PHASE1
Conditions: Dental Diseases
Sponsor: Cairo University

 
Antimicrobial effect of Tropolone 
containing versus Tropolone free 
mouthwash - Randomized clinical trail    
ال تأثير
ال
مضاد ل لميكروبات لغسول الفم المحتوي على 
تروبول
و ن
مقابل التي ال تحتوي ع
لى تروبول
و ن :
 
 دراسة سريرية عشوائية 
 
Protocol 
Submitted to the Faculty of Oral and Dental Medicine, 
Cairo University for partial fulfillment of the Requirements of 
Master Degree in Conservative Dentistry 
 
 
By  
Mohamed Awad Mohamed Abdelkadir 
B.D.S (Misr University for science & technology) 2011 
 
 
Under supervision of  
Prof. Dr. Mohamed Adel Ezzat   
Prof. of Conservative Dentistry, Cairo University 
 
Associated prof. Dr. Eman Ali Abouauf  
Conservative Dentistry Department, Cairo University 
 
 
NCT03384537 -  February 17, 2019  
 
 
 
 

 
Contents table 
Introductory cover page …..………………………………….. 
Contents table ……...…………………………………………. 
Administrative information ……………………………….1-2 
1. Trial registration.   
2. Protocol version. 
3. Funding. 
4. Roles and responsibilities 4.1-4.5 
Scientific Background ………………………………………..3 
Statement of the problem …………………………………....3 
Rationale …………………………………………………...4-5 
Benefits for patient/clinician ……………...…………………6 
 For the patient. 
 For the clinician. 
Objectives ………………………………….………………...6 
Hypotheses …………………………………………………..6 
Trial design …………………………………………………..7 
Trial type …………………………………………………….7 
Allocation ratio ……………………………………………...7  
Search strategy ………………………………………………7 
 Source 
Index terms & Mesh terms table ….………..……….……...7 
Inclusion and exclusion criteria table ...…………….………8 
Review of Literature …………………………………….8-15 
 Antimicrobial effect of Tropolone containing mouthwash.  
Aim of study …………….…………………………….........16  
PICOTS ……………………………………………………..16  
Research question …………………………………………..16 
 

 
Material ……………………………………………………..17 
1. Steps. 
2. Methods. .………………………………………...17- 27 
2.1 Study setting.  
2.2 Eligibility criteria. 
 Inclusion and Exclusion criteria. 
2.3 Variables of study 
 Variables of study table (1). 
 Interaction of variables of study table (2). 
2.4 Intervention / control assessment 
 Examination. 
 Criteria 
for 
discontinuing 
or 
modifying 
allocated 
interventions for a Participant. 
 Strategies that will be used to improve adherence to 
interventions protocol. 
2.5 Outcome  assessment 
 Procedure of viability count measurement. 
2.6  Clinical relevance of the outcome 
2.7  Sample size calculation  
2.8  Recruitment 
2.9  Randomization  
 Implementation. 
 Blinding. 
2.10  Data collection methods 
 Baseline data collection. 
 Outcome data collection. 
 Patient retention. 

 
2.11  Data management 
2.12  Statistical methods 
2.13  Monitoring 
A. Data monitoring. 
B. Harms. 
C. Auditing. 
2.14  Ethics and dissemination 
a. Research ethics approval. 
b. Protocol amendments. 
c. Consent or assent. 
d. Confidentiality. 
e. Declaration of interests. 
f. Access to data. 
g. Ancillary and post-trial care. 
h. Dissemination policy. 
 
 
 

1
 
 
Administrative information: 
1. Trial registration:   
        www.clinicaltrials.gov (NCT03384537) 
 
2. Protocol version: 
              August - 2017, First version. (will be finalized after registration )  
 
3. Funding:  
               The trial will be Self-funding by the main researcher.  
 
4. Roles and responsibilities:  
4.1. Mohammed Awad Mohamed Abdulkadir   
 Role: main researcher, assessor data enterer and writing the 
research. 
 Affiliation:  Master degree candidate in Conservative Dentistry 
Department, Faculty of dentistry, Cairo University Egypt. 
 
4.2. Prof. Dr. Mohamed Adel Ezzat 
 Role: Main supervisor, assessor, data monitoring, auditing.  
 Affiliation: Professor Conservative Dentistry Department, 
Faculty of dentistry, Cairo University, Egypt. 
 
4.3. Associated prof. Dr. Eman Ali Abuauf  
 Role: Co-supervisor, assessor, data entry and auditing. 
 Affiliation:  Conservative Dentistry Department, Faculty of 
dentistry, Cairo University, Egypt. 
 
 
 

2
 
 
4.4. Eman Desouky  
 Role: Sample size calculation. 
 AFFILIATION; Statistician, Faculty of dentistry, Cairo 
University, Egypt. 
 
4.5. Research Ethics Committee (CREC) 
 Role:  Protocol reviewer of the clinical trial in order to 
protect the right, safety, dignity and well-being of the 
participants. 
 Affiliation:  Faculty of dentistry, Cairo University, Egypt.  
 Approval No: 18 2 12 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3
 
 
Scientific Background:  
     Removal of dental bio-film is important as it may become acidic 
causing demineralization of the teeth (dental caries) or harden into dental 
calculus (tartar). 
     Calculus cannot be removed through mouthwash, tooth-brushing or 
with inter-dental aids and can only be removed through professional 
cleaning.  
     Therefore, removal of the dental bio-film will prevent the development 
of dental caries and gum diseases. ( Haider et al, (2013).  
 
Statement of the problem: 
     Oral health has great impact on for prevention of oral diseases. 
Therefore, people have used different methods to promote their oral 
health, among which mechanical techniques such as flossing and 
brushing, are the most well-known ones.  
     However, these methods are not able to eliminate all disease causing 
factors alone, especially in interproximal areas.   
     On the other hand, many people with specific physical and 
psychological conditions are required to use an antiseptic in order to do 
away with many deficiencies of eliminating the mechanical plaque.  
    Thus, using chemical materials (such as mouth rinses) are taken more 
into account.  
    As an aid along with mechanical plaque control, mouth rinses play a 
pivotal role in supra-gingival plaque control and gingivitis control.                      
    An appropriate mouthwash, in addition to antimicrobial spectrum, 
should have low pharmaceutical interaction and cause less damage to the 
normal micro-flora of oral cavity. E Marchetti et al., 2017 
 
 

4
 
 
Rationale:  
      Dental 
Caries 
disease 
prevented 
through 
the 
antibacterial 
mouthwash.                               
     The prevalence of dental caries disease continues to be a challenge 
for oral health care professionals to this day.  
      In fact an alarming 92% of US adults (aged 20 to 64 years) have a 
history of dental caries.   
     While strides have been made since the early 1970s the problem 
persists. 
      From the mid1990s until 2004 according to the National Health and 
Nutrition examination survey there was a small but significant increase in 
primary decay.  
      This trend was even more severe in younger.  
       Dental Caries disease are not being treated can eventually turn into 
more serious issues.  
       Fluoride has long been known as one of the key components to 
good oral health and the prevention of dental caries disease.  
       It is a naturally occurring mineral that makes tooth enamel more 
resistant to acid producing bacteria that cause dental caries disease while 
also repairing teeth in the very early microscopic stages.  
       While the use of fluoride in toothpaste has been responsible for a 
drop in dental caries disease since 1960 this significant problem still 
persists. 
       Yet, many people do not realize that also using a mouth rinse can 
result in 50% stronger teeth than brushing with fluoride toothpaste alone.        
        The American Dental Association (ADA) recommends the use of 
mouth rinses with fluoride to help resist tooth decay.  
       However, while it may appear so on the surface, not all fluoride 
mouth rinses are created the same.   

5
 
 
      At the Listerine brand, our commitment to innovation led to the 
creation of unique anti-cavity mouth rinses powered by breakthrough 
science called rapid fusion technology a unique fluoride delivery system 
which binds calcium ions with fluoride to create fluoride reservoirs that 
attach to tooth enamel and are then released over time for a greater 
fluoride uptake and greater enamel content of fluoride to the tooth 
surface. 
      Rapid fusion technology provides fluoride in a safe acidic 
environment that rapidly liberates calcium and phosphate ions.  
      These ions combine to create millions of tiny fluoride reservoirs on 
tooth enamel and in saliva increasing the amount of fluoride on the tooth 
surface.  
      Throughout the day as enamel is exposed to acids from dietary 
sugars, the reservoirs dissolve and release fluoride to re-mineralize the 
teeth.  
       The fluoride then binds to areas of weak demineralized enamel and 
attracts calcium and phosphate from saliva.  
       These ions penetrate the enamel and combine with fluoride to create 
a new stronger and more acid resistant mineral surface.  
        In this way Rapid fusion technology enhances re-mineralization 
and inhibits demineralization of tooth enamel to provide. 
     Stronger teeth than brushing with fluoride toothpaste alone and 
greater re-mineralization of enamel  May 11, 2016. Johnson & Johnson 
Consumer Inc. 
 
 
 
 
 
 
 
 

6
 
 
Benefits for patient/clinician:  
 For the Patient: The antimicrobial mouthwash is proved to 
prevent the development of dental plaque.  
 The use of antimicrobial agents will lead to the avoidance of 
side effects complained by patients associated with the use of 
chlorhexidine.  
 And proving their potency against mutans streptococci will 
dramatically improve the oral health of patients as it is the 
major cause of dental plaque. 
 
 For the clinician: Dental plaque is reported to be a major 
health problem in public.  
 Therefore, reducing the incidence of caries disease through the 
use of antimicrobial agents will decrease the number of visits 
of patients complaining of plaque and associated health 
problems.  
 Also, complaints of patients regarding side effects of will be of 
no concern to dentists. 
 
Objectives:  
    The objective of this study is to compare the effect of Tropolone 
containing mouthwash versus CHX 0.2% mouthwash in reducing 
intraoral microorganism. Randomized clinical trial study 
 
Hypotheses:  
   The null hypotheses tested is that there is no different between using 
alcohol-free essential oils containing mouth rinse and other mouth rinse 
regarding anti-plaque and anti-gingivitis microbial agent.  
 

7
 
 
Trial design:  
      The study design in this investigation will be an in vivo diagnostic 
study. 
 
Trial type:  
       This study will be a parallel study design. 
 
Allocation ratio: 
       In this study, the allocation ratio will be 1:1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

8
 
 
Review of Literature: 
 
Search strategy:  
 Source: Database used in searched are pub-med, Cochrane 
library database and Google Scholar. 
Index terms 
Mesh terms 
Antimicrobial dental plaque 
 
Index, Dental Plaque 
 
Dental Plaque Indexes 
 
Indexes, Dental Plaque 
 
Dental Plaque Indices 
 
Indices, Dental Plaque 
Anti-Infective agents 
(Tropolone containing ) 
Supplementary Concept 
Anti-Infective agents 
(Chlorhexidine ) 
 Chlorhexidine Hydrochloride 
 Hydrochloride, Chlorhexidine 
 Tubulicid 
 Novalsan 
   Sebidin A 
 Chlorhexidine Acetate 
 Acetate, Chlorhexidine 
Bacterial count 
 Bacterial Loads 
 Load, Bacterial 
 Loads, Bacterial 
 Bacterial Count 
 Count, Bacterial 
 Counts, Bacterial 
 Bacterial Count 
 
Inclusion and exclusion criteria: 
Inclusion 
Exclusion 
In vivo study 
Patient with systemic disease that 
affect study result  
Articles published in English language 
Study for ages below 18 and above 54 
years 
Article published after 2010 
Intervention study other than Listerine 
total care mouthwash   

9
 
 
Antimicrobial Tropolone containing 
mouthwash (Total care zero) 
Study that include product other than 
mouthwash or rinsing  
Chlorhexidine mouthwash   
Study 
didn’t 
measurement 
dental 
plaque (bacterial count) 
 
 
 Antimicrobial effect of Tropolone containing mouthwash : 
     E Marchetti et al.,  2017 The aim of this study was to evaluate the 
antiplaque effects of an alcohol-free essential oil (alcohol-free EO) 
mouthwash and an amine fluoride compared to a positive control of 
chlorhexidine they found there was less of an effect compared to the 
CHX group, with an overall plaque index of 1.41. 
     The differences of 0.96 between alcohol-free EO and CHX were all 
statistically significant (P < 0.001).  
      Conclusion alcohol-free (EO) mouthwash has the same effect of CHX 
control on an inhibiting plaque regrowth.  
 
      Mogharehabed et al. 2016 .The current study was aimed to compare 
the effectiveness and side effects of chlorhexidine mouth rinses with and 
without alcohol. 
   Chlorhexidine mouth rinses have widely been recognized for their 
contribution in maintaining plaque control. Most of them contain alcohol 
that makes them impractical for many patients. 
    They found both mouthwashes significantly reduced the mean scores 
of plaque (P < 0.0001) and gingival (P < 0.032).  
     The extent of stain was the comparable in both groups. While Epimax 
mouth rinse caused severe stains on the teeth, Hexidine mouth rinse 
caused burning mouth.  

10
 
 
     Conclusion Epimax mouthwash it was less suitable and caused more 
dental stain. Ethanol-free Hexidine mouthwash seems to be more proper 
for gingivitis, but its side effects are required to be taken into 
consideration.  
 
C. Vlachojannis et al . 2015. The aim of this study was to get 
preliminary information about the antimicrobial activities of individual 
Listerine components and their mixtures against Streptococcus mutans.                               
They found thymol was the most effective against S. mutans and 
phenols and their concentrations increase their general effectiveness that 
do not induce harm.  
Conclusion based on our experiments and considering the 
antimicrobial effects against the resident physiological micro-flora in the 
oral cavity, we suggest optimizing the phenolic composition of Listerine 
and the concentrations of those phenols that were found to have a 
beneficial effect within the combination. 
 
    BR Charugundla et al 2015. The objective of this study was to 
compare the effectiveness of fluoride essential oil (EO) and chlorhexidine 
(CHX) mouth rinses on dental plaque and gingivitis and to compare their 
relative efficacy in patients with and without dental caries.  
      They found significant reduction in plaque after use of mouth rinses 
(P < 0.05) and no significant differences were observed with respect to 
each other in reducing gingivitis (P > 0.05).  
      Further significant differences were found in reducing plaque and 
gingivitis in dental caries-free subjects in comparison to those with dental 
caries (P < 0.05).  
      Conclusion all the three mouth rinses significantly reduced plaque 
accumulation and gingivitis especially in dental caries-free subjects in 

11
 
 
comparison to those with dental caries, and amongst the three fluoride 
and CHX proved to be more effective than EO mouth rinse. 
 
    Christian Vlachojannis et al . 2013. The studies support the claim 
that ListerineW shows benefit for oral health, but the concerns over its 
safety remain to be clarified.  
     They found until these have been addressed, high risk populations 
(children, alcohol addicts, patients with genetic deficiencies in ethanol 
metabolism) should use alcohol-free mouthwashes for the maintenance of 
oral health.  
      Conclusion the mouthwash ListerineW is not a medicinal plant 
product. 
       The mixture of four essential oil ingredients in an aqueous ethanol 
solution showed benefit for oral health, in terms of gingivitis and dental 
plaque reduction.  
       However, the concern over its safety should be clarified.  
       Since the use of ListerineW is not restricted, rigorous preclinical and 
human long-term pharmacological safety data are warranted to exclude a 
possible link with cancer. 
        Until the safety concerns have been addressed, high risk populations 
(children, alcohol addicts, patients with genetic deficiencies in ethanol 
metabolism, oral cancer or smokers) should use alcohol-free 
mouthwashes for the maintenance of oral health. 
 
    HANS RAGNAR PREUS et al 2013. The aim of the present study 
was to test the clinical effect of Listerine on plaque formation as primary 
and gingivitis as secondary end point, with or without mechanical oral 
hygiene in a modified experimental gingivitis model.  

12
 
 
     They found when comparing the gingival condition in the proximal 
sites only, there were no differences between the mouth rinses.   
      When tooth brushing, flossing and rinsing were performed in the 
same quadrant the plaque scores were very low and gingival scores 
showed no statistically significant differences between the mouth rinses.         
        Conclusion clinical efficacy of Listerine total care on plaque 
formation and gingivitis in this modified experimental gingivitis model, 
with 22% hydro-alcohol and 0.2% CHX solutions as controls, no 
statistically significant antibacterial effect of Listerine over its placebo 
vehicle was found. 
         Neither Listerine nor alcohol had any effect of clinical value to the 
user, since the amount of accumulated plaque after rinsing was still 
enough to cause any of the dental plaque related diseases. 
 
   Sharukh S. Khajotia et al .2013. The objective of this study was to 
report a methodology for quantification and comparison of the concurrent 
three dimensional distributions of three cellular and extracellular 
components of biofilms.  
     The method consists of distinct but interconnected steps involving 
biofilm growth, staining, Confocal laser scanning microscopy (CLSM)   
imaging of biofilms, biofilm structural analysis and visualization and 
statistical analysis of structural parameters.  
       The biofilm growth assay permits biofilm growth on relevant 
substrates and produces biofilm structures that are reproducible.  
       The 
combination 
of 
novel 
simultaneous 
staining 
of 
exopolysaccharides (EPS) proteins and nucleic acid components with the 
measurement of three dimensional biofilm structural parameters results in 
quantifiable distributions of components within biofilms.  

13
 
 
        Statistical analysis of the biofilm structural parameters facilitates 
evaluation of biofilms under specific experimental conditions 
 
    Van Strydonck DAC et al 2012. The aim of study to systematically 
evaluate the efficacy of chlorhexidine (CHX) mouth rinses on dental 
plaque, gingival inflammation and staining in gingivitis patients.  
     They found CHX molecule result in a broad bactericidal and   
bacteriostatic spectrum of action and a high substantively of up to 12 hour 
within the oral cavity.  
       Because CHX binds strongly to tissues, it is poorly absorbed from 
the gastrointestinal tract and therefore lacks systemic toxicity. 
       Conclusion to demonstrate the efficacy of the different CHX mouth 
rinse formulations in the inhibition of dental plaque, gingivitis and stain 
formation, multiple comparative clinical studies have been performed. 
 
    Anthony L. Neely et al. 2012. This systematic review studies had to 
be randomized clinical trials in healthy human subjects comparing the 
effects of essential-oil mouthwash (EOMW) with chlorhexidine on dental 
plaque accumulation, tooth staining and gingival inflammation.  
      Studies could be either short-term less than four weeks duration or 
long-term more than four weeks duration.  
      Studies were required to include a specific formulation of EOMW 
(Listerine).  
        They reportedly selected this standard formula of EOMW because it   
was representative of essential oil based mouthwashes and because it has 
the American Dental Association seal of approval.  
        Conversely, there were no restrictions on the concentration of CHX 
used in studies.  

14
 
 
         They found nineteen controlled clinical trials were included in this 
systematic review. 
          In five of the seven studies of plaque index, CHX was found to be 
significantly better than EOMW at reducing plaque accumulation.  
           Stain development was assessed in five long-term brushing trials. 
CHX was significantly associated with more staining than EOMW in the 
systematic review.  
          The calculus index was significantly greater among CHX users 
versus EOMW users. 
           Gingivitis levels four of five studies provided statistical data that 
could be used in the systematic review.  
          Two of these investigations showed significantly lower gingival 
inflammation with CHX. Bleeding indices were assessed in five short-
term and four long-term studies.  
           Only one of the short-term studies showed a significant difference, 
whereas three of four long-term studies showed no difference between 
CHX and EOMW.  
            Meta-analyses were included for plaque index, gingival index, and 
tooth staining index. In two of three meta-analyses of plaque index trials, 
CHX was shown to be significantly more effective than EOMW at 
reducing plaque. 
             One of the significant meta-analyses involved plaque regrowth in 
less than four weeks of no oral hygiene measures.  
       The overall weighted mean difference (WMD) for the plaque index 
was 0.46 (95% (CI) = 0.09, 0.84)) confidence interval.  
       Other meta-analysis was a comparison of more than four weeks in 
which either daily EOMW or CHX supplemented normal oral hygiene 
measures.  

15
 
 
        This long-term study of plaque accumulation showed significantly 
less plaque with CHX than with EOMW.  
        The WMD for the long-term plaque control studies was smaller than 
for the short-term studies (0.19; 95% CI = 0.08, 0.30). 
         No significant differences were found in meta-analyses for gingival 
inflammation (gingival index17) or stain accumulation (stain index12).  
         Significant heterogeneity was identified in one of two of the meta- 
analyses for both gingival index and stain index. 
         Conclusion CHX was significantly better at reducing plaque   
accumulation than EOMW in short-and long-term studies.   
         Staining and calculus accumulation were greater among CHX users 
compared to EOMW. 
         CHX and EOMW were not different with respect to long-term 
control of gingival inflammation.  
          They concluded that EOMW might be a reliable alternative to CHX 
for controlling gingival inflammation in cases where a dental professional 
deems that anti-inflammatory oral care is beneficial.  
 
    Enrico Marchetti et al. 2011. The study was held to evaluate the 
antiplaque effect of a new alcohol free essential oil (zero alcohol) 
mouthwash with respect to a control of an essential oil with alcohol 
mouthwash.  
     They found the amounts of mouthwashes used indicated good 
compliance with the instructions.  
     No adverse events or side effects were reported or observed.  
     With regard to the subjects rating of the rinsing time the results 
demonstrated a difference between the test and control groups (6.50 and 
4.23 respectively, p < 0.005). 

16
 
 
      This suggest than the rinsing time of EO with alcohol seemed longer 
than EO without alcohol, probably due to the typical burning effect of EO 
with alcohol. 
They also considered EO with alcohol (visual analogue scale, VAS 
6.07) as more effective in reducing plaque in the mouth compared to the 
EO without alcohol (visual analogue scale, VAS 4.97; p < 0.05).                                          
 However, duration of taste, alteration in taste perception and 
convenience, the statistically significant differences were not noted 
between the groups.  
Conclusion Epidemiological studies however are often inconsistent 
and many reviews conclude there are no data demonstrating the direct 
correlation between alcohol containing mouthwashes and oral cancer. 
 Recently, Werner and Seymour have reviewed the two most recent 
revisions on the role of alcohol in the onset of oral cancer stating that 
there is evidence showing the existence of this association, but these are 
still weak and inconclusive and randomized clinical trials would be 
needed on a large sample to verify this hypothesis.  
These authors concluded that the benefit of alcohol in mouthwashes is 
negligible and it may carry a risk of oral cancer which is difficult to 
quantify and so it is preferable not to prescribe or recommend them.  
This three day plaque regrowth study showed that the EO containing 
mouthwash without alcohol was a less potent plaque inhibitor than the 
traditional alcohol containing EO mouthwash.  
It appears that the subjects appreciated the effect on plaque reduction 
of the traditional mouthwash better. 
 
 
 
 

17
 
 
Aim of study: 
    The study will be conducted to evaluate the antimicrobial effect of 
recent rapid fusion technology mouthwash in reducing intra-oral 
cariogenic microorganism. 
  
PICOTS:  
   P: intra-oral cariogenic microorganism.   
   I: (Recent rapid fusion technology mouthwash) Tropolone containing 
mouthwash. 
  C: Chlorhexidine Mouthwash (0.2%).  
  O: bacterial count.   
Type Outcome  
Outcome name  
Measuring device Measuring unit 
Primary 
 
Viability counts 
Counts 
Colony-forming 
unit per milliliter 
(CFU/ml) 
 
T= T0 = base line   
      T1= immediate after using mouthwash.  
      T2= one week after regular use of mouthwash. 
      T3 = two weeks after regular use of mouthwash.   
S= Randomized Clinical Trial 
 
Research question: 
Will a Tropolone containing mouthwash decrease the cariogenic 
microorganism than Chlorhexidine mouthwash (0.2%)?  
 
 
 
 

18
 
 
1. Material:  
Steps:  
1.1. Tropolone containing mouthwash (Listerine total care zero). 
1.2.   Chlorhexidine 
mouthwash 
as 
a 
positive 
control                          
(Hexitol). 
 
2. Methods  
2.1.Study setting 
The study will be conducted in outpatient clinic of the 
Conservative department of the Faculty of Dentistry, Cairo University. 
The bacterial count test will be conducted in the Microbiology 
department, Faculty of Medicine, Cairo University Egypt.  
The researcher will bear ultimate responsibility for all activities 
associated with the conduct of a research project including recruitment of 
patients, explaining and performing the procedures to them. 
 
2.2 Eligibility criteria 
 Inclusion Criteria: 
 Patients should be between 18- 45 years of age. 
 All the volunteers participated in this study will be healthy looking 
with free medical history. 
 The volunteers will be asked to suspend their usual oral hygiene 
practice from two to four days before experiment studying.  
 
 

19
 
 
 Exclusion criteria: 
 Patients with a compromised medical condition. 
 Volunteers that receive any antimicrobial agent during at least two 
weeks prior to study. 
 Volunteer with fixed, removable prosthesis or orthodontics 
appliance.  
 Volunteers with DMF above will be excluded. 
 
2.3. Variables of the study: 
 A total of 30 Volunteers patients will be assigned in this study. 
     Thirty participants in this study will be randomly divided into two 
groups (n= 15) according to the application of the mouth rinse agent  
(M); the first group (M1) will use Tropolone containing mouthwash as a 
mouth rinse, the second group (M2) will use chlorhexidine 0.2% as a 
mouth rinse. Then each patient will be monitoring at the base line T0: 
before using any mouthwash, T1: immedially after using tested 
mouthwash, T2: after One week and finally, T3: after Two weeks of 
using the tested mouthwash.  
      Each patient will be the reference/counted for self as a record. 
 
 
 
 
 
 
 
 

20
 
 
 Table 1: Variables of the study 
Variable 
Symbol 
Refer to 
Mouth rinsing agent 
 
M1 
Tropolone containing 
mouthwash 
M2 
Chlorhexidine mouthwash  
Time relation to 
rinsing 
T0 
 
Base line 
T1 
 
Immediate time 
T2 
After one week 
T3 
After two weeks 
 
Table 2 : Interaction of Variables of the study  
Rinsing agent 
M1 
M2 
Total 
Time 
T0 
M1 T0 
M2 T0 
15 
T1 
M1 T1 
M2 T1 
15 
T2 
M1 T2 
M2 T2 
15 
T3 
M1 T3 
M2 T3 
15 
Total 
30 
30 
60 
N=15  
(N represent a number of subjects in each groups ) 
 
 
 
 
 

21
 
 
2.4. Intervention/control assessment: 
             The participants in this study will be randomly divided into two 
groups according to the application of the mouth rinse agent (M); the first 
group M1) will use Tropolone containing as a mouth rinse, the second 
group (M2) will use chlorhexidine 0.2% as a mouth rinse.  
    Then each patient will be monitoring at the base line T0: before using 
any mouthwash, T1: immedially after using tested mouthwash, T2: after 
one week and finally, T3: after two weeks of using the tested mouthwash.  
      Each patient will be the reference/counted for self as a record. 
 
Examination: 
 
    Patients will be selected and examined according to inclusion and 
exclusion criteria.  
 
Criteria for discontinuing or modifying allocated interventions for a 
Participant: 
   At the given percentage of antimicrobial Tropolone containing 
mouthwash no complications should be anticipated. 
 
Strategies that will be used to improve adherence to interventions 
protocol: 
    The antimicrobial Tropolone containing mouthwash will be kept sealed 
until use.  
 
 
 
 
 
 

22
 
 
2.5. Outcome assessment: 
                     Procedure of viability count measurement 
           Effects of antimicrobial Tropolone containing mouthwash 
versus Chlorhexidine mouthwash 0.2% on viability counts of Mutans 
Streptococci and lactobacilli: 
      Each volunteer will be given a piece of Arabic gum and asked to 
chew it for one minute only, then stimulated saliva was collected in 
sterilized screw capped bottles.  
      After one minute, each volunteer will be asked to rinse with 10 ml of 
test agent for one minute then expectorate.  
      Stimulated saliva will be recollected in the following points: after 30 
minutes of rinsing, one hour, and, two hours.  
      During this time, the volunteers will be asked not to eat or drink 
anything except water.  
      Salivary samples will be dispersed for two minutes by vortex mixer, 
then 0.1 ml of saliva transferred to 0.9 ml of sterile phosphate buffer 
saline (pH 7.0), and tenfold dilutions will be performed.  
      From the dilution 10-3, 0.1 ml will be taken and spread in duplicate on 
Mitis Salivarius Bacitracin agar plates, these plates will be incubated 
anaerobically for 48 hour at 37 ºC then aerobically for 24 hour at room 
temperature.  
       The number of colonies will be expressed as colony forming units 
multiplied by the dilution factor per milliliter of saliva (CFU/ml) and 
compared before and after rinsing.  
 
2.6. Clinical relevance of the outcome: 
       Reduction in the number of the streptococcus mutans and lactobacilli 
after application of the intervention which is the main cause of dental 

23
 
 
caries will mean that this material is an adjuvant to other mouthwash 
rinses and could be used in the clinical practice as its easily use with not 
reported side effects. 
 
2.7. Sample Size calculation: 
 
     The aim of this study is to compare the antimicrobial effects of 
Tropolone containing mouthwash versus antimicrobial Chlorhexidine 
mouthwash 0.2% ( Hexitol) on oral streptococcus mutans and lactobacilli 
with repeated measures bacterial count test of variance will be performed 
to study the effect of different solutions at different times.  
      Based on previous papers by Weli & Mohammed 2013 and Rashad 
2008 a large effect size is expected (f=0.4).  
     A minimum total sample size of 30 patients (15 in each group) will be 
sufficient with the power of 85% and a significance level of 5%. 
       The sample size was calculated using G*Power program 
(University of Dusseldorf, Dusseldorf , Germany). 
 
2.8. Recruitment: 
        Patient will be recruited from the outpatient clinic of the 
Conservative department of the Faculty of Dentistry, Cairo University 
Egypt.  
     After explaining the benefits from the application of the interventions 
to their oral hygiene which is alcohol free .      
    Unlike other mouth rinses that has complications such as staining, 
burning sensation and dry mouth from which eligible patients will be 
recruited to fulfill the eligibility criteria. 
 
 
 

24
 
 
2.9. Randomization: 
         Sequence generation: Randomization will be computer generated 
using (www.randomization.com).  
        Allocation concealment: Allocation of rinsing agents to groups will 
be done through sealed black opaque envelopes to ensure complete 
concealment. 
 
Implementation: 
     A third party will perform the allocation sequence and assign the 
participants to rinsing agent in sequentially numbered opaque envelop. 
 
Blinding: 
     The allocation group will be blinded from those assessing the 
outcome, data collectors, and data analysts.  
    The microbiologist will be blinded by labeling the specimens with non-
identifying terms. 
 
2.10. Data collection methods: 
 
                Baseline data collection: 
      The main researcher will collect the baseline data through a chart 
which is composed of medical, dental history and clinical examination for 
every patient will be filled.  
     The report will be anonymous where patients identified by their serial 
numbers (the first letter of the first and last name and date of birth) only 
will be registered. 
   The main researcher will write the full detailed personal data of the 
patient in a separate sheet having the patient's serial number for further 
contact with patient. 
    This sheet can only be seen by main researcher and co-supervisor.  

25
 
 
Outcome data collection: 
     A Microbiological technician will assess the viability counts of 
Mutans Streptococci and lactobacilli in patient’s saliva at baseline, 
immediate time, after one week and after two weeks of rinsing. 
 
Patient retention: 
     The main researcher should understand the participant the final result 
will be expected and how it will differ in his/her oral hygiene.  
   The main researcher will told the participant that the whole procedure 
will take a short time and it will be painless. 
 
2.11. Data management: 
           
       Data entry will be carried out by the main researcher and revised by 
co-supervisor. 
     All data will be stored on computer and will be encrypted using a 
password. 
      This will be done to allow accurate data entry through revision and 
protect data from being incorrectly used.  
    Data will be packed up on another storage device to prevent it from 
being lost. 
 
2.12. Statistical methods: 
        Data will be analyzed using IBM SPSS advanced statistics 
(Statistical Package for Social Sciences, version 21 (SPSS Inc. 
Chicago.IL). 
    Numerical data will be described as mean and standard deviation or 
median and range, while qualitative data will be described as number and 
percentage.  

26
 
 
    Two ways with repeated measures bacterial count test of variance 
(ANOVA) will be performed. A p-value less than or equal to 0.05 will be 
considered statistically significant. All tests will be two tailed. 
 
2.13. Monitoring: 
 
         A.  Data monitoring: 
 
          The main supervisor will monitor this study. His role is to monitor 
any risk of bias could be done from participants, operator or assessors, 
blinding of the assessors and patient safety, outstanding benefits or 
harms. 
 
         B. Harms: 
   
           The main researcher should inform participants about the possible 
harms, if present.  
           Participants are allowed to contact the operator at moment through 
telephone. The data will be reported to main researcher.  
 
        C. Auditing: 
 
          In the present trial, auditing will be done by the main and co-
supervisors to assure quality of the research frequency procedures.  
 
 
 
 
 
 
 

27
 
 
2.14. Ethics and dissemination 
 
a.  Research ethics approval:   
           Application forms for carrying out the clinical trial, checklist and 
informed consent of Research Ethics Committee (CREC) Faculty of  Oral 
and Dental Medicine, Cairo University will be retrieved and filled, then 
will be delivered for (CREC) committee for approval, this is done to 
prevent any ethical problems during the study or any harm for any of the 
participants. 
                     Research ethics approval:  18 2 12 
 
   b. Protocol amendments: 
       If a new protocol will be used a protocol amendment will be 
submitted; containing a new copy of the new protocol and brief 
explanation about the differences between it and the previous protocols. 
    If there is a change in the existing protocol that affects safety of subject 
investigation scope or scientific quality of the trial, an amendment 
containing a brief explanation about the change will be submitted.   
     If a new author will be added to accomplish the study, an amendment 
including the investigator’s data and qualifications to conduct the 
investigation will be submitted to prevent ghost authorship. 
 
c. Consent or assent: 
 
     The operator (Mohamed Awad Abdulkadir ) is responsible for 
admitting and signing the written consents during the enrolment 
day.(Fig.1). 
 
 

28
 
 
d. Confidentiality: 
         Name, personal data and pictures of the participants will not appear 
on the protocol form and will be maintained secured for ten years after 
the trial. This is done for protection of participant's privacy and civil 
rights. 
 
   e. Declaration of interests: 
 
      There is no conflict of interest, no funding or material supplying from 
any parties. 
 
   f. Access to data: 
 
      Access to final data will be allowed to the operator, the main and co-
supervisors of the study who are involved in assessment of the outcome. 
 
   g. Ancillary and post-trial care: 
 
       Patients will not be followed up after intervention as there is no harm 
from our material we used. 
 
   h. Dissemination policy: 
      Full protocol will be published online in www.clinicaltrials.gov to 
avoid repetition and to keep the integrity of the research work. Thesis will 
be discussed in front of judgment committee. 
   The study will be published to report the results of this clinical trial.