J Clin Periodontol 2013; 40: 1007–1015 doi: 10.1111/jcpe.

12158

Efficacy of a new mouth rinse Xavier Costa1, Estefan
ıa Laguna1,

David Herrera2, Jorge Serrano2,
Bettina Alonso2 and Mariano Sanz2

formulation based on 0.07% 1

Section of Graduate Periodontology –
Faculty of Odontology, University
Complutense, Madrid, Spain; 2Etiology and

cetylpyridinium chloride in the Therapy of Periodontal Diseases (ETEP)

Research Group – Faculty of Odontology,
University Complutense, Madrid, Spain

control of plaque and gingivitis: a

6-month randomized clinical trial
Costa X, Laguna E, Herrera D, Serrano J, Alonso B, Sanz M. Efficacy of a new
mouth rinse formulation based on 0.07% cetylpyridinium chloride in the control of
plaque and gingivitis: a 6-month randomized clinical trial. J Clin Periodontol 2013;
40: 1007–1015. doi: 10.1111/jcpe.12158.

Abstract
Aim: To assess the efficacy of a 0.07% cetylpyridinium chloride (CPC) mouth
rinse in the control of plaque and gingival inflammation during a 6-month
period.
Material and Methods: Adult subjects with moderate gingivitis were selected
[≥40% bleeding on marginal probing (BOMP)]. After retrieving microbiological
samples and evaluating the clinical parameters (plaque, BOMP and stain indexes),
a professional prophylaxis was performed and subjects were randomly assigned to
the test (CPC mouth rinse) or to the placebo group. Subjects were re-assessed
after 3 and 6 months.
Results: A total of 67 patients (35 test, 32 placebo) were included in the analysis.
At 6 months, intra-group significant plaque reductions were observed in the test
group (0.691, p < 0.001), but not in the placebo (0.181, p = 0.653). At 6 months,
the mean BOMP values were lower in the test group (p = 0.052). Changes
between baseline and 6 months were significantly higher in the test group both
for plaque (p = 0.002) and BOMP (p = 0.037) when compared with the placebo.
A microbiological impact was observed in the test group, especially for Prevotella
intermedia. Key words: antiseptics; cetylpyridinium
Conclusion: The evaluated 0.07% CPC-based mouth rinse, used three times per chloride; dental plaque; gingivitis; mouth rinse
day adjunctively to mechanical tooth cleaning, prevents plaque accumulation and
gingival inflammation, as compared to the placebo, for at least 6 months. Accepted for publication 7 August 2013

Conflict of interest and source of funding statement Efficient removal of dental plaque is
essential in the prevention and main-
The authors declare that they have no conflict of interests.
tenance of oral health as plaque
This study was supported by Dentaid (Barcelona, Spain) through a grant to the
accumulation is a critical factor in
ETEP Research Group. The study was designed, conducted and analysed by
researchers belonging to the ETEP Research Group.
the aetiology of caries, gingival
inflammation and periodontitis (Loe
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1007
1008 Costa et al.
et al. 1965, Theilade et al. 1966,
Timmerman & van der Weijden
2006). Mechanical methods, such as
tooth brushing and dental floss, are
the cornerstone in supragingival pla-
que control (Wilkins 1999), but lack
of compliance, poor motivation or
inadequate skills, as well as presence
of hard-to-reach areas may compro-
mise the maintenance of periodontal
health, and in these situations, the
adjunctive use of chemotherapeutic
agents, such as mouth rinses, may
enhance the plaque removal efficacy
and hence, assist in the maintenance
of periodontal health (Barnett 2003,
Paraskevas & van der Weijden
2006).
Cetylpyridinium chloride (CPC)
is a quaternary ammonium com-
pound sharing the antimicrobial
properties of cationic surfactants,
being bactericidal on Gram-positive
pathogens and against fungi and
yeasts (Pitten & Kramer 2001). Its
efficacy, however, can be compro-
mised by interactions with excipients
present in the formulation and is not
uncommon to find two CPC mouth-
washes containing the same concen-
tration of CPC and differing
significantly in their relative efficacy,
bioavailability, biofilm penetrability
and substantivity (Sheen et al. 2001,
Barnett 2003, Mankodi et al. 2005,
Haps et al. 2008). CPC has been
considered by the Food and Drug
Administration Plaque Subcommit-
tee as safe and effective for the treat-
ment of plaque-induced gingivitis
when formulated in mouth rinses in
concentrations ranging 0.045–0.10%
(Food and Drug Administration &
Department of Health and Human
Services 2003). Although the most
frequently used CPC concentration
is 0.05%, a newly formulated 0.07%
CPC mouth rinse has been recently
marketed and its use has shown
effectiveness to inhibit “de novo”
plaque formation in a short-term
3-day plaque accumulation model
(Versteeg et al. 2010). To confirm
these preliminary results, long-term
home-use studies are needed and
hence, this investigation hypothe-
sized that the adjunctive use of a
high-bioavailability 0.07% CPC rinse
would reduce plaque and gingivitis
levels, when compared to a placebo
rinse, over a 6-month period. Fur-
thermore, its use would not develop
a negative microbiological impact or
the occurrence of significant adverse
effects.
Material and Methods
Study population
Consecutive subjects (111 subjects,
Fig. 1) were screened at the Post-
Graduate Periodontal Clinic in the
University Complutense, Madrid,
and enrolled in this clinical trial
if they fulfilled the following criteria:
Inclusion criteria
• ≥18 years old.
• Systemically healthy.
• Presence of at least five evaluable
teeth in each quadrant.
• Moderate gingivitis (≥40% bleed-
ing on marginal probing, BOMP)
(Van der Weijden et al. 1994a).
• Absence of probing pocket depths
(PPD) ≥ 5 mm.
• No orthodontic banding or remov-
able prosthesis.
• Subjects willing to participate
and comply with the objectives
of the study.
Assessed for eligibility (n = 111)
Randomized (n = 74)
Allocated to TEST intervention (n = 37)
♦ Received allocated intervention (n = 35)
♦ Did not receive allocated intervention (n = 2):
Reasons: not attended baseline visit.
Lost to follow-up 3 & 6 months (n = 2):
Reasons: forget, lack of time.
Analysed (n = 35)
♦ Excluded from analysis (n = 2): not received
allocated intervention (not attended baseline
visit).
Fig. 1. Study flow diagram according to CONSORT 2010.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Exclusion criteria
• Dental students.
• Regular users of antiplaque
rinses.
• Antibiotics intake within the pre-
vious month.
• Pregnant women.
• Any adverse medical history or
long-term medication influencing
gingival conditions.
Study design
A parallel, randomized, 6-month,
placebo-controlled, double-blind clin-
ical trial was designed.
Screening visit
After completing a questionnaire
including the subject’s medical con-
ditions, medication and smoking
habits [a smoker was defined when
currently smoked ≥9 cigarettes per
day (Dietrich et al. 2004)], a careful
oral health evaluation was carried
out including full-mouth BOMP. If
subjects met the inclusion/exclusion
Excluded (n = 37)
♦ Not meeting inclusion criteria (n = 35)
♦ Declined to participate (n = 2)
♦ Other reasons (n = 0)
Allocated to CONTROL intervention (n = 37)
♦ Received allocated intervention (n = 32)
♦ Did not receive allocated intervention (n = 5):
Reasons: not attended baseline visit.
Lost to follow-up 3 & 6 months (n = 4):
Reasons: impossible to contact (2) family
problem, hypersensitivity.
Lost to follow-up 6 months (n = 2):
Reasons: not willing to continue.
Analysed (n = 32)
♦ Excluded from analysis (n = 5): not received
allocated intervention (not attended baseline
visit).

criteria, the investigator informed
them about the objectives and details
of the study and requested their
voluntary participation. Once they
accepted by signing the IRB
approved informed consent form,
they were entered into the study.
Baseline visit
At this visit the oral health of every
participant, identified by a unique
trial number, was examined. Clinical
parameters were recorded around all
teeth, except the third molars, in two
randomly chosen (coin toss) contra-
lateral quadrants (one upper and
one lower quadrant) (Bentley &
Disney 1995). Microbiological samples
were also collected. After this clinical
examination, a professional dental
prophylaxis and polishing were pro-
vided.
Subjects were then randomly
assigned to one of two groups,
test or placebo. Randomization
was performed using true random
numbers from a computer-generated
list:
• The experimental group used three
times daily a provided manual
toothbrush with a sodium fluoride
dentifrice (Binacaâ, GlaxoSmith-
Kline Consumer Healthcare, Tres
Cantos, Madrid, Spain) combined
with a high-bioavailability, non-
alcohol containing, 0.07% CPC
mouth rinse (Vitis Xtra Forte;
Dentaid, Barcelona, Spain).
• The control group used three
times daily the same toothbrush
and dentifrice, combined with a
placebo mouth rinse containing a
coloured saline-based solution,
with no alcohol.
All subjects were masked to their
group allocation and received their
assigned products kit containing: the
manual toothbrush (Vitis Suave;
Dentaid), toothpaste (Binacaâ), a
waxed dental tape (Vitis; Dentaid)
and nine 500-ml bottles of the
assigned mouth rinse (coded A or B)
with dose caps. They were instructed
to brush with the toothpaste for
2 min. and then rinse for 30 s with
15 ml of the mouth rinse three times
daily. No specific instructions were
given for flossing. Each subject
received standardized verbal and
written instructions on how to use
the assigned products and was
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Antiplaque efficacy of a CPC-based mouth rinse
prompted to fill in the provided
compliance forms, where they regis-
tered each day the times they have
used their allocated products during
the study.
Three-month visit
After 3 months of product use, an
oral examination was performed.
The subjects were asked for the
occurrence of any adverse effects
and the study coordinator collected
the compliance forms as well as the
empty and unused bottles. A new
subgingival microbiological sample
was then collected and the clinical
parameters were evaluated in the
same quadrants and by the same
examiner as at baseline. Subjects
were provided with a new kit of
assigned products and compliance
forms.
Six-month visit
After 6 months, a similar clinical
examination was performed, includ-
ing subgingival microbiological sam-
pling and the registration of clinical
variables in the same quadrants and
by the same examiner as at baseline.
The subjects were interviewed about
occurrence of adverse effects and the
study coordinator collected the
compliance forms and the empty
and unused bottles. All subjects
were provided with a professional
dental prophylaxis at the end of the
study.
Clinical parameters
Examinations were performed by
two calibrated examiners, blinded to
the treatment allocation and to data
from previous visits. Plaque index
(PI) was assessed using a disclosing
solution (Plac-Controlâ tablets;
Dentaid) with the Turesky (Turesky
et al. 1970) modification of the
Quigley and Hein index (Quigley &
Hein 1962) scored at six sites per
tooth. The gingival condition was
assessed using the BOMP index by
recording the presence or absence of
bleeding within 30 s of probing on a
scale 0–2 (Van der Weijden et al.
1994a,b, Lie et al. 1998). Staining of
teeth was scored using the Gr€ unde-
mann modification of the stain index
(GMSI) (Grundemann et al. 2000),
recorded at nine areas per tooth
(three mesial, three medial and three
distal). Stain was graded using the
1009
intensity stain index of Lobene
(1968).
Microbiological sampling
Four sites were selected, one per
quadrant, based on presence of
gingivitis during the screening visit.
The same sites were sampled at the
follow-up visits. These sites were
isolated with cotton rolls and dried
gently with sprayed air. Two consec-
utive sterile paper points (medium
size, Maillefer, Ballaigues, Switzer-
land) were inserted as deep as possi-
ble into the sulcus, and left in place
for 10 s. The paper points were
transferred to a vial containing
1.5 ml of reduced transport fluid,
and pooled with the other paper
points. The vial was sent to the labo-
ratory and processed within 24 h. At
the laboratory, the samples were
vortexed (30 s), serially diluted and
plated in different media: blood agar
medium (No. 2 of Oxoid; Oxoid
Ltd, Basingstoke, UK), with 5%
horse blood, and haemin (5 mg/l)
and menadione (1 mg/l); and a
selective medium for Aggregatibacter
actinomycetemcomitans (Alsina et al.
2001).
The blood agar plates were stud-
ied after 7 and 14 days of anaerobic
incubation (80% N2; 10% H2; 10%
CO2 at 37°C), and the selective
plates after 3–5 days of 37°C incuba-
tion in air with 5% CO2.
Plates were carefully examined for
the identification of A. actinomyce-
temcomitans, Porphyromonas gingiva-
lis, Prevotella intermedia/nigrescens,
Tannerella forsythia, Parvimonas mi-
cra, Capnocytophaga spp., Eikenella
corrodens, Eubacterium spp. and Fuso-
bacterium nucleatum, based on the
morphology of the colony and using
different standard biochemical tests
to confirm the initial identification
(RAPID ANA II). Other relevant col-
onies (those representing an impor-
tant proportion of the microbiota)
were also isolated for further charac-
terization. The total number of colo-
nies, as well as the number of each
bacterial species, was counted in a
representative plate (containing
between 30 and 300 colonies). Counts
of A. actinomycetemcomitans were
performed on the selective plates,
based on its typical colony morphol-
ogy, a catalase reaction and a set of
specific enzymes.
1010 Costa et al.
Data analysis
The a priori sample size calculation
of 60 subjects (30 per arm) was
based on an expected difference
between the experimental and pla-
cebo groups in the plaque index of
0.25, with a standard deviation (SD)
of 0.34 for an alpha of 0.05 and a
power of 80% (Paraskevas et al.
2005).
The demographic characteristics
of the groups (gender and smoking
habits) were assessed in contingency
tables and compared with the Fish-
er’s exact test. Age distribution was
compared with the t-test.
The subject was used as the
experimental unit and the primary
outcome variables (mean PI and
BOMP indexes) were generated at
each visit, first for patient, then for
treatment group. Means, standard
errors and 95% confidence intervals
(CI) were calculated at each visit as
well as the changes between baseline
and follow-up visits. After checking
for the normality of the distribution
in the outcome variables (skewness
and kurtosis) the analysis of variance
(ANOVA) was used for assessing the
inter-group differences at each visit,
using the treatment group as the fac-
tor and the gender, age, smoking
habit and examiners as the cofactors.
For assessing the intra-group com-
parisons (changes between baseline–
3 months and baseline–6 months),
the same model was repeated, but
adding the baseline value as cofac-
tor. As plaque levels were signifi-
cantly different at baseline, they were
included as cofactors in all models.
For intra-group comparisons, ANOVA
was used, with the multiple rank test
(MRT) as post-hoc test.
The following microbiological
variables were calculated at each
visit: total anaerobic counts, counts
of each pathogen, proportions of
flora (counts of the pathogen/total
counts) and presence/absence of each
pathogen. Total anaerobic counts
were log transformed and compared
by ANOVA; log-transformed counts
(given an arbitrary value of 1 to the
counts with a 0 value) and propor-
tions of flora were compared by
Kruskal–Wallis; presence or absence
of pathogens was compared by a
chi-square test.
Compliance was assessed from
the collected diaries and the
measurement of the liquid remaining
in the returned bottles. The follow-
ing relative variables were defined:
percentage of brushing and of rins-
ing in relation to the maximum
expected (three times per day); and
percentage of liquid used in relation
to the maximum expected. The
quantitative data were compared by
the unpaired t-test and the relative
data compared by the chi-square
analysis from 2 9 2 contingency
tables.
Presence of staining was assessed
in each patient in the upper and
lower anterior buccal sites. Percent-
ages were calculated for all sites, or
by area (gingival, proximal or inci-
sal). Comparisons between groups
were performed by mean of unpaired
t-test.
A level of p < 0.05 was prede-
fined as the level of statistical
significance. All tests were carried
out with Statgraphics software
(Statgraphic 5.1, Statpoint Technolo-
gies, Inc., Warrenton, VA, USA).
An intention to treat analysis was
performed.
Results
The study patient flow and reasons
for exclusion or dropout are detailed
in Fig. 1. From the 111 subjects who
were screened between January 2010
to February 2011, 74 fulfilled the cri-
teria and were randomized, including
37 in each study group: 35 received
the test intervention and 32 the pla-
cebo. Some patients attended the
screening visit and, as they fulfil the
inclusion criteria, were invited to
participate and initially accepted;
however, before attending the base-
line visit, they rejected to participate
on the study. The final sample distri-
bution by age, gender and smoking
habits is presented in Table 1, with
Table 1. Demographic data
Group Total
N 67
Age Mean  SD 25.5  6.0
Range 19–62
Gender Female 25
Male 42
Smoking status Non-smoker 50
Smoker ≥ 10 17
SD, standard deviation.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
no significant differences between
groups.
Clinical outcome variables
Mean PI (Table 2) showed signifi-
cantly higher levels at baseline in the
test group (p = 0.046). Significantly
higher plaque scores in the control
group were also observed at the
3-month (p = 0.001) and 6-month
(p = 0.002) visits.
The reductions in PI, between
baseline and 3 months, were signifi-
cant for both test and control
groups. The changes between base-
line and 6 months, however, were
only significant in the test group
(0.691, p < 0.001).
Mean BOMP (Table 3) values
were comparable between groups at
baseline (p = 0.391) and no signifi-
cant differences were observed at
3 months (p = 0.212). At 6 months,
the mean BOMP values were lower
in the test group with a tendency
towards significance (p = 0.052). The
changes in baseline–3 months also
showed a higher level of bleeding
score reduction in the test group,
although non-significant (p = 0.084).
The changes between baseline and
6 months were significantly different
between groups (p = 0.037), with a
bleeding score reduction of 0.410 in
the test group, as compared with a
0.236 in the control group.
Intra-group assessments revealed
significant changes in the test (p <
0.001) and the control (p = 0.003)
groups, and identified as significant
differences between baseline and
3- and 6-month visits.
Subgingival microbiological outcome
variables
Some samples could not be pro-
cessed due to technical problems
related to the anaerobic incubation.
Test Placebo p-value
35 32
24.6  3.4 26.4  7.6 t-test
19–32 20–62 p = 0.233
12 13 Fisher test
20 22 p = 1.000
21 29 Fisher test
11 6 p = 0.159
 Antiplaque efficacy of a CPC-based mouth rinse 1011

Table 2. Mean plaque index values, with standard error (SE) and 95% confidence intervals (95% CI), at each visit and in changes between
visits. Intra-group comparisons were performed with ANOVA and inter-group comparisons with ANCOVA, with baseline plaque levels as cofac-
tor. Multiple rank test (MRT) was used as post hoc test
Visit Control group Test group Inter-group

n Mean SE 95% CI n Mean SE 95% CI ANCOVA

Baseline 32 2.206 0.076 2.099 2.313 35 2.465 0.117 2.301 2.629 0.046
3 months 28 1.946 0.081 1.832 2.060 33 1.586 0.120 1.418 1.755 0.001
6 months 27 2.044 0.083 1.927 2.160 33 1.736 0.120 1.567 1.905 0.002
Base versus 3 m* 28 0.268 0.110 0.112 0.424 33 0.840 0.102 0.696 0.984 0.001
Base versus 6 m* 27 0.181 0.104 0.034 0.329 33 0.691 0.094 0.557 0.825 0.002
Intra-group ANOVA: p = 0.653 ANOVA: p < 0.001
MRT: baseline versus 3 m MRT: baseline versus 3 m and 6 m

*For changes, positive changes mean decrease in the index between baseline and 3-month or 6-month visits.

Table 3. Mean bleeding on marginal probing (BOMP) values, with standard error (SE) and 95% Confidence Intervals (95% CI), at each
visit and in changes between visits. Intra-group comparisons were performed with ANOVA and inter-group comparisons with ANCOVA, with
baseline plaque levels as cofactor. Multiple rank test (MRT) was used as post hoc test
Visit Control group Test group Inter-group

n Mean SE 95% CI n Mean SE 95% CI ANCOVA

Baseline 32 0.894 0.049 0.824 0.963 35 1.014 0.043 0.954 1.074 0.391
3 months 28 0.660 0.053 0.586 0.734 33 0.596 0.044 0.534 0.658 0.212
6 months 27 0.695 0.054 0.620 0.770 33 0.595 0.044 0.533 0.657 0.052
Base versus 3 m* 28 0.253 0.054 0.176 0.330 33 0.409 0.050 0.339 0.480 0.084
Base versus 6 m* 27 0.236 0.053 0.160 0.311 33 0.410 0.048 0.342 0.479 0.037
Intra-group ANOVA: p = 0.003 ANOVA: p < 0.001
MRT: baseline versus both 3 m and 6 m MRT: baseline versus both 3 m and 6 m

*For changes, positive changes mean decrease in the index between baseline and 3-month or 6-month visits.

In the test group, 34 samples were
processed at baseline, 33 after
3 months and 29 after 6 months.
The corresponding figures for the
control group were 32, 27 and 24
respectively.
Frequency of detection of target
bacterial species
No differences were detected
between the groups and minor
changes were observed for most tar-
get bacterial species, except P. inter-
media, which showed clear reductions
after 3 months and 6 months (p =
0.030) in the test group, which led to a
significant inter-group difference at
6 months (p = 0.033) (Table 4).
Bacterial counts
Total subgingival bacterial counts
demonstrated a significant reduction
after 3 (p = 0.047) and 6 (p = 0.003)
months in the test group, without
relevant changes in the placebo
group. These changes lead to a ten-
dency towards significant inter-group
differences at 6 months (p = 0.069).
No overgrowth of opportunistic spe-
cies was detected (Table 5).
The counts of the target bacterial
species were comparable at baseline,
and minor changes (less than 0.50
logs) were observed for most bacte-
rial species. P. gingivalis revealed
clear reductions both in the test
(p = 0.059) and placebo (p = 0.042)
groups, from baseline to 3 months.
Significant reductions in the counts
of P. intermedia were observed after
3 (p = 0.006) and 6 (p = 0.030)
months in the test group, with a ten-
dency towards significant inter-group
differences in the changes in base-
line–3 months (p = 0.050) and base-
line–6 months (p = 0.051).
Proportions of total microbiota
No inter-group differences were
detected at baseline. Significant
intra-group changes were observed
in the test group, baseline–6 months,
with a reduction in P. micra
(p = 0.029) and an increase in
F. nucleatum (p = 0.047) (Table 6).
For P. gingivalis, from baseline to
6 months, a clear reduction was
observed in the placebo group and
an increase in the test, leading to
significant inter-group differences
in the changes in baseline–6 months
(p = 0.026).
Patient-based variables
No adverse effects were observed,
however, a few complaints were
reported in both groups, such as
changes in taste perception (n = 1),
strange flavour but not disgusting
(n = 1) and itchy feeling when rins-
ing (n = 2) after 3 months, and
staining (n = 1) after 6 months in
the test group. Taste alterations
(n = 1), taste of blood and bad
breath (n = 1) and transparent
appearance and salty flavour (n = 1)
were reported in the control group
after 3 months. At the 6-month visit,
no comments were reported.
Staining
Results were available from 33 test
and 27 control patients. Significant

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1012 Costa et al.

Table 4. Microbiological outcomes: frequency of detection (Freq.) and number of positive samples [n(+)] of different bacterial species
Visit Group Variable Aa Pg Pi Tf Pm Cr Fn Capno Ec Eu

Baseline Control N 32 32 32 32 32 32 32 32 32 32
n(+) 1 17 26 3 14 4 28 6 7 0
Freq. (%) 3.1 53.1 81.3 9.4 43.8 12.5 87.5 18.8 21.9 0.0
Test N 34 34 34 34 34 34 34 34 34 34
n(+) 0 20 30 4 13 5 32 6 7 1
Freq. (%) 0.0 58.8 88.2* 11.8 38.2 14.7 94.1 17.6 20.6 2.9
3 months Control N 27 27 27 27 27 27 27 27 27 27
n(+) 1 10 23 1 13 5 25 3 7 0
Freq. (%) 3.7 37.0 85.2 3.7 48.1 18.5 92.6 11.1 25.9 0.0
Test N 33 33 33 33 33 33 33 33 33 33
n(+) 2 14 22 2 16 8 32 4 7 0
Freq. (%) 6.1 42.4 66.7 6.1 48.5 24.2 97.0 12.1 21.2 0.0
6 months Control N 24 24 24 24 24 24 24 24 24 24
n(+) 0 12 22 1 10 4 22 4 7 1
Freq. (%) 0.0 50.0 91.7† 4.2 41.7 16.7 91.7 16.7 29.2 4.2
Test N 29 29 29 29 29 29 29 29 29 29
n(+) 0 13 18 3 9 7 27 2 5 0
Freq. (%) 0.0 44.8 62.1*,† 10.3 31.0 24.1 93.1 6.9 17.2 0.0

*Intra-group difference: p = 0.033.

†
Inter-group difference: p = 0.030.
Aa, A. actinomycetemcomitans; Pg, P. gingivalis; Pi, P. intermedia; Tf, T. forsythia; Pm, P. micra; Cr, C. rectus; Fn, F. nucleatum; Capno,
Capnocytophaga spp.; Ec, E. corrodens; Eu, Eubacterium spp.

higher levels of staining were
observed in the test group after 3
(p = 0.007) and 6 months (p < 0.001)
(Table 7).
Compliance
Results were available from 31 test
and 26 control patients. Subjects
complied with three times daily
brushing with a mean of 73–76%.
No differences were detected in any
of the groups between the first or
second trimester or between the
groups. The corresponding outcome
for three times daily rinsing was
80–86%, with similar findings with
regard to intra-group or inter-group
differences. The evaluation of the
remaining liquid in the bottles
showed that in the first trimester, the
test group used less of the assigned
mouth rinse than the placebo group
subjects (71% versus 83%, respec-
tively, p = 0.037), but the reverse
was observed during the second tri-
mester (86% versus 80%, respec-
tively, p = 0.094) (Table 8).
Discussion
The results of this 6-month random-
ized clinical trial have shown a clear
beneficial effect of the adjunctive use
of a mouth rinse containing 0.07%
CPC when compared with a placebo,
in terms of plaque and gingival
inflammation reduction. These
results are in agreement with a previ-
ous study using a different formula-
tion of CPC with the same
concentration (Mankodi et al. 2005)
that reported reductions in plaque,
gingival inflammation and gingival
bleeding of 15.8%, 15.4% and
33.3%, respectively, when compared
with a placebo rinse. The present
study observed in the control group
an 8.2% reduction in PI, versus a
28% in the test group, the differ-
ence being 19.8%. In terms of
BOMP, the control group showed a
26.4% bleeding score reduction and
the test group 40.5%, with a differ-
ence of 14.1% between groups. This
is not as substantial as the 33.3%
reduction in bleeding scores reported
in the cited study (Mankodi et al.
2005).
Stookey et al. (2005) reported
that two mouth rinses containing
CPC concentrations of 0.075% and
0.1% provided statistically signifi-
cant plaque inhibitory and antipl-
aque benefits over 6 months of use,
but there were not statistically signif-
icant differences between them. In
another study (Allen et al. 1998)
using a lower concentration (0.05%)
of CPC in a mouth rinse also
reported statistically significantly less
supragingival plaque and gingivitis
than the control group, after the 3
and 6 months. At the 6-month
examination, the magnitude of these
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
differences met or exceeded 24% for
plaque and gingivitis. Previous
shorter term studies, comparing
0.05% CPC mouth rinses to placebo,
have shown lower differences
between groups (Nelson et al. 1991).
The variability in the results may be
explained by the differences in over-
all composition and the formulation
of each mouth rinse, which may
have affected the bioavailability of
the CPC.
In the present study, changes in
the subgingival microbiota were
associated with the use of the test
mouth rinse (although no significant
inter-group differences were
observed), showing significant reduc-
tions in total bacterial counts, in
counts of P. intermedia and P. gingi-
valis, in proportions of P. micra and
in the frequency of detection of P.
micra and P. intermedia. Concomi-
tantly, an increase in proportions of
F. nucleatum and P. gingivalis was
observed. These results contrast with
those of a similar study evaluating a
0.07% CPC mouth rinse (Albert-
Kiszely et al. 2007), in which no
impact in the subgingival microbiota
was observed, except a limited
decrease in total counts. Similarly, a
CPC containing mouth rinse showed
limited effect in bacterial vitality in
an in vitro biofilm model (Pan et al.
2010). Other in vitro studies, how-
ever, have shown a wide spectrum
 Antiplaque efficacy of a CPC-based mouth rinse 1013

antimicrobial activity for 0.07%

0.00
0.91
0.00
0.00
0.90
0.00
0.00
0.94
0.90
1.01

Aa, A. actinomycetemcomitans; Pg, P. gingivalis; Pi, P. intermedia; Tf, T. forsythia; Pm, P. micra Cr, C. rectus; Fn, F. nucleatum; Capno, Capnocytophaga spp.; Ec, E. corrodens; Eu, Eubacte-
SD
CPC formulations (Witt et al. 2005)

Eu
although not based on biofilm mod-

0.00
0.16
0.00
0.00
0.18
0.00
0.00
0.17
0.18
0.19
Mean
els. In vivo salivary counts, models
have also confirmed the antimicro-

1.11
1.27
1.43
1.39
1.33
1.20
1.74
1.62
1.72
1.73
bial efficacy of 0.05% CPC formula-

SD
tions (Santos et al. 2004, Boyd et al.
Ec 2008). This high heterogeneity in the

0.57
0.63
0.80
0.67
0.81
0.52
0.33
0.03
0.16
0.02
Mean
antimicrobial efficacy observed in
the studies with CPC-based products
1.25
1.52
0.98
1.19
1.38
0.91
1.21
1.96
1.42
1.67
highlights the importance of an ade-
SD
Capno

quate formulation and CPC bio-

availability (Gunsolley 2006, Haps
Mean

0.57
0.68
0.32
0.41
0.60
0.24
0.36
0.15
0.01
0.26
et al. 2008).
Few adverse effects were
1.38
1.23
1.15
1.07
1.38
1.16
1.70
1.30
2.10
1.75
SD

reported by the participating sub-

jects after using the 0.07% CPC
Fn

3.05
3.48
3.18
3.32
3.50
3.05
0.20
0.05
0.59
0.27
Mean

mouth rinse formulation. Only

three patients complained of
changes in taste, itchy feeling or
1.24
1.22
1.28
1.55
1.24
1.35
1.54
2.08
1.56
2.13
SD

tooth staining. These results are in

agreement with those from a previ-
Cr

0.46
0.49
0.58
0.85
0.52
0.71
0.15
0.35
0.03
0.14
Mean

ous study (Versteeg et al. 2010)

reporting fewer side effects with the
use of a 0.07% CPC mouth rinse
1.79
1.90
1.90
1.91
1.93
1.61
2.73
2.25
2.33
2.51
SD

when compared with a positive

Table 5. Microbiological outcomes: log of colony-forming units, expressed as mean and standard deviation (SD)

control (a hexetidine mouth rinse).

Pm

1.50
1.42
1.72
1.75
1.50
1.03
0.19
0.30
0.19
0.54
Mean

Adverse effects are rarely reported

in studies evaluating CPC-based
mouth rinses, as shown in a sys-
1.17
1.34
0.71
0.95
0.84
1.06
1.26
1.75
1.10
1.93
SD

tematic review (Haps et al. 2008)

where only one of five trials
Tf

0.37
0.48
0.14
0.23
0.17
0.35
0.14
0.27
0.03
0.22
Mean

reported side effects, with staining

being one of the most relevant
adverse effects. In this study, stain-
1.61
1.59
1.55
1.88
1.37
1.90
2.06
1.99
1.80
2.43
SD

ing of teeth was scored according

to the GMSI, revealing significant
Pi

2.89
3.48
2.93
2.37
2.97
2.25
0.03
1.03
0.13
1.05
Mean

higher levels of staining after 3 and

6 months in both groups and sig-
nificantly more in the test group,
2.22
2.30
2.01
1.71
1.94
2.10
2.32
2.88
3.00
2.08
SD

although only one patient com-

Pg

plained (after 6 months). When a

Mean

2.18
2.54
1.46
1.33
1.77
1.80
0.96
1.00
0.41
0.42

less specific evaluation method was

used (questionnaire), no differences
in staining between groups were
0.51
0.00
0.45
0.57
0.00
0.00
0.73
0.58
0.59
0.00
SD

observed when comparing a 0.07%

Aa

CPC mouth rinse and a placebo

0.09
0.00
0.09
0.14
0.00
0.00
0.02
0.15
0.12
0.00
Mean

(Versteeg et al. 2010). The lack of

adverse effects may have favoured
compliance, since patients in both
0.73
0.78
0.68
0.75
0.81
0.68
0.85
0.83
0.92
0.82
SD

groups demonstrated a high level of

Total

compliance for rinsing at 6 months,

Mean

5.14
5.48
5.02
5.10
5.08
4.88
0.14
0.30
0.05
0.50

86% in the test group and 80% in

*Number of samples processed.

the control group, which is similar

to the 89% reported in another
Control (32*)

Control (27)

Control (24)

Control (27)

Control (24)

6-month study (Blenman et al.

Group

Test (34*)

Test (33)

Test (29)

Test (32)

Test (28)

2005). From a microbiological per-

spective, no adverse effects were
detected, with the lack of growth of
opportunistic species.
Baseline –

Baseline –
3 months

6 months

rium spp.
Baseline

In spite of some identified limita-

3m

6m

tions of this clinical trial, such as the

Visit

limited sample size and the signifi-

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1014 Costa et al.

Table 6. Microbiological outcomes: proportions of bacterial species, expressed as mean and standard deviation (SD)
Group Visit Aa Pg Pi Tf Pm

Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%)

Control Baseline 0.50 2.83 8.70 16.69 3.85 4.75 0.27 1.02 1.91 3.54
3 months 0.00 0.01 4.04 8.51 6.12 11.30 0.09 0.48 2.64 4.31
6 months 0.00 0.00 2.68 8.14 4.44 10.15 0.04 0.20 6.85 25.62
Test Baseline 0.00 0.00 4.61 8.35 5.33 8.53 0.28 1.05 3.59 8.21
3 months 0.10 0.52 2.58 7.95 3.45 7.43 0.09 0.39 4.00 10.98
6 months 0.00 0.00 9.00 18.22 4.83 9.55 0.35 1.14 0.65 2.03
Group Visit Cr Fn Capno Ec Eu

Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%) Mean (%) SD (%)

Control Baseline 0.50 1.79 3.01 3.57 0.11 0.32 0.14 0.35 0.00 0.00
3 months 0.42 1.64 3.30 3.99 0.06 0.18 0.69 2.31 0.00 0.00
6 months 0.26 0.64 8.34 13.52 0.28 0.68 0.40 0.91 0.94 4.59
Test Baseline 1.51 8.53 2.08 1.73 0.25 0.79 0.17 0.43 1.49 8.70
3 months 1.03 4.00 4.15 6.25 0.27 1.13 0.44 1.32 0.00 0.00
6 months 0.45 1.16 3.68 4.23 0.22 0.99 0.32 1.31 0.00 0.00

Aa, A. actinomycetemcomitans; Pg, P. gingivalis; Pi, P. intermedia; Tf, T. forsythia; Pm, P. micra; Cr, C. rectus; Fn, F. nucleatum; Capno,
Capnocytophaga spp.; Ec, E. corrodens; Eu, Eubacterium spp.

Table 7. Patient-based variables: staining (test patients, n = 33; control patients, n = 27)
Visit Group Gingival Proximal Incisal Upper jaw Lower jaw All sites

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

3 months Test 0.118 0.208 0.152 0.246 0.006 0.020 0.099 0.165 0.115 0.202 0.105 0.166
Control 0.013 0.044 0.017 0.042 0.010 0.032 0.021 0.053 0.008 0.025 0.015 0.028
Inter-group p-value 0.013 0.007 0.539 0.021 0.008 0.007
6 months Test 0.124 0.199 0.203 0.262 0.011 0.032 0.116 0.178 0.155 0.211 0.140 0.166
Control 0.016 0.041 0.015 0.032 0.000 0.000 0.011 0.030 0.012 0.033 0.011 0.024
Inter-group p-value 0.008 0.000 0.080 0.004 0.001 0.000

Table 8. Patient-based variables: compliance (test patients, n = 31; control patients, n = 26)
Mean (%) SD (%) n > 70% Freq > 70 (%) Inter-group p-value

Baseline–3 months % brushing Control 73 19 14 53.8 0.858

Test 74 17 18 58.1
% rinsing Control 81 14 20 76.9 0.284
Test 85 15 25 80.6
% liquid Control 83 28 16 61.5 0.037
Test 71 16 17 54.8
3–6 months % brushing Control 76 16 16 61.5 0.937
Test 76 19 20 66.7
% rinsing Control 80 16 18 69.2 0.094
Test 86 14 27 90.0
% liquid Control 83 30 16 61.5 0.627
Test 80 26 19 61.3

SD, standard deviation; Freq, frequency.

cant differences in plaque levels at
baseline, it can be concluded that
this high-bioavailability 0.07%
CPC-based mouth rinse significantly
prevented plaque accumulation and
reduced gingival inflammation, when
compared with a placebo mouth
rinse, for at least 6 months, when
used three times per day adjunctively study design and their suggestions
to mechanical tooth cleaning. during manuscript preparation.
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Address:
David Herrera
Facultad de Odontolog
ıa – Ciudad
Universitaria
Plaza Ram
on y Cajal, s/n. 28040 Madrid
Spain
E-mail: davidher@ucm.es
mation over 6 months, when com-
pared to a placebo.
Practical implications: This 6-month
randomized clinical trial has
demonstrated the efficacy of this
CPC formulation in the prevention
of gingivitis, when used three times
per day adjunctively to mechanical
tooth cleaning.