International Dental Journal 2013; 63 (Suppl.

2): 39–47
ORIGINAL ARTICLE
doi: 10.1111/idj.12072

A randomised clinical study to evaluate the effect

of brushing duration on fluoride levels in dental
biofilm fluid and saliva in children aged 4–5 years
Evelyn E. Newby1, Esperanza A. Martinez-Mier2, Domenick T. Zero2, Sue A. Kelly2,
Nancy Fleming3, Mairead North1 and Mary Lynn Bosma1
1
GlaxoSmithKline Consumer Healthcare, Weybridge, UK; 2Indiana University School of Dentistry, Indianapolis, IN, USA; 3GlaxoSmithKline
Consumer Healthcare, Parsippany, NJ, USA.

Objectives: To compare the effect of 40 seconds versus 2 minutes brushing on saliva and dental biofilm fluid fluoride in
children ages 4–5 years over 1 hour. Design: This was a single-blind, cross-over, randomised, two-period clinical study
in healthy children. Three days before the start of each treatment subjects received a thorough brushing and then
refrained from all oral hygiene procedures. At treatment visits, after collecting baseline biofilm and saliva samples, staff
brushed the occlusal surfaces of the subject’s posterior teeth with a pea-sized amount (0.25 g) of NaF/silica toothpaste
for the randomised time. Samples were taken at 5 minutes, 15 minutes, 30 minutes and 60 minutes after brushing and
analysed for fluoride using a microanalytical methodology. There was a minimum 4-day washout period between treat-
ments. Results: Log changes from baseline biofilm fluid and saliva fluoride were statistically significant (P < 0.05) for
both brushing times at all post-brushing time-points [except 60 minutes saliva where P = 0.06 (t-test)]. Statistically sig-
nificantly greater ln-AUC (area under the curve) was found for biofilm fluid and salivary fluoride after brushing for
2 minutes compared with brushing for 40 seconds over the 1-hour test period. There was a statistically significantly
higher concentration of fluoride in the log change from baseline saliva levels after 5, 15, 30 and 60 minutes for the
2-minute brushing time compared with 40 seconds brushing time. There was no statistically significant difference in con-
centration of log change from baseline fluoride levels in biofilm fluid at each individual time-point (5, 15, 30 and
60 minutes) for the 2-minute brushing time compared with the 40-second brushing time, but significant differences were
observed for 15, 30 and 60 minutes in favour of 2-minute brushing time when log biofilm fluid value was analysed. Con-
clusion: The findings provide further evidence for the benefits of increased duration of brushing with respect to fluoride
delivery.

Key words: Biofilm-fluid, fluoride, retention, saliva, toothpaste, children, brushing time

is the key solvent in which minerals are exchanged at

INTRODUCTION
Dental caries occurs as a result of dissolution of
enamel, caused by acids produced by the dental bio-
film. Topical fluoride delivered by brushing with
toothpaste is accepted as effective in the prevention
of caries1, mainly because it decreases the rate of
enamel demineralisation and enhances the rate of
enamel remineralisation2,3. Numerous in vitro studies
have shown that the rate of caries progression and
remission is directly affected by the fluoride content
of the liquid phase of the biofilm overlying the
lesion4–6.
In practice, a tooth surface is only vulnerable to
caries if it is covered by a biofilm2. Thus biofilm fluid
© 2013 FDI World Dental Federation
the tooth surface. The level of fluoride in biofilm,
more specifically in biofilm fluid, appears to be
directly related to its anti-caries effect7.
Fluoride in the biofilm fluid exists in equilibrium
with fluoride in the saliva8, which in turn exists in
equilibrium with fluoride bound to the oral soft tis-
sues9, which are believed to be the principal fluoride
reservoir after brushing. As fluoride levels in saliva
and biofilm fluid are mutually interdependent, they
are both important in the prevention of caries.
These complex exchange processes and nature of
saliva flow over oral surfaces10 mean that oral deliv-
ery and clearance of fluoride is extremely difficult to
model11, so clinical studies are critical to understand
39
Newby et al.
fully the impact of brushing duration and quantity of
dentifrice on the process.
There have been several studies on the kinetics of
fluoride in biofilm fluid after topical application. The
pioneering work of Tatevossian12 reported increased
fluoride levels in pooled biofilm fluid for 2 hours,
after rinsing with 20 mmol/l NaF rinse. Vogel et al.13
found a strong linear correlation between salivary and
biofilm fluid fluoride after administration of a 0.2%
NaF rinse. Direct comparisons have also been made
of fluoride in biofilm fluid after NaF and monofluoro-
phosphate (MFP) rinses14,15.
Starting from the work of Aasenden et al.16, there
have been many studies of the kinetics of fluoride in
saliva after topical application. The studies of Duck-
worth8,9,17–19 showed that fluoride clearance is bipha-
sic: an initial rapid loss of fluoride in the first
approximately 15 minutes followed by a much slower
rate of loss for the ensuing hours. The rapid clearance
phase has been attributed to loss of fluoride present in
free solution in the oral fluids; the slow clearance
phase has been attributed to loss of fluoride bound to
oral surfaces, which must first dissociate into free
solution before clearance.
Children are particularly vulnerable to caries. Teeth
emerging during childhood will create certain ‘stagna-
tion sites’, especially occlusal surfaces and interproxi-
mal areas, where cariogenic biofilm will accumulate
even with regular toothbrushing habits20,21. If the
overall remineralisation–demineralisation balance at
these sites tends to favour demineralisation, it is likely
that dental caries will progress to the cavitation stage
within a few years (i.e. during childhood)21. Addi-
tional caries risk factors for children may include low
dexterity/enthusiasm for brushing compared with
adults; a diet higher in frequency and quantity of fer-
mentable carbohydrates also predisposed to car-
ies22,23.
Recent studies have shown the importance in adults
of brushing duration, and quantity of dentifrice used,
on in-situ fluoride uptake and consequent reminerali-
sation by early enamel lesions24,25. This suggests that
caries protection from a dentifrice is dependent on
both duration of brushing and quantity of paste used.
To our knowledge, only one published study exists on
saliva fluoride levels as a function of quantity of den-
tifrice used in children26; this concluded that quantity
of dentifrice is very important.
This currently reported study used methodology uti-
lised in previously reported studies evaluating fluoride
in biofilm fluid in an adult population24,25 with a
number of adaptations in order to conduct the study
in a younger population. Based on a pilot study to
evaluate feasibility of conducting a study of this nat-
ure in a population of children aged 4–5 years we
determined that the ability of children of this age to
40
understand the concept of ‘spitting’ or to have the
physical ability to provide saliva samples was varied.
Consequently, the saliva collection method for this
study has been modified in order to collect saliva sam-
ples from all subjects regardless of their ability to spit
by using a modified mucus trap method instead of
requesting that the subjects expectorate into a con-
tainer.
The quantity of paste (0.25 g) was chosen from the
near-universal dental professional body recommenda-
tion that children aged 6 years and under should
brush with a ‘pea-sized’ quantity of paste. The only
guidance (of which we are aware) as to the actual
weight of a pea-size amount of paste is a European
Union Committee recommendation which states that
a ‘pea-sized’ amount should be taken as 0.25 g27.
DenBesten and Ko26 reported that following brushing
with 0.25 g of toothpaste, fluoride levels in saliva had
returned to baseline levels within 45 minutes.
The purpose of the present study was to observe
the fluoride clearance curve in children following two
different brushing times was measured over a 1-hour
period.
The brushing times used have been chosen in the
light of existing data on how long children brush
versus how long professionals recommend. A recent
study of children’s brushing time found that the aver-
age time spent was 39 seconds28, which is in agree-
ment with a previous study indicating approximately
40 seconds29. A general consensus among oral health-
care professionals is that individuals should spend at
least 120 seconds brushing their teeth with an effec-
tive technique at least twice a day.
MATERIALS AND METHODS
Study design
This was a single-blind (fluoride analyst), cross-over,
randomised, two-period study in healthy children aged
4–5 years (inclusive) to evaluate the effect of two dif-
ferent brushing times (40 seconds and 2 minutes) on
biofilm fluid fluoride and salivary fluoride levels over
a 1-hour period after use of an experimental sodium
fluoride/silica children’s toothpaste containing
1150 ppm fluoride. The study protocol and consent
form was reviewed and approved by the Indiana Uni-
versity Institutional Review Board and the study con-
formed to the guidelines laid out in the Declaration of
Helsinki.
Parental/legal guardian informed consent was col-
lected before any subject’s participation in this study –
because of the age of the children no child assent was
collected. A total of 157 healthy subjects between the
ages of 4 and 5 years were screened from 21 October
2011 to 28 February 2012, with 49 subjects randomised
© 2013 FDI World Dental Federation

to study treatment. The study completed in March
2012. The flow of the subjects through the study is
shown in Figure 1.
Subjects were enrolled into the study if they:
• Had a sufficient number of primary teeth that were
fully erupted and not noticeably loose (at least one
tooth per quadrant) to obtain an adequate biofilm
sample
• Produced at least 5 mg of biofilm at the required
screening visits
• Had adequate salivary flow (0.2 ml/minute using
the mucus trap method) at the required screening
visits
• Subjects were excluded from the study if they were:
Assessed for eligibility (n = 157)
Enrolment
Randomised (49)
Sequence 1: 40 seconds
then 120 seconds
Allocated to intervenƟon (n = 24)
Received allocated intervenƟon
(n = 24)
Did not receive allocated
intervenƟon (n = 0)
Lost to follow-up (n = 1)
Withdrawal of consent (n = 2)
Other (n = 1)
Analysed ITT (n = 25)
Subjects excluded from ITT analysis
(n = 0)
Missing data led to fewer than 25
subjects being analysed for certain
endpoints and Ɵmepoints
Figure 1. Trial profile.
© 2013 FDI World Dental Federation
Effect of brushing time on fluoride level
• Undergoing therapy with products or drugs that in
the opinion of the investigator or dental practi-
tioner may influence salivary flow
• Taking fluoride supplements or other fluoride prod-
ucts for health reasons except that naturally occur-
ring in usual diet
• Had been placed under the control or protection of
an agency, organisation, institution or entity by the
courts, the government or a government body, act-
ing in accordance with powers conferred on them
by law or regulation. This included a child cared
for by foster parents or living in a care home or
institution. This does not include a child who is
adopted or has an appointed legal guardian.
Excluded (n = 108)
Not meeƟng inclusion criteria
(n = 105)
Protocol violaƟon (n = 2)
Withdrawal of consent (n = 1)
Sequence 2: 120 seconds
then 40 seconds
Allocated to intervenƟon (n = 25)
Received allocated intervenƟon
(n = 25)
AllocaƟon Did not receive allocated
intervenƟon (n = 0)
Lost to follow-up (n = 1)
Withdrawal of consent (n = 1)
Other (n = 1)
Follow-Up
Analysis Analysed ITT (n = 23)
Subjects excluded from ITT analysis
(n = 0)
Missing data led to fewer than 23
subjects being analysed for certain
endpoints and Ɵmepoints
41
Newby et al.
Oral soft tissue (OST) and oral hard tissue (OHT)
examinations were performed at the initial screening
visit. Subjects then received a thorough brushing and
flossing of all teeth using a marketed children’s tooth-
paste (Aquafreshâ Kids toothpaste, GlaxoSmithKline
Consumer Healthcare, L.P., Pittsburgh, PA, USA) and
toothbrush (Aquafreshâ Kids toothbrush) to remove
all biofilm. Eligible subjects were instructed to refrain
from all oral hygiene products and procedures for the
next 3 days to allow sufficient growth of biofilm at
their next visit.
The second screening visit was to assess whether
subjects were likely to form sufficient amounts of bio-
film during treatment visits, had sufficient salivary
flow and could willingly sit for the collection pro-
cesses, an OST examination was also performed at
this visit by one of two trained dentists. At the end of
the second screening visit subjects had their teeth
flossed and thoroughly brushed with the marketed
children’s toothpaste by one of three trained dental
hygienists. For those that qualified to participate, the
same toothpaste was provided to use at home for the
duration of the study except during the 3-day no oral
hygiene periods before each treatment visit.
Approximately 1 mg of biofilm was required for
each sample in order to perform the analytical mea-
surement (five samples were required in total at each
visit: baseline, 5 minutes, 15 minutes, 30 minutes and
60 minutes after brushing). Therefore, at each treat-
ment visit the subject was required to have approxi-
mately 5 mg of biofilm (following the 3-day brushing
abstinence). Repeat screening and treatment visits
were permitted for subjects who were unable to pro-
duce the required amount of biofilm or who were
unable to attend both the brushing and subsequent
biofilm collection visit within the 3-day time limit.
Subjects attended the study site 3 days before each
treatment visit, where they received an OST assess-
ment followed by having their teeth flossed and thor-
oughly brushed with the washout toothpaste by a
trained dental hygienist. Subjects then abstained from
oral hygiene until the treatment visit 3 days later.
At the treatment visits subjects received a pretreat-
ment OST examination, a saliva sample was then col-
lected followed by collection of an interproximal and
buccal biofilm sample from the posterior teeth by a
trained dental hygienist. The occlusal surfaces of the
teeth were brushed by a trained dental hygienist with
0.25 g  0.03 g (a pea-sized amount) of the test den-
tifrice (an experimental sodium fluoride/silica chil-
dren’s toothpaste containing 1150 ppm fluoride) for
the randomized time (40 seconds or 120 seconds).
Subjects then expectorated the toothpaste slurry and
rinsed with 10 ml of deionised water for 10 seconds
and expectorated. Only the occlusal surfaces were
brushed to avoid disturbing the biofilm. Saliva sam-
42
ples were collected 5  1 minutes, 15  5 minutes,
30  5 minutes and 60  5 minutes after brushing.
Approximately 1 mg of interproximal/buccal biofilm
was collected immediately after each saliva sample.
Thorough brushing, treatment brushing, plaque and
saliva collection was conducted throughout the study
by one of three trained hygienists for each subject.
Subjects were required to remain at the site for the
entire treatment phase.
At the end of each treatment period the hygienist or
dentist thoroughly brushed the subject’s teeth using
the commercial children’s toothpaste and study tooth-
brush. Subjects received a post-treatment OST exami-
nation. There was a minimum of 4 days before the
next treatment period.
Randomisation procedure
Subjects were assigned randomisation numbers by a dis-
penser at the study site using a randomisation schedule
list generated by the study sponsor. Randomisation
numbers were assigned sequentially in chronological
order as each subject was deemed to be fully eligible.
Subjects received the corresponding two study treat-
ments in the sequence order specified by a randomisa-
tion schedule using a Williams Latin Square design.
Saliva collection procedure (mucus trap method)
A mucus trap is a hand-powered suction device with
an attached collection vial for collecting mucus or
saliva, and a suction catheter that is often used with
tracheotomy tubes. In this study, the suction source
was the saliva ejector and a saliva ejector tip was
inserted into the end of the catheter tube. The child
was instructed to swallow all saliva at the beginning
of the 5-minute collection and was then instructed to
open their mouth while the saliva ejector tip was
moved around in the child’s mouth to collect the sal-
iva. The child was then instructed to close their mouth
and the procedure was repeated periodically (every
10–20 seconds) for the 5-minute collection period.
Biofilm collection procedure
Immediately before dental biofilm collection, the sub-
ject was instructed to swallow all remaining saliva
and then keep their mouth open. Interproximal and
buccal biofilm samples were collected by one of the
trained dental hygienists. Approximately 1 mg of den-
tal biofilm was collected from the interproximal and
buccal surfaces of the posterior teeth of all four quad-
rants. Biofilm samples were collected using a standar-
dised approach. Using a stainless steel periodontal
scaler (S. McCall 17/18 or IU 17/18, Hu-Friedy Mfg.
Co, Inc., Chicago, IL, USA), pooled biofilm samples
© 2013 FDI World Dental Federation

were collected from each interproximal area (buccal
aspect) and buccal area starting from the upper right
quadrant to the upper left, lower left and ending in
the lower right quadrants. The pooled biofilm sample
was then transferred to a preweighed plastic strip.
Biofilm-fluid fluoride analysis
The microanalytical method of Vogel et al.13 was
used to analyse biofilm fluid samples for fluoride con-
tent:
• Samples were placed, under mineral oil, on the sur-
face of a specially constructed inverted F electrode.
The mineral oil was used to prevent evaporation
• Total ionic strength-adjusting solution (TISAB III)
was added to the samples in a ratio of 9:1
• The tip of a microreference electrode was placed in
contact with the sample to complete the circuit.
Triplicate analyses was performed on each pooled
biofilm fluid sample. Biofilm fluid fluoride was
expressed as lg F/g, which was calculated by compar-
ison with a standard fluoride curve constructed the
same day of the analysis.
Saliva sample analysis
The concentration of fluoride was measured in all sal-
iva samples. Samples were centrifuged for 10 minutes
at 10,000 rpm (4,000 g gravity) at 7°C and the
supernatant was stored at 4°C for immediate analysis
or at 20°C for later fluoride analysis. Analysis of
saliva was conducted using a modification of the hex-
amethyl-disiloxane (HMDS) microdiffusion method of
Taves30 as modified by Martinez–Mier, et al.31. One
millilitre of centrifuged saliva sample was pipetted into
plastic Petri dishes (Falcon 15-cm plastic Petri dishes),
adding enough deionised water to bring final volume
in each Petri dish to 3.0 ml. A 0.05 M sodium hydrox-
ide analytical reagent (NaOH) 50 ll trap solution was
placed in five drops on the Petri dish lid and after the
addition of 1 ml of sulphuric acid (H2SO4) saturated
with HMDS through a small hole in the lid of the Petri
dish; each dish was immediately tightly sealed with
petroleum jelly. During overnight diffusion, fluoride
was released by acid hydrolysis and trapped in the
NaOH. The trap was recovered and buffered to pH
5.2 with 25 ll acetic acid (CH3COOH). The recovered
solution was adjusted to a final volume of 100 ml with
total ionic strength buffer (TISAB II). Fluoride was
measured using a fluoride combination electrode
(Orion Specific Ion Combination Fluoride Electrode,
96-909-00, Precision Instruments, Sarasota, FL, USA)
and pH/ion meter (Orion EA940). The fluoride content
(lg F) of the samples was calculated from a standard
curve constructed from fluoride standards and micro-
diffused at the same time as the samples.
© 2013 FDI World Dental Federation
Effect of brushing time on fluoride level
The amount of total fluoride in the samples was
calculated based on the amount of fluoride divided by
the volume of the sample and expressed as lg F/ml of
sample.
The amount of fluoride delivered by the dentifrice
to the saliva over the 1-hour post brushing period (i.e.
the area under the salivary F clearance curve) was cal-
culated via trapezoidal rule.
Criteria for evaluation of efficacy
For the primary objective, the criterion of success was
whether there was a statistically significant increase in
the biofilm fluid area under the curve (AUC) between
brushing for 120 seconds compared with 40 seconds.
Safety
The safety profile of the experimental sodium fluoride
children’s toothpaste was assessed with respect to the
incidence of treatment–emergent adverse events (AEs)
and OST abnormalities.
Calculation of sample size
The sample size was calculated for the primary effi-
cacy parameter, AUC, for fluoride concentration in
biofilm fluid in the 1 hour following a single use.
With 50 completed subjects, it was calculated that
there would be an estimated 80% power at the 0.05
level of significance, using two-sided testing, to detect
a mean treatment difference on the natural logarithm
scale of 0.285 for AUC (with a standard deviation of
the difference estimated to be 0.705). This translated
to a standardized effect size of 0.404. The estimate of
standard deviation was based on a two similar, previ-
ous studies conducted on adults over a 4-hour period
using a larger amount of toothpaste24,32.
Statistical methods
The primary efficacy variable was the biofilm fluid
AUC, the area under the biofilm fluid fluoride clear-
ance curve, measured using the trapezoidal method.
Subjects without fluoride data for at least three time-
points after brushing, including the 1-hour time-point,
did not have a sufficient number of data points for an
accurate estimate of AUC to be calculated. In this case
the AUC value was set to missing. Where the baseline
value was missing, it was set as zero for calculation of
AUC. Where the 1-hour value was missing, the AUC
was also set to missing as it is not possible to calcu-
late AUC accurately without this information. The
efficacy analysis required natural logarithm transfor-
mation of AUC values before analysis because of fail-
ure of the assumptions of normality of the model
43
Newby et al.

residuals. Statistical analysis for natural logarithm-
transformed AUC was performed with statistical
analysis system (SAS) software (SAS Cary, NC, USA)
using a linear model. An analysis of covariance (AN-
COVA) model included subject (as a random effect),
treatment and study period as factor and two baseline
terms as covariates; (1) the subject-level baseline bio-
film fluid concentration calculated as the mean baseline
across both periods within a subject, and (2) the per-
iod-level baseline biofilm fluid concentration minus the
subject-level baseline biofilm fluid concentration. Mean
differences between the duration of brushing are pre-
sented with 95% confidence intervals. All tests were
two-sided and performed at the 5% significance level.
The secondary efficacy variables were:
• Biofilm fluid fluoride concentration at each post-
brushing time-point (5, 15, 30 and 60 minutes)
• Saliva fluoride AUC
• Change in fluoride concentration in biofilm fluid
from baseline at each time-point (5, 15, 30 and
60 minutes)
• Change in fluoride concentration in saliva from
baseline at each time-point (5, 15, 30 and 60 min-
utes).
The variables 1 and 2 were analysed in the same
way as primary efficacy but with biofilm fluid and sal-
iva fluid at baseline as the covariates for the biofilm
and saliva endpoints, respectively. Natural logarithm
transformations were required.
The variables 3 and 4 were analysed in the same
way as primary efficacy but with biofilm fluid and sal-
iva fluid at baseline as the covariates for the biofilm
and saliva endpoints, respectively. Within-treatment
change from baseline were assessed comparing the
adjusted mean value observed from the ANCOVA
model versus zero using a t-test. Natural logarithm
transformations were required.
During the blinded data review, it was noted that
there were some instances where the baseline levels of
fluoride were not obtained owing to an inability to
extract fluid from the total biofilm. This sensitivity
analysis analysed the biofilm fluid fluoride in AUC
without baseline levels as a covariate. The model
terms included subjects (as a random effect), treat-
ment and study periods in the analysis of variance
(ANOVA) model.
As the primary endpoint was predefined, there has
been no adjustment for multiplicity.
assessments), 49 subjects were randomised. A total of
42 subjects completed the study. A total of 48 sub-
jects were included in an intent to treat (ITT) popula-
tion and 47 subjects were included in the per protocol
(PP) population. Efficacy analyses were based on the
ITT population; PP population analyses were not per-
formed. Seven of the randomised subjects did not
complete the study, three were because of withdrawal
of consent, two could not grow sufficient biofilm and
two were lost to follow up.
Demographics
The baseline demographics for the study population
are summarised in Table 1.
Efficacy results
The mean (SE) fluoride concentrations in biofilm fluid
at each time-point are shown in Figure 2. The values
observed for 40-second and 2-minute brushing times,
respectively, in lg F/g were: baseline 0.182 (0.016),
0.212 (0.025); 5 minutes 0.616 (0.206), 0.635
(0.116); 15 minutes 0.321 (0.037), 0.521 (0.108);
30 minutes 0.256 (0.023), 0.395 (0.058) and 60 min-
utes 0.239 (0.030), 0.327 (0.046).
The mean (SE) fluoride concentrations in saliva at
each time-point are shown in Figure 3. The values
observed for 40-second and 2-minute brushing times,
respectively, in lg F/g were: baseline 0.032 (0.004),
0.037 (0.006); 5 minutes 0.510 (0.050), 0.797
Table 1 Demographics of intent to treat study popu-
lation
Number of subjects Age (years)
Male Female Overall Mean Range
Overall intent to treat 25 23 48 4.4 4–5

RESULTS
It was planned to randomise 60 subjects to have 50
subjects complete both treatment periods of the study.
One hundred fifty-seven subjects were screened, how-
ever, owing to entry criteria challenges (predomi- Figure 2. Summary of mean biofilm fluid fluoride concentration over
nantly the inability to provide sufficient biofilm for all time.

44 © 2013 FDI World Dental Federation

 Effect of brushing time on fluoride level

No statistically significant difference in log change

Figure 3. Summary of mean saliva fluoride concentration over time.
ITT, intent to treat.
(0.102); 15 minutes 0.155 (0.014), 0.182 (0.017);
30 minutes 0.072 (0.006), 0.107 (0.013) and 60 min-
utes 0.039 (0.006), 0.049 (0.006).
A statistically significant difference based on log-
transformed AUC at 1 hour for both biofilm fluid fluo-
ride (P = 0.0279) and saliva fluoride (P = 0.0033) was
observed in favour of a 120-second brushing time com-
pared with a 40-second brushing time (Tables 2 and 3).
Both the treatment groups showed a statistically sig-
nificant change from baseline at each post-brushing
time-point in terms of log biofilm fluid fluoride con-
centration and log saliva fluoride concentration except
for 1 hour in the 40-second brushing time experimen-
tal group for saliva (P = 0.0630) (Tables 3 and 4).
from baseline biofilm fluid fluoride concentration was
observed at any individual post-brushing time-point
comparing a 40-second brushing with a 120-second
brushing (Table 2), but significant differences were
observed for 15, 30 and 60 minutes in favour of the
120-second brushing time when the log biofilm fluid
value was analysed rather than the change from base-
line value (Table 5).
Statistically significant differences were observed for
log change from baseline in saliva fluoride concentra-
tion at each individual post-brushing time-point com-
paring 40 seconds of brushing with 120 seconds of
brushing (Table 6). The sensitivity analysis for log bio-
film fluoride AUC gave consistent results (P = 0.0123)
in favour of the 120-second brushing time (Table 5).
Safety results
A total of 49 subjects were included in the safety popu-
lation. A total of nine subjects reported 10 treatment-
emergent AEs. There were no oral AEs reported in this
study. None of the AEs were treatment related and all
were mild in intensity; no serious AEs were reported.
DISCUSSION
This single-blind (fluoride analyst), crossover, randomised,
two-period study in healthy children aged 4–5 years
(inclusive) evaluated the effect of two different brushing

Table 2 Log biofilm fluid fluoride concentration between-treatment (40 seconds vs. 120 seconds brushing time)
comparison
Treatment Parameter Log biofilm fluid fluoride concentration of change ln AUC* AUC†
from baseline (lg F/g)

5 minutes 15 minutes 30 minutes 60 minutes

40 seconds Difference (mean) 0.0976 0.1524 0.1991 0.1704 0.23 0.79

versus 120 seconds 95% CI (lower, upper) 0.42, 0.23 0.39, 0.08 0.45, 0.05 0.43, 0.09 0.44, 0.03 0.65, 0.97
P-value 0.5460 0.1985 0.1197 0.1852 0.0279

*Difference (Mean).
†
Ratio of geometric mean.
P-value <0.05 is significant and bold.

Table 3 Log biofilm fluid fluoride concentration within-treatment change from baseline
Treatment Parameter Log biofilm fluid fluoride concentration of change
from baseline (lg F/g)

5 minutes 15 minutes 30 minutes 60 minutes

40 seconds Change from baseline (adjusted mean*) 0.8324 0.5466 0.3323 0.2143
Standard error* 0.1201 0.0930 0.0899 0.0855
P-value <0.0001 <0.0001 0.0005 0.0148
120 seconds Change from baseline (Adjusted Mean*) 0.9299 0.6990 0.5314 0.3847
Standard error* 0.1238 0.0945 0.0901 0.0928
P-value <0.0001 <0.0001 <0.0001 0.0001

*Based on log transformed data.

P-values <0.05 are significant and bold.

© 2013 FDI World Dental Federation 45

Newby et al.

Table 4 Log biofilm fluid fluoride concentration between-treatment (40 seconds vs. 120 seconds brushing time)
comparison (sensitivity analysis for AUC)
Treatment Parameter Log biofilm fluid fluoride concentration (lg F/g) ln AUC* AUC†

5 minutes 15 minutes 30 minutes 60 minutes

40 seconds versus Difference (mean) 0.1400 0.2206 0.2852 0.2443 0.29 0.75
120 seconds 95% CI (lower, upper) 0.45, 0.17 0.43, 0.01 0.48, 0.09 0.48, 0.01 0.52, 0.07 0.59, 0.93
P-value 0.3619 0.0391 0.0063 0.0411 0.0123

*Difference (mean).
†
Ratio of geometric mean.
P-values <0.05 are significant and bold.

Table 5 Log saliva fluoride concentration between-treatment (40 seconds vs. 120 seconds brushing time) comparison
Treatment Parameter Log saliva fluoride concentration of change from baseline (lg F/ ln AUC* AUC†
g)

5 minutes 15 minutes 30 minutes 60 minutes

40 seconds versus Difference (mean) 0.4882 0.3073 0.4266 0.3195 0.27 0.76
120 seconds 95% CI (lower, upper) 0.77, 0.20 0.58, 0.03 0.69, 0.16 0.64, 0.00 0.44, 0.10 0.64, 0.91
P-value 0.0012 0.0299 0.0023 0.0496 0.0033

*Difference (mean).
†
Ratio of geometric mean.
P-values <0.05 are significant and bold.

Table 6 Log saliva fluoride concentration within-treatment change from baseline

Treatment Parameter Log saliva fluid fluoride concentration of change from baseline (lg F/g)

5 minutes 15 minutes 30 minutes 60 minutes

40 seconds Change from baseline 2.8527 1.6885 0.9465 0.2244

(adjusted mean*)
Standard error* 0.1241 0.1058 0.1041 0.1191
P-value <0.0001 <0.0001 <0.0001 0.0630
120 seconds Change from baseline 3.3408 1.9958 1.3731 0.5439
(adjusted mean*)
Standard error* 0.1245 0.1062 0.1045 0.1195
P-value <0.0001 <0.0001 <0.0001 <0.0001

*Based on log transformed data.

P-values <0.05 are significant and bold.

times (40 seconds and 2 minutes) on biofilm fluid fluoride

and salivary fluoride levels over a 1 hour period in chil-
dren aged 4–5 years using 0.25 g of toothpaste.
The results of the current study are in agreement
with previous studies that have reported statistically
significant differences on the AUC for both biofilm
fluid fluoride and saliva fluoride24,33. Also in agree-
ment with those previous reports is the fact that both
treatment groups in the current study demonstrated a
statistically significant (marginally significant in one
case) change from baseline within treatment at each
post-brushing time-point.
Our results provide evidence in support of the impor-
tance of the duration of brushing reported for adults24,25.
Careful analysis of the fluoride values observed in this
study shows that fluoride clearance is biphasic with an
initial rapid loss of fluoride, followed by a much slower
rate of loss for the ensuing 60 minutes, as previously
reported by Duckworth and colleagues8,9,17–19.
CONCLUSION
The results of the present clinical study provide fur-
ther evidence to support the fluoride delivery benefits
of 2 minutes brushing in children.
Conflict of interest
Authors Newby, Fleming, North and Bosma are
employed by GlaxoSmithKline Consumer Healthcare.
Authors Martinez-Mier, Zero and Kelly were funded
by GlaxoSmithKline Consumer Healthcare to conduct
this study.
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Correspondence to:
Evelyn E. Newby,
GlaxoSmithKline Consumer Healthcare,
St George’s Avenue,
Weybridge,
Surrey KT13 0DE, UK.
Email: Evelyn.E.Newby@gsk.com
47
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