J. Periodontal Res.

14: 403-410, 1979

An in vitro study of the role of

dietary factors in the aetiology
of tooth staining associated
with the use of chlorhexidine
M. ADDY, S. PRAYITNO, L. TAYLOR AND S. CADOOAN
Department of Periodontology, Dental School, Welsh National School of Medicine,
Heath Park, Cardiff, S. Wales, United Kingdom.

A spectrophotometric measurement technique was employed to investigate the role of

dietary factors in the aetiology of chlorhexidine staining. Standard solutions were prepared
from a range of dietary components. Test perspex specimens were maintained in the stan-
dard solutions throughout a five day period being removed from the solutions three times a
day and soaked in a 0.2 % chlorhexidine gluconate solution. Control specimens were
similarily treated but not exposed to chlorhexidine gluconate. Daily optical density record-
ings demonstrated some staining of specimens by all dietary components. However for most
there was no significant difference between test and control specimens and in visual
terms such staining was very minimal. Nevertheless, tea, red wine and port produced
rapid and heavy staining on test specimens which was highly significantly increased when
compared to control specimens. Coffee similarly produced more staining of test specimens
compared to control specimens; however, the intensity of staining was considerably less.
Employing the same experimental method, both cigarette smoke and cigarette smoke in
solution produced significantly more staining of control specimens compared to test
specimens.

(Accepted for publication November 28, 1978)

initial clinical experiments with chlor-

Introduction
There is now considerable evidence that
bacterial plaque is the major aetiological
factor in the development of chronic gingi-
vitis (Loe, Theilade & Jensen 1965, Waer-
haug 1967). The observations concerning the
antiplaque activity of chlorhexidine gluco-
nate (Loe & Rindom Schiott 1970 a, b) not
only tended to confirm the role of plaque
in chronic gingivitis but also demonstrated
the possibility of preventing gingival disease
by the use of a chemical agent. Since these
hexidine, many studies concerning the
properties and clinical uses of chlorhexidine
have been reported (Review Nagle & Turn-
bull 1978). Of prime importance were the
long term trials demonstrating the safety of
the antiseptic with no systemic or serious
local side effects observed after two years
continuous use (Nuki et al. 1976, Rindom
Schiott, Loe & Briner 1976). Throughout
the test period however a reduction in both
plaque and gingivitis was recorded (Loe et
al. 1976). Although much of the published
404 ADDY, PRAYITNO,
work has demonstrated the potential value
of chlorhexidine as a preventive and thera-
peutic agent; nevertheless, local side effects,
in particular staining of the teeth and
restorative materials, tend to limit the long
term usage of chlorhexidine (Flotra et al.
1971, Eriksen & Gjermo 1973). The stain-
ing is typically brown in colour varying
from individual to individual (Heyden
1973), is not readily removed and often
requires a professional cleaning which may
be time consuming (Hoyos, Murray &,
Shaw 1977).
The chemical nature of the staining and
the mechanism underlying its formation is
not fully understood. However, a direct
relationship between the presence of the
stain and local concentration of chlor-
hexidine was established (Heyden 1973).
Suggestions which seek to explain the
aetiology of the staining associated with the
use of chlorhexidine have included denatu-
ration of the chlorhexidine molecule on the
tooth surface, or the formation of coloured
reaction products with aldehydes and
ketones (Nordbo 1971). However, none of
these hypotheses have been borne out in
clinical studies. The role of dietary factors
as aetiological agents have been investi-
gated. Thus, chlorhexidine in the presence of
food dyes, has been shown to produce
coloured compounds on hydroxyapatite
(Jensen 1977). Furthermore, beverages, in
particular tea and coffee, produced staining
of tooth and acrylic specimens previously
exposed to chlorhexidine (Addy & Jenkins
1977). The aim of this in vitro study was
to obtain more information concerning the
staining associated with the use of chlor-
hexidine gluconate by employing a spec-
trophotometric measuring technique.-
Method and Materials
A range of dietary components which are
normally found in the diet were chosen for
TAYLOR AND CADOGAN
this study and are shown in Table 1. All of
the preparations chosen were those which
would normally be in solution iti the diet.
Standard solutions were prepared from the
dietary components where necessary. Thus
a standard solution of tea was prepared
from 8 grams of a commercial brand boiled
in 800 millilitres of distilled water for 2
minutes and allowed to cool to room tem-
perature. The liquid was then decanted
from the tea leaves. For coffee, the stan-
dard solution was prepared from 9.7 grams
of instant coffee granules dissolved in 1
litre of water and boiled for two minutes
and allowed to cool as before. For the re-
maining dietary components requiring dis-
solution or suspension in water, the propor-
tions are shown in Table 1. All of the re-
maining components were used as supplied.
In the case of tea and coffee which were
used in more than one experiment a fresh
standard solution was prepared in each case
and used throughout a single experiment.
Clear methyl methacrylate (perspex)
(Perspex. LCI. Ltd. Macclesfield, Cheshire,
England) rectangular blocks measuring 30
mm X 10 mm X 5 mm were prepared.
This size coincided with the width of the
sample chamber of a Beckman (Beckman.
Beckman Instrument Inc. FuUerton, Cali-
fornia 80566, U.S.A.) series DB spectro-
photometer. Three test and three control
perspex specimens were placed in 25 milli-
litre volumes of each standard solution of
the dietary components under study. Three
times each day the test specimens were
removed from their respective standard
solution and placed into an 0.2 % chlor-
hexidine gluconate solution for 2 minutes.
At the end of 2 minutes they were removed
and allowed to dry and then returned to
the standard solutions. At the same time
the eontrol specimens were removed to
distilled water for 2 minutes before return-
ing to the respective standard solution. On
5 consecutive days between 9 a.m. and 10
 DIETARY FACTORS IN CHLORHEXIDINE STAINING 405

a.m. the specimens were removed from the
test solutions, rinsed in distilled water and
allowed to dry. The optical density of the
specimens was then measured on the spec-
trophotometer, at the previously determined
lambda maximum for the respective dietary
component (Table 1). A further experiment
employing tea and coffee as the standard
solutions was carried out using the same
methodology but with the experiment ex-
tending to 10 days.
A similar experimental method was used
to determine the staining effects of ciga-
rette smoke in the presence of chlor-
hexidine. First, 3 test and 3 control spec-
imens were placed in separate glass jars
containing 100 millilitres of distilled water.
The cigarette smoke from a heavy tar non-
tipped cigarette was sucked through the
distilled water in each jar by negative pres-
sure using a Venturi pump, on 3 occasions
each day. The specimens remained within
the water throughout a 5 day period except
that 3 times each day the test specimens
were removed for 2 minutes to a 0.2% chlor-
hexidine gluconate solution as before and
the control specimens removed to distilled
water. Again, at 9 a.m. - 10 a.m. each day
the optical density of the specimens was
recorded using a spectrophotometer at the
wavelength corresponding to the lambda
maximum for the solution of cigarette
smoke in water.
The same experiment was repeated with-
out water, in the glass jar containing the
specimens. Again, the smoke from 3 heavy
tar cigarettes was sucked into the contain-
ers at periods throughout each day. When
the jar was full of smoke the inlet and out-
let tubes were sealed. Test and control
specimens were removed, as before, 3 times
a day to chlorhexidine gluconate or distilled
water. The optical densities for each spec-
imen were determined each day for a 5 day
period.
Statistical analysis of the results was car-
ried out using the Student 't' test.
Results
The lambda maxima values of the standard
solutions used in the study, together with

Table 1
The preparation, pH and lambda maxima of the dietary components.

Dietary Lambda maxima

Method of preparation pH
Components m,u

Beer As supplied 4.4 312

Martini As supplied 3.6 310
Port As supplied 4.0 312
Sherry As supplied 3.9 310
Red wine As supplied 3.9 275
Ribena (Biaciccurrant) As supplied 3.3 260
Coca-Coia As supplied 3.2 300
Orangeade As supplied 3.0 300
Raspberryade As supplied 3.3 265
Coffee 9.7 gms in 1 litre of water 5.4 290
Drini<ing chocolate 80 mis in 800 mis of water 7.3 253
Ovaitine 80 mis in 800 mis of water 6.5 310
Tea 8 gms in 800 mis of water 5.6 295
Bovril (Meat extract) 80 mis in 800 mis of water 5.8 255
Gravy Browning 15 mis in 600 mis of water 4.9 314
Soup (Tomato) 1 pack in 800 mis of water 4.8 312
Soy Sauce 300 mis in 300 mis of water 4.8 310
406 ADDY, PRAYITNO. TAYLOR AND CADOGAN

2-0
2-0
1-8 Experiment Control
1-8
1-6 Tea *' • •- — . «
1-4 1-6
ft Experiment Cotteeo o o. —-_o
-' Control
Io 1-2 2 1-4

f 1-2
O-B a
0-a I 1-0
0-4 0-8
0-2 0-6
ll Mxau 0-4
cf., _.O" ^
0-2

5 6 8 9 10
Fig. 1. The mean optical density readings for test and Days
control specimens exposed to standard solutions at 5 Fig. 2. The mean optical density readings for test and
days. control specimens exposed to tea and coffee over a 10
day period.

their pH values, are shown (Table 1). The

pH range of the dietary components was in Table 2. Considerable variation in stain-
3—7.3, a lower pH being common to all of ing by the different dietary factors was ap-
the soft non alcoholic drinks. The mean parent. Thus at 5 days, all of the standard
optical density values for test and control solutions produced staining of the chlor-
specimens measured in absorbance units at hexidine treated specimens as evidenced by
day 5 of the experimental period for each the recordable absorbance figures. How-
dietary component are shown in Fig. 1. and ever, the degree was slight and for many,
the significance of the differences are given this was not significantly different from the
staining recorded on the control specimens.
Moreover, in the case of Ovaitine and Bov-
Table 2 ril, significantly more staining was apparent
Significance of differences between experi- on the control specimens compared to the
mental and control specimens exposed to test specimens (p = < 0.05). However,
dietary compounds. port, tea and red wine produced marked.
Beer P > 0-4 NS
Martini P > 0-2 NS
Port P < 0-001 S" 1-60 Experlmetit
Sherry P>0-2 NS 1*40 . Control
Wine P < 0-001 S*
Ribena P > 0-2 NS 1-20
Coca Cola P > 0-7 NS <u

Orangeade
Raspberryade
P > 0-3
P>0-9
NS
NS
1^
1 0-80
Coffee P < 0-001 S* •^ n*fiA
Drinkling Chocolate P > 0-1 NS
Ovaitine P < 0-05 S** 0-40
Tea P < 0-001 s*.
Bovril P > 0-05 s** 0-20
Gravy Browning P>0-5 NS
Soup P>0-9 NS 1 2 3 4 5
Soy Sauce P>0-2 NS Days
Pin
Fig. .?3. The mean optical density readings for test
* In favour of the experiment specimens and control specimens exposed to orangeade over a
** In favour of the control specimens 5 day period.
 DIETARY FACTORS
RIBENA COFFEE
Fig. 4. Appearance of test and control specimen exposed to different standard solutions at 5 days.
and highly significantly more staining on
the test than the control specimens (p = <C
0.001). Furthermore, the staining occurred
very rapidly with the maximum absorbance
figure for the spectrophotometer achieved
by day 2 for port and tea and day 3 for
red wine. Coffee also caused significantly
more staining of chloi'hexidine treated
specimens than control specimens at 5 days
(p = << 0.001). Directly comparing the
development of tea and coffee staining over
a 10 day period (Fig. 2) demonstrated that
the staining of the test specimens by coffee
was slower and significantly less at all time
periods (p = < 0.001). The staining by
other dietary components measured at 5
days was very slight and for some, in par-
ticular soft drinks, the development of
staining did not appear progressive (Fig. 3).
Visually, the difference in the degree of
IN CHL ORHEX 1 D i NE STAINING 407
PORT TEA RED WINE
staining of test and control specimens can
be seen (Fig. 4). Thus by 5 days the
staining of test specimens by port, red
wine and tea, was dramatic in com-
parison with the respective control spec-
imens. Furthermore, coffee staining of
test specimens was noticeably less than tea,
red wine or port. Ribena, shown as an
example of the other dietary components,
produced little change in optical density;
the test and control perspex specimens ap-
peared almost completely clear.
The optical density readings of the test
and control specimens either soaked in a
solution of cigarette smoke or exposed
directly to the cigarette smoke throughout
the 5 day period are shown in Table 3. The
optical density readings increased pro-
gressively throughout the 5 day period for
the test and control specimens for both
408 ADDY, PRAYITNO, TAYLOR AND CADOGAN

Table 3
The effect of chlorhexidine and cigarette smoke on the optical density of perspex
specimens throughout a 5 day period.

Day Absorbance
Specimens 3

Cigarette Test 0.04 0.08 0.12 0.14 0.15

Smoke
Solution
Control 0.15 0.30 0.46 0.55 0.66
Cigarette Test 0.67 0.58 0.89 1.04 1.33
Smoke
Control 0.73 1.19 1.07 1.23 1.77

groups. However, the optical density read- Since chlorhexidine has been shown to
ings were significantly higher for the con- adsorb to methyl methacrylates, as well as
trol specimens when compared to the test teeth (Eriksen & Gjermo 1973, Heyden,
specimens both for the cigarette smoke Nordbo & RoUa 1971) the results of these
solution and the cigarette smoke alone laboratory findings would thus infer that
(p = < 0.01 and < 0.05 respectively). certain dietary factors are able to interact
with this locally adsorbed chlorhexidine.
The staining observed was consistent with
Discussion
previous in vitro studies into the staining
In this study the role of dietary compo- by beverages (Addy & Jenkins 1977) and
nents in the aetiology of staining associated food dyes (Jensen 1977) of specimen ma-
with the use of chlorhexidine gluconate was terials previously exposed to chlorhexidine
investigated. Thus, this in vitro experiment gluconate. All of the dietary components
demonstrated that dietary components examined under both test and control situ-
stained perspex specimens previously ex- ations demonstrated an affinity for the
posed to a 0.2 % chlorhexidine gluconate perspex as measured by the spectrophoto-
solution. Although many of the solutions meter. Thus it is likely that the staining ob-
prepared from the dietary components served clinically may result from a combi-
produced staining on perspex, in most cases nation effect of many components. How-
this was slight with no significant difference ever, the dramatic difference in both the
between the test and control specimens. degree of staining and the rate of stain
However, tea, red wine and port produced development for tea, red wine and port
both rapid and marked staining on speci- would indicate that a few dietary factors
mens exposed to chlorhexidine, and this play a major role in the staining observed
staining in visual terms was dramatically clinically.
greater than control specimens soaked in Both the cigarette smoke solution and
these beverages. Coffee was, of the other the cigarette smoke alone produce consid-
dietary components, the only one to-produce erable staining of both test and control
significantly more staining on test as op- specimens. However, chlorhexidine treat-
posed to control specimens. However the ment of specimens did not significantly
rate of stain development and the degree increase the staining; in fact, the reverse
of staining observed was markedly reduced was observed. Aldehydes and ketones may
when compared to tea. complex with adsorbed chlorhexidine to
 DIETARY FAOTORS IN CHLORHEXIDINE
produce staining on enamel in vitro (Nord-
00 1971). The results from this study would
suggest that the aldehydes in cigarette
smoke (Egle 1970) did not play a major
role in the staining observed.
The use of a spectrophotometric method
of analysis appears to have some advan-
tages. In particular, quantitative pieasure-
ment of the staining was possible, which
permitted a direct comparison of staining,
thus eliminating the subjective interpreta-
tion of colour and shade. Colorimetric
methods have been used previously to as-
sess stain on acrylic resins (BonesvoU &
Olsen 1974), and this related spectrophoto-
metric technique may be of future value in
assessing potential antiplaque agents both
for possible side effects of staining and for
adsorption properties. However, the use of
perspex limits the spectrophotometric anal-
ysis to the visible light spectrum. The ad-
sorption of chlorhexidine on to the perspex
can only be inferred from the difference in
staining observed between test and control
specimens. Nevertheless acrylic has been
demonstrated to be capable of adsorbing
chlorhexidine (Budtz-Jorgensen & Loe
1972). The observed interaction of chlor-
hexidine both in solution and on surfaces
with the dye azocarmine B (Heyden, Nord-
bo & RoUa 1971) and food dyes (Jensen
1977) would support the hypothesis that
staining occurs as a result of a cationic-
anionic interaction between chlorhexidine,
and negatively charged ions in the oral
environment (Jensen 1977, Addy & Jenkins
1977). The di-cationic nature of chlor-
hexidine (Leach 1977) at physiological pH
would facilitate such a reaction on the
tooth surface.
The most chromogenic dietary factors
determined from the spectrophotometric
analysis method employed in this study all
contained tannin or tannin like derivatives
(Pearson 1976). Tea, perhaps the most
commonly consumed hot beverage in the
STAINING 409
United Kingdom contains considerable
quantities of tannin-like substances, all of
which are gallic acid derivatives. Further-
more the ratio of the derivatives theaflavin
(yellow) and theambigin (brown) determine
the quality and colour of the tea solution
(Harler 1963). It is possible therefore that
such derivatives at the pH or the test solu-
tions employed would be dissociated with
the anionic and chromogenic part of the
molecule negatively charged. Since the pH
of most of the dietary solutions used in
this study lay between the range of 3-5 the
differences in staining observed with chlor-
hexidine would thus appear primarily to
arise from a difference in chemical com-
position of the dietary factors. Neverthe-
less at pH levels below 2, irrespective of
the chemical nature of the dietary compo-
nent, adsorption of chlorhexidine (Bones-
voU, Lokken & RoUa 1974), and therefore
the staining of the perspex, may be re-
duced.
Acknowledgments
We are grateful to Dr. T. Khosla, Senior
Lecturer in Medical Statistics, Welsh Na-
tional School of Medicine, Cardiff, for sta-
tistical analysis of the results.
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Address:
Department of Periodontology
Dental School
Welsh National School of Medicine
Heath Park
Cardiff, S. Wales, United Kingdom