Chlorhexidine and its applications in Endodontics: A literature review

Abstract

This study aims at presenting the properties of chlorhexidine used as an auxiliary chemical substance for endodontic instrumentation: structure and mechanism of action, substantivity, tissue solvent effect, chlorhexidine x sodium hypochlorite interaction, cytotoxicity, action over biofilm, antibacterial activity, antifungal activity, intracanal dressing, rheological action and allergic reactions. In Dentistry, chlorhexidine has been proved effective and safe against bacterial plaque since 1959. In Endodontics, it has been recommended in liquid or gel form, at different concentrations (usually 2%), as root canal irrigant and dressing (alone or associated with other substances). Additionally, it may be applied as an antimicrobial agent at all stages of root canal preparation, including disinfection of the operative field, removal of necrotic tissues before determining the root length, chemical- mechanical preparation before foraminal clearance and enlargement, disinfection of obturation cones; to shape the main cone with gutta-percha, to remove gutta-percha during retreatment, to disinfect the prosthetic space; etc. It is reasonable to conclude that chlorhexidine, at different concentrations, has an antimicrobial activity against Gram-positive as well as gram-negative bacteria and fungus. Its antimicrobial activity, increased by the substantivity effect, does not have the ability of solving tissues, which is overcome by the rheological action of its gel form that lubricates the endodontic instrumentation used. Its biocompatibility is acceptable with relative absence of cytotoxicity.

Full text

Dental Press Endod. 2013 Sept-Dec;3(3):36-54
© 2013 Dental Press Endodontics 36
Jefferson MARION1
Kathielli PAVAN2
Márcia Esmeralda Bis Franzoni ARRUDA3
Lauro NAKASHIMA4
Carlos Alberto Herrero de MORAIS5
Literature review
Chlorhexidine and its applications in Endodontics:
A literature review
1 Professor, Department of Endodontics, Brazilian Dental Association (ABO) and Ingá College
(UNINGÁ)
2 Specialist in Endodontics, Brazilian Dental Association (ABO) and Maringá Dental Association (AMO)
3 MSc in Health Sciences, State University of Maringá (UEM)
4 Specialist in Endodontics, School of Dentistry — University of São Paulo/Bauru
5 Phd in Dentistry/Endodontics, University of São Paulo (USP).
Contact address: Jefferson José de Carvalho Marion
Rua Néo Alves Martins, 3176 – 6º andar – sala 64 – Centro
CEP: 87.013-060 – Maringá/PR — Brazil
Email: jefferson@jmarion.com.br
Submitted: September 04, 2013. Revised and accepted: September 06, 2013.
How to cite this article: Marion J, Pavan K, Arruda MEBF, Nakashima L, Mo-
raisCAH. Chlorhexidine and its applications in Endodontics: A literature review.
Dental Press Endod. 2013 Sept-Dec;3(3):36-54.
The authors inform they have no associative, commercial, intellectual property
or nancial interests representing a conict of interest in products and compa-
nies described in this article.
ABSTRACT
This study aims at presenting the properties of chlorhexidine
used as an auxiliary chemical substance for endodontic in-
strumentation: structure and mechanism of action, substan-
tivity, tissue solvent effect, chlorhexidine x sodium hypochlo-
rite interaction, cytotoxicity, action over biofilm, antibacterial
activity, antifungal activity, intracanal dressing, rheological
action and allergic reactions. In Dentistry, chlorhexidine
has been proved effective and safe against bacterial plaque
since 1959. In Endodontics, it has been recommended in
liquid or gel form, at different concentrations (usually 2%),
as root canal irrigant and dressing (alone or associated
with other substances). Additionally, it may be applied as
an antimicrobial agent at all stages of root canal prepara-
tion, including disinfection of the operative field, removal of
necrotic tissues before determining the root length, chemi-
cal-mechanical preparation before foraminal clearance and
enlargement, disinfection of obturation cones; to shape the
main cone with gutta-percha, to remove gutta-percha during
retreatment, to disinfect the prosthetic space; etc. It is rea-
sonable to conclude that chlorhexidine, at different concen-
trations, has an antimicrobial activity against Gram-positive
as well as gram-negative bacteria and fungus. Its antimicrobi-
al activity, increased by the substantivity effect, does not have
the ability of solving tissues, which is overcome by the rheo-
logical action of its gel form that lubricates the endodontic
instrumentation used. Its biocompatibility is acceptable with
relative absence of cytotoxicity.
Keywords: Chlorhexidine. Microorganism control agent.
Endodontics.

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 37
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
Introduction
Most bacteria found in infected root canals can
be removed by the simple mechanical action of
endodontic instrumentation. Nevertheless, despite
thorough mechanical instrumentation, organic res-
idues and bacteria located deeply inside the den-
tin tubules cannot be reached due to the anatomic
complexity of root canals.1,2 Irrigation solutions are
indicated to aid mechanical preparation and pulp
space disinfection. Thus, several substances have
been used not only to remove debris and necrotic
pulp tissue during and immediately after root canal
preparation, but also to help eliminate the microor-
ganisms that could not be reached by mechanical
instrumentation.3 The search for an ideal substance
for root canal irrigation has motivated researchers
since the beginning of Dentistry. Chemical agents
chosen to function as endodontic irrigants have four
major properties: antimicrobial activity; organic
tissue dissolution that favors debridement of the
root canal system; and absence of toxicity against
periapical tissues.1,2,4 Most substances used to irri-
gate the root canal are liquid: sodium hypochlorite
(NaOCl), chlorhexidine gluconate — also known as
chlorhexidine digluconate or simply chlorhexidine
(Chlorhexidine) —, 17% EDTA, citric acid, MTDA
and 37% phosphoric acid solution.5
Sodium hypochlorite is the most popular irriga-
tion solution due to its antimicrobial and physico-
chemical properties.6,7 The antimicrobial effica-
cy of NaOCl is due to its high pH (the action of
hydroxyl ions) similar to the mechanism of action
of calcium hydroxide.8 The high pH of NaOCl inter-
feres in the integrity of the cytoplasmic membrane
with an irreversible enzymatic inhibition that causes
biosynthetic alterations in cellular metabolism and
destruction of phospholipids, observed during lipid
peroxidation. The antimicrobial activity of NaOCl
leads to an irreversible enzymatic inhibition of bac-
teria, which originates hydroxyl ions, as well as to
chloramination action.2 Despite being an effective
antimicrobial agent and an excellent organic sol-
vent,9 NaOCl is known for being highly irritant to
periapical tissues,10 especially at high concentra-
tions.11 For this reason, the search for another irri-
gation solution, with lower potential in inducing ad-
verse effects, proves feasible.2,12
Thus, irrigation solutions with antibacterial activ-
ity and biocompatibility, as it is the case of chlorhex-
idine, have been recommended to treat infected root
canals. The antibacterial effect and long-term action
of 2% chlorhexidine digluconate13-17 led researchers
to indicate its use for endodontic treatment.15,16,18
Chlorhexidine is a cationic biguanide that acts by
adsorption in the bacterial wall of a microorganism,
causing leakage of the intracellular components.
Due to being a strong base, low-concentration
chlorhexidine has a bacteriostatic effect; however,
at higher concentrations, it produces a bactericid-
al effect. Chlorhexidine digluconate has a slightly
acidic pH that varies from 5.5 to 6.0, with the ability
to donate protons.19
Chlorhexidine was first introduced in the late 40s,
when scientists, in the search for new agents against
malaria, formulated a group of compounds with a
broad antibacterial spectrum, known as polibigu-
anides.20,21 Chlorhexidine was registered in 1954 by
the Imperial Chemical Co. Ltd. (Macclesfield, Unit-
ed Kingdom), under the trademark Hibitane. Due to
its biocompatibility and broad antibacterial activity,
it was the first antiseptic internationally accepted
for skin, wound and mucosa cleansing.22 Since then,
chlorhexidine has been used for several medical
purposes, namely: gynecology, urology and ophthal-
mology, as well as for the treatment of skin burns
and disinfection.23
In Dentistry, chlorhexidine has been proved ef-
fective and safe against bacterial plaque since 1959.
In the 70s, it was commercialized in Europe as a
0.2% mouthwash solution and in 1% gel.21,23
Chlorhexidine may be applied as an antimicro-
bial agent at all stages of root canal preparation,
including disinfection of the operative field, during
root canal instrumentation, removal of necrotic tis-
sues before determining the root length, chemical-
mechanical preparation before foraminal clearance
and enlargement, as an intracanal dressing (alone
or in association with other substances), disinfection
of obturation cones; to shape the main cone with
gutta-percha, to remove gutta-percha during retreat-
ment, to disinfect the prosthetic space; etc.5
Viscous irrigants, such as glycerin-based ones,
have low solubility. As a result, they leave residues at
the dentin walls, which hinders the final obturation

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 38
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
of the root canal system.12,24 However, Natrosol is a
highly efficient non-ionic inert gel that is hydro-solu-
ble and broadly used in cosmetic products based on
cationic substances.2
Chlorhexidine gel has been widely used in Den-
tistry. It yields satisfactory results for cavity control,
reducing Streptococcus mutans and Lactobacillus, act-
ing as auxiliary in periodontal therapy, and control-
ling the growth of Gram-positive and Gram-negative
bacteria.25
Ferraz et al12 demonstrated that 2% chlorhexi-
dine gel is highly advantageous in comparison to
2% chlorhexidine solution, even though both of
them have similar antimicrobial, substantivity and
biocompatibility properties. Chlorhexidine gel lu-
bricates the root canal walls, which reduces friction
between the endodontic file and the dentin surface.
As a result, it favors instrumentation, improves file
performance and reduces the risk of file fracture in-
side the root canal. Additionally, chlorhexidine gel
allows better debridement and, as a consequence,
compensates its inability in organic tissue dissolu-
tion.2,26 Chlorhexidine gel leaves the majority of den-
tin tubules open as a result of its viscosity that keeps
debris in suspension and reduces the formation of
smear layer, which does not occur with chlorhexi-
dine liquid. Furthermore, the active ingredient of
chlorhexidine gel establishes long-term contact with
microorganisms and, as a consequence, inhibits
their growth.27 When chlorhexidine gel is used for
the mechanical preparation of a root canal, the ir-
rigant solution of choice must be saline solution or
distilled water.
In this context, this study aims at conducting
a literature review that presents the properties of
chlorhexidine used as an auxiliary chemical sub-
stance for endodontic instrumentation.
literature review
Microorganisms have been broadly recognized as
the main etiologic factor of periapical bone lesions.28
Their persistence in the apical area of obturated root
canals is responsible for the majority of endodontic
treatment failures.29,30 Thus, microbial control is of
paramount importance for an effective endodontic
treatment,28 of which success relies on the elimina-
tion of microorganisms from infected root canals.1
Most bacteria found in infected root canals can
be removed by the simple mechanical action of
endodontic instrumentation. However, despite thor-
ough mechanical instrumentation and the several
techniques available, organic residues and bacteria
located deeply inside the dentin tubules cannot be
reached due to the anatomic complexity of root ca-
nals.2,31 For this reason, chemical treatment of the
root canal system proves necessary.
According to several authors,11,32,33,34 the ide-
al auxiliary substance must have the property of:
leaving debris in suspension, lubricating endodon-
tic instruments, dissolving organic tissue, develop-
ing antibacterial activity during instrumentation,
substantivity, exerting chelating action, promoting
cleaning of inaccessible areas, being biocompatible
at concentrations that fulfill these properties with-
in a viable clinical time, removing the smear layer
formed during instrumentation, having low surface
tension, neutralizing action and bleaching effect,
having no color alterations, being of easy applica-
tion, removal, handling and storage, accessible, in-
expensive and of extended useful life.
Several substances have been used to irrigate
the root canal system, namely: sodium hypochlo-
rite (NaOCl), chlorhexidine gluconate — also
known as chlorhexidine digluconate or simply
chlorhexidine —, 17% EDTA, citric acid, MTDA and
37% phosphoric acid solution.5 Sodium hypochlo-
rite, at different concentrations, is the most com-
monly used substance due to its triple mode of ac-
tion: necrotic tissue dissolving ability attributed to
its high alkalinity; antibacterial properties related to
hypochlorous acid formation in chlorine solution;
and fat saponification.35
Sodium hypochlorite is a halogenated compound
of which first use was registered in 1972 under the
name of “Javele’s water”. It was obtained by mixing
NaOCl with potassium. In 1820, Labarraque obtained
sodium hypochlorite at a concentration of 2.5% of
active chlorine. In the early XX century, during World
War I, sodium hypochlorite was used to treat infected
wounds. In 1915, Dakin36 proposed a new concen-
tration for the solution (0.5%) because, according to
the author, wounds treated with 2.5% sodium hypo-
chlorite took too long to heal due to the high content
of sodium hydroxide.36,37 In Endodontics, its use was

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 39
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
first proposed by Coolidge, in 1919; first employed by
Walker, in 1936, due to its excellent tissue dissolv-
ing ability as well as its antimicrobial efficacy,39 and
disseminated by Grossman.38,40 It has been employed
in Endodontics for more than 60 years as an irriga-
tion solution during chemo-mechanical preparation
of the root canal system.9 Despite NaOCl excellent
antimicrobial activity and tissue dissolution ability, it
causes irritation to periapical tissues,41 it is caustic
and causes clothes stain and instruments corrosion,42
especially at high concentrations.11 According to Ra-
mos and Bramante,43 biocompatibility is one of the
main desirable properties of an irrigation solution.
For this reason, the search for another irrigation solu-
tion with lower potential in inducing adverse effects
proves feasible.2,12
Among different alternatives, chlorhexidine has
proved to be an effective antimicrobial agent acting
inside root canals, showing a great potential to be
used as irrigant or intracanal dressing. It is also rec-
ommended for cases of incomplete root formation
or hypersensitivity to sodium hypochlorite due to its
low toxicity. Chlorhexidine is found in the form of
liquid (water solution) or gel, at concentrations that
vary from 0.2 to 2%.35,44
It is characterized as a cationic detergent of the
biguanide class. It is available as acetate, hydrochlo-
ride and digluconate which is the most used for-
mat.45 Chlorhexidine was first introduced in the late
40s when scientists were searching for new agents
against malaria.20,21 In 1954, it was first used as an
antiseptic to treat skin wounds46 under the trade-
mark Hibitane registered by the Imperial. Ltd. (Mac-
clesfield, United Kingdom).22
In Dentistry, chlorhexidine has been proved ef-
fective and safe against bacterial plaque since 1959.
It was first tested by Löe and Schiott47 who dem-
onstrated that 0.2% chlorhexidine mouthwash twice
a day is effective to decrease biofilm growth and
gingivitis development for a period of 21 days.45
Initially, it was commercialized in Europe, in the 70s,
as a 0.2% mouthwash solution and in 1% gel.21,25
Due to its broad antibacterial spectrum, it has
been widely used in Periodontology. In Endodontics,
it has been recommended as digluconate salt, liquid
or gel at different concentrations, as well as root ca-
nal irrigant13,15,18,23,48,50,51 or as intracanal dressing.13,53-57
In this context, this literature review highlights
11 major points related to chlorhexidine, so as to
facilitate understanding. The extensive literature on
chlorhexidine determined that discussions should
be restricted to factors commonly focused by in vivo
studies and literature reviews. To this end, the fol-
lowing databases were used for research: MEDLINE,
PubMed, BBO, Lilacs, SciELO, websites available on
the internet and the library archives of the School of
Dentistry / Piracicaba (FOP-UNICAMP).
Structure and mechanism of action
The structural formula of chlorhexidine consists
of two symmetric 4-chlorophenyl rings and two bi-
guanide groups connected by a central hexameth-
ylene chain.22 Classified as a cationic detergent, this
biguanide is a strong base which is practically in-
soluble in water. For this reason, it is prepared in
the form of salt,23 which increases its solubility. In
Dentistry, its most commonly used form is chlorhex-
idine digluconate salt in water solution.13,22 The
bactericidal effect of the drug is due to its cationic
molecule binding to extra-microbial complexes and
negatively charged microbial cell walls, entering in
the cell by active or passive transportation.58 At high
concentrations (2%), chlorhexidine has a bacteri-
cidal effect due to precipitation and/or coagulation
of thecytoplasm of bacterial cells, probably caused
by proteincross-linking, resulting in cell death.59,60
At lower concentrations (0.2%), chlorhexidine has a
bacteriostatic effect, which causes inhibition of the
membrane function. This effect remains for several
hours after application due to its excellent substan-
tivity (residual effect).49 Solutions are usually color-
less as well as odorless.
When aqueous, chlorhexidine seems to be more
stable for pH varying from 5 to 8. pH values above 8
lead to precipitation. In an acidic pH, chlorhexidine
solution loses stability and, as a consequence, de-
terioration of its properties occurs. Its antibacterial
effect is excellent for pH values varying from 5.5 to
7.48,61 Chlorhexidine is found in the form of solution,
dentifrices, varnishes and gel.62
Tasman et al63 assessed the surface tension of dif-
ferent irrigation solutions: distilled water; 2.5% sodium
hypochlorite; 5% sodium hypochlorite; 17% EDTA;
3% hydrogen peroxide; 3% citanest-octapressin and

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 40
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
0.2% chlorhexidine. The ring method was employed
to this end. The authors yielded the following results
in ascending order: chlorhexidine; 2.5% hypochlorite;
5% hypochlorite; 17% EDTA; 3% citanest-octapressin;
hydrogen peroxide; saline solution and distilled wa-
ter. The authors concluded that the low surface ten-
sion of chlorhexidine favors its penetration into the
dentin tubules.
According to Ferraz et al,2 chlorhexidine gluco-
nate had lower surface tension in comparison to so-
dium hypochlorite and EDTA. The use of chlorhexi-
dine associated with a gel vehicle provides dentin
walls free of waste produced by instrumentation as
a result of the mechanical properties of gel.
Substantivity
According to Hortense et al,64 substantivity is
the capacity chlorhexidine has to remain active in
the surface where it is applied (tooth, gingiva and
oral mucosa surfaces negatively charged). It is slow-
ly released, avoiding salivary flow to neutralize its
action. Substantivity is an important property for
treatment of dental plaque infections, since anti-
microbial agents need some time to neutralize/kill
a microorganism.22
In Endodontics, the residual antibacterial ef-
fect of chlorhexidine is due to its capability to bind
to hydroxyapatite.65 Therefore, a gradual release of
chlorhexidine could maintain a constant level of mol-
ecules, which is enough to create a bacteriostatic sce-
nario inside the root canal for a long period of time.
Parsons et al48 conducted one of the first stud-
ies recommending the use of chlorhexidine for end-
odontic purposes. The authors observed the adsorp-
tion and release of chlorhexidine solution by bovine
pulp and dentin samples, as well as its antibacterial
properties after a deliberate contamination caused
by
Streptococcus faecalis
. Results revealed that, af-
ter the samples were treated with chlorhexidine, no
contamination was observed within 48 and 72 hours
of bacterial exposure. This confirmed the residual
effect of chlorhexidine.
Other studies have been conducted to assess the
substantivity of chlorhexidine. Their results showed
that this activity can last 48 hours,18 72 hours,16
7 days (chlorhexidine liquid and gel),66 21 days17
or 4 weeks.67 Rosenthal, Spangberg and Safavi68
assessed the substantivity of 2% chlorhexidine in
root canal system and its long-term efficacy in com-
parison to its antimicrobial effect. Their results re-
vealed that chlorhexidine remains in the dentin of
root canals with its antimicrobial effect for more
than 12 weeks.
According to Messer and Chen,69 this property
differs chlorhexidine from other disinfectants that
quickly dissipate and have no residual antibacterial
effect. Khademi, Mohammadi and Havaee67 high-
light that only chlorhexidine and tetracycline have
the aforementioned property.
Tissue dissolving effect
Several studies have searched for a product that
meets the properties necessary for a root canal ir-
rigant: antimicrobial activity, non-toxic to periapi-
cal tissues, soluble in water and organic matter dis-
solving ability.31 In 1941, Grossman and Meiman70
demonstrated the importance of tissue dissolving
ability of an endodontic irrigant, determining that
success of endodontic treatment relies on pulp tis-
sue elimination from the root canal. Zehnder19 cor-
roborates Grossman and Meiman70 and asserts that
the ideal cleaning of root canals is crucial for end-
odontic treatment, given that removal of tissues
and bacterial residue would prevent the tooth from
becoming a source of infection. Therefore, the ne-
crotic tissue dissolving ability of irrigation agents
was assessed. An
in vitro
study revealed that 1%
sodium hypochlorite had a substantial dissolution
capacity, unlike 10% chlorhexidine.71 According to
Moorer and Wesselink,72 tissue dissolution depends
on the frequency of agitation, the amount of or-
ganic matter in relation to the irrigant, and on the
tissue surface area available for contact with the
irrigant. Okino et al73 assessed the tissue dissolving
ability of sodium hypochlorite at different concen-
trations, 2% chlorhexidine digluconate water so-
lution, chlorhexidine gel and distilled water. Frag-
ments of bovine pulp were submerged in 20 mL of
each solution. Both distilled water and chlorhexi-
dine solutions did not dissolve the pulp during the
six hours of the experiment.
Considering the experiments performed, it can
be concluded that a disadvantage of chlorhexidine
is its inability to dissolve tissues.31

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 41
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
Interaction between chlorhexidine and
sodium hypochlorite
An in vivo study conducted by Zamany74 employed
two therapeutic protocols in which, after chemo-
mechanical preparation with NaOCl, a final irriga-
tion with 4 mL of saline solution or 2% chlorhexi-
dine was performed during 30 seconds. Evaluation
was carried out by means of culture mediums and
biological indicators collected from tooth canals.
The chlorhexidine protocol produced a positive cul-
ture in one out of 12 cases, whereas the saline solu-
tion protocol produced a positive culture in seven
out of 12 cases. The use of 2% chlorhexidine diglu-
conate as an extra irrigant used after biomechanical
preparation improved the efficiency of endodontic
therapy with regard to antimicrobial activity.
For treatment before root canal filling, Zehnder19
recommends irrigation with sodium hypochlorite
to dissolve organic tissue, irrigation with EDTA
to eliminate the smear layer and irrigation with
chlorhexidine to increase antimicrobial spectrum
and substantivity. Despite the visible increase in an-
timicrobial efficacy produced by the combination of
irrigants,41 chemical interactions, such as precipita-
tion and color change that result from a combination
between NaOCl and chlorhexidine, must be taken
into account.19,26,75 This corroborates the study con-
ducted by Basrani et al76 who sought to determine
the minimum concentration of sodium hypochlorite
causing pigmentation and precipitation when associ-
ated with 2% chlorhexidine. The resultant precipitate
was qualified and quantified. All sodium hypochlo-
rite solutions in combination with 2% chlorhexidine
digluconate led to color alterations, even with Na-
OCl at low concentrations (0.023%). The formation
of precipitate was also observed until the sixth di-
lution (0.19%). Both pigmentation and precipitation
were directly proportional to the concentration of
sodium hypochlorite. By-products were formed in
the mixtures with 3% and 6% sodium hypochlorite.
One example is the formation of parachloraniline, a
fragment that results from hydrolysis of chlorhexi-
dine digluconate. In other words, a by-product that
theoretically forms another by-product. Fragmenta-
tion occurs in the bond between carbon and nitrogen
(guanidine group) of which dissociation requires lit-
tle energy. The clinical importance of these findings
relies on the pathological potential of parachlorani-
line, as well as on other by-products that result from
the mixture. Parachloraniline has a carcinogenic
potential and causes methemoglobinemia and cya-
noses, being cytotoxic.77 Other by-products might
have pathological action related to their own mo-
lecular character, as it is the case of action exerted
by higher reactivity (free radicals). The formation of
precipitate may be explained by the acid-base re-
action that results from mixing sodium hypochlorite
and chlorhexidine.31
The precipitate that results from mixing sodium
hypochlorite and chlorhexidine is also known as flu-
conation.78 Basrani et al76 observed that it produces
an orangish-brown solution which, once in the pulp
chamber, chemically stains the dentin tubules and, as
a consequence, changes tooth color78-81 and interferes
in root canal filling.28,82 A spectrophotometric analysis
revealed the presence of calcium, iron, magnesium,
copper, zinc and manganese in the precipitate.78
According to Heling and Chandler,83 associating
chlorhexidine with EDTA also forms a milky-white
precipitate. When combined with saline solution and
ethanol, they produce salt. Thus, when sodium hypo-
chlorite is used as an irrigation solution during me-
chanical preparation, chlorhexidine may be used as a
final irrigant or intracanal dressing only after sodium
hypochlorite is completely removed from the root ca-
nal,82 so as to avoid interaction between solutions.5
As complementary irrigation solutions, distilled water
and saline solution are recommended.
Cytotoxicity
Chlorhexidine is stable and has low citotoxicity.6 It
is minimally absorbed by the mucosa and skin, it is well
tolerated in animals, when administered via parenteral
and intravenously, it seems not to cross the placental
barrier, it does not cause systemic toxic side effects
or alterations in the oral microbiota.84-88 With regard
to the metabolic pathways of chlorhexidine, whenever
ingested, it reduces plasma levels and is excreted in
feces (90%) and urine (10%). The frequency of meta-
bolic segmentation by oral intake is also low, with no
evidence of parachloraniline formation. When carried
in the bloodstream of dogs, it is metabolized by the
liver and kidney, producing polar metabolites, while
chlorhexidine remains intact in the bile.87

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 42
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
Tanomaru Filho et al6 assessed the inflammato-
ry response of different endodontic solutions used
in rats. 0.5% sodium hypochlorite, 2% chlorhexidine
digluconate and saline solutions were injected in the
peritoneal cavity of the animals which were killed af-
ter 4h, 24h, 48h and seven days. Results revealed that
sodium hypochlorite induced inflammatory response,
whereas chlorhexidine digluconate did not provoke
any significant response. In 2005, Ribeiro et al89 as-
sessed the genotoxicity (potential damage to DNA)
of formocresol, paramonochlorophenol, calcium hy-
droxide and chlorhexidine against the ovary cells of
Chinese hamsters. The results revealed that none of
the agents damaged the DNA. Faria et al90 assessed
the cytotoxicity of chlorhexidine digluconate by
means of observing tissue lesions (edema/inflam-
mation) in rats’ paws. Assessment was complement-
ed by histopathological examination and analysis of
cell death and stress in culture of fibroblasts. Edema
(inflammation) was observed as a result of exposing
the lesions to chlorhexidine digluconate at differ-
ent concentrations (0.125; 0.25; 0.5 and 1%). Edema
subsided after 14 days at the two lowest concentra-
tions. At 0.125%, no tissue necrosis was observed
despite mild inflammation, whereas at 0.25%, small
foci of necrosis were found. Edema persisted after
14 days at the two highest concentrations. Inflam-
mation and larger foci of tissue necrosis were also
observed. The authors concluded that chlorhexidine
digluconate may produce an adverse effect on the
resolution of apical periodontitis. Additionally, their
results point to higher biocompatibility in concen-
trations equal to or less than 0.25%. Furthermore,
lower concentrations are characterized by promot-
ing cell apoptosis, whereas higher concentrations
cause stress and cellular necrosis.
Thus, the concentrations of chlorhexidine clini-
cally used have acceptable biocompatibility,31 with
relative absence of cytotoxicity.15
The first studies about the toxicology of chlorhexi-
dine were conducted by Foukes91 who established the
lethal dose of chlorhexidine orally and intravenous-
ly taken, and tolerance to chronic administration.
The author concluded that chlorhexidine has unusu-
ally low toxicity for both, animals and humans. Addi-
tional research conducted by Davies and Hull84 con-
firmed the findings of other authors, determining
the lethal dose of 50 (LD 50) for chlorhexidine ap-
plied by intravenous injection (22 mg/Kg/day) , and
LD 50 (1800 mg/kg/day) for oral administration.
These results were obtained from experiments car-
ried out with species of rodents (rabbits and mice)
and ruminants (cattle). Hugo and Longworth93 found
no harmful effect for chlorhexidine digluconate
orally taken. To test the carcinogenic potential, four
groups with 224 rats each were used. The animals
received doses of 5, 25 or 50 mg/kg of body weight
and were tested for two years. By the end of the dos-
age, peak levels dropped by half within one to two
weeks. Chlorhexidine levels in the brain, lung, liver,
kidney, mesenteric nodes and other lymph nodes,
as well as in the blood were determined at regular
intervals during the experiment and after the end
of administration during three, six and nine weeks.
No histological changes were found. The concentra-
tion of chlorhexidine in the liver was high in the final
controls, but decreased to half after one and two
weeks. There was no incidence of neoplasm in the
control and treated groups. The extremely low acute
oral toxicity found in animals has been confirmed
in humans in the last 30 years of experience, with
unrestricted use. Pereira94 conducted a research on
acute and chronic toxicity of chlorhexidine digluco-
nate orally taken by mice and found an increase in
weight gain in comparison to the control group, sig-
nificant reduction in the number of deaths attribut-
able to the inhibition of intercurrent infections in the
treated groups and absence of teratogenic effects.
Case85 and Rushton86 concluded that percutaneous
absorption is practically null.
action over biofilm
According to Costerton, Stewart and Greenberg,95
biofilm is a structured community of microorganisms
surrounded by a matrix of polysaccharides produced
and adhered to live or inert surfaces. The cells com-
prising the biofilm structure are phenotypically differ-
ent from planktonic cells (microorganisms presented
in a free and disorganized form), since they are less
susceptible to antimicrobial substances.96
Biofilm control occurs as a result of the anti-
septic property of chlorhexidine associated with
adsorption (ability to be retained on an oral sur-
face and be slowly released), assuring an extended

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 43
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
antimicrobial environment.60,97 Adsorption is ex-
plained by electrostatic interaction. Due to its cat-
ionic characteristic, chlorhexidine has a strong affin-
ity for anions, such as phosphate ions from the cell
wall of oral microbiota which normally colonizes the
tooth surfaces,98 thus reducing adherence and colo-
nization of tooth surfaces. This process enhances
cell wall permeability and, as a consequence, leads
to cytoplasm rupture and causes cell death.98 Due to
its bactericide and bacteriostatic effect, chlorhexi-
dine inhibits the development of microbial plaque
development.64 This anti-plaque effect is probably
the most significant property of chlorhexidine.99
One of the major mechanisms of resistance of
biofilm is associated with failure of agents in pen-
etrating its extension. Polymeric substances, such as
those found in biofilm matrix, reduce the diffusion
of chemical substances and antibiotics. Solutes tend
to diffuse more slowly. The speed of penetration
varies according to the type of microorganism and
the composition of the exopolysaccharide matrix.
A second mechanism of resistance is associated
with the ability of a microorganism present in biofilm
to survive after long periods of food shortage which
decreases its growth rate. Microorganisms with re-
duced growth rate, or no growth, are less sensitive
to chemical substances.95,99,100,101 Mohammadi and
Abbott31 reported that a microorganism growing in
biofilms is two to 1,000 times more resistant than its
correspondent planktonic form.
Studies conducted with biofilm composed by
a single species102,103 and apical dentin biofilm104
revealed that an increase in sodium hypochlorite
concentration (varying from 2.25 to 6%) and 2%
chlorhexidine solution were effective against the mi-
croorganisms tested. Mechanical agitation enhances
antimicrobial activities of chemical substances, par-
ticularly favoring liquid agents such as 5.25% sodium
hypochlorite and 2% chlorhexidine.102 Chlorhexidine
has a significantly lower effect on microbial biofilm
in comparison to hypochlorite.31
Tyler et al105 assessed the distribution and trans-
port of chlorhexidine digluconate and glucose in
Candida albicans
biofilm. Their results confirmed
the diffusion capacity of chlorhexidine digluconate
through biofilm, which is not uniform, thus suggest-
ing that chlorhexidine preferentially binds to sites
of microbial cells and/or passes through microca-
nals present in biofilm. The presence of microca-
nals suggests that biofilm is somehow organized or
at least has a complex structure, since microcanals
allow the entrance of nutrients and excreta output.
Additionally, the authors concluded that the action
of chlorhexidine is directly proportional to con-
centration that tends to decrease as chlorhexidine
goes deeper into the biofilm. Glucose does not dif-
fuse uniformly either, which results in areas with
nutrients shortage.
Clegg et al104 assessed the efficacy of disaggre-
gating and removing polymicrobial biofilm produced
by sample collected from teeth of patients diag-
nosed with periapical lesion 3-mm in diameter asso-
ciated with pulp necrosis and who were not treated
by antibiotic drugs. The samples were seeded in
culture medium and evaluated microscopically. 2%
chlorhexidine proved not to affect biofilm or elimi-
nate bacteria. Nevertheless, it generated absence of
microbial growth (culture medium). 6% sodium hy-
pochlorite was the only substance that favored ab-
sence of bacteria, removed biofilm and promoted
absence of microbial growth (culture medium).
Antibacterial activity
Its antibacterial activity is explained by the ability
of chlorhexidine to be rapidly attracted by the nega-
tive charge of bacterial surface, and adsorbed to the
cell membrane by electrostatic interactions, prob-
ably by hydrophobic bindings or hydrogen bridges.
Adsorption is concentration-dependent. In higher
concentrations, it causes not only precipitation and
coagulation of cytoplasmic proteins, but also bacte-
rial death; whereas in low concentrations, cell mem-
brane integrity is altered, resulting in extravasation
of low molecular weight bacteria components.60,93,106
Thus, the molecule cationic end binds to the pellicle
with negative charge (anionic), whereas the other
cationic end is free to interact with bacteria that aim
at colonizing the tooth.45 In Endodontics, chlorhexi-
dine is recommended for root canal irrigation dur-
ing chemo-mechanical preparation,106 since it inhib-
its bacterial growth in endodontic infections.51,56,107
The action of chlorhexidine depends on the sus-
ceptibility of microorganisms; Gram-positives have
higher susceptibility to chlorhexidine in comparison

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 44
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
to Gram-negatives.107 Some species of Streptococci
seem to retain an additional amount of chlorhexi-
dine in their extracellular polysaccharide capsules,
which might be related to the high sensitivity of
Streptococci to chlorhexidine.108
In 1982, Delany et al13 conducted an
in vitro
study
on the antimicrobial action of 0.2% chlorhexidine
gluconate solution used as irrigant and intracanal
dressing on root canal microbiota of recent extract-
ed necrotic pulp of human teeth. Bacterial growth
was observed by inoculation of dentin debris on
agar, which caused a significant reduction in the
number of bacteria in both endodontic procedures.
Heling et al53 conducted an
in vitro
study to as-
sess the antibacterial effect of 2% chlorhexidine glu-
conate at 20% used, in a in a slow release system, as
intracanal dressing in bovine incisors contaminated
with S. faecalis. The slow release system consisted of
strips containing glutaraldehyde as vehicle and 1.2
mg of 20% chlorhexidine as active agent. The mi-
crobiological analysis of dentin removed from canal
walls revealed that both forms of dressing were ef-
fective for depth of 0.5 mm in experimental periods
of 24, 48 hours and seven days.
Siqueira Jr. and Uzeda56 assessed the antibacte-
rial activity of 0.12% chlorhexidine digluconate gel,
10% metronidazole gel, calcium hydroxide with dis-
tilled water, calcium hydroxide with PMCC camphor-
ated paramonochlorophenol and calcium hydroxide
with glycerin applied on strict and facultative anaer-
obic bacteria commonly found in endodontic infec-
tions. Their results revealed that calcium hydroxide
paste with PMCC and chlorhexidine were effective
for all species of bacteria tested (strict anaerobic —
Porphyromonas endodontalis, P. gingivalis, Actinomyce-
sisraelis, Fusobacterium nucleatum, Propionibacterium
acnes and Campylobacter rectus; and facultative an-
aerobic — Staphylococcus aureus, Streptococcus mu-
tans, S. sanguis, S. salivarius, Enterococcus faecalis and
Actinomyces viscosus). Metronidazole inhibited the
growth of all strict anaerobic species, whereas cal-
cium hydroxide with distilled water or glycerin were
ineffective.
Lindskog, Pierce and Blomlöf57 assessed the ef-
fect of 10% chlorhexidine gluconate gel used as in-
tracanal dressing during one month on inflammato-
ry root resorption induced in monkeys. The authors
found a reduction in the resorption process due to
the antimicrobial action of chlorhexidine inside den-
tin tubules and on periodontal ligament cells.
Ferraz51 conducted an
in vitro
research on
chlorhexidine gel used as endodontic irrigant in com-
parison to other irrigants commonly used in End-
odontics. The author concluded that 2% chlorhexi-
dine gel or solution showed the highest averages of
inhibition halos against all microorganisms tested by
the agar diffusion test. Chlorhexidine gel produced,
in vitro
, higher inhibition halos of microbial growth
when compared to chlorhexidine solution at equiva-
lents concentrations. However, with no statistically
significant differences. Similarly to 5.25% sodium
hypochlorite, 2% chlorhexidine solution produced
negative cultures after 45 seconds of contact with
Enterococcus faecalis, acting more rapidly than other
irrigants. Teeth irrigated with 2% chlorhexidine gel
had a higher number of negative microbiological
cultures (80%); after
in vitro
instrumentation, 2%
chlorhexidine gel significantly reduced smear layer
in comparison to 2% chlorhexidine solution and
5.25% sodium hypochlorite.
Menezes et al52 conducted an
in vitro
study to as-
sess the efficacy of sodium hypochlorite and 2%
chlorhexidine used as irrigation solution. Teeth had
been contaminated by Enterococus faecalis. The authors
concluded that chlorhexidine was more effective.
Haapasalo et al44 conducted a literature review
in which they highlight that the use of chlorhexidine
at 0.2 to 2% might offer an additional advantage
against resistant microorganisms disseminated by
the root canal system. This is due to the ability of
chlorhexidine to increase bacterial cell or cell wall
permeability; act inside fungi cytoplasm membrane;
cause coagulation of intracellular constituents at
high concentrations. Other advantages include re-
sidual antimicrobial action and substantivity; rela-
tively low toxicity, wide spectrum of action and
efficacy against Enterococcus faecalis and Staphylo-
coccus aureus. According to the authors, chlorhexi-
dine efficacy decreases in contact with organic mat-
ter, mycobacteria, bacterial spores and virus, all of
which are resistant. Additionally, chlorhexidine has
cytotoxicity at high concentrations; chlorhexidine
gel is less effective against Enterococcus faecalis in
comparison to solution; chlorhexidine combinations

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 45
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
are so or less effective than its compounds alone;
when in contact with tooth dentin (organic com-
pounds), chlorhexidine efficacy decreases, but is not
completely neutralized; albumin from bovine plasma
neutralizes chlorhexidine action and does not act as
a tissue solvent.
Dametto et al66 conducted an
in vitro
study to as-
sess the antimicrobial activity of 2% chlorhexidine
gel against Enterococcus faecalis in comparison to
other endodontic irrigants (2% chlorhexidine solu-
tion and 5.25% sodium hypochlorite). 2% chlorhexi-
dine gel and 2% chlorhexidine solution significantly
reduced E. faecalis at post-treatment and final phases.
5.25% sodium hypochlorite also reduced E. faecalis
immediately after root canal instrumentation. How-
ever, it did not completely eliminate E. faecalis from
the root canal. The authors concluded that 2%
chlorhexidine gluconate (gel and solution) had high-
er antimicrobial capacity against E. faecalis in com-
parison to 5.25% sodium hypochlorite seven days
after biomechanical preparation.
In 2006, the results of a research conduct-
ed by Fachin, Nunes and Mendes92 agreed with
Jeansonne et al15 who affirmed that 2% chlorhexi-
dine is an effective antimicrobial that produces
results statistically similar to 5.25% sodium hy-
pochlorite, and of which substantivity increases
antimicrobial performance.
Wang et al109 assessed the clinical efficacy of 2%
chlorhexidine gel with regard to the reduction of in-
tracanal bacteria during root canal instrumentation.
The additional antibacterial effect of calcium hy-
droxide associated with 2% gel used as an intracanal
dressing was also assessed. The authors concluded
that 2% chlorhexidine gel effectively decontami-
nates the root canal, and, when used as intracanal
dressing, does not produce additional significant ef-
fects on bacterial reduction.
Pretel et al110 concluded that 2% chlorhexidine is
a feasible irrigation solution due to its specific char-
acteristics of substantivity and high antibacterial ef-
fect. According to the authors, chlorhexidine proves
more effective considering its penetration and sub-
stantivity inside dentin tubules.
Its bactericidal activity is faster than its fungicide
activity and strongly depends on pH. Its maximum
activity can only be achieved with pH 8 (Neobrax111).
Antifungal activity
Chlorhexidine digluconate has a wide spectrum of
action59,112 with potent antifungal action against
Can-
dida albicans
.113,114 Fungi (or yeast) represent a small
portion of oral microbiota. Candida is the species of
fungi most commonly found in healthy (30 to 45%)
as well as in medically compromised individuals
(95%).115 These fungi might be involved in cases of
persistence and secondary infection associated with
relapse of periapical lesions, given that they are mi-
croorganisms strongly associated with therapeutic
failures.17,59,65,74,75,114,116-119 For this reason, endodon-
tic irrigants should include these microorganisms in
within their spectrum of activity.31 According to Wal-
timo et al,113 the presence of fungi in infected root
canals varies between 1 to 17%.
In 1999, Sen, Safavi and Spangberg120 assessed
the antifungal effects of 0.12% chlorhexidine and 1
to 5% sodium hypochlorite on root canals. They per-
formed root sections and removed smear layer in half
of the specimens. Root canals were inoculated with
Candida albicans
for 10 days. Subsequently, root sec-
tions were treated with 3 mL of the irrigation solution
during one, five, 30 and 60 minutes. The authors ob-
served that, in the presence of smear layer, the anti-
fungal activity of all irrigants started after 60 minutes,
only. Antifungal activity was higher in teeth of which
the smear layer was removed. After 30 minutes, 5%
sodium hypochlorite showed antifungal activity of
70% and after one hour, it was totally effective. 0.12%
chlorhexidine and 1% sodium hypochlorite proved to
be totally effective after an hour.
Waltimo et al113 assessed the antifungal action of
calcium hydroxide, 0.5% chlorhexidine acetate, 0.05%
iodinated potassium iodide and sodium hypochlorite,
alone and in combination. To this end, they used ab-
sorbent paper points contaminated with
Candida albi-
cans
, directly exposed to the disinfectants, for periods
of 30 seconds, five minutes, one and 24 hours. In com-
parison to calcium hydroxide associated with distilled
water, 0.5% and 0.05% chlorhexidine proved more ef-
fective. After 24h, the association of 0.5% chlorhexi-
dine with calcium hydroxide P.A. was also more effec-
tive than calcium hydroxide associated with distilled
water and less effective than 0.5% chlorhexidine alone.
Alexandra et al121 conducted an in vitro study
in which the efficacy of four chemical substances

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 46
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
used as intracanal dressing were compared: cal-
cium hydroxide, chlorhexidine gel, PerioChip (As-
tra Zeneca) and chlorhexidine gel associated with
calcium hydroxide. Saline solution was used as the
control group. The substances were tested in three
different periods (three, eight and 14 days) using hu-
man teeth previously contaminated with E. faecalis.
Calcium hydroxide eliminated Enterococcus faecallis
within three to eight days, but it was effective in the
14-day group, probably due to a pH drop. The dif-
ferent formulations of chlorhexidine were effective
in eliminating E. faecalis from dentin tubules, with
chlorhexidine gel showing the best results.
Siqueira Jr. et al122 assessed the efficacy of four
intracanal dressings in decontaminating the root
canal of bovine teeth experimentally infected with
Candida albicans
. Infected dentin cylinders were
exposed to four different dressings: calcium hy-
droxide and glycerin; calcium hydroxide and 0.12%
chlorhexidine digluconate; calcium hydroxide with
camphorated paramonochlorophenol and glycerin;
0.12% chlorhexidine digluconate with zinc oxide.
Specimens were in contact with the dressings during
1 hour, 2 and 7 days.
Candida albicans
viability after
exposure was evaluated by means of incubating the
sample in culture medium to compare the efficacy of
the dressing in dentin disinfection. Results revealed
that specimens treated with calcium hydroxide as-
sociated with camphorated paramonochlorophenol
and glycerin, or with chlorhexidine combined with
zinc oxide were completely decontaminated after
1-hour exposure. Calcium hydroxide with glycerin
eliminated C. albicans after 7 days, only. Calcium hy-
droxide associated with chlorhexidine proved inef-
fective to disinfect dentin, even after one week of
exposure. Calcium hydroxide with camphorated par-
amonochlorophenol and glycerin, as well chlorhexi-
dine digluconate associated with zinc oxide proved
to be the most effective in eliminating C. albicans.
Ruff, McClanahan and Babel123 compared the
antifungal efficacy of 6% sodium hypochlorite, 2%
chlorhexidine, 17% EDTA and MTDA BioPuro with
final rinse as canal preparation, in which teeth were
contaminated with
Candida albicans
. Teeth were di-
vided into four groups: Group 1 – 1 mL of 6% so-
dium hypochlorite for 1 min; Group 2- 0.2 mL of 2%
chlorhexidine for 1 min; Group 3 -5 mL of MTDA
BioPuro for 5 min, following the manufacturer’s in-
structions; Group 4 – 1 mL of 17% EDTA for 1 min.
Results showed that 6% sodium hypochlorite and
2% chlorhexidine were equally effective and signifi-
cantly superior to the other groups. MTDA was sig-
nificantly superior to 17% EDTA.
Ballal et al124 analyzed the antiseptic action of dif-
ferent intracanal dressings. They used
Candida albi-
cans
and Enterococcus faecalis as microbiological in-
dicators and conducted an observation on inhibition
halos of microbial growth in solid medium culture.
All intracanal dressings tested exhibited inhibition
halos. Within 24 hours of action against C.albicans,
calcium hydroxide paste in water proved to be
the most effective, whereas against E.faecalis, 2%
chlorhexidine gel had the best action. After 72 hours,
2.% chlorhexidine gel was the most effective dress-
ing against C.albicans and E.faecalis, whereas the
combination of the two substances yielded the
worst results against both biological indicators. The
authors concluded that 2% chlorhexidine gel is more
efficient than calcium hydroxide paste, whether as-
sociated with water or 2% chlorhexidine gel.
Intracanal dressing
Chemo-mechanical preparation significantly re-
duces microbiota in infected root canals. However,
Bystrom, Claesson and Sundqvist;126 Sjögren et al127
as well as Ando and Hoshino125 highlighted the need
for intracanal dressing use to prevent those bacteria
surviving to chemo-mechanical preparation in a suf-
ficient number and adequate environment from multi-
plying between treatment sessions. Thus, the need for
root canal disinfection through chemo-mechanical
preparation is clear. It can be achieved not only by
the proper use of an intracanal dressing that has anti-
microbial properties and functions as a physical bar-
rier,3,127-130 but also by proper filling of the root canal
system and appropriate coronal sealing.132 Addition-
ally, intracanal dressing aims at reducing periapical
lesions, solubilizing organic matter, neutralizing toxic
products, controlling persistent exudate, controlling
inflammatory external root resorption and stimulat-
ing repair by means of mineralized tissue.133
Chlorhexidine has been highly recommended as
intracanal dressing due to its immediate antimicro-
bial action; wide antibacterial spectrum of action

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 47
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
against Gram-positive and Gram-negative bacteria,
whether anaerobic, facultative and aerobic; yeast and
fungi20,23,59,112 (especially
Candida albicans
);113,120 rela-
tively absence of toxicity;49,86 dentin adsorption ca-
pacity and slow release of its active substance, which
extends its residual antimicrobial activity.15,16,53,54,134
Delany et al13 demonstrated the effect of 0.2%
chlorhexidine gluconate used as intracanal dressing
on the reduction of remaining antimicrobial popula-
tion after root canal instrumentation. Due to its wide
antimicrobial spectrum, chlorhexidine has been
largely used in Endodontics. It has been recom-
mended as digluconate salt, liquid or gel at different
concentrations, as well as intracanal dressing.13,53-57
Ohara et al14 assessed the antimicrobial effects of
six irrigants against anaerobic bacteria and highlighted
that chlorhexidine was the most effective. With regard
to the elimination of E.faecalis from inside of dentin tu-
bules, chlorhexidine used as intracanal dressing yield-
ed better results than calcium hydroxide.53
Lenet et al135 conducted an
in vitro
study to com-
pare the residual antimicrobial activity of 0.2 and 2%
chlorhexidine gel in a system of controlled release,
and calcium hydroxide associated with saline solu-
tion used as intracanal dressing in bovine incisors,
during seven days. After the experimental period,
the specimens were inoculated in E.faecalis during
21 days. Results revealed that 2% chlorhexidine gel
had no viable bacteria in all dentin depths.
According to Vianna,134 2% chlorhexidine gel had
higher antimicrobial activity. The combination be-
tween calcium hydroxide and 2% chlorhexidine gel
decreased the antimicrobial activity of chlorhexidine,
however, it increased the activity of calcium hydroxide.
Gomes et al136 assessed the efficacy of 2%
chlorhexidine digluconate gel and calcium hydrox-
ide used as intracanal dressing at different time
intervals (one, two, seven, 15 and 30 days). To this
end, roots from bovine teeth previously infected with
E.faecalis were used. 2% chlorhexidine gel; calcium
hydroxide associated with polyethylene glycol 400;
and 2% chlorhexidine gel associated with calcium
hydroxide were used as intracanal dressing. The au-
thors observed that 2% chlorhexidine gel inhibited
bacterial growth in the infected dentin samples in all
periods tested. The combination of calcium hydrox-
ide and polyethylene glycol 400 was inefficient in
eliminating bacteria during all periods. Absence of
dentin contamination was found in periods of one
and two days for samples comprising the associa-
tion of 2% chlorhexidine gel and calcium hydroxide.
As for periods of seven and 15 days, there was a de-
crease in antimicrobial activity and, after 30 days, all
samples from this group were contaminated. In con-
clusion, 2% chlorhexidine gel has a wide antimicro-
bial activity against E.faecalis. However, the authors
highlighted that this property might decrease with
time if the medication is used for long periods.
Pinheiro et al137 conducted an
in vitro
study to
assess the antimicrobial activity of 50% calcium
hydroxide and 2% chlorhexidine gel used alone
or in combination. The following microorganisms
were tested: Enterococcus faecalis,
Candida albi-
cans
, Escherichia coli, Sthaphylococcus aureus, Stah-
phylococcus epidermis and Pseudomonas aeruginosa.
After 24 and 48 hours, they assessed the inhibition
halos. The halos formed against E. coli, S. aureus and
S. epidermis were discrete and of similar dimension.
Calcium hydroxide and 2% chlorhexidine gel used
alone showed antimicrobial activity against all mi-
croorganisms tested. When combined, the substanc-
es showed higher inhibition halos against E.faecalis
and C.albicans in comparison to calcium hydroxide
used alone. However, the combination of substances
showed smaller halos, for both microorganisms, in
comparison with 2% chlorhexidine gel used alone.
In 2006, Montagner et al138 assessed the anti-
microbial action of intracanal dressings on exter-
nal surface root against different microorganisms.
288 roots extracted from upper canines were divided
into two groups, with and without cementum. The
following microorganisms were isolated from clini-
cal samples and analyzed: Enterococcus faecalis,
Can-
dida albicans
, Actinomyces viscosus and Porphyromon-
as gingivalis. 2% chlorhexidine gel; 2% chlorhexidine
gel and calcium hydroxide (1:1); 2% chlorhexi-
dine gel, calcium hydroxide and zinc oxide (1:1:1);
calcium hydroxide and saline solution; saline solu-
tion (positive control) were used as intracanal dress-
ings. The best antimicrobial effect was produced by
2% chlorhexidine gel, followed by 2% chlorhexidine
gel and calcium hydroxide; 2% chlorhexidine gel,
calcium hydroxide and zinc oxide; and calcium hy-
droxide and saline solution. A. viscosus (2.85 mm)

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 48
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
was most sensitive to the medications, followed by
E. faecalis (1.84 mm), C. albicans (0.95 mm) and P.
gingivalis (0.82 mm). Presence or absence of ce-
mentum did not interfere in the substance capac-
ity of reaching the outer root surface and exerting
its antimicrobial action. The authors concluded that
intracanal dressings associated with chlorhexidine
were able to diffuse through the dentin and reach
the outer root surface. The combination between
calcium hydroxide and saline solution did not show
antimicrobial activity in the outer root surface within
72 hours. Conversely, 2% chlorhexidine gel associ-
ated with calcium hydroxide and zinc oxide revealed
rapid diffusion capacity in root dentin, causing inhi-
bition of bacterial growth.
Gomes et al139 investigated the antimicrobial ac-
tivity of intracanal dressings by means of the agar
diffusion test as well as by direct contact. The follow-
ing biological indicators, which represent endodontic
infection, were included: Enterococcus faecalis,
Can-
dida albicans
, Staphylococcus aureus, Porphyromo-
nas endodontalis, Porphyromonas gingivalis and Pre-
votella intermedia. Agar diffusion and direct contact
tests revealed that 2% chlorhexidine digluconate gel
(1% Natrosol “hydroxyethil cellulose” with pH 7.0)
had the highest efficacy; calcium hydroxide in 2%
chlorhexidine digluconate gel, intermediate efficacy;
and calcium hydroxide with sterile water as vehicle,
the worst. The latter did not produce inhibition halos.
There was susceptibility of Enterococcus faecalis and
Candida albicans
to intracanal dressings, following
the order previously related, as well as inactivity of
calcium hydroxide in water in the agar diffusion test.
The authors explained that the inability of calcium
hydroxide in water to diffuse throughout agar is due
to the low solubility of hydroxide, as well as the buf-
fer effect and protein coagulation action occurring
in the agar. These effects are liable to occur in vivo,
which avoids penetration of the intracanal dressing
into the dentin tubules and irregularities of the root
canal. The antimicrobial action of 2% chlorhexidine
digluconate gel is reduced when the substance is as-
sociated with calcium hydroxide.
Fachin, Nunes and Mendes92 assessed the ef-
ficacy of four intracanal dressings (camphor-
ated paramonochlorophenol, calcium hydroxide,
2% chlorhexidine gel and 1% sodium hypochlorite)
in cases of pulp necrosis with periapical lesion, by
means of clinical and radiographic control. All solu-
tions were effective to decrease the size of apical
lesions. Initial results reveal that, after three months,
the highest percentages of reduction in lesion diam-
eter occurred with 2% chlorhexidine gel.
The results of this research are encouraging with
regard to the use of 2% chlorhexidine gel as intraca-
nal dressing in cases of pulp necrosis. Thus, these re-
sults corroborate Heling et al,54 Barbosa et al,55 Lenet
et al135 and Rosa et al140 and confirm the efficacy of
2% chlorhexidine used as intracanal dressing.
Ballal et al124 analyzed the antiseptic action of dif-
ferent intracanal dressings. They used
Candida albi-
cans
and Enterococcus faecalis as microbiological in-
dicators and conducted an observation on inhibition
halos of microbial growth in solid medium culture. All
tested intracanal dressings exhibited inhibition halos.
Within 24 hours of action against C.albicans, calcium
hydroxide paste in water proved to be the most effec-
tive, whereas against E.faecalis, 2.0% chlorhexidine gel
had the best action. After 72 hours, 2.0% chlorhexi-
dine gel was the most effective medication against
C.albicans and E.faecalis, whereas the combination of
the two substances yielded the worst results against
both biological indicators. The authors concluded
that 2% chlorhexidine gel is more efficient than cal-
cium hydroxide paste, whether associated with water
or 2% chlorhexidine gel.
Marion et al141 reported a case conducted by means
of a new therapeutic protocol, in which calcium hy-
droxide was associated with 2% chlorhexidine gel and
zinc oxide and used as filling paste for avulsed tooth.
The combination between calcium hydroxide, 2%
chlorhexidine gel and zinc oxide was also assessed by
Souza-Filho et al,142 Almeida et al143 and Montagner et
al144 in an
in vitro
study that revealed the antimicrobial
action and capacity to keep an alkaline pH of the sub-
stance. Other case reports found in the literature138,145
reveal that this association has a fast diffusing capacity
in root dentin, causing inhibition of bacterial growth
on the outer surface of the root canal. The case report
conducted by Marion et al141 revealed absence of signs
and symptoms in tooth treated with filling paste, which
remained after a three-year follow-up, thus proving the
efficiency of this medication in the treatment of trau-
matized permanent teeth.

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 49
Marion J, Pavan K, Arruda MEBF, Nakashima L, Morais CAH
Rheological action
This action is found in chlorhexidine gel, since it
refers to the capacity of maintaining debris in sus-
pension inside the root canal.5
When the pulp chamber and root canal are flooded
with chlorhexidine gel and mechanical preparation
of root canal system is initiated (instrumentation),
both inorganic and organic debris (smear layer)
— detached from root canal walls — accumulate
in the amorphous mass of gel which captures and
keep them suspended. Subsequently, active irriga-
tion with saline or distilled water removes the de-
bris, preventing them from accumulating in the root
canal walls and, as a result, exposing the entrance
of dentin tubules. In other words, it considerably re-
duces the formation of smear layer, thus improving
the efficacy of EDTA as a chelating substance and
increasing treatment prognostic.2,5,27,146,147
Ferraz et al2 investigated the antimicrobial action
of chlorhexidine gel and solution over Enterococcus
faecalis and its capacity of cleaning the root canal
wall, in comparison to 5.25% sodium hypochlorite.
To this end, 70 recently-extracted single-rooted
teeth were selected. They were prepared up to the
apical foramen with file #40, submitted to a 17%
EDTA wash with ultrasound, sterilized and infected.
Subsequently, root canals underwent instrumenta-
tion with 2% chlorhexidine gel, chlorhexidine so-
lution or 5.25% sodium hypochlorite. Water and
Natrosol gel were used as control. As for suppres-
sion of bacterial growth, no statistical differences
were found between groups. Nevertheless, with re-
gard to cleaning, the highest number of open dentin
tubules was found in chlorhexidine gel, followed by
chlorhexidine solution and 5.25% sodium hypochlo-
rite, which confirmed the capacity of chlorhexidine
gel in preventing smear layer formation, probably as
a result of the mechanical action of Natrosol gel.
Allergic reactions
No adverse effects have been published regard-
ing the use of chlorhexidine as irrigant or intracanal
dressing.5 On the other hand, animal studies have
shown that 2.0% chlorhexidine used as intracanal
dressing did not induce intense inflammatory re-
sponse when injected into the peritoneal cavity of
mice.148,149 Chlorhexidine has a limited number of
adverse effects, such as desquamative gingivitis, tooth
and tongue discoloration or dysgeusia (distortion of
the sense of taste). Contact sensitivity to chlorhexi-
dine was first described by Calnan.150 Contact with the
conjunctiva may cause permanent damage, whereas
accidental contact with the tympanum might cause
ototoxicity.151 It may also cause contact urticaria,
photo-sensibility, fixed drug eruption and occupation-
al asthma. Patients with leg ulcers and eczema have
particular risks of contact allergy (besides doctors
and dentists). Contact sensitivity to chlorhexidine
seems to be generally rare. Some studies have dem-
onstrated a high rate of sensitization, around 2%.152,153
Ohtoshi, Yamauchi and Tadokoro155 described even
rarer reactions caused by chlorhexidine, in which case
immediate anaphylactic reactions were observed and
IgE antibodies were found in patients’ serum.
The major side effects of chlorhexidine are as
follows: tooth discoloration (in the cervical third and
proximal surfaces),156 restorations, prosthesis and
tongue; dental calculus accumulation, taste altera-
tion (especially to salt), oral desquamation, supragin-
gival calculus formation and occasional parotid glad
swelling dyspnea and anaphylaxis.157-161 Among these
effects, tooth discoloration stands out as patients’
chief complaint,162 since it affects 30 to 50% of pa-
tients.88,153 It is considered as the main limiting factor
of chlorhexidine when used for long periods of time.
Concentration and volume of chlorhexidine inter-
fere in the prevalence and severity of discoloration.
Thus, despite having similar efficacy and effective-
ness,164 lower concentrations, in larger volumes,
proved to cause less tooth discoloration.165 However,
these unpleasant effects are reversible once the use
of chlorhexidine is suspended.161
Although sensitivity to chlorhexidine may be
rare, the possibility of complications should be kept
in mind during its application.118
Final considerations
Based on this literature review on the applica-
tions of chlorhexidine for endodontic purposes, it is
reasonable to conclude that:
» Chlorhexidine, liquid or gel, may be used dur-
ing all phases of root canal preparation, in
which case the concentration of 2% is most
frequently used.

Dental Press Endod. 2013 Sept-Dec;3(3):36-54© 2013 Dental Press Endodontics 50
Chlorhexidine and its applications in Endodontics: Literature review
[ Literature review ]
» Its wide antimicrobial spectrum (Gram-pos-
itive and Gram-negative bacteria), including
fungi, is improved due to substantivity, which
may last from 48 hours to 12 weeks.
» Chlorhexidine does not solve organic tis-
sue, however, chlorhexidine gel may favor
it as a result of rheological reaction and lu-
brication of endodontic instruments during
mechanical action.
» Sodium hypochlorite associated with
chlorhexidine results in an orangish-brown
solution (parachloraniline) that requires fur-
ther investigation.
» Chlorhexidine has been recommended as an
alternative to sodium hypochlorite. It is con-
sidered a biocompatible solution, however, the
possibility of further complications should be
taken into account during its application.
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