1

An In Vitro Evaluation of the

Cytotoxicity of Various
Endodontic Irrigants On Human
2
Gingival Fibroblasts
JOE Volume 31, Number 8, August 2005
3 Introduction
The principles of root canal preparation are to remove all
organic debris and microorganisms from the root canal
system, and to shape the walls of the root canal to facilitate
that cleaning and the subsequent obturation of the entire
root canal space.

However, a tooth root rarely contains a single simple root

canal. Accessory canals, lateral canals, fins, anastomoses
between canals, and an apical delta all contribute to the
root canal system
4

An irrigant solution must be used which can be flushed

through this system, will destroy the microorganisms
and preferably dissolve organic debris at the same
time.

Thus the current concept of root canal preparation is

not cleaning and shaping, but shaping and cleaning.
 The main root canals should
5
be rapidly and efficiently
shaped with instruments to
permit thorough and
extended cleaning of the
entire pulpal system with
the irrigant solution.

The majority of these

anatomical features are not
accessible to
instrumentation
6 ROOT CANAL SYSTEM INFECTION
Microorganisms and their byproducts play an
essential role in the induction, progression,
and perpetuation of pulpal and periradicular
pathoses.

3 More than 150 microbial species have

been isolated from infected root canal
systems (infected pulps, biofilm, and
infected dentin), usually in mixed infections
with predominance of obligate anaerobic
bacteria
 Microorganisms of probable pathogenic significance in
7 endodontic infections include Porphyromonas species,
Prevotella species, Fusobacterium nucleatum, species
of the Streptococcus anginosus group, Bacteroides
forsythus, Treponema denticola, Peptostreptococcus
species, Eubacterium species, and Actinomyces
species.

7 In addition, enterococci, pseudomonas, yeasts, and

some enteric rods may be involved in persistent or
secondary root canal infections.
8 ROOT CANAL INFECTIONS VERSUS
OTHER INFECTIONS
Once established, a root canal system infection cannot be
eliminated by the host defense mechanisms or by
systemic antibiotic therapy. This is explained by the fact
that microorganisms present in root canal system
infections are in a protected sanctuary, where the
absence of a blood supply in a necrotic pulp impedes the
transport of defense cells and molecules as well as
systemically administrated antibiotics to the infected site.
9 On the other hand, although host
defense mechanisms and
systemic antibiotics are
ineffective against
microorganisms within the root
canal system, if microorganisms
gain access to the highly
vascularized periradicular tissues,
they are usually effectively
eliminated and thereby prevented
from spreading to other sites
 Thus, the endodontic treatment involves 3 important
10
steps to control of the root canal system infection: (1)
the chemomechanical preparation; (2) the intracanal
medication (in retreatment and infected cases); and
(3) the root canal obturation
11 MECHANICAL INSTRUMENTATION
Infected root canal systems can harbor between <
102 to > 108 bacterial cells. Mechanical
instrumentation is a critical step in the microbial
control phase of the root canal treatment.

Bystrm and Sundqvist found that using 6 to 10 mL

per canal of physiologic saline solution during
instrumentation can reduce the number of bacteria in
infected root canals by 100 to 1,000 fold.

.
12 Ingle and Zeldowfound that

immediately after instrumentation,

using sterile water as an irrigant,

80% of the initially infected root

canals yielded positive cultures. At

the beginning of the second

appointment, 48 hours later, this

number increased to 95.4%.

 Wu, et al reported that in oval canals, even
13
circumferential hand filing could only remove the
inner layer of dentin from 58% of the circumference of
the canal wall, leaving 42% unprepared.

.
14

On the whole, when using advanced bacteriological

techniques, it has been shown that the number of
bacteria can be significantly reduced, but not to an
extent that negative culture can be obtained at the
end of the first appointment. This means that
mechanical instrumentation should be supplemented
with antimicrobial irrigant.
15
16 In both vital and nonvital cases
extrusion of irrigant occurs even in
teeth with fully mature, intact
apexes . Blunderbuss canals,
perforations, and improper
techniques can also allow the
solution to permeate into
surrounding periodontal tissues.
Tissue cytotoxicity is therefore of
great concern when choosing an
endodontic irrigant.
17 Aim
The purpose of this study was to measure the
cytotoxicity of six endodontic irrigants on cultured
gingival fibroblasts using the CyQuant assay.

The following irrigants were tested at various

concentrations: Sodium hypochlorite (NaOCl); iodine
potassium-iodide (IKI); Betadine scrub (BS); calcium
hydroxide [Ca(OH)2]; chlorine dioxide (SCD).
18 NaOcl
NaOCl has been a popular endodontic irrigant . It has
been shown that 5.25% NaOCl provides immediate
sterilization in vitro.

NaOCl has the additional quality of necrotic tissue

dissolution . Although negative findings regarding
toxicity prompted recommendations to dilute 5.25%
NaOCl to lower concentrations , diluting NaOCl may
undermine its positive attributes.

.
19
Limitations

Unfortunately, even though NaOCl has many ideal

properties, it has some limitations such as being toxic,
nonsubstantive, ineffective in smear layer removal
and corrosive. It may cause discoloration and has
unpleasant odor. When NaOCl is used as a fi nal rinse,
bonding of the sealer to the dentin may be altered.
20 Stabilized Chlorine Dioxide (SCD)
Has recently come under consideration as a possible
root canal irrigant because of its reported antibacterial
activity.

According to the manufacturer, this product is

tuberculocidal, bactericidal, virucidal, and fungicidal.
The recent detection of Cytomegalovirus and Epstein-
Barr virus associated with periradicular lesions may
promote the use of SCD, which kills both enveloped
and nonenveloped viruses.
21 SCD is currently used in water purification, surface
disinfection, and some commercially available mouth
rinses. There is potential for this material to be used as
an endodontic irrigant, but data on cytotoxicity is
lacking.

It is used as a disinfectant in a concentration of 0.1 to

15mm.
22 Calcium hydroxide
Ca(OH)2 is widely accepted as an endodontic intracanal
medicament

Initial investigations into the antimicrobial properties of

saturated Ca(OH)2 demonstrated its total ineffectiveness
as an irrigant .

A later study found the addition of a detergent to be

synergistic and eliminated all test organisms within 30
min.
23 Povidine iodine
Several studies have evaluated the antimicrobial
efficacy of different iodine compounds against root
canal infections

Application of PVP-I solution as an endodontic irrigant

was proposed based on its rapid antiseptic action
against a broad range of microorganisms, low toxicity,
hypoallergenicity, and greatly reduced tendency to
stain dentin than other iodine containing antiseptics.
24

In 1976 Torneck advocated the use of povidone-iodine

solution as an endodontic irrigant.

Safavi, et al showed that in 2% IKI-treated root canals

in human teeth, a period of one to 2 hours was
required to prevent growth of Enterococcus faecalis in
dentinal tubules,
25 Materials and Methods
Human ginigival fibroblasts (hGFs) at passage three or four were
obtained.

There were five experimental groups: Group 1: Saturated CaOH2;

group 2: 2% IKI; group 3: 5.25% NaOCl; group 4: 7.5% Betadine
scrub (BS) (a proprietary 7.5% povidone iodine preparation which
contains surfactant); and group 5: 28% SCD (Clidox-S.

A 200l volume of each test irrigant was added to each well at the
predetermined concentration. The plates were incubated at 37C
in 5% CO2 and 95% air for 15 min and plates were frozen at70C
for a minimum of 24 h.
26

The cell number was then quantified using the

CyQUANT Cell Proliferation Assay Kit (#C-7026,
Molecular Probes, Eugene, OR) according to the
manufacturers instructions
27 Results
The results of this study demonstrated IKI and Ca(OH)2
were the least toxic irrigants tested with SCD appearing
slightly more toxic.

The LD50 for IKI and Ca(OH)2 were 1.92% and 8.93%,
respectively, which closely approximates their respective
clinical concentrations.

These findings demonstrate that 2% IKI and 10%Ca(OH)2

are relatively nontoxic alternatives for irrigation.
28 Discussion
The disadvantage of 10% Ca(OH)2 is its poor antimicrobial
properties when used for short periods of time, such as during
intra-appointment irrigation.

Dentinal tubules infected with S. faecalis are not disinfected with

calcium hydroxide (Heling et ah 1992). However, Bystrom et al
(198 5) and Sjogren et al. (1991) demonstrated that calciuim
hydroxide effectively reduces cultivable microorganisms from root
canals after 1 week of dressing. These authors suggested that the
slow-acting antimicrobial properties of calcium hydroxide were
due to a slow release of hydroxyl ions.
29

In the present experiment, however, saturated

calcium hydroxide irrigation solutions had limited
action after 60 min against most aerobic
microorganisms commonly found in the root canal.
30

IKI has shown promise as an effective antimicrobial

but its use requires mixing from stock chemicals and
special storage conditions. However, IKIs low
cytotoxicity and high antimicrobial properties give
merit to its use as an endodontic irrigant.
31 Stabilized chlorine dioxide was used in this study because of
its reported antimicrobial properties and its wide use in
water purification and surface disinfection. The
manufacturers maximum concentration of SCD, 28%, was
used because an effective clinical concentration has not yet
been determined. However, the less cytotoxic LD50
concentration of 15% may be an effective antimicrobial
irrigant. Therefore, if a lower effective concentration is
determined, this irrigant may prove to be a relatively
nontoxic alternative for irrigation.
32 Conclusion

2% IKI and 10% Ca(OH)2 appear to be irrigants that

are reasonably well tolerated by hGFs. Both may
provide safe alternatives for irrigation, but 2% IKI has
superior antimicrobial properties. SCD was found to be
significantly more toxic than IKI or CaOH2, but the
high concentration used in this study may be
unwarranted.
33
Further investigation into the effective clinical
concentration of SCD may disclose a lower concentration
that is both effective against endodontic pathogens and
relatively nontoxic to the periapical tissue.

It must be kept in mind that the effectiveness of all

irrigants has mostly been measuredin vitroenvironments.
More research that relates to endodontic success with
irrigant types and methods used is required
 Sodium hypochlorite is still the most effective gold
34
standard irrigant. Unlike with sodium hypochlorite, the
extrusion of iodine and chlorhexidine is thought to be more
forgiving to the soft tissues as they do not dissolve organic
tissue. Chelators in liquid form are not a replacement for
antimicrobial irrigants like NaOCl. The antimicrobial
properties of chelators are low yet they can be used to
remove the smear layer, increasing the penetration of other
irrigants such as NaOCl and hence increasing their
antimicrobial effects.
35 References

An In Vitro Evaluation of the Cytotoxicity of Various Endodontic Irrigants

On Human Gingival Fibroblasts JOE Volume 31, Number 8, August
2005.
Goldman M, Kronman JH, Goldman LB, Clausen H, Grady J. New method
of irrigation during endodontic treatment. J Endod 1976;2:257 60.
Siqueira JF, Lopes HP. Mechanisms of antimicrobial activity of calcium
hydroxide: a critical review. Int Endod J 1999;32:3619.
Sabeti M, Simon JH, Nowzari H, Slots J. CMV and Epstein-Barr virus active
infection in periapical lesions of teeth with intact crowns. J Endod
2003;29:3213.
Chemical Disinfection of the Root Canal System first edition.
36