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TITLE PAGE
Title of the article: Comparative evaluation of the cytotoxicity of MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite on human red blood corpuscles.
Contributors
1. Dr. Krishna Prasad Shetty MDS
Professor and Head
Department of Conservative dentistry and Endodontics
Navodaya Dental College & Hospital. Raichur
Karnataka. India
Email: [email protected]
2. Dr. Sarvepalli Venkata Satish MDS
Professor
Department of Conservative dentistry and Endodontics
Navodaya Dental College & Hospital. Raichur
Karnataka. India
Email: [email protected]
3. Dr. Krishnarao Kilaru MDS, DNB
Reader
Department of Conservative dentistry and Endodontics
Navodaya Dental College & Hospital. Raichur
Karnataka. India.
Email: [email protected]
4. Dr. Ratnakar P. MDS
Reader
Department of Conservative dentistry and Endodontics
H K E S Dental College. Gulbarga
Karnataka. India
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Email: [email protected]
5. Dr. Vijay Reddy Venumuddala MDS
Professor and Head
Hitech Dental College and Hospital. Bhubneswar
Orissa. India
Email: [email protected]
6. Dr. Kalyana Chakravarthi Ponangi BDS
Post Graduate Student.
Department of Conservative dentistry and Endodontics
Navodaya Dental College & Hospital. Raichur
Karnataka. India
Email: [email protected]
Department and institution
Department of Conservative dentistry and Endodontics.
Navodaya Dental College & Hospital. Raichur
Karnataka. India
Running title: cytotoxicity of three endodontic irrigants on human red blood corpuscles:
MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite.
Key-words: Cytotoxicity-Endodontic irrigants; Red Blood Corpuscles; MTAD-
cytotoxicity; Chlorhexidine-cytotoxicity; Sodium Hypochlorite-cytotoxicity.
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Corresponding Author:
Name:Dr. Krishna Prasad Shetty
Address: Department of Conservative dentistry and Endodontics
Navodaya dental college & hospital. Navodaya nagar.
Mantralayam road. Raichur. PIN – 584103
Karnataka. India.
Phone numbers: 919611282811
Facsimile numbers: 08532-223043
E-mail address: [email protected]
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Title of the article: Comparative evaluation of the cytotoxicity of 5.25% Sodium
hypochlorite, 2% Chlorhexidine & MTAD on human red blood corpuscles: An in-vitro
study
Abstract:
Aim: The aim of this study was to analyse the cytotoxicity of various volumes of 5.25%
Sodium hypochlorite, 2% Chlorhexidine gluconate and MTAD by checking for
haemolysis of human red blood corpuscles.
Methodology: Hundred micro litres of diluted Red Blood Corpuscles obtained through
centrifugation was added to three groups (Group I: 5.25% Sodium hypochlorite, Group
II: 2% Chlorhexidine, Group III: MTAD) of six test tubes each. Individual irrigants per
group were added in increasing volume starting from the second test tube keeping the
first one as the control. After incubation for three minutes haemoglobin content was
measured using an automated haemoanalyser. Data was analyzed using one sample t
test.
Results: Sodium hypochlorite is the most cytotoxic followed by MTAD and
chlorhexidine the least.
Conclusions: This study suggests that these irrigating fluids do cause detrimental
effects on vital tissues. Sodium hypochlorite though an irrigant of choice should be used
cautiously. Chlorhexidine should be used when excessive extrusion is expected. MTAD
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is the irrigant of choice because of its antimicrobial efficacy and smear layer removing
ability.
Key Messages: 2% Chlorhexidine is less cytotoxic when compared to 5.25% Sodium
Hypochlorite and MTAD. Therefore 2% Chlorhexidine is the irriganting solution of choice
in teeth with incomplete root development and perforations.
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Introduction:
Successful endodontic treatment depends on the ability of the operator to mechanically
and chemically clean the root canal system and subsequently obturate it three
dimensionally (1). Endodontic therapy is primarily based on the removal of potentially
noxious stimuli from the complex root canal system (2).
The complexity of the root canal system is, however, one of the determining factors in
the failure of root canal treatment even in properly treated teeth, due to irregular and
sometimes unpredictable spaces that characterize root canals (3, 4). The persistence of
residual pulp tissue, infected dentin or bacteria in the root canal system is responsible
for treatment failure (5). Irrigation, therefore, plays a crucial role in determining the
outcome of root canal treatment (6). Irrigants flush debris from canals and assist in
reducing microbial flora of infected canals and help to dissolve the necrotic tissues.
An endodontic irrigant should be non toxic when it comes in contact with vital tissues
and non caustic to the periodontal tissues (7). A potential complication of irrigation is the
forced extrusion of the irrigant and debris through the apex. Tissue cytotoxicity is
therefore a major concern when choosing an endodontic irrigant for root canal
treatment.
Sodium hypochlorite has solvent activity for both necrotic and vital tissues (2). It is
known in medical fields since the beginning of 20th century and was introduced as a part
Text
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of endodontic treatment in 1936 by Walker. Literature is full of studies showing severe
irritations when concentrated solution were inadvertently forced into the periapical
tissues beyond the apex of the tooth under endodontic treatment (8, 9).
Chlorhexidine was developed in the late 1940s in the research laboratories of Imperial
Chemical Industries Ltd (7). Chlorhexidine is a cationic biguanide that possesses broad
antibacterial activity in combination with relatively low toxicity and the ability to bind to
skin and mucous membranes.
MTAD was introduced as an alternative to EDTA to remove the smear layer by
Torabinejad et al. It is a mixture of 3% doxycycline, 4.25% citric acid and detergent-
Tween 80. It has a combined chelating and antibacterial property (10).
So the main objective of this study is to compare the cytotoxicity of 5.25%sodium
hypochlorite, 2%chlorhexidine and MTAD.
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Materials and Methods:
The informed consent of all human subjects who participated in the experimental
investigation reported or described in this manuscript was obtained after the nature of
the procedure and possible discomforts and risks had been fully explained. And the
institutional IRB approved the protocol.
Red Blood Corpuscles are chosen as the convenient cell type as they are readily
available and whose intracellular haemoglobin content can be easily measured. Fresh
blood was drawn from a human volunteer and collected in EDTA bottles. Centrifugation
of the blood is done, plasma removed and packed cell volume of Red Blood Corpuscles
is obtained. Red Blood Corpuscles are washed with saline and centrifuged several
times to remove white cells and any traces of plasma. One millilitre of packed cell
volume is added to four millilitres of saline to increase the volume of blood to five
millilitres. Hundred micro litres of this diluted Red Blood Corpuscles is added to 18 test
tubes with six test tubes in each group. The groups are as mentioned below.
Group I: 5.25% Sodium hypochlorite (VENSONS INDIA)
Group II: 2% Chlorhexidine gluconate (RC-CHLOR, DEOR, INDIA)
Group III: MTAD (DENTSPLY Tulsa Dental, United States)
For all the three groups, the first test tube is kept as a control in which no irrigant is
added. In the second test tube ten micro litres of the irrigant is added. Twenty micro
litres is added to third test tube, thirty micro litres to the fourth test tube, forty micro litres
to the fifth test tube and fifty micro litres to the sixth test tube.
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After an incubation time of three minutes, haemoglobin percentage after haemolysis of
red blood corpuscles is noted using an automated haemoanalyser (ABX MICROS 60).
The haemoanalyser measures the haemoglobin content of the remaining red blood
corpuscles after haemolysis. For all the three groups the experiment is repeated three
times and the mean value is taken.
Data obtained in the present study is subjected to statistical analysis using one sample t
test. A p value of less than .05 is considered as significant. Statistical analysis is done
using Minitab V.14 software.
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Results:
Group I (5.25% Sodium Hypochlorite) showed significant cytotoxicity in comparison to
the control group. The cytotoxicity of the irrigant is directly proportional to the increase
in the volume and the exposure time. At a volume of fifty micro litres, Sodium
Hypochlorite showed complete haemolysis of the Red Blood Corpuscles as shown in
Table 1.
GROUP I – 5.25% Sodium HypochloriteVolume of
the irrigant in
micro litres
Percentage of haemoglobin
Trial I
Percentage of haemoglobin
Trial II
Percentage of haemoglobin
Trial III
0(control) 4.4 4.4 4.410 1.9 2.0 2.020 1.0 0.9 1.130 0.3 0.4 0.340 0.1 0.2 0.150 0 0 0
Table 1: Sodium Hypochlorite shows complete haemolysis at a volume of 50 micro
litres.
Group II (2% Chlorhexidine gluconate), regarded as the safest among the three irrigants
showed cytotoxicity which is statistically significant. The cytotoxicity is least when
compared to MTAD and Sodium Hypochlorite. The test results are shown in Table 2.
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GROUP II – 2% Chlorhexidine gluconateVolume of irrigant in
micro litres
Percentage ofhaemoglobin
Trial I
Percentage of haemoglobin
Trial II
Percentage of haemoglobin
Trial III0(control) 4.4 4.4 4.4
10 3.7 3.5 3.720 3.5 3.5 3.430 3.3 3.3 3.240 3.0 2.8 3.050 2.8 2.6 2.8
Table 2: Reduction in the haemoglobin percentage as the irrigant volume increases
Group III (MTAD) showed cytotoxicity which is statistically significant but lesser when
compared to sodium hypochlorite and more when compared to chlorhexidine. The test
results are shown in Table 3.
GROUP III – MTADVolume of irrigant in
micro litres
Percentage of haemoglobin
Trial I
Percentage of haemoglobin
Trial II
Percentage of haemoglobin
Trial III0(control) 4.4 4.4 4.4
10 3.5 3.6 3.620 3.5 3.4 3.430 3.1 3.2 3.040 2.9 3.0 2.950 2.7 2.6 2.7
Table 3: Reduction in the haemoglobin percentage as the irrigant volume increases.
Chlorhexidine is the least cytotoxic of the compared irrigants.
The mean and standard deviation of the percentage of haemoglobin for all the three
irrigants using one sample t test, depicting significance is shown in Tables 4, 5 and 6.
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Group I - 5.25% Sodium Hypochlorite
Volume of the irrigant
in micro litres
Min MaxMean ± SD
Mean differe
nce
95% CI differenc
e
t-value
p-value Remarks
10 1.9 2.01.97
± 0.06
2.432.29 – 2.57
73P<0.000
2Significanc
e
20 0.9 1.11 ± 0.1
3.403.15 – 3.65
58.89
P<0.0003
Significance
30 0.3 0.40.33
± 0.06
4.073.92 – 4.21
122P<0.000
1Significanc
e
40 0.1 0.20.13
± 0.06
4.274.12 – 4.41
128P<0.000
1Significanc
e
Table 4: Statistically significant cytotoxicity shown by sodium hypochlorite at all the
volumes tested.
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Group II - 2% Chlorhexidine gluconate
Volume of the irrigant
in micro litres
Min MaxMean ± SD
Mean differenc
e
95% CI differenc
e
t-value
p-value Remarks
10 3.5 3.73.63
± 0.11
0.760.47 - 1.05
11.5 P<0.007 Significance
20 3.4 3.53.47
± 0.06
0.930.79 – 1.07
28 P<0.001 Significance
30 3.2 3.33.27
± 0.06
1.130.99 – 1.27
34 P<0.0009 Significance
40 2.8 3.02.93
± 0.11
1.471.18 – 1.75
22 P<0.002 Significance
50 2.6 2.82.73
± 0.11
1.671.38 – 1.95
25 P<0.002 Significance
Table 5: Statistically significant cytotoxicity shown by chlorhexidine gluconate at all the
volumes tested.
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Group III – MTAD
Volume of the
irrigant in
micro litres
Min MaxMean ± SD
Mean differenc
e
95% CI differenc
e
t-value
p-value Remarks
10 3.5 3.63.57
± 0.06
0.830.68 – 0.97
25 P<0.002 Significance
20 3.4 3.53.43
± 0.06
0.970.82 – 1.11
29 P<0.001 Significance
30 3.0 3.23.1 ± 0.06
1.301.05 – 1.55
22.52 P<0.002 Significance
40 2.9 3.02.93
± 0.06
1.471.32 – 1.61
44 P<0.0005 Significance
50 2.6 2.72.63
± 0.06
1.771.62 – 1.91
53 P<0.0004 Significance
Table 6: Statistically significant cytotoxicity shown by MTAD at all the volumes tested.
Graphical representation of the fall in the percentage of haemoglobin with the increase
in the volume of the irrigant for all the three groups is shown in Graph 1.
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Discussion:
Sodium hypochlorite is considered as an ideal irrigant in endodontics (Grossman et al.,
1941) (11). Hypochlorite preparations are sporicidal, virucidal and show far greater
tissue dissolving effects on necrotic than on vital tissues. These features prompted the
use of aqueous sodium hypochlorite in endodontics as the main irrigant as early as
1920 (12).
Various concentrations of sodium hypochlorite from 0.5% to 5.25% have been tried out
(13, 14). Higher the concentration better will be the antimicrobial effect and the tissue
dissolving capacity. At the same time higher concentration also carries the risk of
toxicity and tissue reaction. It is shown that 1% sodium hypochlorite is sufficient to
dissolve the pulp tissue (15). Higher concentrations of sodium hypochlorite are proven
to have greater bactericidal effect (16). Sodium hypochlorite (5.25%) in 1:1000 dilution
caused complete hemolysis of Red blood cells in vitro (Pashley et al., 1985) (17). On
investigating various concentrations of Sodium Hypochlorite (0.25%, 0.025 % and
0.0125%) for antimicrobial activity and tissue toxicity at varying time intervals it is found
that 0.025% of Sodium Hypochlorite is bactericidal and not tissue toxic and 0.25%
concentration of Sodium Hypochlorite showed tissue toxicity (Heggers et al., 1991)
(18).Sodium Hypochlorite in a concentration of 0.55% induced an inflammatory
response (M.Tanomaru Filho et al., 2002) (19). Sodium hypochlorite is toxic enough to
cause complete haemolysis according to this study.
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Chlorhexidine is recommended as an endodontic irrigant because of its antimicrobial
activity, presumed non toxicity and intracanal substantivity (20). At higher
concentrations, Chlorhexidine results in extensive cell damage, coagulation of
cytoplasm, and precipitation of proteins and nucleic acids (21).The concentration often
used in endodontic therapy is 2% as it is more effective in less time when compared to
other concentrations of Chlorhexidine ranging from 0.002% to 2% (22).
Chlorhexidine is toxic to human gingival cells and the toxic potency is dependent on the
length of exposure and the composition of the exposure medium (23). On comparing the
inflammatory response of 0.5% Sodium Hypochlorite, 2% Chlorhexidine digluconate,
and phosphate buffered saline, it is found that 2% Chlorhexidine injection is similar to
the phosphate-buffered saline control at all times tested, while the 0.5% Sodium
Hypochlorite injection resulted in significant inflammation (Tanamaru Filho et al., 2002)
(20).On treating fifty pieces of bovine pulp tissue with normal saline, MTAD, 2%
Chlorhexidine digluconate and 2.5% Sodium Hypochlorite it is found that Chlorhexidine
has the weakest tissue dissolution capacity(Khademi et al., 2007) (24).
Chlorhexidine is recommended as an alternative in patients allergic to sodium
hypochlorite or in teeth with incomplete root formation (22) and crestal perforations to
prevent inflammatory response in proximity to the epithelial attachment (25).
Bio PureMTAD (Dentsply, Tulsa, OK) is designed to be used as a final root canal rinse
before obturation (11). Tetracycline has many unique properties of low pH and acts as a
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calcium chelator and cause enamel and root surface demineralization (26). MTAD is
effective in removing the smear layer along the whole length of the root canal and does
not produce any signs of erosion or physical changes in dentin (27, 28 and 29). MTAD
is effective against Enterococcus faecalis, and it is less cytotoxic than a range of
endodontic medicaments, including eugenol, hydrogen peroxide (3%), EDTA, and
calcium hydroxide paste (30). In this study MTAD is found to be less cytotoxic than
sodium hypochlorite and more cytotoxic than chlorhexidine.
Acknowledgement
We affirm that we have no financial affiliation or involvement with any commercial
organization with direct financial interest in the subject or materials discussed in this
manuscript, nor have any such arrangements existed in the past three years. We deny
any conflict of interest related to this study.
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Conclusion:
The ideal functions of an irrigant are to dissolve both the organic as well as inorganic
components, render the root canal free of microbes and be non toxic to the tissues. No
single irrigant accomplishes all the functions.
This study suggests that these irrigating fluids do cause detrimental effects on vital
tissues. The clinical situation, concentration used, exposure time to the agent and the
exposure surface area are important factors which affect the cytotoxicity of the irrigating
solution. Therefore taking into account the limitations & merits of the irrigating solutions,
if used judiciously, it adds to the success of root canal therapy.
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References:
1. Schilder H. Cleaning and shaping the root canal. Dental Clinics of North
America 1974; 18:269-96.
2. Cemil Yesilsoy, Eugene Whitaker, Deborah Cleveland, Eric Phillips and Martin
Trope. Antimicrobial and toxic effects of established and potential root canal
irrigants. Journal of Endodontics 1995; 20:513-5.
3. Nair PN. On the causes of the persistent apical periodontitis. A review.
International Endodontic Journal 2006; 39:249-81.
4. Gulabivala K, Patel B, Evans G. Effects of mechanical and chemical
procedures on root canal surfaces. Endodontic Topics 2005; 10:103–22.
5. Moorer WR, Wesselink PR. Factors promoting the tissue dissolving capability
of sodium hypochlorite. International Endodontic Journal 1982; 15:187–96.
6. Filippo Santarcangelo. Root Canal Irrigation in Modern Endodontics: From
routine to complex Part One: The Routine [WWW document]. URL
http://en.zerodonto.com/2012/10/root-canal-irrigation-in-modern.html
[Accessed on 27 Nov 2012].
7. M.Zehnder. Root Canal Irrigants. Journal of Endodontics 2006; 32:389-98.
8. Witton R, Henthorn K, Ethunandan M, et al. Neurological complications
following extrusion of sodium hypochlorite solution during root canal treatment.
International Endodontic Journal 2005; 38:843-8.
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9. Gursoy UK, Bostanci V, Kosger HH. Palatal mucosa necrosis because of
accidental sodium hypochlorite injection instead of anesthetic solution.
International Endodontic Journal 2006; 39:157-61.
10.Torabinejad M, Khademi A A, Babagoli j, Cho Y, Johnson W B, Bozhilov K
(2003) A new solution for removal of smear layer. Journal of Endodontics 2003;
29:170-5.
11.Grossman L I, Meiman B W (1941) Solution of pulp tissue by chemical agents.
Journal of American Dental Association 1941; 28:223-5.
12.Kandaswamy D, Venkateshbabu N. Root canal irrigants. Journal of
Conservative Dentistry 2010; 13:256-64.
13.Gomes BPFA, Ferraz CCR, Vianna MEVB, Berber FB. In vitro antimicrobial
activity of several concentrations of sodium hypochlorite and Chlorhexidine
Gluconate in the elimination of Enterococcus Faecalis. International
Endododontic Journal 2001; 34:24-8.
14.M E Vianna, BPFA Gomes. In vitro evaluation of the antimicrobial activity of
Chlorhexidine and sodium hypochlorite. Oral surgery Oral Medicine Oral
Pathology 2004; 97:79-84.
15.Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effect of temperature on
sodium hypochlorite short-term stability, pulp dissolution capacity, and
antimicrobial efficacy. Journal of Endodontics 2005; 31: 669-71.
16.Arieh Y Kaufman, Senia Keila. Hypersensitivity to sodium hypochlorite. Journal
of Endodontics 1989; 15:224-6.
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17.EL Pashley, N.L Birdsong, k Bowman and D.H pashley. Cytotoxic effects of
Sodium hypochlorite on vital tissue. Journal of Endodontics 1985; 11:525-8.
18. Heggers JP, Sazy A J, Stenberg BD, Strock LL, Mc Cauley RL, Hernon DN,
Robson MC. Bacterial and wound healing properties of sodium hypochlorite
solutions. Journal of Burn Care Rehabilitation 1991; 12:420-4.
19.M Tanomaru Filho MR Leonardo L .A .B Silva, F .F Anibal and L.H Faccioli.
Inflammatory response to different endotoxic irrigating solutions. International
Endodontic Journal 2002; 35:735-9.
20.Leonard MR, Tanomaru Filho, M.Silva CAB, Nelson Filho N, Bonifacio KC. In
vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating
solution. Journal of Endodontics 1999; 25:167-71.
21.M E Vianna, BPFA Gomes. In vitro evaluation of the antimicrobial activity of
Chlorhexidine and sodium hypochlorite. Oral surgery Oral Medicine Oral
Pathology 2004; 97:79-84.
22.Lessa, F C Rosetti, Nogueira,F Silveira. Direct and transdentinal antibacterial
activity of chlorhexidine. American Journal of Dentistry 2010; 25:255-9.
23.Babich H, Wurzburger BJ, Rubin Y L, Sinensky MC, Blau L (1995) An in Vitro
study on the cytotoxicity of Chlorhexidine digluconate to human gingival cells.
Cell Biol Toxicol 1995; 11:79-88.
24.Abbasali Khademi, Ehsan usefian, Mahboobe F. Tissue dissolving ability of
several endodontic irrigants on bovine pulp tissue. Iranian Endodontic Journal
2007; 2:65-67.
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25.Fuss Z, Trope M. Root perforations classification and treatment choices based
on prognostic factors. Endodontics and Dental Traumatology 1996; 12: 225-64.
26.K. Bjorvatn, N. Skaug, and K. A. Selvig. Tetracycline-impregnated enamel and
dentin: duration of antimicrobial capacity. Scandinavian Journal of Dental
Research 1985; 93:192–7.
27.M. Torabinejad, Y. Cho, A. A. Khademi, L. K. Bakland, S. Shabahang. The
effect of various concentrations of sodium hypochlorite on the ability of MTAD
to remove the smear layer. Journal of Endoddontics 2003; 29:233–9.
28.R. E. Beltz, M. Torabinejad, M. Pouresmail. Quantitative analysis of the
solubilizing action of MTAD sodium hypochlorite, and EDTA on bovine pulp and
dentin. Journal of Endodontics 2003; 29:334–7.
29.T. K. Machnick, M. Torabinejad, C. A. Munoz, S. Shabahang. Effect of MTAD
on flexural strength and modulus of elasticity of dentin. Journal of Endoddontics
2003; 29:747–50.
30.S. Shabahang, M. Pouresmail, M. Torabinejad. In vitro antimicrobial efficacy of
MTAD and sodium hypochlorite. Journal of Endodontics 2003; 29:450–2.
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FIGURE LEGENDS:
Figure 1 (graph 1): Graphical representation of the fall in the percentage of
haemoglobin with the increase in the volume of the irrigant for all the three groups is
shown in Graph 1.