Basic Research—Technology
Effect of Lowering the pH of Sodium Hypochlorite on
Dissolving Tissue in Vitro
Chad E. Christensen, DMD,* Sandre F. McNeal, MPH,† and Paul Eleazer, DDS, MS*
Abstract
Sodium hypochlorite (NaOCl), a common antimicrobial
and tissue-dissolving irrigant, comes from the manu-
facturer at pH 12. When the pH is lowered, NaOCl
becomes more antimicrobial. The aim of this study was
to examine what effect lowering the pH has on the
property of tissue dissolution. Seven groups were tested
for dissolving porcine muscle tissue at varying pH,
concentrations, and times. When groups were exam-
ined solely by pH, there was no significant difference
between the pH 12 and 9 groups, but a statistically
significant level was found between pH 12 and 9 versus
the pH 6 groups (P ⬍ .05). Higher concentrations and
greater time periods all led to greater amounts of tissue
dissolution. (J Endod 2008;34:449 – 452)
Key Words
pH, sodium hypochlorite, tissue dissolution
From the *Department of Endodontics and †Department of
Diagnostic Sciences, University of Alabama at Birmingham,
Birmingham, Alabama.
Address requests for reprints to Dr Paul Eleazer, University
of Alabama at Birmingham, Department of Endodontics, 1530
3rd Ave S, SDB 406, Birmingham, AL 35294. E-mail address:
eleazer@uab.edu.
0099-2399/$0 - see front matter
Copyright © 2008 by the American Association of
Endodontists.
doi:10.1016/j.joen.2008.01.001
JOE — Volume 34, Number 4, April 2008
S uccessful root canal therapy depends on thorough chemomechanical debridement
of pulpal tissue, dentin debris, and infective microorganisms (1–3). Irrigants can
augment mechanical debridement by flushing out debris, dissolving tissue, and disin-
fecting the root canal system. Chemical debridement is especially needed for teeth with
complex internal anatomy, such as fins, and other irregularities that might be missed by
instrumentation (4).
Sodium hypochlorite (NaOCl) is popular today and has an extensive history in
medicine and dentistry. Dakin (5) first recommended the use of buffered and diluted
NaOCl (0.5% at pH 9) as an antiseptic solution to irrigate wounds during World War I
(5). Walker (6) and Grossman and Meiman (7) introduced chlorinated soda solution
to endodontics because of its antibacterial properties and its ability to effectively dis-
solve pulp tissue. NaOCl is an extremely effective antimicrobial agent that can also
detoxify bacterial products (8, 9). NaOCl exerts a nonspecific, noncoagulating digestive
effect on vital and necrotic tissues (10, 11).
Studies have shown that both the antibacterial properties and tissue-dissolving
properties of 5.25% NaOCl decrease when it is diluted (8, 11, 12). When NaOCl is added
to water, it undergoes the following reaction:
NaOCl ⫹ H2O → NaOH ⫹ HOCl (hypochlorous acid) (1)
In aqueous solution, hypochlorous acid partially dissociates into the anion hypochlorite
(OCl⫺):
HOCl ↔ H⫹ ⫹ OCl⫺ (2)
The “available” chlorine is the sum of the HOCl and OCl⫺ concentrations in the
solution (13). Available chlorine might be defined as a measurement of oxidizing
capacity and is expressed in terms of the amount of elemental chlorine. HOCl is con-
sidered to be a stronger oxidant than the hypochlorite ion. The hypochlorous acid
(HOCl) molecule is responsible for strong chlorinating and oxidizing action on tissue
and microorganisms.
Hypochlorous acid dissociation (equation 2) depends on pH, with the clinical
equilibrium between HOCl and OCl⫺ being maintained as HOCl is consumed through its
germicidal function (14). Baker (14) plotted the relationship between HOCl, OCl⫺, and
pH. At pH 10, basically all chlorine is in the OCl⫺ form, and the reverse occurs at a 4.5
pH, where all chlorine is in the form of HOCl. The disinfecting properties decrease with
higher pH, paralleling the concentration of dissociated hypochlorous acid. Bloomfield
and Miles (13) confirmed that hypochlorites at a lower pH possess greater antimicro-
bial activity. Andrews and Orton (15) were among the early workers suggesting hypo-
chlorous acid was responsible for the destruction of microorganisms. In 1966, Morris
(16) found that OCl⫺ ion possesses approximately 1/80 the germicidal potency of HOCl
in killing Escherichia coli.
The effect of lower pH on tissue dissolution effect is unknown. The purpose of this
study was to evaluate the effects of tissue-dissolving capability of NaOCl at pH 9 and pH
6 versus the commercially supplied pH 12.
Materials and Methods
Porcine muscle tissue was used to test the dissolving capabilities of NaOCl at
differing pH, concentration, and time. The tissue was kept refrigerated in 100% humid-
ity and divided into tissue pieces ranging in size from 0.4 –1.0 g. Ten tissue pieces were
used for each time period within each group. The time periods tested were 5, 15, and
Effect of Lowering the pH of NaOCl on Dissolving Tissue In Vitro 449
Basic Research—Technology
30 minutes. All test procedures were done at room temperature (25°C). TABLE 2. Mean and Standard Deviations for the Percent of Weight Loss for the
Saline controls were run for the 5-minute time period only. This gave a 7 Groups Regardless of Time
total of 190 samples. Mean, pct Standard deviation,
The groups were as follows: group 1 (control): saline; group 2: N
loss pct loss
5.25% NaOCl, pH 12; group 3: 2.6% NaOCl, pH 12; group 4: 5.25% 1 (Control) 10 1.22 0.76
NaOCl, pH 9; group 5: 2.6% NaOCl, pH 9; group 6: 5.25% NaOCl, pH 6; 2 (5.25%, pH 12) 30 22.77 12.80
group 7: 2.6% NaOCl, pH 6. 3 (2.6%, pH 12) 30 4.12 4.42
The tissue samples were preweighed by using a Sartorius analytical 4 (5.25%, pH 9) 30 18.96 8.34
digital scale (Sartorius, Edgewood, NY). After the weights were re- 5 (2.6%, pH 9) 30 4.22 3.66
6 (5.25%, pH 6) 30 6.46 2.97
corded, the samples were placed in 10 mL of the solution being tested 7 (2.6%, pH 6) 30 6.75 3.28
for the allotted time while periodically swirling the cup to agitate the
solution and tissue sample. The sample pieces were then removed,
blotted dry, and weighed.
The test solutions were prepared by adjusting the pH of 5.25% statistically significant difference between 5.25% and 2.6% concentra-
NaOCl (A-1 Bleach; James Austin Co, Mars, PA) by adding hydrochloric tions of NaOCl. These results reinforce the concept of NaOCl concen-
acid (HCl) that had been adjusted to either a 5.25 mol/L or 2.6 mol/L tration as an important factor in the dissolution of tissue at pH levels of
concentration corresponding to the 5.25% or 2.6% NaOCl concentra- 12 and 9.
tions. All experiments were done under a vacuum hood. Pipettes were
used to add the HCl to a stirred beaker of NaOCl to titrate the solution pH
until a digital UB-10 pH meter (Denver Instruments, Denver, CO) gave
Table 4 presents means and standard deviations for combined
the desired reading. The pH meter was calibrated before mixing test
groups according to pH regardless of time and concentration. Table 5
solutions. All solutions were made fresh just before the tests were per-
shows statistical comparisons for these combined groups. When exam-
formed.
ined solely by pH, there was no significant difference between the pH 12
The data from the above experiments were recorded as the percent
and pH 9 groups, but a statistically significant difference was found
of tissue weight loss. An analysis of variance test was done on the data by
between both pH 12 and pH 9 groups versus the pH 6 group.
using a computerized statistics program (SAS Institute Inc, Cary, NC). A
value of P ⬍.05 was considered statistically significant.
Time
Results When times were grouped for evaluation, it was shown that longer
The control samples showed only a very slight loss in tissue weight. exposure times resulted in improved tissue dissolution. Comparing time
The mean and standard deviations for the percentage weight loss for all periods within groups of the same pH value, longer exposure times
groups at all times are presented in Table 1. within the pH 12 group showed a greater percent of tissue weight loss
Table 2 shows means and standard deviations for the percent of (P ⬍ .05). With all pH 9 groups, no statistically significant difference
weight loss for the 7 groups regardless of time. Table 3 presents statis- was found between the 5- and 15-minute time periods, but a difference
tical comparisons between groups regardless of time interval. was found when comparing the 5-minute with 30-minute period and
also the 15-minute with 30-minute period. In lowering the pH further to
Concentration 6, the only statistically significant time difference found was between the
When comparing full-strength (5.25%) and half-strength (2.6%) 5-minute and 30-minute time periods.
concentrations of NaOCl within groups with the same pH and regardless These results showed that the further the pH was lowered, the
of time interval, the percent of tissue loss was statistically significant more time that was required for solution contact to get similar tissue
(P ⬍ .05) at pH 12 and 9. When the pH was titrated to 6, there was no dissolution.

TABLE 1. Mean and Standard Deviations for the Percent of Weight Loss for All Groups at All Time Intervals
Time (min) Level of group N Mean, pct loss Standard deviation, pct loss
5 1 (Control) 10 1.22 0.76
5 2 (5.25%, pH 12) 10 7.03 2.47
15 2 10 24.48 3.08
30 2 10 36.79 3.99
5 3 (2.6%, pH 12) 10 0.54 0.30
15 3 10 2.08 1.18
30 3 10 9.75 2.72
5 4 (5.25%, pH 9) 10 10.72 1.10
15 4 10 16.67 2.37
30 4 10 29.50 3.26
5 5 (2.6%, pH 9) 10 1.35 1.34
15 5 10 2.54 2.23
30 5 10 8.77 1.07
5 6 (5.25%, pH 6) 10 4.40 1.15
15 6 10 5.23 2.10
30 6 10 9.76 2.07
5 7 (2.6%, pH 6) 10 3.97 1.02
15 7 10 6.19 1.72
30 7 10 10.07 3.09
The group with greatest mean weight loss was the 5.25%/pH 12 group at 30 minutes followed by the 30-minute 5.25%/pH 9 group. The group with lowest mean was the 2.6%/pH 12 group at the 5-minute interval.

450 Christensen et al. JOE — Volume 34, Number 4, April 2008


TABLE 3. Group Statistical Comparisons Regardless of Time Interval
Group 2 3 4 5 6 7
2 ⬍.05 ⬍.05 ⬍.05 ⬍.05
3 ⬍.05 ⬍.05 .8670 ⬍.05
4 ⬍.05 ⬍.05 ⬍.05 ⬍.05
5 ⬍.05 .8670 ⬍.05 ⬍.05
6 ⬍.05 ⬍.05 ⬍.05
7 ⬍.05 ⬍.05 ⬍.05 ⬍.05 .6299
Note that all groups showed statistically significant differences except groups 3 versus 5 and groups 6
versus 7. Control differences were statistically significant but were omitted for clarity.
Discussion
Many authors have reported on the significant influence of con-
centration, time, temperature, tissue-irrigant contact area, canal prep-
aration size, volume, tissue type, and mechanical action on the ability of
NaOCl to dissolve both necrotic and vital tissues. This study focused on
the effect of concentration, time, and pH (4 –7, 10 –12, 17–22).
This study confirmed the notion that stronger concentrations of
alkaline NaOCl resulted in greater tissue dissolution (8, 11–13). How-
ever, when the pH was titrated to a level of 6, full-strength and half-
strength concentrations were both poor at tissue dissolution.
We found that the variables of concentration, time, and pH were all
important factors in the capacity for NaOCl to dissolve tissue. The more
concentrated the NaOCl solution, the longer the solution was left in contact
with tissue, and higher pH levels all resulted in greater tissue dissolution.
In this study, we used HCl to lower the pH of our NaOCl solution
through a neutralization reaction that produced a salt (NaCl). Salt
formed during the reaction between NaOCl and HCl could be the whitish
deposit that developed on the surface of the tissue while testing the pH
6 solutions. When blotting these samples dry, it was noted the tissue was
firm and almost crust-like. This surface layer of “salt” might have acted
as a physical barrier preventing fresh tissue-solution contact. Also, the
weight of the salt could have obscured some tissue dissolution, although
this was considered to cause a negligible difference.
To confirm that the salt reaction did not significantly decrease
effective chlorine levels, an analytical chemistry lab (Guardian Systems,
Inc, Leeds, AL) examined freshly mixed solutions for total residual
chlorine. At pH level of 6, with EPA test #330.5, the total residual chlo-
rine of the 5.25% solution was 32,000 ppm. The 2.6% solution at pH 6
gave a total residual chlorine value of 20,000 ppm. These numbers
demonstrated a significant amount of available chlorine, even with the
neutralization reaction and formation of a salt.
Zehnder et al. (23) compared unbuffered solutions of 2.5% and
0.5% NaOCl and 0.5% solutions buffered to pH 12 and pH 9. They used
1% sodium bicarbonate (NaHCO3) and 5% sodium carbonate
(Na2CO3) as buffers to change the pH of NaOCl. They found the 2.5%
NaOCl solution substantially more effective than either of the 0.5% so-
lutions in dissolving tissue. They found buffering the 0.5% solution to pH
9 had little effect on tissue-dissolving properties when compared with
the unbuffered (pH 12) 0.5% solution. This study confirmed the above
results, failing to show a statistically significant difference between pH
12 and pH 9. Clinicians might consider the difference important, albeit
TABLE 4. Mean and Standard Deviations for Combined Groups According to
pH Regardless of Time and Concentration
Groups with Mean, pct Standard deviation,
N
same pH loss pct loss
2 ⫹ 3 (pH 12) 60 13.45
4 ⫹ 5 (pH 9) 60 11.59
6 ⫹ 7 (pH 6) 60 6.60
JOE — Volume 34, Number 4, April 2008
Basic Research—Technology
TABLE 5. Statistical Comparisons of Combined pH Groups
Groups with 2ⴙ3 4ⴙ5 6ⴙ7
⬍.05 same pH (pH 12) (pH 9) (pH 6)
⬍.05 2 ⫹ 3 (pH 12) .2033 ⬍.05
⬍.05 4 ⫹ 5 (pH 9) .2033 ⬍.05
⬍.05 6 ⫹ 7 (pH 6) ⬍.05 ⬍.05
.6299
When groups were examined solely by pH, there was no significant difference between the pH 12 and
pH 9 groups, but a statistically significant difference was found between both pH 12 and 9 versus the
pH 6 groups.
not statistically so. The conclusion by Zehnder et al. that greater tissue
dissolution occurs with higher concentrations of NaOCl was also con-
firmed by this study.
This study confirmed the advantage of using longer contact time for
tissue removal. Of course, risks of periapical tissue damage from inadver-
tent extrusion also increase. As the pH was decreased from 12 to levels of 9
and 6, statistical significance was lost between the 5-minute and 15-minute
intervals but was typically significant at the 30-minute period. Although sta-
tistically not significant, these differences might be very significant clinically.
Further areas of study might include what gases are produced from
the neutralization reaction, would these gases be harmful if inhaled, and
what cytotoxic effects would these solutions have on the vital tissues of
the periapex.
Other unanswered questions concern the effects of this solution on
tissue and bacteria within dentinal tubules, how easily can these solutions be
removed from the canal space before obturation, how will they react with
restorative and obturation materials, and what effects do other irrigants
such as ethylenediaminetetraacetic acid have on the pH values of NaOCl.
In conclusion, concentration, time, and pH all play important roles
in determining the amount of tissue dissolution. Higher concentration
and greater time periods all lead to greater amounts of tissue dissolu-
tion. Although antibacterial powers increase as pH decreases, tissue
dissolution decreases.
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JOE — Volume 34, Number 4, April 2008