Detection of Organochlorine Compounds Formed During the Contact of Sodium Hypochlorite with Dentin and Dental Pulp

Abstract

This study used gas chromatography-mass spectrometry (GC-MS) to detect the products formed during the contact of sodium hypochlorite (NaOCl) with bovine pulp and dentin. For analysis of the products formed in the volatile phase, 11 mg of bovine pulp tissue were placed in contact with 0.5%, 2.5% and 5.25% NaOCl until complete tissue dissolution occurred. The solid phase microextraction (SPME) fiber was exposed inside the container through the cover membrane and immediately injected into the GC-MS system. 30 mg of the of dentin were kept in contact with NaOCl, and then the SPME fiber was exposed inside the container through the cover membrane for adsorption of the products and injected into the GC-MS system. The same protocol was used for the aqueous phase. For analysis of the volatile compounds, the final solution was extracted using pure ethyl ether. The suspended particulate phase of the mixture was aspirated, and ether was separated from the aqueous phase of the solution. The ether containing the products that resulted from the chemical interaction of dentin and pulp with the NaOCl was filtered and then injected into the GC-MS system for analysis of the aqueous phase. The aqueous and volatile phases of both dentin and pulp showed the formation of chloroform, hexachloroethane, dichloromethylbenzene and benzaldehyde. In conclusion, organochlorine compounds are generated during the contact of dentin and pulp with NaOCl at concentrations of 0.5%, 2.5% and 5.25%.

Full text

This study used gas chromatography-mass spectrometry (GC-MS) to detect the products
formed during the contact of sodium hypochlorite (NaOCl) with bovine pulp and
dentin. For analysis of the products formed in the volatile phase, 11 mg of bovine pulp
tissue were placed in contact with 0.5%, 2.5% and 5.25% NaOCl until complete tissue
dissolution occurred. The solid phase microextraction (SPME) fiber was exposed inside
the container through the cover membrane and immediately injected into the GC-MS
system. 30 mg of the of dentin were kept in contact with NaOCl, and then the SPME
fiber was exposed inside the container through the cover membrane for adsorption of
the products and injected into the GC-MS system. The same protocol was used for the
aqueous phase. For analysis of the volatile compounds, the final solution was extracted
using pure ethyl ether. The suspended particulate phase of the mixture was aspirated,
and ether was separated from the aqueous phase of the solution. The ether containing
the products that resulted from the chemical interaction of dentin and pulp with the
NaOCl was filtered and then injected into the GC-MS system for analysis of the aqueous
phase. The aqueous and volatile phases of both dentin and pulp showed the formation of
chloroform, hexachloroethane, dichloromethylbenzene and benzaldehyde. In conclusion,
organochlorine compounds are generated during the contact of dentin and pulp with
NaOCl at concentrations of 0.5%, 2.5% and 5.25%.
Detection of Organochlorine
Compounds Formed During the
Contact of Sodium Hypochlorite
with Dentin and Dental Pulp
Tiago Gilioli Varise1, Carlos Estrela2, Débora Fernandes Costa Guedes1, Manoel
Damião Sousa-Neto1, Jesus Djalma Pécora1
1Department of Restorative Dentistry,
School of Dentistry of Ribeirão
Preto, USP - University of São
Paulo, Ribeirão Preto, SP, Brazil
2Federal University of Goiás,
Goiânia, GO, Brazil
Correspondence: Prof. Dr. Jesus
Djalma Pécora, Avenida do Café, S/N,
Monte Alegre, 14040-904 Ribeirão
Preto, SP, Brasil. Tel: +55-16-3602-
4114. e-mail: pecora@forp.usp.br
Key Words: sodium
hypochlorite, organochlorine,
chloroform, hexachloroethane,
dichloromethylbenzene.
Introduction
The irrigation of infected root canals is an important
factor in the success of endodontic therapy. Sodium
hypochlorite is a well-studied irrigant because of its
antimicrobial effect, tissue dissolution capacity and
acceptable biological compatibility at lower concentrations.
Accidents with sodium hypochlorite may occur when the
irrigant is in contact with periapical tissue or other soft
tissues, which leads to severe inflammation (1-5).
The molecular formula of sodium hypochlorite is NaOCl,
its molar mass, 74.44 g/mol, its density, 1.07-1.14 g/cm³
and its boiling point, about 101°C. It is totally miscible
in water, and its surface tension (about 70 dynes/cm)
is high. Its concentration is directly proportional to its
antimicrobial effect and tissue dissolution capacity and
inversely proportional to its biological compatibility (3).
In dilution tests, its minimal inhibitory concentration was
lower than 1% for important microorganisms (S. aureus,
E. faecalis, P. aeruginosa and C. albicans) (4). Its high pH
explains its antimicrobial mechanism of action, similar to
that of calcium hydroxide (5). It also affects the integrity
of the cytoplasm membrane due to irreversible enzymatic
inhibition, biosynthetic changes in cell metabolism
and phospholipid destruction in lipid peroxidation. The
formation of amino acid chloramine affects cell metabolism.
Oxidation results in irreversible enzymatic inhibition of
bacteria and replaces hydrogen with chlorine. Enzymes
may be inactivated during the reaction of chlorine with
amino groups and the irreversible oxidation of sulfhydryl
groups of bacterial enzymes. The antimicrobial effect of
NaOCl is explained by its action upon essential bacterial
enzymes, which results in irreversible inactivation due to
the action of hydroxyl ions and chloramines. Dissolution
of organic tissues is observed during saponification, when
NaOCl destroys fatty acids and lipids, which generates soap
and glycerol (5).
Organochlorine compounds, usually found in small
amounts in nature, originate from the contact of chlorine-
based substances with organic tissue composed of carbon
chains. They are neurotoxic, highly lipophilic, chemically
stable and persistent in nature. Toxic to some plants and
insects, they may be produced synthetically by the action
of elemental chlorine upon petroleum hydrocarbons,
and many have been widely used as pesticides. In the
last decades, governmental agencies and environmental
groups have made efforts to document contamination by
organochlorine compounds and regulate their use, which
has avoided dangerous concentrations, particularly in
ISSN 0103-6440
Brazilian Dental Journal (2014) 25(2): 109-116
http://dx.doi.org/10.1590/0103-6440201302404

Braz Dent J 25(2) 2014
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T.G. Varise et al.
human foods (6,7).
Because of their toxicity, studies have investigated
their formation during water treatment (8-12) and their
use as pesticides in agriculture (13,14). Although the
environmental burden of some organochlorine pesticides
has slowly decreased in several areas due to the restrictions
to its use and production (15), the accumulation of these
substances in the human organism still raises concerns
(16). Some organochlorine-based pesticides, known as
persistent organic pollutants, are hydrophobic, have long
half-lives and tend to accumulate in the fatty tissue of
animals and humans (17).
Chlorine (Cl2) is the most common disinfectant
in water treatment because it is easy to use and has
effective germicide properties and a low cost. However,
chlorine also reacts with dissolved organic material and
produces disinfection byproducts, such as trihalomethanes
(chloroform) and haloacetic acids. Water disinfection,
one of the main advances in public health, is responsible
for decreases in mortality due to infectious diseases (18),
but some disinfectant agents, such as chlorine, ozone,
chlorine dioxide and chloramines, produce compounds
that react with natural organic material during clean water
production (19). Daily exposure to chlorinated water may
be dangerous to human health because of the carcinogenic
and mutagenic properties of these compounds. The
consumption of chlorinated has been correlated with
cancer risks (20). Baird (6) reported on the association
between chlorinated water and cancer rates in several
American communities. Public health problems caused
by water disinfection are a major current concern in the
entire world. Some studies demonstrated the health risks
of organochlorine compounds for human beings, such as
changes in cell metabolism and vital functions that lead
to death by necrosis or in vivo apoptosis (8,9,19).
During root canal treatment, NaOCl is in direct
and constant contact with dental pulp and dentin. As
organochlorine compounds play in important role in human
health, the toxic byproducts of the reaction of NaOCl and the
organic matter inside the root canals should be investigated.
This study used gas chromatography-mass spectrometry
(GC-MS) to determine what compounds were produced as
a result of the contact of different concentrations of NaOCl
with bovine dental pulp and dentin.
Material and Methods
Organic Substrate Preparation
Freshly extracted bovine teeth were decoronated using
a flexible mono-faced diamond disk (7010, KG Sorensen,
São Paulo, SP, Brazil) and a N270 straight handpiece (Dabi
Atlante, Ribeirão Preto, SP, Brazil). After that, the pulp
tissue was removed from the roots using Hedströem files
(Dentsply-Maillefer, Ballaigues, Switzerland), weighed
(analytical scale, Mettler Toledo AG245, Canton, MA),
separated into portions of about 11 mg each, stored
in Eppendorf tubes (Quimis, Campinas, SP, Brazil) with
deionized water (Quimis) and kept under refrigeration. The
cement layer of the external surface of the root sections was
removed using diamond tips (2068G, KG Sorensen), and the
samples were then cut into small fragments and powdered
using a ball mill (MLW, Germany) to obtain dentin micro-
particles. After that, bovine dentin was weighed (analytical
scale, Mettler Toledo AG245), separated into portions of 30
mg and stored in Eppendorf tubes at room temperature.
Extraction Method for GC-MS
Dental pulp analysis: volatile extraction
Previously weighed pulp samples, separated and stored
to preserve their features, were placed into capped glass
bottles containing 2 mL of NaOCl at concentrations of 0.5%,
2.5% and 5.25%. The NaOCl samples were obtained from
10% NaOCl dilution in deionized water and immediately
titrated using the iodometric method. The samples were
divided into the following groups: (a) bovine pulp (11 mg) +
0.5% NaOCl (2 mL); (b) bovine pulp (11 mg) + 2.5% NaOCl
(2 mL); (c) bovine pulp (11 mg) + 5.25% NaOCl (2 mL). All
the samples were prepared in triplicate.
The bottles containing bovine pulp and the NaOCl
solutions were kept under mixing (magnetic mixer without
heating, Fisatom, São Paulo, Brazil) to ensure that the liquid
was in contact with the entire external surface of the
organic material until its total dissolution was confirmed
by the presence of halogenated compounds. After that,
still under mixing, SME fiber (65-µm PDMS/DVB SUPELCO,
Bellefonte, PA, USA) attached to a holder was inserted into
the recipient through a membrane in the cap, without
touching the aqueous phase, and kept there for 15 min
for the adsorption of the volatiles generated during the
dissolution of organic material that was in touch with
NaOCl at different concentrations.
After exposure (15 min), the fiber was removed from
the bottle and immediately injected into the CG-MS
system (QP 2010 plus; high injector AOC-20i Shimadzu,
Tokyo, Japan) and kept there for 5 min for the analysis of
volatile compounds.
Dentin analysis: volatile extraction
Dentin samples previously prepared, weighed and stored
were placed in capped glass bottles with 2 mL of NaOCl at
the concentrations described above and divided into the
following groups: (a) bovine dentin (30 mg) + 0.5% NaOCl
(2 mL); (b) bovine dentin (30 mg) + 2.5% NaOCl (2 mL);
(c) bovine dentin (30 mg) + 5.25% NaOCl (2 mL). All the
samples were prepared in triplicate.

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Organochlorine formed by NaOCl and substrate
The bottles containing bovine dentin and NaOCl
solutions were kept under mixing (magnetic mixer without
heating, Fisatom, São Paulo, SP, Brazil) for 15 min to ensure
that the liquid was in contact with the entire external
surface of the material. After that, still under mixing, SPME
fiber (65-µm PDMS/DVB SUPELCO, Bellefonte, PA, USA)
attached to a holder was inserted into the bottle using the
membrane in the bottle cap, without touching the aqueous
phase, and kept there for 15 min for the adsorption of the
volatiles generated by dissolution of organic material in
contact with NaOCl at different concentrations.
After exposure (15 min), the fiber was removed from the
bottle, immediately injected into the CG-MS system and
kept there for 5 min for the analysis of volatile compounds.
Dental pulp anD Dentin analyses - aqueous phase extraction
The same protocol was used to analyze the aqueous
phase of pulp and dentin. After fiber removal for the analysis
of volatile compounds, the final solution was extracted
using pure ethyl ether at a standardized mixture of 2 mL
of solution + 2 mL of ether (Merck Millipore, Darmstadt,
Germany). The sample was kept under mixing (magnetic
mixer without heating, Fisatom, São Paulo, Brazil) for 5
min to make sure the liquid was adequately mixed.
After mixing, the suspended particulate phase was
aspirated using a micro-syringe (2-mL # 100² syringe,
Hamilton CO. Gastight, Reno, NV) to separate ether from the
aqueous phase of the solution. The ether, which contained
the material resulting from the chemical interaction of
the organic compounds of the pulp and dentin with the
different NaOCl concentrations, was filtered (15-mm 0.45-
µm organic regenerated cellulose filter, Ministart, Sartorius,
Goettingen, Germany) and injected into the CG-MS system
for analysis of the aqueous phase of the sample.
CG-MS Optimization to Detect Byproducts of Organic
Material Decomposition by NaOCl
Different temperatures were used to ensure the best
possible separation of decomposing products of organic
material in NaOCl. Of the conditions assessed, the ideal
chromatographic resolution was: initial oven temperature:
40 °C; injector temperature: 250°C; ramp: 40 °C for 2.5 min
> 130 °C (at 35 °C/min) > 145 °C for 4 min.; flux control:
linear speed; pressure: 49.7 kPa; total flux: 14 mL/min;
split ratio: 10:1.
Sample Calibration Curve
The calibration curve was built using a standard method.
Control 1 (SPME fiber): the fiber was injected for CG-
MS analysis as soon as removed from casing. The results
indicated that no substances that might affect results were
detected in the SPME fiber; Control 2 (5.25% NaOCl): the
fiber was placed into a closed environment containing
5.25% NaOCl, exposed for 15 min without contact with
liquid, and injected into the CG-MS system for analysis.
The results revealed that low oscillations were within the
standard deviation, which showed that 5.25% NaOCl had
no substances that might affect results; Control 3 (bovine
pulp): the fiber was placed into a closed environment
containing bovine pulp, kept there for 15 min without
contact with the sample, and then injected into the CG-
MS system. The results indicated that the bovine pulp had
no substances that might affect results; Control 4 (bovine
dentin): the fiber was placed into a closed environment
containing bovine dentin, kept there for 15 min without
contact with the sample, and then injected into the CG-MS
system. There were no substances in bovine dentin that
might affect results; Control 5 (ethyl ether): the fiber was
placed into a closed environment containing ethyl ether,
kept there for 15 min without contact with the sample and
then injected into the CG-MS system. The peak retention
time of 1.6 min indicated the presence of volatized ethyl
ether adsorbed by fiber; no other substances or products
that might affect results were found.
Results
The GC-MS results of compounds formed during the
contact of NaOCl with organic material (pulp and dentin)
will be described in three sections: 1) volatile extraction; 2)
aqueous phase extraction; 3) analysis of GC peaks of volatile
compounds (NaOCl concentration and products generated).
Volatile Extraction
Dentin
The GC-MS results of volatile extraction of compounds
formed during the contact of NaOCl at different
concentrations with bovine dentin are shown in Figure 1A.
Peak retention time (RT) was 2.15 min, which corresponds
to chloroform, and mass load (m/z) was 83.47, which
corresponds to an organochlorine (Fig. 1B). The second
peak RT, 5.32 min, corresponds to benzaldehyde (m/z =
106.77.51) (Fig. 1C). After that, there was a third peak at
RT = 6.30 min, which indicated the detection of another
organochlorine, hexachloroethane (m/z = 201.166.117.94)
(Fig. 1D). A last peak retained at RT = 6.7 min corresponded
to another organochlorine formed during the contact
of NaOCl with the organic material of bovine dentin:
dichloromethylbenzene (m/z = 125.89.63) (Fig. 1E).
pulp
The GC-MS results of volatile extraction of compounds
formed during the contact of NaOCl at different
concentrations with bovine pulp are shown in Figure 2. The
compounds formed during the contact of NaOCl with bovine

Braz Dent J 25(2) 2014
11 2
T.G. Varise et al.
Figure 1. A: Initial chromatographic analysis of originated products in reactions of NaOCl and bovine dentin. B: Mass spectrum of peak TR = 2.15
min from chloroform, obtained after fiber desorption. C: Mass spectrum of peak TR = 5.32 min from benzaldehyde, obtained after fiber desorption.
D: Mass spectrum of peak TR = 6.3min from hexachloroethane, obtained after fiber desorption. E: Mass spectrum of peak TR = 6.7min from
dichloromethylbenzene, obtained after fiber desorption.
pulp and dentin were the same: chloroform, benzaldehyde,
hexachloroethane and dichloromethylbenzene.
Aqueous Phase Extraction
The GC-MS results of the extraction of the aqueous
phase of compounds formed during the contact of NaOCl
at different concentrations with bovine dentin and pulp are
shown in Figure 3. A peak between 1-2min corresponded
to ethyl ether. The black line corresponds to the control
graph of the solvent used, which confirmed its identification

Braz Dent J 25(2) 2014
11 3
Organochlorine formed by NaOCl and substrate
by CG-MS. After identification, GC-MS detected no other
response to ethyl ether.
The results of the analysis of volatile compounds in both
bovine dentin and pulp samples revealed that, in aqueous
phase, the same compounds were formed in the different
substrates: chloroform, benzaldehyde, hexachloroethane
and dichloromethylbenzene at RT of 2.15 min, 5.32 min,
6.3 min and 6.70 min.
Analysis of GC Peaks of Volatile Compounds - NaOCl
Concentration and Compounds Formed
Dentin
Table 1 shows the areas of each peak of Figure 1A,
which correspond to the compounds formed during
the contact of bovine dentin with NaOCl at different
concentrations. The height of peaks in Figure 1A does not
correspond to the amount of each compound formed. The
values in the y-axis of Figure 1A are the intensity (uV) of
each compound detected using CG-MS. Thus, the amount
formed of each compound is expressed by peak area. At RT
= 2.15 min in Figure 1A, which corresponds to chloroform,
the highest peak was found for 2.5% NaOCl, followed by
that for 5.25% NaOCl, and the lowest, for 0.5% NaOCl,
but the amount of compound formed was proportional
to NaOCl concentration. In other words, the lowest NaOCl
concentration (0.5%) had a smaller area (10681137) of
chloroform formed during the reaction, followed by the
area of 24501360, which corresponded to chloroform
formed during contact of 2.5% NaOCl with bovine dentin,
whereas the largest area was 24856101, which corresponded
to the organochlorine formed for 5.25% NaOCl, as shown
in Table 1. Therefore, the formation of chloroform was
directly proportional to NaOCl concentration.
The analysis of benzaldehyde revealed that at RT = 5.32
min (Fig. 1A) the peaks generated according to the variation
of each NaOCl concentration were proportional to peak
area. The lowest peak (0.5% NaOCl) also had the smallest
area (2045827), followed by the area of the 2.5% NaOCl
peak (8417137), whereas the highest peak (12298806)
corresponded to 5.25% NaOCl. The peaks of benzaldehyde,
hexachloroethane and dichloromethylbenzene were
also proportional to NaOCl areas and concentrations.
Hexachloroethane (RT = 6.30 min) had the lowest peak
when 0.5% NaOCl was used, and its area was 511172.
NaOCl at 2.5%, the second highest concentration, had the
second highest peak and the second largest area (8887362).
The solution with the highest concentration, 5.25%, had
the highest peak in Figure 1 and an area of 12298806.
Dichloromethylbenzene detected at RT = 6.7 min had the
lowest peak when NaOCl concentration was 0.5%, and its
area was 1208900; when 2.5% NaOCl was used, the area
was 3045170; and the largest area (11691997) was found
for 5.25% NaOCl.
pulp
Table 2 shows the areas of the peaks in Figure 2, which
correspond to the compounds formed during the contact
with NaOCl at different concentrations. In the first peak of
Figure 2 (RT = 2.15 min), which corresponds to chloroform,
the highest peak is the GC-MS result for 2.5% NaOCl,
followed by the peak for organochlorine formation when
in contact with 0.5% NaOCl, whereas the lowest peak
corresponded to bovine dentin in contact with 5% NaOCl;
however, the amount of chloroform does not correspond to
the peaks, as it increases with concentration. The smallest
area (7442200) corresponded to the second highest
peak (0.5% NaOCl), followed by the second largest area
(12190135), seen in Table 2, and whose peak, the highest,
is shown in Figure 2, whereas the largest area (12382353)
in Table 2 corresponds to the lowest peak. The peaks in
Figure 2 that correspond to benzaldehyde are seen at RT =
5.32 min and confirm that the higher the peak, the larger
the area in Table 2 and the higher the concentration of
NaOCl. The area of the lowest peak, which corresponded
Figure 3. Extraction of ethyl ether of aqueous phase of originated
products in reaction between 5.25% NaOCl / bovine dentin and 5.25%
NaOCl + bovine pulp.
Figure 2. Immediate analysis of volatile products originated in reactions
of NaOCl and bovine pulp.

Braz Dent J 25(2) 2014
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T.G. Varise et al.
to 0.5% NaOCl, was 1837003, followed by that of 2.5%
NaOCl (area = 4872823); and the highest peak had an area
of 6867971 and corresponded to benzaldehyde formed
during the contact with 5.25% NaOCl.
The GC-MS peaks of hexachloroethane at RT = 6.30
min also vary according to area and NaOCl concentration.
The smallest area (1393254) and lowest peak corresponded
to the lowest concentration of NaOCl in this study (0.5%
NaOCl). The second highest peak corresponded to 2.5%
NaOCl (2791955), and the largest area (8957649) was
found for the highest peak when the 5.25% concentration
was used.
At RT = 6.70 min (Fig. 2), which corresponded to peaks
of dichloromethylbenzene, the amount of organochlorine
did not increase with NaOCl concentration, differently
from all the other peaks in the results. The highest peak
with the largest area (4943370) was formed when the
sample was in contact with 2.5% NaOCl. When using
0.5% NaOCl, the lowest concentration in this study, a
second highest peak and second largest area were found
for dichloromethylbenzene (1574741). The use of 5.25%
NaOCl corresponded to the lowest peak and smallest area
(1115776), as shown in Figure 2.
Discussion
When treating infected root canals, cleaning and
shaping, together with the action of NaOCl, reduce the
remaining microbiota and ensure better prognoses. NaOCl
has been studied and indicated at different concentrations
(1-5).
The results of this study showed that the contact
of NaOCl with dentin and pulp results in the formation
of organochlorine compounds. Regardless of NaOCl
concentration (0.5%, 2.5% or 5.25%), the same
compounds were formed, and concentration was directly
proportional to amount of each compound. Analysis of
the aqueous and volatile phases of both dentin and pulp
revealed the formation of chloroform, hexachloroethane,
dichloromethylbenzene and benzaldehyde.
GC-MS was effective in detecting products formed
during the contact of NaOCl with bovine dentin and pulp.
Chromatography is a physical separation method in which
compounds are distributed into two phases, one stationary
and one that moves in a defined way. The mixture with
the compounds to be separated is dissolved during the
mobile phase. As the mobile phase passes through the
stationary phase, some compounds are substantially
retained, and, therefore, move slowly. Meanwhile, other
compounds interact weakly with the stationary phase
and are transported more easily by the mobile phase.
These differences in mobility are used to separate
compound mixtures and analyze them qualitatively or
quantitatively, using, for example, spectrophotometry or
mass spectrometry (10). In a GC-MS system, samples are
bombarded by electrons and broken, generating positive
and negative ions and radicals. The differences in the
Table 2. Areas of the peaks in Figure 2, which correspond to the
compounds formed during the contact with NaOCl at different
concentrations
Peak NaOCl (%) Retention
time (min) Area
Chloroform
0.5
2.15 (peak 1)
7442200
2.5 12190135
5.25 12382353
Benzaldehyde
0.5
5.32 (peak 2)
1837003
2.5 4873283
5.25 6867971
Hexachloroethane
0.5
6.30 (peak 3)
32675
2.5 2791955
5.25 8957649
Dichloro-methyl-
benzene
0.5
6.70 (peak 4)
1574741
2.5 4943370
5.25 1115776
Table 1. Areas of each peak of Figure 1A, corresponding to the
compounds formed during the contact of bovine dentin with NaOCl
at different concentrations
Peak NaOCl (%) Retention
time (min) Area
Chloroform
0.5
2.15 (peak 1)
10681137
2.5 24501360
5.25 24856101
Benzaldehyde
0.5
5.32 (peak 2)
2045827
2.5 8417137
5.25 11931805
Hexachloroethane
0.5
6.30 (peak 3)
5111 72
2.5 8887362
5.25 12298806
Dichloro-methyl-
benzene
0.5
6.70 (peak 4)
1208900
2.5 3045170
5.25 11691997

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11 5
Organochlorine formed by NaOCl and substrate
Figure 4. Chemical reactions between pulp tissue and NaOCl. A:
saponification reaction. B. Neutralization reaction. C: Chloramination
reaction. D: Byproducts generated: chloroform, benzaldehyde,
hexachloroethane and dichloromethylbenzene).
relation of mass/load of ions generated separate them
(21,22).
This study identified organochlorine compounds
(chloroform, hexachloroethane and dichloromethylbenzene)
formed during the contact of NaOCl with bovine pulp and
dentin. Chloroform, or trichloromethane, an organochlorine
unduly called formaldehyde trichloride, is highly refractive,
nonflammable and volatile and has a high molecular
weight, characteristic smell and, when in its liquid state,
a sweet taste. It solidifies at -63.5 °C and hits boiling
point at 59 °C (6-10). This study found a greater amount
of chloroform in the analysis of the volatile phase than of
the aqueous phase. This is explained by the high volatility
of this organochlorine. Smyth et al. (23) studied the effect
of chloroform on rats and found that it is toxic to living
organism, and that the lethal oral dose for that species
was 2.18 g/kg. When inhaled in high doses, it may cause
hypotension, respiratory and cardiac depression and even
death. Although this substance is carcinogenic, it is still used
in fats, oils, rubber, waxes, gutta-percha solvent, cleaning
agents and fire extinguishers, where it reduces the freezing
temperature of carbon tetrachloride. Hexachloroethane,
an organochlorine that smells like camphor, is soluble
in chloroform, alcohol, benzene, ether and oils, and is
insoluble in water. Found in its crystal form, it sublimates
without melting (6-10). The IV lethal dose in dogs is 325
mg/kg (24). In humans, it may cause moderate irritation
to skin and mucous membranes. Dichloromethylbenzene
is an organochlorine that should be further evaluated,
although benzenes are toxic substances. Benzaldehyde,
another substance formed during this study, is not an
organochlorine and is found in grain kernels. In its liquid
state, it is yellow, has a high refraction value and a
characteristic smell and must be kept in a closed recipient
and protected from light. Its boiling point is about 25° C,
and its freezing point, 56.5 °C. The lethal oral dose in rats
is 1300/1000 mg/kg (25). It is used in the production of
dyes and perfume and as solvents and aromatic agents. At
high concentrations, it may cause contact dermatitis (6-10).
Sodium hypochlorite has a dynamic balance, as shown
in NaOCl + H2O « NaOH + HOCl « Na+ + OH- + H+ + OCl
(3,5). The analysis of some chemical reactions of organic
tissues and NaOCl conducted so far are shown in Figure
4A-C (5). The interpretation of these chemical reactions
demonstrates that NaOCl acts as an organic and fat
solvent that degrades fatty acids and transforms them
into fatty acid salts (soap) and glycerol (alcohol), which
reduces the surface tension of the remaining solution
(saponification, Fig. 4A). NaOCl neutralizes amino acids
and forms water and salt (neutralization, Fig. 4B). As
hydroxyl ions are lost, its pH is lowered. Hypochlorous
acid, a substance found in NaOCl solutions, acts as a
solvent when in contact with organic tissues and releases
chlorine, which, when combined with the protein amino
group, forms chloramines (chloramine formation, Figure
4C). Hypochlorous acid (HOCl-) and hypochlorite ions (OCl-)
lead to amino acid degradation and hydrolysis (5). This study
found that, in the volatile and the aqueous phases, the
contact of NaOCl with bovine pulp and dentin led to the
formation of four byproducts: chloroform, benzaldehyde,
hexachloroethane and dichloromethylbenzene. Three of
these products have chemical structures that characterize
organochlorine compounds: chloroform, hexachloroethane
and dichloromethylbenzene (Fig. 4D).
NaOCl has been one of the most common irrigant
agents in root canal treatment for more than a century. The
positive aspects of its use are associated with its organic
and necrotic tissue dissolution properties, whitening action,
antimicrobial potential, saponification, transformation of
amines into chloramines and deodorization. On the other
hand, the complete disruption and elimination of the
bacterial biofilm remains a challenge for new studies, as
well as the fact that no operator is immune to accidents
that may occur during the use of this substance not in
the root canal (1-5).
Therefore, its use in dental clinical procedures should
be reviewed frequently because both the dentist and
the patient may accidentally inhale the volatile phase

Braz Dent J 25(2) 2014
11 6
T.G. Varise et al.
of byproducts formed during the contact of NaOCl with
organic substrates and be exposed to organochlorine
compounds that represent a threat to human health, as they
tend to accumulate in human adipose tissue (8-10). Further
studies should be conducted to analyze the potential tissue
damage caused by organochlorine compounds formed
during the contact of NaOCl with organic substrates. One
of aspects that should be investigated is the effect of the
amount of organochlorine compounds produced during
the irrigation and manipulation of NaOCl, which can be
associated with actual health risks for dentists and patients.
This study showed that organochlorine compounds are
formed during the contact of NaOCl with organic substrates
(pulp or dentin). The generation of these byproducts
occurred at all concentrations (0.5%, 2.5% and 5.25%). The
amounts of all byproducts were directly associated with
NaOCl concentrations, except for dichloromethylbenzene
in the volatile phase of the test for bovine pulp.
Resumo
Este estudo utilizou a cromatografia gasosa acoplada a espectrometria
de massa (CG-MS) para detectar os produtos que se formaram durante o
contato de hipoclorito de sódio (NaOCl) com polpa dental bovina e dentina.
Para a análise dos produtos formados na fase volátil, 11 mg de polpa bovina
foram colocados em contato com 0,5 % , 2,5 % e 5,25 % de NaOCl, até à
dissolução completa dos tecidos. A fibra de microextração em fase sólida
(SPME) era exposta dentro do recipiente através da membrana da tampa,
por 15 minutos, para a adsorção dos produtos formados e imediatamente
injetada no CG-MS para análise. Para a análise da dentina, 30 mg do de
amostras foram mantidas em contacto com o NaOCl, por 15 min, e então a
fibra de SPME era exposta no interior do recipiente através da membrana
de cobertura para a adsorção dos produtos e injectado no sistema de GC-
MS. O mesmo protocolo foi utilizado para a fase aquosa. Para a análise
dos compostos voláteis, a solução final foi extraída com éter etílico puro.
A fase de partículas em suspensão da mistura foi aspirada, e o éter foi
separado da fase aquosa da solução. O éter contendo os produtos que
resultaram da interacção química da dentina e polpa com hipoclorito de
sódio foi filtrado e, em seguida, injectado no sistema GC-MS para análise
da fase aquosa. As fases aquosas e voláteis de dentina e polpa mostraram
a formação de clorofórmio, hexacloroetano, dichloromethylbenzene e
benzaldeído. Compostos organoclorados são gerados durante o contacto
da dentina e polpa com hipoclorito de sódio em concentrações de 0,5
% , 2,5 % e 5,25 %.
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Received November 11, 2013
Accepted March 18, 2014