Chloramine T: an effective disinfectant for reducing bacterial contamination and preserving dentinABSTRACT
Objective: To assess the effectiveness of dentin decontamination using chloramine T and its impact on the structural integrity of dentin, comparing it with alternative chemical and physical decontamination methods.
Methods: Forty-eight sound human third molars were sectioned at the occlusal surface, and the roots were randomly assigned to four disinfection methods: deionized water, UV light, 0.1% thymol, and 1% chloramine T. Dentin slices were incubated in Brain-Heart Infusion broth to assess disinfection (n=3). Knoop hardness was measured (n=6), and scanning electron microscopy was used to observe surface morphology post-disinfection (n=3). Quantitative data analysis involved ANOVA and post hoc Games-Howell tests (α=5%).
Results: Deionized water, thymol, and UV light exhibited high absorbance values (2,112±19, 764±67, and 866±75, respectively), while chloramine T displayed no bacterial growth. None of the decontamination methods led to a reduction in dentin hardness (p>0.05), and no significant changes in surface morphology were identified by scanning electron microscopy.
Conclusion: Chloramine T demonstrated strong antimicrobial activity while preserving dentin morphology, suggesting it could be a promising disinfectant for dentin decontamination and may improve clinical practices in dental procedures..
Indexing terms
Anti-bacterial agents; Dentin; Decontamination
RESUMO
Objetivo: Avaliar a eficácia da descontaminação da dentina utilizando cloramina T, comparando-a com métodos alternativos de descontaminação químicos e físicos, e também avaliar seu impacto na integridade estrutural da dentina.
Métodos: Quarenta e oito terceiros molares humanos hígidos foram seccionados separando a superfície oclusal e as raízes, e foram distribuídas aleatoriamente entre quatro métodos de desinfecção: água deionizada, luz UV, 0.1% timol, e 1% cloramina T. As fatias de dentina foram incubadas em caldo infusão cérebro coração para avaliação da desinfecção (n=3). A dureza Knoop foi mensurada (n=6), e a microscopia eletrônica de varredura foi utilizada para observar a morfologia superficial após a desinfecção (n=3). A análise quantitativa dos dados foi realizada por meio de ANOVA e testes post hoc de Games-Howell (α=5%).
Resultados: A água deionizada, timol e a luz UV, apresentaram altos valores de absorbância (2,112±19, 764±67, and 866±75, respectivamente), enquanto a cloramina T não apresentou crescimento bacteriano. Nenhum dos métodos de descontaminação causou redução da dureza da dentina (p>0,05), e não foram identificadas alterações significativas na morfologia superficial pela microscopia eletrônica de varredura.
Conclusão: A cloramina T demonstrou forte atividade antimicrobiana, preservando a morfologia da dentina, sugerindo que pode ser um desinfetante promissor para a descontaminação dentinária e contribuir para a melhoria das práticas clínicas em procedimentos odontológicos..
Termos de indexação
Antibacterianos; Dentina; Descontaminação
INTRODUCTION
As the use of extracted teeth is a common practice in dental research and education to mimic tooth decay when simulating oral conditions [1], evaluating different decontamination methods is essential. However, to carry out the assays without cross-contamination to the environment, people and equipment, exogenous factors must be controlled [2].
Dentin is a heterogeneous composite, morphologically composed of peritubular dentin, dentinal tubules surrounded by mineral content, and intertubular dentin, located between the canaliculi, which consists of an organic network composed of type I collagen fibrils, water and non-collagenous proteins [3]. Their decontamination is challenged by the variability of methods, high costs, long term tools, and the shortage of removal of microorganisms from the dentin surface by chemical substances and physical decontamination methods [4]. Besides the effectiveness of decontamination, the methods might keep the original structural characteristics of the substrate, not compromising or causing damage to the dentin morphological, biochemical or physical properties [5]. Therefore, dentin decontamination is one of the biggest challenges when performing caries models.
Among physical decontamination methods, sterilization through wet heat under pressure and the ionization radiation by gamma rays are considered the gold standard for dentin decontamination [5,6]. While the high heat of the first physical method could cause obliteration of dentinal tubules [7] which influences the bond of adhesive restorations, the use of the second one is limited because it is time consuming, it demands need of reactors in nuclear facility, besides the high costs for the researchers [6]. Those limitations remain as determining factors for the search for other equally efficient and low-cost methods. On the other hand, Ultraviolet Irradiation (UV light) is a nonionizing radiation well-established as a decontamination method used to inactivate pathogenic microorganisms [8]. Basically, the direct application of a long wavelength, low energy, and low penetration UV radiation eliminates pathogens on surfaces by causing damage to the genetic material in their cell nucleus or nucleic acids [9].
Chloramine T (CH₃C₆H₄SO₂NClNa) is an active chlorine compound widely used control the growth of microorganisms on tissues [10] because of its ability to interrupt bacterial metabolism by the hypochlorite portion that oxidizes the DNA structure, inhibiting the growth and reproduction of microorganisms [11]. Another chemical substance with biological activity is thymol (2-isopropyl-5-methylphenol; C₁₀H₁₄O), a monoterpenoid phenol found in essential oils extracted from plants of the Lamiaceae family that has important antimicrobial activity [12]. The mechanism of the antimicrobial action of thymol is based on its ability to integrate itself into the lipid layer of the cell membrane of microorganisms, causing rupture of the bacterial membrane and leakage of intracellular content [13]. However, decontamination methods can affect bond strength in different ways for different adhesive systems [10]. A previous study demonstrated that chloramine T and Thymol did not interfere with bond strength when stored for 24 h, whereas for 3 months they decreased the bond strength of self-etching and etch and rinse adhesive systems. Then, the specimen’s sterilization is a critical step on bond strength evaluations [10].
Given the challenges of controlling exogenous factors and preserving dentin integrity during decontamination, comparing chemical methods like chloramine T and Thymol with physical methods such as UV light provides valuable insights. This comparison would offer clear information on efficient, low-cost decontamination approaches that minimize damage to dentin, thereby enhancing the accuracy and reliability of caries models and other dental research applications.
To identify the effectiveness for reducing bacterial contamination without damaging the dentin surface, this study compared chemical (chloramine T and Thymol) and physical (UV light) decontamination methods. The hypotheses of this study were: (1) Chloramine T would inhibit bacterial growth on dentin; (2) Chloramine T would keep the mechanical properties of dentin unchanged; (3) Chloramine T decontamination preserves the integrity of dentin.
METHODS
Forty-eight sound human third molars were collected after the patients had signed the informed consent form. Inclusion criteria encompassed intact third molars recently extracted for other reasons than the study, without any cracks, fractures, or structural defects in the enamel and/or dentin. Teeth exhibiting any cracks, fractures, or structural defects in the enamel and/or dentin, as well as those with signs of carious lesions, significant wear or previous restorative treatments were excluded.
The teeth were fixed on acrylic plates with a stick wax (Asfer, São Caetano do Sul, São Paulo, Brazil) and green stick godiva (Lysanda, Vila Prudente, São Paulo, Brazil), and the set was positioned on an Isomet 1000 automatic cutter (Buehler, Illinois, United States) and the occlusal surface and the roots of the teeth were sectioned to obtain the dentin slices, using a high-precision diamond disc under constant cooling at 275 rpm and 250 g. After that, all slices were rinsed with deionized water in an ultrasonic bath for 20 min (Ultrasound Ultrason 1440 D-Odontobrás Ind. E Com. Med. Odont. Ltda, Rio Preto, SP, Brazil) and the specimens were randomly distributed into the groups shown in the flowchart (figure 1).
Evaluation of decontamination
Dentin slices were randomly divided according to the groups: (1) Control group − Deionized water, (2) 1% chloramine T (Sigma Aldrich, Jurubatuba, São Paulo, Brazil), (3) 0.1% thymol (Êxodo Científica, Sumaré, São Paulo, Brazil) and (4) UV light (LucMatLamp, Vila Medeiros, São Paulo, Brazil).
The dentin slices were placed in falcon tubes, containing the solutions to be tested, and were stored for 7 days [14]. After the decontamination period, the solution was removed using a pipette, and Brain Heart Infusion broth (Kasvi, Pinhais, Paraná, Brazil) was added to it. After that, the tubes were stored for 24 h in the microaerophilic oven with 20% CO2 and 37 oC.
For the group in which UV light was used, the slices were placed in petri dishes and sterilized using UV light (LucMatLamp, Vila Medeiros, São Paulo, Brazil) for 30 min (15 min on top/bottom), with an intensity of 30 W/m2 [15]. After the decontamination process, the slices were placed in falcon tubes containing Brain-Heart Infusion broth (BHI) (Kasvi, Pinhais, Paraná, Brazil) and taken to a microaerophilic oven with 20% CO2 and 37 oC for a 24 h.
After the incubation periods of all groups, a qualitative analysis of the bacterial viability was performed, and decontamination was validated when there was no turbid growth in the medium [16]. To complement the qualitative evaluation, the quantitative analysis of the BHI broth was carried out. An aliquot of 300 ml of medium was vortexed for 5s and the bacterial growth was evaluated by measuring the absorbance at 550 nm (Thermo GENESYS™ 10UV, Thermo Scientific™, Massachusetts, United States) [16].
Surface hardness
Dentin slices were polished with sandpaper of grit #600, #800, #1200, #2400 and #4000 (Carborundum abrasives, Guarulhos, São Paulo, Brazil). Between each change in the sandpaper granulation, all slices were rinsed with deionized water in an ultrasonic bath for 30 min (Ultrasound Ultrason 1440 D-Odontobrás Ind. E Com. Med. Odont. Ltda, Rio Preto, SP, Brazil) to remove debris. (Ultrasound Ultrason 1440 D-Odontobrás Ind. E Com. Med. Odont. Ltda, Rio Preto, SP, Brazil), The slices were randomly divided into the following groups: (1) Control group: Deionized water; (2) Chloramine T 1%; (3) Thymol 0.1%; and (4) UV light.
The hardness of the dentin surface was evaluated using a Shimadzu microhardness tester (HMV 2000, Shimadzu, Tokyo, Japan) coupled to a software consisting of a Knoop pyramidal diamond indenter, with a load of 0.05 g, applied for 15 s. Five indentations were performed for each sample, with distances of 100 µm between them [17]. The arithmetic average from each slice was considered as an experimental unit for statistical purposes.
Evaluation of the surface morphology of dentin using a Scanning Electron Microscope
Dentin slices were polished with sandpaper with grit #600, #800, #1200, #2400 and #4000 (Carborundum Abrasivos, Guarulhos, São Paulo, Brazil). Between each change of the sandpaper granulation, all slices were rinsed with deionized water in an ultrasonic bath for 30 min (Ultrasound Ultrason 1440 D-Odontobrás Ind. E Com. Med. Odont. Ltda, Rio Preto, SP, Brazil) to remove debris, and each was randomly divided according to the study group: (1) Control group: Deionized water; (2) Chloramine T 1%; (3) Thymol 0.1%; and (4) UV light.
After the dentin slices had received the treatments, they were fixed in stubs with double-sided carbon tape (Electron Microscopy Sciences, Washington 19034 – USA) and dehumidified for 2 h in a closed plastic container with silica gel. After that, these stubs were coated with gold (SCD 050; Balzers, Schaan, Liechtenstein) for 120 s at 40 mA, and analyzed in a scanning electron microscope (SEM) (JSM 5600 LV; JEOL, Tokyo, Japan) at a voltage acceleration of 15 kV at 4000x and 6000x magnifications by the same previously calibrated operator.
Statistical analysis
The data values of surface hardness were submitted to Levene’s test to assess the homogeneity of variances and the Shapiro-Wilk test to assess the normality of data distribution. To determine the statistical differences in the decontamination of the dentin slices, the one-way ANOVA test and the Games-Howell post hoc test were used, as the variances were not homogeneous. Jamovi software version 2.2 was used to perform the tests, and statistical significance was set at α=5%.
RESULTS
Chloramine T was the only method that inhibited bacterial growth, as evidenced by the absence of turbidity in the BHI broth, indicating the decontamination of dentin on qualitative analysis. Deionized water-control group, the thymol solution and the UV light showed bacterial growth, as evidenced by the presence of turbidity in the BHI broth.
As a second form of growth control in the culture medium (BHI), absorbance readings were performed as a quantitative analysis. Sterile BHI was used as a control. Table 1 shows that the deionized water (control group), thymol, and UV light decontamination methods had high absorbance values compared to chloramine T, confirming the qualitative evaluation of bacterial growth indicated by the turbidity of the BHI broth. High absorbance values indicate a small amount of light passing through the sample, indicating bacterial growth.
The means and standard deviations of the surface hardness are presented in table 2. No significant differences were observed among the groups (ANOVA; p<0.05). The surface hardness was not affected by the surface treatment, regardless of the decontamination method used.
The Scanning Electron Microscopy (SEM) micrographs (figure 2) showed that the deionized water and UV light group had some occluded dentinal tubules (Pointer), while the thymol and chloramine groups demonstrated all the unoccluded tubules. Additionally, there was a clear differentiation between the intertubular and peritubular dentin among the groups. It was observed that none of the physical or chemical methods used caused any morphological alteration to the dentin.
DISCUSSION
Decontamination is the act of removing most of the pathogenic microorganisms present in a structure, and sterilization is the complete removal of the pathogenic microorganisms [18]. The present study comparatively evaluated the decontamination capacity and the effect of different low-cost physical and chemical dentin decontamination methods and the ability of the structural integrity of the dentin. The first hypothesis was accepted as chloramine T demonstrated the highest effectiveness in inhibiting bacterial growth on dentin compared to deionized water, UV light, and 0.1% thymol. The results of decontamination evaluation showed that the chloramine T group was the only method to inhibit bacterial growth, which can be explained by its composition. Its mechanism of action starts when the powder is dissolved in water, the chlorine-nitrogen bonds are gradually broken, allowing the chlorine to dissolve slowly, exerting its potential for disinfecting action against microorganisms [19], not having an adverse effect on dentin collagen [20]. The result obtained from this study corroborates that one obtained by Khvostenko et al. [14] using chloramine T solution to disinfect molars for 7 days. Indeed, the authors evidenced that when a freshly prepared chloramine solution is used, its effectiveness is guaranteed by the slow release of chlorine, providing prolonged antiseptic activity [21]. In the current study, fresh solutions were used, and results of the microbiological evaluation demonstrated the low absorbance values obtained by the spectrophotometer.
Both Chloramine and thymol can disinfect, despite using different mechanisms of action for this purpose. However, the results of the microbiological evaluation showed that thymol could not disinfect the dentin slices. This information corroborates the literature review by Nawrocka et al, who reported that thymol when used as a disinfectant did not demonstrate satisfactory results [1]. The choice of disinfectants will depend on factors such as the type of target microorganism and the physical characteristics of the material to be disinfected. Although Thymol has an antimicrobial activity described in the literature, it could not fully disinfect the dentin slices, unlike chloramine. However, this study does not invalidate thymol’s disinfectant properties in other situations.
UV light was also unable to decontaminate the dentin sections. Although it is a commonly used technology for certain procedures and environments, the main limitation of UV light is related to its inefficiency in shading areas, and therefore, it may not provide efficient decontamination results [22]. Another point of view that we must take into consideration is the distribution of light that can occur in dentin, precisely through dentinal tubules, resulting in low light absorption and a low rate of light transmission. The linearity between light absorption and material concentration is limited if the medium is highly dispersive [23]. Thus, dentin has structural characteristics that prevent decontamination through exposure to UV light, precisely because it has areas where light cannot penetrate.
In vitro studies must consider the composition and structure of dentin specimens to simulate oral conditions accurately [5]. The surface hardness assay is commonly used to determine the degree of structural variation in dentin, as it relies on the mineral content of the specimen. The results of the surface hardness test did not show any alteration in the dentin surface following exposure to any of the decontamination models tested in this study. Thus, the hypothesis that the dentin hardness would not be significantly reduced by decontamination with chloramine T was accepted. These findings are consistent with a previous study by Aydin et al. [24], where dentin slices were stored in deionized water and thymol for 2 months, and no significant structural loss or variation was observed following surface hardness measurements.
The protocol used in this study involved a short storage period of 7 days [14], which allowed for efficient decontamination while minimizing any potential effects on dentin structure. A similar study by Haller et al. [25], also used a storage period of 14 days and found no significant effects on dentin. However, Mobarak et al. [26] even long-term storage in chloramine, did not significantly affect dentin mechanical properties, suggesting that chloramine can be used for extended storage times of up to 2 years. The same result was confirmed when evaluating the dentin surface using SEM, as no changes in surface morphology were observed in the control group or any of the other groups. However, using a 7-day storage period provided the advantage of efficiently balancing decontamination with minimal impact on dentin, while also allowing for a practical and manageable timeframe for experimental procedures.
As in vitro study, the small sample size could be a limitation of the study, which may impact the generalizability of the findings and reduce statistical power. Variations in experimental conditions, such as storage temperature or chloramine T concentration, could also influence the results and their applicability to different clinical settings. Additionally, the study focuses on a 7-day storage period, leaving a short and long-term effects of chloramine T on dentin morphology unaddressed. Regarding the substrate, the variability in dentin among different teeth was not accounted for, potentially affecting the outcomes. However, as dentin morphology was not affected by chloramine T, the results could be broadly applicable to other types of substrates and dental materials samples. Lastly, specific microorganisms inhibited by chloramine T were not isolated, limiting the understanding of its broad-spectrum efficacy in a clinical context.
The third hypothesis demonstrated that chloramine T decontamination would preserve the integrity of dentin. Surface hardness tests using the Knoop microhardness method did not show differences between the chloramine T treated groups and the control group, corroborating the hypothesis proposed above.
The SEM analysis showed no alterations in dentin surface morphology, including the integrity of the dentinal tubules in both intertubular and peritubular dentin. This corroborates with previous studies that demonstrated chloramine T did not cause significant damage to dentin even after prolonged storage [24,26]. Also, chloramine T offers practical advantages such as ease of preparation without the need for specialized equipment like UV irradiation or gamma radiation, making it a low-cost and effective option for dentin decontamination in dental research.
Chloramine T was effective and safe over a 7-day period, allowing its utilization in further experiments, but additional research is necessary to assess its long-term effects and applicability to various dental tissues. Also, identifying and isolating specific microorganisms inhibited by chloramine T could enhance understanding and applicability of its clinical use.
CONCLUSION
The findings of this study suggest that chloramine T is an effective method for decontaminating dentin without causing any structural damage. Therefore, dentin samples treated with chloramine T can be used in experiments that require prior decontamination. The other physical and chemical decontamination methods tested in this study were not effective in decontaminating dentin. The use of chloramine T can be a cost-effective and safe alternative for decontaminating dentin samples in experimental settings.
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How to cite this article
Marcelino L, Castilho ARF, Franco EM, Mattos-Graner RO, Pfeifer CSC, Puppin-Rontani RM. Chloramine T: an effective disinfectant for reducing bacterial contamination and preserving dentin. RGO, Rev Gaúch Odontol. 2026;74:e20260005. http://dx.doi.org/10.1590/1981-86372026000520250040
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Support
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Data Availability
The research data are available in the body of the manuscript.
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Edited by
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Assistant editor
Luciana Butini Oliveira
Note: A) Deionized Water x4000, A1) Deionized Water x6000 (black pointer demonstrates occluded dentinal tubules). B) UV Light x4000, B1) UV Light x6000 (black pointer demonstrates occluded dentinal tubules). C) thymol x4000, C1) thymol x6000 (images demonstrates that there is no occlusion of the dentinal tubules). D) chloramine T x4000, D1) chloramine T x6000 (images demonstrates that there is no occlusion of the dentinal tubules). SEM: Scanning Electron Microscopy; UV: Ultraviolet Irradiation.