# Study of Compounds Released from Experimental Dental Composite Formulated Through In Situ Sol-Gel Process

Rayenne Latoui, Djallel Bouzid, Elodie Fromentin, Laurent Vanoye, Olivier Boyron

► To cite this version:

Rayenne Latoui, Djallel Bouzid, Elodie Fromentin, Laurent Vanoye, Olivier Boyron. Study of Compounds Released from Experimental Dental Composite Formulated Through In Situ Sol-Gel Process. Macromolecular Chemistry and Physics, 2025, 226 (20), pp.e00291. 10.1002/macp.202500291. hal-05448782

HAL Id: hal-05448782

https://hal.science/hal-05448782v1

Submitted on 8 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Study of Compounds Released from Experimental Dental Composite Formulated Through In-situ Sol-gel Process

Rayenne Latoui<fnref n="1" />, Djallel Bouzid<fnref n="1" />*, Antoine Vauchez<fnref n="2" />, Elodie Fromentin<fnref n="2" />, Laurent Vanoye<fnref n="3" />, Olivier Boyron<fnref n="3" />*

Email*:olivier.boyron@univ-lyon1.fr

**Abstract**

The development of an experimental dental composite requires an assessment of the possible
release of molecules used in the manufacture of this composite, such as unpolymerized
monomers, photoinitiator and coupling systems. The unpolymerized monomers are due to the
incomplete conversion rate, and these molecules can degrade and react with each other to give
other products.

The aim of this study was to evaluate, in particular, the release of Bisphenol A-glycidyl methacrylate (Bis-GMA) and Triethylene glycol dimethacrylate (TEGDMA) monomers, bisphenol A (BPA) and camphorquinone (CQ) from an experimental dental composite composed of TEGDMA and Bis-GMA monomers and 50 wt.% fillers, formulated using an in-situ sol-gel process. The aim was to understand the degradation process and provide valuable information on the long-term biocompatibility of in situ sol-gel composites.

Release was assessed using a protocol based on variation in the medium and release time.
Qualitative and quantitative analysis of the release products was carried out using high-
performance liquid chromatography-mass spectrometry (LC-MS).

The products released are essentially the two monomers Bis-GMA and TEGDMA, the initiator
(CQ), as well as BPA which is a degradation product of Bis-GMA, other products were also
detected, particularly the hydrolysis products of the Bis-GMA and TEGDMA monomers, as
well as additional compounds likely resulting from the degradation and/or reactions of the
composite components.

<footnote n="1">¹LGPDDPS, Ecole Nationale Polytechnique de Constantine, 25000 Constantine, Algérie.</footnote>

<footnote n="2">²Univ Lyon, CNRS, UMR5246, ICBMS, 69622 Villeurbanne, France.</footnote>

<footnote n="3">³Univ Lyon, CPE Lyon, CNRS, UMR 5128, CP2M, Equipe PCM, 69100 Villeurbanne, France.</footnote>
The quantities of products released vary according to the release medium and the nature of the
molecules, relative to the base composition, CQ released the most in both media, around 48%
in ethanol and 1.6% in saliva, and BPA the least, around 0.01% in ethanol and less than 0.002%
in saliva. Meanwhile, more TEGDMA was released in the artificial saliva (0.2%), while more
Bis-GMA was eluted in the ethanol/water mixture (14%).

**Keywords:** Dental composite, monomer release, unpolymerized monomer, BPA, Camphorquinone, LC-MS
Introduction

Dental composites have become the cornerstone of modern restorative dentistry, offering
aesthetic and functional solutions for a wide range of clinical applications. [1] However,
ongoing research is aimed at further optimizing these materials, particularly in terms of
mechanical properties and polymerization shrinkage.

The in-situ sol-gel process holds promise for enhancing dental composites by creating a more interconnected network between the resin matrix and reinforcing particles, leading to improved properties like strength, hardness, and thermal stability. A study by Latoui et al. has validated its effectiveness in achieving these benefits, making it a valuable tool for developing high-quality dental materials. [2] However, the effectiveness and biocompatibility of a material depends not only on its chemical or physical nature, nor on the duration of exposure and the type of tissue exposed, but also on the substances released. [3] Therefore, a truly comprehensive assessment of biocompatibility must go beyond basic material characteristics and include analysis of released substances, and long-term monitoring to ensure the safety and effectiveness of the material for its intended biomedical application.

Resin-based dental restorative materials are complex composites consisting of inorganic filler
particles and an organic resin matrix, bonded together by a coupling agent. The matrix is a
network of cross-linked polymers formed by the polymerization of various types of monomers
using photoinitiators. [4] The photopolymerization reaction is never complete, and the degree
of conversion ranges from 55% to 75% at best. [4, 5, 6] Therefore, the presence of unreacted
monomers, can lead to the continuous release of residues with potentially cytotoxic and
genotoxic effects on the human organism. [7]

Research by Bakopoulou et al. [8] has identified over 30 chemical compounds released from dental composites into the external environment. These released substances encompass a wide range, including monomers, resin co-monomers and oligomers, hydrolysis products derived from these components, filler particles, initiators, catalysts and stabilizers involved in the polymerization reaction, and even metal ions.

The selection of the main target compounds was based on the composition of resin-based dental materials studied, the consistently high prevalence of certain monomers, their specific function in the material and their potential adverse health effects, as briefly described below. Specifically, we focused on the release of TEGDMA, Bis-GMA, bisphenol A (BPA), and camphorquinone (CQ).
Since the early 60s, the most commonly used monomers in composites, crucial for strength and durability are dimethacrylates such as bisphenol-A glycol dimethacrylate (Bis-GMA). [9] This monomer is very viscous and require a diluent to be workable. The most commonly used diluent is triethyleneglycol dimethacrylate (TEGDMA). [9] Occasionally, it is claimed that BPA can be found in dental composites, present as an impurity from the synthesis process and/or possibly as a degradation product of Bis-GMA [10, 11]. CQ is the most commonly used photo-initiator and ensures the initiation of the polymerization reaction [12].

Moreover, almost all targeted monomers and additives have been shown to be cytotoxic in vitro, though often only at high concentrations [13, 14]. However, even sub-cytotoxic levels of common monomers can significantly disrupt crucial cellular functions, including immune responses, differentiation, or induction of cell death by apoptosis [15, 16]. Furthermore, the presence of endocrine disruptors, such as BPA and its derivatives, in the saliva of patients with resin-based dental sealants raises serious concerns [17]. These compounds can exhibit estrogenic activity in vitro, interfering with endocrine function by altering hormone levels and mimicking or blocking endogenous hormones.

The aim of this study was to evaluate the qualitative and quantitative analysis of components released from a new dental composite based on Bis-GMA/TEGDMA resin and silica nanoparticles, manufactured using the in-situ sol-gel approach. The analysis was conducted in two simulating media and at different storage times to better understand the release process and provide valuable insights into the biocompatibility of in-situ sol-gel composites. High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) was first employed in full scan mode to identify all released products. Subsequently, selected ion monitoring (SIM) mode was utilized to quantify the target components, Bis-GMA, TEGDMA, BPA, and CQ.

# 1. Experimental section

## 1.1. Materials and methods

### 1.1.1. Dental composite manufacture

The material studied is a dental composite prepared [2] by an in-situ sol-gel process, containing 50 wt.% inorganic fillers (silica nanoparticles), as well as monomers (Bis-GMA and TEGDMA), a coupling agent and photoinitiators (DMAEMA and camphorquinone).

Details about composite preparation are given in the Supporting Information (SI) section, and the characteristics of the composites are summarized in Table 1.
<table><caption>**Table 1:** Main characteristics of the experimental resin composite used in the study</caption><thead><tr><th>Filler content</th><th>Conversion degree<sup>b)</sup></th><th>Shrinkage<sup>c)</sup></th><th>Hardness<sup>d)</sup></th><th>Elasticity modulus<sup>d)</sup></th><th>Water sorption (WSP)</th><th>solubility (WSL)</th></tr></thead><tbody><tr><td>50%</td><td>60%</td><td>0.46%</td><td>72 HV</td><td>10 Gpa</td><td>1.35 wt%</td><td>0.1 wt%</td></tr></tbody></table>

### 1.1.2. Artificial saliva

Artificial saliva was prepared according to the Ringer protocol [18, 19, 6] A volume of 606 mL was prepared with various compounds in pure water at the concentrations shown in Table 2.

<table><caption>**Table 2.** Artificial saliva composition.</caption><thead><tr><th>Chemicals</th><th>Na<sub>2</sub>HPO<sub>4</sub></th><th>KH<sub>2</sub>PO<sub>4</sub></th><th>NaCl</th><th>KHCO<sub>3</sub></th><th>CaCl<sub>2</sub></th><th>MgCl<sub>2</sub></th><th>Citric acid</th></tr></thead><tbody><tr><th>Concentration (mM)</th><td>0.39</td><td>0.41</td><td>0.16</td><td>0.24</td><td>0.24</td><td>0.024</td><td>0.00002</td></tr></tbody></table>

### 1.1.3. Chemicals

Dibasic sodium phosphate dihydrate was purchased from Honeywell (Germany), monobasic potassium phosphate and citric acid monohydrate were acquired from SIGMA-ALDRICH (Spain), potassium carbonate, calcium chloride, magnesium chloride and sodium chloride were supplied by BIOCHEM (France).

Bisphenol A (BPA), triethylene glycol dimethacrylate (TEGDMA), and bisphenol A diglycidyl methacrylate (Bis-GMA) were of analytical grade and were supplied by Sigma-Aldrich (Germany), camphorquinone (CQ) was obtained from Alfa cesar (Germany). Methanol and water (LC-MS grade) were acquired from Fisher Scientific (United Kingdom).

Ammonium acetate was acquired from Merck (Germany) and Ethanol absolute (99%) was purchased from Carlo Erba reagents (France). Pure water was prepared from demineralized water with an Elga Purelab Flex system (Veolia Water Solutions and Technologies, Belgium).

## 1.2. LC-MS analysis

To achieve both qualitative and quantitative analysis of the compounds released from the composite, two complementary LC-MS approaches were employed in succession.

<footnote>a) determined by TEM microscopy; b) determined by FTIR spectroscopy; c) determined using a pychnometer; d) determined by nanoindentation test.</footnote>
## First Stage – Screening and Quantification (HPLC- QMS)

An initial screening of all extractable compounds was performed using a high-performance liquid chromatography system HPLC (Agilent technologies, 1260 Infinity II) coupled to a single quadrupole mass spectrometer (QMS) operated in electrospray ionization mode (ESI), both in positive (ESI+) and negative (ESI-) modes. The system was run in full-scan mode (mass range: 20–1000 Da), which allowed untargeted detection of a broad range of ions. Chromatographic separation was achieved on an XBridge BEH-C18 column (50 mm × 2.1 mm, 2.5 µm, Agilent Technologies) using a binary mobile phase composed of 2 mM ammonium acetate buffer, pH 5.6 (A), and methanol (B), with a gradient elution. The injection volume was 10 µL, and the flow rate was 0.175 mL/min. The column temperature was maintained at 40 °C.

Following full-scan analysis, four representative compounds were selected based on their abundance and relevance. These were then quantified using the Selected Ion Monitoring (SIM) mode on the same QMS instrument, in both ESI+ and ESI- as appropriate. The use of SIM on the quadrupole system enhanced specificity and reproducibility for these target analytes.

## Second Stage – Structural Identification (UHPLC-QTOF)

To further identify additional compounds observed in the first scan analysis but not quantified, a second, more sensitive and high-resolution method was employed. This involved a UHPLC system (U3000, Thermo Fisher Scientific) coupled to a high-resolution hybrid quadrupole time-of-flight (QTOF) mass spectrometer (Impact II, Bruker). Ionization was achieved via electrospray in positive mode (ESI+). The source temperature was set at 200 °C, capillary voltage at 4500 V, and nebulizer pressure at 35 psi. The mass range covered was 20–1000 Da. The system was calibrated using sodium formate solution, and data were processed using DataAnalysis 6.1 (Bruker).

UHPLC separation used the same column type and conditions as in the HPLC setup. Instead of SIM, Extracted Ion Chromatograms (EIC) were used on the QTOF instrument to selectively visualize ions of interest from the full-scan data, offering both accurate mass information and retention time confirmation. This high-resolution approach allowed tentative identification of additional compounds based on their exact masses, isotope patterns, and fragmentation profiles.

The chemical structures of compounds selected for quantification using the SIM mode are shown in Table 3, and their corresponding MS parameters and retention times are detailed in Table 4.
<table><caption>**Table 3.** Chemical structures of the examined compounds.</caption><thead><tr><th>Compound</th><th>Function</th><th>Structure</th><th>Molecular formula</th><th>Molar mass (g/mol)</th></tr></thead><tbody><tr><td>Bis-GMA<sup>a)</sup></td><td>Monomer</td><td><img src="bisd_gma_structure.png" alt="Chemical structure of Bis-GMA"></td><td>C<sub>29</sub>H<sub>36</sub>O<sub>8</sub></td><td>512.6</td></tr><tr><td>TEGDMA<sup>b)</sup></td><td>Monomer</td><td><img src="tegdma_structure.png" alt="Chemical structure of TEGDMA"></td><td>C<sub>14</sub>H<sub>22</sub>O<sub>6</sub></td><td>286.3</td></tr><tr><td>BPA<sup>c)</sup></td><td>Degradation by-products</td><td><img src="bpa_structure.png" alt="Chemical structure of BPA"></td><td>C<sub>15</sub>H<sub>16</sub>O<sub>2</sub></td><td>228.3</td></tr><tr><td>CQ<sup>d)</sup></td><td>Initiator</td><td><img src="cq_structure.png" alt="Chemical structure of CQ"></td><td>C<sub>10</sub>H<sub>14</sub>O<sub>2</sub></td><td>166.2</td></tr></tbody></table>

**1.3. Quantification method**

The analysis of composite samples was conducted using LC-MS with both UV and MS detection. Linearity of the analytical method used was investigated by constructing calibration curves using stock of each compound Bis-GMA, TEGDMA, BPA, and CQ with a concentration of 1000 ppm were prepared by dissolving the appropriate amount of each monomer in ethanol/water (1:1, v/v). Eleven calibration points were used at concentrations ranging from 0.005 to 100 ppm in order to ensure coverage of the full studies concentration range. Standard solutions were stored at 4°C. Chemical structures of the four compounds studies are represented in Table 3. Calibration curves were constructed from the peak areas of the analytes. The slope, y-intercept and correlation coefficient of each calibration line were calculated using linear regression analysis.

The HPLC-MS system with a UV-Vis detector and a MS detector were used to analyze the four products. The UV-Vis detector was set to 220 nm, the absorption wavelength of the four analyzed products, as illustrated in Figure 1-SI. Separation of the compounds Bis-GMA, TEGDMA, BPA and CQ was achieved in less than 8 minutes. The retention times for the compounds examined were 2.1 min for camphorquinone, 4 min for BPA, 4.2 min for

<footnote><sup>a)</sup>Bisphenol A-Glycidyl Methacrylate, <sup>b)</sup>Triethylene Glycol Dimethacrylate, <sup>c)</sup>Bisphenol A, <sup>d)</sup>Camphorquinone</footnote>
TEGDMA, and 7.4 min for bis-GMA. A typical chromatogram of standard solutions using both detectors is shown in Figure 1.

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Chromatograms recorded by a) HPLC-DAD at 210 nm, b) MS of the pure Bis-GMA, TEGDMA, BPA and CQ.</figcaption></figure>

Mass spectrometry was chosen as the detection method for quantifying Bis-GMA, TEGDMA, BPA and camphorquinone released from dental resin. Compared with UV detection, mass spectrometry offers significantly higher specificity, sensitivity and selectivity. This enables each compound to be unambiguously identified because of its unique mass spectrum, even in complex matrices. In addition, the Selected Ion Monitoring (SIM) mode offers excellent selectivity, enabling precise quantification without interference from other compounds. This is particularly advantageous for analysis, especially as TEGDMA and BPA peaks are seen to overlap in UV detection (Figure 1-a). Likewise, MS is far more sensitive than UV detection. It can detect extremely small quantities of compounds, which is interesting in our case.

<table><caption>**Table 4:** MS parameters for the analysis of the target compounds</caption><thead><tr><td>Compound</td><td>Retention time (min)</td><td>Ionization mode</td><td>m/z</td></tr></thead><tbody><tr><td>Bis-GMA</td><td>7.4</td><td>ESI<sup>+</sup></td><td>530.271</td></tr><tr><td>TEGDMA</td><td>4.2</td><td>ESI<sup>+</sup></td><td>304.168</td></tr><tr><td>BPA</td><td>4.0</td><td>ESI<sup>-</sup></td><td>227.097</td></tr><tr><td>CQ</td><td>2.1</td><td>ESI<sup>+</sup></td><td>184.080</td></tr></tbody></table>

## 1.4. Linearity and limits of quantification

Standards were newly prepared and analyzed on the same day. Calibration curves were fitted by linear regression analysis on the peak area of the target compounds, as a function of the
different nominal concentrations of the target compounds. The accuracy of the calibration curves was evaluated using the error of the calculated concentrations expressed as a percentage of the target concentrations (% of target) and the correlation coefficient R² of the calibration curves. According to the above-mentioned international rules, the deviation between measured and nominal concentrations must not exceed 20%. [22] The error is shown in Figure 2-SI.

<figure><img src="image_3.png" /><figcaption>**Figure 2:** Mass calibration curves for Bis-GMA (a), TEGDMA (b), BPA(c) and CQ (d).</figcaption></figure>

Responses as a function of nominal concentrations fitted well to a straight line as shown in Figure 2.

All calibration data as well as LOD and LOQ are presented in Table 5.

<table><caption>**Table 5:** Calibration and sensitivity data of the four examined compounds.</caption><thead><tr><td>Compound</td><td>Equation</td><td>R<sup>2</sup><sub>adjusted</sub></td><td>Error<sup>a)</sup> (%)</td><td>LOD<sup>b)</sup> (ppm)</td><td>LOQ<sup>c)</sup> (ppm)</td></tr></thead><tbody><tr><td>Bis-GMA</td><td>Y= 0.79X+5.4</td><td>0.997</td><td>13.2</td><td>0.0005</td><td>0.005</td></tr><tr><td>TEGDMA</td><td>Y=0.56X+7.2</td><td>0.996</td><td>9.0</td><td>0.0005</td><td>0.005</td></tr><tr><td>BPA</td><td>Y=0.93X+5.1</td><td>0.998</td><td>10.8</td><td>0.0005</td><td>0.005</td></tr><tr><td>CQ</td><td>Y=0.93X+6.0</td><td>0.999</td><td>6.4</td><td>0.0005</td><td>0.001</td></tr></tbody></table>
The regression equations for all compounds revealed good correlation coefficients R² ranging from 0.996 to 0.999 and precision (RSD lower than 20%). These results indicated a good correlation between the measured response (peak area) and the nominal concentration of the target compounds.

**1.5. Samples preparation**

Two composites were prepared in a polyamide mold, 10 mm in diameter and 2 mm thick, then were irradiated at room temperature with a LED lamp for 40 seconds using an Eighteen curing pen LED dental lamp with an intensity of 1500 mW cm⁻² at a distance of 5 mm. [6] One composite was immersed in 3 mL of artificial saliva, and the other was immersed in ethanol/water (75 :25, v/v). **Both composites were kept at 37 °C and incubated in a shaker at 100 rpm to ensure gentle agitation during the incubation process.** They were measured after 1, 24 hours, and 3, 7, 14, 28, 45 days. The immersion solution was refreshed after every measurement. By continuously replacing the release medium, the experiment accurately simulates the *in vivo* dilution of released monomers in saliva, preventing measurement distortion from saturation and providing a more reliable assessment of initial monomer release kinetics.

A volume of the samples, from the monomer leaching experiment was taken and diluted to reach a mixture of ethanol/water or ethanol/artificial saliva (1 :1, v/v) to avoid any variations caused by the different solvents used in release experiments [20, 21] and the need to prepare different standard calibration curves for each solvent tested individually [22]. The artificial saliva samples were further acidified with 0.1% formic acid to have a similar pH value as for the mobile phase. [22] Finally, 200µL of the samples were transferred to an amber LC-vial with insert and 10 µL were injected into the LC/MS system.

**2. Results and discussion**

**2.1. Release experiments on composites**

**2.1.1. Identification**

The suitability of the developed method LC-MS was evaluated by analyzing the released compounds from the experimental dental composite submitted to an in vitro release experiment using 3 mL ethanol/water (75/25) and artificial saliva as aging media.

<footnote>a) Relative error, Y: log (Area), X=log (concentration), b), c) LOD and LOQ values were determined using standard solutions in solvent and represent instrumental detection limits.</footnote>
Artificial saliva was used in this study to simulate a real relevant situation for testing of composite material. Nevertheless, in literature the use of ethanol as simulating medium is often to detect the maximum amount of released monomers, whereas a mixture of ethanol/water is described to be more relevant [23].

The LC-MS analysis was initially performed in scan mode to identify the full spectrum of compounds released during the aging process. The chromatograms presented in Figure 3 showcase the detected peaks at different point of time, corresponding to various compounds. These compounds were further analyzed, classified, and summarized in Table 6, which provides their retention times, molecular masses, names, and origins.

<figure><img src="image_4.png" /><figcaption>**Figure 3:** MS chromatograms of compounds released into ethanol/water medium at different time point using scan mode.</figcaption></figure>

<table><caption>**Table 6:** Identified compounds released in Ethanol/water and artificial saliva medium.</caption><thead><tr><th>Peak</th><th>Tr<br>(min)</th><th>Formula</th><th>M[μ]</th><th>Compound</th><th>Origin</th></tr></thead><tbody><tr><td>1</td><td>1.0</td><td>C<sub>6</sub>H<sub>14</sub>O<sub>4</sub></td><td>150.084</td><td>TEG<sup>a</sup></td><td>Hydrolysis product of TEGDMA</td></tr><tr><td>2</td><td>1.5</td><td>C<sub>10</sub>H<sub>18</sub>O<sub>5</sub></td><td>218.115</td><td>TEGDMA<sup>b</sup></td><td>Hydrolysis product of TEGDMA</td></tr></tbody></table>
<table><tr><td>3</td><td>2.3</td><td>C<sub>10</sub>H<sub>14</sub>O<sub>2</sub></td><td>166.099</td><td>CQ</td><td>Initiator</td></tr><tr><td>4</td><td>3.8</td><td>C<sub>21</sub>H<sub>28</sub>O<sub>6</sub></td><td>376.188</td><td>Bis-HPPP<sup>c</sup></td><td>Hydrolysis product of Bis-GMA</td></tr><tr><td>5<sup>d</sup></td><td>4.0</td><td>C<sub>15</sub>H<sub>16</sub>O<sub>2</sub></td><td>228.115</td><td>BPA</td><td>Degradation by-product of Bis-GMA</td></tr><tr><td>6</td><td>4.3</td><td>C<sub>14</sub>H<sub>22</sub>O<sub>6</sub></td><td>286.141</td><td>TEGDMA</td><td>Monomer</td></tr><tr><td>7</td><td>5.3</td><td>C<sub>19</sub>H<sub>24</sub>O<sub>5</sub></td><td>334.162</td><td>-</td><td>-</td></tr><tr><td>8</td><td>6.0</td><td>C<sub>25</sub>H<sub>32</sub>O<sub>7</sub></td><td>444.219</td><td>-</td><td>Hydrolysis product of Bis-GMA</td></tr><tr><td>9</td><td>7.3</td><td>C<sub>29</sub>H<sub>36</sub>O<sub>8</sub></td><td>512.241</td><td>Bis-GMA</td><td>Monomer</td></tr><tr><td>10</td><td>9.3</td><td>C<sub>18</sub>H<sub>35</sub>NO</td><td>281.271</td><td>-</td><td>-</td></tr></table>

<sup>a</sup>Triethylene glycol, <sup>b</sup>Triethylene glycol monomethacrylate, <sup>c</sup>2,2-Bis[4-(2,3-dihydroxypropoxy) phenyl] propane, <sup>d</sup>compound detected in negative ionization mode (chromatogram not shown).

The compounds identified include unpolymerized monomers, unreacted initiators, monomer hydrolysis products and monomer and polymer degradation by-products. Specifically, we detected Bis-GMA and TEGDMA as unpolymerized monomers, camphorquinone as an unreacted initiator and BPA as a degradation by-product of Bis-GMA. BPA was detected in negative ionization mode (chromatogram not shown). BPA (bisphenol A) serves as a precursor to Bis-GMA (glycidyl methacrylate and bisphenol A). The release of BPA during the degradation of Bis-GMA in dental composites is a complex process. Bis-GMA does not degrade directly to free BPA; instead, its breakdown can produce fragments that resemble parts of the BPA molecule without necessarily releasing the intact BPA [10, 11].

In addition, we observed the presence of TEG/TEGDMA (hydrolysis products of one ester and two esters of TEGDMA, respectively), C<sub>25</sub>H<sub>32</sub>O<sub>7</sub> and Bis-HPPP (hydrolysis products of one ester and two esters of Bis-GMA, respectively), as shown in Figure 4. Despite these results, some compounds could not be identified, probably due to the complexity of the chemical reactions involved, such as polymer decomposition, oxidation and cross-linking.
<figure><img src="image_5.png" /><figcaption>**Figure 4:** Mechanism of hydrolysis processes of Bis-GMA (a, b) and TEGDMA (c, d).</figcaption></figure>

### 2.1.2. Quantification

After an initial identification of the compounds released by the dental composite, four compounds: Bis-GMA, TEGDMA, BPA and CQ were accurately quantified using the SIM mode to evaluate their release levels under the experimental conditions. Figure 5 illustrates the MS chromatograms of these compounds released into ethanol/water medium after 14 days as an example.
<figure><img src="image_6.png" /><figcaption>**Figure 5:** MS chromatograms of composite 14 days after its immersion in ethanol/water in SIM mode (a, b, c).</figcaption></figure>

In full scan mode, the entire m/z range was scanned to identify all compounds present in the sample. This provided an overview of the composition (Figure 5). Next, SIM mode was used to target specific compounds of interest, Bis-GMA, TEGDMA, BPA and CQ. By focusing on these predefined m/z values (Table 4), the analysis gained in sensitivity and specificity, enabling accurate quantification.

Compounds were ionized in either positive or negative mode based on their chemical structures. As shown in Figures 5-a, b, c, BPA was ionized in negative mode due to its phenolic nature. Bis-GMA, TEGDMA and CQ were ionized in positive mode. SIM mode allowed for the isolation of specific ions for each compound, minimizing interferences, particularly between TEGDMA and camphorquinone.

The data obtained from the LC-MS analysis of released Bis-GMA, TEGDMA, BPA and CQ are presented in Table 7.

**Table 7:** Mean values (n=3) with standard deviation (SD) for concentration (µg/mL) of Bis-GMA, TEGDMA, BPA, and CQ released by composite materials after incubation in ethanol/water and saliva for different time points.
<table><thead><tr><th rowspan="2">Medium</th><th rowspan="2">Monomer</th><th colspan="8">Concentration (SD) (µg/mL)<sup>a)</sup></th></tr><tr><th>1h</th><th>1 day</th><th>3 days</th><th>7 days</th><th>14 days</th><th>21 days</th><th>28 days</th><th>45 days</th></tr></thead><tbody><tr><td rowspan="7">Ethanol/water</td><td>Bis-GMA</td><td>33.9<br>± 0.5</td><td>79.2<br>± 0.4</td><td>73.5<br>± 0.2</td><td>35.3<br>± 0.7</td><td>68.9<br>± 0.1</td><td>51<br>± 0.3</td><td>46.8<br>± 0.6</td><td>32.2<br>± 0.5</td></tr><tr><td>TEGDMA</td><td>14.6<br>± 2.3</td><td>10.7<br>± 1.5</td><td>1.8<br>± 0.3</td><td>0.5<br>± 0.1</td><td>0.8<br>± 0.1</td><td>0.9<br>± 0.15</td><td>0.3<br>± 0.02</td><td>0.1<br>± 0.04</td></tr><tr><td>BPA</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>0.265<br>± 0.02</td><td>0.016<br>± 0.006</td><td>0.05<br>± 0.0003</td><td>0.028<br>± 0.004</td></tr><tr><td>CQ</td><td>0.2<br>± 0.04</td><td>0.6<br>± 0.09</td><td>1.7<br>± 0.2</td><td>0.7<br>± 0.02</td><td>2.9<br>± 0.6</td><td>6<br>± 0.3</td><td>5.7<br>± 0.6</td><td>1.9<br>± 0.3</td></tr><tr><td>Bis-GMA</td><td>0.017<br>± 0.003</td><td>0.12<br>± 0.01</td><td>0.016<br>± 0.002</td><td>0.04<br>± 0.006</td><td>0.05<br>± 0.003</td><td>0.36<br>± 0.01</td><td>0.05<br>± 0.001</td><td>0.002<br>± 0.0008</td></tr><tr><td>TEGDMA</td><td>0.46<br>± 0.2</td><td>1.53<br>± 0.1</td><td>0.21<br>± 0.02</td><td>0.15<br>± 0.03</td><td>0.016<br>± 0.005</td><td>0.1<br>± 0.001</td><td>0.14<br>± 0.003</td><td>0.0004<br>± 0.0001</td></tr><tr><td>BPA</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>0.046<br>± 0.003</td><td>0.05<br>± 0.001</td><td>nd</td><td>nd</td></tr><tr><td rowspan="2">Artificial saliva</td><td>CQ</td><td>0.008<br>± 0.0003</td><td>0.02<br>± 0.006</td><td>0.02<br>± 0.001</td><td>0.04<br>± 0.003</td><td>0.2<br>± 0.001</td><td>0.1<br>± 0.006</td><td>0.2<br>± 0.03</td><td>0.08<br>± 0.003</td></tr></tbody></table>

As the degree of conversion refers to the percentage of monomers that have been successfully converted into the polymer network during the polymerization process, so, a higher degree of conversion is associated with reduced monomer release, as more monomers are incorporated into the polymer structure, leaving fewer unreacted monomers that can leach out. Conversely, a lower degree of conversion results in a higher amount of residual monomers, which are more likely to be released into the surrounding environment. In the composite material studied, the degree of conversion was found to be 60%, which indicates a moderate level of polymerization. At this level, there is a substantial amount of unreacted monomer that may leach out. Besides Berghaus et al. reported that release was determined not only by the amount of residual monomer present, but also by other factors such as, possibly, density and network structure.[11]

All four compounds were detected in both media within the first hour, except for bisphenol A (BPA), which was only observed after 14 days (Table 6). This delayed detection contrasts with findings by Małkiewicz et al. [24], who reported an earlier release of BPA from six commercial

<footnote><sup>a)</sup> Concentration (µg/mL) of compounds released from the composite in ethanol and artificial saliva calculated using the calibration curve. <sup>nd</sup>: not detected, under the LOD. <sup>C concentrations</sup> are not cumulative. Each value corresponds to the specific time point after replacing the solvent.</footnote>
dental composites, using HPLC analysis. Their study observed BPA concentrations ranging from 0.02 to 0.08 µg/mg during the first hour of incubation in water, with a peak release of 1.8 µg/mL from the Gradia Direct Anterior composite after 24 hours. Additionally, their analysis identified the release of TEGDMA, with a maximum concentration of 8.11 µg/mL from the Charisma composite after 7 days. Similarly, Archegas et al. [9] evaluated the release of monomers from three dental composites with resin compositions comparable to ours and filler contents of 79–81% by weight. Their HPLC analysis revealed the maximum release of TEGDMA (6.39 µg/mL) from the Cearm Teric composite after 7 days, and Bis-GMA (2.2 µg/mL) from the Herculic XRV composite within 1 hour. LCMS analysis of the monomers released by Filtek Suprem and Venus Pearl dental composites after 24 hours of incubation in artificial saliva medium revealed the presence of CQ, with concentrations of 1.44 µg/mL and 1.79 µg/mL, respectively, Bis-GMA (0.97 µg/mL and 2 µg/mL) and TEGDMA (0.76 µg/mL, 2 µg/mL).[22]

In our study, the in-situ sol-gel process can help minimize monomer release. It promotes the uniform distribution of organic and inorganic components, enhancing the overall stability of the material and preventing the aggregation of unreacted monomers. In addition, the molecular-level coupling achieved during the sol-gel process effectively binds the monomers to the silica network. This strong attachment makes it significantly harder for the monomers to be released quickly, contributing to the long-term stability of the composite material.

The quantity of released Bis-GMA, TEGDMA, BPA and CQ in ethanol/water and artificial saliva normalized to the composite's initial mass (mg/mg) at different time point is presented in Table 2-SI and the cumulated release (mg/mg) over 120 days of the four compounds is shown in Figure 6.
<figure><img src="image_7.png" /><figcaption>**Figure 6:** Amounts of released Bis-GMA, TEGDMA, BPA and CQ normalized to the composite's initial mass (mg/mg) in ethanol/water and artificial saliva as release media at different time point.</figcaption></figure>

As the detected concentration of compounds is set as a function of composite initial mass [mg/mg], the numbers can be compared directly between the two media. As expected, release in ethanol/water mixture is much more prominent than in saliva. These findings are consistent with previous research, confirming the higher release of compounds in ethanol/water [22-25].

The results show that the compounds Bis-GMA, TEGDMA and CQ are released to a significantly greater extent in an ethanol/water mixture than in artificial saliva. In contrast, BPA release was significantly lower and delayed in both media. Its detection was only possible from day 14 onwards in artificial saliva and in the ethanol/water mixture, and then only in very small quantities. There are several reasons why its release is generally not excessive. Although BPA is a precursor of the monomer Bis-GMA, the strong chemical bonds that bind it to other molecules make it difficult to release by simple dissolution or diffusion. In addition, the three-dimensional structure of the composite prepared in situ, with its interpenetrating network of polymers and mineral fillers, forms a physical barrier that limits outward diffusion of BPA.

As shown in Figure 6, the monomer most released in terms of quantity in ethanol/water was Bis-GMA, followed by TEGDMA and the initiator CQ (7.2 mg/mg, 0.6 mg/mg, 0.3 mg/mg,
respectively), while in artificial saliva, TEGDMA was the most released (0.05 mg/mg), followed by CQ (0.013 mg/mg) and Bis-GMA (0.012 mg/mg).

The slightly detectable release of residual monomers in saliva could already be confirmed by
several studies [26-28]. The low tendency of the monomers to dissolve in water can be
explained by their hydrophobic structures and their molecular size, which are particularly
pronounced in Bis-GMA [29]. Smaller molecules are presumed to have increased mobility and
might therefore be released faster than larger molecules. [9] This was observed in the present
study by the higher release of TEGDMA in saliva, when compared to Bis-GMA, which was in
agreement with other studies. [9,22, 30, 31] In addition to molecular size and hydrophobicity,
the solvent environment plays a crucial role in monomer release. Ethanol is known to act as a
plasticizer, promoting swelling of the polymer network more effectively than water-based
media such as saliva. This swelling increases the free volume within the matrix, facilitating the
diffusion and elution of residual monomers.

In addition, Table 2-SI and Table 3-SI lists the release of the four compounds over a period of 120 days in correlation to the initial amount of monomers and initiator [wt.%]. and Figure 7 represent the cumulated release of the four compounds after 120 days in ethanol/water and artificial saliva.
<figure><img src="image_8.png" /><figcaption>**Figure 7:** Cumulated release of Bis-GMA (a) and TEGDMA (b), BPA (c) and CQ (d) over a period of 120 days in correlation to the compounds's initial mass [wt.%]. Left Y-axis (black): % of compounds released in ethanol; Right Y-axis (red): % of compounds released in saliva.</figcaption></figure>

A rapid increase in the percentage of monomers released was observed in the first two months of the experiment. Then a stabilization phase began to emerge. This plateau suggests that the majority of easily releasable monomers were released during this period, leaving room for slower residual release thereafter.

Figure 7 shows that the initial release of TEGDMA was very rapid, reaching a plateau in 10 days, reflecting its high solubility in aqueous solutions. Its small molecular size enables it to diffuse easily through the polymer matrix of the composite and into the surrounding environment. On the other hand, its simpler, smaller and less functionally complex structure contributes to a more homogeneous, densely cross-linked polymer network. The result is a higher degree of conversion for TEGDMA, leading to better integration into the composite matrix and making it less likely to release over time. [28]
In contrast, Bis-GMA, with its larger molecular size and more complex structure, has a slower release profile, taking almost a month to reach plateau. Its lower conversion rate and impeded diffusion result in prolonged leaching from the composite. These contrasting release profiles highlight the influence of molecular structure and polymerization characteristics on the long-term behavior of dental materials.[22]

We can observe that initial BPA release is delayed, starting in both environments after day 14, and reaching a plateau within a relatively short time due to the stabilizing composite structure.

Camphorquinone (CQ) is the most released **compound** from composites, this observation can be attributed to several factors related to its chemical structure and its use in formulations. Due to its relatively high solubility, CQ can more easily migrate from the composite, particularly when used at high concentrations for efficient polymerization. In addition, incomplete polymerization can lead to the presence of residual CQ in the material. CQ's tendency to diffuse into the polymer matrix also contributes to its release, which explains its continuous liberation over time.

### 3. Comparative study

Given the potential cytotoxicity of released monomers, a conservative approach is adopted for assessing their release kinetics. A 4-month duration is selected for the release study, representing a significant portion of the typical lifespan of a dental restoration. Ethanol/water is chosen as the release solvent to simulate a worst-case scenario, as ethanol enhances the solubility of many organic compounds. The resulting data have been compared with existing literature on cell experiments conducted in water-based media, allowing for contextualization of the findings and identification of potential discrepancies between in vitro and in vivo release behavior. This comprehensive approach aims to provide a reliable and relevant assessment of the long-term release of monomers from dental composites and their potential impact on human health.

For instance, Urcan et al. [32] analyzed the EC50 value of TEGDMA and Bis-GMA monomers on human gingival fibroblasts. The EC50 values of the TEGDMA, and Bis-GMA monomers were 3.60 (0.20) mmol/l and 0.09 (0.01) mmol/l, respectively.

In similar study, a EC50 value of 1.83 mmol/L for TEGDMA was stated cytotoxic against human bronchoalveolar carcinoma derived A549 cells [33]. In the present study the maximum
TEGDMA released reaches concentrations of 0.051 mM and 0.0053 mM in ethanol and water respectively.

Kraus et al. [34] tested the cytotoxicity of Bis-GMA and TEGDMA against two osteoblast-like cell lines from tumor origin and immortalized human fetal osteoblasts. They found Bis-GMA to be more cytotoxic than TEGDMA. All concentrations used had a cytotoxic effect on the cells, with the lowest concentration to be 0.05 mM.

When these mmol values were converted to µg, it was found that only Bis-GMA monomer release from composite after immersion in ethanol/water was above the EC50 dose in the present study.

BPA release in ethanol was measured at 0.001 ng/kg/d, which is 200 times lower than the limit of 0.2 ng/kg/d set by EFSA in 2023.[35] As for saliva, the quantity released was even lower, at just 0.00003 ng/kg/d, or around 6666 times less than the tolerable daily intake (TDI). These results suggest that exposure to BPA under the conditions studied is well below the new strict standards defined by EFSA.

To evaluate the efficacy of the in-situ sol-gel method in reducing chemical leaching, the release of Bis-GMA, TEGDMA, BPA, and CQ from the experimental composite was compared to that of various commercial composites after 7 days of immersion in saliva.[9, 22, 24]

<table><caption>**Table 8:** Release of Bis-GMA, TEGDMA, BPA, and CQ from commercial and experimental composites after 7 days of immersion in saliva.</caption><thead><tr><th rowspan="2">Composite</th><th rowspan="2">Filler content (%.wt)</th><th colspan="4">Concentration (mg mL<sup>-1</sup>)</th><th rowspan="2">Ref</th></tr><tr><th>Bis-GMA</th><th>TEGDMA</th><th>BPA</th><th>CQ</th></tr></thead><tbody><tr><td>Herculite XRV</td><td>79</td><td>2.012</td><td>5.202</td><td>0.65</td><td>-</td><td></td></tr><tr><td>Tetric Ceram</td><td>81</td><td>0.539</td><td>6.394</td><td>-</td><td>-</td><td></td></tr><tr><td>Filtek Z250</td><td>80</td><td>0.340</td><td>1.939</td><td>-</td><td>-</td><td></td></tr><tr><td>Gradia Direct Anterior</td><td>76</td><td>-</td><td>0</td><td>1.564</td><td>-</td><td></td></tr><tr><td>Arkon</td><td>70</td><td>-</td><td>2.234</td><td>0.122</td><td>-</td><td></td></tr><tr><td>Charisma</td><td>70</td><td>-</td><td>8.112</td><td>0.137</td><td>-</td><td></td></tr></tbody></table>
<table><tr><td>Filtek Supreme XTE</td><td>78</td><td>1.91</td><td>2.66</td><td>-</td><td>8.43</td></tr><tr><td>Venus Pear</td><td>78</td><td>5.69</td><td>7.3</td><td>-</td><td>10.08</td></tr><tr><td>Experimental composite</td><td>50</td><td>0.04</td><td>0.15</td><td>0</td><td>0.04</td></tr></table>

This table compares the release of Bis-GMA, TEGDMA, BPA, and CQ from various commercial composites and an experimental composite after 7 days of immersion in saliva. The experimental composite consistently shows significantly lower release levels compared to the commercial products. Specifically, Bis-GMA release is lower by a factor of 50 compared to Herculite XRV, which exhibits the highest release. TEGDMA release is reduced by a factor of about 40 compared to Tetric Ceram. BPA is detected only in Herculite XRV and Arkon, whereas it is entirely absent in the experimental composite, demonstrating enhanced biocompatibility. CQ release from the experimental composite is lower by a factor of over 200 compared to Venus Pear, which shows the highest photoinitiator release. These significant reductions in chemical release suggest that the experimental composite has a more tightly crosslinked polymer network, thereby offering improved biocompatibility compared to the commercial counterparts after 7 days of immersion in saliva.

## Conclusion

This study provides a comprehensive assessment of the release of specific compounds from an experimental dental composite formulated by an in-situ sol-gel process. The focus was on the release of Bis-GMA, TEGDMA, BPA and CQ, which are essential components of dental composites.

The study also highlighted the influence of the release medium on the quantity and nature of the compounds released. Ethanol/water mixtures facilitated a greater release of monomers and initiators, while artificial saliva showed a more controlled release, mimicking in vivo conditions. This differential release model is crucial for assessing the performance and safety of dental composites.

Furthermore, the delayed and minimal release of BPA, a known endocrine disruptor, is a significant finding. The composite's structure effectively limited BPA diffusion, ensuring that its release remained well below regulatory limits. This highlights the potential of the in-situ sol-gel process in mitigating the risks associated with BPA exposure.
The in situ sol-gel process has demonstrated its potential to improve the stability and biocompatibility of dental composites. The uniform distribution of organic and inorganic components, together with the strong coupling at the molecular level, has significantly reduced the release of unpolymerised monomers, initiators, leachable ions and hydrolysis by-products. The release of harmful compounds is lower than that of commercial composites.

By quantifying the release of these compounds over time, this study seeks to provide valuable insights into the biocompatibility of sol-gel in situ composites. The findings will not only contribute to a better understanding of the long-term behavior of these materials but also guide the development of safer and more durable restorative dental materials.

## References

1. Samanidou V. F, Kerezoudi C, Tolika E, Palaghias G. A Simple Isocratic HPLC Method for the Simultaneous Determination of the Five Most Common Residual Monomers Released from Resin-Based Dental Restorative Materials, *J LIQ CHROMATOGR R T.* 2015, 38:6, 740-749, https://doi.org/10.1080/10826076.2014.968662

2. Latoui R, Bouzid D, Dugas P.Y, Espinosa E, Boyron O. In-situ sol-gel process for the formulation of Bis-GMA/TEGDMA/silica dental nanocomposite. *Polymer*. 2024, 313. https://doi.org/10.1016/j.polymer.2024.127731

3. Delaviz, Y.; Finer, Y.; Santerre, J. P. Biodegradation of Resin Composites and Adhesives by Oral Bacteria and Saliva: A Rationale for New Material Designs That Consider the Clinical Environment and Treatment Challenges. *Dent. Mater.* 2014, 30 (1), 16–32. https://doi.org/10.1016/j.dental.2013.08.201

4. Michelsen V. B, Kopperud H. B. M, Lygre G. B, Bjoerkman L, Jensen E, Kleven IS, Svahn J, Lygre H. Detection and quantification of monomers in unstimulated whole saliva after treatment with resin-based composite fillings in vivo. *Eur J Oral Sci.* 2012 120: 89–95.

5. Cohn-Inostroza N, De la Rosa-Varela C, Moreno-Villoslada I. Determination of BisGMA by ethanol release from dental composites. *Int. Dent. J. Stud. Res.* 2014

6. Latoui R., Bouzid, D., Belhani, Ma. Boyron, O. Enhancing photopolymerization and modeling kinetic degradation in dental composites. *J Polym Res.* 2024, 31, 273. https://doi.org/10.1007/s10965-024-04116-y

7. Bakopoulou, A.; Papadopoulos, T.; Garefis, P. Molecular Toxicology of Substances Released from Resin-Based Dental Restorative Materials. *Int. J. Mol. Sci.* 2009, 10 (9), 3861–3899. https://doi.org/10.3390/ijms10093861
8. Bakopoulou A, Papadopoulos T, Gerefis P. Molecural toxicology of substances released from resin based dental restorative materials. *Int J Molec Sci.* 2009, 10: 3861–3899.

9. Archegas L. R. P, Rached R. T, Ignacio S. A, Carvalho de Vasconcelos E, Ramos D. T and Machado de Souza E. Identification and Quantification of Monomers Released from Dental Composites Using HPLC. *Braz. arch. biol. technol.* 2009, 52, 4. https://doi.org/10.1590/S1516-89132009000400009.

10. Lewis J.B., Rueggeberg F.A., Lapp C.A., Ergle J.W., Schuster G.S., Identification and characterization of estrogen-like components in commercial resin-based dental restorative materials. *Clin. Oral Investig.* 1999, 3, 107–113.

11. Fleisch A.F., Sheffield P.E., Chinn C., Edelstein B.L., Landrigan P.J., Bisphenol A and related compounds in dental materials. *Pediatrics*. 2010, 126, 760–768.

12. Santini A., Gallegos I.T., Felix C.M., Photoinitiators in dentistry: a review. *Prim. Dent. J.* 2013, 2, 30–33.

13. Reichl F.-X., Simon S., Esters M., Seiss M., Kehe K., Kleinsasser N.H., Hickel R., Cytotoxicity of dental composite (co)monomers and the amalgam component Hg²⁺ in human gingival fibroblasts. *Arch. Toxicol.* 2006, 80, 465–472.

14. Engelmann J., Volk J., Leyhausen G., Geurtsen W., ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone. *J. Biomed. Mater. Res. B. Appl. Biomater.* 2005, 75, 272–276.

15. Schweikl H., Spagnuolo G., Schmalz G., Genetic and cellular toxicology of dental resin monomers. *J. Dent. Res.* 2006, 85, 870–877

16. Krifka S., Hiller K.-A., Spagnuolo G., Jewett A., Schmalz G., Schweikl H., The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA). *Biomaterials.* 2012, 33, 5177–5186.

17. Olea N., Pulgar R., Pérez P., Olea-Serrano F., Rivas A., Novillo-Fertrell A., Pedraza V., Soto A.M., Sonnenschein C., Estrogenicity of resin-based composites and sealants used in dentistry. *Environ. Health Perspect.* 1996, 104, 298–305

18. Pinto MM, Cesar PF, Rosa V, Yoshimura HN. Influence of pH on slow crack growth of dental porcelain. *Dent Mater*, 2008; 24: 814–823.

19. Mohammed, I., Alwahab, Z. N. A comparison of the effect of artificial saliva with different pH values on surface roughness of veneering ceramic to metal and zirconia substructure (in vitro study). *World J. Pharm. Res.* 2018, 7(17), 107. https://doi.org/10.20959/wjpr201817-13326
20. Moharamzadeh K., Van Noort R., Brook I.M., Scutt A.M., HPLC analysis of components released from dental composites with different resin compositions using different extraction media. *J. Mater. Sci. Mater. Med.* 2007, **18**, 133–137.

21. Polydorou O., Huberty C., Wolkewitz M., Bolek R., Hellwig E., Kümmerer K., The effect of storage medium on the elution of monomers from composite materials. *J. Biomed. Mater. Res. B. Appl. Biomater.* 2012, **100**, 68–74.

22. Eveline Putzeys, Stevan M. Cokic, Hui Chong, Mario Smet, Jeroen Vanoirbeek, Lode Godderis, Bart Van Meerbeek, Kirsten L. Van Landuyt and Radu Corneliu Duca. Simultaneous analysis of bisphenol A based compounds and other monomers leaching from resin-based dental materials by UHPLC-MS/MS. *J. Sep. Sci.* 2017, 40(5):1063-1075.
https://doi.org/10.1002/jssc.201601153

23. Pawlowska E, Poplawski T, Ksiazek D, Szczepanska J, Blasiak J. Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate. *Mutat. Res.* 2010, 696, 122–129

24. Małkiewicz K, Owoc A, Kluska M, Grzech-Leśniak K, Turło J. HPLC analysis of potentially harmful substances released from dental filing materials available on the EU market. *Ann Agric Environ Med.* 2014; 21(1): 86–90.

25. Urcan E., Scherthan H., Styllou M., Haertel U., Hickel R., Reichl F.-X., Induction of DNA double strand breaks in primary gingival fibroblasts by exposure to dental resin composites. *Biomaterials* 2010, 31, 2010–2014.

26. Fleischer H., Vorberg E., Warkentin M., Thurow K., Qualitative and Quantitative Determination of Methacrylates in Dental Filling Materials. Am. Lab. 2014, 46, 10-15.

27. Tokay U., Koyuturk A.E., Aksoy A., Ozmen B., Do the monomers release from the composite resins after artificial aging? Microsc. Res. Tech. 2015, 7 8, 255–259.

28. Tsitrou E., Kelogrigoris S., Koulaouzidou E.A., Antoniades -H alvatjoglou M., Koliniotou - Koumpia E., van Noort R., Effect of extraction media and storage time on the elution of monomers from four contemporary resin composite materials. Toxi col. Int. 2014, 21, 89–95.

29. Schweikl H., Spagnuolo G., Schmalz G., Genetic and cellular toxicology of dental resin monomers. J. Dent. Res. 2006, 85, 870–877.

30. Ortengren U.; Wellendorf H.; Karlsson S. and Ruyter I. E. Water sorption and solubility of dental composites and identification of monomers released in an aqueous environment. J. Oral Rehabil. 2001, 28, 1106-1115.

31. Pelka M.; Distler W. and Petschelt A. Elution parameters and HPLC-detection of single components from resin composite. Clin. Oral Invest. 1999, 3, 194-200.
32. E. Urcan, H. Scherthan, M. Styllou, U. Haertel, R. Hickel, and F. X. Reichl. Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites,” Biomaterials, vol. 31, no. 8, pp. 2010–2014, 2010.

33. Emmler J, Seiss M, Kreppel H, Reichl FX, Hickel R, Kehe K. Cytoxicity of the dental composite component TEGDMA and selected metabolic by-products in human pulmonary cells. Dent Mater. 2008; 24:1670–5. https://doi.org/10.1016/j.dental.2008.04.001

34. Kraus D, Wolfgarten M, Enkling N, Helfgen E-H, Frentzen M, Probstmeier R, et al. In-vitro cytocompatibility of dental resin monomers on osteoblast-like cells. J Dent. 2017; 65:76–82. https://doi.org/10.1016/j.jdent.2017.07.008

35. EFSA. Bisphenol A in food is a health risk. European Food Safety Authority; 2023. Available from: https://www.efsa.europa.eu/en/news/bisphenol-food-health-risk