In this in vitro study, the amount of BIS-GMA, TEGDMA, HEMA, and UDMA monomers released into 75% ethanol solution in the 1st hour and on the 1st, 7th, 14th, and 21st days after polymerization of compomers in different colors (a shade A2 compomer and 8 different color packable compomers), composites (shade A2) in different hybridization (nano-hybrid, micro-hybrid), and RMGIC, which are frequently used in pediatric dentistry, was investigated by using HPLC–PDA method.
It is recommended to irradiate the resin materials in layers of 2 mm to increase the polymerization efficiency . In this study, 2 mm high teflon molds were used, taking into account the manufacturer's recommendations for the product use.
Some of the studies measuring the effectiveness of LCUs on the polymerization of resin based restorative materials indicated that Halogen LCUs were more effective [2, 25, 28, 29], while others stated that LED LCUs were more effective [18, 30]. In line with the manufacturer's recommendation, an LED LCU was preferred for polymerization in this study.
It is known that the oxygen inhibition layer formed on the surface of the restorative resin material after polymerization is rich in residual monomers . Bezgin et al.  reported that Mylar strips did not prevent the formation of the oxygen inhibition layer, and finishing-polishing is still essential for the elimination of the resin-rich outer layer that can be the source of the unreacted monomers eluted to the oral cavity. Although transparent strips were used in our study, finishing and polishing processes were applied to the upper surfaces of the discs to remove the oxygen inhibition layer.
The most commonly used chromatographic methods for distinguishing the components released from resin-based materials are HPLC and Gas Chromatography / Mass Spectrometry (GC/MS) , and HPLC was preferred in this study.
It is known that saliva is the main factor in the dissolution of resin-based dental materials in the oral environment over time after application . This dissolving effect of saliva in the oral environment is tried to be imitated with solvents such as acitonitrile, artificial saliva, water, ethanol and methanol in different proportions in vitro studies [31,32,33]. The US Food and Drug Administration (FDA) recommends a 75% ethanol-water solution, which is similar to the oral conditions, for the detection of residual monomers . While artificial saliva barely penetrates the polymer network of the resin-based material [20, 21], ethanol has been used by many researchers because it penetrates the polymer network of the material, widening the gaps between the polymer chains and facilitating the release of unreacted monomers over time [8, 16, 20,21,22, 35]. Therefore, in this study, 75% ethanol—25% deionized water was used as the extraction medium to measure the release of the monomers.
The ethanol solution was not changed from the beginning to the end of the study in our previous study . However, in this study, the solution was changed at the end of each time period based on the previous studies [20,21,22, 35]. Shahabi et al.  evaluated the effect of the volume (1 mL or 3 mL) and renewing of storage media (ethanol/water solution) on monomer (UDMA, BIS-GMA, TEGDMA) leachability from two dental composites using HPLC. They reported that saturation makes the storage media volume important factor in monomer elution and refreshing of storage media had significant effect on monomer release before the elution of 50% of total released monomer.
It has been reported that monomer release is high in resin based restorative materials at the beginning and this amount continues to decrease over time [21,22,23, 36]. The first mechanism of monomer elution is elution from the composite surface that occurs in the first 24 h. Subsequently monomer elution continues with a slower rate, since increasing the volume of polymeric chains and release of unreacted monomers from composite take substantial time . There are studies examining the release of monomers from restorative resin materials in different time periods [21, 35, 37, 38]. Moreira et al.  indicated that the release of residual monomer continued until the 30th day. In the previous study conducted in our clinic, residual monomer release was evaluated at the end of 10 min, 1 h, 6 h, 1, 7, and 14 days . Unlike our previous study, the 10th minute was eliminated from the evaluation periods and the final evaluation period was extended to 21 days. In this study, the measurement periods for the amount of residual monomer released from the resin-based materials were determined as 1 h, 1, 7, 14, and 21 days.
Although monomer elution from the resin-composites has been widely assessed [7, 15, 17,18,19,20,21,22,23,24], studies evaluating monomer elution from the compomers are very limited [8, 25, 26]. To the best of our knowledge, there are very few studies in the literature examining the amount of residual monomer released after polymerization of colored compomers and RMGIC. Botsali et al.  reported that there was a higher amount of HEMA release than TEGDMA release in their study with one RMGIC (Ketac N100) and two compomers (Dyract Extra and Twinkystar), and this release damaged fibroblast cells. Tunç et al.  stated that compomers are potentially toxic to human pulp fibroblasts and the type of curing unit will affect compomer toxicity. In our previous study, it was determined that BIS-GMA was the most released residual monomer, despite its high viscosity which makes it difficult to release in packable and flowable compomers, while TEGDMA was the least released monomer . Residual monomer release continued on the 14th day and the compomer with the highest residual monomer release was the gold-colored compomer. It was concluded that color and viscosity affected the residual monomer release in compomers. Although that study was the first to examine the release of residual monomer according to the color of the compomers, it showed that not all the colors in the compomer color scale were examined as a limitation of the study . In this study, which was carried out with all the colors in the compomer color scale in order to eliminate this limitation in the literature, it was concluded that color is important in residual monomer release. Dark colored compomers absorbing blue light are thought to have a greater depth of polymerization. Vandenbulcke et al.  reported that the polymerization depth of colored compomers could be affected by the amount and type of pigment. They found that the relatively darker shades (blue and green) had the greatest polymerization depths.
According to the previous studies, the toxicity for the following monomers was ranked as BIS‐GMA > UDMA > TEGDMA > HEMA (least toxic) [39, 40]. In this study, these four monomers were also investigated.
BIS-GMA was the most released monomer in all the groups except RMGIC. Ranasathien et al.  found the cytotoxic effect value of BIS-GMA as 9.35 μM/L (4.78 µg/mL) in their study on mouse fibroblasts. In a study, exposure of dental pulp cells to BIS-GMA at concentrations of 0.075 mmol/L markedly affected the viable cell number with 40% of inhibition , while in another study, it was reported that BIS-GMA at concentration of 0.087 mmol/L causes 50% reduction (half maximal effect concentration: EC50) of cell viability on human gingival fibroblasts . In this study, BIS-GMA concentration in gold-colored compomer (Twinky Star) – in the 24th hour, which was the highest concentration with 35.731 µg/mL (0.0697 mmol/L), was found to be either lower or greater than the toxic concentrations obtained in some previous studies [14, 39, 40].
HEMA release showed a maximum increase on the 7th day in all the groups. Altıntaş and Üşümez,  investigated the residual monomer release from resin cements, and reported the HEMA release amount from Nexus 2 (Kerr/Italy) cement to be 117 µg/mL in the 10th minute, and 440 µg/mL on the 21st day. In the same study, they were measured as 98.15 µg/mL in the 10th minute and 142.61 µg/mL on the 21st day for Rely X Arc (3 M ESPE/Germany). Botsali et al.  found HEMA release from RMGIC (Ketac N100) to be 7.1 µm/L in the 4th hour and 16.8 µm/L in the 24th hour. On the other hand, in our study, the amount of HEMA released from the resin cement of Ionolux (VOCO, Germany) was found to be 0.006 µg/mL in the 1st hour, 0.025 µg/mL (0.0002 mmol/L) on the 1st day, and 0.010 µg/mL (0.0008 mmol/L) on the 21st day. HEMA release from RMGIC is less than other materials in all time periods. This situation may be due to the interaction of HEMA molecules with water, considering that HEMA is highly hydrophilic and the solution consists of 75% ethanol- 25% water . Although HEMA is listed by the manufacturers as a component of RMGIC, it is not listed as a component of compomer. However, Geurtsen et al. , Bezgin et al. , and Botsali et al.  confirmed its presence in compomers. In this study, HEMA release was determined from compomers, and the highest HEMA release was from the gold-colored compomer. (0.203 ± 0.032 µg/mL–0.0016 mmol on the 7th day). Bezgin et al.  explained the presence of HEMA in the compomer by stating that manufacturers may keep the components with concentrations lower than 1% in their products confidential as it is a trade secret, and also ingredients in the Material Safety Data Sheet (MSDS) are sometimes insufficient. However, HEMA release could be a degradation product from UDMA, which is an ingredient in restorative materials [8, 16, 24]. Toxic concentration 50 (TC50) of HEMA ranged from 3.6 to 11.2 mmol/L with different cell lines in various studies [42,43,44]. In this study, no material reached the toxic concentrations obtained for HEMA in some previous studies [42,43,44].
TEGDMA is a low molecular weight monomer used to reduce the viscosity of BIS-GMA and UDMA. Sonkaya et al.  reported that the use of TEGDMA (co)monomer in dental composites reduced the monomer release. Of the resin-based dental materials used in this study, compomers contain less than 2.5% and composites contain between 2.5–5% TEGDMA. In our previous study, the most TEGDMA-releasing material among the packable compomers was the gold-colored compomer, and A2 shade compomer was found to be statistically much higher than the blue and pink-colored packable compomers . In this study, the gold-colored compomer was the material that released the most TEGDMA with 10.410 µg/mL (0.0364 mmol/L) at the end of the 1st hour. TEGDMA release from RMGIC and nanohybrid resin composite-GrandioSO could not be detected at the end of 1 h and 21 days, respectively. Reichl et al.  reported the EC50 values decreased from about 5 mmol/L (6 h) to about 0.6 mmol/L (48 h) for HEMA and from about 3 mmol/L (6 h) to about 0.4 mmol/L (48 h) for TEGDMA in their cytotoxicity study. The effective dose that reduced the number of cell viability to 50% for TEGDMA was reported to be 0.26 mmol/L on human pulp fibroblasts  and 3.46 mmol/L on human gingival fibroblasts . The TEGDMA concentrations of all the materials were found to be lower than the toxic concentrations obtained for TEGDMA in some previous studies [14, 39].
Although its molecular weight is close to that of BIS-GMA (512 g/mol), UDMA (470 g/mol) is highly viscous. In addition, despite having the same proportion as BIS-GMA in compomers, UDMA release is much lower than BIS-GMA. This is associated with its viscous structure. Reichl et al.  reported that UDMA at concentration of 0.106 mmol/L causes 50% reduction of cell viability (EC50) on human gingival fibroblasts. In this study, the highest UDMA release was observed on the 1st day in gold-colored compomers with 0.060 mmol/L.
Nanotechnology is the creation of macroscale structures by various processes of materials. This brings a more homogeneous matrix distribution with smaller particles and reduces the monomer matrix volume. As a result, the negative properties of the composite such as residual monomer release and polymerization shrinkage are reduced . In general, residual monomer release was greater in microhybrid composites than in nanohybrid composites in this study. For TEGDMA, the situation was opposite in the first hour and on the first day. This difference in the residual monomer release taking place between micro-hybrid and nano-hybrid composites is thought to be due to the differences in filler particle type and monomer ratios specified by the manufacturer. De Angelis et al.  measured eluted monomer from GrandioSO (VOCO) nanohybrid composite after one day and 14 days using HPLC. They reported that the observable levels of TEGDMA were found only after 24 h (7.9 ng/mL), while the levels of BIS-GMA were about 4500 ng/mL after 24 h and 3500 ng/mL after 14 days. In this study, the levels of TEGDMA released from GrandioSO composite were 1.854 and 0.314 μg/mL after 24 h and 14 days, respectively, and it could not detected on the 21st day. Morever, the levels of BIS-GMA released were 3.137 and 1.123 μg/mL after 24 h and 14 days, respectively.
This study, in which all existing colors of resin based colored compomers were evaluated in terms of residual monomer release, will shed light on future studies. The limitation of the previous study on compomers , in which a limited number of colors were included, was resolved in this study. However, only one commercial company's filling materials and LED LCU were used in this study (VOCO®). The studies using the products of different companies should also be included. However, the strength of the study was the evaluation of compomers in all colors, composites with different hybridization properties, and RMGIC commonly used in pediatric dentistry in terms of residual monomer release by HPLC for the first time. In addition, the fact that it was conducted in vitro is another limitation of the study, and long-term clinical studies to be performed in saliva and gingival crevicular fluid will add valuable information to the literature.