Browsing by Author "Vasconcelos, Ana Luísa"
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- Contribution to hazard characterization of the Next-Generation Plasticizer DINCH®: cytotoxicity and genotoxicity assessment in human cellsPublication . Vasconcelos, Ana Luísa; Silva, Maria João; Louro, HenriquetaThe chemical diisononyl cyclohexane-1,2-dicarboxylate (Hexamoll® DINCH®), a cyclohexanoate plasticizer, has been employed as a safer alternative to restricted phthalates, to increase flexibility and elasticity of many consumer products made of PVC or polystyrene, namely food packaging, children’s toys, and medical devices. Concomitantly, the detection of DINCH in human surroundings and in biological matrices has increased during the last decade. This prompt the establishment of biomonitoring guidance values for DINCH metabolites in urine, as a measure of precaution; however, the studies about potential adverse effects of DINCH in humans are still scarce. DINCH is not classified as reprotoxic nor genotoxic and mutagenic however, there are limited data available regarding safety assessment, especially regarding cytotoxic and genotoxic effects. Since liver and kidney are DINCH target organs in animal models, the aim of this study was to assess DINCH cytotoxic and genotoxic effects in a human liver (HepG2) and kidney cell lines (HK-2). The methodology included the MTT cell viability, micronucleus, conventional and FPG-modified comet assays to detect cytotoxicity and genotoxicity. The results showed that DINCH was moderately cytotoxic for kidney cells exposed for 48h, but not for liver cells. No chromosomal damage was induced after short-term or longer exposures of both cell lines. However, DINCH was able to induce oxidative DNA damage in liver cells exposed for 3h, which decreased after a more prolonged exposure. The occurrence of oxidative lesions, even transiently, implies that mutation fixation may occur leading to adverse effects in liver. Overall, the present work provides new insights into the potential toxicity of this next-generation plasticizer in kidney and liver cells, in spite of public reports in which DINCH is classified as non-genotoxic agent. On the other hand, human biomonitoring studies are fundamental to confirm the current levels of human internal exposure to DINCH, as well as to detect early biologic effects.
- In vitro exposure to the next-geenration plasticizer DINCH: cytotoxicyy and genotoxicity assessment in human cellsPublication . Vasconcelos, Ana Luísa; Silva, Maria João; Louro, HenriquetaThe chemical Di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH) has been applied since 2002 as a non-aromatic plasticizer and a substitute for unsafe phthalates. This non-regulated chemical is a potential carcinogen to humans using (Q)SAR approaches, thus its increasing presence in human households and body is a major concern. Although in humans DINCH is rapidly metabolized and excreted a continuously exposure may lead to adverse health effects, e.g. in occupational environments [1]. Recently, this substance was included in a priority group by the Human Biomonitoring for Europe Initiative (HBM4EU), meaning that human exposure and effects need evaluation. Several in vivo studies revealed that DINCH did not induce toxicity, at low concentrations, although was able to activate phase I and II liver enzymes and triggered cell proliferation in liver, kidney and thyroid glands. In contrast, nephrotoxicity was reported as a critical endpoint at high exposure concentrations [2]. Therefore, this work aimed to investigate DINCH hazardous potential thereby adding information to the limited knowledge about its biological effects. The study comprised a short and longer exposure of human liver cells (HepG2) to a range of concentrations (1-500 μg/mL). Cytotoxicity was determined by the MTT assay. Potential DNA lesions were evaluated by the comet assay 3h or 24h after exposure, while the clastogenic and aneugenic effects were determined using the cytokinesis-block micronucleus assay following 6h and 48h of exposure, including in the presence of exogenous liver metabolic enzymes (S9 mix). The results indicate that DINCH treatment did not induce cytotoxicity in HepG2 cells for the tested concentrations. Neither it was able to induce DNA lesions or chromosomal damage. Moreover, the plasticizer did not cause oxidative DNA damage, as measured using modified comet assay. In conclusion, the next-generation plasticizer DINCH did not cause cytotoxicity and genotoxicity in HepG2 cells. Furthermore, the results reported for genotoxicity are in line with the ones reported by Anses report [3] and in ECHA database. However, recent in vitro studies showed that DINCH is cytotoxic at 100 μg/mL after a 7-day period exposure of L929 murine cells [4]. Given the reported nephrotoxicity, the current analysis focuses on the potential in vitro toxic outcomes using human kidney (HK-2) cells.[1] H.M. Koch, et. al., Archives of Toxicology 87(5), 799-806 (2013). [2] V.S. Bhat, et. al., Journal of Toxicology and Environmental Health 17(2), 63-94 (2014). [3] Avis de l'Anses n°2015-RE-0003 relatif à une analyse de la meilleure option de gestion des risques pour le DINCH ® (1,2-cyclohexanedicarboxylic acid, diisononyl ester, n° CAS 166412-78-8). https://www.anses.fr/fr/system/files/REACH2015re0003.pdf. [4] T. Eljezi, et. al., Chemosphere 173, 452-459 (2017).
- In vitro exposure to the next-generation plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH): cytotoxicity and genotoxicity assessment in human cellsPublication . Vasconcelos, Ana Luísa; Silva, Maria João; Louro, HenriquetaPlasticizers are currently present in many consumer products, particularly food packaging, children's toys, and medical devices. There are concerns regarding potential leaching to environment or food, thus increasing the risk of human exposure by inhalation, ingestion and/or dermal absorption potentially leading to adverse health consequences. Hexamoll diisononyl cyclohexane-1,2-dicarboxylate (Hexamoll® DINCH®), a non-phthalate plasticizer, has been used as a safer alternative to hazardous phthalates. In contrast to phthalates, evidence indicates that DINCH did not produce endocrine disruption, reproductive dysfunctions, genotoxicity or mutagenicity. However, there are limited data available regarding safety assessment, especially with respect to genotoxicity in human cells. The aim of this study was to assess DINCH cytotoxic and genotoxic effects in human liver and kidney cell lines following several exposure periods. For this purpose, the MTT cell viability, micronucleus, conventional and formamidopyrimidine DNA glycosylase (FPG)-modified comet assays were employed to detect cell death and genotoxicity, respectively. Data demonstrated that DINCH induced cytotoxicity in kidney cells exposed for 48hr, but not in liver cells. No marked chromosomal damage was noted after short-term or longer following treatment of both cell lines. However, DINCH produced oxidative DNA damage in liver cells exposed for 3 h, which decreased after a more prolonged incubation period. The occurrence of oxidative lesions, even transiently, indicates that mutation fixation may occur leading to adverse effects in liver. Therefore, these findings suggest that DINCH may be hazardous to humans and that further investigation is necessary to warrant its safety.