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Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen’s egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC–MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

Achyrocline satureioides (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NEASE) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen’s egg chorioallantoic membrane test (HET-CAM). NEASE exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of −40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NEASE (up to 5 μg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NEASE did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NEASE was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NEASE. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.

The aim of this study was to design and evaluate novel cyclodextrin (CD)-based aggregate formulations to efficiently deliver nepafenac topically to the eye structure, to treat inflammation and increase nepafenac levels in the posterior segment, thus attenuating the response of inflammatory mediators. The physicochemical properties of nine aggregate formulations containing nepafenac/&gamma, CD/hydroxypropyl-&beta, (HP&beta, )-CD complexes as well as their rheological properties, mucoadhesion, ocular irritancy, corneal and scleral permeability, and anti-inflammatory activity were investigated in detail. The results were compared with a commercially marketed nepafenac suspension, Nevanac&reg, 3 mg/mL. All formulations showed microparticles, neutral pH, and negative zeta potential (&ndash, 6 to &ndash, 27 mV). They were non-irritating and nontoxic and showed high permeation through bovine sclera. Formulations containing carboxymethyl cellulose (CMC) showed greater anti-inflammatory activity, even higher than the commercial formulation, Nevanac&reg, 0.3%. The optimized formulations represent an opportunity for topical instillation of drugs to the posterior segment of the eye.

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P

Objective: The aim of this study was to investigate the effects of naringin, a known flavonoid, on in vitro cytotoxicity, irritation and in vivo potential efficacy when topically applied to the diabetic wound. Material and Method: In vitro direct contact assay and hen's egg chorio-allantoin membrane tests were used to evaluate irritation, and cytotoxicity potential of Naringin. In vitro antimicrobial activity was also tested. Topical treatments were administered once a day on the wound. Wound lesions were photographed and statistically analyzed. After the 10th day, histopathological parameters of tissues were assessed. Result and Discussion: The results showed that naringin gel has no cytotoxicity and irritation potential. Also, it displayed a weak antimicrobial effect. Aside from these, in vivo studies revealed that naringin gel showed statistically higher wound contraction and re-epithelialization, the thickness of granulation tissue, angiogenesis scores. The results indicated that naringin gel effectively enhanced diabetic wound healing, and so this formulation could be considered a promising therapeutic alternative for treating diabetic wounds. Amaç: Bu çalışmanın amacı, bir flavonoid olan Naringin’in in vitro sitotoksisite, irritasyon ile diyabetik yara üzerindeki in vivo potansiyel etkilerini araştırmaktı. Gereç ve Yöntem: Naringin'in irritasyon ve sitotoksisite potansiyelini değerlendirmek için in vitro doğrudan temas deneyi ve tavuk yumurtası korio-allantoin membran testleri kullanıldı. Aynı zamanda in vitro antimikrobiyal etkinlikte test edildi. Topikal tedaviler günde bir kez yaraya uygulandı. Yara lezyonları fotoğraflandı ve istatistiksel olarak analiz edildi. 10. günden sonra dokuların histopatolojik parametrelerideğerlendirildi.Sonuç ve Tartışma: Sonuçlar, naringin jelin sitotoksisite ve tahriş potansiyeli olmadığını göstermiştir. Ayrıca, zayıf bir antimikrobiyal etki gösterdi. Bunların yanı sıra, in vivo çalışmalar naringin jelin istatistikselolarak daha yüksek yara kapanması ve re-epitelyalizasyon, granülasyon dokusunun kalınlığı ve anjiyogenezskorları gösterdiğini ortaya koymuştur. Sonuçlar, naringin jelin diyabetik yara iyileşmesini etkili bir şekilde arttırdığını gösterdi ve bu nedenle bu formülasyon, diyabetik yaraların tedavisi için umut verici bir terapötik alternatif olarak düşünülebilir.

The design and development of ceramic structures based on 3D scaffolding as dental bone substitutes has become a topic of great interest in the regenerative dentistry research area. In this regard, the present study focuses on the development of two scaffold-type structures obtained from different commercial dental ceramics by employing the foam replication method. At the same time, the study underlines the physicochemical features and the biological profiles of the newly developed scaffolds, compared to two traditional Cerabone® materials used for bone augmentation, by employing both the in vitro Alamar blue proliferation test at 24, 48 and 96 h poststimulation and the in ovo chick chorioallantoic membrane (CAM) assay. The data reveal that the newly developed scaffolds express comparable results with the traditional Cerabone® augmentation masses. In terms of network porosity, the scaffolds show higher pore interconnectivity compared to Cerabone® granules, whereas regarding the biosafety profile, all ceramic samples manifest good biocompatibility on primary human gingival fibroblasts (HGFs), however only the Cerabone® samples induced proliferation of HGF cells following exposure to concentrations of 5 and 10 µg/mL. Additionally, none of the test samples induce irritative activity on the vascular developing plexus. Thus, based on the current results, the preliminary biosecurity profile of ceramic scaffolds supports the usefulness for further testing of high relevance for their possible clinical dental applications.

The validation of novel target-specific radioligands requires animal experiments mostly using mice with xenografts. A pre-selection based on a simpler in vivo model would allow to reduce the number of animal experiments, in accordance with the 3Rs principles (reduction, replacement, refinement). In this respect, the chick embryo or hen&rsquo, s egg test&ndash, chorioallantoic membrane (HET-CAM) model is of special interest, as it is not considered an animal until day 17. Thus, we evaluated the feasibility of quantitative analysis of target-specific radiotracer accumulation in xenografts using the HET-CAM model and combined positron emission tomography (PET) and magnetic resonance imaging (MRI). For proof-of-principle we used established prostate-specific membrane antigen (PSMA)-positive and PSMA-negative prostate cancer xenografts and the clinically widely used PSMA-specific PET-tracer [68Ga]Ga-PSMA-11. Tracer accumulation was quantified by PET and tumor volumes measured with MRI (n = 42). Moreover, gamma-counter analysis of radiotracer accumulation was done ex-vivo. A three- to five-fold higher ligand accumulation in the PSMA-positive tumors compared to the PSMA-negative tumors was demonstrated. This proof-of-principle study shows the general feasibility of the HET-CAM xenograft model for target-specific imaging with PET and MRI. The ultimate value for characterization of novel target-specific radioligands now has to be validated in comparison to mouse xenograft experiments.

Despite the standard approaches to treat the highly aggressive and invasive glioblastoma (GBM), it remains incurable. In this sense, cannabinoids highlight as a promising tool, because this tumor overexpresses CB1 and/or CB2 receptors and being, therefore, can be susceptible to cannabinoids treatment. Thus, this work investigated the action of the cannabinoid agonist WIN55-212-2 on GBM cell lines and non-malignant cell lines, in vitro and in vivo. WIN was selectively cytotoxic to GBM cells. These presented blebbing and nuclear alterations in addition to cell shrinkage and chromatin condensation. WIN also significantly inhibited the migration of GAMG and U251 cells. Finally, the data also showed that the antitumor effects of WIN are exerted, at least to some extent, by the expression of p53 and increased cathepsin D in addition to the decreased expression of HSP70.This data can indicate caspase-independent cell death mechanism. In addition, WIN decreased tumoral perimeter as well as caused a reduction the blood vessels in this area, without causing lysis, hemorrhage or blood clotting. So, the findings herein presented reinforce the usefulness of cannabinoids as a candidate for further evaluation in treatment in glioblastoma treatment., Minas Gerais State Research Foundation (FAPEMIG) and NationalCouncil for Scientific and Technological Development (CNPq) bothfrom Brazil, provided thefinancial support to this study

The essential oils obtained from leaves of Piper duckei and Piper demeraranum by hydrodistillation were analyzed by gas chromatography-mass spectrometry. The main constituents found in P. demeraranum oil were limonene (19.3%) and β-elemene (33.1%) and in P. duckei oil the major components found were germacrene D (14.7%) and trans-caryophyllene (27.1%). P. demeraranum and P. duckei oils exhibited biological activity, with IC(50) values between 15 to 76 μg mL(-1) against two Leishmania species, P. duckei oil being the most active. The cytotoxicity of the essential oils on mice peritoneal macrophage cells was insignificant, compared with the toxicity of pentamidine. The main mono- and sesquiterpene, limonene (IC(50) = 278 μM) and caryophyllene (IC(50) = 96 μM), were tested against the strains of Leishmania amazonensis, and the IC(50) values of these compounds were lower than those found for the essential oils of the Piper species. The HET-CAM test was used to evaluate the irritation potential of these oils as topical products, showing that these oils can be used as auxiliary medication in cases of cutaneous leishmaniasis, with less side effects and lower costs.

Poly(lactide-co-glycolide) acid (PLGA) nanospheres incorporating flurbiprofen (FB) were produced by the solvent displacement technique, for ocular applications aiming to avoid/minimize inflammation induced by surgical trauma. In this work, a PLGA of low viscosity has been tested and the results obtained were compared with those previously reported by Vega et al. The physicochemical properties of the developed formulations were evaluated by measuring particle size, zeta potential and FB entrapment efficiency, showing no significant differences. Release studies demonstrated that the formulation produced with PLGA of higher viscosity revealed a slower drug release rate. Stability analysis, for a period of 75 days, was performed using three complementary methods: (i) turbidity experiments using a Turbiscan optical analyzer, (ii) particle size measurements, and (iii) zeta potential analysis. The results revealed long-term physicochemical stability suitability for ophthalmic use, being independent from the polymer viscosity. The ocular tolerance was assessed by an alternative in vitro method to animal experimentation, the HET-CAM. For all developed formulations no ocular irritancy has been detected.