2-Methyloxolane as an effective bio-based solvent for the removal of ßN-alkanoyl-5-hydroxytryptamines from Arabica green coffee beans.

ßN-alkanoyl-5-hydroxytryptamines (Cn-5HTs) are the main constituents of coffee wax and may be responsible for the increased severity of gastric disorders in sensitive consumers. Their removal from green coffee beans can result in a "stomach-friendly" brew. This work presents a green approach to Cn-5HTs extraction using the bio-based solvent 2-methyloxolane (2-MeOx). HPLC/DAD analyses on Arabica Brazil samples show that mild conditions (30 min at 50 °C) extract about 90% of the wax, without affecting the caffeine content of the beans, whereas almost complete removal is achieved in 60 min at reflux. 2-MeOx forms an azeotrope with water, its possible re-use has been demonstrated using aqueous 2-MeOx (95.5%) as the solvent. These preliminary results make 2-MeOx a possible candidate for the replacement of dichloromethane (DCM) in coffee dewaxing. The importance of fermentation in reducing Cn-5HTs by about 36% has been demonstrated in an analysis of green beans subjected to different post-harvest treatments.

Antihypertensive Potential of Japanese Quail (Couturnix Couturnix Japonica) Egg Yolk Oil (QEYO) in Sprague Dawley Rats.

Oils from animal sources have been used for centuries in the management of diseases. This research was conducted to screen the ex vivo and in vivo toxicity of quail egg yolk oil (QEYO) extracts and assess their effects on the management of hypertension in rats. QEYO was extracted using gentle heating (GH) and n-hexane (NHN). The extracts were subjected to toxicity testing using the hen's egg test on chorioallantoic membrane (HET-CAM) and bovine corneal histology test. Acute and sub-chronic toxicity (28 days) were evaluated in rats. Hypertension was induced in rats by administering 80 mg/kg of Nω-L-Arginine Methyl Ester (L-NAME) per day for 28 days. Treatments commenced on the 14th day; Nifedipine at 30 mg/kg and 1 mL of distilled water were administered as positive and negative controls. Blood pressure (BP), lipid profiles, and oxidative stress markers were quantified. No irritation was observed using the HET-CAM test in the egg treated with both extracts. Bovine corneal histology showed no lesions in all treated groups. No signs of toxicity were observed in either acute or sub-chronic toxicity studies. A significant reduction in blood pressure was observed in rats treated with the extracts (p < 0.05). Changes in total cholesterol (TC), triglycerides (TGs), low-density lipoproteins (LDLPs), and high-density lipoproteins (HDLPs) were not significant compared to the control (p > 0.05). Oxidative stress markers (SOD and CAT) increased significantly in the treated groups compared to the control, while the malondialdehyde levels decreased (p < 0.05). QEYO was safe in both ex vivo and in vivo studies and can be said to have the potential to lower blood pressure as well as cardio-protective effects in hypertensive rats. This research provides evidence based on which QEYO could be used safely as an adjuvant therapy in eye drops and cosmetics and can be considered an effective choice for preventing hypertension.

Carcinoma de células escamosas y el rol discutido de la irritación mecánica crónica. Informe de un caso / Squamous oral cell carcinoma and the discussed role of chronic mechanical irritation. Report of a case

Resumen Objetivo Describir el desarrollo de un carcinoma oral de células escamosas, en el que la irritación mecánica crónica aparenta tomar un rol protagonista en la carcinogénesis. Caso clínico Un paciente de 41 años de edad, argentino, con antecedentes de fisura labio alvéolo palatina, diabetes mellitus, convulsiones, consumo de cocaína y marihuana, enolismo crónico y tabaquismo, acude al Servicio de Odontología del Hospital Central de Mendoza para la evaluación de una úlcera dolorosa en el dorso de su lengua, de varias semanas de evolución, en íntima relación con un primer premolar superior derecho y una pieza supernumeraria. Se realizó una biopsia y de la anatomía patológica resultó el diagnóstico de carcinoma oral de células escamosas. Se ofreció al paciente posibles tratamientos que rechazó, por lo que se inició terapia paliativa y sintomática. Al avanzar su mal estado general, falleció por complicaciones relacionadas a la deglución. Si bien no está definido el rol de la irritación mecánica crónica en la etiología de la carcinogénesis, ejerce un efecto promotor del daño causado por el tabaco y el alcohol. Si bien el paciente era fumador y bebía alcohol, se puede observar que desarrolló un carcinoma de células escamosas en evidente relación a un trauma crónico, ya que la lesión en la cara dorsal de lengua está en íntimo contacto con el factor irritante. Aun así, la evidencia actual disponible es limitada y discute el protagonismo del trauma crónico por lo que se necesitan más estudios para evaluar y definir la posible relación causal de la irritación mecánica crónica en la carcinogénesis.

Abstract Aim To describe the development of an oral squamous cell carcinoma, in which chronic mechanical irritation appears to play a significant role in carcinogenesis. Clinical case A 41-year-old patient, from Argentina, with a history of cleft lip and palate, diabetes mellitus, seizures, cocaine and cannabis use, chronic alcoholism and smoking, comes to the Dentistry Service of the Central Hospital for the evaluation of a painful ulcer on the dorsum of the tongue, which had been developing for several weeks, in close relation to an upper right first premolar and a supernumerary tooth. A biopsy was performed, and the pathological anatomy resulted in the diagnosis of oral squamous cell carcinoma. Possible treatments were offered to the patient, which he rejected, so palliative and symptomatic therapy was initiated instead. As his poor general condition progressed, he died due to complications related to swallowing. Although the role of chronic mechanical irritation in the development of carcinogenesis is not yet fully defined, it has been shown to have a promoting effect on the damage caused by tobacco and alcohol. Although the patient was a smoker and drank alcohol, it can be observed that he developed a squamous cell carcinoma in obvious relation to a chronic trauma, since the lesion develops on the dorsal face of the tongue in close contact with the irritant factor. Still, the current evidence available is limited and discusses the role of chronic trauma, so more studies are needed to evaluate and define the possible causal relationship of chronic mechanical irritation in the development of carcinogenesis.

Carcinoma de células escamosas y el rol discutido de la irritación mecánica crónica: informe de un caso / Squamous oral cell carcinoma and the discussed role of chronic mechanical irritation: report of a case

Objetivo: Describir el desarrollo de un carcinoma oral de células escamosas, en el que la irritación mecánica crónica aparenta tomar un rol protagonista en la carcinogénesis. Caso clínico: Un paciente de 41 años de edad, argen- tino, con antecedentes de fisura labio alvéolo palatina, diabe- tes mellitus, convulsiones, consumo de cocaína y marihuana, enolismo crónico y tabaquismo, acude al Servicio de Odonto- logía del Hospital Central de Mendoza para la evaluación de una úlcera dolorosa en el dorso de su lengua, de varias sema- nas de evolución, en íntima relación con un primer premolar superior derecho y una pieza supernumeraria. Se realizó una biopsia y de la anatomía patológica resultó el diagnóstico de carcinoma oral de células escamosas. Se ofreció al paciente posibles tratamientos que rechazó, por lo que se inició terapia paliativa y sintomática. Al avanzar su mal estado general, fa- lleció por complicaciones relacionadas a la deglución. Si bien no está definido el rol de la irritación mecánica crónica en la etiología de la carcinogénesis, ejerce un efecto promotor del daño causado por el tabaco y el alcohol. Si bien el paciente era fumador y bebía alcohol, se puede observar que desarrolló un carcinoma de células escamosas en evidente relación a un trauma crónico, ya que la lesión en la cara dorsal de lengua está en íntimo contacto con el factor irritante. Aun así, la evi- dencia actual disponible es limitada y discute el protagonismo del trauma crónico por lo que se necesitan más estudios para evaluar y definir la posible relación causal de la irritación me- cánica crónica en la carcinogénesis (AU)

Aim: To describe the development of an oral squamous cell carcinoma, in which chronic mechanical irritation ap- pears to play a significant role in carcinogenesis. Clinical case: A 41-year-old patient, from Argenti- na, with a history of cleft lip and palate, diabetes mellitus, seizures, cocaine and cannabis use, chronic alcoholism and smoking, comes to the Dentistry Service of the Central Hospi- tal for the evaluation of a painful ulcer on the dorsum of the tongue, which had been developing for several weeks, in close relation to an upper right first premolar and a supernumerary tooth. A biopsy was performed, and the pathological anatomy resulted in the diagnosis of oral squamous cell carcinoma. Possible treatments were offered to the patient, which he re- jected, so palliative and symptomatic therapy was initiated instead. As his poor general condition progressed, he died due to complications related to swallowing. Although the role of chronic mechanical irritation in the development of carcino- genesis is not yet fully defined, it has been shown to have a promoting effect on the damage caused by tobacco and alco- hol. Although the patient was a smoker and drank alcohol, it can be observed that he developed a squamous cell carcinoma in obvious relation to a chronic trauma, since the lesion devel- ops on the dorsal face of the tongue in close contact with the irritant factor. Still, the current evidence available is limited and discusses the role of chronic trauma, so more studies are needed to evaluate and define the possible causal relation- ship of chronic mechanical irritation in the development of carcinogenesis (AU)

Anomie, irritation, and happiness in the Chilean society post-social outbreak.

On 18 October 2019, the Chilean people witnessed an unprecedented social outbreak across most of their country. We argue that a state of anomie is a factor associated with the weakening of states, and an anomic state might negatively influence people's well-being through an increased feeling of irritation. Convenience recruitment via social networks allowed us to form a sample of 194 Chilean participants from the center-south region of the country (M = 36.53 years old, SD = 17.48; 56.7% women). All participants completed testing instruments to measure anomie, irritation, happiness, and political beliefs. Descriptive scores suggest situating Chile in the quadrant of high anomie. Two mediation analyses were conducted. The main results showed a negative indirect effect of the breakdown of the social fabric and leadership on happiness through irritation, although the findings for the former dimension were more robust. Additionally, the breakdown of the social fabric was positively related to the belief that left and right-wing democratic governments are helpless when it comes to fighting delinquency. The breakdown of leadership, on the other hand, was negatively related to political interest. The results should be interpreted with caution due to the limitations of the sample type and the construction validity of some instruments.

Benefits of the Dermocosmetic Mineral 89 Probiotic Fractions Adjunct to Topical Retinoids for Anti-Aging Benefits.

Purpose: Tretinoin is a topical gold standard for photoaging treatment. However, patient adherence can be impaired by local tolerability in the first 1-2 weeks of treatment. Mineral 89 Probiotic Fractions® (M89PF) containing Vichy volcanic mineralizing water®, probiotic fractions, hyaluronic acid, niacinamide and tocopherol was developed to fulfill the need for adjunctive products that can reinforce skin barrier and manage retinoid induced irritation. Patients and Methods: The study included 38 women, aged 44-60 years, phototype II-VI, applying 0.025% tretinoin gel once nightly for 84 days. For 28 days, one hemi face was treated with M89PF and sunscreen SPF 50+ while other hemi face received sunscreen only. Then, M89PF application was changed to full face. Evaluations were performed at days 0, 7, 28 and 84. Erythema, dryness, fine lines, skin tone, radiance and pore appearance were assessed by a dermatologist. Tolerability was evaluated through self-assessment questionnaire. Skin hydration levels, inflammatory and oxidative stress biomarkers were analyzed by immunological assay: Interleukin(IL)-8, IL1-alpha, IL1-Receptor Antagonist (IL-1Ra), Prostaglandin E2 (PGE2), Catalase and Superoxide Dismutase (SOD). Results: Hemiface analysis showed that erythema, fine lines, skin tone, radiance, pore appearance, hydration, tightness, dryness, burning, itching and stinging sensations were improved (p<0.05) on the M89PF side. At full face analysis on D84, erythema, fine lines, skin tone, radiance and pore appearance were improved compared to D0 (p<0.001). Tightness, dryness, burning, itching and stinging were reduced when compared to D7 (p<0.05). Dermatology Life Quality Index (DLQI) and Skindex 16 showed improvement in quality of life (p<0.05). IL-1RA increased at D28 (p=0.003) and PGE2 decreased at D28 and D84 compared to D0 (p<0.01). Conclusion: M89PF reduced retinoid induced irritation with a good tolerability profile and, used as an adjunct to topical tretinoin, significantly improved skin hydration, erythema, fine lines, skin tone, radiance and pore appearance.

Oral squamous cell carcinoma arising from chronic traumatic ulcers.

BACKGROUND: A proportion of oral squamous cell carcinoma (OSCC) is preceded by oral potentially malignant disorders (OPMD), and the rest might not be associated with any well-established OPMD. Chronic traumatic ulcer (CTU) has been suggested as a lesion with potential for malignant transformation, but the evidence is inconclusive. OBJECTIVE: The aim of this study was to describe clinicopathological features of non-healing CTU (NHCTU) that later developed to OSCC. MATERIALS AND METHODS: A retrospective cohort study was performed of patients attending the Facultad de Odontología Universidad Nacional de Córdoba (2013-2019). The study included patients with a provisional diagnosis of CTU. CTUs without complete healing after removal of traumatic factors-non healing CTU (NHCTU)-were investigated by biopsy. A detailed description of cases where a malignancy was diagnosed is presented. RESULTS: A total of 107 CTU were included. Of these, 32 CTUs were considered NHCTU and two cases showed histological features of OSCC in the background of NHCTU. In both cases, lesions were found on the lateral tongue. Both were non smoking, non alcohol drinking individuals. Absence of any well-known OPMD was also registered. CONCLUSIONS: The possibility of malignant transformation in a subset of tongue NHCTU should not be underestimated. CLINICAL RELEVANCE: In a subgroup of patients, NHCTU could be considered an OPMD. Although the potential malignant transformation rate is low, these conditions should be always followed up.

Soap or alcohol-based products? The effect of hand hygiene on skin characteristics during the COVID-19 pandemic.

BACKGROUND: Different strategies for hand skin hygiene have been used to prevent the spread of SARS-CoV-2. However, frequent hand sanitization has been associated with skin damage. The present study aimed to evaluate hand hygiene habits during the COVID-19 pandemic and the effect of the repetitive use of soap or alcohol-based products on skin characteristics. METHODS: We conducted a survey regards hand hygiene habits acquired during the COVID-19 pandemic. Also, we performed cutometry in a cohort of individuals who cleansed their volar forearms every 30 min, during 4 h, using soap or alcohol-based products. RESULTS: We received 138 responses from people with medium-high educational level who reported a 2.5-time increase in the frequency of hand cleansing (p < 0.0001) that resulted in skin damage. An in vivo analysis of skin moisture and elasticity was also performed among 19 health workers and students. In general, skin moisture decreased with every cleansing, mainly after 2 h of washing with soap (p < 0.01), while skin elasticity only reduced after 4 h of treatment (p < 0.05). Alcohol-based solution or alcohol-based gel (70% ethanol, both) did not affect skin moisture or elasticity during testing. CONCLUSION: It is known that the excessive use of soap or alcohol-based products causes dermatological issues. The present study demonstrates that non-medicated soap significantly affects skin moisture and elasticity, probably because the soap removes the hydrolipidic protective barrier, favoring transepidermal water loss, where the lack of the appropriate stratum corneum hydration also affects skin elasticity, mainly associated with changes in epidermal structure.

The potential use of kraft lignins as natural ingredients for cosmetics: Evaluating their photoprotective activity and skin irritation potential.

The cosmetics market has recently undergone changes as consumers increasingly seek sustainable products. In this context, agro-derived lignins have the potential for cosmetics applications. Thus, this study investigated the photoprotective activity and skin irritation potential (OECD TG 439 with SkinVitro-RHE, an in-house reconstructed human epidermis (RHE) model) of a kraft lignin (LE) and two LE-derived lignins modified by enzymatic reactions to achieve higher molecular weight (Mw) (R1: intermediate Mw and E60: highest Mw). Results showed that LE and R1 lignins present adequate photoprotective activity with averages Sun Protection Factor (SPF) of 33.8 ± 0.02 and 22.7 ± 0.04, respectively. The E60 also present adequate SPF (22.4 ± 0.2); however, due to its poor solubility, this lignin has potential application as a physical filter. In terms of safety, these lignins did not cause skin irritation or cellular and structural damage to the epidermis. Additionally, using an analysis based on the autofluorescence feature of lignin, no sign of the tested lignins was found in exposed RHE models, indicating that these three lignins did not penetrate the skin. Altogether, the results indicate a promising application of kraft lignins for sunscreen products regarding safer alternatives and product sustainability. Also, the SkinVitro-RHE showed to be a good model for evaluating the skin irritation potential of substances, including natural cosmetic ingredients.

Development of a novel ex vivo model for chemical ocular toxicity assessment and its applicability for hair straightening products.

This study developed an air-liquid interface (ALI) corneal model using explants bovine eyes for ocular toxicity assessment of ten chemicals and seven hair straightening mixtures. It was successfully maintained physiologically viable and normal for six days. Both eye damage (GHS cat. 1) and irritating (GHS cat. 2) chemicals induced corneal injury in our model. However, cat. 2 irritants triggered moderate damage when compared to cat. 1 agents, which induced a marked cytotoxicity profile. The mixtures were also able to trigger viability reduction associated with histopathological changes in the corneal tissues, especially when the exposure was via aerosol particles. Thus, the chemical exposure microenvironment simulation seemed to provide more reliable toxicological data. Moreover, mixture-induced corneal damage correlated with increased ROS levels, suggesting a close correlation between tissue death and oxidative stress. Besides mixtures showing the potential to induce moderate/mild ocular toxicity, we could verify that the corneal tissue damage showed reversibility due to the recovery from the injury after exposure to some of the mixtures. Hence, our ex vivo corneal model seems to be a simple and cost-effective approach for future studies related to further investigating the reversibility of damage in the cornea triggered by chemicals and their mixtures.

Toxicological Evaluation of Silver Nanoparticles Synthesized with Peel Extract of Stenocereus queretaroensis.

Silver nanoparticles (AgNPs) synthesized with plants are widely used in different industries, such as the medical, industrial, and food industries; however, their hazards and risks remain unclear. Here, we aimed to evaluate the toxicological effects of AgNPs in both in vitro and in vivo models. Previously, we developed and characterized green synthesized AgNPs based on Stenocereus queretaroensis (S. queretaroensis). The present study evaluates the toxicity of these AgNPs through cytotoxicity and mutagenicity tests in vitro, as well as genotoxicity tests, including the evaluation of acute oral, dermal, and inhalation toxicity, along with dermal and ocular irritation, in vivo, according to guidelines of The Organization for Economic Co-operation and Development (OECD). We evaluated cell cytotoxicity in L929 cells, and the half-maximal inhibitory concentration was 134.76 µg/mL. AgNPs did not cause genotoxic or mutagenic effects. Furthermore, in vivo oral, dermal, and acute inhalation toxicity results did not show any adverse effects or mortality in the test animals, and after the dermal and ocular irritation assessments, the in vivo models did not exhibit irritation or corrosion. Therefore, the results show that these previously synthesized S. queretaroensis AgNPs do not represent a risk at the tested concentrations; however, little is known about the effects that AgNPs induce on physiological systems or the possible risk following long-term exposure.

Chronic mechanical irritation enhances the effect of tobacco and alcohol on the risk of oral squamous cell carcinoma: a case-control study in Argentina.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is a multifactorial disease. The individual effect of each risk factor for OSCC may be conditioned by the frequency of other factors. The objective of this study was to identify the association between chronic mechanical irritation (CMI) and OSCC and to analyse the influence of CMI on other important risk factors for OSCC. MATERIALS AND METHODS: A prospective and age/sex-matched case-control study was performed in two institutions from Argentina between 2009 and 2019, with consecutive and newly diagnosed OSCC. The frequencies of tobacco, alcohol, and CMI were analysed using conditional logistic regression. Cumulative tobacco consumption and the presence of CMI were analysed using the Mann-Whitney test. RESULTS: CMI and OSCC were associated with an OR of 7.02 (95% CI 3.57-13.78, p < 0.001). The combination of CMI and alcohol demonstrated the highest risk of OSCC (OR 53.83, CI 95% 8.04-360, p < 0.0001), followed by the combination of CMI, tobacco, and alcohol (OR 48.06, CI 95% 8.47-272, p < 0.0001). The combination of CMI and tobacco was also significant (OR 5.61, CI 95% 1.07-29.54, p = 0.042). Patients with CMI developed OSCC with less cumulative tobacco use compared with those without CMI. CONCLUSION: CMI is an independent risk factor for OSCC, and it could act as a risk modifier among tobacco and alcohol users having an enhancing effect. CLINICAL RELEVANCE: Elimination of CMI could decrease the risk of OSCC.

Strategies for the evaluation of the eye irritation potential of different types of surfactants and silicones used in cosmetic products.

The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen's Egg Test - Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as "severe irritants", it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as "no prediction can be made". However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.

Gingival irritation in patients submitted to at-home bleaching with different cutouts of the bleaching tray: a randomized, single-blind clinical trial.

OBJECTIVES: This split-mouth randomized, single-blind clinical trial evaluated the gingival irritation (GI) of at-home bleaching with individual trays of different cutouts, as well as the tooth sensitivity (TS) and color change. MATERIALS AND METHODS: One hundred and twenty patients were randomized as to which side would receive the type of bleaching tray cutout: scalloped (in the gingival margin) and nonscalloped (extended from the gingival margin). The at-home bleaching was performed for 30 min with 10% hydrogen peroxide (HP) for 2 weeks. The absolute risk and intensity of GI and TS were assessed with a visual analog scale. Color change was assessed using a digital spectrophotometer and a color guide (α = 0.05). RESULTS: The proportion of patients who experienced GI was 57.5% (odds ratio 95% CI = 1.1 [0.7 to 1.8]), with no significant difference between groups (p = 0.66). The proportion of patients who experienced TS was 64.1% (odds ratio 95% CI = 1.0 [0.6 to 1.6]), with no significant difference between groups (p = 1.0). There is equivalence of scalloped and noscalloped groups for GI intensity (p < 0.01). Significant whitening was detected for both groups. Although some differences were observed between groups (CIELab and CIEDE00; p < 0.02), these were below of the considered clinically noticeable. CONCLUSIONS: The different cutouts of trays proved to be equivalent when regarding gengival irritation and tooth sensitivity when 10% HP for at-home bleaching was used. Significant color change was observed in both groups. However, significant differences detected between groups are not considered clinically noticeable. TRIAL REGISTRATION: Brazilian Clinical Trials Registry (RBR-2s34685). CLINICAL RELEVANCE: Scalloped or not, the individual trays for at-home bleaching could be considered a clinician's decision.

Development and evaluation of topical formulations that contain hydroethanolic extract from Schinus terebinthifolia against HSV-1 infection

Abstract The therapeutic drugs to treat Herpes simplex virus (HSV) infections have toxic side effects and there has been an emergence of drug-resistant strains. Therefore, the search for new treatments for HSV infections is mounting. In the present study, semi-solid formulations containing a crude hydroethanolic extract (CHE) from Schinus terebinthifolia were developed. Skin irritation, cutaneous permeation, and in vivo therapeutic efficacy of the formulations were investigated. Treatment with the ointment formulations did not result in any signs of skin irritation while the emulsions increased the thickness of the epidermis in Swiss mice. The cutaneous permeation test indicated that the CHE incorporated in the formulations permeated through the skin layers and was present in the epidermis and dermis even 3 h after topical application. In vivo antiviral activity in BALB/c mice treated with the CHE ointments was better than those treated with the CHE emulsions and did not significantly differ from an acyclovir-treated group. Taken together, this suggests that the incorporation of CHE in the ointment may be a potential candidate for the alternative topical treatment of herpetic lesions.

Ex Vivo Human Skin: An Alternative Test System for Skin Irritation and Corrosion Assays.

Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.

Comparison between HET-CAM protocols and a product use clinical study for eye irritation evaluation of personal care products including cosmetics according to their surfactant composition.

The hen's egg test on chorioallantoic membrane (HET-CAM) is one of the most frequently used alternative tests for prediction of ocular irritation of cosmetic products. There are different HET-CAM protocols widely accepted, but there is no information about which of the protocols better correlates with the results obtained in product use clinical study under the conditions of use. Two Fix Time Methods (FTM) -Lüepke and the ICCVAM guideline - and two Reaction Time Methods (RTM) -ECVAM DBALM Prot. No. 47 and No. 96- were employed to test 18 cosmetic products. Simultaneously, they were evaluated by an ophthalmological clinical test. A unified classification system was used, and products were classified into four irritation levels: non-irritant, weak, moderate and severe irritant. The duration of use (rinse-off or leave-on), and the concentration and type of surfactants were taken into account in the analysis. All the products that were classified as non-irritant by any HET-CAM protocols were also safe in the product use clinical study. The product that was found to be non-safe in the product use clinical evaluation was also unsuitable by most of the HET-CAM protocols. These results were employed to develop an algorithm that allows selecting the appropriate HET-CAM protocol for each type of product to be tested.

Strategy Combining Nonanimal Methods for Ocular Toxicity Evaluation.

Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) have been proposed, including a tiered testing approach combining different nonanimal testing with different endpoints, which have been used for regulatory purposes, on a case-by-case basis and within integrated approaches to testing and assessment (IATA), to identify materials according to their ability to trigger eye damage. In particular, the top-down and bottom-up approaches have been recommended for the C&L of materials, which cause serious eye damage or eye irritation, respectively. This chapter describes detailed protocols for eye irritation testing based on cells (Short Time Exposure-STE, OECD No. 491/2017), a vascularized membrane (the Hen's Egg Test-Chorioallantoic Membrane-HET-CAM) and corneal tissue (Bovine Corneal Opacity and Permeability-BCOP, OECD No. 437/2017), which can be applied using top-down or bottom-up approaches. In addition, it suggests making a corneal histomorphometric evaluation as an additional parameter in the BCOP method to differentiate materials that cause serious eye tissue damage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).

Immortalized keratinocytes cells generates an effective model of Epidermal Human Equivalent for irritation and corrosion tests.

Three-dimensional skin models, also named 3D skin models, human skin equivalents (HSEs), or Human Epidermal Equivalents (HEEs), have been increasingly used for chemical assessments in terms of efficacy and safety. Considering this, we developed an HEE model using immortalized HaCaT cells, aiming to overcome the limitation of primary tissue source. Our 3D model (HaCaT-HEE) exhibited important markers of cell differentiation (CK10, CK14, involucrin, and filaggrin), although the stratum corneum was shown to be modest. Besides, the model showed a good prediction potential considering membrane permeability, sensitivity, specificity, and accuracy in distinguishing irritant and corrosive effects after exposure to selected chemicals recommended by the OECD protocols. We also validated the formazan determination for the MTT method using High-Performance Liquid Chromatography (HPLC). For that, we considered carry over, linearity, reproducibility/robustness, accuracy, precision, selectivity, and matrix effect, according to the Food and Drug Administration (FDA) guideline. Based on our results, we can conclude that our model has an acceptable predictive value for the safety evaluation of compounds after skin exposure, with the great advantage of being constructed using immortalized cells.

Summer bloom of Vulcanodinium rugosum in Cienfuegos Bay (Cuba) associated to dermatitis in swimmers.

The marine dinoflagellate Vulcanodinium rugosum produces powerful paralyzing and cytotoxic compounds named pinnatoxins (PnTX) and portimines. Even though, no related human intoxication episodes following direct exposure in seawater or the ingestion of contaminated seafood have been documented so far. This study aimed at investigating a dinoflagellate bloom linked to acute dermatitis cases in two recreational beaches in Cienfuegos Bay, Cuba. We used epidemiological and clinical data from 60 dermatitis cases consisting of individuals in close contact with the bloom. Seawater physical-chemical properties were described, and the microorganism causing the bloom was identified by means of light and scanning electron microscopy. Morphological identification was confirmed genetically by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA region. Toxic compounds were identified from a bloom extract using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS), and their concentrations were estimated based on low-resolution tandem mass spectrometry (LC-MS/MS). Sixty people who had prolonged contact with the dinoflagellate bloom suffered acute dermal irritation. Most patients (79.2%) were children and had to be treated with antibiotics; some required >5-day hospitalization. Combined morphological and genetic characters indicated V. rugosum as the causative agent of the bloom. rDNA sequences of the V. rugosum genotype found in the bloom aligned with others from Asia, including material found in the ballast tank of a ship in Florida. The predominant toxins in the bloom were portimine, PnTX-F and PnTX-E, similar to strains originating from the Pacific Ocean. This bloom was associated with unusual weather conditions such as frequent and prolonged droughts. Our findings indicate a close link between the V. rugosum bloom and a dermatitis outbreak among swimmers in Cienfuegos Bay. Phylogenetic evidence suggests a recent introduction of V. rugosum from the Pacific Ocean into Caribbean waters, possibly via ballast water.