Neutral Red Uptake Phototoxicity Test Research Articles

Cellular damage photosensitized by dasatinib, radical-mediated mechanisms and photoprotection in reconstructed epidermis

Dasatinib (DAS) is an anticancer drug employed in the treatment of certain hematological malignancies. Although DAS has been mainly developed for oral administration, it has recently garnered attention for its possible topical application. The use of topical drugs can cause photosensitivity, which is not listed as an adverse reaction for DAS. Since DAS absorbs UVA, it could potentially induce photosensitivity reactions and lead to oxidative damage to cellular targets. This study aims to investigate whether DAS exhibits phototoxic reactions on primary cellular targets in both solution and artificial skin, mimicking topical drug administration. It also examines the potential generation of highly reactive intermediates like organic radicals and ROS, which could trigger photosensitivity reactions. Upon DAS irradiation in the UVA region, the first transient species detected was the diradicaloid triplet excited state (3DAS∗) with an absorption maximum of around 490 nm, which was quenched by oxygen to produce singlet oxygen. Quenching experiments with linoleic acid and 3-methylindole indicated that radical-mediated (Type I) photosensitized damage to lipids and proteins is possible. However, the lack of triplet quenching with guanosine suggests that the Type II mechanism also plays a role in the photooxidation of biomolecules. Accordingly, the neutral red uptake phototoxicity test (photoirritation factor of 5) and the comet assay, revealed that this drug is photo(geno)toxic to cells. Moreover, investigations on lipid photoperoxidation, and protein and DNA photooxidation strongly support that different cellular compartments are potential targets for DAS-induced phototoxicity.Regarding its potential application in topical dermatological formulations, an O/W emulsion of DAS was prepared and tested in reconstructed human epidermis, and a significant phototoxicity was also demonstrated. Fortunately, this undesired side effect disappeared upon formulation of DAS along with a sunscreen. Thus, for topical treatments, the photosensitivity reactions induced by DAS can be prevented by using formulations including appropriate UVA filters.

Chlorin E6-Curcumin-Mediated Photodynamic Therapy Promotes an Anti-Photoaging Effect in UVB-Irradiated Fibroblasts.

Skin photoaging due to ultraviolet B (UVB) exposure generates reactive oxygen species (ROS) that increase matrix metalloproteinase (MMP). Chlorin e6-photodynamic therapy (Ce6-PDT), in addition to being the first-line treatment for malignancies, has been shown to lessen skin photoaging, while curcumin is well known for reducing the deleterious effects of ROS. In the current study, PDT with three novel Ce6-curcumin derivatives, a combination of Ce6 and curcumin with various linkers, including propane-1,3-diamine for Ce6-propane-curcumin; hexane-1,6-diamine for Ce6-hexane-curcumin; and 3,3'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(propan-1-amine) for Ce6-dipolyethylene glycol (diPEG)-curcumin, were studied for regulation of UVB-induced photoaging on human skin fibroblast (Hs68) and mouse embryonic fibroblast (BALB/c 3T3) cells. We assessed the antiphotoaging effects of Ce6-curcumin derivatives on cell viability, antioxidant activity, the mechanism of matrix metalloproteinase-1 and 2 (MMP-2) expression, and collagen synthesis in UVB-irradiated in vitro models. All three Ce6-curcumin derivatives were found to be non-phototoxic in the neutral red uptake phototoxicity test. We found that Ce6-hexane-curcumin-PDT and Ce6-propane-curcumin-associated PDT exhibited less cytotoxicity in Hs68 and BALB/c 3T3 fibroblast cell lines compared to Ce6-diPEG-curcumin-PDT. Ce6-diPEG-curcumin and Ce6-propane-curcumin-associated PDT showed superior antioxidant activity in Hs68 cell lines. Further, in UVB-irradiated in vitro models, the Ce6-diPEG-curcumin-PDT greatly attenuated the expression levels of MMP-1 and MMP-2 by blocking mitogen-activated protein kinases (MAPKs), activator protein 1 (AP-1), and tumor necrosis factor-α (NF-κB) signaling. Moreover, Ce6-diPEG-curcumin effectively inhibited inflammatory molecules, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, while accelerating collagen synthesis. These results demonstrate that Ce6-diPEG-curcumin may be a potential therapy for treating skin photoaging.

Cellular photo(geno)toxicity of gefitinib after biotransformation.

Gefitinib (GFT) is a selective epidermal growth factor receptor (EGFR) inhibitor clinically used for the treatment of patients with non-small cell lung cancer. Bioactivation by mainly Phase I hepatic metabolism leads to chemically reactive metabolites such as O-Demethyl gefitinib (DMT-GFT), 4-Defluoro-4-hydroxy gefitinib (DF-GFT), and O-Demorpholinopropyl gefitinib (DMOR-GFT), which display an enhanced UV-light absorption. In this context, the aim of the present study is to investigate the capability of gefitinib metabolites to induce photosensitivity disorders and to elucidate the involved mechanisms. According to the neutral red uptake (NRU) phototoxicity test, only DF-GFT metabolite can be considered non-phototoxic to cells with a photoirritation factor (PIF) close to 1. Moreover, DMOR-GFT is markedly more phototoxic than the parent drug (PIF = 48), whereas DMT-GFT is much less phototoxic (PIF = 7). Using the thiobarbituric acid reactive substances (TBARS) method as an indicator of lipid photoperoxidation, only DMOR-GFT has demonstrated the ability to photosensitize this process, resulting in a significant amount of TBARS (similar to ketoprofen, which was used as the positive control). Protein photooxidation monitored by 2,4-dinitrophenylhydrazine (DNPH) derivatization method is mainly mediated by GFT and, to a lesser extent, by DMOR-GFT; in contrast, protein oxidation associated with DMT-GFT is nearly negligible. Interestingly, the damage to cellular DNA as revealed by the comet assay, indicates that DMT-GFT has the highest photogenotoxic potential; moreover, the DNA damage induced by this metabolite is hardly repaired by the cells after a time recovery of 18h. This could ultimately result in mutagenic and carcinogenic effects. These results could aid oncologists when prescribing TKIs to cancer patients and, thus, establish the conditions of use and recommend photoprotection guidelines.

Development of a GCN-based model to predict <i>in vitro</i> phototoxicity from the chemical structure and HOMO-LUMO gap

The interaction between sunlight and drugs can lead to phototoxicity in patients who have received such drugs. Phototoxicity assessment is a regulatory requirement globally and one of the main toxicity screening steps in the early stages of drug discovery. An in silico-in vitro approach has been utilized mainly for toxicology assessments at these stages. Although several quantitative structure-activity relationship (QSAR) models for phototoxicity have been developed, in silico technology to evaluate phototoxicity has not been well established. In this study, we attempted to develop an artificial intelligence (AI) model to predict the in vitro Neutral Red Uptake Phototoxicity Test results from a chemical structure and its derived information. To accomplish this, we utilized an open-source software library, kMoL. kMoL employs a graph convolutional neural networks (GCN) approach, which allows it to learn the data for the specified chemical structure. kMoL also utilizes the integrated gradient (IG) method, enabling it to visually display the substructures contributing to any positive results. To construct this AI model, we used only the chemical structure as a basis, then added the descriptors and the HOMO-LUMO gap, which was obtained from quantum chemical calculations. As a result, the assortment of chemical structures and the HOMO-LUMO gap produced an AI model with high discrimination performance, and an F1 score of 0.857. Additionally, our AI model could visualize the substructures involved in phototoxicity using the IG method. Our AI model can be applied as a toxicity screening method and could enhance productivity in drug development.

Evaluation of phototoxicity induced by the anticancer drug rucaparib

Rucaparib (RCP) is a potent selective inhibitor of both PARP-1 and PARP-2 enzymes that induces synthetic lethality in cancer cells. It is used for the treatment of breast and ovarian tumors harboring deleterious germline or somatic cancer susceptibility genes mutations. Although RCP has an indole chromophore in its structure, it displays a bathochromic shift of the absorption band towards the UVA region of sunlight, thus extending the active fraction of solar light able to produce photosensitivity reactions. In this context, it is highly interesting to study the photo(geno)toxicity disorders associated with this drug, bearing in mind that, for dermatologists it is crucial to understand the toxicity mechanism to improve clinical management. In the present work, RCP has shown to be potentially phototoxic, as observed in the neutral red uptake phototoxicity test. Moreover, this significant phototoxicity is attributed to both proteins and genomic DNA, as revealed in the protein photooxidation and comet assays. The results obtained are highly relevant concerning RCP photosafety and become clinically important in the context of identification of the cutaneous adverse events that can be associated with the targeted therapies. Interestingly, this is the first example of a PARP inhibitor able to induce photosensitized damage to biomolecules.

Safety and efficacy of combined essential oils for the skin barrier properties: In vitro, ex vivo and clinical studies.

To evaluate the safety and the synergistic effects of tea tree, lavender, eucalyptus and tangerine essential oils in combination on the skin using in vitro, ex vivo and clinical studies. The phototoxicity was predicted using 3T3 neutral red uptake phototoxicity test (OECD TG 432). Skin penetration was evaluated by confocal Raman microspectroscopy using direct application of essential oils to pig ears. For the clinical studies, 40 participants were enrolled and randomized in three groups: (1) lavender, eucalyptus and tangerine, (2) the same essential oils plus melaleuca and (3) placebo group. The skin was evaluated by noninvasive techniques before and after a 90-day period of topical use. The essential oils were non-phototoxic, but the tangerine oil showed dose-dependent cytotoxicity (IC50: 33.1µg/ml), presenting 35% of penetration in the viable epidermis. On the contrary, 17.7µg/ml in combination was applied per day in the clinical study and the penetration rate for the combinations (10%, 1.77µg/ml achieving the viable epidermis) guaranteed the safety, since in the clinical study, the application of the four essential oils improved skin barrier and morphologic skin characteristics, as well as increased skin hydration and decreased sebum levels, with no unwanted effects reported. All essential oils studied were considered non-cytotoxic or non-phototoxic separately except tangerine, which present a dose-dependent cytotoxicity. Finally, the essential oils in combination in an appropriate amount were safe and effective in the improvement of the hydrolipidic balance and morphological properties of the skin.

In Vitro Evaluation of the Photoreactivity and Phototoxicity of Natural Polyphenol Antioxidants.

Polyphenols are a large family of natural compounds widely used in cosmetic products due to their antioxidant and anti-inflammatory beneficial properties and their ability to prevent UV radiation-induced oxidative stress. Since these compounds present chromophores and are applied directly to the skin, they can react with sunlight and exert phototoxic effects. The available scientific information on the phototoxic potential of these natural compounds is scarce, and thus the aim of this study was to evaluate the photoreactivity and phototoxicity of five phenolic antioxidants with documented use in cosmetic products. A standard ROS assay was validated and applied to screen the photoreactivity of the natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxyphenylacetic acid (DOPAC), and rutin. The phototoxicity potential was determined by using a human keratinocyte cell line (HaCaT), based on the 3T3 Neutral Red Uptake phototoxicity test. Although all studied phenolic antioxidants absorbed UV/Vis radiation in the range of 290 to 700 nm, only DOPAC was able to generate singlet oxygen. The generation of reactive oxygen species is an early-stage chemical reaction as part of the phototoxicity mechanism. Yet, none of the studied compounds decreased the viability of keratinocytes after irradiation, leading to the conclusion that they do not have phototoxic potential. The data obtained with this work suggests that these compounds are safe when incorporated in cosmetic products.

Safety Assessment of Ethanolic Extract of Padina gymnospora as a Cosmetic Ingredient

Purpose: In a previous study, we identified the skin-whitening effect of the ethanolic extract of Padina gymnospora. The present study was performed to confirm the safety of the extract in animal replacement tests.Methods: To evaluate the safety of the extract of Padina gymnospora, the photosensitivity test (Harber test), in vitro 3T3 neutral red uptake (3T3 NRU) phototoxicity test, local lymph node assay (5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay), acute oral toxicity test, and reconstructed human epidermis (RHE) test were used. All experiments followed the guidelines of the Korean Ministry of Food and Drug Safety and were conducted by a GLP-certified organization (Chemon Inc.).Results: The extract of Padina gymnospora was not photosensitive: 0% photosensitization was detected (I grade: very weak). In the 3T3 NRU phototoxicity test, the relative viability of the extract-treated cells was higher than the guideline level; thus, the extract was classified as non-phototoxic. Treatment with the extract did not trigger skin irritation in the RHE test model and did not cause skin sensitization in the local lymph node assay. Finally, oral administration of the extract to rats indicated that it was not a harmful material as the LD<sub>50</sub> was estimated at >2,000 mg/kg.Conclusion: The ethanolic extract of Padina gymnospora was demonstrated to be safe when applied to the skin. Taken together with our previous study of its efficacy, we conclude that this extract has the potential for use as a cosmetic ingredient.

Comparison of Phototoxicity Sensitivity by the Neutral Red Uptake Method for BALB/c 3T3, HaCaT, and HDFa Cells In Vitro

ABSTRACT Introduction Phototoxicity is an acute photoinduced reaction. The 3T3 neutral red uptake (NRU) phototoxicity test has high sensitivity for the determination of phototoxic substances. To further optimize the method, this study mainly focused on comparing the phototoxicity sensitivity by using the NRU method for BALB/c 3T3, HaCaT, and HDFa cells in vitro. Methods The NRU method was used to evaluate the phototoxicity of chlorpromazine hydrochloride (CPZ), amiodarone hydrochloride (Amiodar), and L-histidine (L-His) on BALB/c 3T3 cells, HaCaT cells, and HDFa cells. The sensitivity of different cells to ultraviolet (UVA) irradiation in vitro was studied. Results L-His showed no phototoxicity, but the phototoxicity of CPZ and Amiodar showed different sensitivities among the three kinds of cells. The in vitro phototoxicity evaluation of HaCaT cells is closer to that of primary human fibroblasts. Conclusion This study provides a reference for cell line selection to optimize the existing in vitro evaluation method of 3T3 NRU phototoxicity.

Current Issues in Photosafety Evaluation

Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance.

Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals. In the present study, we attempted to develop a new photosafety test method, named the electron spin resonance-based photosafety test (ESR-PT), that would be applicable even to hydrophobic chemicals based on the detection of singlet oxygen generation after irradiation using ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine as a spin trap reagent. To achieve a quantitative evaluation, the singlet oxygen formation (SOF) value, which can be calculated as the increment in relative intensity after irradiation of the test mixture normalized by the increment in relative intensity after irradiation of the vehicle control solution, was calculated. The performance of the ESR-PT was evaluated by testing all the proficiency chemicals of the ROS assay plus additional chemicals, including hydrophobic chemicals and chemicals that tested false negative in the 3T3-NRU PT and ROS assay. SOF values were successfully calculated for all the chemicals tested including the hydrophobic chemicals, and the accuracy of the ESR-PT using a tentative cutoff value of 2.8 against the photosafety information was 100%. Therefore, the SOF value could be an effective parameter for photosafety evaluations, suggesting that the newly developed ESR-PT is a promising non-animal test applicable even to hydrophobic chemicals.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

Evaluation of phototoxicity of tattoo pigments using the 3 T3 neutral red uptake phototoxicity test and a 3D human reconstructed skin model

Phototoxicity due to dermally impregnated compounds exposed to ultraviolet radiation is associated with skin inflammation. The phototoxicity potential of active substances applied to the skin should be evaluated. Pigments are widely used for tattoos, and hypersensitivity reactions, such as photoallergic dermatitis, are possible tattoo-related complications. However, the phototoxicity of these chemicals is not well known. In this study, we evaluated the phototoxicity potential of six tattoo pigments, cadmium sulfide, carbazole, cadmium selenide, mercury (II) sulfide, chromium oxide, and cobalt aluminate, using in vitro methods—3 T3 neutral red uptake (NRU) phototoxicity test (PT) and a 3D human reconstructed skin model (EpiDerm). The validated 3 T3 NRU PT indicated the phototoxicity potential of carbazole and cadmium sulfide. The 3D human skin model confirmed that only carbazole was phototoxic. The 3 T3 NRU PT data corresponded well with those from the 3D skin model and suggested the need to employ several test systems for final phototoxicity assessment. In addition to the results obtained using 3 T3 NRU PT, further testing on 3D skin models may better reflect the bioavailability of a given chemical in the skin.

Implementation of an in vitro methodology for phototoxicity evaluation in a human keratinocyte cell line

To assess photoxicity, several in vitro methods using different cellular models have been developed for preclinical testing. Over prediction of the in vivo photosafety hazard has been however appointed. Herein, we describe the implementation and validation of an in vitro methodology for phototoxicity evaluation based on the 3T3 neutral red uptake phototoxicity test using the HaCaT human keratinocyte cell line, and UVA/UVB radiation. Known positive (5-methoxypsoralen, chlorpromazine, and quinine) and negative (acetyl salicylic acid, hexachlorophene, and sodium lauryl sulphate) controls were tested together with a set of chemical currently used in cosmetic/pharmaceutical formulations. Apart from the advantage of using a cell line of human origin, these cells were generally more resistant to the cytotoxic effects of the test substances relative to the 3T3 mouse fibroblasts when exposed to an UVA irradiation dose of 1.7 mW/cm2. Therefore, this HaCaT NRU assay provides a more realistic experimental model that overcomes the over/high sensitivity frequently noted with the 3T3 NRU assay and that is more consistent with the human in vivo situation. Using a more representative method can prevent time-consuming and expensive in vivo testing in both animal models and humans that can significantly delay the clinical development of new chemicals.

Photocatalytic production of hydroxyl radicals by commercial TiO2 nanoparticles and phototoxic hazard identification

This study identifies the phototoxic potential of commercial titanium dioxide nanoparticles (TiO2 NPs) used in sunscreens and consumer products by employing a tiered testing approach comprising physicochemical, in vitro and ex vivo tests. Our results revealed that all the test samples of TiO2 NPs, varied in surface coating, crystallinity and primary particle size, produced hydroxyl radicals upon UVA photoexcitation as determined by electron spin resonance (ESR) spectroscopy. Their phototoxic potentials were assessed first by combining the validated 3T3 neutral red uptake phototoxicity test and red blood cell phototoxicity test and subsequently in ex vivo models of chick chorioallantoic membrane (CAM) and reconstructed human 3D skin model (H3D). Crystalline structure and particle size of TiO2 NPs were found to exert a major influence on the photocatalytic activity and the associated phototoxic effects. Besides, a medium-sized sample with silica/alumina also exhibited high phototoxic potency with no obvious relevance to the enhanced hydroxyl radicals and lipidperoxidation. This effect might be taken place through the interaction of harmful metal ions released from the oxide coating. However, no phototoxicity was observed on a H3D skin model probably due to the lack of efficient percutaneous absorption of TiO2 NPs. This study demonstrates the efficacy of a tiered testing strategy for identifying phototoxic hazards of TiO2 NPs and suggests the need for a comprehensive assessment that takes account of the effects of different coating materials and potential interactions between multiple mechanisms.

Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance.

Safety assessment of chemicals, pharmaceuticals, food and food ingredients, cosmetics, industrial products is very crucial prior to their approval for human uses. Since the commencement of toxicity testing (about 500 years ago, since 1520), significant advances have been made with respect to the 3Rs (reduction, refinement and replacement) alternative approaches. This review is focused on the update in acute systemic toxicity testing of chemicals. Merits and demerits of these advances were also highlighted. Traditional LD50 test methods are being suspended while new methods are developed and endorsed by the regulatory body. Based on the refinement and reduction approaches, the regulatory body has approved fixed dose procedure (FDP), acute toxic class (ATC) method and up and down procedure (UDP) which involves few numbers of animals. In terms of replacement approach, the regulatory body approved 3T3 neutral red uptake (NRU), the normal human keratinocyte (NHK), and the 3T3 neutral red uptake (NRU) phototoxicity test for acute phototoxicity. However, other promising replacement alternatives such as organ on chip seeded with human cells for acute systemic toxicity and 3T3 neutral red uptake (NRU) cytotoxicity test for identifying substances not requiring classification, as well as the in silico approaches are yet to receive regulatory approval. With this backdrop, a collaborative effort is required from the academia, industries, regulatory agencies, government and scientific organizations to ensure speedily regulatory approval of the prospective alternatives highlighted.

The Phototoxic Potential of the Flavonoids, Taxifolin and Quercetin.

Quercetin, one of the most abundant polyphenols in the plant kingdom has been shown to be photodegraded on exposure to UV light. Despite the fact, it is a component of several dermatological preparations. Its phototoxic potential has not been evaluated to date. The aim of this study was to assess whether photo-induced degradation of quercetin is linked to phototoxic effects on living cells. Its dihydro derivative, taxifolin, was included in the study. For evaluation, the 3T3 Neutral Red Uptake Phototoxicity Test according to OECD TG 432 was used. To better approximate human skin, HaCaT keratinocytes, normal human epidermal keratinocytes and dermal fibroblasts were used, apart from the Balb/c 3T3 cell line. Quercetin showed a dose-dependent photodegradation in aqueous and organic environments and a phototoxic effect on all used cells. Quercetin pretreatment and following UVA exposure resulted in increased reactive oxygen species production and intracellular glutathione level depletion in human dermal fibroblasts. Taxifolin was found completely nonphototoxic and photostable. As only in vitro methodology was used, further studies using 3D skin models and/or human volunteers are needed to confirm whether exposure to sunlight, tanning sunbeds and/or phototherapy in people using cosmetics containing quercetin is a health risk.

In vitro safety and efficacy evaluations of a complex botanical mixture of Eugenia dysenterica DC. (Myrtaceae): Prospects for developing a new dermocosmetic product

In the context of developing a new natural product-based cosmetic, the in vitro efficacy and safety evaluations of a complex botanical mixture based on Eugenia dysenterica leaf hydroalcoholic extract (EDE) (2.5–1000μg/mL) were carried out. Chromatographic analysis demonstrated the presence of the tannin (ellagic acid) and flavonoids (quercetin and gallic acid) which characterize the EDE as a polyphenol-rich mixture. Using HFF-1 fibroblasts, it was shown that EDE promoted cell regeneration after UVA exposure. It also led to the inhibition of the collagenase, elastase and tyrosinase enzymes, which are involved in skin-related disorders. In terms of toxicological evaluation, the EDE was classified as non-phototoxic through the 3T3 Neutral Red Uptake Phototoxicity Test (OECD N° 432, 2004) and non-eye irritant by Bovine Corneal Opacity and Permeability (OECD N° 437, 2013) assay, in conjunction with corneal histomorphometric analysis. Furthermore, the EDE has no skin sensitization potential as demonstrated by a two-out-of-three prediction model [protein-binding/haptenization (OECD N° 442C, 2015), keratinocyte and dendritic cell activations]. In addition, it was shown that the EDE seems to be non-genotoxic through the cytokinesis-block micronucleus assay (OECD N° 487, 2014) using HepG2 cells. When considered together, these findings support the use of EDE botanical mixture in cosmetic/pharmaceutical products.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

Novel phototoxicity assay using human embryonic stem cell-derived retinal pigment epithelial cells.

Some chemicals are harmful in to light-exposed tissues such as skin and eyes. The 3T3 Neutral Red Uptake Phototoxicity Test has been validated and adopted by the Organization of Economic and Community Development (OECD) as a method of evaluating chemical phototoxicity using mouse 3T3 fibroblasts. However, the high rate of false positive results associated with this test eventually led to increased laboratory animal usage. Although the eye is vulnerable to light damage because of constant exposure to environmental radiation, few approaches are available to predict ocular phototoxicity in humans. Here, we propose a tier one test that identifies the potential ocular phototoxicity of chemical substances. Using a three-dimensional culture technique, human embryonic stem cells (hESCs) were differentiated to retinal pigment epithelial cell (RPE) precursors. The precursors after prolonged treatment with FBS formed a uniform hexagonal lattice of cells with well-developed tight junctions and time-dependent elevation of melanin content and RPE maturation marker levels. Hierarchical clustering of gene transcripts revealed that hESC-derived RPEs were very similar to tissue-derived adult RPEs. Interestingly, there were a high percentage of chemicals eliciting a positive response in 3T3 cells and negative in hESC-derived RPEs under the experimental conditions used in the phototoxicity test. The response to treatment of hESC-derived RPEs with these negative chemicals became positive at a higher dose of UVA irradiation; however, the biological responses to these chemicals differed between the two cells. Taken together, we conclude that hESC-derived RPEs are novel tool for future toxicological and mechanistic studies of ocular phototoxicity in humans.