In vitro Safety Assessment of Extracts and Compounds From Plants as Sunscreen Ingredients.

This work investigated the safety of extracts obtained from plants growing in Colombia, which have previously shown UV-filter/antigenotoxic properties. The compounds in plant extracts obtained by the supercritical fluid (CO2) extraction method were identified using gas chromatography coupled to mass spectrometry (GC/MS) analysis. Cytotoxicity measured as cytotoxic concentration 50% (CC50) and genotoxicity of the plant extracts and some compounds were studied in human fibroblasts using the trypan blue exclusion assay and the Comet assay, respectively. The extracts from Pipper eriopodon and Salvia aratocensis species and the compound trans-ß-caryophyllene were clearly cytotoxic to human fibroblasts. Conversely, Achyrocline satureioides, Chromolaena pellia, and Lippia origanoides extracts were relatively less cytotoxic with CC50 values of 173, 184, and 89 µg/mL, respectively. The C. pellia and L. origanoides extracts produced some degree of DNA breaks at cytotoxic concentrations. The cytotoxicity of the studied compounds was as follows, with lower CC50 values representing the most cytotoxic compounds: resveratrol (91 µM) > pinocembrin (144 µM) > quercetin (222 µM) > titanium dioxide (704 µM). Quercetin was unique among the compounds assayed in being genotoxic to human fibroblasts. Our work indicates that phytochemicals can be cytotoxic and genotoxic, demonstrating the need to establish safe concentrations of these extracts for their potential use in cosmetics.

Influence of TiO2 nanocomposite UV filter surface chemistry and their interactions with organic UV filters on uptake and toxicity toward cultured fish gill cells.

Aquatic environments have been found to be contaminated with a variety of inorganic and organic UV filters. This includes novel nano-sized titanium dioxide (TiO2) composite particles, which have been increasingly developed and incorporated into commercial sunscreens in recent years. So far, relatively little is known about the effects of this novel class of UV filters on aquatic life. Therefore, this study aimed to determine and compare the toxicity of three such nanoparticulate TiO2 UV filters with different surface coatings, namely Eusolex® T-Avo (SiO2-coated), T-Lite™ SF (Al(OH)3/PDMS-coated), and Eusolex® T-S (Al2O3/stearic acid-coated) either alone, or in the presence of selected organic UV filters (octinoxate, avobenzone, octocrylene), toward fish using RTgill-W1 cell cultures as an in vitro experimental model. Besides standard exposure protocols, alternative approaches (i.e., exposure to water accommodated fractions (WAFs), hanging-drop exposure) were explored to account for nanoparticle (NP)-specific fate in the medium and obtain additional/complementary information on their toxicity in different conditions. The AlamarBlue, CFDA-AM and Neutral Red Retention (NR) assays were used to measure effects on different cellular endpoints. Transmission electron microscopy (TEM) was used to examine NP uptake. Our results showed that none of the TiO2 NP UV filters were cytotoxic at the concentrations tested (0.1-10 µg/mL; 24 h) but there were differences in their uptake by the cells. Thus, only the hydrophilic T-AVO was detected inside cells, but the hydrophobic T-Lite SF and T-S were not. In addition, our results show that the presence of NPs (or the used dispersant) tended to decrease organic UV filter toxicity. The level of combination effect depended on both NP-type (surface chemistry) and concentration, suggesting that the reduced toxicity resulted from reduced availability of the organic UV filters due to their adsorption to the NP surface. Thus, mixtures of TiO2 NP UV filters and organic UV filters may have a different toxicological profile compared to the single substances, but probably do not pose an increased hazard.

Effect of Combined Prenatal and Adult Benzophenone-3 Dermal Exposure on Factors Regulating Neurodegenerative Processes, Blood Hormone Levels, and Hematological Parameters in Female Rats.

Benzophenone-3 (BP-3), the most widely used UV chemical filter, is absorbed well through the skin and gastrointestinal tract and can affect some body functions, including the survival of nerve cells. Previously, we showed that BP-3 evoked a neurotoxic effect in male rats, but since the effects of this compound are known to depend on gender, the aim of the present study was to show the concentration and potential neurotoxic action of this compound in the female rat brain. BP-3 was administered dermally to female rats during pregnancy, and then in the 7th and 8th weeks of age to their female offspring. The effect of BP-3 exposure on short-term and spatial memory, its concentrations in blood, the liver, the frontal cortex, and the hippocampus, and the effect on selected markers of brain damage were determined. Also, the impact of BP-3 on sex and thyroid hormone levels in blood and hematological parameters was examined. It has been found that this compound was present in blood and brain structures in females at a lower concentration than in males. BP-3 in both examined brain structures increased extracellular glutamate concentration and enhanced lipid peroxidation, but did not induce the apoptotic process. The tested compound also evoked hyperthyroidism and decreased the blood progesterone level and the number of erythrocytes. The presented data indicated that, after the same exposure to BP-3, this compound was at a lower concentration in the female brain than in that of the males. Although BP-3 did not induce apoptosis in the hippocampus and frontal cortex, the increased extracellular glutamate concentration and lipid peroxidation, as well as impaired spatial memory, suggested that this compound also had adverse effects in the female brain yet was weaker than in males. In contrast to the weaker effects of the BP-3 on females than the brain of males, this compound affected the endocrine system and evoked a disturbance in hematological parameters more strongly than in male rats.

Preparation and characterisation of organic UV filters based on combined PHB/liposomes with natural phenolic compounds.

In this study preparation and characterization of new UV-protecting systems based on liposomes/polyhydroxybutyrate (PHB) with encapsulated coffee extracts are presented. Green and roasted coffee extracts with high phenolics content, high antioxidant activity and sun protection factor (SPF) value 40-50 were used as model organic UV filters and encapsulated into liposomes and PHB-liposomes. Particle size and colloid stability was observed by dynamic light scattering and zeta-potential. Toxicity of particles was tested by MTT and LDH assay on HaCaT cell line. All prepared samples showed moderate or high encapsulation efficiency. Addition of PHB up to 50 % of lecithin led to increased size and stability. As optimal addition of 20 % PHB into liposome particles was found leading to optimum size and processing of particles, to high UV-protective effect as well as to increased colloid stability and SPF value during long-term storage. Significant differences in cell viability were found in cytotoxicity studies after exposure of keratinocytes to liposomes with different PHB content. Newly fabricated PHB-liposome particles with coffee extract were not found as toxic for HaCaT cells and in LDH test up to 12 %. These particles can act as active carriers for organic sunscreen components in combination with UV-protective effect of PHB.

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

A Bioscreening Technique for Ultraviolet Irradiation Protective Natural Substances.

Ultraviolet radiation (UV-R) causes genotoxic and aging effects on skin, and sunscreens are used to alleviate the damage. However, sunscreens contain synthetic shielding agents that can cause harmful effects in the environment. Nature-derived substances may have potential as replacement materials for the harmful sunscreen chemicals. However, screening of a broad range of samples is tedious, and often requires a separate genotoxicity assessment. We describe a simple microplate technique for the screening of UV protective substances using a recombinant Escherichia coli biosensor. Both absorbance-based and bioactivity-based shields can be detected with simultaneous information about the sample genotoxicity. With this technique, a controversial sunscreen compound, oxybenzone offers physical or absorbance-based shield but appears genotoxic at higher concentrations (3.3 mg/mL). We also demonstrate that pine needle extract (PiNe ) shields the biosensor from UV-R in a dose-dependent manner without showing genotoxicity. The physical shield of 5 mg/mL PiNe was similar to that of one of the most common UV-shielding compound TiO2 concentration 0.80 mg/mL. The bioactivity-based shield of PiNe also reaches the extent of the physical shield with the highest concentration (3.3 mg/mL). We conclude that our technique is suitable in detecting the UV-shielding potential of natural substances, and gives simultaneous information on genotoxicity.

Safety assessment and toxicological profiling of a novel combinational sunprotective dermal formulation containing melatonin and pumpkin seed oil.

Ultraviolet (UV) radiation exposure has been known to cause irreparable damages to human skin. The daunting risk of UV radiation exposure faced by military personnel led to the development of a sunscreen formulation which has superior sun protection factor combined with the ability to counteract reactive oxygen species. The present work deals with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil, via OECD protocols of assessing acute oral and dermal toxicity; skin sensitizing; skin irritating; ocular irritating and genotoxic potential. Both oral and dermal LD50 values were found to be ˃2000 mg/kg body weight in adult Wistar albino rats using acute dermal and oral toxicity tests. The sunscreen formulation was found to be non-sensitizing to the skin of guinea pigs and non-irritating to both skin and eyes of rabbits. The sunscreen formulation was also found to be non-mutagenic which was affirmed by a battery of genotoxicity and muagenicity assays. The results obtained from this preclinical study indicated that the sunscreen formulation is non toxic and safe in animal models. This study along with additional preclinical evaluations may serve as a basis for considering the formulation as a potential candidate for further trials to establish its efficacy, tolerability and applicability.

Toxicity evaluation of the photoprotective compound LQFM048: Eye irritation, skin toxicity and genotoxic endpoints.

A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.

Protection against UV-induced toxicity and lack of mutagenicity of Antarctic Sanionia uncinata.

Antarctica moss Sanionia uncinata (Hedw.) Loeske is exposed in situ to damaging levels of ultraviolet (UV) radiation. This moss has the ability to respond to UV radiation exposure producing secondary metabolites such as flavonoids, and has been recommended as a potential source of photoprotective compounds and antioxidants. The aim of the present paper was to investigate the free-radical scavenging activity and mutagenic and photomutagenic properties of methanolic (ME), hydroethanolic (HE) and ethanolic (EE) extracts of S. uncinata. The phenolic contents were evaluated by high-performance liquid chromatography (HPLC) and spectrophotometry. The findings showed that ME and EE presented the highest phenolic contents and inhibited free radical-scavenging activity against 2,2'-diphenyl-1 picrylhydrazyl (DPPH) and the HPLC analysis indicated several classes of phenolic acids and flavonoids. The sun protection factors (SPF) were determined by an in vitro method and the results showed significant values. The SPF values of BZ-3 at 50µg/mL increased significantly in association with ME, HE and EE. The extracts did not induce mutagenicity in auxotrophic Salmonella typhimurium histidine and photomutagenicity was not detected in the TA102 and TA104 strains after exposure to UV-A at doses of up to 6.5J/cm2 for the TA102 strain and up to 0.24J/cm2 for the TA104 strain. In addition, with the exception of ME, all the extracts induced photoprotective effects in the presence of the TA104 strain at 0.04J/cm2. The present results suggest that S. uncinata extracts did not induce photomutation and showed promise for photoprotection against the photobiological and ROS-inducing effects of the UV-A radiation.

Effect of flexing and massage on in vivo human skin penetration and toxicity of zinc oxide nanoparticles.

AIM: We assessed the effects of flexing and massage on human skin penetration and toxicity of topically applied coated and uncoated zinc oxide nanoparticles (˜75 nm) in vivo. MATERIALS & METHODS: Noninvasive multiphoton tomography with fluorescence lifetime imaging was used to evaluate the penetration of nanoparticles through the skin barrier and cellular apoptosis in the viable epidermis. RESULTS: All nanoparticles applied to skin with flexing and massage were retained in the stratum corneum or skin furrows. No significant penetration into the viable epidermis was seen and no cellular toxicity was detected. CONCLUSION: Exposure of normal in vivo human skin to these nanoparticles under common in-use conditions of flexing or massage is not associated with significant adverse events.

A new hair follicle-derived human epidermal model for the evaluation of sunscreen genoprotection.

Induction of skin cancer is the most deleterious effect of excessive exposure to sunlight. Accurate evaluation of sunscreens to protect the genome is thus of major importance. In particular, the ability of suncare products to prevent the formation of DNA damage should be evaluated more directly since the Sun Protection Factor is only related to erythema induction. For this purpose, we developed an in vitro approach using a recently characterized reconstituted human epidermis (RHE) model engineered from hair follicle. The relevance of this skin substitute in terms of UV-induced genotoxicity was compared to ex vivo explants exposed to solar-simulated radiation (SSR). The yield of bipyrimidine photoproducts, their rate of repair, and the induction of apoptosis were very similar in both types of skin samples. In order to evaluate the protection afforded by sunscreen against DNA damage, bipyrimidine photoproducts were quantified in tissue models following SSR exposure in the presence or absence of a SPF50+ formula. A rather high DNA protection factor of approximately 20 was found in RHE, very similar to that determined for explants. Thus, RHE is a good surrogate to human skin, and also a convenient and useful tool for investigation of the genoprotection of sunscreens.

Topically applied ZnO nanoparticles suppress allergen induced skin inflammation but induce vigorous IgE production in the atopic dermatitis mouse model.

BACKGROUND: Metal oxide nanoparticles such as ZnO are used in sunscreens as they improve their optical properties against the UV-light that causes dermal damage and skin cancer. However, the hazardous properties of the particles used as UV-filters in the sunscreens and applied to the skin have remained uncharacterized. METHODS: Here we investigated whether different sized ZnO particles would be able to penetrate injured skin and injured allergic skin in the mouse atopic dermatitis model after repeated topical application of ZnO particles. Nano-sized ZnO (nZnO) and bulk-sized ZnO (bZnO) were applied to mechanically damaged mouse skin with or without allergen/superantigen sensitization. Allergen/superantigen sensitization evokes local inflammation and allergy in the skin and is used as a disease model of atopic dermatitis (AD). RESULTS: Our results demonstrate that only nZnO is able to reach into the deep layers of the allergic skin whereas bZnO stays in the upper layers of both damaged and allergic skin. In addition, both types of particles diminish the local skin inflammation induced in the mouse model of AD; however, nZnO has a higher potential to suppress the local effects. In addition, especially nZnO induces systemic production of IgE antibodies, evidence of allergy promoting adjuvant properties for topically applied nZnO. CONCLUSIONS: These results provide new hazard characterization data about the metal oxide nanoparticles commonly used in cosmetic products and provide new insights into the dermal exposure and hazard assessment of these materials in injured skin.

Physiological hepatic response to zinc oxide nanoparticle exposure in the white sucker, Catostomus commersonii.

Liver toxicity of commercially relevant zinc oxide nanoparticles (nZnO) was assessed in a benthic freshwater cypriniform, the white sucker (Catostomus commersonii). Exposure to nZnO caused several changes in levels of liver enzyme activity, antioxidants, and lipid peroxidation end products consistent with an oxidative stress response. Aconitase activity decreased by ~65% but tended to be restored to original levels upon supplementation with Fe(2+), indicating oxidative inactivation of the 4Fe-4S cluster. Furthermore, glucose-6-phosphate dehydrogenase activity decreased by ~29%, and glutathione levels increased by ~56%. Taken together, these suggest that nZnO induces hepatic physiological stress. Each assay was then validated by using a single liver homogenate or plasma sample that was partitioned and treated with nZnO or Zn(2+), the breakdown product of nZnO. It was found that Zn(2+), but not nZnO, increased detected glutathione reductase activity by ~14% and decreased detected malondialdehyde by ~39%. This indicates that if appreciable nZnO dissolution occurs in liver samples during processing and assay, it may skew results, with implications not only for this study, but also for a wide range of nanotoxicology studies focusing on nZnO. Finally, in vitro incubations of cell-free rat blood plasma with nZnO failed to generate any significant increase in malondialdehyde or protein carbonyl levels, or any significant decrease in ferric reducing ability of plasma. This suggests that at the level tested, any oxidative stress caused by nZnO is the result of a coordinated physiological response by the liver.

Ecotoxicological effects of an aged TiO2 nanocomposite measured as apoptosis in the anecic earthworm Lumbricus terrestris after exposure through water, food and soil.

Titanium dioxide nanoparticles seem to have a low toxicity to terrestrial organisms, though few studies are published in this area. TiO(2) used in sunscreens are nanocomposites where TiO(2) has been coated with magnesium, silica or alumina, as well as amphiphilic organics like polydimethyl siloxane (PDMS), and these coatings are modified by ageing. We assessed the ecotoxicity and propensity for bioaccumulation of an aged TiO(2) nanocomposite used in sunscreen cosmetics, and its potential effect on the frequency of apoptosis in different earthworm tissues. The earthworm Lumbricus terrestris was exposed to the TiO(2) nanocomposite for 7 days in water or 2-8 weeks in soil with the nanocomposite mixed either into food or soil at concentrations ranging from 0 to 100 mg kg(-1). Apoptosis was then measured by immunohistochemistry and Ti localized by XRF microscopy. Results showed no mortality, but an enhanced apoptotic frequency which was higher in the cuticule, intestinal epithelium and chloragogenous tissue than in the longitudinal and circular musculature. TiO(2) nanoparticles did not seem to cross the intestinal epithelium/chloragogenous matrix barrier to enter the coelomic liquid, or the cuticule barrier to reach the muscular layers. No bioaccumulation of TiO(2) nanocomposites could thus be observed.

A water soluble extract from Uncaria tomentosa (Cat's Claw) is a potent enhancer of DNA repair in primary organ cultures of human skin.

Cat's Claw (Uncaria tomentosa) water extracts, essentially free of oxindole alkaloids, have been shown to possess a broad spectrum of biological activity including DNA repair enhancement and antiinflammatory properties. These two biological mechanisms are key molecular targets to develop treatments that protect skin exposed to ultraviolet light from the sun. Because C-Med-100, a Cat's Claw water extract, is the only documented natural source of components that can up-regulate simultaneously both DNA repair and antiinflammation, its ability to modulate DNA repair in human skin organ cultures was undertaken. For this purpose skin cultures were treated with or without 5 mg/mL C-Med-100, irradiated with 0-100 mJ/cm2 UVB, and microscopically analysed for necrosis as well as the level of pyrimidine dimers using immunofluorescent TT-dimer antibody staining. The data clearly demonstrated that co-incubation with C-Med-100 reduced skin cell death from UV exposure, and this protection was accounted for by a concomitant increase in DNA repair. Based on these results, it was concluded that C-Med-100 was a natural plant extract worthy of further consideration as a sunscreen product.

[Systemic photoprotection through carotenoids]. / Systemische Photoprotektion durch Karotinoide.

Nutritional supplements are increasingly used to protect human skin against environmentally-induced damage, most importantly as a consequence of ultraviolet radiation exposure. beta-carotene is a major constituent of comercially available products administered for systemic photoprotection. Studies on the systemic use of beta-carotene provide evidence that 15-30 mg/d over a period of about 10-12 wk produces a protective effect against UV-induced erythema. Similar effects have been attributed to mixtures of carotenoids or after long-term intake of dietary products rich in carotenoids. Supplementation with carotenoids contributes to basal protection of the skin but is not sufficent to obtain complete protection against severe UV irradiation.