Effects associated with exposure to the emerging contaminant octyl-methoxycinnamate (a UV-B filter) in the aquatic environment: a review.

Given the increasing concern surrounding ultraviolet (UV) radiation-induced skin damage, there has been a rise in demand for UV filters. Currently, UV-filters are considered emerging contaminants. The extensive production and use of UV filters have led to their widespread release into the aquatic environment. Thus, there is growing concern that UV filters may bioaccumulate and exhibit persistent properties within the environment, raising several safety health concerns. Octyl-methoxycinnamate (OMC) is extensively employed as a UV-B filter in the cosmetic industry. While initially designed to mitigate the adverse photobiological effects attributed to UV radiation, the safety of OMC has been questioned with some studies reporting toxic effects on environment. The aim of this review to provide an overview of the scientific information regarding the most widely used organic UV-filter (OMC), and its effects on biodiversity and aquatic environment.

Triplet stabilization for enhanced drug photorelease from sunscreen-based photocages.

Recently, sunscreen-based drug photocages have been introduced to provide UV protection to photoactive drugs, thus increasing their photosafety. Here, combined experimental and theoretical studies performed on a photocage based on the commercial UVA filter avobenzone (AB) and on the photosensitizing non-steroidal anti-inflammatory drug ketoprofen (KP) are presented unveiling the photophysical processes responsible for the light-triggered release. Particular attention is paid to solvent stabilization of the drug and UV filter excited states, respectively, which leads to a switching between the triplet excited state energies of the AB and KP units. Most notably, we show that the stabilization of the AB triplet excited state in ethanol solution is the key requirement for an efficient photouncaging. By contrast, in apolar solvents, in particular hexane, KP has the lowest triplet excited state, hence acting as an energy acceptor quenching the AB triplet manifold, thus inhibiting the desired photoreaction.

Substitution effect on the nonradiative decay and trans → cis photoisomerization route: a guideline to develop efficient cinnamate-based sunscreens.

Cinnamate derivatives are very useful as UV protectors in nature and as sunscreen reagents in daily life. They convert harmful UV energy to thermal energy through effective nonradiative decay (NRD) including trans → cis photoisomerization. However, the mechanism is not simple because different photoisomeirzation routes have been observed for different substituted cinnamates. Here, we theoretically examined the substitution effects at the phenyl ring of methylcinnamate (MC), a non-substituted cinnamate, on the electronic structure and the NRD route involving trans → cis isomerization based on time-dependent density functional theory. A systematic reaction pathway search using the single-component artificial force-induced reaction method shows that the very efficient photoisomerization route of MC can be essentially described as "1ππ* (trans) → 1nπ* → T1 (3ππ*) → S0 (trans or cis)". We found that for efficient 1ππ* (trans) → 1nπ* internal conversion (IC), MC should have the substituent at the appropriate position of the phenyl ring to stabilize the highest occupied π orbital. Substitution at the para position of MC slightly lowers the 1ππ* state energy and photoisomerization occurs via a slightly less efficient "1ππ* (trans) → 3nπ* → T1 (3ππ*) → S0 (trans or cis)" pathway. Substitution at the meta or ortho positions of MC significantly lowers the 1ππ* state energy so that the energy barrier of IC (1ππ* → 1nπ*) becomes very high. This substitution leads to a much longer 1ππ* state lifetime than that of MC and para-substituted MC, and a change in the dominant photoisomerization route to "1ππ* (trans) → C[double bond, length as m-dash]C bond twisting on 1ππ* → S0 (trans or cis)". As a whole, the "1ππ* → 1nπ*" IC observed in MC is the most important initial step for the rapid change of UV energy to thermal energy. We also found that the stabilization of the π orbital (i) minimizes the energy gap between 1ππ* and 1nπ* at the 1ππ* minimum and (ii) makes the 0-0 level of 1ππ* higher than 1nπ* as observed in MC. These MC-like relationships between the 1ππ* and 1nπ* energies should be ideal to maximize the "1ππ* → 1nπ*" IC rate constant according to Marcus theory.

Sodium cationization can disrupt the intramolecular hydrogen bond that mediates the sunscreen activity of oxybenzone.

A key decay pathway by which organic sunscreen molecules dissipate harmful UV energy involves excited-state hydrogen atom transfer between proximal enol and keto functional groups. Structural modifications of this molecular architecture have the potential to block ultrafast decay processes, and hence promote direct excited-state molecular dissociation, profoundly affecting the efficiency of an organic sunscreen. Herein, we investigate the binding of alkali metal cations to a prototype organic sunscreen molecule, oxybenzone, using IR characterization. Mass-selective IR action spectroscopy was conducted at the free electron laser for infrared experiments, FELIX (600-1800 cm-1), on complexes of Na+, K+ and Rb+ bound to oxybenzone. The IR spectra reveal that K+ and Rb+ adopt binding positions away from the key OH intermolecular hydrogen bond, while the smaller Na+ cation binds directly between the keto and enol oxygens, thus breaking the intramolecular hydrogen bond. UV laser photodissociation spectroscopy was also performed on the series of complexes, with the Na+ complex displaying a distinctive electronic spectrum compared to those of K+ and Rb+, in line with the IR spectroscopy results. TD-DFT calculations reveal that the origin of the changes in the electronic spectra can be linked to rupture of the intramolecular bond in the sodium cationized complex. The implications of our results for the performance of sunscreens in mixtures and environments with high concentrations of metal cations are discussed.

Survey of cytotoxic and UV protection effects of biosynthesized cerium oxide nanoparticles.

Cerium oxide nanoparticles (CeO2 NPs) are among the important nanoparticles that are extensively utilized in cosmetics, automotive industries, ultraviolet (UV) filtration, gas sensors, and pharmaceutical products. In this study, CeO2 NPs were synthesized using an aqueous extract of Ziziphus jujube fruit. The synthesized nanoparticles were characterized using UV-visible spectroscopy, powder X-ray diffraction, Fourier transform infrared spectroscopy, energy-dispersive spectroscopy, field energy scanning electron microscopy, and Raman methods. The results indicated that the size of synthesized nanoparticles is between 18 and 25 nm, and they have a spherical shape. UV absorbance of the synthesized nanoparticles was measured through spectrophotometric method in the range of 290 to 320 nm. The cytotoxic activity of synthesized CeO2 NPs against colon (HT-29) cancer cell line was surveyed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that synthesized nanoparticles are nontoxic on HT-29 cells under 400 µg/mL concentrations after 24 hours of treatment time periods. The increase in treatment time cases increases cytotoxic activity of synthesized nanoparticles. Sun protection factor of CeO2 NPs, as a criterion for amount of sunlight radiation protection, was determined by applying Mansur equation. The results demonstrated that synthesized CeO2 NPs have excellent UV protection and sunscreen physical absorption properties.

The Influence of Short-Wave and Long-Wave Radiation Spectrum on the Photostability of Sunscreens.

BACKGROUND: Sunscreen products aim to help protect the skin against UV radiation and consequently reduce the risk of early skin ageing and skin cancer. However, it is well known that some sunscreen ingredients are not photostable, but this usually refers to irradiation with UV light. Moreover, it has to be mentioned that a relative cumulative erythema effectiveness compliant light source is used for the in vivo sun protection factor (SPF) testing. Here, UV simulators equipped with a xenon arc lamp use filters such as WG320 and UG11 (thickness 1 mm) to minimize infrared (IR) radiation and wavelength below 300 nm. However, under practical conditions, the sunscreen product is not only exposed to UVA/B light, but also to visible light (VIS) and IR light. In fact, the spectrum of solar radiation is composed of approximately 7% UV, 39% VIS and 54% IR. AIMS: To investigate the influence of short-wave and long-wave radiation on the photostability of sunscreens. METHODS: Irradiation was performed with the Suntest CPS+ that is considered to closely imitate solar radiation. The filter UG11 (thickness 1 mm), which absorbs much of the VIS and IR light, and the glass filter WG320 (thickness 2 mm), which effectively absorbs radiation of wavelengths less than 300 nm, were used in the Suntest CPS+ both individually and in combination and were inserted between the light source and the samples. The following transmission measurements were carried out with Labsphere's UV-2000s device. Here, the effectiveness (percentage change of SPF before irradiation to SPF after irradiation) as a measure of the photostability was calculated. RESULTS: As expected after total solar spectrum irradiation, the effectiveness in all tested sunscreens is lower compared to relative cumulative erythema effectiveness light used for in vitro testing of SPF. In the reference sunscreen formula S2 as well as in the two different sunscreen products, especially long-wave radiation (>400 nm) had an effect on photostability, whereas short-wave radiation had only a minor impact. In contrast, in the BASF sun care gel line only short-wave radiation below 300 nm had an effect on photostability, and blocking VIS and IR light had no effect at all. CONCLUSION: Based on these data, we can conclude that short waves and/or VIS + IR light have an influence on the photostability of sunscreens.

Unravelling the Photoprotective Mechanisms of Nature-Inspired Ultraviolet Filters Using Ultrafast Spectroscopy.

There are several drawbacks with the current commercially available ultraviolet (UV) filters used in sunscreen formulations, namely deleterious human and ecotoxic effects. As a result of the drawbacks, a current research interest is in identifying and designing new UV filters. One approach that has been explored in recent years is to use nature as inspiration, which is the focus of this review. Both plants and microorganisms have adapted to synthesize their own photoprotective molecules to guard their DNA from potentially harmful UV radiation. The relaxation mechanism of a molecule after it has been photoexcited can be unravelled by several techniques, the ones of most interest for this review being ultrafast spectroscopy and computational methods. Within the literature, both techniques have been implemented on plant-, and microbial-inspired UV filters to better understand their photoprotective roles in nature. This review aims to explore these findings for both families of nature-inspired UV filters in the hope of guiding the future design of sunscreens.

Discovery of Novel UV-Filters with Favorable Safety Profiles in the 5-Arylideneimidazolidine-2,4-dione Derivatives Group.

Effective protection from the harmful effects of UV radiation may be achieved by using sunscreens containing organic or inorganic UV filters. The number of currently available UV filters is limited and some of the allowed molecules possess limitations such as systemic absorption, endocrine disruption properties, contact and photocontact allergy induction, and low photostability. In the search for new organic UV filters we designed and synthesized a series consisting of 5-benzylidene and 5-(3-phenylprop-2-en-1-ylidene)imidazolidine-2,4-dione (hydantoin) derivatives. The photoprotective activity of the tested compounds was confirmed in methanol solutions and macrogol formulations. The most promising compounds possessed similar UV protection parameter values as selected commercially available UV filters. The compound diethyl 2,2'-((Z)-4-((E)-3-(4-methoxyphenyl)allylidene)-2,5-dioxoimidazolidine-1,3-diyl)diacetate (4g) was characterized as an especially efficient UVA photoprotective agent with a UVA PF of 6.83 ± 0.05 and favorable photostability. Diethyl 2,2'-((Z)-4-(4-methoxybenzylidene)-2,5-dioxo- imidazolidine-1,3-diyl)diacetate (3b) was the most promising UVB-filter, with a SPFin vitro of 3.07 ± 0.04 and very good solubility and photostability. The main photodegradation products were geometric isomers of the parent compounds. These compounds were also shown to be non-cytotoxic at concentrations up to 50 µM when tested on three types of human skin cells and possess no estrogenic activity, according to the results of a MCF-7 breast cancer model.

Comparison of UVB effects on growth and induction of UVB screening compounds in isolates of metaphytic algae from temperate zone streams and ponds.

Filaments in the surface layers of metaphytic mats are exposed to high photon flux densities of PAR and UVBR. We investigated the effect of UVBR exposure on growth of eight isolates of common metaphytic algae (Cladophora, Mougeotia, Oedogonium, Pithophora, Spirogyra, and Zygnema) acclimated to either high or low PAR levels prior to UVBR exposure. All isolates acclimated to low PAR exhibited significant reductions in growth rate caused by the UVBR exposure (P < 0.05). Acclimation to high PAR resulted in seven of the isolates being more tolerant of the UVB exposure. The two Zygnema isolates exhibited the most pronounced effect of high PAR acclimation with growth rates of UVB exposed treatments being equal to that of controls (P > 0.05). High PAR acclimation also protected chlorophyll a levels in the Zygnema isolates. Absorption of UVB by methanol extracts increased 322%-381% for the two Zygnema isolates when high PAR acclimated. The broad absorption peak at 270 nm suggests that phenolic compounds were responsible. Previous studies have shown that Zygnema isolates from extreme environments tolerate UVBR and contain UVB screening compounds, but our results extend these adaptions to Zygnema from typical temperate zone habitats. Although none of the other metaphytic algae produced UVB absorbing compounds, they all exhibited higher growth rates under UVBR exposure following high PAR acclimation. This suggests that the algae evaluated have inducible defenses against UVBR exposure that coupled with their mat structure would provide an adaption to the challenging light environment in shallow-water habitats.

Theoretical insights into the photo-protective mechanisms of natural biological sunscreens: building blocks of eumelanin and pheomelanin.

Eumelanin (EM) and pheomelanin (PM) are ubiquitous in mammalian skin and hair--protecting against harmful radiation from the sun. Their primary roles are to absorb solar radiation and efficiently dissipate the excess excited state energy in the form of heat without detriment to the polymeric structure. EU and PM exist as polymeric chains consisting of exotic arrangements of functionalised heteroaromatic molecules. Here we have used state-of-the-art electronic structure calculations and on-the-fly surface hopping molecular dynamics simulations to study the intrinsic deactivation paths of various building blocks of EU and PM. Ultrafast excited state decay, via electron-driven proton transfer (in EU and PM) and proton-transfer coupled ring-opening (in PM) reactions, have been identified to proceed along hitherto unknown charge-separated states in EU and PM oligomers. These results shed light on the possible relaxation pathways that dominate the photochemistry of natural skin melanins. Extrapolation of such findings could provide a gateway into engineering more effective molecular constituents in commercial sunscreens--with reduced phototoxicity.

BB creams and their photoprotective effect.

BB creams appeared on the market quite recently. These creams, which give a perfect complexion by covering up the skin's blemishes, have a photoprotective effect in the majority of cases. An SPF value ranging from 10 to 45 concerning the products we tested is displayed on the packaging. The 21 commercially-available BB creams were tested to assess their efficacy (determination of the SPF) and their photostability (determination of their efficacy after UV irradiation). It was shown that 70% of the products tested have an SPF determined in vitro by us which matches the SPF displayed on the product. For the remaining 30%, it can be seen that products have SPF values of between 2 and 10 times lower than those indicated on the products. It can also be noted that there is a large disparity in terms of photostability since, under the same experimental conditions, however, some products only lose 5% of their photoprotective efficacy, whereas others lose 60%.

Photon Harvesting in Sunscreen-Based Functional Nanoparticles.

The ultraviolet light component in the solar spectrum is known to cause several harmful effects, such as allergy, skin ageing, and skin cancer. Thus, current research attention has been paid to the design and fundamental understanding of sunscreen-based materials. One of the most abundantly used sunscreen molecules is Avobenzone (AB), which exhibits two tautomers. Here, we highlight the preparation of spherically shaped nanoparticles from the sunscreen molecule AB as well as from sunscreen-molecule-encapsulated polymer nanoparticles in aqueous media and study their fundamental photophysical properties by steady-state and time-resolved spectroscopy. Steady-state studies confirm that the AB molecule is in the keto and enol forms in tetrahydrofuran, whereas the enol form is stable in the case of both AB nanoparticles and AB-encapsulated poly(methyl methacrylate) (PMMA) nanoparticles. Thus, the keto-enol transformation of AB molecules is restricted to a nanoenvironment. An enhancement of photostability in both the nanoparticle and PMMA-encapsulated forms under UV light irradiation is observed. The efficient excited energy transfer (60 %) from AB to porphyrin molecules opens up further prospects in potential applications as light-harvesting systems.

Photochemical degradation of the UV filter octyl methoxycinnamate in solution and in aggregates.

The photodegradation of the ultraviolet (UV) filter octyl methoxycinnamate (OMC) is investigated in both dilute solution and in aggregated form. In dilute solution, the ratio of trans and cis isomers achieved at the photostationary state is solvent-dependent because of variations in the isomerization quantum yield. The two isomeric forms at the photostationary state are highly resistant to further photodegradation and no other UVA-absorbing species are formed. Aggregation of OMC, either in a neat film or in aqueous colloidal suspensions, leads to irreversible photodegradation of the molecule and the formation of multiple photoproducts. In addition to previously identified photoproducts like the UVB-absorbing cis and trans isomers and photodimers, we find photoproduct species whose absorption extends into the UVA. Characterization of the photophysical properties of these species indicates that they have long-lived excited-states (τf > 1 ns, 400 nm), unlike the isomeric forms of OMC (τf < 30 ps, 266 nm), and that excitation at 405 nm can sensitize the formation of singlet oxygen. These results show that the environment of OMC affects the photochemistry of the molecule and that the environmental conditions must be taken into account when considering the molecule's stability. In particular, aggregation of OMC molecules results in complex photochemistry that can produce species whose absorption extends into UVA and are capable of generating reactive oxygen species.

Sunscreens as a source of hydrogen peroxide production in coastal waters.

Sunscreens have been shown to give the most effective protection for human skin from ultraviolet (UV) radiation. Chemicals from sunscreens (i.e., UV filters) accumulate in the sea and have toxic effects on marine organisms. In this report, we demonstrate that photoexcitation of inorganic UV filters (i.e., TiO2 and ZnO nanoparticles) under solar radiation produces significant amounts of hydrogen peroxide (H2O2), a strong oxidizing agent that generates high levels of stress on marine phytoplankton. Our results indicate that the inorganic oxide nanoparticle content in 1 g of commercial sunscreen produces rates of H2O2 in seawater of up to 463 nM/h, directly affecting the growth of phytoplankton. Conservative estimates for a Mediterranean beach reveal that tourism activities during a summer day may release on the order of 4 kg of TiO2 nanoparticles to the water and produce an increment in the concentration of H2O2 of 270 nM/day. Our results, together with the data provided by tourism records in the Mediterranean, point to TiO2 nanoparticles as the major oxidizing agent entering coastal waters, with direct ecological consequences on the ecosystem.

Structural, energetic, and UV-Vis spectral analysis of UVA filter 4-tert-butyl-4'-methoxydibenzoylmethane.

The growing awareness of the harmful effects of ultraviolet (UV) solar radiation has increased the production and consumption of sunscreen products, which contain organic and inorganic molecules named UV filters that absorb, reflect, or scatter UV radiation, thus minimizing negative human health effects. 4-tert-Butyl-4'-methoxydibenzoylmethane (BMDBM) is one of the few organic UVA filters and the most commonly used. BMDBM exists in sunscreens in the enol form which absorbs strongly in the UVA range. However, under sunlight irradiation tautomerization occurs to the keto form, resulting in the loss of UV protection. In this study we have performed quantum chemical calculations to study the excited-state molecular structure and excitation spectra of the enol and keto tautomers of BMDBM. This knowledge is of the utmost importance as the starting point for studies aiming at the understanding of its activity when applied on human skin and also its fate once released into the aquatic environment. The efficiency of excitation transitions was rationalized based on the concept of molecular orbital superposition. The loss of UV protection was attributed to the enol → keto phototautomerism and subsequent photodegradation. Although this process is not energetically favorable in the singlet bright state, photodegradation is possible because of intersystem crossing to the first two triplet states.

Electromagnetic sunscreen model: implementation and comparison between several methods: step-film model, differential method, Mie scattering, and scattering by a set of parallel cylinders.

Sunscreens protect from UV radiation, a carcinogen also responsible for sunburns and age-associated dryness. In order to anticipate the transmission of light through UV protection containing scattering particles, we implement electromagnetic models, using numerical methods for solving Maxwell's equations. After having our models validated, we compare several calculation methods: differential method, scattering by a set of parallel cylinders, or Mie scattering. The field of application and benefits of each method are studied and examples using the appropriate method are described.

In vitro assessment of commercial sunscreens available in Latin America.

In Latin America, people have largely abandoned the practice of wearing hats and traditional clothing that provided skin protection. Sunscreen application has therefore become essential to protect against the increased sun exposure. The physician-prescribed medical-grade sunscreens provide sufficient sun protection but the requirement for regular use puts a financial burden on the patient that is often not sustainable. An appropriate sunscreen should provide a high and broad ultraviolet (UV) protection against UVB and UVA. Several over-the-counter (OTC) sunscreens have been developed for sale at affordable prices and are available for purchase in convenient locations, such as local grocery stores. The aim of this study was to assess the in vitro UV protection of 34 popular OTC sunscreens found in the Latin American market. UV absorbance/transmittance was quantified by diffusion transmission spectroscopy using coarse silica plaques. Photostability was tested by irradiating them with simulated solar light and calculating the sun protection factor (SPF), critical length of absorption (C lambda ), UVA/UVB ratio, and the spectral uniformity index (SUI). The results indicated that the in vitro SPFs were significantly lower than the value declared on the labels, particularly for those claiming high SPF values; however, the majority of these sunscreens offered high levels of UV protection. Considering the advantages of low cost and ample accessibility, we concluded that this sample of OTC sunscreens can be beneficial to the general public by providing some level of skin protection from solar radiation, and may be promoted to improve compliance with recommended photoprotection behavior.

Disparate SPF testing methodologies generate similar SPFs.

Regulatory agencies throughout the world have developed exclusive methodologies for assessing and classifying sunscreen product efficacy in their respective markets. Three prevalent methods, the Food and Drug Administration-Final Monograph (FDA-FM) method, the Australia/New Zealand (Aus/NZ) method, and the COLIPA International (International) method, contain procedural and statistical dissimilarities with undefined significance. The objective of our clinical trials was to evaluate the influence of these disparities on sun protection factor (SPF) values. Our clinical trials evaluated the SPF of 59 test materials, using two or all three of the aforementioned methods in simultaneous trials, providing two or three SPF values for each formulation. A total of 135 trials were conducted. The consequent mean SPF values generated per trial were used to compare methods in a correlation and variance analysis. The correlation coefficients for each method pair, International vs. FDA-FM, Aus/NZ vs. FDA-FM, and International vs. Aus/NZ, were each ≥0.94. The difference in least square mean SPF for each method pair was 0.12, 0.62, and 0.81, respectively. Our juxtaposition of the mean SPFs produced by these methods clearly illustrate that any disparities between average SPF values produced by these methods are not clinically or statistically significant and that using one method should be sufficient for SPF labeling in all three respective markets.

Biomineralization: A new tool for developing eco-sustainable Ti-doped hydroxyapatite-based hybrid UV filters.

It is well known that the prolonged exposure to UV radiation from sunlight can compromise human health and is particularly damaging to the skin, leading to sunburn, photo-aging and skin cancer. Sunscreen formulations containing UV-filters present a barrier against solar UV and help to mitigate the harmful effects however, concern about their safety for both human and environmental health is still a much-debated topic. EC regulations classify UV-filters depending on their chemical nature, particle size, and mechanism of action. Furthermore, it regulates their use in cosmetic products with specific limitations in terms of concentration (organic UV filters) and particle size and surface modification to reduce their photo-activity (mineral UV filters). The regulations have prompted researchers to identify new materials that show promise for use in sunscreens. In this work, biomimetic hybrid materials composed of titanium-doped hydroxyapatite (TiHA) grown on two different organic templates, derived from animal (gelatin - from pig skin) and vegetable (alginate - from algae) sources. These novel materials were developed and characterized to obtain sustainable UV-filters as a safer alternative for both human and ecosystem health. This 'biomineralization' process yielded TiHA nanoparticles that demonstrated high UV reflectance, low photoactivity, good biocompatibility and an aggregate morphology which prevents dermal penetration. The materials are safe for topical application and for the marine environment; moreover, they can protect organic sunscreen components from photodegradation and yield long-lasting protection.

The Impact of Flavonoid-Loaded Nanoparticles in the UV Protection and Safety Profile of Topical Sunscreens.

Excessive UV radiation exposure is harmful to skin cells since sunburn is accompanied by oxidative burst, leading to a rapid increase in skin cancer. However, the insufficient UV photoprotection of approved sunscreens and the negative impact of their compositions on ecosystems and human health makes the utility of sunscreen a questionable recommendation. Therefore, discovering UV filters with significant antioxidant activity and improved topical performance and photostability is an urgent need. Recently, the use of nanosized natural molecules incorporated in sunscreens has been a scientific hot topic, as it has been suggested that they provide a synergistic effect with synthetic UV filters, improving overall SPF and antioxidant activity, higher retention on the epidermis, and less toxicity. The aim of this review was to verify the usefulness of sunscreens incorporating flavonoid-loaded nanoparticles. A literature review was performed, where original and review articles published in the last 6 years were analyzed. Formulations containing nanosized flavonoids with improved UVA photoprotection and safer toxicological profiles, associated or not with synthetic filters, are promising sunscreens and more clinical investigation must be performed to validate these findings.