Phototoxic assessment of a sunscreen formulation and its excipients: An in vivo and in vitro study

Accepted Manuscript Phototoxic assessment of a sunscreen formulation and its excipients: An in vivo and in vitro study

Bryan Hudson Hossy, Alvaro Augusto da Costa Leitão, Elisabete Pereira dos Santos, Monique Matsuda, Laura Barros Rezende, Janine Simas Cardoso Rurr, Alicia Viviana Pinto, Marcia Ramose-Silva, Marcelo de Pádula, Nádia Campos de Oliveira Miguel PII: DOI: Reference:

S1011-1344(17)30477-3 doi: 10.1016/j.jphotobiol.2017.06.043 JPB 10904

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

10 April 2017 27 June 2017 28 June 2017

Please cite this article as: Bryan Hudson Hossy, Alvaro Augusto da Costa Leitão, Elisabete Pereira dos Santos, Monique Matsuda, Laura Barros Rezende, Janine Simas Cardoso Rurr, Alicia Viviana Pinto, Marcia Ramos-e-Silva, Marcelo de Pádula, Nádia Campos de Oliveira Miguel , Phototoxic assessment of a sunscreen formulation and its excipients: An in vivo and in vitro study, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.06.043

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ACCEPTED MANUSCRIPT Phototoxic assessment of a sunscreen formulation and its excipients: an in vivo and in vitro study

Bryan Hudson Hossy1 Alvaro Augusto da Costa Leitão2 Elisabete Pereira dos Santos3 Monique Matsuda4 Laura Barros Rezende5,6 Janine Simas Cardoso Rurr2 Alicia Viviana Pinto7 Marcia Ramos-e-Silva1# Marcelo de Pádula5# Nádia Campos de Oliveira Miguel6#(*)

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Running title: Phototoxic effect of sunscreen excipients

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Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil;

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1 – Programa de Pós-Graduação em Clínica Médica, Faculdade de Medicina – Serviço de Dermatologia,

2 – Laboratório de Radiobiologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade

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Federal do Rio de Janeiro, Rio de Janeiro, Brasil;

3 – Departamento de Medicamentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

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4 - Laboratório de Investigação em Oftalmologia (LIM-33), Universidade de São Paulo, Faculdade de Medicina, São Paulo, Brasil;

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5 – Laboratório de Microbiologia e Avaliação Genotóxica, Departamento de Análises Clínicas e

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Toxicológicas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; 6 – Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro Federal, Rio de Janeiro, Brasil.

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Brasil;

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7 – Instituto Nacional de Controle de Qualidade em Saúde, Fundação Oswaldo Cruz, Rio de Janeiro,

ACCEPTED MANUSCRIPT ABSTRACT Background: Cosmetic preservatives are used to protect cosmetic formulations and improve its shelf-life. However, these substances may exert phototoxic effects when used under sunlight. Objective: To assess safety, efficacy and putative phototoxic effects of a sunscreen formulation SPF 30 and its excipients. Materials/Methods: Irradiation was performed with solar simulated light (SSL) and the sunscreen from

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the School of Pharmacy/UFRJ/Brazil. We used albino hairless mice in different groups (control (G1), only irradiated (G2), sunscreen plus irradiation (G3) and vehicle plus irradiation (G4) for morphological

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assessment and immunefluorescence detection to OKL38. In vitro analyses were with a Saccharomyces

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cerevisiae (SC) strain plus SSL in the presence of methylparaben , propylparaben , imidazolidinyl urea , aminomethyl propanol and their association .

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Results: G3 and G4 displayed photosensitization leading to thickening of the epidermis and increased dermal cellularity. G4 displayed strong OKL38 labeling when compared with other groups. Aminomethyl

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propanol, methylparaben and propylparaben are endowed with phototoxic activity against SC. Propylparaben displayed the highest phototoxic effect, followed by excipients association. Conclusions: The sunscreen´s vehicle is endowed with phototoxic activity. Propylparaben was the most

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phototoxic agent, increasing the overall phototoxicity of excipient association, pointing to a critical

Key Words:

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concern regarding vehicle associations intended to cosmetic purposes. Methylparaben, Propylparaben, Aminomethyl propanol, Simulated

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Saccharomyces cerevisiae

*Corresponding author:

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Dr. Nádia Campos de Oliveira Miguel, Associate Professor Programa de Pesquisa em Biologia Celular e do Desenvolvimento Instituto de Ciências Biomédicas, CCS Universidade Federal do Rio de Janeiro – UFRJ Av. Carlos Chagas Filho 373- Bloco F2-008 e F2-026 Ilha do Fundão, Rio de Janeiro, RJ, Brazil CEP-21949-902 fone +55 (21)25426430/ [email protected] CV: http://lattes.cnpq.br/0240414887390653

Solar Light and

ACCEPTED MANUSCRIPT 1 - INTRODUCTION To protect human skin from sunlight adverse effects as sunburn, wrinkles, aging spots, etc, sunscreens have been used as an almost mandatory strategy [1]. As most cosmetics, sunscreen formulations also contain preservatives, which are supposed to maintain product’s integrity, to increase shelf life and to prevent microbial contamination [2] . However, they may induce skin reactions, such as dermatitis and, in certain circumstances; cosmetic excipients can react with sunlight and exert phototoxic,

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photoallergic or both effects on human skin [3].

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The phototoxic phenomenon is described as excitation of a chromophore with subsequent free radical formation which may directly or indirectly result in cell membrane damage, lipid peroxidation,

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DNA damage, etc. Photoallergic reactions are based on free radical formation upon light absorption by a chromophore or its oxidation leading to a new immunogenic hapten, which may sensitize the immune

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system leading to inflammation and cell damage [4, 5]

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To minimize those effects and to promote the rational use of preservatives, health agencies and researchers around the world have been studying preservatives adverse reactions in vivo and in vitro to

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assess their safety and efficacy of them.[2].

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One of the most adopted biological model for in vivo analyses of cosmetics formulations involving safety and efficacy assessment is the albino hairless mice [6]. Seong-Hoon et al. (1997) used its back skin to determine methylparaben (MP) skin permeation [7]. Hossy et al. (2013) observed that a

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sunscreen vehicle, containing MP, propylparaben (PB) and another cosmetic ingredients, was capable to induce morphological alteration (induction of angiogenesis, cell proliferation, and epidermal thickness)

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on the back skin of albino hairless mice when irradiated with simulated solar light (SSL) [8]. Parabens are the most common used preservative in cosmetics, and a number of studies reported its allergenic potential in clinical trials pointing that 1% of the population is sensitive to them [9, 10]. Handa et al. (2006) displayed methylparaben (MP) photosensitizing effects in vitro using human skin keratinocytes and UVB as light source. MP was capable to induce oxidative stress, nitric oxide production, cellular lipid peroxidation and cell death [11]. Okamoto et al. (2008) showed that when MP was exposed to sunlight irradiation two photoproducts are formed (p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH)).

ACCEPTED MANUSCRIPT Although these molecules were inactive in an in vitro DNA damage assay, an active metabolite from MP3OH was capable to generate oxidatively-induced DNA damage [12] . The propylparaben (PB) toxic effects were studied by Martín et al. (2010) in vitro using kidney cells from a green monkey. The researchers observed cytotoxicity and genotoxicity in addition to cell proliferation decline, DNA breaks and oxidative damage [13].

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Free radical induction by UV light or by photoreactive substances play a role in the connective tissue degradation, increasing neutrophils and macrophages cell number in skin leading to skin

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inflammation and photodamage [14, 15]. It is possible to monitor this phenomenon using the OKL 38

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expression. This biomarker (a tumor suppressor gene that inhibits tumor cell growth) has been involved in DNA damage by oxidative processes mostly associated with inflammatory profiles [16]. OKL38 is

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expressed in all tissues regulating the differentiation and proliferation of normal cells, including the regulation of the cell death. The expression of OKL 38 is a p53 dependent marker for mitochondrial

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structure, and function, oxidative stress and apoptosis (with cytochrome C release) [17-18] Here, we conduct an in vitro and in vivo study to assess phototoxicityy and photosensitization induced by a vehicle sunscreen formulation, its preservatives (MP, PB, imidazolidinyl urea (IU)) and pH

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adjusting agent aminomethyl propanol (AMeP).

2 - MATERIALS AND METHODS

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2.1 - Animals

Male adult albino hairless mice (HRS /J - UFRJ) (n = 36), 12 weeks old, were from the colony of

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the Faculty of Pharmacy of the Federal University of Rio de Janeiro – UFRJ). The animals were maintained in cages at room temperature (25-28°C) and a constant 12/12 h photoperiod, under artificial light. The animals received food and water ad libitum. They were placed in groups (control (G1), only irradiated (G2), sunscreen + irradiation (G3) and vehicle + irradiation (G4) according to Hossy et al. (2013) [8]. This study was approved by the Evaluation Committee for Use of Animals in Research of the Center for Health Sciences of UFRJ, DAHEICB, protocol 65. 2.2 - Sunscreen formulation We used a sunscreen formulation (SPF 30 determined by in vitro and in vivo methods thermostable and photostable tests [19, 20] from Faculty of Pharmacy, UFRJ, Brazil (lot number 69 in

ACCEPTED MANUSCRIPT gel cream galenic) on albino hairless mice back side to study the photoprotection profile (semithin for optical microscopic routine and immunofluorescence). This formulation contents only chemical filters (UVA and UVB protection spectrum) (9% octocrylene (Galena, Brazil), 8 % benzophenone 3 (Farmos, Brazil) and 9%octyl methoxycinnamate (Mapric, Brazil).We also studied morphological changes to the sunscreen vehicle formulation (see [8]).

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2.3 - Simulated Solar Light (SSL)

The SSL irradiation was performed using a Solar Light Simulator (Oriel Model 91192, 1000W;

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Newport Corp., USA) emitting 104.0 J.m-2. s-1 (92.63%) UVA and 8.15 J.m-2.s-1 UVB (7.26%).

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Atmospheric attenuators AMO and 81017 were used, resulting in a final UVB/UVA emission ratio of 1/5. To determine the total doses of UVA and UVB, a Model VLX-3-W dosimeter (Vilber Lourmat, France)

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was used, with appropriate photocells. The SSL was at a distance of 40 cm from the receptor base. The SSL conditions were as previously described [8]. The protocol used in our study mimics the

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characteristics of a sunny summer day in Rio de Janeiro at 12:00 h (22.9° S and 043.17° W) [8].

2.4 - Skin photodamage assay

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2.5 - Histological Analyses

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The irradiation conditions used in this study were describe before [8].

One week after the last exposure to SSL, the animals were deeply anesthetized until death with

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ketamine (0.1 mg/mL) (União Química Farmacêutica Nacional, Brazil) and xylazine (0.2 mg/mL) (Vetbrands, Brazil). Then the back skin was completely removed [8].

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The fragments of the skin were first fixed by paraformaldehyde 4% immersion for 10 minutes. Thereafter, they were immersed in a solution containing 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) over night. The next day, the biological material were rinsed in sodium cacodylate buffer and post fixed for 1 hour in 1% osmium tetroxide in cacodylate buffer plus 1% potassium ferricyanide and 5 mM calcium chloride for 1:30 hour. Then, the biological samples were rinsed again in sodium cacodylate buffer and stained en bloc with 1% uranyl acetate in water overnight. Subsequently, samples were dehydrated with a series of acetone solutions at increasing concentrations (70%, 80%, 90% and 100%) and embedded in Poly/Bed 812 resin (Ted Pella, Inc.). The semithin sections (0.5 μm) were mounted on glass slides, stained with Toluidine Blue (TB) 1% , and observed under a Zeiss

ACCEPTED MANUSCRIPT Axioskop 2 Plus microscope (Carl Zeiss, Baltimore, Maryland, USA), and photographed with a Zeiss Axiocam MRC camera, using the Axiovision program, version 4.5 (Zeiss) for image acquisition [21, 22]. 2.6 - Immunofluorescence analyses of the OKL38 The scarified conditions, skin biopsies and fixation were the same as previously described [8]. The tissue cross sections (7.0 µm thick ) were made by Leica CM 3000 cryostat, mounted on poly-L-

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lysine- (Sigma, USA) coated slides and stored at -20°C until processing [23]. For immunodetection of OKL38 by immunofluorescence, we used the same protocol as before

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[8] to prepared the sections for the primary antibody incubation (1: 200, Santa Cruz USA; ‘‘polyclonal

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Goat Anti-Human OKL38 (G16)’’code sc-82002; 200 µg in 1 ml of BSA with < 0,1% sodium azide and 0,1% gelatin) dilutes in PBS overnight at 4º C. Further processing was done with a specific fluorescent

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secondary antibody (1:400 donkey anti-goat IgG-FITC: sc-2024, Santa Cruz, USA) for 2 h at room temperature. Next, the preparations were mounted in mounting medium (Fluoromount Aqueous Mounting

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Medium; (Sigma Aldrich Corp.) and observed under the confocal fluorescence microscope (Leica TCSSP5; Leica Microsystems) at 526-nm wavelength of excitation for the fluorochrome used using a 40x objective. All conditions for image acquisition were those described by Miguel et al. (2012) [23].

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Negative and positive control sections for the reactions were prepared (data not shown).

2. 7 - Assay using S. cerevisiae

2.7.1 - Preservatives and alkalizing agent

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We evaluated, singly, each preservative from the sunscreen formulation (Methylparaben (MB) (0.1%) (Pharma Nostra, Brazil), Propylparaben (PB) (0.1%) (DEG, Brazil), Imidazolidinyl urea (IU)

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(0,2%) (Fagron, Brazil)) and the alkalizing agent (Aminomethyl propanol 95 % (AMeP) (0.3%), (Mapric, Brazil)) and association (ASC) (MP, PB, IU and, AMeP) and their phototoxic effects on Saccharomyces cerevisiae cells.

2.7.2 - Yeast Strain, Media and Growth Conditions Saccharomyces cerevisiae FF18733 cells (MAT a, leu2-3–112, trp1-289, his7-2, ura3-52, lys11) were grown in YPD medium (1% yeast extract, 1%bacto peptone, 2% glucose with 2 % agar for plates) or YNBD (yeast nitrogen base-dextrose) medium (2% glucose, 0,7% yeast nitrogen base without amino acids with 2% agar for plates) supplemented with apposite amino acids. Supplemented YNBD

ACCEPTED MANUSCRIPT medium without arginine but containing canavanine (Sigma-Aldrich, USA) (60 mg/mL) was used for the selection of canavanine-resistant (CanR) mutants at 30ºC [24].

2.7.3 - Cell Treatment with SSL and excipients Cell growth conditions were previously described [25]. Briefly, FF18733 cells were grown to a cell density approximately 1x108 cells/mL at 30ºC under agitation to stationary phase (~48). After that

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cells were rescued by centrifugation, washed twice and resuspended in sterile ultrapure water (SWU). Cells (1x107 cells/mL) were exposed to SSL for increasing times (to doses ranging from 0 to 561.6 kJ/m2

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for UVA and 0 to 44 kJ/m2 for UVB) under agitation with different treatments: MP 0.1%, PB 0,1%, IU

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0,2%, AMeP 0,3% and their association in a glass petri dish (0.5 cm diameter and SWU final volume 10 mL). During irradiation aliquots were taken after each time interval and plated on YPD and YNBD CanR

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medium to determine cell survival and induced mutation frequencies (IMF) at 30ºC. Colonies were scored after 72 hours. For yeast treatment, irradiation took place at a 15ºC room temperature. Cell suspension

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was initially at 20ºC (monitored with a clinical thermometer). At the end of SSL irradiation (longest treatment) cell suspension attained 28ºC, avoiding heat shock to yeast. The experiments were performed at least in triplicate.

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For comparison of IMF, treatments were standardized to equivalent number of cell survivors at

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2.8 - Statistical analyses

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37%, which correspond to one lethal hit per cell, maximizing mutagenic events [25, 26].

For immunofluorescence, statistical analyses were performed by means of a Kruskal–Wallis test

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plus Dunn’s multiple compressions. Significance was established at p<0.001 using GraphPad 247 Prism version 5.00 from GraphPad Software, Inc., USA. In vitro experiment statistical analyses were performed ANOVA and Kruskal – Wallis test to evaluate statistical differences (p<0.05), using the free statistical software R version 2.15.3.

3 - RESULTS 3.1 - Histological Analyses

ACCEPTED MANUSCRIPT According to images we observed the G1 (control) group displayed an epidermis with up to 3 keratinocytes layers and a dermis with normal aspects (Fig. 1a). The G2 (irradiated) group showed thickening of the epidermis and dermis inflammatory infiltrate (Fig. 1b). The G3 (sunscreen + SSL exposed) group displayed the same aspect as the G1control groups indicating the photoprotective effect of the SPF formulation (Fig. 1c). The G4 (vehicle + SSL exposed) group presented thickening of the

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epidermis and dermis inflammatory infiltrate (Fig. 1d) in higher extent than the G2 group.

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3.2 - Immunofluorescence technique for the OKL 38

The G2 (Fig. 1f) and G4 (Fig. 1h) groups displayed stronger OKL 38 expression than the G1

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control group (Fig. 1e) and group G3 (Fig. 1g). The results obtained for the G4 group showed difference in comparison to the groups G1, G2 and G3 (p<0.001) indicating that the vehicle was capable to induce

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OKL38 expression, through mitochondrial cell damage by oxidative stress. The G2 groups displayed statistical difference between G1 and G3 at p<0,001. Between the groups G1 and G3 no significant

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differences in OKL 38 expression were observed (Fig. 2).

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3.3 - Phototoxicity analyses

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MB, PB, AMeP and ASC groups displayed phototoxic effects when compared to the control group (yeast cells irradiated with SSL in the absence of any preservative or alkalizing agents) (Fig. 3). IU did not show phototoxic effects (data not shown). PB displayed the highest phototoxic effect, leading to

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absolute no cell survivors after 17’55’’ of SSL irradiation. MP, AMeP and ASC displayed phototoxic effects to a lesser extent than PB alone. Cell survival was dramatically lowered (p<0.05) when exposed to

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high doses of SSL (71’25’’, i.e. 445.6 kJ/m2 of UVA and 34.9 kJ/m2 for UVB). ASC, after PB alone, was the second most phototoxic treatment, pointing to the intrinsic phototoxicity conferred by the presence by PB itself. (Fig. 3).

3.4 - Excipients mutagenicity and photoinduced mutagenicity In our conditions, none of the preservatives were mutagenic to yeast cells when incubated for 2 hours in the absence of light. Also, they were not more photomutagenic upon exposure to SSL when compared to SSL-induced mutagenesis alone (p<0,05) . .

ACCEPTED MANUSCRIPT 4- DISCUSSION Cosmetic adverse reactions, as contact dermatitis, photoallergic or phototoxic effects may take place when human skin is exposed to a variety of cosmetic ingredients, and parabens are known for such problems [27]. The term Cosmetovigilance was firstly used in France, in the 90’s to help researchers and health

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agencies to assess safety, undesirable effects and the quality of cosmetic products, etc. [28]. Reports from dermatologists, pharmacists and consumers had been shown that facial care, body care, perfumes and eye

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care products can cause allergic or irritant contact dermatitis [29]. However, when solar light interacts

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with a cosmetic product and the skin phototoxic or photoallergic reactions may be induced [3]. When we irradiated the albino hairless mice back skin covered with vehicle sunscreen

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formulation, the morphological aspect was worse than that of the other groups. The skin became thicker, and the cellularity of the dermis was higher than in the control group. Nowadays, parabens may represent

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the main putative species at the origin of phototoxic effects involving cosmetics [11]. Here, we conducted an in vivo and in vitro study focused on vehicles, specifically on MP, PB, IU

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and AMeP phototoxic assessment. In vivo analyses were performed using two techniques: (i) routine

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histological semithin sections and (ii) immunofluorescence for OKL38. The morphological and immunofluorescence analyses corroborated those of Hossy et al. (2013) [8]. We observed that the sunscreen formulation FPS 30 used was effective for albino hairless mice photoprotection and the vehicle

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was phototoxic [8].

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Potential phototoxic compounds can induce selective damage to some cell structures such as the nucleus, plasma membrane and organelles [30, 31]. Free radical generation is a common route that leads to cell damage in phototoxic and photoallergic mediated reactions [3]. p53-dependent expression of OKL38 has been shown to be involved in mitochondrial dysfunction and the increase of cellular levels of reactive oxygen species; and there is a close interaction between p53 and OKL 38 in regulating mitochondrial structure and function [17]. We used OKL38 to measure oxidative stress induced among the groups, and we observed that the group 4 (vehicle + SSL exposure) induced strong expression of OKL38 indicating photooxidative effects of the vehicles. These were not observed in the groups 1 and G3 (with sunscreen). Thus the sunscreen formulation could protect the skin against the possible harmful

ACCEPTED MANUSCRIPT effects of the vehicle, which may still be occurring in some extent in the formulation, and ultimately demand expression of cell antioxidant defenses [32]. In fact, although effective, sunscreen formulation may not be unequivocally considered safe. The induction of petite mutants was also monitored in yeast cells treated with vehicles plus SSL. However, no significant increase was observed in comparison with the control group (data not shown).

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This slight increase does mean that mitochondria is not suffering phototoxic effects, as yeast is known to contain high level of inducible antioxidants that are able to protect against quite a number of oxidative

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species [33, 34]

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Since 1968 alterative cosmetic testing methodologies have been used to reduce animals use and suffering. Skin culture, Ames test, reconstituted skin culture, Franz cell, mammalian cells (keratinocytes

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or others) and microorganisms as Escherichia coli and Saccharomyces cerevisiae compose the alternative test arsenal in cosmetic and university laboratories around the world [35].

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To study the phototoxic effects of vehicle ingredients from our formulation we used Saccharomyces cerevisiae cells. They are widely used to study effects on DNA, mutagenesis, aging

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mechanisms and safety and efficacy of a series of substances including cosmetic ingredients such as

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preservatives, colorants, perfumes, photoprotectors and formulations, etc. [25, 26, 36, 37]. PB was found to be the most phototoxic excipient according to our assay, followed by ASC, MB and AMeP. Ahn et al. (2005) showed in in vitro study that PB is more cytotoxic than other parabens,

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triclosan and phenoxyethanol [38]. Martín et al. (2010) showed that PB caused changes in cell proliferation rates, cell viability, oxidative cell damage and DNA double-strand breaks in kidney

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epithelial cells (Vero) derived from an African green monkey [13]. Our study indicated that PB contributes, in a great extent, to the phototoxic effect of ASC, which can be partially attenuated by the presence of the other three substances, AMeP, MP and IU, that are also competing for light absorption. A series of studies have been performed in cosmetic science to observe MP side effects [10-12]. Handa et al. (2006) studied MP phototoxic effects in keratinocytes using UVB (0,15 or 0,3kJ/m²) as light source. They exposed cells for 24h with MP, followed by UVB irradiation, and then further cultured for another 24h. They observed particular deleterious effects such as cell death [11]. Here, we could observe

ACCEPTED MANUSCRIPT that the MP is phototoxic at equivalent 30 and 90 kJ/m² (UVB) exposure, but with SSL. Although our total UVB dose was higher than that used by Handa's group, our conditions denote the exposure of a sunny summer day in Rio de Janeiro at noon (see Material and Methods). Our protocol allowed the detection of MP-induced phototoxic effects within a few minutes in the actual conditions in which sunscreens are used. The AMeP is widely used as pH adjusting cosmetic agent. Although it displayed phototoxic

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effect, it was not photomutagenic. AMeP toxicology studies demonstrated in a skin sebum model, that it could penetrate, react and accumulate above the sebum layer [39]. In 1974, the Cosmetic Toiletry, and

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Fragrance Association (CTFA) studied the AMeP primary irritation in rabbits. It showed that two

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formulations containing AMeP (0,26%) induced erythema, desquamation and edema after 72 h exposure [40]. Ball et al. (2014) showed that the AMeP embryotoxicity by oral but not upon dermal exposure.

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AMP displayed an increased postimplantaion loss in rats, uterine histological alterations that resulted in lowered ability of the uterus to support an implanted embryo, but no carcinogenic or mutagenic effect

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[41].

As sunscreen formulation spreading on the skin is not homogeneous, photosensitive molecules

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that are present in these formulations may be available to start phototoxic reactions. FPS determination in

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silico has been preconized to help cosmetic developers to understand how the distribution of sunscreens actually occurs on a given surface [42].

Using Saccharomyces cerevisiae, we were able to determine both the different degrees of

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phototoxicity of the substances alone as well as the substances in association among each other. This confirms again the usefulness of Saccharomyces cerevisiae as an important tool for preclinical studies

[43-48].

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and the effective screening for safer and more suitable substances that can be used in cosmetic industry

PB cannot be used alone as a preservative system in a cosmetic formulation at low exposition time to SSL because it may be phototoxic. In our experiments, also the association of preservatives ingredients displayed a phototoxic profile but less than each ingredient alone. The results obtained in vitro studies are in line with this, and thus can at least in part reduce animal use and suffering in phototoxicity testing.

ACCEPTED MANUSCRIPT 5-ACKNOWLEDGMENTS: This study was funded by CNPq – Brazil and FAPERJ – Rio de Janeiro – Brazil. B.H.H. received a scholarship from CAPES (2013-2014) and FAPERJ (2015-2016). 6 - CONFLICT OF INTEREST: None

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Figure 1: The albino hairless mouse back skin images showing histological analyses (a, b, c and d) and immunofluorescence for the OLK38 (e, f, g and h) beyond of different experimental groups G1 (a and e), G2 (b and f), G3 (c and g) and G4 (d and h). The histological sections showing epidermal thickness (black arrow) and dermal inflammatory infiltrate (white dotted square), immunofluorescence for the von OKL38 factor (white arrowhead). Bars 20 µm.

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Figure 2: Statistical analyses of OKL 38 immunohistochemistry labeling among different groups (G1, G2, G3 and G4). (*) p<0,001.

Figure 3: Survival of yeast cells to SSL alone () and in the presence of preservatives (MP () and PB

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(■)), alkalizing agent (AMeP (○)) alone and to their association (▼). *, different from control (p<0,05).

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temperature did not exceed 30oC (see materials and methods)

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**, different from control and between each other (p<0,05). During SSL exposure, cell suspension

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 Studies in vivo and in vitro results revealed that sunscreen´s vehicle is endowed with phototoxic activity.  Sunscreen excipients display phototoxic risk  -Propylparaben is the strongest phototoxic agent

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 Phototoxic mechanism involves oxidative stress