Fatma Sahli

Marta Silva

Sousa

Bertrand Vileno

Jutta Lichter

Jean-Pierre Lepoittevin

Brunhilde Blömeke
email: bloemeke@uni-trier.de

Elena Giménez-Arnau

Understanding the skin sensitization

Keywords: Skin sensitization, dendritic cell activation, THP-1 cells, keratinocytes, ascaridole, reactivity, EPRspectroscopy, spin-trapping, reconstructed human epidermis

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment

Fatma Sahli, Marta Silva E. Sousa, Bertrand Vileno, Jutta Lichter, Jean-Pierre Lepoittevin, Brunhilde Blömeke, Elena Giménez-Arnau

To cite this version:

Fatma Sahli, Marta Silva E. Sousa, Bertrand Vileno, Jutta Lichter, Jean-Pierre Lepoittevin, et al.. Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment. Archives of Toxicology, 2019, 10.1007/s00204-019-02444-3. hal-02119633

Introduction

Allergic contact dermatitis (ACD) is the clinically relevant outcome of skin sensitization, one of the most important occupational and environmental health issues associated with chemicals. It is a delayedtype hypersensitivity reaction caused by a wide range of reactive chemicals referred to as contact allergens, natural or synthetic man-made, after repeated contact with the skin (Lepoittevin 2011). ACD is considered the most frequent expression of immunotoxicity in humans [START_REF] Pesonen | Patch test results of the European baseline series among patients with occupational contact dermatitis across Europe - analyses of the European Surveillance System on Contact Allergy network, 2002-2010[END_REF]) and its prevalence is rising worldwide. The risk of skin sensitization resulting from exposure to allergens present in consumer products is a major regulatory issue within industry. It is thus crucial to predict the sensitization potential of chemicals and therefore perform reliable risk assessment before their introduction to the market. In this context, fundamental upstream investigations are required aiming at understanding molecular mechanisms involved in allergic-inflammatory reactions induced by sensitizers to cover all categories of molecules for risk assessment.

Ascaridole (Asc) is considered to be one of the compounds responsible for ACD to tea tree oil (TTO). Fresh TTO is a weak-moderate sensitizer (SCCP 2008) but air exposure leads to a 3-fold increase in its sensitization potential [START_REF] Hausen | Degradation products of monoterpenes are the sensitizing agents in tea tree oil[END_REF]). In the presence of atmospheric oxygen, the TTO constituents α-terpinene, γ-terpinene and terpinolene are oxidized to p-cymene, which is a good measure of TTO oxidative degradation, but oxidation processes also lead to the formation of endoperoxide Asc (de Groot and Schmidt 2016). Asc is classified as a moderate-strong sensitizer by the local lymph node assay with an EC 3 value of 1.8 [START_REF] Rudbäck | α-Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure[END_REF]. Also, we studied the elicitation potential of Asc in individuals with eczema and suspected fragrance allergy after topical exposure [START_REF] Christoffers | Co-sensitization to ascaridole and tea tree oil[END_REF]2014). The finding that every positive reaction to oxidized-TTO was accompanied by a positive reaction to Asc supported the hypothesis that Asc might be a contact allergen in oxidized-TTO. Furthermore, we studied [START_REF] Krutz | Activation of the endoperoxide ascaridole modulates its sensitizing capacity[END_REF] the capacity of Asc to react with proteins and evaluated its potential to mature monocyte-derived dendritic cell (DC), two key events of chemical-induced sensitization (OECD, 2012). Some reactivity toward cysteine-but not lysinecontaining peptides was observed in the direct peptide reactivity assay (DPRA), the regulatory agencies approved in chemico method to assess the sensitizing potential of chemicals. Treatment of human monocyte-derived DC with Asc supported full DC maturation and induced significant upregulation of co-stimulatory CD86, CD80, CD40 molecules and the adhesion molecule CD54 in a time-dependent manner.

As for sensitizing allylic hydroperoxides derived from autoxidation of terpenes [START_REF] Kao | Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway[END_REF]2014), and from evidence for radical mechanisms as triggers for protein reactivity of endoperoxides [START_REF] Tang | Dispiro-1,2,4-trioxane analogues of a prototype dispiro-1,2,4-trioxolane: mechanistic comparators for artemisinin in the context of reaction pathways with iron (II)[END_REF], a radical mechanism could be proposed for the protein-binding or other steps of the sensitization process to Asc including activation of the innate immune system. In the DPRA, the depletion of the cysteine peptide was much higher when Asc was treated with iron as radical inducer, compared to Asc itself. This activated Asc showed furthermore increased potential for induction of the Nrf2 target gene heme oxygenase 1 and upregulation of CD86 and CD54 on THP-1 cells, an established DC surrogate [START_REF] Krutz | Activation of the endoperoxide ascaridole modulates its sensitizing capacity[END_REF]. Moreover, the high-throughput screening dansyl cysteamine method (HTS-DCYA) applied to Asc and activated Asc with iron (II) revealed the formation of electrophilic species via radical degradation as a potential pathway for the peptide reactivity of aged TTO [START_REF] Chittiboyina | What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization[END_REF]. However, the link between radicals actually formed in the epidermis and their mechanism of action are far from being fully unraveled.

The aim of the present study was to broaden knowledge about the contribution of free radical intermediates derived from Asc to 2 key events in the process of sensitization. We approached this by assessing the reactivity profile towards amino acids prone to radical reactions, by identifying whether Asc derived free radicals are formed in a reconstructed human epidermis 3D model (RHE) which is believed to be closer to what may happen in vivo, and by examination of its properties to activate the immune system, namely dendritic cells (DCs) in their natural context of keratinocytes and in the RHE epidermal skin environment.

Materials and methods

Synthesis of ascaridole (Asc)

Chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) and used as received.

The reactions were followed by TLC (0.25 mm silica gel plates; 60F 254 ). After migration, the plates were inspected under UV light (254 nm) and sprayed with a solution containing phosphomolybdic acid (5 g), cerium (IV) sulfate (2 g) and sulfuric acid (12 mL) in water (188 mL). Column chromatography purification was performed using silica 60 (Geduran, 40-63 µm). 1 H and 13 C NMR spectra were recorded in a Bruker Avance spectrometer at 300 and 75 MHz, respectively. Chemical shifts (δ) are reported in ppm, indirectly referenced to TMS via the solvent signal (CDCl 3 : δ 1 H 7.26; δ 13 C 77.16).

Multiplicities are denoted as s (singlet), d (doublet), hept (heptuplet) and m (multiplet). The spectroscopic data agree with reported literature data [START_REF] Chittiboyina | What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization[END_REF].

A microemulsion was prepared by adding sodium molybdate (2.3 g in 12 mL of distilled water)

dropwise into a suspension of sodium dodecyl sulfate (19 g) in butanol (23 mL) and dichloromethane (133 mL). The microemulsion became clear after 25 min of stirring. A solution of α-terpinene (3.3 g, 24.5 mmol, 1 equiv) in the microemulsion (50 g) was treated at room temperature with a first portion of hydrogen peroxide (35% wt in water, 1 mL). The brick red mixture was stirred for 10 min until it became light yellow. Thirteen additional portions of hydrogen peroxide (35% wt in water, 1 mL each)

were successively added every 10 min. A total of 14 mL of aqueous hydrogen peroxide (35%) was used (160 mmol, 6.5 equiv). The light yellow mixture was stirred overnight at room temperature until it became completely clear. The solvent was removed under reduced pressure and the crude product obtained was dissolved in dichloromethane (100 mL). 3,17.4,21.5,25.7,29.6,32.2,74.5,79.9,133.2,136.5;].

-6-Reactivity with amino acids: general procedure Asc (100 mg, 0.59 mmol) was dissolved in deareated 1:1 (v/v) H 2 O/CH 3 CN (15 mL). The amino acids

(2 equiv) were added together with a catalytic amount (0.1 equiv) of FeSO 4 heptahydrate. The reaction mixtures were continuously stirred at room temperature and kept under argon atmosphere to minimize competing reactions with oxygen. Reactions were monitored by TLC (0.25 mm silica gel plates; 60F 254 ). After migration, TLC plates were inspected under UV light (254 nm) then sprayed with the previously described phosphomolybdic acid solution followed by heating. Reactions were stopped after 7 days if Asc was still present in the reaction mixtures and the reactions did not evolve after that time.

The mixtures were filtered on Celite (Celite 545) in order to remove iron salts and the filtrate solvent was removed in vacuo. Crude products were analyzed by LC-ESI-MS associated with ESI-MS/MS.

LC-ESI-MS/MS analyses were performed using an ion trap mass Agilent QToF spectrometer connected to an Agilent 1200 HPLC system. The samples were subjected to reverse phase chromatography on a Zorbax SB-C18 column (50 mm × 2.1 mm, 1.8 µm particle size) at a flow rate of 0.5 mL/min. They were eluted from the column using a mobile phase A (0.05% formic acid in acetonitrile) and a mobile phase B (0.05% formic acid in H 2 O) with the following gradient: started at 98% B and decreased to 0% B after 8 min, kept at 0% B during 12.5 min, and increased again to 98% B after 12.5 min, kept at 98% B during 13 min at the end. After passing through the diode array absorbance detector, the eluent was directed to the connected ion trap mass spectrometer with a standard electrospray source. The ionization method used was ESI in the positive ion mode (heated capillary temperature 340°C, gas flow 8 L/min, nebulizer gas 30 psi). Full scan mass spectra were acquired in the profile mode scanning m/z 50 to 1000. For the ESI-MS/MS studies, the mass spectrometer was equipped with the ESI source used for ESI-MS studies described above and with the same source parameter settings. The ionization mode was ESI in the positive ion mode. The isolation width was 4 m/z. Data were processed using Agilent MassHunter Qualitative Analysis software. Only molecular ions detected by LC-ESI-MS having an m/z value in accordance with potential reactivity were considered as molecular species provided that they were relatively abundant. Even if unlikely, the eventuality of these ions being fragments of other molecular species cannot be excluded.

EPR studies

EPR spectra were recorded on an EPR X-band spectrometer (ESP300E, Bruker Biospin GmbH, Germany) equipped with a high sensitivity resonator (4119HS-W1, Bruker Biospin GmbH, Germany).

The g calibration was performed using Bruker standard (strong pitch) with known isotropic g factor of 2.0028. The principal experimental parameters values were: microwave power of circa 5 mW, modulation amplitude 1 G, and conversion time of circa 160 ms with 1024 points per spectrum resulting in a sweep time of circa 3 min per scan. Several spectra were accumulated to ensure a good signal-to-noise ratio (S/N). Spectra were recorded at room temperature (295K±1K). All experimental EPR spectra were analyzed by means of computer simulation using labmade scripts based on Easyspin toolbox under Matlab (Mathworks) environment [START_REF] Stoll | EasySpin, a comprehensive software package for spectral simulation and analysis in EPR[END_REF]. Spin-trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) was synthesized as reported in the literature [START_REF] Barbati | Free Radicals in Biology and Environment[END_REF].

For preliminary studies in solution, HEPES buffer (10 mM, pH 6.8) was prepared with 1. EpiSkin TM (Lyon, France) is an ex vivo reconstructed human epidermis (RHE) from normal human keratinocytes cultured for 13 days on a collagen matrix at the air-liquid interface. The small 0.38 cm 2 format was chosen for these studies. Immediately after arrival in the laboratory, the RHE was removed from the agarose-nutrient solution in the shipping multiwell plate under a sterile airflow. It was immediately placed in a plate in which each well was previously filled with 2 mL EpiSkin TM maintenance or growth medium at room temperature. Samples were placed in the incubator at 37 °C, 5% CO 2 and saturated humidity for at least 24 h before incubation. Afterwards, the RHE were treated with DEPMPO in dimethylsulfoxide/HEPES 1:1 (250 mM, 20 µL) and incubated for 15 min (37 °C, 5% CO 2 ). After that time, treated RHE were placed in an EPR tissue cell equipped with a silica window (Willmad, #ER162TC-Q) and Asc (100 mM, 50 mM, 25 mM, 10 mM or 5 mM in acetone, 20 µL) was topically applied to the epidermis taking care to ensure that the solution was only applied to the skin surface. It was necessary to act quickly as the tissue cultures dry out rapidly if not in contact with medium. EPR spectra were then recorded. The principal experimental parameters values were:

microwave power 5 mW, modulation amplitude 1 G, and resulting sweep time of ca. 328 s for a single scan. Up to 10 scans were accumulated to improve the signal-to-noise ratio.

Activation of THP-1 cells cocultured with HaCaT keratinocytes

HaCaT cells were cultured in media (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% antibiotic/antimycotic solution. THP-1 monocytes were established as surrogate for monocyte-derived DC by us [START_REF] Tietze | Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1)[END_REF] and others [START_REF] Bocchietto | Human monocytoid THP-1 cell line versus monocyte-derived human immature dendritic cells as in vitro models for predicting the sensitizing potential of chemicals[END_REF]). THP-1 cells were cultured in RPMI 1640 containing 4 mM L-glutamine, 25 mM HEPES, 50 µM β-mercaptoethanol, 10% FBS and 1% antibiotic/antimycotic solution as we described [START_REF] Hennen | Cross talk between keratinocytes and dendritic cells: impact on the prediction of sensitization[END_REF]Hennen and Blömeke 2017). THP-1 cells (8 × 104 in a 96-well plate) were placed on top of confluent HaCaT keratinocytes and exposed to increasing concentrations of Asc (7.8-1000 µM, 0.2% DMSO) and to 20 µM DNCB (0.2% DMSO) for varying times (0.5 h-48 h). After the indicated time points, the exposure medium (containing the test compound) was replaced by fresh medium without chemicals and cells were further incubated for 24 h or 48 h at 37 °C, 5% CO 2 in a humidified atmosphere. The number of experiments performed for each time point were: n = 1-3 for 0.5 h; n = 3-5 for 1 h; n = 4-5 for 2.5 h and 24 h; n = 2-3 for 4 h and n = 3 for 48 h.

Activation of THP-1 cells cocultured with RHE

Here reconstructed human epidermis SkinEthic TM (Lyon, France) model of 0.5 cm 2 format mounted on a semipermeable membrane was used. This design allowed us to place the THP-1 cells underneath the RHE (the details are described by Schellenberger et al. in revision). After removal of the agarosenutrient solution each of the inserts was placed overnight in a 6-well plate with EpiSkin maintenance medium at 37 °C, 5% CO 2 . THP-1 cells were seeded in the basolateral compartment underneath the RHE model, in a 12-well culture plate containing 500 µL of THP-1 culture medium without βmercaptoethanol. Increasing concentrations of Asc 10 mM, 60 mM, 100 mM and 200 mM, dissolved in acetone-olive oil (AOO, 4:1) were applied on top of the skin models (volume of 6 µL) and incubated

for 24 h at 37 °C and 5% CO 2 in a humidified incubator.

Cell surface expression of CD86 and CD54 on cocultured

Results

Reactivity with amino acids prone to radical reactions

Reactivity towards cysteine, tryptophan and tyrosine (N-acetylated, ester form) was studied. Cysteine is probably the most labile residue involved in radical reactions induced by peroxides. A well-known example is the Fe(II)/Fe(III) induced radical mechanism proposed for the interaction of cysteine with antimalarial endoperoxide artemisinin involving C-centered radicals [START_REF] Wang | Further evidence for the participation of primary carbon-centered free radicals in the antimalarial action of the qinghaosu (artemisinin) series of compounds[END_REF] with weak H-bonds or β-scission reactions producing fragmentation of side-chains or backbones from proteins [START_REF] Carroll | Superoxide radicals react with peptide-derived tryptophan radicals with very high rate constants to give hydroperoxydes as major products[END_REF]. Tyrosine modification by biological oxidants depends essentially on the formation of Tyr-radicals located in carbon-centered positions of the benzene ring, as for the wellknown nitration of protein tyrosine residues occurring through free radical-mediated pathways [START_REF] Bartesaghi | Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration[END_REF].

Asc and the amino acids (2 equiv) in a deareated 1: described previously for other thiol reagents [START_REF] Chittiboyina | What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization[END_REF]). Peak at m/z 345 could match with both, 2 and 3. Isoascaridole (IsoAsc, Fig. 1c) is a known byproduct obtained after decomposition of Asc [START_REF] Satyal | Bioactivities of volatile components from Nepalese Artemisia species[END_REF]. Further nucleophilic thiol ring opening of the oxirane moieties should be at the origin of 2/3. Due to the inherent electrophilic feature of ketone 4 and IsoAsc we studied the -10-reactivity of activated Asc against N-Ac-Lys-OMe and this in order to check its observed non-reactivity toward DPRA lysine-containing peptide [START_REF] Krutz | Activation of the endoperoxide ascaridole modulates its sensitizing capacity[END_REF]) confirming the result.

1 (v/v) H 2 O/CH 3 CN
The fact that only oxidation/degradation/dimerization derivatives from N-Ac-Trp-OEt and N-Ac-Tyr-OEt were observed indicated the oxidative strength of the media, inducing the production of a number of reactive radical species involved in a permanent game of redox reactions able to chemically modify certain amino acids themselves as previously observed in the case of some sensitizing allylic hydroperoxides [START_REF] Kao | Mechanistic studies on the reactivity of sensitizing allylic hydroperoxides: investigation of the covalent modification of amino acids by carbon-radical intermediates[END_REF].

EPR studies

Electron paramagnetic resonance spectroscopy (EPR)-spin trapping (ST) is the most efficient technique for the identification of transient radicals in chemical and biological systems. Basically, radical intermediates that are too short-lived or of too low concentration to be directly detected react with a spin-trap to form more persistent radicals (spin-adducts) observed by EPR with a signature depending on the trapped transient reactive species [START_REF] Lauricella | Détection et caractérisation de radicaux libres après piégeage de spins[END_REF]. We recently developed a new EPR-ST methodology with the potential to investigate in situ the formation and behaviour of free radicals derived from xenobiotics in a reconstructed human epidermis 3D model (RHE) [START_REF] Kuresepi | Potential of EPR spintrapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxyde as proof of concept[END_REF]. RHE consist of a normal multi-layered keratinocytes culture, the major cell type in the epidermis playing a key role in skin inflammatory reactions. Using this approach, several O-and Ccentred radicals were identified in this complex environment using cumene hydroperoxide as proof of concept. Thus, to study the basic triggers for activation of Asc in the skin, we employed here this EPR-ST methodology applied to RHE EpiSkin TM .

Studies in solution were firstly carried out to set up the optimal experimental conditions while providing a good S/N and keeping the Asc concentrations as low as possible, suitable with physiological conditions. DEPMPO was chosen as spin-trap, based on (i) our previous studies decreasing the Fe(II) content available [START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF][START_REF] Kuresepi | Potential of EPR spintrapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxyde as proof of concept[END_REF]. EPR spectra recorded after activation of Asc with Fe(II) in solution are shown in Fig. 2. The spectrum shown in Fig. 2d was assigned to a mixture of spin-adducts formed by trapping two C-centred radicals with characteristic g factor and hyperfine coupling constants (hfccs; Gauss) values of g=2.0056, a N =14.7, a H = 21.5, a P =47.2 and g=2.0057, a N =15.0, a H = 25.8, a P =47.7, respectively. The use of higher Fe(II) and Asc concentrations did not provide significant additional information (Fig. S3).

In a second phase, experiments were carried out in RHE based on a topical application procedure to get closer to real life sensitization. EpiSkin TM RHE is a very good replicate of human epidermis architecture. It is used for irritation and penetration tests of cosmetic and chemical compounds as replacement of in vivo testing. In OECD guidelines on in vitro skin irritation assessment by using such RHE system it is specified that test chemicals must be applied topically to the RHE model (OECD 2015). In order to carry out these experiments, the skin samples were initially loaded with DEPMPO to ensure that it is present at the site of radical generation before application of Asc [START_REF] Timmins | Free radical formation in murine skin treated with tumour promoting organic peroxides[END_REF]. To cause this loading, RHE were pre-treated with DEPMPO in 50% DMSO, this way facilitating penetration of the spin-trap through the stratum corneum. When RHE pre-treated with ST solution studies, some differences are however observed, especially for one of the radicals trapped.

This could be partially explained because the environment of the spin-adducts is necessarily different in solution compared to that of a complex heterogeneous tissue such as the epidermis. The magnitude of the hfccs indicating the extent of delocalization of the unpaired electron over the molecule, this may be affected by changes in the matrix environment and polarity. Finally, the incubation in the RHE of the sole Asc or DEPMPO (Figs. 3a,3b) gave no signal, indicating that the C-radicals originate rather from Asc than from RHE biomolecules. Worthy of note, after penetrating the RHE, Asc radical initiation should have been induced by RHE itself as no Fe(II) was added in RHE experiments.

Activation of THP-1 cells cocultured with HaCaT keratinocytes

Since cross-talk between structural cells like keratinocytes and immune cells is likely to be critical in determining the outcome of sensitizer exposure, our aim was to co-expose keratinocytes and immune cells and evaluate the impact of this environment on the activation of dendritic cells. For this purpose -12-we evaluated the activation of THP-1 cells cocultured with HaCaT keratinocytes and exposed to several concentrations of Asc for varying time points without adding iron as a radical starter (t = 0.5-48 h). A low (non-toxic), medium and high (toxic) concentration was selected in order to understand how cell death has impact on the dendritic cell phenotype. Results are summarized in Fig. 4. Treatment of cells with the positive control (20 µM DNCB) promoted upregulation of CD86 and CD54 already after 1 h. For Asc, low concentrations (62.5 µM and lower) promoted CD54 upregulation after exposure for 24 h and 48 h. This pattern could not be overcome by using higher concentrations (up to 1000 µM) for shorter exposure times. These data are in line with the common believe that it is the concentration of reactive intermediates and/or protein antigens and not the parent compound, which determines whether cells of the innate immune system are activated.

Activation of THP-1 cells cocultured with RHE

The chemical and biological microenvironment can have an intense effect on dendritic cell phenotype and function and on the nature of the adaptive immune response that follows antigen presentation to Tcells. Therefore, we aimed to evaluate the microenvironment provided by a RHE model and placed the THP-1 cells underneath the RHE. This allows studying dendritic cell activation after topical exposure of keratinocytes thereby simulating more realistic skin conditions [START_REF] Christoffers | The optimal patch test concentration for ascaridole as a sensitizing component of tea tree oil[END_REF]. Based on the EPR results, we used concentrations of Asc (10-80 mM) that all generate radicals, thereby allowing us to monitor their effects on keratinocyte/dendritic cell signaling. The results are depicted in Fig. 5. With respect to concentration needed, we observed no clear upregulation of CD54 and CD86 after application of up to 30 mM Asc, while clear upregulation was observed after 24 h and application of 60 mM. In line, short exposure time (0.5 h) with 60 mM and for which we detected already radicals, did not result in THP-1 activation (data not shown). Important, we found no impact of the tested concentrations on THP-1 cell variability, thus the data generated provide specific information on the signaling molecules released from keratinocytes and how keratinocyte-derived signals affect the activation of the innate immune system. These data suggest that possibly not the radicals but likely the rearranged products that build up in time, may be responsible for the activation of the cells, although this needs clearly further investigations.

Discussion

Asc has been this last decade object of multiple studies to elucidate its mechanism(s) of action, not only in the context of ACD to tea tree oil, but also as the anthelmintic principle of Chenopodium oil. It was used for centuries to treat parasitic diseases but it is irritant to the gastrointestinal tract and genotoxic [START_REF] Lepoittevin | Toxic effects of carvacrol, caryophyllene oxide, and ascaridole from essential oil of Chenopodium ambrosioides on mithocondria[END_REF], or in the treatment of cutaneous leishmaniasis for example [START_REF] Geroldinger | Mechanism of ascaridole activation in Leishmania[END_REF]. In these studies, evidence for Asc C-centered radical intermediates arising from Fe(II)-induced degradation was obtained by using EPR-ST. On the basis of artemisinin's mode of action, important antimalarial drugs, cleavage of the peroxide bond via reduction by Fe(II) is the accepted pathway to initially form highly reactive alkoxyl radicals that further rearrange giving long half-life reactive Ccentered radicals. Although this mechanism might be of relevance for the skin sensitizing action of Asc, to our knowledge so far, there is no publication demonstrating directly the activation of this endoperoxide in the skin to C-centered radicals, as well as the relationship with associated immune responses in skin cells and keratinocyte/dendritic cell signaling. In the work reported herein, the use of a recently developed EPR-ST methodology to elucidate whether radicals can be formed from xenobiotics in the EpiSkin TM 3D RHE model showed for the first time that C-radicals derived from Asc are formed in this epidermal-like skin environment. As showed previously in EPR studies by irradiating TTO (436 nm) in presence of Fe(II) and flavin mononucleotide, combined with HPLC-EPR analysis, two C-radicals could be formed from Asc, the isopropyl (1-methyl ethyl) and the methyl radical [START_REF] Mori | Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion[END_REF]. In our studies we also detected in RHE two DEPMPO spin-adducts with hfccs characteristic of C-radicals trapped. DEPMPO is known to produce stereo-specific isomers when trapping superoxide or alkylperoxyl radicals on both planar sides of the spin-trap leading to two slightly different and superimposed EPR fingerprints [START_REF] Clément | Deuterated analogues of the free radical trap DEPMPO: synthesis and EPR studies[END_REF]. However, this has not been well defined in the case of trapped C-radicals. If we could thus hypothesize that the two C-radicals trapped were of different nature, the trapping of a single C-radical species on both sides of DEMPO molecule giving such mixed EPR signature couldn't be excluded. No spin-adduct was observed for control experiments performed in absence of Asc, suggesting that DEPMPO trapped radicals most probably originated from Asc rather than through biomolecules present in RHE. Last but not least, no Fe(II) was used for radical initiation when using RHE to mimic real life skin allergy situation. One can hypothesize that reaction of organic peroxides in the skin in the presence of one-electron donor agents (e.g. amino and thiol groups present in amino acids, metal complexes, enzymes) may start the electron transfer processes under radical oxidation conditions and further skin proteins haptenation [START_REF] Fujimoto | Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation controls[END_REF].

-14-To be a sensitizer, Asc needs to covalently bind to skin proteins to form the antigenic entity for the interaction of dendritic cells with the T cell receptor of naïve T cells. In addition, sufficient activation and maturation of dendritic cells is needed to provide co-stimulation and thereby influencing the strength of T cell receptor signalling and consequently the nature of the adaptive immune response [START_REF] Steinman | Dendritic cells: understanding immunogenicity[END_REF]. Even if we have proved that C-radicals are formed in the skin, this does not corroborate that they are the reactive intermediates towards skin proteins and maturation of dendritic cells. Radical degradation of Asc is known to yield electrophilic reactive species such as IsoAsc and α,β-unsaturated ketone 4 that could react with nucleophilic residues present on skin proteins. Indeed, we have shown in this work that both can react with the thiol group of N-Ac-Cys-OMe through a nucleophile-electrophile mechanism. We observed 1 derived from reaction of 4 formed after loss of stable isopropyl radical, in agreement with the observation of C-radicals of the isopropyl kind in RHE EPR-ST studies. However, we did not observe reactivity derived from reaction of intermediates formed after loss of methyl radical. In parallel, IsoAsc electrophilic epoxide chemical functions could also react with thiol groups and form antigenic structures. We have thus the impression that radicals formed in RHE are not themselves the reactive species towards amino acids, but the electrophilic intermediates derived from radical degradation of Asc. Formation of electrophilic species via radical degradation in the skin is thus also one possible pathway to trigger sensitization to Asc. Further, both danger signals from formed C-radicals and chemical-induced and oxidative stress signalling (ROS) may jointly have promoted activation of keratinocytes and dendritic cells following Asc exposure. For Asc these aspects still need to be further elucidated.

Conclusion

In this study we have demonstrated that Asc is activated in the skin resulting in the formation of high reactive alkoxyl radicals that further rearrange into C-radicals, and that under these conditions dendritic cell activation is promoted. However, we could not directly link C-radicals as being reactive intermediates towards skin proteins. The fact that time plays a crucial role in dendritic cell activation after exposure to Asc leads us to hypothesize that this could be promoted as a combined effect of formation of electrophilic reactive species together with a depletion of keratinocytes scavenging properties. Fig. 5

  19 g HEPES in 400 mL deionized water, 4 g NaCl and 0.1 g KCl. Deionized water was added to a final volume of 500 mL. Stock solutions were prepared for Asc (10 mM, HEPES/CH 3 CN 9/1 v/v), DEPMPO (100 mM in HEPES) and FeSO 4 .7H 2 O (10 mM in HEPES). 25 µL of spin-trap solution were mixed with FeSO 4 .7H 2 O (i. e. 0.5 µL for a final concentration in the reaction mixture of 0.1 mM), 5 µL Asc solution added and the final volume completed to 50 µL. This way, final concentrations in the reaction mixture were 50 mM DEPMPO, 1 mM Asc and 0.1 mM Fe(II). The reaction mixture was subjected to stirring, introduced into a glass capillary tube (Hirschamnn, 20 µL) sealed on both ends and EPR spectra registered.

  THP-1 cells THP-1 cells were harvested and cell surface expression (mean fluorescence intensities, MFI) of CD86 and CD54 was analyzed on THP-1 cells (PI or DAPI negative) via flow cytometry. Briefly, THP-1 cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD86 (clone 2331 [FUN-1]), APC-labeled anti-CD54 (clone HA58) monoclonal antibodies (all mouse IgG1, obtained from BD Pharmingen, Heidelberg, Germany) or corresponding isotype controls. Then, the chemical-induced MFIs were calculated or the difference in cell surface levels of CD86 or CD54 (ΔMFI) was calculated for each sample.

  mixture were treated with a catalytic amount of FeSO 4 (radical initiator) at room temperature. TLC indicated that Asc was not completely consumed even after 7 days. Reactions were then stopped, the solvent removed under reduced pressure and the crude products analysed by LC-ESI-MS/MS. Asc only reacted with N-Ac-Cys-OMe to afford 1-3 (Fig.1), whereas oxidation/degradation products of N-Ac-Trp-OEt (Fig.S1) and dimerization of N-Ac-Tyr-OEt (Fig.S2) were observed. In the case of N-Ac-Cys-OMe the reactivity was already noticed in the blank reactions conducted without Fe(II) albeit this was very limited. LC-MS of the reaction Asc/N-Ac-Cys-OMe/FeSO 4 crude product showed one major peak with associated m/z of 352 corresponding to the cysteine disulfide dimer, and two other peaks with m/z 303 and 345.MS fragmentation of 303 displayed a fragment ion at m/z 178 due to the loss of N-Ac-Cys-OMe. Also, the 303 m/z value indicated loss of the Asc isopropyl group. Mechanistically, activation of the endoperoxide with Fe(II) facilitates the cleavage of the O-O bond to form oxygenated-radicals. Then, formation of α,β-unsaturated ketone 4 is possible through fragmentation and loss of a stable isopropyl radical. 4 can further undergo Michael addition with the nucleophilic thiol group of cysteine, as

(

  [START_REF] Kuresepi | Potential of EPR spintrapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxyde as proof of concept[END_REF]) (ii) its ability to clearly distinguishO-and C-centred radicals (Stolze et al. 2000) (iii) the high persistency of its superoxide and alkyl-peroxyl radicals spin-adducts[START_REF] Frejaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro spin trapping of oxygen-centered radicals[END_REF]. Spin-adducts are formed by the addition of transient short-lived radicals to the α-C of the nitronyl group of the spin-trap. Radical initiation was triggered by catalytic Fe(II)-induced endoperoxide cleavage. DEPMPO in large excess (50 mM) with respect to Asc (1 mM) was mixed with Fe(II) (0.1 mM) and the mixture subjected to conventional field-swept continuous wave-EPR spectroscopy (cw-EPR) in a sealed glass capillary. HEPES was preferred to phosphate buffer to avoid the formation of insoluble Fe(III) through the autoxidation of Fe(II) catalysed by phosphate and thus

  DEPMPO were exposed topically to different concentrations of Asc in acetone, EPR spectra of DEPMPO radical trapping was observed from a 10 mM concentration, with the best signal-to-noise ratio obtained at 50 mM(Fig. 3d, Fig S4). As for the investigations in solution, the fingerprints of two C-centred radical DEPMPO spin-adducts were detected. These spin-adducts could be assigned to Cradical trapping based on their hfccs (a N =14.8 G, a H =21.6 G, a P =47.4 G, g=2.0049 and a N =14.8 G, a H =22.8 G, a P =50.5 G, g=2.0048). When comparing these hfccs values with those obtained in the EPR-

Fig. 1 Fig. 2

 12 Fig.1 Reaction Asc/N-Ac-Cys-OMe/FeSO 4 studied by LC-ESI-MS/MS. (a) LC chromatogram of the reaction. (b) ESI-MS/MS of m/z 303 at t R 3.48 min. (c) Mechanistic pathway suggested for the formation of 1-3. Fig.2 Reaction Asc (1 mM)/DEPMPO (50 mM)/Fe (II) (0.1 mM) in HEPES (10 mM, pH 6.8): (a) possible reaction paths for the formation of isopropyl and methyl carbon radicals; (b) control EPR spectrum with single DEPMPO; (c) control EPR spectrum with DEPMPO and Fe(II) (1 mM); (d) EPR spectrum of the reaction (circles DEPMPO-carbon R1, triangles DEPMPO-carbon R2); (e) computer simulation; (f-g) deconvolution of (e) with: (f) DEPMPO-carbon R1 spin-adduct (a N (G) = 14.7, a H (G) = 21.5, a P (G) = 47.2, g = 2.0056, 83%) and (g) DEPMPO-carbon R2 spin-adduct (a N (G) = 15.0, a H (G) = 25.8, a P (G) = 47.7, g = 2.0057, 17%).

Fig. 3

 3 Fig.3 EPR spectra obtained in RHE after topical application of Asc: (a) RHE control experiment with single Asc in acetone; (b) RHE control experiment with single DEPMPO in vehicle; (c) RHE spectra after incubation of DEPMPO (15 min, 37°C, 5% CO 2 ) followed by Asc (5 mM) addition; (d) RHE spectra after incubation of DEPMPO (15 min, 37°C, 5% CO 2 ) followed by Asc (50 mM) addition. Labels identify each spin-adduct formed (circles DEPMPO-carbon R1, triangles DEPMPOcarbon R2); (e) computer simulation with DEPMPO-carbon R1 spin-adduct (a N (G) = 14.8, a H (G) = 21.6, a P (G) = 47.4, g = 2.0049, 83%) and (g) DEPMPO-carbon R2 spin-adduct (a N (G) = 14.8, a H (G) = 22.8, a P (G) = 50.5, g = 2.0048, 17%).

Fig. 4

 4 Fig.4 Impact of concentration and exposure time on CD86 and CD54 expression on cocultured THP-1 cells with HaCaT exposed to Asc and DNCB. THP-1 cells cocultured with HaCaT were exposed to increasing concentrations of Asc (7.8-1000 µM) for increasing times (t = 0.5-48 h). DNCB (20 µM) served as positive control. Cell surface expression of CD86 (squares), CD54 (triangles) on collected THP-1 cells (viability, grey dots) was analyzed by flow cytometry. Upregulation of CD86 and CD54 on the surface of THP-1 cells was calculated as difference in mean fluorescence intensities between chemical-treated and solvent-exposed cells and given in ΔMFI. Mean ± SE, n = 5, except n= 3 (t = 0.5 h, 4 h and 48 h).

Fig. 5

 5 Fig.5 Impact of Asc concentration on CD86 and CD54 expression on cocultured THP-1 cells underneath RHE. RHE/THP-1 were treated with acetone-olive oil 4:1 (AOO) and Asc for t = 24 h (mean ± SE, n=5) and 0.5 h (n=1). CD86 (A and C) and CD54 (B and D) expression was analyzed on viable THP-1 cells by flow cytometry. MFI, mean fluorescence intensity. § THP-1 reduction of viability higher than 50%.

  Fig.1

Fig. 2

 2 Fig.2

  ; O'Neill et al. 2010). Tryptophan, redox-active, plays a key role in several biological multistep electron transfer processes. Oxidation results in formation of Trp-derived indolyl radicals which can undergo further reactions, i.e. with O 2 giving peroxyl radicals, dimerization, hydrogen abstraction from compounds

Acknowledgements

This work was part of the DEFCHEMSKALL Franco-German Collaborative International Research Project financially supported by the ANR (Agence Nationale de la Recherche; project no. ANR-15-CE15-0023-01) and the DFG (Deutsche Forschungsgemeinschaft; project no. DFG, BL340/6-1). The Reseau NAtional de Rpe interDisciplinaire (RENARD, Fédération IR-RPE CNRS #3443) is also acknowledged. Fig. 3 -24-

Fig. 4

Compliance with ethical standards

The manuscript does not contain clinical studies or patient data.

Conflict of interest

The authors declare that they have no conflicts of interest.


Andreas V Thomsen
email: andreas.vinther.thomsen.01@regionh.dk

Jakob F Schwensen

Rossana Bossi

Piu Banerjee

Elena Giménez-Arnau

Jean-Pierre Lepoittevin

Carola Lidén

Wolfgang Uter

Ian R White

Jeanne D Johansen

Isothiazolinones are still widely used in paints purchased in five European countries: a follow-up study

Keywords: benzisothiazolinone, dichlorooctylisothiazolinone, environmental label, methylchloroisothiazolinone, methylisothiazolinone, octylisothiazolinone, paint, safety data sheet

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The isothiazolinones methylisothiazolinone, methylchloroisothiazolinone, the mixture methylchloroisothiazolinone/methylisothiazolinone 3:1, benzisothiazolinone, octylisothiazolinone and dichlorooctylisothiazolinone (see Table 1 for further information) are preservatives used in a wide range of industrial chemical products (as biocides) and partly (MCI, MI) in cosmetic products due to their activity against bacteria and fungi (1-5).

Although isothiazolinones have been recognized since the 1970s as contact allergens and MCI/MI (e.g. Kathon® CG) resulted in an epidemic of contact allergy in the 1980s, it is the recent and unprecedented epidemic of contact allergy to MI that has raised awareness of products containing MI [START_REF] Bruze | Incompetence and failure to regulate methylisothiazolinone[END_REF][START_REF] Fischer | On Benzisothiazolinones: A Series with a range of bacteriostatic and fungistatic activity[END_REF][START_REF] De Groot | Isothiazolinone preservative cause of a continuing epidemic of cosmetic dermatitis[END_REF][START_REF] Gonçalo | While Rome burns: the epidemic of contact allergy to methylisothiazolinone[END_REF]. The epidemic of contact allergy to MI was mainly driven by exposures to cosmetic products; however, a substantial number of the cases have been due to exposures to occupational products, for example water-based paints [START_REF] Uter | Risk factors associated with methylisothiazolinone contact sensitization[END_REF][START_REF] Aerts | The dramatic increase in the rate of methylisothiazolinone contact allergy in Belgium: a multicentre study[END_REF][START_REF] Lundov | Methylisothiazolinone contact allergy -a growing epidemic[END_REF][START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. A recent multi-centre prospective study of European dermatitis patients has additionally shown that 7.3% of MI-allergic patients have experienced symptoms, for example flare-up episodes of airborne allergic contact dermatitis, when being in newly-painted rooms [START_REF] Schwensen | The epidemic of methylisothiazolinone: a European prospective study[END_REF]. In accordance, an experimental study showed that MI may evaporate from newly painted walls for at least 42 days [START_REF] Lundov | Emission of isothiazolinones from water-based paints[END_REF] In 2015, a comprehensive study on the use of MI, MCI and BIT in water-based paints purchased in five European countries was published [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. 71 water-based paints were purchased from December 2013 to January 2014. It was found by HLPC-MS-MS that a total of 93.0% of the paints contained MI in concentrations of up to 180.9 ppm, whereas BIT was found in 95.8% of the paints in concentrations up to 462.5 ppm [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. Notably, a few manufacturers managed to preserve 7% (n=5) of the purchased paints without the use of MI [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF].

In March 2016, the Committee for Risk Assessment (RAC) of the European Chemicals Agency (ECHA) recommended that MI should be classified (harmonised classification, legally binding) as a skin sensitiser ('Skin Sens 1A, H317') with a specific concentration limit of 0.0015% [START_REF]Committee for risk assessment, RAC. Opinion, proposing harmonised classi cation and labelling at EU level of 2-methylisothiazol-3(2H)-one (ISO)[END_REF]. The final decision on this recommendation is still awaited, and until MI has a harmonised classification as a skin sensitiser, MI adheres to the rules of self-classification: all non-harmonized skin sensitizing substances and mixtures (chemical products) (Skin Sens 1, 1A or 1B, H317) must be labelled on the product and safety data sheet if the concentration exceeds one-tenth of the concentration limit for classification and labelling (Table 1) [START_REF]Guidance on labelling and packaging in accordance with regulation (EC) No. 1272/2008[END_REF].

In this multi-centre follow-up study, we wanted to investigate the use of selected isothiazolinones in European purchased water-based paints and compare this to the results of the similar 2015 investigation in order to detect trends in the use of especially MI, but also related isothiazolinones (MCI, BIT, OIT and DCOIT).

Materials and methods

Paint collection and samples

A total of 60 white wall paints and wet room paints intended for consumer and/or professional use were purchased in retail stores in Denmark (Copenhagen), France (Strasbourg), Germany (Erlangen), Sweden (Stockholm) and the United Kingdom (London) from 1 June to 31 July 2016. As far as possible, the same products as in 2014 were purchased. All paints were sent by courier to the Department of Environmental Science, Aarhus University in Roskilde, Denmark, where the cans were opened for the first time and analyzed. The paints were thoroughly mixed before sampling. If a thin layer of transparent liquid was visible on top in the paint can, a sample was taken before mixing [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. A portion of 5 ml was taken with a disposable plastic syringe.

Safety data sheets

At the time of purchase, safety data sheets were obtained as far as available at the retail outlet or subsequently from the producer's or retailer's website. The sheets were meticulously searched for any information, warnings or listings referring to isothiazolinones or for warnings concerning allergy risks. The paint cans were inspected for information on isothiazolinone content and environmental labelling. The environmental labels found consisted of 'EU Ecolabel' ('European Flower'; EU) [START_REF]Commission decision of 28 May 2014 establishing the ecological criteria for the award of the EU Ecolabel for indoor and outdoor paints and varnishes ([END_REF], 'Svanen' (The Nordic Swan label; Denmark and Sweden) (18), 'Der Blaue Engel' (The Blue Angel; Germany) [START_REF] Gmbh | [END_REF], 'Svalanmärkt' (Asthma and Allergy Association; Sweden) (20), 'TÜV NORD: Für Allergiker geeignet, Freiwillife Materialprüfung' (optional material testing; Germany) and volatile organic compound (VOC) labelling (low VOC content, minimal VOC content).

The TÜV NORD and VOC labels were not included in the analysis.

Analysis of isothiazolinones in samples

The concentration of isothiazolinones was measured in all collected paint samples. As described elsewhere [START_REF] Aerts | The dramatic increase in the rate of methylisothiazolinone contact allergy in Belgium: a multicentre study[END_REF][START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF], a sample of 1 g (+/-0.1 g) from each paint was extracted in [START_REF] Aerts | Airborne and systemic dermatitis, mimicking atopic dermatitis, caused by methylisothiazolinone in a young child[END_REF] [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF][START_REF] Lundov | Emission of isothiazolinones from water-based paints[END_REF] The recovery of the extraction method for paint was calculated by spiking five different paints with the analytes. The samples were spiked at three different concentrations: 0.1 µg/ml, 1.0 µg/ml or 10 µg/ml. Average recoveries obtained for MI, MCI, BIT, and OIT were 86, 83, 74, and 53%, respectively. The recovery of DCOIT was very low (5%), thus the results for this compound are probably characterised by a large uncertainty. The limit of detection and limit of quantification for all isothiazolinones were 1 ppm and 5 ppm, respectively.

Labels

The 'EU Ecolabel' allows for combined isothiazolinone concentrations of up to 500 ppm in indoor paints with a maximum MI concentration of 200 ppm, a maximum BIT concentration of 500 ppm, a maximum OIT concentration of 500 ppm and a maximum MCI/MI concentration of 15 ppm [START_REF]Commission decision of 28 May 2014 establishing the ecological criteria for the award of the EU Ecolabel for indoor and outdoor paints and varnishes ([END_REF].

The 'Nordic Swan label' limits the combined concentrations of isothiazolinones to 500 ppm with a maximum MI concentration of 100 ppm and a maximum MCI/MI concentration of 15 ppm [START_REF]Nordic Ecolabelling of Indoor paints and varnishes[END_REF].

'Der Blaue Engel' limits the use of MCI/MI to 15 ppm, BIT to 200 ppm and MI/BIT (1:1) to 200 ppm [START_REF] Gmbh | [END_REF]. Products labelled with 'Svalan' are recommended by the Swedish Asthma and Allergy Association. The products must be "free from allergens, perfumes and irritants in amounts so that no reported medical cases are known", except paints, for which only respiratory problems are considered. For paints, the criteria come into force 2 weeks after application [START_REF] Och | [END_REF].

Statistics

The data were processed with SPSS TM (SPSS TM Statistics, Chicago, IL, USA; IBM PASW Statistics) for Windows TM , edition 22.0, and R statistical software (www.r-project.org), version 3.2.0.

The Mann-Whitney U-test was applied for (i) analysis of differences in MI, MCI and BIT concentrations found in the previously tested paints [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF] and the newly found MI, MCI and BIT concentrations, (ii) analysis of differences between the MI, MCI, BIT, OIT and DCOIT concentrations in paints with environmental labels and paints with no environmental labelling and (iii) analysis of differences between the MI, MCI, BIT, OIT and DCOIT concentrations in wet room paints and white wall paints. The distribution of the measured values for MI and BIT were graphically represented by a strip chart with an overlay box plot. The Kruskal-Wallis H-test for global heterogeneity was applied for analysis of differences in MI, MCI, BIT, OIT and DCOIT concentrations across countries. The threshold for statistical significance was predefined as a pvalue of < 0.05.

Results

Sixty paints were analyzed for their contents of five isothiazolinones. MI was identified in 91.7% (n = 55) of the purchased paints, with concentrations ranging from 1.1 to 142.7 ppm. MCI was in 20.0% (n = 12) of the paints, ranging from 0.5 to 3.5 ppm. BIT was in 88.3% (n = 53) of the paints, ranging from 0.4 to 331.0 ppm. OIT was in 26.7% (n = 16) of the paints, ranging from 0.03 to 16.01 ppm. DCOIT was identified in 50.0% (n = 30) of the purchased paints, ranging from 0.01 to 156.2 ppm. Concomitant presence of MI, MCI and BIT is visualized in Fig. 1B. Supplemental Table S1 shows a detailed description of the sixty paints with regards to product names, analysis results, environmental labelling and safety data sheet information. As in the previous study, samples were taken from a thin layer of surface liquid when obviously present (seven paints). Concentrations of MI in this surface layer were up to 2 times larger than the concentration in the paint itself after mixing.

The distribution of MI concentrations is presented in Fig. 2A. No statistically significant heterogeneity between countries was observed for MI or MCI. However, BIT (presented in Fig. 2B), OIT and DCOIT concentrations all varied statistically significantly between countries. In paints containing DCOIT, the concentration was found to be up to 5.64 ppm with a single outlier of 156.2 ppm.

Three paints (two Danish and one German) did not contain any detectable amount of isothiazolinones. Another German paint claiming to be free of preservatives did however contain MI at a concentration of 1.05 ppm. Nine paints had an MI concentration of < 5 ppm, and seven of these paints contained no detectable MI. MCI was found in twelve of the purchased paints, of which eight were purchased in Sweden. None of the paints purchased in France or Germany were found to contain MCI. The MI concentrations in paints containing MCI ranged from 2.85 -96.7 ppm, and was significantly lower than the MI concentrations in paints without MCI (Mann-Whitney U-test, p = 0.027). The results suggest a higher use of MCI/MI in paints purchased bought in Sweden than in other countries. The MI concentration was always higher than the MCI concentration, suggesting that MI is used together with MCI/MI, which now is banned in leave-on cosmetics.

Eleven of the purchased paints were wet room paints. MI and MCI contents did not differ statistically significantly between wall paints and wet room paints, but BIT, OIT and DCOIT were all found in significantly higher concentrations in wet room paints.

Thirty-one paints featured an environmental label of interest on the can (Table 2). MI, MCI and DCOIT contents did not differ statistically significantly between paint with or without environmental labels. OIT was found in lower concentrations in paints with environmental labels (p = 0.034), whereas BIT concentrations were higher in paints with environmental labels (p = 0.048).

The Mann-Whitney U-test did not show any statistically significant differences in MI, MCI or BIT concentrations between the paints purchased for the 2014 study as a whole or for any individual country.

Presence of MI, MCI and BIT according to chemical analysis, information on paint cans, and in safety data sheets was compared for each substance and between the present study and the study 2014. Agreement between the three sources of information is visualized by areaproportional Euler diagrams in Fig. 1. The agreement was generally stronger in the current study (Fig. 1D,F,H) than in the previous study (Fig. 1 C,E,G) where it was poor. In both studies, MCI was reported on cans and in safety data sheets, also when no MCI was detected by chemical analysis (Fig. 1E,F). concentrations, and relatively frequently, in our analyzed paints. This indicates that DCOIT may be a relevant contact allergen in especially painters with suspected contact allergy to paints. Seven paints were found to have a thin surface layer of liquid, known as anti-skinning agent, which is added to the surface to prevent skinning during storage of the paint. The same types of isothiazolinones were found in the surface layer as in the paint itself, but in generally higher concentrations. MI concentrations in the surface layer were up to 2 times larger than the concentration found in the paint after mixing as in accordance with the previous study on analyzed paints [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. Consumers and painters using such paints may be exposed to particularly high concentrations of MI upon mixing the paints.

Labelling of paints that contain MI, regardless of concentration, is essential to avoid flare-up episodes in already sensitized individuals. There is a remarkable improvement concerning information on cans and in safety data sheets between 2014 and 2016. The explanation must be the entry into force by June 2015 of the CLP requirement to give information according to EUH208 (e.g. "Contains methylisothiazolinone. May produce an allergic reaction.") for concentrations down to 1/10 of the concentration limit. However, our data showed that only 38.3% (n = 23) was either labelled to contain MI on the paint can, or information was given in the safety data sheet. It is, however, unlikely that the average consumer examines safety data sheets. Hence, if information is provided only in safety data sheets, secondary and tertiary prevention is not fully addressed.

Hitherto, MI needs to be labelled according to the rules self-classification. Often, self-classification applies considerably less protective classification limits than harmonized classification does, although the same criteria and guidelines shall be applied. Notwithstanding several years of delay, the Committee for Risk Assessment (RAC) concluded in its opinion that MI should be classified as 'Skin Sens 1A, H317', with a specific concentration limit of 0.0015% [START_REF]Committee for risk assessment, RAC. Opinion, proposing harmonised classi cation and labelling at EU level of 2-methylisothiazol-3(2H)-one (ISO)[END_REF]. This recommendation still awaits final decision, however. We suggest, for health protection, that industry applies this classification now, before required by law.

In conclusion, the proposed classification of MI as a skin sensitizer with a specific concentration limit of 15 ppm is, without doubt, an important step forward. However the continued extensive use of MI warrants restrictions in the use of MI in paint due to the risk of contact allergy, airborne and direct contact. It is of special concern that the paints carrying an environmental label do not provide any extra safety. The current study as well as the previous one [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF] showed that paints without MI, MCI or BIT do exist and thus that it is possible to preserve paint without the use of isothiazolinones.

  ml methanol/0.4 % formic acid (20:80 v/v) by means of ultrasound for 10 min. The suspension was filtered through a Phenex-GF/CA (fibreglass/cellulose) filter and analysed by high-performance liquid chromatography (HPLC) coupled to UV detection and confirmed by HPLC-MS-MS previous dilution of sample extracts. The analytes were separated on a Kinetex C18 (100 x 2.1 mm) HPLC column and detected at 275 nm wavelength. LC-MS-MS confirmation was performed with electrospray ionization (ESI) in positive mode. The MS was operated in multiple reaction monitoring (MRM) with two mass transitions (parent ion/product ion) for each analyte (m/z 116/101 and 116/71 for MI; m/z 150/87 and 150/135 for MCI; m/z 152/109 and 152/134 for BIT; m/z 214/102 and 214/71 for OIT; m/z 282/170 and 282/71 for DCOIT). Detection of the analytes was based on retention time and the most abundant mass transition corresponding to an authentic standard. Confirmation of analyte identity was based on the relative response of the secondary mass transition to the primary mass transition. Quantification of the analytes was done using response factors calculated from a four-point calibration curve

Conflicts of interests:

All authors declare no conflicts of interests pertinent to the present study.

Discussion

In this multi-centre follow-up study we investigated the concentrations of MI, MCI, BIT, OIT and DCOIT in water-based paint purchased in five European countries: Denmark, France, Germany, Sweden and the United Kingdom. The results show a continued extensive use of methylisothiazolinone in water-based wall paints. We found MI in 91.7% of the purchased paints in a maximum concentration of 142.7 ppm with no statistical difference between the present data and the study from 2014, where 93% of paints contained MI in up to 180.9 ppm [START_REF] Schwensen | Methylisothiazolinone and benzisothiazolinone are widely used in paint: a multicentre study of paints from five European countries[END_REF]. The use of MI in paint has throughout the last decade been shown to be a relevant exposure in patients with MI contact allergy, and it is well recognized that MI may evaporate from newly painted walls [START_REF] Lundov | Emission of isothiazolinones from water-based paints[END_REF][START_REF] Kaae | Presumed primary contact sensitization to methylisothiazolinone from paint: a chemical that became airborne[END_REF][START_REF] Lundov | Two cases of airborne allergic contact dermatitis caused by methylisothiazolinone in paint[END_REF][START_REF] Lundov | Methylisothiazolinone in paint forces a patient out of her apartment[END_REF][START_REF] Alwan | Presumed airborne contact allergy to methylisothiazolinone causing acute severe facial dermatitis and respiratory difficulty[END_REF][START_REF] Aerts | Airborne and systemic dermatitis, mimicking atopic dermatitis, caused by methylisothiazolinone in a young child[END_REF][START_REF] Kaur-Knudsen | Systemic allergic dermatitis following airborne exposure to 1,2-benzisothiazolin-3-one[END_REF][START_REF] Tokunaga | Occupational airborne contact dermatitis by isothiazolinones contained in wall paint products[END_REF]. Additionally, a large prospective observational study showed in 2016 that approximately 7% of all patients with MI contact allergy previously had experienced allergic symptoms (dermatitis, rhinitis and/or conjunctivitis) when being in newly painted rooms [START_REF] Schwensen | The epidemic of methylisothiazolinone: a European prospective study[END_REF]. Currently, it is unknown to which extent consumers are primarily sensitized due to exposure to paint preserved with MI, however, a Belgium case showed that 53 ppm MI in a paint was enough to elicit airborne allergic contact dermatitis in a girl [START_REF] Aerts | Airborne and systemic dermatitis, mimicking atopic dermatitis, caused by methylisothiazolinone in a young child[END_REF]. Notably, 32 (53.3%) of the paints analyzed contained MI in concentrations > 50 ppm.

Although some focus throughout the past years has been on reducing the use of MI in paint, no significant change could be detected in our study. This highlights the need for sufficient legislation restricting the content of MI in particular in paint, since self-regulation has proven unsuccessful.

The concentration of OIT in paint was much lower than anticipated. A former Danish registrybased study from 2014 showed that OIT was found in relatively high concentrations in chemical products for occupational use, including varnishes and paints [START_REF] Friis | Isothiazolinones in commercial products at Danish workplaces[END_REF]. Observational studies have additionally shown that OIT contact allergy is frequently found in painters [START_REF] Mose | Occupational contact dermatitis in painters: an analysis of patch test data from the Danish Contact Dermatitis Group[END_REF][START_REF] Mose | Allergic contact dermatitis from octylisothiazolinone[END_REF]. The recovery of OIT in the present study was only 53%, which may have led to some under-estimation of occurrence in paints.

DCOIT is used in antifouling paints for use to impede growth of barnacles, algae and other marine organisms as the outer layer to the hull of a ship [START_REF] Guardiola | Risks of using antifouling biocides in aquaculture[END_REF]. However, DCOIT was found in high


Jennifer Hennen

Marta Silva E Sousa

Fatma Sahli

Jutta Lichter

Jean-Pierre Lepoittevin

Elena Giménez-Arnau

Prof. Dr Brunhilde Blömeke
email: bloemeke@uni-trier.de

Sensitization potential and potency of terpene hydroperoxides in the COCAT method

Keywords: in vitro, human skin sensitization potential, sensitization potency, fragrances, hydroperoxides, Cocultured Activation Test, COCAT

Background: Positive patch tests to mixtures of oxidized terpenes containing allergenic hydroperoxides are frequently reported. However, human sensitization data for these hydroperoxides are not available.

Objectives:

To analyze and evaluate human sensitization potential and potency of hydroperoxides in vitro using human cells.

Materials/Methods:

Limonene-1-hydroperoxide, limonene-2-hydroperoxide, citronellol-7hydroperoxide, cumene hydroperoxide, 1-(1-hydroperoxy-1-methylethyl)cyclohexene and mixtures of citronellol hydroperoxides (isomers at positions 6/7) and linalool hydroperoxides (isomers at positions 6/7) were studied. All compounds were synthesized with the exception of cumene hydroperoxide that was commercial. Their potential and potency to activate dendritic cells (DC) was evaluated by measuring the upregulation of CD86 and CD54 on THP-1 cells upon exposure in the cocultured activation test (COCAT) consisting of HaCaT (human keratinocyte cell line) and THP-1 monocytes (as surrogate for DC).

Results: Hydroperoxides upregulated CD86 and/or CD54 on cocultured THP-1 cells in a concentration-dependent manner. Obtained results are comparable with their sensitization potency ranking in predictive animal models.

Conclusions:

For the first time, human sensitization potential and potency of several hydroperoxides were determined using human cells by the COCAT method.

Introduction

Allergic contact dermatitis is the clinical expression of contact allergy, an altered immune status of an individual induced by a particular sensitizing substance, a contact allergen. Upon uptake into the living epidermis, a contact allergen needs to provide an antigen and strongly activate the innate immune system of the skin, providing inflammatory and danger signals from keratinocytes [START_REF] Banchereau | Dendritic cells and the control of immunity[END_REF][START_REF] Iwasaki | Toll-like receptor control of the adaptive immune responses[END_REF][START_REF] Banchereau | The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming[END_REF]. These signals allow dendritic cells to leave the skin and mature in the course of their migration to skin-draining lymph nodes where they activate specific naïve T cells, resulting in expansion of allergen-responsive T cells. At this point, an individual is immunologically sensitized. Only upon re-exposure with the same, or a cross-reacting, contact allergen, the elicitation phase is triggered, leading to specific T cell activation with clinical symptoms.

Fragrance compounds remain the most commonly detected allergens in consecutive dermatitis patients, although they are typically moderate sensitizers [START_REF] Liden | Comparative sensitizing potencies of fragrances, preservatives, and hair dyes[END_REF]. The prevalence rates of contact allergy to fragrances in women are about twice of those in men [START_REF] Diepgen | Prevalence of fragrance contact allergy in the general population of five European countries: a cross-sectional study[END_REF]. Specifically terpene fragrances are ingredients in up to 60% of cosmetic and household products, and occur naturally in essential oils.

Contact allergy to oxidation products of fragrance terpenes such as linalool and limonene are frequently recorded [START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC* prospective study[END_REF][START_REF] Wlodek | Recommendation to test limonene hydroperoxides 0.3% and linalool hydroperoxides 1.0% in the British Baseline patch test series[END_REF][START_REF] Nath | Contact Allergy to Hydroperoxides of Linalool and D-Limonene in a US Population[END_REF][START_REF] Audrain | Allergy to oxidized limonene and linalool is frequent in the U.K[END_REF][START_REF] Bråred Christensson | Air-oxidized linalool: a frequent cause of fragrance contact allergy[END_REF][START_REF] Bråred Christensson | An international multicentre study on the allergenic activity of air-oxidized R-limonene[END_REF], while patch test studies for air-oxidized citronellol are absent. Linalool, limonene and citronellol are not contact allergens based on human and animal data [START_REF] Kern | Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation[END_REF][START_REF] Api | Correlation between experimental human and murine skin sensitization induction thresholds[END_REF].

The sensitizing potency on the basis of the murine local lymph node assay (LLNA) is categorized into extreme (EC3 value of ≤0.2%), strong (>0.2% to ≤2%), and moderate (>2%) . The allylic hydroperoxides derived from air-oxidized linalool [START_REF] Sköld | Contact allergens formed on air exposure of linalool. Identification and quantification of primary and secondary oxidation products and the effect on skin sensitization[END_REF], limonene [START_REF] Johansson | Carbon-and oxygencentered radicals are equally important haptens of allylic hydroperoxides in allergic contact dermatitis[END_REF], citronellol [START_REF] Rudbäck | Characterization of skin sensitizers from autoxidized citronellol -impact of the terpene structure on the autoxidation process[END_REF] as well as the other air-oxidized compounds were evaluated in the LLNA and results showed that the hydroperoxides are the most potent sensitizers among all oxidation products derived from airoxidation of the parent terpenes. Consequently, limonene-1-hydroperoxide, limonene-2-hydroperoxide [START_REF] Johansson | Carbon-and oxygencentered radicals are equally important haptens of allylic hydroperoxides in allergic contact dermatitis[END_REF] and the mixture of linalool-hydroperoxides [START_REF] Sköld | Contact allergens formed on air exposure of linalool. Identification and quantification of primary and secondary oxidation products and the effect on skin sensitization[END_REF] are categorized as strong sensitizers, and the mixture of citronellol hydroperoxides as moderate [START_REF] Rudbäck | Characterization of skin sensitizers from autoxidized citronellol -impact of the terpene structure on the autoxidation process[END_REF] (see Tab. 2). Furthermore, exposure to hydroperoxides may also occur due to their use as synthetic chemicals. For instance, cumene hydroperoxide is used in the production of acetone and phenol and as polymerization initiator.

Overall, the most frequently studied fragrance hydroperoxides are limonene-2-hydroperoxide (lim-2-OOH), linalool-6-hydroperoxide (lin-6-OOH) and linalool-7-hydroperoxide (lin-7-OOH). The quantification of hydroperoxides in consumer products is challenging due to low thermal stability and their lack of chromophores [START_REF] Kern | Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products[END_REF][START_REF] Kern | Stability of limonene and monitoring of a hydroperoxide in fragranced products[END_REF][START_REF] Rudbäck | Essential oils can contain allergenic hydroperoxides at eliciting levels, regardless of handling and storage[END_REF][START_REF] Ramzi | A two-dimensional non-comprehensive reversed/normal phase high-performance liquid chromatography/tandem mass spectrometry system for determination of limonene and linalool hydroperoxides[END_REF]. The link between exposure to the hydroperoxides and allergic contact dermatitis is well documented only in few cases. For example, a 7-year-old girl developed severe eyelid dermatitis after using a shampoo, which contained linalool and low levels (0.2 µg/g) of lin-OOH [START_REF] Elliott | Severe intractable eyelid dermatitis probably caused by exposure to hydroperoxides of linalool in a heavily fragranced shampoo[END_REF].

The aim of the present study was to investigate the sensitizing potency of a series of hydroperoxides by using our Cocultured Activation Test (COCAT), developed to identify the human skin sensitization hazard and determination of skin sensitization potency in vitro [START_REF] Hennen | Cross talk between keratinocytes and dendritic cells: impact on the prediction of sensitization[END_REF][START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF]. COCAT is based on the use of cocultures of HaCaT (human keratinocyte cell line) and THP-1 cells (surrogate of antigen presenting cells). Individually, these cell lines are used to address key steps in the process of sensitization, namely activation of keratinocytes (key event 2) and activation and maturation of dendritic cells (key event 3) of the skin sensitization Adverse Outcome Pathway . We found that exposure on the cocultured cells has the potential to increase their response to sensitizing chemicals, enabling the detection of prohaptens and supporting the identification of skin sensitization potency [START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF][START_REF] Hennen | Assessment of skin sensitization potency of hair dye molecules in vitro[END_REF][START_REF] Hennen | Ranking skin-sensitizing hair dye molecules according to their potency by the use of human cells[END_REF].

Additionally, a 79.5% (31/39) concordance was found when estimating potency based on a prediction of 4 potency subcategories (extreme/strong, moderate, weak, non-sensitizer) by linear interpolation of the lowest concentration needed to reach positivity for estimated markers (CD54 or CD86) as compared to LLNA EC3 values [START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF].

Materials and Methods

Chemicals

The following compounds were studied: limonene The synthesis of the hydroperoxides was performed in the Dermatochemistry Laboratory (Strasbourg, France). Lim-1-OOH and lim-2-OOH hydroperoxides were synthesized as reported previously in the literature [START_REF] Calandra | An HPLC method for hydroperoxides derived from limonene and linalool in citrus oils, using post-column luminol-mediated chemiluminescence detection[END_REF][START_REF] Kao | Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway[END_REF]. For the mixture citr-6/7-OOH we have followed exactly the procedure we described for lin-6/7-OOH and obtained for both mixtures an isomer ratio of 6:4 [START_REF] Kao | Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway[END_REF]. Cyc-hex-OOH was synthesized following also a method described in the literature [START_REF] Lepoittevin | Interactions of Allergenic Hydroperoxides with Proteins -a Radical Mechanism[END_REF]. All synthesized compounds were pure according to the 1 H and 13 C NMR characterizations, in agreement with the literature mentioned.

-1-hydroperoxide [(1S, 4R)-4-isopropenyl-1- methyl-2-cyclohexene-1-hydroperoxide (lim-1-OOH)], limonene-2-hydroperoxide [(5R)-5- isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (lim-2-OOH)], citronellol-7-hydroperoxide [(E)-7-hydroperoxy-3,7-dimethyloct-5-ene-1-ol (citr-7-OOH)],
Chemical structures of studied hydroperoxides are given in Fig. 1.

Cocultured activation test (COCAT)

THP-1 cells (8 × 10 [START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF][START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF].

Results

We investigated the human sensitization potential and potency using the COCAT method.

Specifically, limonene-1-hydroperoxide (lim-1-OOH), limonene-2-hydroperoxide (lim-2-OOH), citronellol-7-hydroperoxide (citr-7-OOH), mixture of citronellol hydroperoxides (citr-6-OOH) and

(citr-7-OOH) in a 6:4 ratio (citr-6/7-OOH), mixture of linalool hydroperoxides (lin-6-OOH) and (lin-7-OOH) in a 6:4 ratio (lin-6/7-OOH), cumene hydroperoxide (cum-OOH), and 1-(1-hydroperoxy-1methylethyl)cyclohexene (cyc-hex-OOH) were investigated (see Fig. 1). Representative flow cytometry histogram plots are shown in Fig. 2. All studied hydroperoxides upregulated both markers for activation of dendritic cells, CD86 and CD54, on cocultured THP-1 cells in a concentrationdependent manner. The corresponding curves for CD86 and CD54 are shown in Fig. 3 A-G.

Furthermore, all compounds reached the threshold for positivity (CD86: ΔMFI of 10.8, CD54: ΔMFI of 300) for one or two markers in at least 2 out of 3 runs. All hydroperoxides were correctly predicted as sensitizers. The lowest concentration needed to reach a threshold for positivity (effective concentration, ECΔ) was used to estimate potency of the molecule [START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF]. Here we found that the tested hydroperoxides needed similar concentrations to reach positivity, values varied between 0.29 and 0.65 mM. In contrast, for DNCB classified as an extreme sensitizer in the LLNA, at least a 10-fold lower concentration was sufficient to reach positivity (Tab. 2). In case of the limonene hydroperoxides, we found lim-1-OOH more potent than lim-2-OOH, similarly to the LLNA data. The values for the lin-6/7-OOH are close to lim-2-OOH. Interestingly, citr-7-OOH alone was less potent than the mixture of citr-6/7-OOH (ratio 6:4). The concentrations needed for positivity of cum-OOH and cyc-hex-OOH were comparable to values found for lim-2-OOH (Tab. 3).

Discussion

In this study, we analyzed the skin sensitization potential and potency of allergenic hydroperoxides in vitro using our COCAT method [START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF][START_REF] Hennen | Assessment of skin sensitization potency of hair dye molecules in vitro[END_REF][START_REF] Hennen | Ranking skin-sensitizing hair dye molecules according to their potency by the use of human cells[END_REF][START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF]. All hydroperoxides were correctly classified as skin sensitizers based on analysis of DC activation in their natural environment of keratinocytes. We found a concentration-dependent upregulation of CD86 and CD54 over a wide range of concentrations. The concentration needed for positivity ranged between 0.29 and 0.65 mM. Thus, our potency ranking is comparable to reported in vivo data from predictive animal testing (Tab. 2 and 3). The highest potency was found for lim-1-OOH followed by lim-2-OOH. Although exploration of the mechanism(s) leading to the upregulation of the two markers CD86 and CD54 was not the focus of this study, it seems reasonable to speculate that the difference might reflect the different capacity of the two molecules to generate radicals. For instance, a higher ability of lim-1-OOH to generate different kinds of radicals was detected using radical trapping experiments [START_REF] Bråred Christensson | Limonene hydroperoxide analogues differ in allergenic activity[END_REF]. For lim-1-OOH both carbon-centered and oxygen-centered radicals were trapped, while experiments with lim-2-OOH retrieved only small amounts of carbon-centered radicals. Instead, the main product trapped in experiments testing lim-2-OOH was carvone. But carvone is much less sensitizing in animal experiments (LLNA EC3 value 0.86 M, 13% w/v [START_REF] Nilsson | An alpha,betaunsaturated oxime identified as a strong contact allergen. Indications of antigen formation via several pathways[END_REF], and cannot account for the results obtained with COCAT. This notion is also supported by the ECΔ value of carvone (0.55 ± 0.11 mM) in COCAT [START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF]. In addition, published prevalence of positive patch test reactions to oxidized fragrance hydroperoxides [START_REF] Bråred Christensson | Limonene hydroperoxide analogues show specific patch test reactions[END_REF] is in accordance with the potency ranking obtained in this study. Specifically, a human patch test study including 763 eczema patients all tested with lim-1-OOH and lim-2-OOH showed that 18 individuals responded to lim-1-OOH and 13 individuals to lim-2-OOH [START_REF] Bråred Christensson | Limonene hydroperoxide analogues show specific patch test reactions[END_REF].

Our results for lim-OOH and lin-OOH are in general agreement with a previous study investigating THP-1 activation in the absence of keratinocytes among further in-depth mechanistically aspects [START_REF] Raffalli | Editor's Highlight: Fragrance Allergens Linalool and Limonene Allylic Hydroperoxides in Skin Allergy: Mechanisms of Action Focusing on Transcription Factor Nrf2[END_REF].

Looking at the correlation of the compound concentrations, we needed much lower concentrations in the presence of keratinocytes. This suggests that keratinocytes support THP-1 activation. A different responsiveness between mono-and coculture of THP-1 cells was already observed with a different set of chemicals [START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF][START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF] where compared to exposing THP-1 alone, coculturing resulted in up to 3-fold enhanced maximal CD86 and/or CD54 upregulation on THP-1.

Cum-OOH and cyc-hex-OOH are quite comparably potent in COCAT. The comparable potency of cum-OOH and cyc-hex-OOH with lim-2-OOH observed in COCAT is in good agreement with in vivo results also indicating comparable potencies of these molecules [START_REF] Lepoittevin | Interactions of Allergenic Hydroperoxides with Proteins -a Radical Mechanism[END_REF]. The lowest potency was detected for citr-7-OOH. Citronellol belongs also to the highly used fragrances in terms of the relative volume sold [START_REF] Schnuch | Risk of sensitization to fragrances estimated on the basis of patch test data and exposure, according to volume used and a sample of 5451 cosmetic products[END_REF]. With respect to its oxidation products it was demonstrated that citr-7-OOH together with citr-6-OOH contributes the most to the sensitizing potency of autoxidized citronellol in the LLNA [START_REF] Rudbäck | Characterization of skin sensitizers from autoxidized citronellol -impact of the terpene structure on the autoxidation process[END_REF]. Comparison of our results for citr-7-OOH alone with the results gained for citr-6/7-OOH indicate that citr-7-OOH may have influenced the results obtained for the mixture. This is in contrast to results reported for lin-6/7-OOH and lin-7-OOH exposing THP-1 alone, where no difference was found for lin-7-OOH and the lin-6/7-OOH [START_REF] Raffalli | Editor's Highlight: Fragrance Allergens Linalool and Limonene Allylic Hydroperoxides in Skin Allergy: Mechanisms of Action Focusing on Transcription Factor Nrf2[END_REF]. Besides the different experimental setups, there is no obvious difference in the structure of citr-7-OOH and lin-7-OOH but clearly more detailed structure-related analysis are necessary to explain the observed differences depending on chemical structures.

At present, comparison of our data for the hydroperoxides with results from other alternative methods explicitly evaluating the potency of these compounds is not possible due to a lack of data. In addition no meaningful results were received from an in silico tool available (DC ITS at https://its.douglasconnect.com).

In sum, this study is the first evaluating the human sensitization potential and potency of several hydroperoxides, mainly fragrance hydroperoxides, in vitro with the COCAT method. The results obtained are in line with the results from the literature using in vivo methods. These data, together with our earlier results [START_REF] Hennen | Cross talk between keratinocytes and dendritic cells: impact on the prediction of sensitization[END_REF][START_REF] Hennen | Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells[END_REF][START_REF] Hennen | Assessment of skin sensitization potency of hair dye molecules in vitro[END_REF][START_REF] Hennen | Ranking skin-sensitizing hair dye molecules according to their potency by the use of human cells[END_REF][START_REF] Hennen | Cocultured activation test (COCAT)-Advanced safety assessment of chemical sensitizers[END_REF] Afterwards, the expression of CD86 and CD54 was analyzed on harvested THP-1 cells via flow cytometry and given as chemical-induced differences in cell surface levels of CD86 or CD54 (mean ± SEM of n=3; citr-6/7-OOH and cum-OOH, n=2).

  mixture of citronellol hydroperoxides [6-hydroperoxy-3,7-dimethyloct-7-ene-1-ol (citr-6-OOH)] and citr-7-OOH (citr-6/7-OOH, ratio 6:4), mixture of linalool hydroperoxides [6-hydroperoxy-3,7-dimethylocta-1,7-diene-3-ol (lin-6-OOH)] and [7-hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol (lin-7-OOH)] in a 6:4 ratio (lin-6/7-OOH), cumene hydroperoxide (cum-OOH, Sigma-Aldrich, Saint Quentin Fallavier, France), 1-(1-hydroperoxy-1methylethyl)cyclohexene (cyc-hex-OOH), sodium dodecyl sulfate (SDS, Carl Roth, Karlsruhe, Germany) and 2,4-dinitrochlorobenzene (DNCB, Sigma Aldrich Taufkirchen, Germany).

Figure legends Fig. 1

 legends1 Figure legends

Fig. 3

 3 Fig. 3 Concentration-dependent upregulation of CD86 and CD54 by hydroperoxides. Cocultured

  4 , 96-well) were placed on top of confluent HaCaT keratinocytes. The maximal test concentration (up to 4000 µM) for each test chemical is determined based on solubility in the culture medium and/or cell viability. Cocultured cells were exposed to increasing concentrations of the Overall 7-9 concentrations were evaluated for each hydroperoxide (see Tab. 1). In each run, 20 µM of 2,4-dinitrochlorobenzene (DNCB) and 144 µM sodium dodecyl sulfate (SDS) were used as positive and negative control, respectively. After 24 h of treatment, floating THP-

	1 cells were harvested and cell surface expression (mean fluorescence intensities, MFI) of CD86 and
	CD54 was analyzed on viable THP-1 cells (DAPI/propidium iodide negative) via flow cytometry
	(FACSVerse TM or FACSCalibur TM , BD Biosciences, Heidelberg, Germany). Briefly, THP-1 cells were
	stained with fluorescein isothiocyanate (FITC)-labeled anti-CD86 (clone 2331 [FUN-1]), APC-labeled
	anti-CD54 (clone HA58) monoclonal antibodies (all mouse IgG1, obtained from BD Pharmingen,
	Heidelberg, Germany) or corresponding isotype controls. Then, the chemical-induced differences in

compounds (7.8-4000 µM) dissolved in final concentration of 0.2%DMSO. Afterwards cell viability was determined and concentrations inducing not more than 30-35 % reduction of THP-1 cell viability were used for the experiments. cell surface levels of CD86 or CD54 (ΔMFI) on viable THP-1 (determined by DAPI/propidium iodide exclusion) were calculated for each sample. A ΔMFI of 10.8 for CD86 and 300 for CD54 were used as thresholds to identify a sensitizer. These thresholds were established by analyzing a set of 21 sensitizers and 12 non-sensitizers in the course of the method development. The lowest ECΔ (either CD54 or CD86) for each run was used to estimate potency. Mean ECΔ and standard error were calculated for each compound

  demonstrate that COCAT can support the assessment of chemicals with as yet limited or absent human sensitization data. Comparison of the skin sensitization potencies of fragrance terpene hydroperoxides and 2,4-dinitrochlorobenzene (DNCB) in vitro (COCAT) and in vivo (LLNA). Comparison of the skin sensitization potencies of lim-2-OOH, cum-OOH and cyc-hex-OOH in vitro (COCAT) with published in vivo results derived from guinea pig test (Freund's complete adjuvant test, FCAT). † n=3, except n=2 for cum-OOH

	Tab. 2: Compound Lim-1-OOH Lim-2-OOH Lin-6/7-OOH, ratio 3:5 Lin-6/7-OOH, ratio 6:4 Citr-6/7-OOH, ratio 6:4 Citr-7-OOH DNCB † n=3, except n=2 for citr-6/7-OOH. n.a., not available COCAT ECΔ (mM, mean ± SEM) † 0.29 ± 0.01 0.35 ± 0.08 n.a. 0.36 ± 0.04 0.29 ± 0.07 0.65 ± 0.17 0.014 ± 0.0037 Compound COCAT ECΔ (mM, mean ± SEM) † Lim-2-OOH 0.35 ± 0.08 Cum-OOH 0.30 ± 0.12 Tab. 3: 1 at 72h after challenge Cyc-hex-OOH 0.38 ± 0.01	Human sensitization category n.a. n.a. n.a. n.a. n.a. n.a. 1 guinea pig test (FCAT) LLNA EC3 (mM; % w/v) 19; 0.33 1 49; 0.83 1 86; 1.6 2 n.a. 100; 2.3 3 n.a. 2.96; 0.06 4 Sensitized animals 1 (%) after challenge with 0.3% Sensitized animals 1 (%) after challenge with 1% 33.3 2 100 2 13.3 3 86.7 3 25 4 91.7 4
	1 Ref. (16) 2 induction was performed with 1.5% lim-2-OOH (38)
	2 Ref. (15) 3 induction was performed with 1% cum-OOH (38)
	3 Ref. (17) 4 induction was performed with 3% cyc-hex-OOH (31)
	4 Ref. (37)

Funding:

The project (DEFCHEMSKALL) is supported by the Deutsche Forschungsgemeinschaft (DFG 284100702, BL360-6) and the Agence Nationale de la Recherche (ANR-15-CE15-0023-01).

Conflict of interest:

The authors declare that there are no conflicts of interest.