Fatma Sahli

Amélie Godard

Bertrand Vileno

Jean-Pierre Lepoittevin

Elena Giménez-Arnau

Pascal Godard

Pascal Gimenez-Arnau

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin

Keywords: cumene hydroperoxide, skin sensitizers, 13C-substitution, EPR spintrapping spectroscopy, reconstructed human epidermis cumene hydroperoxide, skin sensitizers, 13 C-substitution, EPR spin-trapping spectroscopy, reconstructed human epidermis

Introduction

Cumene hydroperoxide (CumOOH) is produced mainly through the oxidation of cumene in the socalled "cumene process", an industrial procedure for synthesizing phenol and acetone from benzene and propylene based on the acid-catalyzed Hock rearrangement [START_REF] Yaremenko | Rearrangements of organic peroxides and related processes[END_REF]. CumOOH is used as catalyst, curing agent, and initiator in the manufacture of polymers and fiber-reinforced plastics. Plus, it is a strong oxidizing agent making it an important intermediate in the fabrication of epoxy and polyester resin coatings. However, dermal exposure to CumOOH during manufacturing processes is an important toxicological issue for the industry, currently lacking of regulatory guidelines for occupational exposure limits [START_REF] Rider | Dermal exposure to cumene hydroperoxide: assessing its toxic relevance and oxidant potential[END_REF]. CumOOH genotoxicity, mutagenic action and ability to promote skin tumor and induce epidermal hyperplasia have been described [START_REF] Lai | Carcinogenic potential of organic peroxides: prediction based on structure-activity relationships (SAR) and mechanism-based short-term tests[END_REF][START_REF] Hanausek | Exposure of mouse skin to organic peroxides: subchronic effects related to carcinogenic potential[END_REF][START_REF] Brink | Biological significance of DNA adducts: comparison of increments over background for various biomarkers of genotoxicity in L5178Y tk(+/-) mouse lymphoma cells treated with hydrogen peroxide and cumene hydroperoxide[END_REF]. CumOOH is also known to cause allergic contact dermatitis (ACD) and has been reported as a significant sensitizer in guinea pigs, with positive specific responses reported in allergy cross-reactivity studies with allergenic cyclohexene hydroperoxides [START_REF] Bråred Christensson | Hydroperoxides form specific antigens in contact allergy[END_REF]. These toxic effects are believed to be mediated through the activation of the hydroperoxide to radical species such as hydroxyl (HO • ), alkoxyl (CumO • ) and alkyl (R • ) radicals.

Electron paramagnetic resonance (EPR) together with spin-trapping (ST) is a powerful combination for the characterization of transient radicals in chemical and biological systems [START_REF] Lauricella | Détection et caractérisation de radicaux libres après piégeage de spins[END_REF].

Basically, the formed radical (either too short-lived or of too low concentration to be directly detected) reacts with a diamagnetic reagent (spin-trap) to form a more persistent radical (spin-adduct), whose EPR signature can help the characterization of the trapped transient reactive species. EPR-ST has been used for dermatological purposes mainly for the detection of free radicals in UV-irradiated skin, melanoma investigation and in vivo EPR imaging directly in skin of human volunteers [START_REF] He | In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin[END_REF][START_REF] Jurkiewicz | EPR detection of free radicals in UV-irradiated skin: mouse versus human[END_REF][START_REF] Plonka | Electron paramagnetic resonance as a unique tool for skin and hair research[END_REF]. EPR-ST has also been used to characterize radicals in isolated mouse keratinocytes and in murine skin treated with CumOOH [START_REF] Taffe | Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes[END_REF][START_REF] Timmins | Free radical formation in murine skin treated with tumour promoting organic peroxides[END_REF][START_REF] Timmins | Free radical formation in isolated murine keratinocytes treated with organic peroxides and its modulation by antioxidants[END_REF]. In addition, EPR-ST proved the formation of in vivo lipid-derived free radicals triggered by CumOOH when mouse skin was exposed causing severe oxidative stress [START_REF] Shvedova | Antioxidant balance and free radical generation in vitamin E-deficient mice after dermal exposure to cumene hydroperoxide[END_REF][START_REF] Shvedova | Pro/antioxidant status in murine skin following topical exposure to cumene hydroperoxide throughout the ontogeny of skin cancer[END_REF]. To understand ACD mechanisms for risk assessment purposes, we have proved that EPR-ST is also a powerful technique for the detection and identification of radical species derived from sensitizing hydroperoxides (ROOHs) present in consumer products [START_REF] Sahli | Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) on the epidermal environment[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][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]. To be skin sensitizers, ROOHs need to covalently bind via radical processes to skin proteins after having penetrated the epidermis, to form this way the antigenic entity trigger of the immunotoxic process. As for the CumOOH tumor promoting action, initial cleavage of the O-O bond forms unstable RO • /ROO • radicals that further rearrange to longer half-life carbon centered radicals R • proposed to be at the origin of the antigen formation [START_REF] Shvedova | Pro/antioxidant status in murine skin following topical exposure to cumene hydroperoxide throughout the ontogeny of skin cancer[END_REF][START_REF] Bråred-Christensson | Limonene hydroperoxide analogues differ in allergenic activity[END_REF][START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC prospective study[END_REF]. Still, it is not still clear which R • are precisely formed in the epidermis and thus involved in the sensitization processes.

We have recently reported an EPR-ST methodology allowing to investigate the formation of free radicals issued from skin allergens in a reconstructed human epidermis (RHE) 3D model, thus much closer to what may happen in vivo, by using CumOOH as proof of concept [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF]. RHE are multi-layered keratinocyte cultures, major cell type in the epidermis playing a key role in skin inflammatory reactions. We disclosed that in the EpiSkin TM RHE model carbon radicals are mainly formed, both by incubating the assay medium and by topical application with CumOOH. Previous in vitro EPR-ST studies reported in keratinocytes cultures suggested these carbon radicals as being methyl radicals issued from -scission fragmentation of tertiary alkoxyl radicals (CumO • ) formed through cleavage of the O-O bond (Scheme 1) [START_REF] Plonka | Electron paramagnetic resonance as a unique tool for skin and hair research[END_REF]. Unlike keratinocytes cultures isolated from skin biopsies, EpiSkin TM RHE is an in vitro reconstructed human epidermis histologically similar to in vivo human epidermis. It is a complex 3D structure including a stratum corneum that allows assessing skin penetration concerns with phospholipids and ceramides content influencing the RHE barrier properties [START_REF] Netzlaff | The human epidermis models EpiSkin ® , SkinEthic ® and EpiDerm ® : an evaluation of morphology and suitability for testing phototoxicity, irritancy, corrosivity, and substance transport[END_REF]. The aim of the present study was to elucidate, in the skin allergy context and in conditions closer to real-life sensitization, if methyl radicals are also formed in EpiSkin TM RHE by using 13 C-substituted CumOOH derivatives 1 ( 13 C-MeCumOOH) and 2 (( 13 C-Me) 2 CumOOH) (Figure 1), and thus be able to suggest their participation in the formation of the immunogenic complex inducing ACD. 13 C-substituted CumOOHs 13 C-MeCumOOH 1 and ( 13 C-Me) 2 CumOOH 2 were synthesized 13 C-substituted at the methyl positions following the synthetic pathway shown in Scheme 2.

Materials and methods

Synthesis of

Chemicals and instrumentation

Starting materials were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) and used as received. 13 C-Labeled reagents and deuterated solvents were purchased from Euriso-Top (Saint Aubin, France). Air-/moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. Solvents were dried on alumina, under argon, using a Glass Technology GTS 100 device. Reactions were followed by thin layer chromatography (TLC) on 0.25 mm silica gel plates (60F 254 ; Merck, Darmstadt, Germany). After migration, the plates were inspected under UV light (254 nm) or sprayed with a solution containing phosphomolybdic acid (5 g), cerium (IV) sulfate (2 g) and sulfuric acid (12 mL) in water (188 mL), followed by heating. Column chromatography purifications were performed on silica gel 60 (Merck, Geduran ® , 40-63 m). Neutralized silica was prepared by adding to a homogenous water solution of silica gel 60 a saturated solution of NaHCO 3 until pH about 8. After decantation the silica precipitate was washed with water to reach a pH of 7, filtered then dried in a oven for at least 24 h. 1 H and 13 C NMR spectra were recorded on a Bruker Avance 500 spectrometer at 500 MHz and 125 MHz, respectively. Chemical shifts () are reported in ppm and are indirectly referenced to TMS via the solvent signal (CDCl 3 :  1 H  7.26,  13 C  77.16). Spectra multiplicities are denoted as d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet).

Synthesis of 1-( 13 C)acetophenone (4)

To a suspension of aluminum chloride (2.07 g, 15.5 mmol, 1.4 eq) in anhydrous benzene (6 mL) at 0°C, 2-( 13 C)acetyl chloride was added drop wise (0.8 mL, 11.07 mmol, 1 eq) under argon atmosphere. Once the addition completed, the mixture was stirred for 17 h at room temperature. After that time, the mixture was placed into an ice bath and distilled water (6 mL) was added drop wise until it became white, followed by addition of HCl 2 M (12 mL). The two phases were separated, and the aqueous phase was extracted with diethyl ether (4 × 40 mL). The organic phases were combined, washed with brine, dried over MgSO 4 , filtered and concentrated to obtain 1-(

Synthesis of 1-( 13 C)2-phenylpropan-2-ol (5)

In a two-necked flask under argon 1-( 13 C)acetophenone (4) (450 mg, 3.71 mmol, 1 eq) was dissolved in 6 mL of anhydrous tetrahydrofuran at -78 °C. Methyllithium (2.8 mL, 4.46 mmol, 1.6 M in Et 2 O, 1.2 eq) was added drop by drop and the mixture was stirred at -78 °C for 21 h then at room temperature for 5 h. Ammonium chloride was added (7 mL) and the solution was extracted with diethyl ether (4 × 20 mL). Organic phases were combined, dried over MgSO 4 , filtered and concentrated under reduce pressure to give an orange oil. The crude product was purified by chromatography on silica gel (pentane/EtOAc 9/1 then 5/5) to afford 1-( 13 C)2-phenylpropan-2-ol (5) as a yellow oil (269.4 mg, 1.96 mmol, 53 % yield). 1 H NMR (CDCl 3 ) δ 1.59 (d, 3H, 1 J H1-C1 = 126.5 Hz, H1), 1.59 (d, 3H, 3 J H3-C1 = 4.1 Hz, H3), 7.24-7.27 (m, 1H, H7), 7.34-7.37 (m, 2H, H6 and H8), 7.49-7.51 (m, 2H, H5 and H9); 13 C NMR (CDCl 3 ) δ 31.9 (2C, C1 and C3), 72.7 (d, 1 J C2-C1 = 39.3 Hz, C2), 124.5 (2C, C5 and C9), 126.8 (C7), 128.4 (2C, C6 and C8), 149.2 (C-4).

Synthesis of 1-( 13 C)cumene hydroperoxide (1)

To an aqueous solution of hydrogen peroxide (30 %, 12.7 mL) at 0°C, one drop of concentrated sulfuric acid was added.

Synthesis of 1,3-( 13 C)2-phenylpropan-2-ol (6)

Previous synthesis of ( 13 C)methylmagnesium iodide: in a 50 mL two-necked flask under argon, magnesium (305 mg, 12.53 mmol, 1.2 eq) was flame-dried before to add a spatula tip of iodine and anhydrous diethyl ether (10 mL). ( 13 C)Methyl iodide (0.65 mL, 10.44 mmol, 1 eq) was then added to the reaction mixture heated slightly with a hairdryer to initiate the reaction. Diethyl ether was added (10 mL) and the reaction heated again slightly. The reaction mixture was stirred at room temperature during 24 h. A solution of ( 13 C)methylmagnesium iodide 0.5 M in diethyl ether was in this way obtained.

In a two-necked flask under argon, zinc chloride (79.1 mg, 0.58 mmol, 0.2 eq) was placed and dried under reduced pressure. Lithium chloride (295.0 mg, 6.94 mmol, 2.4 eq) was added and the flask was again dried under reduced pressure. To the mixture, (trimethylsilyl)methylmagnesium chloride (2.30 mL, 1.2 mmol, 0.4 eq) was added drop wise at room temperature and the reaction was stirred for 15 min. ( 13 C)Methylmagnesium iodide (10 mL, 4.9 mmol, 0.5 M in diethyl ether, 1.7 eq) was then added drop wise and the reaction stirred for 45 min and then cooled to 0°C. 1-( 13 C)Acetophenone (4) (350 mg, 2.89 mmol, 1 eq) was added during 1 h with the use of a syringe pump and the reaction was stirred for 3 h at 0°C. After this time, the reaction mixture was quenched with ammonium chloride (10

Synthesis of 1,3-( 13 C)cumene hydroperoxide (2)

To an aqueous solution of hydrogen peroxide (30 %, 7.5 mL) was added at 0°C one drop of concentrated sulfuric acid. After 30 min, 1,3-( 13 C)2-phenylpropan-2-ol (6) (155.9 mg, 1.13 mmol, 1 eq) was added and the reaction mixture was vigorously stirred at 0°C during 4 h, followed by extraction with ethyl acetate (3 × 10 mL). The organic layers were combined, dried over MgSO 4 , filtered, and concentrated under reduce pressure. The crude product was purified by flash chromatography on neutralized silica gel (pentane/EtOAc 5/1 and 1/1) to obtain 1,3-( 13 C)cumene hydroperoxide (2) as an orange oil (36.7 mg, 0.24 mmol, 21 % yield). 1 H NMR (CDCl 3 ) δ 1.62 (d, 3H, 1 J H1/3-C1/3 = 123.8 Hz, H1 or H3), 1.62 (d, 3H, 1 J H1/3-C1/3 = 132.1 Hz, H1 or H3), 7.29-7.32 (m, 1H, H7), 7.37-7.41 (m, 2H, H6 and H8), 7.47-7.49 (m, 2H, H5 and H9); 13 C NMR (CDCl 3 ) δ 26.2 (2C, C1 and C3), 84.1 (t, 1 J C2-C1/3 = 39,9 Hz, C2), 125.5 (2C, C5 and C9), 127.6 (C7), 128.7 (2C, C6 and C8), 144.7 (C4).

EPR studies in solution

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

DEPMPO was synthesized as reported in the literature [START_REF] Barbati | Minisci F. Ed. Free Radicals in Biology and Environment[END_REF]. HEPES (≥ 99.5 %), ferrous sulfate heptahydrate FeSO 4 .7H 2 O and acetonitrile CH 3 CN (99.8%) were acquired from Sigma-Aldrich (Saint-Quentin Fallavier, France). Aqueous solutions were prepared with deionized water. HEPES buffer

EPR studies in reconstructed human epidermis

EpiSkin TM (Lyon, France; 0.38 cm 2 ) is a RHE from normal human keratinocytes cultured for 13 days on a collagen matrix at the air-liquid interface. Immediately after arrival in the laboratory, the RHE were removed from the agarose-nutrient solution in the shipping multiwell plate under a sterile airflow.

They were 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, at least 24 h before incubation. EpiSkin TM (Lyon, France) furnished the assay medium used for incubations. RHE were topically treated first with DEPMPO (20 L, 250 mM in HEPES) and post incubated (37 °C, 5% CO 2 ) during 15 min. After the incubation time, the RHE were placed in the EPR tissue cell and the hydroperoxide (20 L, 50 mM in acetone) was applied to the epidermis taking care to ensure that the solution was only applied to it. EPR spectra were then recorded. 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 5 mW, modulation amplitude 0.5-1 G, and conversion time of ca. 160 ms resulting sweep time of ca. 3 min s for a single scan. Spectra were recorded at room temperature (295K±1K). Regarding the investigations in solution, samples were introduced into glass capillaries (Hirschmann, 25 L), sealed at both ends and transferred into the EPR cavity for measurement as fast as possible after reagents mixing. RHE were placed in an EPR tissue cell equipped with a silica window (Willmad, #ER162TC-Q) and resulting EPR spectra recorded in situ right afterwards. Up to 10 scans were accumulated to improve the signal-to-noise ratio (S/N).

EPR instrumentation

EPR simulations

All experimental 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]. Relevant 14 N, 1 H, 31 P, 13 C hyperfine coupling constants (hfccs) of detected spin-adducts are listed in Table 1. Coupling constants in RHE were calculated following the best simulation attempts for such S/N.

Results

EPR studies in solution

Analyses in solution were initially carried out to evaluate radicals formed from 13 C-substitued CumOOHs in comparison with previous studies we achieved with non-substituted CumOOH [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF].

DEPMPO was chosen as the spin-trap since we observed previously that it allowed a broad view of all sort of radicals deriving from CumOOH [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF]. Radical initiation was triggered by a catalytic amount of Fe(II) (0.1 mM) inducing a Fenton-like reaction. DEPMPO was employed in an excess concentration Computer simulation of the signal displayed a mixture of different spin-adducts formed by addition of transient short-lived radicals derived from 1 to the -carbon of DEPMPO nitronyl group. In agreement with our previous investigations on CumOOH [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF], spin-adducts were identified through their hyperfine coupling constants (hfccs) [START_REF] Frejaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals[END_REF][START_REF] Karoui | DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water[END_REF] as originating from HO • (Figure 2(e)), carbon centred radicals, probably methyl (Figure 2(f)), methoxy and/or possible peroxyl radicals (Figure 2(h)). But here, when compared to the non-substituted sample, an additional hyperfine splitting appeared (Figure 2(g)), attributed to the presence of the paramagnetic probe of a 13 C nucleus with nuclear spin I = ½.

Analysis of the spectrum leaded to hfccs values of a N (G) = 15.5, a H (G) = 22.6, a P (G) = 48.3 and a new value a 13C of 6.3 G (Table 1). As only half of methyl radicals were 13 C-substituted, it was possible to observe two spin-adducts corresponding to two types of carbon radicals trapped with similar a N , a H and a P hfccs, being either non-substituted (Figure 2(f)) or 13 C-substituted (Figure 2(g)). There are only few examples in the literature describing hfccs for 13 C atoms, for example the description of p-benzyne radicals [START_REF] Usuki | Spin trapping of 13 C-labeled p-benzynes generated by Masamune-Bergman cyclization of bicyclic nine-membered enediynes[END_REF], -substituted ethyl radicals [START_REF] Griller | On the conformation of β-substituted ethyl radicals[END_REF] or some vinyl and allyl radicals [START_REF] Griller | Kinetic applications of electron paramagnetic resonance spectroscopy. XVIII. Persistent vinyl, alkyl, and allyl radicals[END_REF]. However, no reference was found on hfccs values for the coupling with 13 C atoms on  position of a nitroxide radical. Thus, to corroborate that 13 C-methyl radicals had been trapped by DEPMPO, studies were conducted with di-substituted ( 13 C-Me) 2 CumOOH 2 having 13 C-substituted methyl groups exclusively.

EPR studies in reconstructed human epidermis

In order to carry out the experiments, the RHE tissue was loaded beforehand with the spin-trap. As described previously in studies with tumour promoting peroxides [START_REF] Timmins | Free radical formation in murine skin treated with tumour promoting organic peroxides[END_REF], radicals formed from CumOOH in the skin model being both short lived and potentially generated at low concentrations, it was desirable for the spin-trap to be as close as possible to the site of radical generation prior to the topical application of the hydroperoxides. If similar EPR fingerprints to those of the experiments in solution were obtained, it was noticeable that other features did not fully overlay with any of the solution studies. Such differences can be explained by the much more complex system formed by the RHE when compared to simplified in solution investigations. Figure 4 shows the spectra obtained in solution along and in comparison with the spectra obtained in RHE for the three hydroperoxides tested. If the appearance of the spin-adduct resulting from trapping the radical 13 C-substituted in RHE was barely visible when using 13 C-MeCumOOH 1 (Figure 4(f)), it became more evident with ( 13 C-Me) 2 CumOOH 2 (Figure 4(g)). Major peaks could be assigned to DEPMPO-OH, DEPMPO-CH 3 and DEPMPO-13 CH 3 spin-adducts by comparison with previous data in solution (Table 1) [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF]. Again, the difference observed when comparing the hfccs values with those obtained in the EPR-ST solution studies could be explained by the distinct environment of the spin-adducts in solution compared to that of a complex heterogeneous tissue such as the RHE. Indeed, magnitudes of the hfccs pointing to the extent of delocalization of the unpaired electron over the molecule may be affected by the local spin environment and polarity. Moreover, control experiments were carried out for each compound by 13 simply incubating DEPMPO in the RHE and showed no EPR signal, corroborating that spin-adducts resulted from trapping radicals issued from the hydroperoxide. Also, after penetrating the RHE, CumOOH radical initiation has been induced by RHE matrix itself as no Fe(II) was supplemented, thus better mimicking real life exposure scenarios. These results suggested methyl radicals being formed in RHE after CumOOH exposure.

Discussion

In a recent work, we employed EPR-ST for the first time in the skin allergy field to study the potential of this methodology to investigate in situ the formation of free radicals from skin allergens in 3D RHE models [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF]. CumOOH was used as proof of concept and we demonstrated the formation of carbon radicals in RHE. However, no evidence could be obtained concerning their specific nature. In the present work, this was investigated by using 13 C-substitution as a tool to unravel which carbon radicals could be formed within the epidermis. As the major mechanism described for the formation of carbon radicals from CumOOH is -scission of cumyl oxyl radicals affording methyl radicals, we developed the synthesis of CumOOH containing either one ( 13 C-MeCumOOH 1) or both (( 13 C-Me) 2 CumOOH 2) methyl groups 13 C-substituted and we further studied the formation of free radicals in solution and in

RHE.

Preliminary studies in solution allowed setting up the optimal experimental conditions providing a decent signal-to-noise ratio while keeping low CumOOHs concentrations, i.e. suitable with physiological conditions. DEPMPO was chosen as an accurate spin-trap based upon (i) the high persistency of its superoxide and alkylperoxyl spin-adducts when compared to other spin-traps (e.g. DMPO); (ii) the clear distinction between oxygen-and carbon-derived spin-adducts [START_REF] Frejaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals[END_REF][START_REF] Stolze | Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase[END_REF]. Radical initiation was performed via a Fe(II) Fenton-like reaction. As for CumOOH, carbon centered, CumO • and HO • radicals were identified for 13 CumOOH. Thus, when all methyl groups were 13 C-substituted, only DEPMPO-13 CH 3 spin-adducts were detected confirming that methyl radicals were the carbon-centered trapped radicals. This information was of high relevance for further studies in RHE. RHE EpiSkin TM shows reasonable similarities to native human tissue in terms if morphology, stratum corneum presence, lipid composition and biochemical markers [START_REF] Netzlaff | The human epidermis models EpiSkin ® , SkinEthic ® and EpiDerm ® : an evaluation of morphology and suitability for testing phototoxicity, irritancy, corrosivity, and substance transport[END_REF]. It is today a useful tool for testing irritation and penetration features of chemicals in contact with the skin as replacement of in vivo testing [START_REF]vitro skin irritation: RHE test method[END_REF]. For our studies on skin allergy the availability of EpiSkin TM RHE is a very good alternative to the use of human and animal tissues as the model amends important legal and ethical issues. Although considerably more permeable than human skin on average, the model appears to be more consistent in permeability and responsiveness than human skin which is highly variable. Similar EPR-ST results were observed in RHE when testing CumOOH, 13 C-MeCumOOH 1 and ( 13 C-Me) 2 CumOOH 2. Though, new features in the RHE samples did not fully overlay with the reference spectra in solution and were ascribed to the complexity of the RHE system. Even if the S/N was significantly lower when compared to solution investigations and thus did not allow quantification of the spin-adducts, we can still suggest that methyl radicals issued from CumOOH decomposition can be formed in RHE EpiSkin TM . Indeed, spectrum of the RHE experiment with twice 13 C-labeled CumOOH (Figure 4(g)) was rather convincing as an additional spin-adduct assigned to DEPMPO-13 CH 3 spinadduct (black circles) was detected. Moreover, control experiments with DEPMPO, in absence of 13 were not the purpose of the present study and will be part of future investigations. Worthy of note, no Fe(II) was used for radical initiation when probing RHE, to mimic real life skin allergy situation, whereas we previously showed that Fe(II) was required to initiate the free radical generation when mixing CumOOH and DEPMPO in solution [START_REF] Kuresepi | Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept[END_REF]. As it is shown in Figure 5(d), no EPR signal was observed in the absence of Fe(II), whereas 0.1 mM Fe(II) was enough to initiate the radical reaction with a Fe(II) concentration dependent answer on the amount of spin-adducts formed. We can then hypothesize that reaction of organic peroxides in the skin in the presence of one-electron donor agents (i.e. amino and thiol groups present in amino acids, metal complexes, enzymes) may initiate the electron transfer processes and further skin proteins haptenation [START_REF] Moriarty-Craige | Extracellular thiols and thiol/disulphide redox in metabolism[END_REF][START_REF] Fujimoto | Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation controls[END_REF][START_REF] Grossi | The mechanism of oxidation of aniline by peroxy-compounds: can EPR spectroscopy help to discriminate between one-electron transfer and a nucleophilic substitution process?[END_REF]. Also, the potential presence in the skin of Fe(III) heme-containing enzymes could be responsible for the peroxide O-O bond cleavage and start radical processes. Reactions of model Fe(III) porphyrin complexes with oxidants, such as peroxyacids and hydroperoxides, have been extensively studied as models. The cleavage has been confirmed but a clear mechanistic consensus has not evolved on the homolytic or heterolytic nature of the O-O bond cleavage by these species, both kinds of mechanisms being possible [START_REF] Traylor | Reactions of hydroperoxides with iron (III) porphyrins: heterolytic cleavage followed by hydroperoxide oxidation[END_REF][START_REF] Almarsson | A Homolytic mechanism of O-O bond scission prevails in the reactions of alkyl hydroperoxides with an octacationic tetraphenylporphinato-iron(III) complex in aqueous solution[END_REF][START_REF] Labeque | Homolytic and heterolytic scission of organic hydroperoxides by (mesotetraphenylporphinato)iron(III) and its relation to olefin epoxidation[END_REF].

While skin allergens (haptens) are unable to stimulate an adaptive immune response after penetration into the epidermis, the immunogenicity is reached by reaction with skin proteins, to form stable antigenic conjugates recognized and processed for presentation to the immune system [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF].

Therefore, the hapten-protein interaction is the primary key step to induce sensitization and was shown to occur relatively quickly in RHE [START_REF] Elbayed | HR-MAS NMR spectroscopy of reconstructed human epidermis: potential for the in situ investigation of the chemical interactions between skin allergens and nucleophilic amino acids[END_REF]. The resulting sequence of events leading epidermal cells to present the antigen so formed to the immune system and its activation take 10 to 15 days in humans.

The fast trapping by DEPMPO allowed EPR-ST characterization of the generated methyl radicals but the reaction with epidermal proteins was not evidenced. Thus, if we cannot testify so far that these radicals are the reacting species for the antigen formation we can strongly suggest their involvement.

Additional tests to decipher their reactivity towards amino acids in the skin could be isolation from

Conclusion

This work shows by using 13 C-substituted compounds and EPR-ST that the production of methyl radicals from CumOOH previously reported in isolated keratinocytes and other model systems is also possible in reconstructed skin tissue through a one-electron reductive pathway, and suggest that these methyl radicals could be involved in the antigen formation responsible for skin sensitization to CumOOH.

F

  After 30 min stirring, 1-( 13 C)2-phenylpropan-2-ol (5) (268.1 mg, 1.95 mmol, 1 added and the reaction mixture was vigorously stirred at 0°C during 4 h, followed by extraction with ethyl acetate (3 × 10 mL). The organic layers were combined, dried over MgSO 4 , filtered, and concentrated under reduce pressure. The crude product was purified by flash chromatography on neutralized silica gel (pentane/EtOAc 85/15 then 50/50) to obtain 1-( 13 C)cumene hydroperoxide (1) as an orange oil (136.0 mg, 0.88 mmol, 32 % yield).1 H NMR (CDCl 3 ) δ 1.62 (d, 3H, 3 J H3-C1 = 4.4 Hz, H3), 1.62 (d, 3H, 1 J H1-C1 = 127.7 Hz, H1), 7.29-7.34 (m, 2 × 1H, H6 and H8), 7.38-7.41 (m, 1H, H7), 7.47-7.49 (m, 2 × 1H, H5 and H9); 13 C NMR (CDCl 3 ) δ 26.2 (2C, C1 and C3), 84.1 (d, 1 J C2-C1 = 39.9 Hz, C2), 125.5 (2C, C5 and C9), 127.6 (C7), 128.7 (2C, C6 and C8), 144.7 (C4).

  extracted with ethyl acetate (3 × 10 mL) and washed with brine (10 mL). The organic phases were combined and dried over MgSO 4 , filtered and concentrated under reduced pressure to obtain an orange oil. The crude product was purified by chromatography on silica gel (pentane/EtOAc 92/8) and 1,3-( 13 C)2-phenylpropan-2-ol[START_REF] Bråred Christensson | Hydroperoxides form specific antigens in contact allergy[END_REF] was obtained as a yellow oil (303.7 mg, 2.19 mmol, 76% yield).1 H NMR (CDCl 3 ) δ 1.59 (dd, 3H, 1 J H1/3-C1/3 = 121.3 Hz, 3 J H1/3-C1/3 = 1.7 Hz, H1 or H3), 1.59 (d, 3H, 1 J H1/3-C1/3 = 130.7 Hz, H-1 or H-3), 7.25-7.28 (m, 1H, H7), 7.34-7.37 (m, 2H, H6 and H8), 7.50-7.51 (m, 2H, H5 and H9);13 C NMR (CDCl 3 ) δ 31.8 (2C, C1 and C3), 74.0 (t, 1 J C2-C1/3 = 38.9 Hz, C2), 124.5 (2C, C5 and C9), 126.8 (C7), 128.3 (2C, C6 and C8), 149.2 (C4).

  mM, pH 6.8) was prepared by dissolving 1.19 g HEPES in 400 mL deionized water, with additional 4 g NaCl and 0.1 g KCl. To attain pH 6.8 NaOH pellets were added. If the pH went too high, it was lowered back by carefully adding HCl (2 M) until pH remained stable to 6.8. Deionized water was added for a final volume of 500 mL. Stock solutions were prepared for CumOOH,13 C-MeCumOOH and ( 13 C-Me) 2 CumOOH (10 mM, HEPES/CH 3 CN 9/1), DEPMPO (100 mM in HEPES) and FeSO 4 .7H 2 O (10 mM in HEPES). 12.5 L of DEPMPO solution were mixed with FeSO 4 .7H 2 O (0.5 L for a final 0.1 mM concentration in the reaction mixture), 5 L of the hydroperoxide solution added and final volume completed to 50 L. This way, final concentrations in the reaction mixture were 25 mM spin-trap and 0.54 mM hydroperoxide. The reaction mixture was subjected to stirring and further introduced into the EPR quartz capillary tube.

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

11 (

 11 25 mM) with respect to13 C-substitued hydroperoxides (0.54 mM). First studies were completed with mono-substituted13 C-MeCumOOH 1. Figure2(c) shows the experimental EPR spectrum obtained.

Figure 3 (

 3 Figure 3(c) shows the experimental EPR spectrum obtained. Here, computer simulation displayed a

  C-MeCumOOH 1 and ( 13 C-Me) 2 CumOOH 2. A new signal case of mono-substituted 1 with hfccs a N (G) = 15.5, a H (G) = 22.6, a P (G) = 48.3 and a 13C (G) = 6.3, whose fingerprint significantly increased in the case of the double-substituted compound 2, fully replacing the signal of carbon centered radicals trapped from non-substituted

C- MeCumOOH 1

 1 or ( 13 C-Me) 2 CumOOH 2, did not exhibit any EPR signal (Figure 4(d)), suggesting that the trapped • CH 3 radicals originate from the hydroperoxide. Initially formed alkoxyl radicals derived from the O-O bond cleavage and giving the • CH 3 radicals by -scission could also react with biomolecules in the RHE before DEPMPO trapping occurred. However, these potential side-reactions

  proteins (reactivity in the absence of spin-trap) by sonication followed by mass spectrometry analysis and will be addressed in a near future.

  Figure 3

Figure 4 Page 26 of 28 Site 28 LegendsScheme 1 .Scheme 2 .

 4282812 Figure 4

Figure 1 .

 1 Figure 1. Chemical structures of 13 C-MeCumOOH 1 and ( 13 C-Me) 2 CumOOH 2.

Figure 2 .

 2 Figure 2. EPR spectra of 13 C-MeCumOOH 1/DEPMPO/Fe(II) in solution. (a) Control with single DEPMPO (25 mM); (b) Control with DEPMPO (25 mM) and Fe(II) (1 mM); (c) Experimental spectrum obtained with 13 C-

Figure 3 .

 3 Figure 3. EPR spectra of ( 13 C-Me) 2 CumOOH 2/DEPMPO/Fe(II) in solution. (a) Control with single DEPMPO (25 mM); (b) Control with DEPMPO (25 mM) and Fe(II) (1 mM); (c) Experimental spectrum obtained with ( 13 C-Me) 2 CumOOH 2 (0.54 mM), DEPMPO (25 mM) and Fe(II) (0.1 mM); (d) Computer simulation of spectrum (c); (e-g) deconvolution of (d) with: (e) DEPMPO-OH spin-adduct (55%); (f) DEPMPO-13 CH 3 spinadduct (42%); (g) DEPMPO-OCH 3 and/or DEPMPO-OOCum spin-adduct (3%).

Figure 4 .

 4 Figure 4. Comparative experimental EPR spectra in solution (a-c) and in RHE (d-g): (a) CumOOH (0.54 mM)/DEPMPO (25 mM)/Fe (II) (0.1 mM) [21]; (b) 13 C-MeCumOOH 1 (0.54 mM)/DEPMPO (25 mM)/Fe (II) (0.1 mM); (c) ( 13 C-Me) 2 CumOOH 2 (0.54 mM)/DEPMPO (25 mM)/Fe (II) (0.1 mM); (d) DEPMPO (250 mM)/RHE; (e) CumOOH (10 mM)/DEPMPO (250 mM)/RHE; (f) 13 C-MeCumOOH 1 (50 mM)/DEPMPO (250 mM)/RHE; (g) ( 13 C-Me) 2 CumOOH 2 (50 mM)/DEPMPO (250 mM)/RHE. Because of the low signal-to-noise ratio, DEPMPO-OCH 3 and/or DEPMPO-OOCum spin-adducts are no indicated for clarification purposes. Spinadducts are indicated as follows: DEPMPO-OH white circle, DEPMPO-CH 3 star and DEPMPO-13 CH 3 black circle. Dotted arrows indicate the formation of signals related to DEPMPO-13 CH 3 spin-adduct in RHE.

Figure 5 .

 5 Figure 5. EPR experimental spectra in solution of CumOOH (0.54 mM)/DEPMPO (25 mM) with varying

Table 1

 1

	o r
	P
	e
	e r
	-
	R
	e v
	i
	e w
	O n
	l y

Table 1 .

 1 Hyperfine coupling constants (hfccs) of spin-adducts identified in solution (sol.) and in RHE*

	Cpd.		Coupling constants		Assignment
		a N (G)	a H (G)	a P (G) a 13C (G)	g
	CumOOH	14.2	13.5	47.4		2.0055	DEPMPO-OH
	(sol.) [21]	15.4	22.6	48.1		2.0053	DEPMPO-carbon R
		11.3	13.7	48.5		2.0055	DEPMPO-OCH 3 and/or
							DEPMPO-OOCum
	1 (sol.)	14.2	13.3	47.3		2.0056	DEPMPO-OH
		15.4	22.6	48.0		2.0053	DEPMPO-carbon R
		15.5	22.6	48.3	6.3	2.0053	DEPMPO-13 CH 3
		11.3	13.7	48.2		2.0055	DEPMPO-OCH 3 and/or
							DEPMPO-OOCum
	2 (sol.)	14.2	13.4	47.4		2.0053	DEPMPO-OH
		15.4	22.6	48.3	6.4	2.0051	DEPMPO-13 CH 3
		11.6	14.4	48.1		2.0055	DEPMPO-OCH 3 and/or
							DEPMPO-OOCum
	CumOOH	12.5	13.6	46.0		2.0064	DEPMPO-OH
	(RHE)	14.8	21.4	48.0		2.0058	DEPMPO-carbon R
	1 (RHE)	12.9	13.7	46.0		2.0064	DEPMPO-OH
		14.5	21.2	47.3		2.0056	DEPMPO-carbon R
		14.9	21.6	47.9	6.2	2.0056	DEPMPO-13 CH 3
	2 (RHE)	13.1	14.0	46.4		2.0063	DEPMPO-OH
		15.1	21.4	48.2	6.4	2.0059	DEPMPO-13 CH 3

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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 authors thank the University of Strasbourg, the CNRS and the REseau NAtional de Rpe interDisciplinaire (RENARD, Fédération IR-RPE CNRS #3443).

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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

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

Background. An increasing incidence of contact allergy to methylisothiazolinone (MI) has been seen caused, in particular, by cosmetic products and paints. A study from 2014 showed that 93.0% of paints bought in five European countries contained MI. New regulations have been discussed for paints in EU, which may have influenced this market.

Objectives. To re-evaluate the use and concentrations of MI and four other isothiazolinones in water-based wall paints.

Methods.

Water-based white wall paints (n = 60) were purchased in retail stores in five European countries: Denmark, France, Germany, Sweden and UK. The paints were analyzed for content of isothiazoliones using high-performance liquid chromatography coupled to UV detection and confirmed with high-performance liquid chromatography-tandem mass spectrometry.

Results. MI was identified in 55 (91.7%) of the paints, with concentrations from 1.1 to 142.7 ppm.

The other isothiazolinones were identified in 20.0% (methylchloroisothiazolinone; MCI) to 88.3% (benzisothiazolinone; BIT) of the paints. BIT concentrations varied significantly between countries, whereas MI and MCI contents did not. There were no statistically significant differences between MI, MCI and BIT concentrations compared to the 2015 study.

Conclusion.

MI and other isothiazolinones are widely used in European purchased paints. Their use does not seem to be waning.

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