Salen Kuresepi

Bertrand Vileno

Jean-Pierre Lepoittevin

Elena Giménez-Arnau

Mechanistic insights on skin sensitization to linalool hydroperoxides: EPR evidence on radical intermediates formation in reconstructed human epidermis and 13 C-NMR reactivity studies with thiol residues

Keywords: Skin sensitization, linalool hydroperoxides, radical mechanisms, reconstructed human epidermis, electron paramagnetic resonance, spin trapping, 13 C-substitution, chemical reactivity

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Mechanistic insights on skin sensitization to linalool hydroperoxides: EPR evidence on radical intermediates formation in reconstructed human epidermis and 13 C-NMR reactivity studies with thiol residues

INTRODUCTION

Linalool is one of the most commonly used fragrance terpenes in consumer products because of its flowery-fresh odor. [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] It is also found in many essential oils as lavender, ylang-ylang and rosemary oils. [START_REF] De Groot | Essential oils, part IV: contact allergy[END_REF] Linalool is one of the 26 compounds (EU Cosmetic Regulation 1223/2009) that is mandatory to label in the packaging of cosmetic products when the concentration exceeds 10 ppm in leave-on products and 100 ppm in rinse-off products. [START_REF]1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (2009) Off[END_REF] Pure linalool is considered as non-allergenic because of its very low skin sensitization potential. [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] Though, it easily autoxidizes once exposed to air. Autoxidation is a free radical chain reaction in which hydrogen atom abstraction with addition of oxygen forms hydroperoxides as primary oxidation products. Primary autoxidation products of linalool Lina-7-OOH and Lina-6-OOH (Chart 1) have been shown to be major skin sensitizers in the oxidation mixtures when tested in the murine local lymph node assay. [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] Besides, high prevalence of allergic contact dermatitis (ACD) to a mixture of these hydroperoxides (herein referred as Lina-OOHs) has been reported in several dermatological multicenter studies when patch testing consecutive dermatitis patients with autoxidized linalool at the suggested optimal patch test concentration (6% in petrolatum, containing Lina-OOHs 1% w/w). [START_REF] Bråred Christensson | Air-oxidized linalool -a frequent cause of fragrance contact allergy[END_REF][START_REF] Audrain | Allergy to oxidized limonene and linalool is frequent in the U[END_REF][START_REF] Bråred Christensson | Oxidized limonene and oxidized linalool -concomitant contact allergy to common fragrance terpenes[END_REF][START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC prospective study[END_REF][START_REF] Nath | Contact allergy to hydroperoxides of linalool and Dlimonene in a US population[END_REF] Also, a repeated open application test study indicated that oxidized linalool can elicit ACD when applied repeatedly in lower concentrations in previously sensitized individuals, as for daily use of cosmetic products. As low a concentration as 0.3% w/w of oxidized linalool (0.056% w/w of Lina-OOHs) gave reactions in participants within the test period of three weeks. [START_REF] Björkman | Air-oxidized linalool elicits eczema in allergic patients -a repeated open application test study[END_REF] ACD results from an adverse reaction of the immune system to low molecular weight chemicals. It is a T-cell mediated delayed-type hypersensitivity reaction based on a sensitization and a further elicitation phase. [START_REF] Martin | Immunological mechanisms in allergic contact dermatitis[END_REF] The OECD has approved the mechanism behind the sensitization phase as an adverse outcome pathway (AOP) built on four "key events". [START_REF]The adverse outcome pathway for skin sensitization initiated by covalent binding to proteins[END_REF] Following entry of the chemical into the skin, the opening key event is chemical. Skin sensitizers are unable to stimulate directly an immune response. Immunogenicity is attained by chemical reaction with skin proteins, forming stable antigenic conjugates that will be recognized and processed for presentation to the immune system. [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] The common process for this reaction is the formation of a covalent bond between the chemical and skin proteins via a two electrons mechanism. Yet, many allergens do not fit this pattern and there is a strong belief that radical mechanisms are involved in the antigen formation. This is especially the case for Lina-OOHs that have the potential to form antigens via radical mechanisms arising from the easy cleavage of the O-O bond when considering its weak dissociation energy (175 kJ.mol -1 ). This process affords unstable alkoxyl (RO • ) radicals, which can lead via intramolecular cyclization, fragmentation or hydrogen abstraction to the formation of more stable carbon centered radicals (RC • ). Consequently, we believe that Lina-OOHs intrinsic chemical reactivity constitutes the needed trigger that provides information to the immune system, alerting defense mechanisms of the organism. A further key event of the skin sensitization AOP is the activation of dendritic cells (DCs), antigen-presenting cells that take up and process the antigenic complex, mature and migrate to the local lymph nodes ensuring the presentation of altered peptides to naïve T-lymphocytes. We have recently shown that the THP-1 cell line, employed in the h-CLAT in vitro test as a surrogate for DCs, [START_REF] Oecd | Key event-based test guideline. In vitro skin sensitization assays addressing the key event on activation of dendritic cells on the adverse outcome pathway for skin sensitization[END_REF] is activated by Lina-OOHs with up regulation of cell surface markers (CD54, CD86) and activation of the Nrf2 pathway. 15 We also demonstrated that RO • , peroxyl (ROO • ) and RC • radicals were formed from iron activated Lina-OOHs by using electron paramagnetic resonance (EPR) and spin trapping (ST), [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] a powerful approach toward the characterization of transient radicals in chemical and biological systems. [START_REF] Lauricella | Detection and characterization of free radicals after spin trapping[END_REF] Further, we studied the reactivity toward amino acids prone to radical reactions by liquid chromatography combined to mass spectrometry, and we proved that RC • radicals issued from Lina-OOHs altered directly the lateral chain of cysteine and the thiol group of glutathione (GSH) forming adducts via radical processes after iron induced radical initiation. [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] We suggested thus the involvement of RC • intermediates in the biological answers related to Lina-OOHs sensitization process. However, EPR-ST studies were at that time carried out in solution (aqueous buffer or semi-organic), yet with experimental conditions being far away from real life sensitization, and reactivity studies did not allow to conclude on the specific carbon centered radical RC • reacting with thiol groups. To overcome these drawbacks, we report herein more evidences and mechanistic insights on Lina-OOHs mechanisms of action by assessing (i) the formation of carbon radicals in a reconstructed human epidermis (RHE) 3D model, much closer to what may happen in vivo, by EPR-ST and (ii) the molecular structure of adducts resulting from reactivity with thiol groups using [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substituted Lina-OOHs at positions precursor of radical intermediates and 13 C-NMR.

MATERIALS AND METHODS

Caution: Linalool hydroperoxides must be handled with caution, as they are skin sensitizers.

EPR Spin Trapping Studies

Studies in Solution.

A mixture of Lina-7-OOH ((5E)-7-hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol) and Lina-6-OOH (6-hydroperoxy-3,7-dimethylocta-1,7-diene-3-ol) (Lina-OOHs, ratio 2:3) was synthesized from linalool as previously described in the literature. [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] 5-Diethoxyphosphoryl-5-methyl-1pyrroline N-oxide (DEPMPO) was synthesized as reported in the literature. [START_REF] Barbati | 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. To prepare a HEPES buffer solution i.e. 10 mM at pH 6.8, 1.19 g HEPES were dissolved in 400 mL deionized water, 4 g sodium chloride and 0.1 g potassium chloride were added. To reach pH 6.8, sodium hydroxide pellets were added. If the pH went too high, it was lowered back by adding hydrochloric acid until the pH remained stable to 6.8. Finally, deionized water was added to a final volume of 500 mL. Stock solutions were prepared for Lina-OOHs (10 mM in HEPES/CH 3 CN 9/1), DEPMPO (100 mM in HEPES) and FeSO 4 .7H 2 O (10 mM in deionized water). 12.5 µL of DEPMPO 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), supplemented by 5 µL Lina-OOHs solution and final volume completed to 50 µL. This way, final concentrations in the reaction mixture were 25 mM DEPMPO and 1 mM Lina-OOHs. The reaction mixture was subjected to stirring and further transferred into a glass capillary (Hirschmann, 25 µL) sealed on both ends prior EPR investigation. EPR spectra were recorded on a X-band spectrometer (EMXplus, Bruker Biospin GmbH, Germany) equipped with a high sensitivity resonator (HSW, Bruker Biospin GmbH, Germany). The g calibration standard was Bruker strong pitch of known g factor 2.0028. The spectrometer was operated at ca. 9.8 GHz and principal spectrometer settings were 100 kHz modulation frequency, microwave power of ca. 4.5 mW, sweep width 150 G, modulation amplitude 1 G, sweep time 120 s, conversion time ca. 130 ms and time constant ca. 40 s. A couple of spectra were accumulated to ensure a decent signal-to-noise S/N ratio. Spectra were recorded at room temperature (295K±1K) as soon as possible after mixing the reagents.

Studies in Reconstructed Human

Epidermis. EpiSkin TM (Lyon, France) is a RHE from normal human keratinocytes cultured for 13 days on a collagen matrix at the air-liquid interface. The 0.38 cm 2 format was chosen. 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 µL, 250 mM in HEPES) and postincubated (37 °C, 5% CO 2 ) during 15 min. After the incubation time, the RHE were placed in an EPR tissue cell equipped with a silica window (Wilmad, #ER162TC-Q) and Lina-OOHs (20 µL, 10 mM in acetone) were applied to the epidermis taking care to ensure that the solution was only applied to it.

EPR spectra were then recorded on a X-band spectrometer (ESP300E, Bruker Biospin GmbH, Germany) equipped with a rectangular Te 102 resonator (Bruker Biospin GmbH, Germany). The g calibration standard was performed with Bruker strong pitch. The spectrometer was operated at ca. 9.8 GHz and spectrometer settings were 100 kHz modulation frequency, microwave power of ca. 5.1 mW, sweep width 160 G, sweep time 328 s, modulation amplitude 1 G and time constant of ca. 164 s.

Spectra were recorded at room temperature (295°K ± 1°K).

EPR Simulations. 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] Synthesis of ( 13 C)-Lina-OOHs Lina-OOHs (mixture Lina-7-OOH/Lina-6-OOH) were synthesized containing a [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substitution at carbon atom positions 5 or 4 (Chart 1). Here the synthesis of 5-( 13 C)-Lina-OOHs is described as it afforded the most important reactivity information. The synthesis of 4-( 13 C)-Lina-OOHs and results obtained, corroborating that information, are detailed in the Supporting Information.

Chemicals and Reagents.

Anhydrous solvents were obtained by distillation under an inert atmosphere of argon and kept on molecular sieves (4 Å). Ethanol was distilled on sodium hydroxide.

Tetrahydrofuran, diethyl ether and dichloromethane were dried through activated alumina columns. All reactions needing anhydrous conditions were carried out under argon atmosphere in flame-dried glassware. 1-( 13 C)-Acetic acid was purchased from Euriso-top (Saint-Aubin, France). All other reagents and solvents were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) or Fisher Scientific (Illkirch-Graffenstaden, France) and were used as received. Reactions were followed by thin layer chromatography (TLC) using silica gel plates (Merck 60 F 254 ; thickness 0.25 mm). After elution, the plates were revealed under short-wave UV radiation (254 nm) or immersed in a phosphomolybdic acid solution (5 g phosphomolybdic acid and 2 g cerium sulfate tetrahydrate in a mixture of 12 mL sulfuric acid and 188 mL distilled water) or a potassium permanganate solution (1.5 g potassium permanganate, 1.25 mL sodium hydroxide 10% and 10 g potassium carbonate in 200 mL water) followed by heating. Column chromatography purifications were performed using silica 60 (Merck; Geduran, 40-63 µm). 1 H and [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR spectra were recorded on Bruker Avance 300 (300 MHz for 1 H and 75 MHz for 13 C) and Bruker Avance 500 (500 MHz for 1 H and 125 MHz for 13 C) spectrometers.

Chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in Hz. Chemical shifts are indirectly referenced to TMS via the solvent signal CHCl 3 (CDCl 3 δ 1 H = 7.26 ppm; δ 13 C = 77.2 ppm). Multiplicity is denoted as s (singlet), sl (singlet large), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet) and m (multiplet).

2-Bromo-1-( 13 C)-ethyl acetate (( 13 C)-1). In a flame-dried double-necked flask under argon, to a solution of 1-( 13 C)-acetic acid (2.0 g, 33.3 mmol, 1 equiv) and phosphorus tribromide (1.60 mL, 16.66 mmol, 0.5 equiv) was slowly added bromine (6.0 mL, 116.56 mmol, 3.5 equiv). The mixture was stirred during 30 min at room temperature, then heated at 60 °C for 6 h. After that time the mixture was cooled to room temperature before anhydrous ethanol (3.6 mL, 63.28 mmol, 1.9 equiv) was added.

After one night at room temperature, the reaction mixture was hydrolyzed with ethanol (10 mL) and distilled water (30 mL). The mixture was extracted with diethyl ether (3 × 30 mL). Collected organic phases were neutralized using a saturated solution of sodium bicarbonate (2 × 30 mL) and washed with a saturated solution of sodium thiosulfate (30 mL, then 20 mL). Organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure. 2-Bromo-1-( 13 C)-ethyl acetate ( 13 C)-1 (3.67 g) was obtained as an orange oil and was used without further purification. [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.28 (t, 3H, 3 J HH =7.5 Hz, OCH 2 CH 3 ), 3.81 (d, 2H, 2 J HC =5.0 Hz, CH 2 ), 4.22 (qd, 2H, 3 J HH =7.0 Hz, 3 J HC =3.0 Hz, OCH 2 CH 3 ). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 14.1 (CH 3 ), 26.3 (d, 1 J CC =64.6 Hz, CH 2 Br), 62.4 (OCH 2 CH 3 ), 167.4 ( 13 C=O).

2-(Diethoxyphosphoryl)-1-( 13 C)-ethyl acetate (( 13 C)-2).

In a flame-dried round bottom flask under argon triethyl phosphite (4.7 mL, 26.21 mmol, 1.2 equiv) was added to ( 13 C)-1 (3.67 g, 21.84 mmol, 1equiv). The mixture was heated at 140 °C for 3 h and then cooled to room temperature. The product was purified by silica gel chromatography (EtOAc/pentane 8/2). 2-(Diethoxyphosphoryl)-1-( 13 C)-ethyl acetate ( 13 C)-2 was obtained as a yellow oil (4.95 g, 90 %). [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.26 (t, 3H, 3 J HH =7.0 Hz, OCH 2 CH 3 ), 1.32 (t, 6H, 3 J HH =7.5 Hz, 2 × OCH 2 CH 3 ), 2.94 (dd, 2H, 2 J HP =22.0 Hz, 2 J HC =7.5 Hz, CH 2 ), 4.06-4.20 (m, 6H, 3 × OCH 2 CH 3 ). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 14.1 (OCH 2 CH 3 ), 16.4 (d, 10

3 J CP =6.5 Hz, 2 × OCH 2 CH 3 ), 34.5 (dd, 1 J CC =59.0 Hz, 1 J CP =134.0 Hz, CH 2 ), 61.6 (OCH 2 CH 3 ), 62.7 (d, 2 J CP =6.5 Hz, 2 × OCH 2 CH 3 ), 166.0 (d, 2 J CP =6.1 Hz, [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF]

C=O).

Ethyl-1-( 13 C)-3-methyl but-2-enoate (( 13 C)-3). Sodium hydride (60 % in mineral oil, 0.87 g, 22.10 mmol, 1 equiv) was introduced in a flamed dried flask under argon. 2-(Diethoxyphosphoryl)-1-( 13 C)-ethyl acetate ( 13 C)-2 (4.95 g, 22.10 mmol, 1 equiv) dissolved in anhydrous tetrahydrofuran (50 mL) was added at 0 °C. The mixture was stirred for 15 min. Anhydrous acetone (1.57 mL, 20.1 mmol, 0.95 equiv) was added to the reaction and the mixture was stirred during 30 min at 0 °C then allowed to warm to room temperature overnight. The reaction was quenched using a saturated solution of ammonium chloride (25 mL). The aqueous phase was extracted with diethyl ether (3 × 80 mL), organic fractions collected, dried over magnesium sulfate , filtered then concentrated under reduced pressure.

The product was purified by silica gel column chromatography (pentane/diethyl ether 97/3), to obtain ( 13 C)-3 as a yellow oil (1.46 g, 51 %). [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.25 (t, 3H, 3 J HH =7.1 Hz, OCH 2 CH 3 ), 1.88 (s, 3H, CH 3 cis), 2.14 (s, 3H, CH 3 trans), 4.15 (dd, 2H, 3 J HH =7.1 Hz, 3 J HC =3.1 Hz, CH 2 ), 5.67 (m, 1H, =CH). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 13.9 (OCH 2 CH 3 ), 19.9 (CH 3 cis), 27.7 (CH 3 trans), 59.2 (OCH 2 CH 3 ), 116.0 (d, 1 J CC =75.4 Hz, =CH), 156.5 ((CH 3 ) 2 C=), 166.7 ( 13 C=O).

1-( 13 C)-3-Methylbut-2-en-1-ol (( 13 C)-4).

A diisobutylaluminum hydride solution in dichloromethane (27.50 mL, 27.50 mmol, 2.4 equiv) was introduced into a flame-dried round bottom flask equipped with an addition funnel and cooled at -78 °C. ( 13 C)-3 (1.46 g, 11.43 mmol, 1 equiv) dissolved in anhydrous dichloromethane (20 mL) was slowly added. The reaction mixture was allowed to warm to room temperature overnight. The reaction was stopped by adding ethyl acetate (10 mL) and water (10 mL). The solution was filtered on celite and the organic phase separated. The aqueous phase was extracted with diethyl ether (2 × 10 mL). Combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure . 1-( 13 C)-3-Methylbut-2-en-1-ol ( 13 C)-4 (1.65 g) was obtained as a transparent oil and used without further purification. [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.69 (s, 3H, CH 3 cis), 1.75 (s, 3H, CH 3 trans), 4.10 (dd, 2H, 3 J HH =7.1 Hz, 1 J HC =142.1 Hz, [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] CH 2 ), 5.41 (m, 1H, =CH). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 17.8 (CH 3 cis), 25.7 (CH 3 trans), 59.3 (HO 13 CH 2 ), 123.6 (d, 1 J CC =47.0 Hz, =CH), 136.3 ((CH 3 ) 2 C=).

1-( 13 C)-1-Bromo-3-methyl but-2-ene (( 13 C)-5).

To a solution of 1-( 13 C)-3-methylbut-2-en-1-ol ( 13 C)-4 (1.65 g, 19.17 mmol, 1 equiv) in dichloromethane (7 mL) cooled at 0 °C was added hydrobromic acid (48%, 24.3 mL, 214.2 mmol, 11.2 equiv). The mixture, protected from daylight, was vigorously stirred for 2 h. Magnesium sulfate (6.69 g, 55.72 mmol, 2.9 equiv) and dichloromethane (30 mL) were then added and the solution was allowed to warm to room temperature. The organic phase was separated and the aqueous phase was extracted using dichloromethane (2 × 20 mL). The organic layers were assembled and dried over magnesium sulfate. The solvent was evaporated under reduced pressure. 1-( 13 C)-1-Bromo-3-methyl but-2-ene ( 13 C)-5 (1.44 g) was obtained as brown oil and used without further purification. [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.73 (s, 3H, CH 3 cis), 1.78 (s, 3H, CH 3 trans), 4.15 (dd, 2H, 3 J HH =8.5 Hz, 1 J HC =153.0 Hz, CH 2 ), 5.48-5.56 (m, 1H, =CH). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 17.5 (CH 3 cis), 25.8 (CH 3 trans), 29.7 ( 13 CH 2 ), 121.6 (d, 1 J CC =47.0 Hz, =CH), 137.9 ((CH 3 ) 2 C=).

Ethyl-3-( 13 C)-2-acetyl-5-methylhex-4-enoate (( 13 C)-6).

In a flame-dried round bottom flask, sodium ethoxide (4.34 mL, 11.62 mmol, 1.2 equiv) was added to a solution of ethyl acetoacetate (2.47 mL, 19.37 mmol, 2 equiv) in anhydrous ethanol (20 mL). The mixture was cooled to 0 °C and a solution of ( 13 C)-5 (1.44 g, 9.6 mmol, 1 equiv) in ethanol (10 mL) was added to the mixture. After stirring 1 h at 0 °C, the solution was refluxed for 4 h. The solvent was evaporated under reduced pressure, then water (5 mL) was added and the aqueous phase was extracted with ethyl acetate (3 × 15 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified using silica gel chromatography (pentane/diethyl ether 9/1) and ethyl-3-( 13 C)-2-acetyl-5-methylhex-4-enoate ( 13 C)-6 was obtained as a colorless oil (1.289 g, 67%). [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.25 (t, 3H, 3 J HH =7.1 Hz, OCH 2 CH 3 ), 1.63 (s, 3H, CH 3 cis), 1.67 (s, 3H, CH 3 trans), 2.22 (s, 3H, COCH 3 ), 2.53 (m, 2H, [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] CH 2 ), 3.40 (m, 1H, CH), 4.19 (q, 2H, 3 J HH =7.1 Hz, OCH 2 CH 3 ), 4.98-5.06 (m, 1H, =CH). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF]

4-( 13 C)-6-Methylhept-5-en-2-one (( 13 C)-7

). An aqueous solution of sodium hydroxide (10%, 18 mL) was added to a solution of ( 13 C)-6 (1.29 g, 6.50 mmol, 1 equiv) in ethanol (20 mL) at 0 °C. The mixture was refluxed during 5 h before allowing warming to room temperature. Then it was acidified using a solution of hydrochloric acid 10% down to pH 4-5. The aqueous phase was extracted with diethyl ether (3 × 50 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. 4-( 13 C)-6-Methylhept-5-en-2-one ( 13 C)-7 (0.26 g) was obtained as colorless oil and was used without further purification. [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR (CDCl 3 ) δ 17.6 (CH 3 cis), 22.5 ( 13 CH 2 CH), 25.7 (CH 3 trans), 29.9 (COCH 3 ), 44.3 (d, 1 J CC =34.5 Hz, CH 2 CO), 122.6 (d, 1 J CC =44.2 Hz, =CH), 132.7 ((CH 3 ) 2 C=), 208.9 (C=O).

5-( 13 C)-3,7-Dimethylocta-1,6-dien-3-ol; 5-( 13 C)-linalool (( 13 C)-8).

In a flame-dried round bottom flask, vinyl magnesium bromide (4.12 mL, 4.12 mmol, 2 equiv) was added to a solution of 4-( 13 C)-6methylhept-5-en-2-one ( 13 C)-7 (0.26 g, 2.06 mmol, 1 equiv) in anhydrous tetrahydrofuran (10 mL) previously cooled to 0 °C. After stirring 1 h at 0 °C, the reaction was stopped using a saturated solution of ammonium chloride (10 mL). The aqueous phase was extracted with diethyl ether (3 × 30 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure.

The crude product was purified by silica gel column chromatography (pentane/diethyl ether 8/2) and 5-( 13 C)-linalool ( 13 C)-8 was obtained as a yellow oil (0.198 g, 63 %). [START_REF] Yazar | Preservatives and fragrances in selected consumer-available cosmetics and detergents[END_REF] H NMR (CDCl 3 ) δ 1.27 (s, 3H, CH 3 ), 1.52-1.57 (m, 2H, =CHCH 2 CH 2 ), 1.60 (s, 3H, CH 3 cis), 1.67 (s, 3H, CH 3 trans), 1.92-2.09 (m, 2H, =CH [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] CH 2 CH 2 ), 5.05 (dd, 1H, 3 J HH =1.1 Hz, 2 J HH =10.8 Hz, CH=CH 2 trans), 5.11 (m, 1H, =CH), 5.20 (dd, 1H, 3 J HH =1.1 Hz, 2 J HH =17.3 Hz, CH=CH 2 cis), 5.90 (dd, 1H, 3 J HH =10.8 Hz, 2 J HC =17.3 Hz,CH [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] CH 2 ). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] The filtrate, partially evaporated under reduced pressure, was washed with distilled water (3 × 50 mL) and the aqueous phase was extracted with dichloromethane (50 mL). Combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a yellow oil. A mixture of hydroperoxides ( 13 C)-9 and ( 13 C)-10 (ratio 2:3) was obtained using silica gel column chromatography (petroleum ether/EtOAc 8/2, then 6/4) as a yellow oil (0.238 g, 1.25 mmol, 99%).

RESULTS

EPR Spin Trapping Studies. EPR-ST is a perfect tool for the identification of transient radicals in chemical and biological systems. Radical intermediates that are too short-lived or of too low concentration to be directly detected react with a spin-trap to form persistent radicals (spin-adducts)

observed by EPR with a signature depending on the trapped transient reactive species. [START_REF] Lauricella | Detection and characterization of free radicals after spin trapping[END_REF] To study Lina-OOHs (mixture Lina-7-OOH/Lina-6-OOH, ratio 2:3), we employed our EPR-ST method that allows investigating in situ the formation of free radicals derived from xenobiotics in the EpiSkin TM reconstructed human epidermis (RHE) model. [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] EpiSkin TM RHE are normal multi-layered keratinocytes cultures, the major cell type in the epidermis playing a key role in skin inflammatory reactions. Initially, the aim was to establish in solution the optimal experimental conditions providing a good S/N ratio while keeping Lina-OOHs concentrations as low as possible, consistent with real life exposure conditions. DEPMPO was the spin-trap chosen based on (i) our previous studies, [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) the aptitude to distinguish oxygen and carbon centered radicals, [START_REF] Stolz | Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase[END_REF] and (iii) the high persistency of the 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] Radical initiation was triggered by the known catalytic Fe(II)-induced reductive cleavage of the hydroperoxide chemical function in a Fenton-like reaction. [START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF] HEPES was preferred to phosphate buffer (PB) to avoid the formation of insoluble Fe(III)

through Fe(II) oxidation catalyzed by PB, decreasing the Fe(II) content available. [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][START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF] Experimental EPR spectrum in Figure 1 was obtained with DEPMPO 25 mM and Lina-OOHs 1 mM, supplemented by a Fe(II) catalytic amount (0.1 mM) to trigger radical initiation. The spectrum showed the spinadduct corresponding to hydroxyl radicals (DEPMPO-OH spin-adduct), together with species testimony of carbon centered radicals (DEPMPO-R1 and DEPMPO-R2 spin-adducts) and potential alkoxyl radicals (DEPMPO-OR spin-adduct), all of them with characteristic hyperfine coupling constant (hfccs) values (Table 1). [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][START_REF] Karoui | DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water[END_REF][START_REF] Dikalov | Characterization of the high resolution ESR spectra of the methoxyl radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)[END_REF] Experiments were further carried out in RHE based on a topical application procedure to get closer to real life sensitization. RHE samples were initially loaded with DEPMPO in HEPES (15 min, 37 °C, 5% CO 2 ) to ensure that the spin-trap is present at the site of radical generation before application of Lina-OOHs. [START_REF] Timmins | Free radical formation in murine skin treated with tumour promoting organic peroxides[END_REF] When pre-treated RHE were then exposed topically to different concentrations of Lina-OOHs in acetone, DEPMPO spin-adducts were observed from a 10 mM concentration (Figure 2). As for previous studies, concentrations of spin-trap and target compounds needed in the case of EPR-ST studies in RHE were ten-fold higher (250 mM and 10 mM, respectively) than those giving a good S/N ratio signal in solution. [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] This is probably due to the fact that RHE are complex 3D structures including a stratum corneum and therefore skin penetration concerns need to be considered, together with the content in phospholipids and ceramides that may influence 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] As for the investigations in solution, the fingerprint of a carbon centered radical DEPMPO spin-adduct (DEPMPO-R, Figure 2) was detected. Moreover, both hydroxyl radical (DEMPO-OH spin-adduct) and alkoxyl radical (DEPMPO-OR spin-adduct) were also detected in the RHE when put in presence of Lina-OOHs. Noteworthy, spin-adducts for RHE were assigned despite of low S/N as being based on the direct comparison with corresponding investigations in solution (Table 1). This is especially the case for the latter one (DEPMPO-OR) that is barely visible in the RHE. Even though we are here at the detection limit, shallow shoulders are noticeable within the experimental spectrum that are compatible with hfccs of DEPMPO-OR spin-adduct. Besides, the incubation of the sole spin-trap in RHE gave no signal, indicating that observed radicals intermediates originate rather from Lina-OOHs than from RHE biomolecules. On another hand, RHE incubation with single Lina-OOHs gave also no signal. Lina-OOHs generate radical intermediates thus pointing out the necessity to use the EPR-ST methodology for these studies. It is important to stress here that no Fe(II) was added in RHE experiments, indicating that Lina-OOHs radical initiation has been induced by the RHE itself.

Synthesis of 5-( 13 C

)-Lina-OOHs. In our laboratory, we have developed NMR techniques in association with [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substituted molecules to probe hapten-protein interaction mechanisms in buffered or semi-organic solutions and in RHE. [START_REF] Debeuckelaere | In situ chemical behaviour of methylisothiazolinone (MI) and methylchloroisothiazolinone (MCI) in reconstructed human epidermis: a new approach to the cross-reactivity issue[END_REF] Herein, to study the reactivity toward thiol groups, Lina-OOHs were synthesized [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substituted at carbon positions 4 or 5 in order to follow the reactivity toward thiol groups by [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR. Indeed, we have showed previously that Lina-7-OOH decomposes in the presence of ferrous salts affording carbon radicals that could potentially be centered at these positions (Scheme 1). [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] The synthetic route developed for the synthesis of 5- Emmons reaction with acetone afforded ( 13 C)-3. [START_REF] Sharma | Exploration of the molecular origin of the azinomycin epoxide: timing of the biosynthesis revealed[END_REF] Further reduction to allylic alcohol ( 13 C)-4 was carried out by using diisobutyl aluminum hydride. The alcohol functionality of ( 13 C)-4 was then converted into a good bromine-leaving group leading to ( 13 C)-5, which reacted with ethyl acetoacetate deprotonated at the α position of the carbonyl group to afford ( 13 C)-6. In order to obtain the key ketone intermediate ( 13 C)-7, β-keto ester ( 13 C)-6 was first saponified and the carboxylic acid function obtained decarboxylated in presence of hydrochloric acid. Linalool [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substitued at position 5 ( 13 C)-8 was then synthesized by treating the ketone with vinyl magnesium bromide. Finally, ( 13 C)-8 was subjected to the ene or "Schenck reaction" allowing the preparation of allylic hydroperoxides from alkenes containing allylic hydrogens via singlet oxygen ( 1 O 2 ) reaction as we described previously. [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] 1 O 2 was quantitatively generated by disproportionation of hydrogen peroxide induced catalytically by sodium molybdate in alkaline aqueous solutions. [START_REF] Aubry | Chemical sources of singlet oxygen. 2. Quantitative generation of singlet oxygen from hydrogen peroxide disproportionation catalyzed by molybdate ions[END_REF] A microemulsion consisting of water, dichloromethane (organic phase), sodium dodecyl sulfate (surfactant), and butanol (cosurfactant) was used as reaction medium to avoid epoxidizing competitive reactions. [START_REF] Nardello | Reactivity, chemoselectivity, and diastereoselectivity of the oxyfunctionalization of chiral allylic alcohols and derivatives in microemulsions: comparison of the chemical oxidation by the hydrogen peroxide/sodium molybdate system with the photooxygenation[END_REF] In a 99% yield reaction, a mixture of ( 13 C)-9 and ( 13 C)-10 was obtained with a 2:3 ratio measured by NMR. Due to the difficulty of isolating these two position isomers, the mixture ( 13 C)-9/( 13 C)-10 was subsequently used for thiol reactivity studies followed by [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR. We considered that the use of the mixture was not a limitation as ( 13 C)-9 contains the [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] Csubstitution on a vinyl carbon atom and ( 13 C)-10 on a methylene group, both giving distinct easily recognizable [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR chemical shifts. Moreover, it is actually the mixture that is used in clinical patch testing studies and repeated open application tests with Lina-OOHs.

13 C-NMR reactivity studies. The reaction of ( 13 C)-9/( 13 C)-10 with N-acetyl-L-cysteine methyl ester (N-Ac-Cys-OMe) in CD 3 CN/H 2 O 1/1 was followed by mono-dimensional [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR at room temperature during one week (Figure 3). A catalytic amount of iron (II) sulfate heptahydrate (0.1 mM) was sufficient to induce radical initiation as for the EPR studies in solution. Control experiments were realized without the presence of iron (II) as initiator.

As shown in Figure 3, [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] ). These data indicated that both vinyl groups of ( 13 C)-9 had been preserved. More interestingly, we could suggest that a cysteine unit was linked as there was also a long-distance correlation with a proton at 2.16 ppm, this chemical shift being characteristic of a >CH-group having a thiol substituent (position c).

However, many of the chemical shifts described above could also match with a structure such as ( 13 C)-12, that could derive from β-scission of the initially formed alkoxyl radical RO • . Consequently, the cohabitation of both compounds cannot be neglected, as we are not able to assign one chemical structure unambiguously upon the [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR data. On another hand, [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-peak at 28.

DISCUSSION

Chemical and molecular understanding on the mode of action of skin sensitizers is necessary to improve risk assessment procedures and thus help to protect producers and consumers. Linalool (CAS:

78-70-6; EC: 201-134-4) is listed in the European inventory as a deodorant and perfuming agent, playing an indispensable role in fragrances and cosmetics, [START_REF]reconstructed human epidermis test method[END_REF] sharing the spotlight with limonene. These terpenes readily oxidize on exposure to air and the resulting hydroperoxides have shown to have high frequencies of positive patch tests reactions in several European and international studies. [START_REF] Bråred Christensson | Oxidized limonene and oxidized linalool -concomitant contact allergy to common fragrance terpenes[END_REF][START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC prospective study[END_REF] Although a great step forward has been made in understanding their mechanism of action involving the intervention of radical mechanisms, essential informations are still lacking to unravel the link between these allergens and the still unknown chemical "radical structural alerts" at the basis of their sensitizing potential.

Our previous studies in solution on Lina-7-OOH demonstrated the formation of oxygen and carbon centered radicals derived from the hydroperoxide, by using the spin trapping technique combined to EPR spectroscopy and Fe(II)/Fe(III) radical initiation. [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] Here, we have evidenced that these radical intermediates are well and truly formed in a RHE model, histologically similar to human epidermis.

EpiSkin TM RHE (Lyon, France) is a very good replicate of human epidermis architecture, and is used for irritation and penetration tests of cosmetic and chemical compounds replacing in vivo testing. 34 As a linalool oxidation mixture containing both Lina-7-OOH and Lina-6-OOH has been used for patch testing in multicenter studies in European dermatological clinics (6.0% oxidized linalool in petrolatum, 1% w/w of Lina-OOHs content), [START_REF] Bråred Christensson | Air-oxidized linalool -a frequent cause of fragrance contact allergy[END_REF][START_REF] Bråred Christensson | Oxidized limonene and oxidized linalool -concomitant contact allergy to common fragrance terpenes[END_REF][START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC prospective study[END_REF] our studies were conducted with a Lina-7-OOH/Lina-6-OOH (2:3 ratio) mixture that we synthesized from linalool following a procedure identical to that described for the obtention of ( 13 C)-9 and ( 13 C)-10 from ( 13 C)-8. EPR technical adjustments needed firstly to be established to obtain EPR fingerprints with reasonable S/N ratio. Preliminary studies were carried out in solution to get a first accurate picture of radical intermediates issued from Lina-7-OOH/Lina-6-OOH. Further, the optimized method in RHE was based on a topical application procedure to get closer to real life sensitization. [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] The fingerprint of a carbon centered radical DEPMPO spin-adduct, formed by addition of transient short-lived carbon radicals to the α-carbon of the nitronyl group of the spin-trap (see Supporting Information), was identified. The experimental data was in good agreement with simulations using the calculated parameters a N =14.8 G, a H =21.2 G and a P =48.2 G, such fingerprint being testimony of trapping carbon radicals. [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] The HO • radical, highly reactive and with a very short life in all kinds of media, was also detected by spin trapping with DEPMPO in treated RHE with hfccs a N =13.8 G, a H =13.7 G and a P =47.1 G. However, one must be careful when interpreting EPR experiments in aqueous media, and keep in mind that detection of the DEPMPO-OH nitroxide does not necessarily mean that HO • has been trapped, as non-spin trapping reactions involving nucleophilic addition of water can lead to similar EPR spectrum. For example, the Forrester-Hepburn reaction, consisting in a nucleophilic addition on the spin-trap followed by oxidation of the so formed hydroxylamine (e.g. by dioxygen present in the medium), [START_REF] Forrester | Spin traps. A cautionary note[END_REF] must be always considered in aqueous media, especially in the presence of traces of metal ions which can by complexation activate the nitrone function making it more sensitive to the addition of water. [START_REF] Finkelstein | Spin trapping of superoxide and hydroxyl radical: practical aspects[END_REF] We performed several control experiments by incubating exclusively the spin-trap (also in the solution experiments with Fe(II)) and any spinadducts were never observed, suggesting that maybe we could rule out this competition reaction with water in our studies. Finally, when considering the hfccs values obtained in RHE and those obtained in solution (Table 1) variances are noticeable. This could be explained by the very different environment of the paramagnetic probe in mere solution when compared to that of a complex heterogeneous tissue such as RHE. Indeed, the magnitude of the hfccs, testimony of the delocalization extent of the nitroxide unpaired electron, is known to be sensitive to its close surrounding especially regarding the local polarity: the spin density on the 14 N (i.e. the corresponding hfcc) increases with the polarity of the solvent. [START_REF] Bordignon | Heterogeneity in the nitroxide micro-environment: polarity and proticity effects in spin-labeled proteins studied by multi-frequency EPR[END_REF][START_REF] Owenius | Influence of solvent polarity and hydrogen bonding on the EPR parameters of a nitroxide spin label studied by 9-GHz and 95-GHz EPR spectroscopy and DFT calculations[END_REF] It is worth to compare the amount of Lina-OOHs topically applied on the RHE in our study with the amounts of Lina-OOHs used in clinical ACD diagnostic tests. An investigation on the optimal patch test concentration for detection of contact allergy to oxidized linalool suggested the mostly nowadays used concentration of 6% in petrolatum, containing 1% w/w of Lina-OOHs. [START_REF] Bråred Christensson | Linalool -A significant contact sensitizer after air-exposure[END_REF] A dose per unit area of approximately 2.4 mg/cm 2 was considered when 20 mg oxidized linalool was tested in small Finn Chambers ® (diameter 8 mm, inner area of 0.5 cm 2 ), which means 400 µg/cm 2 of Lina-OOHs. Further, to identify threshold concentrations for elicitation of ACD caused by oxidized linalool in allergic individuals with repeated exposures, repeated open application tests were performed on 6 patients simulating daily exposure over a period of three weeks on their forearms (circular area 3.6 cm diameter). [START_REF] Björkman | Air-oxidized linalool elicits eczema in allergic patients -a repeated open application test study[END_REF] Two types of model product containing oxidized linalool were tested in parallel, solutions in ethanol to mimic a 'fine fragrance', and cream base formulations with 15% glyceryl stearate in water to mimic a scented cosmetic 'cream'. 5/6 participants reacted to the 'cream' containing 3% oxidized linalool, thus 0.56% w/w of Lina-OOHs. The correspondent calculated dose per unit area was of 273 µg/cm 2 for oxidized linalool, thus approximately 50 µg/cm 2 for Lina-OOHs. With preparations containing 0.3% oxidized linalool, i.e. 0.056% w/w of Lina-OOHs, 2/6 participants reacted to the 'cream' and 1/6 to the 'fine fragrance'. The correspondent calculated dose per unit area was of 27 µg/cm 2 for oxidized linalool, thus about 5 µg/cm 2 for Lina-OOHs. In our studies, 20 µL Lina-OOHs (10 mM in acetone) were topically applied to 0.38 cm 2 RHE, meaning a dose per unit area of approximately 100 µg/cm 2 . This is four times less than the dose per unit area used in patch test clinical studies. It is necessary to mention here that the optimal patch test concentration established was considerably high to get the maximum number of answers and thus avoid missing false positives.

Indeed, concentrations used in patch testing are often much higher than concentrations found in commercial products. However, with repeated exposure to a hapten in everyday products, low concentrations may be sufficient to cause or worsen eczema in allergic individuals. 100 µg/cm 2 is approximately two times more than the dose per unit area used for the more concentrated solution in the repeated open application tests and 20 times more than the dose per unit area used for the less concentrated one. This would mean that in our studies we are approaching the elicitation dose per unit area when exposing patients to a repeated exposure, and that the EPR-ST methodology in RHE has sensitivity in good agreement with what could happen in vivo. Though, further experiments will be carried out to improve the sensitivity of the methodology in order to highlight the formation of radicals with lower doses, which have clinically proven elicitation of allergy to Lina-OOHs, and this under repeated exposure.

It is important to note that after penetrating the RHE, Lina-OOHs radical initiation should have been induced by RHE itself as no extra Fe(II) was added in RHE experiments. The reaction of organic hydroperoxides 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] In this sense, we have conducted some very preliminary studies of reactivity with the system Lina-OOHs/N-Ac-Cys-OMe/DEPMPO in a Fe(II) free solution. Spin-adducts EPR fingerprints pointing to HO • , carbon centered or alkoxyl based radicals were detected (see Supporting Information) indicating at a first sight that cysteine induces radical generation form the target compounds.

Knowing that carbon centered radicals can be formed in the epidermis, our thereafter objective was to identify which of these radicals are able to react with amino acids and form potential antigenic entities. Reactivity of ( 13 C)-Lina-OOHs towards cysteine, probably the most labile residue involved in radical reactions triggered by peroxides, was studied. We had reported previously the reactivity of Lina-7-OOH towards N-Ac-Cys-OMe and GSH triggered by Fe(II) and followed by liquid chromatography combined to mass spectrometry. [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] We validated that carbon radicals issued from Lina-7-OOH are able to directly alter thiol chemical groups, forming adducts via radical processes, and we suggested chemical structures for some of the adducts observed (Figure 5). Regardless of the experimental conditions used we observed prevailing oxidation of N-Ac-Cys-OMe to form a cysteine dimer together with suggested adducts 14 and 15. We proposed that 14 and 15 could result from reaction of the thiol group with carbon radicals derived from allylic hydrogen abstraction by initially formed Lina-7-O • (RO • ), possible at a single position for Lina-7-OOH, and from a β-scission process, respectively. The same kind of chemistry was proposed with GSH (adduct 16) together with a nucleophilic addition of GSH on the α,β-unsaturated ketone derived from β-scission of Lina-7-O • (adduct 17). Findings reported herein, where reactivity towards N-Ac-Cys-OMe has been followed by [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR using [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substitued Lina-OOHs, are in agreement with these previous results in the case of Lina-7-OOH (( 13 C)-9 in the mixture studied). [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR data, and more precisely direct and long distance 1 H-13 C correlations, were in line with compounds ( 13 C)-11 and/or ( 13 C)-12. The possibility to form adduct ( 13 C)-11 is a very good evidence that allylic hydrogen abstraction is a major radical pathway to form carbon radicals from Lina-7-OOH. A suggestion for a mechanistic pathway explaining the formation of ( 13 C)-11 is given in Scheme 3, based on a probable concerted mechanism. Initially, 5- to form an allylic alcohol could be detected. This would probably mean that Lina-7-OOH is much more reactive than Lina-6-OOH towards cysteine and thiol containing peptides. This could be essential information pointing the radical at carbon atom 4 of Lina-7-OOH as a potential "radical structural alert" that could give an explanation to its sensitizing potential. Reactivity studies carried out with 4-( 13 C)-Lina-OOHs corroborated the preferential reactivity of Lina-7-OOH compared to Lina-6-OOH (see Supporting Information). It is worth noting that 5-( 13 C)-Lina-OOHs reactivity studies gave better information on radical reactivity at the hypothetically preferential position 4 than 4-( 13 C)-Lina-OOHs, the 1 H-13 C NMR long distance correlations having a higher resolution in the spectra.

Resuming, EPR-ST confirmed the formation of carbon radicals issued from Lina-OOHs in RHE. [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR reactivity studies with [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substitued compounds suggested carbon atom position 4 as a potential "radical structural alert" reacting with thiol groups. It is worthy to note that [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR studies allowed detection only of diamagnetic adducts. However, as EPR studies in solution showed, other radical (paramagnetic species) are coexisting in solution and it is not excluded that they could react with other amino acids prompt to radical reactions. It would be essential to study by EPR-ST which carbon radical is specifically formed in RHE. As we have shown here, nitrones such as DEPMPO are excellent traps for carbon radicals derived from Lina-OOHs in RHE. We have shown that spin-adducts formed are particularly stable, though the S/N ratio is relatively poor. It would be further necessary to use spin-traps more lipophilic than DEPMPO, able for instance to pass through the cell membrane and so allowing to distinguish if radical generation is intra or intercellular in the case of Lina-OOHs.

Though, nitrones rarely allow the different carbon radicals formed to be distinguished (carbon-radical at position 4 versus position 5), as the coupling patterns observed in the spectrum are always very similar. A very good tool to overcome this drawback is the introduction of a 13 C-substitution in the positions of the molecule where a carbon radical can be formed such as for 5-( 13 C)-Lina-OOHs and 4-( 13 C)-Lina-OOHs. If the trapped radical is centered on a 13 C-substituted carbon, an additional hyperfine coupling will be observed on the EPR spectrum due to its nuclear spin, which is then in the beta position, as we have shown recently when studying the sensitizing cumene hydroperoxide. [START_REF] Sahli | Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis : an EPR spin-trapping confirmation by using 13 C-substitution[END_REF] This property can thus be used to elucidate reaction mechanism(s).

CONCLUSION

To complement previous studies in solution on the identification of reactive radical intermediates issued from linalool sensitizing hydroperoxydes, we have now taken a step forward demonstrating that these are indeed formed in a reconstructed human epidermis 3D model, much closer to what may happen in vivo. For this purpose, we demonstrated that EPR-ST is a very powerful tool. Studying the reactivity of these radicals towards amino acids prone to radical reactions such as cysteine has given new insights on the mechanisms most likely to be involved in the reaction with thiol groups that might be present in the skin environment. The use of [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substituted sensitizing compounds at positions precursor of carbon radicals and further follow up of the reactions by [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR is thus also an efficient tool to give innovative understanding on the reactivity of allylic hydroperoxides towards proteins and thus their sensitizing potential.

Chart 1. Chemical structures of linalool, Lina-7-OOH ((5E)-7-hydroperoxy-3,7-dimethylocta-1,5diene-3-ol) and Lina-6-OOH (6-hydroperoxy-3,7-dimethylocta-1,7-diene-3-ol).

Scheme 1. Potential carbon centered radicals resulting from Lina-7-OOH justifying the synthesis of [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-linalool at positions 5 and 4, further oxidized to the corresponding target ( 13 C)-Lina-OOHs.

  C NMR (CDCl 3 ) δ 17.7 (CH 3 cis), 22.8 ( 13 CH 2 CH), 25.7 (CH 3 trans), 27.9 (CH 3 ), 42.0 (d, 1 J CC =34.4 Hz, CH 2 COH), 73.5 (COH), 111.7 (=CH 2 ), 124.3 (d, 1 J CC =43.6 Hz, =CH), 131.9 13 ((CH 3 ) 2 C=), 145.0 (CH 2 =CH). 5-( 13 C)-(5E)-7-Hydroperoxy-3,7-dimethylocta-1,5-dien-3dimethylocta-1,7-dien-3-ol (( 13 C)-10). A microemulsion was prepared by slowly adding a sodium molybdate solution (0.239 g in 1.27 mL distilled water) to a suspension of sodium dodecyl sulfate (1.975 g) in butanol (2.41 mL) and dichloromethane (11.76 mL). At the end of the addition, the microemulsion became clear after 5 min stirring. 5-( 13 C)-Linalool ( 13 C)-8 (0.198 g, 1.26 mmol, 1 equiv) was introduced in the microemulsion at room temperature. Hydrogen peroxide (aqueous solution 35%, 71 µL one fraction) was then added. The solution was instantly red colored. After 20 min stirring the solution became yellow. Fourteen other fractions of hydrogen peroxide (aqueous solution 35%, 71 µL each) were added successively every 10 min. The resulting yellow solution was stirred overnight at room temperature. When the reaction was thoroughly translucent, 5-( 13 C)-linalool ( 13 C)-8 was completely oxidized. The solvent was removed under reduced pressure at 40 °C. The semi-solid white product obtained was suspended in dichloromethane (65 mL) and vigorously stirred for 48 h. The suspension was filtered to recover sodium molybdate and sodium dodecyl sulfate.

13 C-NMR reactivity studies 5 -

 5 ( 13 C)-Lina-OOHs ( 13 C)-9 and ( 13 C)-10 (ratio 2:3) (1 mg, 5.37 µmoles, 1 equiv) dissolved in 300 µL of deuterated acetonitrile were incubated with N-acetyl-L-cysteine-methyl ester (1.94 mg, 10.74 µmoles, 2 equiv) dissolved in 300 µL of deionized water, and in the presence of a catalytic amount of iron sulfate heptahydrate (0.5 µmoles, 0.1 equiv). The whole mixture was introduced into an NMR tube to follow the reaction by[START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR. The reactions were followed by mono-dimensional[START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR on a Bruker Avance 500 spectrometer at 125 MHz. Chemical shifts (δ) are reported in ppm in comparison to tetramethylsilane, using the residual signal of acetonitrile (δ 1 H = 21.94 ppm; δ 13 C = 118.26 ppm) as internal standard. The potential structure of the products formed during the reactions was assigned by heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments. Chemical shifts were compared with those calculated using ACD/CNMR and ACD/HNMR Predictor software. Same methodology was employed in the case of 4-( 13 C)-Lina-OOHs (Supporting Information).

  ( 13 C)-Lina-OOHs is illustrated in Scheme 2 (synthesis of 4-( 13 C)-Lina-OOHs with a 13 C-substitution at position 4 is detailed in the Supporting Information). Initially, ( 13 C)-1 was synthesized from 1-( 13 C)-acetic acid in a two-step's reaction. 1-( 13 C)-Acetic acid (source to introduce the 13 C-substitution) was submitted to a Hell-Vollhardt-Zellinsky reaction allowing bromination of the α-carbon to the carbonyl chemical function in presence of bromine and phosphorous tribromide, followed by esterification using ethanol. A nucleophilic substitution of bromine by triethyl phosphite afforded 2-(diethoxyphosphoryl)-1-( 13 C)-ethylacetate ( 13 C)-2 through a Michaelis-Arbuzov reaction. Based on the literature, the stabilized phosphonate carbanion of ( 13 C)-2 was then obtained by treatment with a strong base and a Horner-Wadsworth-

  C peaks of 5-( 13 C)-Lina-7-OOH ( 13 C)-9 at 125.7 ppm and of 5-( 13 C)-Lina-6-OOH ( 13 C)-10 at 24.3 ppm disappeared with time and new peaks appeared at 122.1 ppm and 28.8 ppm. Prediction of 13 C-NMR chemical shifts by computer simulation suggested that 13 C-peak at 122.1 ppm could correspond to compounds ( 13 C)-11 or ( 13 C)-12, derived from ( 13 C)-9, and 13 C-peak at 28.8 ppm to compound ( 13 C)-13, derived from ( 13 C)-10 (Figure 4). Compounds ( 13 C)-11 and ( 13 C)-12 have theoretically similar chemical shifts for several parts of the terpene unit. We observed by HSQC bidimensional NMR a direct correlation between the 13 C-peak at 122.1 ppm (position d) and a hydrogen atom of a vinyl group at 5.54 ppm. Also, HMBC bi-dimensional NMR showed long-distance correlations with protons at 1.18 ppm (methyl group, position b in Figure 4), 5.60 ppm (hydrogen atom of a vinyl group, position a) and 5.70 ppm (hydrogen atom of a vinyl group, position e

8 ppm (position β in ( 13 C)- 13 )( 13 C)- 10 .

 13131310 was correlated directly to protons at 1.45 ppm, this being characteristic of a methylene -CH 2 carbon. Also, the HMBC bi-dimensional NMR spectrum showed long-distance correlations with protons at 1.19 ppm (methyl group, position δ), 1.35 ppm (methylene -CH 2 , position γ) and, more precisely, with a -CH proton at 3.93 ppm. This chemical shift was distinctive of the secondary alcohol methyl allyl function (position α) of compound ( 13 C)-13, itself resulting from radical degradation of When using a large excess of N-Ac-Cys-OMe (10 equiv) the results were identical and the reaction was much faster (a few hours). However, the intensity of carbon peaks corresponding to N-Ac-Cys-OMe was much more important and prevented a good interpretation of NMR mono-and bidimensional spectra. Complementary studies were carried out with 4-( 13 C)-Lina-OOHs, having a tendency to corroborate results above concerning the hypothetical formation of compounds ( 13 C)-11 and ( 13 C)-12 via radical degradation processes (see Supporting Information).

( 13 C

 13 )-Lina-7-OOH ( 13 C)-9 is able to oxidize Fe(II) to Fe(III) generating RO • radicals through a Fentonlike reaction, and the thiol group of N-Ac-Cys-OMe can then be oxidized by reduction of Fe(III) to Fe(II). Then, N-Ac-Cys-OMe dimer can be formed. Concomitantly, RO • radicals may abstract an allylic hydrogen to form carbon centered radicals at position 4 that afterwards attack on the cysteine dimer to form adduct ( 13 C)-11 and release a thiyl radical. On another hand, hypothetical compound ( 13 C)-12 confirms that β-scission processes could occur in parallel and must not be neglected, though addition of the thiol moiety as in 15 and 17 was not observed here. No reactivity with the thiol group was observed in the case of Lina-6-OOH (( 13 C)-10 in the mixture studied). Only radical decomposition

Scheme 2 .Scheme 3 .

 23 Scheme 2. Synthesis of Lina-OOHs[START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-substituted at position 5.Scheme 3.Possible mechanistic pathway for the formation of ( 13 C)-11 through the involvement of a carbon radical at position 4.[START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C-NMR only allows detection of diamagnetic adducts formed by reaction with the thiol group of cysteine. This does not mean there are not in the solution other radical intermediates, as EPR studies have shown, that could potentially react with other amino acids in the skin.

Figure 1 .

 1 Figure 1. EPR experimental spectrum (Exp) of Lina-OOHs (1 mM)/DEPMPO (25 mM)/Fe(II) (0.1 mM) in solution, together with computer simulation (Sim) and deconvolution affording spectra of spinadducts DEPMPO-OH, DEPMPO-R1, DEPMPO-R2 and DEPMPO-OR. Values of the hfccs are shown in Table1. All g factors were measured and found to be of ca. 2.0055±0.0002. Control experiments with single Lina-OOHs or single DEPMPO did not give any signal.

Figure 2 .

 2 Figure 2. EPR experimental spectrum (Exp) of Lina-OOHs (10 mM)/DEPMPO (250 mM) in RHE, together with computer simulation (Sim) and deconvolution affording spectra of spin-adducts DEPMPO-OH, DEPMPO-R and DEPMPO-OR. Values of the hfccs are shown in Table1. All g factors were measured and found to be of ca. 2.0055±0.0002. Control experiments with single Lina-OOHs or single DEPMPO did not give any signal.

Figure 3 .

 3 Figure 3. 13 C-NMR spectra obtained from the reaction ( 13 C)-9/( 13 C)-10 (5.37 µmoles, 1 equiv)/ N-Ac-Cys-OMe (10.74 µmoles, 2 equiv)/Fe(II) (0.1 equiv) in CD 3 CN/H 2 O: (a) ( 13 C)-9/( 13 C)-10 control with 13 C peaks at 125.7 ppm and 24.30 ppm corresponding to ( 13 C)-9 and ( 13 C)-10, respectively; (b) N-Ac-Cys-OMe control; (c) reaction mixture at day 0; (d) reaction mixture at day 3; (e) reaction mixture at day 7.

Figure 4 .

 4 Figure 4. Hypothetical chemical structures of compounds ( 13 C)-11, ( 13 C)-12 and ( 13 C)-13 based on HMBC interpretation.

Figure 5 .

 5 Figure 5. Comparison of the chemical structures of the products suggested in previous liquid chromatography-mass spectrometry studies (14-17) 18 with those identified in this 13 C-NMR study (( 13 C)-11, ( 13 C)-12).

  Scheme 3

Figure 3 H

 3 Figure 3

  C NMR (CDCl 3 ) δ 14.0 (OCH 2 CH 3 ), 17.6 (CH 3 cis), 25.8 (CH 3 trans), 27.1 ( 13 CH 2 ), 59.8 (d, 1 J CC =34.2 Hz, CH), 60.4 (COCH 3 ), 61.4 (OCH 2 CH 3 ), 120.1 (d, 1 J CC =44.1 Hz, =CH), 134.9 ((CH 3 ) 2 C=), 169.6 ((O)C=O), 203.0 (C=O).

  H NMR (CDCl 3 ) δ 1.61 (s, 3H, CH 3 cis), 1.67 (s, 3H, CH 3 trans), 2.13 (s, 3H, COCH 3 ), 2.36 (m, 2H, CH 2 CO), 2.53 (m, 2H, 13 CH 2 CH), 5.01-5.08 (m, 1H, =CH).

  Hz, 2 J HH =17.4 Hz, CHC(OH)), 5.88 (dd, 1H, 3 J HH =10.8 Hz, 2 J HH =17.4 Hz, CHC(OH)).[START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C NMR CH 3 (C)CH(OOH)), 25.1 (d, 1 J CC =3.5 Hz, 2 × 13 CH 2 (CH)OOH), 28.2 and 28.4 (2 × CH 3 C(OH)), 37.7 and 37.8 (d, 1 J CC =23.8 Hz, 2 × CH 2 C(OH)), 73.1 (2 × C(OH)), 89.2 and 89.7 (d, 1 J

	(CDCl 3 ): Isomer ( 13 C)-9 (mixture of two enantiomers) δ 24.4 (2 × (CH 3 ) 2 C(OOH)), 27.8 (CH 3 C(OH)),
	45.1 (d, 1 J CC =43.5 Hz, CH 2 C(OH)), 72.8 (C(OH)), 82.1 (C(OOH)), 112.3 (CH 2 (CH)C(OH)), 126.7
	(CH 2 ( 13 CH)CH), 138.0 (d, 1 J CC =70.9 Hz, CHC(OOH)), 144.8 (CHC(OH)); Isomer ( 13 C)-10 (mixture of
	four diastereoisomers) δ 17.1 (2 ×
	31 (s, 6H,
	(CH 3 ) 2 C(OOH)), 2.28 (m, 2H, CH 2 C(OH)), 5.05 (dd, 1H, 3 J HH =1.1 Hz, 2 J HH =10.8 Hz,
	CH 2 (CH)C(OH)), 5.21 (dd, 1H, 3 J HH =1.1 Hz, 2 J HH =17.4 Hz, CH 2 (CH)C(OH)), 5.50-5.64 (m, 2H,

1 H NMR (CDCl 3 ): Isomer ( 13 C)-9 (mixture of two enantiomers) δ 1.27 (s, 3H, CH 3 C(OH)), 1.13 CHCH), 5.92 (dd, 1H, 3 J HH =10.7 Hz, 2 J HH =17.3 Hz, CHC(OH)); Isomer ( 13 C)-10 (mixture of four diastereoisomers) δ 1.29 (s, 6H, 2 × CH 3 C(OH)), 1.51-1.66 (m, 4H, 2 × 13 CH 2 CH 2 ), 1.73 (s, 6H, 2 × CH 3 (C)CH(OOH)), 4.29 (m, 2H, 2 × CH(OOH)), 5.00 (m, 4H, 2 × CH 2 (C)CH(OOH)), 5.07 (dd, 2H, CC =20.5 Hz, 2 × CH(OOH)), 112.3 (2 × CH 2 (CH)C(OH)), 114.2 and 114.3 (2 × CH 2 (C)CH(OOH)), 143.6 (2 × C(CH)OOH), 144.7 and 144.8 (2 × CHC(OH)).

Table 1

 1

Table 1 .

 1 Hyperfine coupling constants (hfccs) of spin-adducts identified in solution and in RHE All g factors were measured and found to be of ca. 2.0055±0.0002. ** Values given on an indicative basis following the best simulations attempts considering the S/N

	Spin-adducts	Coupling constants*
		aN (G)	aH (G)	aP(G)
	In solution
	DEPMPO-OH	14.1	13.3	47.6
	DEPMPO-R1	15.0	22.5	47.2
	DEPMPO-R2	14.4	20.9	47.1
	DEPMPO-OR	13.6	8.5	48.2
	In RHE
	DEPMPO-OH	13.8	13.7	47.1
	DEPMPO-R	14.8	21.2	48.2
	DEPMPO-OR	15.2**	7.2**	45.9**

*

  [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF] C)-3

								NaH, acetone
								THF
								0°C to rt
								51%
			Br			OH
			13 CH 2	HBr, 2 h		13 CH 2	DIBAL-H, CH 2 Cl 2
					CH 2 Cl 2 , 0°C			10 h, -78°C to rt
			( 13 C)-5			( 13 C)-4
			CH 3 C(O)CH 2 C(O)OEt
			EtONa, EtOH, 4 h, reflux
			67% over three steps
		O	O				O		HO
	EtO	13 CH 2	NaOH 10%, EtOH then HCl 10% 5 h, reflux	H 2	13 C	63% over two steps CH 2 CHMgBr, THF 1 h, 0°C	H 2	13 C
		( 13 C)-6				( 13 C)-7		( 13 C)-8
								H 2 O 2 , MoO 4	2-cat.
								microemulsion, rt
								99%

J HH =1.1 Hz, 2 J HH =10.8 Hz, 2 × CH 2 (CH)C(OH)), 5.17 (dd, 1H, 3 J HH =1.1 Hz, 2 J HH =17.4 Hz,

Funding Sources

This research was supported by the Chemistry Research Foundation (Fondation ciRFC, Strasbourg, France, Grant Number JL-FRC-0001).

Notes

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of this article.

ASSOCIATED CONTENT Supporting Information.

Experimental data on synthesis and reactivity of 4-( 13 C)-Lina-OOHs; General mechanism for the detection of free radicals by spin trapping; Data on EPR spin trapping experience with cysteine.

AUTHOR INFORMATION

Corresponding Author * Elena Giménez-Arnau e-mail: egimenez@unistra.fr Tel: +33 3 68 85 15 25

ACKNOWLEDGEMENT

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


Simon G Potts

Peter Neumann

Bernard Vaissière

Nicolas J Vereecken

Robotic bees for crop pollination: Why drones cannot replace biodiversity

a b s t r a c t

The notion that robotic crop pollination will solve the decline in pollinators has gained wide popularity recently (Fig. 1), and in March 2018 Walmart filed a patent for autonomous robot bees. However, w present six arguments showing that this is a technically and economically inviable 'solution' at present and poses substantial ecological and moral risks: (1) despite recent advances, robotic pollination is far from being able to replace bees to pollinate crops efficiently; (2) using robots is very unlikely to be economically viable; (3) there would be unacceptably high environmental costs; (4) wider ecosystems would be damaged; (5) it would erode the values of biodiversity; and, (6) relying on robotic pollination could actually lead to major food insecurity.

Throughout the Anthropocene, biodiversity has underpinned a wide range of ecosystems goods and services providing multiple benefits to people and improving human wellbeing [START_REF] Díaz | The IPBES conceptual framework-connecting nature and people[END_REF]. Often these services are unrecognized or perceived to be 'free' [START_REF] Daly | Ecological Economics: Principles and Applications[END_REF], and the increasing threats to their ongoing provision poses major challenges for society on how to protect and manage biodiversity (Global Biodiversity Outlook 4, 2014).

Nature has elegantly solved many problems, and scientists and engineers can learn a great deal by studying biodiversity and nature. For instance, the growing field of biomimicry, and its many success stories, is a direct testimony of how innovative engineering solutions derived from the study of adaptations in the natural world can benefit our societies from transport to architecture, swimsuits or even military camouflage [START_REF] Benyus | Biomimicry: Innovation Inspired by Nature[END_REF]. Prototype robots are being developed as autonomous weeding machines for agricultural crops [START_REF] Reuters | Robots fight weeds in challenge to agrochemical giants[END_REF]. A recent study by Harvard researchers [START_REF] Chechetka | Materially engineered artificial pollinators[END_REF], and the breaking news of Walmart filing a patent for autonomous robot bees (Business Insider, 2018), both propose bringing together the fields of biomimetic science and miniature robotics to address the looming crop pollination crisis as the need for insect pollination increases while the population of managed and wild pollinator dwindle [START_REF] Aizen | Expanding the limits of the pollen-limitation concept: effects of pollen quantity and quality[END_REF][START_REF] Potts | Safeguarding pollinators and their values to human well-being[END_REF]. Public concern for pollinators, and the pollination service they provide, has grown rapidly and a quick 'technological fix' to the problem seems quite appealing, especially when the developers of prototype robotic bees claim that they will be able to safeguard crop pollination in the near future. In contrast, the Intergovernmental Platform on Biodiversity and Ecosystem Services global assessment of pollinators, pollination and food production (IPBES, 2016) and the Convention on Biological Diversity [START_REF] Cbd | Convention on Biological Diversity, Subsidiary Body on Scientific, Technical and Technological Advice 22nd meeting of SBSTTA "Conservation and Sustainable use of Pollinators[END_REF] found no evidence to include robotic bees as a credible response option to loss of pollinators or pollination. In this discussion, we recognise that nature can inspire cutting edge technology, but put forward robust arguments showing that robotic pollination is currently incapable of delivering crop pollination to sustain production on a world scale, and even if the technology was developed sufficiently, then there are strong economic, ecological and social reasons not to pursue this route (Fig. 1). Given the increasing scientific, political and public interest in the plight of pollinators, it is important we separate the evidence-based response options from those which are highly speculative and make unrealistic claims as to their putative effectiveness. The aim of this piece is to present a considered view on the opportunities and risks for securing crop pollination services with and without robotic bees. We highlight that emerging technologies have many beneficial roles to play in society, but in this case there is no justification for needlessly trying to replace a key component of biodiversity which can readily be protected and enhanced.

Despite recent advances, robotic pollination is far from being able to replace bees to pollinate crops efficiently

While technology is moving in the direction of unmanned flying robots able to make complex decisions, they are still extraordinarily clumsy and unsophisticated compared to real bees. Flowers represent multimodal sensory billboards involving shape, colour, scent and even iridescence that are detected, approached and manipulated by bees for the collection of pollen, nectar and other floral rewards through neurological and behavioural responses that are still poorly understood [START_REF] Cresswell | Manipulation of female architecture in flowers reveals a narrow optimum for pollen deposition[END_REF]. Delivering efficient cross-pollination at the level of species-rich communities of co-occurring plant species, or even in a more homogeneous field of cultivated flowering plants, involves a lot ⁎ Corresponding author. URL's: s.g.potts@reading.ac.uk, (S.G. Potts), peter.neumann@vetsuisse.unibe.ch, (P. Neumann), bernard.vaissiere@inra.fr, (B. Vaissière), nicolas.vereecken@ulb.ac.be (N.J. Vereecken). more than taking up the technological challenge of designing a miniature drone flying towards a flower and picking up a fraction of the available pollen grains. There are more than 350,000 species of flowering plants on the planet [START_REF] Ollerton | How many flowering plants are pollinated by animals?[END_REF], and they all interact in very unique ways with animals as pollen vectors to bring about sexual reproduction, fruit and seed production, and evolution. Moreover, there are many floral visitors, but only few are actually effective pollinators [START_REF] King | Why flower visitation is a poor proxy for pollination: measuring single-visit pollen deposition, with implications for pollination networks and conservation[END_REF], and their ecological/behavioural traits diversity, not the sheer abundance of one species of particular bee, has been shown to be a significant driver of pollination efficiency and crop yields [START_REF] Hoehn | Functional group diversity of bee pollinators increases crop yield[END_REF][START_REF] Albrecht | Diverse pollinator communities enhance plant reproductive success[END_REF][START_REF] Fründ | Bee diversity effects on pollination depend on functional complementarity and niche shifts[END_REF][START_REF] Garibaldi | Wild pollinators enhance fruit set of crops regardless of honey-bee abundance[END_REF][START_REF] Martins | Pollination services are mediated by bee functional diversity and landscape context[END_REF]. Technology has taken tiny steps to try to address the pollination process of a few 'easy' crops such as sunflowers (Helianthus annuus) which has large disk-shaped easily accessible inflorescence, but is still barely out of the starting gates, while evolution, through the high levels of functional biodiversity and complex ecological, crossed the finishing line millions of years ago.

Using robots is very unlikely to be economically viable

There are many billions of individual bees and other pollinators across the planet already doing an effective job of crop pollination. Given some of them are declining, the most cost efficient strategy to secure production is to safeguard the pollinators we already have and sustainably manage landscapes to increase their numbers further (IPBES, 2016). Trying to replace this existing pollination service with fleets of robots is economically inviable: even if the technology was up to the job, the cost will likely be totally prohibitive. Even at a modest $10 per bee for example, the total cost would be many 100 s of billions of dollars to pollinate the area of insect-pollinated crops that is currently grown over the world. Further, there are the costs of hardware repair and maintenance, command and control infrastructure. For a fraction of the cost of robot pollination, society could implement well-established solutions [START_REF] Dicks | Ten policies for pollinators[END_REF]IPBES, 2016) to protect pollinator habitats, reduce threats to pollinators and promote biodiversity-friendly cities and landscapes, thereby protecting nature's heroes instead of trying to replace them at exorbitant costs. This is not to say that in the future private individuals and businesses should not invest in developing such technologies for target niches, as it could potentially have a role to play in food production for a small number of specialist crops for which we currently do not have manageable pollinators (such as for hybrid seed production of nectarless crops such as tomato Lycopersicon esculentum or lettuce Lactuca sativa; [START_REF] Liu | Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers[END_REF]. Robotic pollinators would likely result in spin-off applications outside pollination, however, to use publically funded research or government subsidies is highly questionable when the outcome is likely to be loss of opportunities to protect existing biodiversity.

There would be unacceptably high environmental costs

There would be a huge energy, carbon, water and material's footprints to extract, transport and process the raw materials, to manufacture, distribute, and operate, maintain and repair all the robot bees and their associated infrastructure, and to ultimately to dispose of or recycle irreparable or broken robots. For instance, what would be the additional environmental impact of mining all the necessary lithium and other rare earth metals, whose current exploitation is already of growing environmental and social concerns? When robots reach the end of their working lives, or become broken or trapped, what is the fate of all their constituent pollutants entering human and wildlife food chains? Characterising the full energetic and environmental costs of robot bee technology though life cycle assessments (LCAs) will likely reveal a carbon footprint and significant negative impacts on the environment that are all incompatible with our aims for a low-carbon, energyefficient future.

Wider ecosystems would be damaged

Populating the world with robotic pollinating machines would be a species invasion of epic proportions. It is well-established for pollinators, wild plants and many elements of biodiversity that alien invasive species cause local/regional extinctions, disrupt species interactions networks, as well as ecosystem functions and services [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions. In networks of invasion: empirical evidence and case studies[END_REF]. Introducing robotic pollinating machines to remove and spread pollen would disrupt the delicate balance of species already in, and reliant on, agricultural and natural ecosystems by displacing existing pollinators, removing pollen forage, while failing to pollinate all the wild flowering plants reliant on biotic pollination [START_REF] Ollerton | How many flowering plants are pollinated by animals?[END_REF].

It would erode the values of biodiversity

Replacing a key component of biodiversity with a technological alternative, while ignoring opportunities to protect it, fails to take account of the multiple values associated with pollinator biodiversity, such as intrinsic (e.g. inherent worth), social (e.g. beekeeping) and cultural (e.g. aesthetic and recreational) values (IPBES, 2016). For instance, the high social value placed upon monarch butterflies in North America has helped drive conservation actions for this and other threatened species [START_REF] Diffendorfer | National valuation of monarch butterflies indicates an untapped potential for incentive-based conservation[END_REF]. Other innovations, such as robotic weeding machine with precision spraying systems, can significantly reduce the amount of herbicides used by thereby helping support wider biodiversity [START_REF] Reuters | Robots fight weeds in challenge to agrochemical giants[END_REF], which is in stark contrast to pollinating robots aiming to replace biodiversity. 6. Relying on robotic bees could actually lead to major food insecurity Reliance on a single pollinator is already a high risk strategy for crop pollination [START_REF] Garibaldi | Wild pollinators enhance fruit set of crops regardless of honey-bee abundance[END_REF] and the same applies for substituting a diversity of pollinators with robotic devices. While Walmart and others may be proposing new technology to secure supply chains and food production, in reality this approach may increase vulnerability through the failure of complex technology or cyber-attack. Further, low income farmers represent more than 2 billion people reliant on smallholder agriculture in developing nations [START_REF] Garibaldi | Mutually beneficial pollinator diversity and crop yield outcomes in small and large farms[END_REF], and it is hard to see how these growers will be able to afford robotic pollination services given they struggle to buy even basic agricultural inputs.

Nature is inspirational and we should seek opportunities to learn from millions of years of evolution. But jumping on the popular pollination band wagon, while deliberately overlooking more than 200 years of research on plant-pollinator interactions, by claiming robots can replace bees, or rather "help counter the decline in honeybee populations" [START_REF] Chechetka | Materially engineered artificial pollinators[END_REF] flies in the face of many local, regional, national and international initiatives aiming to safeguard pollinators and their critical values to human well-being (IPBES, 2016). The proposed technology is embryonic, the cost prohibitive, and the wider environmental and societal risks unacceptable.

Risks to crop pollination need to be addressed, and there are a wide range of options open to society going from well-proven practical interventions (e.g. managing habitats to support wild pollinators and/or augmenting populations of managed pollinators), to new food production systems (e.g. ecological intensification of agriculture to embed pollinators into farming, or breed and/or genetically engineer crops that are parthenocarpic or more self-compatible and self-fertile to produce plants less reliant on biotic pollination) (IPBES, 2016;[START_REF] Knapp | Re-evaluating strategies for pollinatordependent crops: how useful is parthenocarpy?[END_REF]. There are well developed practices and policies to reduce the immediate risks to pollinators from pesticides, pest and diseases, and climate change, and also more ambitious approaches to transform societies relationship with nature. Together these provide a portfolio of effective tools and solutions to safeguard crop pollination. One has to wonder whether robotic bees simply represent a technological solution desperately looking for a relevant real-world problem to solve. Miniature flying robots, as with other digital technologies, may have many potentially important uses [START_REF] Arts | Digital technology and the conservation of nature[END_REF]. Robotic pollination, however, is simply not the answer to securing widespread crop pollination, and encouraging its development diverts time, money, and other resources that could be directed towards national and international pollinator initiatives and policies [START_REF] Dicks | Ten policies for pollinators[END_REF], striving to secure both biodiversity conservation and food production in a sustainable manner.

Fig. 1 .

 1 Fig. 1. Could robotic drones replace pollinator biodiversity to sustainably deliver pollination services to wild and cultivated flowering plants? The answer is no: as it is currently technically and economically inviable and poses substantial environmental and moral risks. Drone image reprinted from Chechetka et al. (2017) with permission from Elsevier.

S.G. Potts et al. / Science of the Total Environment 642 (2018) 665-667