Moreira Luís

João Grosso Gonçalves
email: luis.goncalves@fc.up.pt

Paulo Jorge Pacheco

José António Magalhães

Aquiles Araújo Rodrigues

Barros

Gonçalves

João Grosso Pacheco

Paulo Jorge Magalhães

José António

Aquiles Araújo Barros

Determination of Free and Total Sulfites in Wine using an Automatic Flow Injection Analysis System with Voltammetric Detection

Keywords: Wine, Sulphur dioxide, Sulfites, Gas-diffusion, Square-wave voltammetry, 18 Flow injection analysis (FIA), Food analysis, Food additives, Analytical chemistry 19 20

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

SO 2 and its source compounds, such as sulfite salts, are widely used as preservatives in 22 the food industry. They are added to foods and beverages, in order to prevent undesirable 23 microbial growth, discoloration and oxidation processes, consequently improving the 24 quality and final appearance of the products. In wine, sulphur dioxide is usually found 25 either free (as HSO 3 -, SO 3 2-and SO 2 , depending on the sample's pH), or bound to carbonyl 26 and unsaturated compounds and/or phenol derivatives [START_REF] Barbe | Role of carbonyl compounds in SO 2 199 binding phenomena in musts and wines from botrytized grapes[END_REF].

27

The relevance of SO 2 determination in food analysis is widely established; in the 28 particular case of wine, its control is essential not only because at high levels it produces an 29 unpleasant aroma and taste but also because of health hazards especially for hypersensitive 30 people (Decnop-Weever et al. 1997). SO 2 levels should be checked before bottling in order 31 to be adjusted for a suitable aging and at the end-product to comply with existing legal 32 limits (Segundo et al. 2001). In the EU the maximum level of total SO 2 permitted is 160 The voltammetric flow cell was manufactured in our laboratory and has been described 81 previously [START_REF] Barros | Processo para a determinação 201 voltamétrica, célula voltamétrica e dispositivo para determinação em fluxo[END_REF], 2002, Rodrigues et al. 2001, Rodrigues et al. 2002).

82

A home-made Perspex gas-diffusion module was used with PTFE membranes, 5.0 µm 83 pore size and 47 mm diameter, from Pall Corporation (Ann Arbor, U.S.A.).

84

The FIA system is depicted in Figure 2: it includes several three way solenoid valves, a 85 peristaltic pump and PTFE tubing. The sample flow reacts on-line with an acid solution, in 86 the "Y" connection in order to turn all sulfites into SO 2 ; then it goes through a thermostated 87 coil that has the function of pre-heating the donor flow before entering the gas-diffusion 88 module; the SO 2 passes through the hydrophobic membrane and is received by the acceptor 89 flow (acetate buffer) that takes it to be instrumentally detected. 90

The FIA system was incorporated in a prototype (Figure 3). This equipment works 91 automatically with its own purposely designed control software. A GPES 4.9 Software The wine sample must be acidified prior to gas diffusion in order to turn all sulfites into 113 SO 2 , and the pH must be very low (pk a1 = 1.77 and pk a2 = 7.22). On-line acidification with 114 sulfuric acid was achieved using a "Y" shaped connection that helped mixing together the 115 sample flow with the acid flow.

116

The acid concentration was optimized, knowing that it should be as low as possible to electron reduction of HSO 3 -has been suggested (1) [START_REF] Reynolds | Polarographic reduction of aqueous sulfur dioxide[END_REF]) along with the 126 possibility that HSO 3 -becomes SO 2 at the drop's surface, and it is this species reduced in 127 the electrode (2) [START_REF] Tolmachev | Electrochemical reduction of bisulfite in mildly acidic buffers: kinetics 258 of sulfur dioxide and bisulfite interconversion[END_REF].

128

HSO 3 -+ H + + e -→ SO 2 -+ H 2 O (1) 129 SO 2 + e -→ SO 2 - ( 2
)
130 Due to matrix effect, standard additions were used to quantify the level of SO 2 in the 131 different wine samples (Figure 5). Since the linearity was quite good for routine analysis 132 one single addition was enough.

133

Two separate determinations must be done for each wine, one for the free sulphur content 134 and another for the total, the difference lays on previous sample treatment with sodium 135 hydroxide for the second case.

136

Study of interferences 137

The level of ethanol in wine varies to a large degree and consequently the influence of The determination of sulfite concentrations in the different wine samples by the 148 developed flow procedure was compared with the reference "Ripper" method and its 149 simplified version generally used in the wine industry (Figure 6). 3). LOD and LOQ [START_REF] Eurachem | The fitness for purpose of analytical methods -A laboratory guide to method validation 220 and related topics[END_REF] [START_REF] Barros | Processo para a determinação 201 voltamétrica, célula voltamétrica e dispositivo para determinação em fluxo[END_REF][START_REF] Barros | Process and cell for voltammetric 203 measurement[END_REF].

  117save chemical resources and to prevent the equipment's corrosion but still strong enough to 118 accomplish its objective. After several experiments (Figure4) concentration chosen was 1 electrochemical reduction of SO 2 in aqueous solution with dropping 122 mercury electrode has reported two waves in acidic medium and no waves in neutral or 123 slightly alkaline media. There has been much disagreement in the interpretation of 124 polarographic reduction of aqueous sulphur dioxide at pH 4[START_REF] Streeter | Cathodic reduction of bisulfite and sulfur dioxide in 255 aqueous solutions on copper electrodes: an electrochemical ESR study[END_REF]. A one-125

  150The "Ripper" method was performed according to the Portuguese regulation(IPQ 1987) 151 based in a procedure from the Organisation Internationale de la Vigne et du Vin (normally uses a simple iodometric methodology for routine analysis: 154 instead of the four titrations in the "Ripper" method (one for the free SO 2 , two for the total 155 SO 2 and one for the interferences) only two are carried out (one for the free and another one 156 for the total SO 2 ). The methodology consists on iodometric titrations of an acidified wine 157 sample. For the free SO 2 determination, 5 mL of 1:4 H 2 SO 4 are added immediately before 158 the titration. To determine the total SO 2 content, 25 mL of 1 mol•L -1 NaOH are added 15 159 minutes prior to analysis, in order to perform alkaline hydrolysis, and then 10 mL of 1established by plotting a calibration curve with eight different 163 standards, from 10 to 250 mg•L -1 , with six repeated measurements of each point (n = 6) 164 (Table

274 275Figure 2 . 280 Figure 3 .Figure 4 .Figure 5 . 290 Figure 6 .Figure 7 .

 274228034529067 Figure 2. Flow system scheme. A -acceptor circuit; B -donor circuit; solid lines -permanent

  mg•L -1 for red wines and 210 mg•L -1 for white and rosé wines (EC 1999).

	73
	74	Instrumentation
	75	Voltammetric measurements (parameters are described in Table 1) were performed with
	76	a Metrohm 663 VA Voltammetric Stand (Herisau, Switzerland) equipped with a hanging
	77	mercury drop electrode with glassy carbon auxiliary electrode and reference AgCl/Ag (3M
	78 79 80	F o r KCl) electrode. The system was connected to an Autolab PGSTAT 10 Voltammetric System F (Eco Chemie, Utrecht, The Netherlands). All measurements were made at room o r temperature.
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
		w w
		O n l O n l
		y y
	33

[START_REF] Barros | Voltammetry of compounds confined at 205 the hanging mercury drop electrode surface[END_REF]

.

71

Materials and methods

92 project was developed for automatic voltammetric data acquisition, data saving, and signal 93 processing. It was possible to interconnect both software using a signal trigger transmission. 94 Chemicals and wine samples

  For the determination of free SO 2 no sample preparation was required. For the 106 determination of total SO 2 an alkaline pre-treatment was essential to destroy the adducts

		F o r
		P
		e
		e r
		R
		e
		v i e
		w
	95 96 97 98	All reagents used were of p.a. grade and were used without further purification. Ultra-pure water from a Millipore Simplicity 185 water purification system (Millipore, Billerica, O n l U.S.A.) was used. The supporting electrolyte (acceptor solution) consisted of acetate buffer y
		pH 4.0 (0.1 mol•L -1 ), sodium acetate was purchased from Merck (Darmstadt, Germany).
	105

99

The buffer's pH was adjusted with hydrochloric acid, 6 mol•L -1 , from Prolabo (Fontenay-100 sous-Bois, France). Na 2 SO 3 (Sigma, St. Louis, U.S.A.) was used as a source of SO 2 . Ethanol 101 was also purchased from Merck. 102 Some of the wine samples were bought in regular supermarkets in Porto while others 103 were kindly supplied by the Instituto dos Vinhos do Douro e Porto (IVDP).

104 Sample preparation 107 (SO 2 bounded to several aldehydes and ketones) formed in wine (Sarudi et al. 1998). For 108 that purpose 10 g of sodium hydroxide were added per liter of sample. The sample was 109 homogenized and left to rest for about 20 min. 110 Results and discussion 111 On-line acidification 112

  ethanol concentration was studied. With that purpose, two SO 2 calibrations curves (10 to 139 100 mg•L -1 ) were compared: one in water and the other one in ethanol 10% (v/v), 140 replicating ethanol concentration in wine. As can be perceived in Table2, ethanol showed

	141
	142	no influence in the analysis.
	143	Carbon dioxide is normally found in wine in the concentration range of 1 to 2 g•L -1 due to
	144	the fermentation process (Santos et al. 2006). Previous studies showed that this compound
	145	does not interfere in the analytical procedures that make use of gas-diffusion (Santos et al.
	146	2006, Segundo et al. 2001).
	147	r o F Method comparison
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
	138

Table 1 .

 1 were adequate to wine analysis, though they can In this paper a new automatic FIA system for the determination of SO 2 in wine is SWV parameters.

	165
	166	be easily decreased with a slight time-consuming change in the procedure. The buffer
	167	solution can be stopped and isolated at the upper chamber by this way allowing an
	168	incremented quantity of SO 2 passage to the upper chamber and consequently an increase in
	169	the analytical signal, i.e. the LOD can be lowered by increasing the time needed for each
		analysis (Figure 7). This makes it possible to analyze matrices with low sulfite content such
	173

170 as beer, juices and other food products. In the present configuration the prototype is able to 171 analyze 15 samples per hour. 172 Conclusions 182 industry needs. The authors also wish to thank the European Regional Development Fund 186 (ERDF), the Portuguese Programa Operacional Ciência Tecnologia Inovação (POCTI) and 187 the Portuguese Programa Operacional da Economia (POE) for all the financial support 188 given to project "BEERVOLT" (POCI 2010 -IDEIA, project no. 13-02-03-FDR-01252). 189 L.M.G. (SFRH/BD/36791/2007), P.J.M. (SFRH/BD/27834/2006) and J.G.P. 190 (SFRH/BD/30279/2006) wish to acknowledge Portuguese Fundação para a Ciência e a

Table 2 .

 2 Evaluation of the possible interference of ethanol.

	268
		water		ethanol 10% (v/v)
		y = mx + b	r 2	y = mx + b	r 2
	Day 1	y = 4.7x -7	0.995	y = 4.6x -16	0.995
	Day 2	y = 4.6x + 8	0.998	y = 4.5x -5	0.999

Table 3 .

 3 Method performance characteristics, I = f ([SO 2 ]).

	Page 11 of 19	Food Additives and Contaminants
	271
	Linear dynamic range RSD (n = 6)	r 2	LOD	LOQ
	10 to 250 mg•L -1	6%	0.9999	2.7 mg•L -1	9.2 mg•L -1
	272

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Chinvongamorn C, Pinwattana K, Praphairaksit N, Imato T, Chailapakul O. 2008. Amperometric

determination of sulfite by gas diffusion-sequential injection with boron-doped diamond electrode.

Sensors. 8: 1846-1857.

Decnop-Weever L, Kraak J. 1997. Determination of sulphite in wines by gas-diffusion flow injection

analysis utilizing spectrophotometric pH-detection. Anal Chim Acta. 337: 125-131.

Dvořák J, Dostálek P, Štěrba K, Čejka P, Kellner V, Čulík J, Beinrohr E. 2006. Determination of total

Sarudi I, Kelemen J. 1998. Determination of sulphur and total sulphur dioxide in wines by an ICP-AES

method. Talanta. 45: 1281method. Talanta. 45: -1284. .

Segundo M, Rangel A. 2001. A gas diffusion sequential injection system for the determination of sulphur

dioxide in wines. Anal Chim Acta. 427: 279-286.

Silva R, Silva C, Nóbrega J, Neves E. 1998. Flow injection spectrophotometric determination of free and

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Acknowledgments 183

The authors thank the IVDP, particularly Eng. Natália Ribeiro and Eng. Maria José 184 Mirão, for gently supplying us with wine samples and helping us understand the wine


Miguel Mauricio-Iglesias

Simon Jansana

Stéphane Peyron

Nathalie Gontard

Valérie Guillard

M Jansana

S Gontard

N Guillard

Effect of HP/T treatments of in-package food on additive migration

Keywords:

Introduction 14

In order to fulfill the increasing demand for high quality food, high pressure (HP) treatments stand as 15 one of the most promising new preservation technologies. Indeed, HP has been proven to be a mild 16 treatment able to render micro organisms inactive as well as the enzymes responsible for shelf-life 17 shortening with almost no modifications of the sensory and nutritional attributes of the product 18 (Rastogi et al. 2007) . In order to extend the applicability of HP, a number of papers have recently 19 appeared proposing the combination of pressure and temperature as a less aggressive method to 20 sterilize food than the conventional thermal sterilization (Ven et al. 2007;Wilson et al. 2008) . Since 21 food is packed before high pressure/thermal (HP/T) batch processing, the packaging material is also 22 exposed to non-conventional conditions of pressure and temperature. The packaging structure may be 3 altered and consequently its mechanical and mass transfer (barrier and migration) properties as well 24 (Guillard et al. 2009) . Therefore, it is essential to assess properly the impact of this novel process on 25 migration in order to ensure its safety and avoid any potential health concern, especially since some Migration of a substance from packaging into food is a subject of growing interest for the scientific 30 and legislative communities. Low and medium molecular weight substances, (e.g. plastic additives, 31 residual monomers) are not chemically bound to the polymer chains and can therefore migrate from 32 the polymer matrix, especially, when packaging undergoes severe conditions of temperature during 33 treatment. To date, research on mass transfer phenomena in packaging submitted to the couple effect 34 of HP/T found that these treatments had no or slight effect on barrier properties of packaging materials 35 e.g. Lopez-Rubio et al (2005) on gas permeation and conditions up to 800 MPa and 75°C; 36 Schauwecker et al.(2002) on permeation of the pressure transmitting fluid (up to 827 MPa and 75°C); 37 Caner et al(2004) and Kübel et al (1996) on aroma sorption (up to 800 MPa and 60°C). To our

( )       + - + - = - - ∑ ∞ = ∞ t D L n n C C C C n in L in t . ) 1 2 ( exp 1 2 1 8 1 2 2 2 0 2 2 , π π (1)

Results and Discussion

188 Uvitex OB is an optical brightener and UV stabilizer approved for use in food contact material (FCM) 189 with a specific migration limit (SML) of 0.6 mg kg -1 (2002/72/EEC). Irganox is an antioxidant 190 approved for FCM with a specific migration limit (SML) of 6 mg kg -1 (2002/72/EEC). in n-hexane, 320 g kg -1 ). For Uvitex OB, a quite complex molecule, it is not easy to find a trend (a 213 solubility scale could be built as chloroform>ethyl acetate>acetone>n-hexane>methanol). Therefore, 214 the solubility was obtained experimentally giving a result of 5±2 g L -1 of Uvitex in olive oil. The migration of Irganox 1076 during the HP/T treatment alone (no further storage), was assessed by 230 comparing the quantity of remaining additive before and after the treatment (Figure 1A). As it can be 231 seen, migration is much higher in olive oil than in the aqueous FSL (in almost all cases lower actually 232 than the limit of quantification for the FTIR method estimated at 0.03 % w/w in the film 1) and the assumption that the FSL surrounding the sample was close to zero.

347

The diffusivity values obtained are not the "actual" diffusivities but for the UTC, since in the other 348 cases the kinetics includes not only the storage but also the treatment (HP/LT and HP/HT). According 349 to Figures 1 and2, this influence would be negligible. Anyway, these values are suitable for the 350 comparison of the whole migration story of the packaging/FSL system. Figure 5

  38 knowledge, only one study (Caner and Harte 2005) was devoted to the impact of HP/T on specific 39 migration of one additive (Irganox 1076 in PP) and did not observe any difference for treated samples 40 at 800 MPa and 40 or 60°C. 41 42 The aim of this paper is to present a complete assessment of migration under pasteurization and 43 sterilization conditions. Therefore, it aims at extending the knowledge on migration including (i) the 44 four food simulating liquids (FSL) set by European Directive 85/572/EEC (1985); (ii) more severe 45 conditions of pressure/temperature coupled (up to 800 MPa and 115°C) and (iii) on new materials as 46 bio-sourced PLA. It is important to point out that even if olive oil is the fatty FSL set in priority by 47 were analyzed by transmission FTIR. Spectra were recorded using a Nexus 5700 117 spectrometer (ThermoElectron Corp.) equipped with HeNe beam splitter and a cooled MCT detector. 118 Spectral data were accumulated from 128 scans with a resolution of 4 cm -1 in the range 800-4000 cm -1 . 119 Three samples were employed for the measure and three spectra were recorded for each sample 120 All spectra pre-treatments were performed using Omnic v7.3 and TQ Analyst v7.3 softwares 121 (ThermoElectron) Processing included: (1) a multipoint linear baseline correction, (2) a normalization 122 according to the area of the LLDPE doublet (1369-1378 cm-1) due to the CH3 symmetric Uvitex OB from the sample was followed by the disappearance of a double bond 126 aromatic peak at 1590 cm -1 A Partial Least Square (PLS) model was calibrated on the basis of the 127 intensity ratio 1579/1378 cm -1 . The regression produced a linear relationship (R 2 = 0.96, RMSE= 128 0.034% w/w) between spectral data and additives concentration measured by UV analysis as release of Irganox 1076 from LLDPE samples was followed by the disappearance of the 132 peak at 1235 cm -1 following a similar procedure. The PLS results for Irganox 1076 were R 2 = 0.97, 133 RMSE= 0.019% w/w. 134 FTIR measurements were successfully validated by measuring migration after 26 days of storage at 135 40°C using the UV method to determine the final content in the film. in the LLDPE were determined as follows. Thin slices of LLDPE were 139 prepared using a razor blade and stuck on a microscope slide. Raman spectra were recorded between 140 cm -1 Raman shift using a confocal Raman microspectrometer Almega (Thermo-Electron) 141 with the following configuration: excitation laser He-Ne λ0 = 633 nm, grating 500 grooves/mm, 142 pinhole 25 µm, objective × 50. The collection time was about 50 s. Measurements were carried out in 143 the depth sample with a step size of 10 µm from the sample center to the interface. All spectra pre-144 treatments were performed with Omnic v7.1 (Thermo-Electron). Processing included: (i) a multipoint 145 linear baseline correction, (ii) normalization according to the area of the LLDPE specific band at 1129 146 cm-1 representing the symmetric C-C stretching of all-trans PE chains. The relative content of Uvitex 147 OB was assessed using the area of the specific doublet (1569-1614 cm -1 ) assigned to the aromatic C=C 148 and C=N bands respectively. in olive oil was measured by a Varian Cary Eclipse Fluorimeter. The 152 measurements of fluorescence intensity were done at two excitation wavelengths, 332 and 373 nm, and 153 detected at an emission wavelength of 434 nm. The collection time was 1 s. The results were 154 averaged on the readings at the two excitations wavelengths. 155 Solutions of olive oil were oversaturated with Uvitex OB and kept for 20 days at 40°C. A small 156 volume of the supernatant was taken and diluted to 1:1000 or 1:2000 and then measured in the 157 fluorimeter (in sextuplicate).

  were developed at the FP5 Foodmigrosure project (Anonymous 2004) . 161 Uvitex OB and Irganox 1076 were extracted from the polymers via dissolution in toluene at 103°C for 162 7 min for LLDPE and 5 min for PLA followed by a precipitation in methanol for LLDPE and in 163 petroleum ether for PLA. The cooled solution was filtered to remove the precipitate and evaporated 164 50°C (for 7 min approximately). Calibration of the extraction procedure showed that 165 these conditions enabled stable and reproducible recovery (88.5% ± 0.6). The calibration and recovery 166 were determined following the same procedure as Nerin et al. (2003) . Dry extracts containing Uvitex 167 OB were then dissolved in 10.0 ml of THF and dry extracts containing Irganox 1076 were dissolved in 168 10.0 ml methanol. The additive content was quantified by UV spectroscopy (Varian Cary 100 Scan, 169 UV-Visible spectrophotometer) at 374 nm for Uvitex OB and 277 nm for Irganox 1076. of a sheet of polymer immersed in a liquid of infinite volume and constant concentration, 173 the evolution of additive content with time is given by (Crank 1980) : 174

  175where C t is average concentration in diffusing substance in the packaging at time t, C in is the initial 176 concentration in additive and C L,∞ is the concentration of the diffusing substance on the surface of the 177 polymer required to maintain equilibrium with the concentration of this substance in the liquid at any 178 time t.179Additive diffusivity was identified from experimental data by minimizing the root mean square error 180 (RMSE) between experimental and predicted data (Equation1) using an optimization method 181 (Levenberg-Marquardt algorithm predefined in Matlab (Mathworks, USA) software). A Monte Carlo 182 sampling was applied to the input parameters (concentration, thickness and time) in order to determine 183 the 95% confidence intervals of the determined diffusivity as detailed elsewhere(Hessler 1997

Food

  were selected to spike the studied samples (LLDPE and PLA) because of (i) their 193 excellent thermal stability (for example, according to the supplier datasheet, the thermogravimetrical 194 analysis on Uvitex OB shows a loss of only 1% weight at 280°C) (ii) their relatively high diffusivities 195 in polymers allowing to easily observe migration kinetics in LLDPE (Dole et al. 2006; Helmroth et al.196 2002) and (iii) their easy detection and quantification using both standard methods (HPLC, UV) and 197 spectroscopic method such as FTIR and Raman (Mauricio-Iglesias et al. 2009) . The objective was to 198 assess the influence of HP/T treatments comparing: 199 -HP sterilization-like treatment (800 MPa, 115°C, 5 min) 200 -HP pasteurization like treatment (800 MPa, 40°C, 5 min) 201 -Conventional sterilization (20 min at 121°C) in autoclave 202 -Conventional mild pasteurization (30 min at 63°C) 203 -Untreated control (abbreviated to UTC, 40°C, 7 min to take into account the time of 204 build-up and release of pressure, approx 2 min) 205 206 Determination of Uvitex OB solubility in olive oil 207 Before tackling the assessment of migration of additives it was essential to gather some information 208 about its solubility. A rather low upper bound for the solubilities in water of both Irganox 1076 and 209 Uvitex OB can be found in the safety data sheet, however there is no indication of their solubility in 210 olive oil. Irganox 1076 is consistently more soluble in non polar solvents than in polar ones, what 211 Page 10 of 30 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk solubility in olive oil as very high (probably of the same order of magnitude as 212

  performed on untreated control (UTC) and treated samples (HP/HT, 218 HP/LT and conventional pasteurization and sterilization) of LLDPE spiked with Uvitex OB (initial 219 quantity of 0.41±0.10 % w/w) or Irganox 1076 (initial quantity of 0.37±0.023 % w/w. The kinetic 220 release of Uvitex OB and Irganox 1076 was monitored with FTIR measurements that supplied the 221 content of Uvitex or Irganox in the polymer strip. Concerning the thermal sterilization treatment, none 222 of the migration results could be obtained since the sterilized samples melted during the sterilization 223 process, which indeed made them useless for packaging purposes. This was not surprising since the 224 melting point of the LLDPE used had been determined near 115°C. However, it should be emphasized 225 that the LLDPE samples, which underwent the HP at high temperature (HP/HT, sterilization-like 226 treatment) kept their integrity in turn. This result underpinned the fact that if a packaging material 227 cannot withstand a thermal treatment; an equivalent treatment might be done with high pressure.228 229

  packaging to foodstuff can be well simulated provided that three parameters are 337 known, diffusivity in the polymer (D), the partition coefficient polymer/foodstuff (KPL) and the mass 338 transfer coefficient (k), although the latter can be ignored in many practical cases as its influence is low 339 compared to the diffusivity in the material (Pocas et al. 2008) . Roughly the KPL represents the values 340 of migration at equilibrium and D, how fast equilibrium is reached. The use of this parameter makes 341 easier the comparison of results inter-studies since it allows to get rid of the influence of variable 342 experimental set-ups (e.g. polymer thickness, polymer/FSL mass ratio). With the scope of fully 343 characterizing this case study, the mass transfer parameters were determined when possible. 344 Otherwise, at least a rough estimation is provided whenever the results prevented an accurate 345 determination of the parameters. The mathematical model used to determine the diffusivity consisted 346 on Fick's law (Equation

Figure 1 .

 1 Figure 1. Comparison of Irganox 1076 losses from LLDPE in contact with four different FSL after

Figure 2 .

 2 Figure 2. Comparison of Uvitex OB losses from LLDPE in contact with four different FSL after high

Figure 3 :Figure 4 :Figure 5 :Figure 1 .

 3451 Figure 3: Raman spectra at different depths of LDPE film

Figure 2 .

 2 Figure 2. Comparison of Uvitex OB losses from LLDPE in contact with four different FSL after high

Figure 3 :Figure 4 :Figure 5 :

 345 Figure 3: Raman spectra at different depths of LDPE film

  On the other hand, the phenomena assessed here are more complex in the case of Uvitex OB in 354 LLDPE because of blooming of the additive. To the best of our knowledge, little has been published 355 on the kinetics of blooming.Spatafore and Pearson (1991) found that the kinetics of blooming of

	Page 17 of 30	Food Additives and Contaminants Food Additives and Contaminants	Page 16 of 30
	401	detected for HP/HT sterilization. For pasteurized samples, there were no differences between Anonymous 2008. Bisphenol A in food packaging in Sense about Figure captions.
	402	conventional pasteurized and HP/LT treated samples.
	356 403
	357 404	Irganox 1076 from polypropylene followed Fickean kinetics, at least apparently since they stated that Concerning PLA, the effect of HP/T could not be properly assessed since migration was too low and
	358 405	the molecular mechanism must be different that simple diffusion; there were evidences that whole within the experimental error to be detected. Diffusivity of Uvitex OB in PLA is then much lower than
	359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 406 407 408 409	F o r P e e r R e v i e w O n l y crystals could migrate instead of disperse molecules. As a consequence, if blooming is controlled by Fickean kinetics, the results obtained are also valuable for the assessment of the effect of HP/T treatments on migration. Uvitex OB diffusivity in LLDPE was determined for each FSL and for treated or non treated polymers using the kinetic profile obtained by FTIR. The diffusivity values found for Uvitex OB (Table 1) were in good accordance with those found in the literature: 3.2×10 -14 m 2 s -1 (measured with Moisan cells) and 5×10 -14 m 2 s -1 (measured with sorption kinetics) in LLDPE (Dole et al. 2006) . Besides the root mean square error (RMSE) was in all the cases of the same order of magnitude of the experimental error. For the same FSL, no significant difference was found between the HP/T treated samples and the control although clear differences were detected between different FSL. Likewise, diffusivity values are in agreement with the migration tests showing that there is no significant difference within FSL for each of the treatments but, that the release is faster in olive oil. Olive oil is widely regarded as a plasticizer of plastics that could increase diffusion and then. However, a number of authors have found that olive oil did not have a significant influence on migration of medium weight F o r P e e r R e v i e w O n l y F o r P e e r R e v i e w O n l y in LLDPE and show good barrier properties. However, both sterilization processes (HP/HT and conventional) affect clearly the structure of PLA what suggests that PLA should be restricted to low temperature treatments. polypropylene (PP) in FP5 Foodmigrosure F o r P e e r R e v i e w O n l y F o r P e e r R e v i e w O n l y F o r F o r P e P e e r e r R e R v e i e v i e w w O n l y O n l QLK1-CT2002-y
	351 374	shows an example of molecules from polyolefins (Helmroth et al. 2002; Mauricio-Iglesias et al. 2009; O'Brien et al. 1999)
	352 375	model fitting for HP/HT treated samples. Therefore, the discrepancy of diffusivity in olive oil and the other FSL can be linked likely to the
	353 376	inaccuracy of the assumption that Equation (1) correctly describes the blooming phenomena.
	377
				17 19 20

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Table 1 .

 1 Diffusivity of Uvitex OB in the system LLDPE/simulant after HP/T and 26 days of

	Page 29 of 30	Food Additives and Contaminants
	storage at 40°C
	Diffusivity (10 -14 ) m 2 s -1 Water	3% Acetic Acid 15% Ethanol	Olive Oil
	UTC	1.8±0.4 a,b	2.3±0.5 b	3.1±0.7 b	8.2±1.7 c
	HP/LT (20-40°C)	1.4±0.3 a,b	1.8±0.5 a,b	2.1±0.4 b	6.2±1.3 c
	HP/HT (90-115°C)	2.3±0.5 b	2.1±0.4 b	2.2±0.4 b	8.3±1.6 c
	Pasteurization (63°C)	1.1±0.3 a	n.d.	n.d.	8.2±1.6 c
	Means with different letters are significantly different (p<0.05)

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Table 2 .

 2 Diffusivity of Irganox 1076 in the system LLDPE/olive oil after HP/T and 26 days of

		Page 30 of 30
	storage at 40°C
		Diffusivity (10 -14 ) m 2 s -1
	UTC	4.6±0.9
	HP/LT (20-40°C)	3.7±0.7
	HP/HT (90-115°C)	4.4±0.9
	Pasteurization (63°C)	5.5±1.2

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Acknowledgments

acid and 15% ethanol). A mass balance was made to check the results between the losses in the films 284 and the migrates in the FSL. However, the quantity of Uvitex OB released could not be found back 285 dissolved in the aqueous FSL. The examination of the tissue sheets used to wipe the polymer strips 286 before the FTIR analysis showed traces of Uvitex OB powder. As an illustration, the amount released 287 by the samples in water for the UTC would represent a concentration of 0.5 g kg -1 FSL, more than ten 288 folds higher than the already overestimated value of solubility (<0.03 g kg -1 ). Actually, Uvitex OB 289 turned out to be stuck to the surface of the polymer film, where it had previously emerged. This 290 phenomenon characterized by a loss of additive from the bulk of the polymer that emerges or "blooms" 291 on the surface is called blooming (sometimes also referred to as bleeding). In principle, blooming 292 occurs when the additive concentration is higher than its solubility in the polymer (Billingham 2001) Authors would like to acknowledge Prof. Salvatore Iannace and co-workers from IMCB (Naples, Italy) 411 for the preparation of the packaging samples used in this study and the analysis of their thermal


Marielle Spanjersberg

André Knulst

Astrid Kruizinga

Gert Van Duijn

Geert Houben

Spanjersberg Mqi

Knulst Ac

Van Duijn

Houben Gf

Concentrations of undeclared allergens in food

Keywords: Analytical Research Houben, Geert Probabalistic modelling, Risk assessment, Risk assessmentmodelling Additives/Contaminants: Allergens Food Types: Cocoa, Processed foods Allergen contamination, Food Allergy, Risk Assessment, Probabilistic, May Contain Labelling, Milk Protein, Dark Chocolate, Risk-Based Concentration Limits

published or not. The documents may come

Introduction

As only strict avoidance of food allergens can prevent an allergic reaction, the allergic consumer relies on the allergy information on the packaging. Declaration of certain allergenic ingredients is obligatory and clearly defined in directives (2003/89/EC and 2005/26/EC). An allergen can also be present due to cross contact, for instance if a production line cannot be cleaned completely after producing an allergen containing product or due to cross contact in the raw material or ingredient supply chains. In such cases, precautionary 'may contain' labelling may alert to the possible presence of the concerned allergen. However, there is no guidance for the use of 'may contain' labeling in most countries. In the absence of such guidance, many producers seem to choose to label products precautionary in case they cannot guarantee full absence of allergens, even if the chance of contamination and/or the potential health impact are negligible. This leads to unnecessary limitation of consumer choice and devaluation of the allergen labelling information (Health Council of the Netherlands (GR) 2007). On the other hand, many products fill the shelves that appear to contain allergens while there is no warning on the labels, as shown by two recent surveys. The Dutch Food and Consumer Product Safety Authority performed a survey in order to make an inventory of the percentage of products either labelled or not to (possibly) contain allergen. Several types of products were analysed for traces of certain allergens. In 32% of 44 products not labelled for peanut, the allergen was detectable at different concentrations. Casein was detected in 45% of 64 products not labelled for milk proteins (VWA 2007). Comparable results were obtained in a survey by Pele et al. Hundreds of different types of cookies and chocolates were purchased in different countries in Europe and analysed for peanut and hazelnut proteins. Cookies not declared to (possibly) contain peanut or hazelnut proteins were analysed positive for these proteins in 11% and 25% of the samples, respectively. For chocolates, these percentages were 25% and 53% (Pele et al 2007).

Both situations -unlabelled contaminated products and labelled products with negligible contamination chance -are undesirable. Only in situations where cross contact is likely and health risks are expected, 'may contain' labelling should be adopted. It is, however, difficult to assess when such a situation applies. We previously developed a risk assessment methodology that calculates the magnitude of a potential public health risk posed by defined concentrations of food allergens. Probabilistic modelling of adequate food consumption, allergen concentration and threshold distributions can establish what proportion of the at-risk population could be expected to react, subdivided into the severity of the reaction (Spanjersberg et al 2007[START_REF] Kruizinga | Probabilistic risk assessment model for allergens in food: sensitivity analysis of the minimum eliciting dose and food consumption[END_REF].

By now, probabilistic modelling is considered to be the most promising approach for use in population risk assessment (Madsen et al 2008) and can be used for establishing risk-based concentration limits for 'may contain' labelling as well.

Recently, a cow's milk protein allergic patient from the outpatient department of the University Medical Centre Utrecht, The Netherlands, experienced a severe allergic reaction to a dark chocolate product that was not declared to contain milk. This case induced us to investigate to what extent allergen concentrations of unlabelled products reach levels that are of public health relevance.

Case description

A 38 year-old female patient, with adult onset cow's milk and hen's egg allergy since her early twenties, followed a strict elimination diet. In 1998 she suffered a severe allergic reaction to a sandwich with salmon due to the hidden presence of casein in the salmon [START_REF] Koppelman | Anaphylaxis caused by the unexpected presence of casein in salmon[END_REF]. Recently she visited the outpatient department of the University Medical Center (UMC) in Utrecht, The Netherlands, because she experienced an unexpected allergic reaction to dark chocolate sprinkles (a favourite sandwich filling in the Netherlands).

Previously she experienced mild tingling in the mouth on several occasions after consuming one sandwich with the chocolate sprinkles, which she did not attribute to the product. Then after consumption of several slices of (milk-free) bread with this product on the same eating occasion, she had a more severe reaction. Within 30 minutes she got tingling lips, redness of the face, swelling of the eyes, sneezing and obstruction of the nose, nausea and itching in the inguinal area. She interpreted this correctly as a systemic allergic reaction, treated herself with oral antihistamines and went to a nearby physician who treated her with intravenous antihistamines. Subsequently the symptoms gradually disappeared. The patient read labels carefully and avoided products labelled to (possibly) contain milk proteins. The product in question was not declared to (possibly) contain milk proteins. The complaint sample was brought along by the patient and was analysed for the presence of cow's milk and hen's egg proteins in order to find out the cause of her allergic reaction. Egg protein was shown to be absent, milk protein was indeed present (see sections below).

Materials and methods

In order to investigate to what extent products not declared to (possibly) contain certain allergens may contain these allergens at concentration levels that pose a risk to allergic consumers, we conducted a probabilistic risk assessment for a collection of the dark chocolate product type the patient reacted to.

The following data had to be collected: the distribution of the concentration of milk proteins in dark chocolate sprinkles, the consumption figures of dark chocolate sprinkles and the distributions of the eliciting doses (thresholds) for milk protein among milk protein allergic individuals.

The concentration of milk protein in the dark chocolate product

Twelve packages of nine different brands of dark chocolate sprinkles from different regional stores were collected. Samples of these packages as well as of the complaint sample were analysed. Three of these packages (from two brands) were labelled precautionarily for the possible presence of milk proteins; the other ten were not labelled. For comparison, three samples of milk chocolate sprinkles were analysed.

Milk protein predominantly consists of casein and β-lactoglobulin (approximately 85% and 10%, respectively). Both of these protein groups were analysed in extracts of the samples.

Because the patient from the case description also was allergic for hen's egg protein, egg proteins were determined as well. The chocolate sprinkles were processed to prepare extracts as follows. To 1 g of chocolate sprinkle sample 10 mL extraction buffer (0.05M sodium carbonate, pH 9.6) was added. Since the presence of cocoa in a sample may inhibit the detection of milk proteins, the extraction procedure has been improved by the addition fish Tepnel and measured with a Spectramax. The limits of detection were 1 mg/kg. The analyses were performed in triplicate.

The concentrations of milk proteins determined for the ten unlabelled products were used for risk assessment.

The consumption of dark chocolate sprinkles

The consumption of dark chocolate sprinkles is represented by two variables: the probability of using dark chocolate sprinkles and the amount of sprinkles consumed. Consumption data were derived from the Netherlands Food Consumption Survey from 2003, in which a representative sample of 352 male and 398 female consumers aged 19-30 years recorded their food consumption for two non-consecutive days [START_REF] Hulshof | Results of the Food Consumption Survey[END_REF]. In the absence of specific food consumption data from allergic consumers, we assumed that the pattern of consumption does not differ between allergic consumers and the general population. The chance of consumption was based on the number of people who consumed the product on measurement day 1 and/or day 2; the amount consumed was based on the highest individual amount consumed on day 1 or 2. Because the consumption pattern differs between males and females, we separated the consumption part of the assessment and calculated their risk separately. The proportion of the population consuming the product and the amounts consumed were established to be 12.5% and 16.1 ± 8.9 g and 10.1% and 10.7 ± 8.2 g for males and females, respectively.

The minimum eliciting doses (thresholds) of milk protein in milk protein allergic adults

Data on minimum eliciting doses of cow's milk protein in cow's milk allergic patients can be derived from several publications. However, most of these provocation studies have been performed in children. Thresholds established in children may not be comparable to those of adults. As we intended to conduct a risk assessment for the adult allergic population, we used the results of a recent provocation study in adult patients in which minimum eliciting doses were established with double-blind placebo-controlled food challenges (DBPCFC). Ten patients with clinically established cow's milk protein allergy were challenged with increasing doses of milk protein in order to determine the minimum eliciting doses for both subjective and objective symptoms (for study details and results see Lam et al 2008).

Subjective symptoms (for instance itch, nausea, abdominal pain and dizziness) can only be reported by the subject being challenged and are thought to be the preliminary stages of the subsequent objective reactions (in this instance urticaria, vomiting, diarrhoea and drop in blood pressure), which can be verified independently by an outside observer. In an individual patient, subjective effects usually occur at lower challenge doses than those eliciting objective symptoms. In our case study we used minimum eliciting dose distributions for both subjective and objective reactions to assess the probability for different types of reactions separately.

Probabilistic modelling

The data distributions of consumption and concentration were defined as cumulative distributions. Threshold data can be fitted as Cumulative, LogLogistic, LogNormal or Weibull distributions. As there is no apparent biological or mathematical basis for choosing one of these statistical distributions, we calculated the results for all four distributions. The parameters of the chosen distributions were defined using SAS (version 9.1) and entered in the software programme @-RISK, with 25 runs of 10000 iterations for each simulation, a procedure reported in detail in a previous publication (Spanjersberg et al 2007).

Results

Milk and egg protein concentrations

Casein and β-lactoglobulin analysis showed similar patterns. As casein is the predominant milk protein and can thus be measured more accurately, concentrations of total milk protein were calculated from the casein results. The results of the analysis and calculation of the casein and total milk protein concentrations in chocolate sprinkles are summarized in table 1.

Egg protein was found to be absent in all samples (<1 mg/kg).

<INSERT Table 1a and 1b and 1c>

Probabilistic risk assessment

The results of the risk assessment were expressed as the percentages of the milk protein allergic adult male or female population expected to experience an allergic reaction due to dark chocolate sprinkles not labelled to (possibly) contain milk proteins. The results are shown in figure 1 Standard deviations were too low to have impact on the outcome and are therefore not reported. After all, for risk management of population risks, a calculated risk of for instance 15% is not expected to differ fundamentally from 14 % or 16% in terms of indicated risk management measures.

For objective symptoms, a cumulative threshold distribution underestimates the risk, because the model assumes that below the lowest eliciting dose for objective reactions observed in the provocation study no reaction can occur. In reality, below this dose reactions do occur, yet these are often subjective and therefore not included in the distribution for objective effects.

Therefore, a cumulative distribution was not included in the calculation of the percentage of the allergic population expected to respond objectively.

<INSERT Table 2>

<INSERT Figure 1> Table 2 shows that a large percentage of milk allergic subjects would react if they ate the product. The sample the patient brought along (complaint sample: brand 1 production code a) was calculated to pose a risk of subjective complaints in 57.5% and 50.3% of the milk allergic male and female consumers of dark chocolate sprinkles, respectively. The expected risk for objective allergic reactions was calculated to be 7.7% and 5.8% of the milk allergic consumers (males and females, respectively).

The sample with the highest contamination level (brand 1 production code c) is expected to cause subjective allergic reactions in 67.9% and 61.3% and objective allergic reactions in 12.2% and 9.2% of the milk allergic consumers (males and females, respectively).

Because not all milk allergic individuals will consume dark chocolate, the risk calculated for the milk allergic population is lower. If non-users of dark chocolate sprinkles are included, the percentage of responders to the complaint sample, for instance, was calculated to be 5.0-7.2% for subjective allergic reactions and 0.6-1.0% for objective symptoms.

The four statistical distributions (Cumulative, LogLogistic, LogNormal and Weibull) in which the thresholds for subjective symptoms were fitted resulted in similar outcomes. For objective symptoms the chosen distribution was found to have consequences for the outcome, as reflected in a broader outcome range.

Discussion

The decision whether or not a potentially contaminated product should be labelled precautionarily is often difficult to make. Labelling of a product contributes to the protection of allergy patients, but, in case of a negligible risk, leads to unnecessary limitation of consumer's food choice and devaluation of the allergen information value on the packaging.

Not labelling, on the other hand, results in an inability for allergic subjects to avoid a potentially contaminated product and would pose a risk to the allergic consumer in case the allergen is present at a quantity that provokes a reaction. The patient case described and analytical results demonstrate that this latter situation indeed occurs. The patient read labels carefully and avoided products labelled to (possibly) contain milk proteins, but was not warned for the possible presence of milk proteins in the product she consumed.

It is not known to what extent allergic consumers suffer from allergic reactions caused by

hidden allergens in food. The results from our study suggest that it might be expected to occur rather frequently. In practice, however, complaints about products are not known to be reported frequently. On the basis of our patient case, it can be postulated that the problem may be underreported, as allergic consumers may often be unable to link complaints to a consumed food or just do not seek medical attention because the response is mild and transitory. Our patient experienced mild tingling in the mouth at several occasions after consuming one sandwich with the chocolate sprinkles, which she did not attribute to the product. Only after consumption of several slices of bread with the product on one eating occasion, she had a more severe reaction and linked it to the sprinkles.

As mentioned before, results of two recent surveys (VWA 2007 andPele et al 2007) indicate a poor reliability of allergy information on products as well. Thus, the presence of unlabelled allergens in foods may be considered as a real health problem.

In our study only a restricted amount of dark chocolate sprinkle samples was assessed as we aimed to investigate to what extent unlabelled products may be contaminated at levels that pose a risk to the allergic consumers and did not aim to perform a complete risk assessment of chocolate sprinkles. For population risk assessment purposes, more samples and aspects such as the prevalence of cow's milk protein allergy, protein levels in products of other brands and batches and the market share of the various brands would be required. The representativeness of the threshold data distributions for milk protein may be discussed, as the number of participants was rather low. This dataset may be improved by including data of additional patients from other studies. An interval-Censoring Survival Analysis (ICSA) may be applied to pool the data from the available studies, as has been done with peanut threshold data by Taylor et al (2009). As many threshold data are available for children, inclusion of these data may strengthen this part of the risk assessment. In addition, keeping a separate data set for children, who generally have milder allergic reactions to milk protein, makes an assessment of their level of risk possible.

In a clinical setting, thresholds may differ from the real life situation. However, this is only speculative. In addition, it is important to realize that the applied risk assessment method uses human data of high quality and does not need any species to species extrapolation which generally is a most uncertain step in risk assessment for chemical substances.

A critical point of our study is the representativeness of the consumption data. Due to lack of consumption data within the allergic part of the population, we had to use consumption data of the general population, which may differ.

Despite the inadequateness of the available consumption data, the applied probabilistic risk assessment model is generally considered to be the most sophisticated approach.

The choices made with regard to the defined statistical models were shown to have little impact on the results. The four statistical distributions in which the thresholds for subjective symptoms were fitted resulted in similar outcomes. This shows that this risk assessment methodology is relatively insensitive to the type of statistical distribution chosen for the threshold data for subjective symptoms. The chosen distribution for objective symptoms had somewhat more impact. Mathematically the reason is obvious: the distribution for objective symptoms needs extrapolation towards the lower dose area (this distribution has no data points in the lower dose area, as most patients show subjective symptoms first) and uncertainty simply increases with the extent of low-dose extrapolation.

Conclusion

This study was conducted in order to investigate to what extent allergen concentrations in unlabelled products are relevant for public health. Milk protein concentrations in unlabelled dark chocolate sprinkles, probably present as a result of cross-contact, were shown to reach levels that can cause serious allergic reactions within the milk allergic population, as evidenced by the patient case as described. Obviously, precautionary labelling needs guidance in terms of concentration levels that indicate when a product should be labeled precautionarily in order to be valuable to the consumer. Probabilistic allergen risk assessment can be an aid to establish risk-based concentration limits and can validate already existing limits. Now and in the future, probabilistic allergen risk assessment is a way to calculate the health risk impact of contaminated products and provides a sound basis for risk management decisions. M/F, males/females; Based on the concentrations of ten samples of dark chocolate sprinkles from packagings not labeled as 'may contain milk protein' and the average consumption levels of this product; Calculated using the software programme @-RISK (version 3.5.2), 25 runs of 10 000 iterations for each simulation; Cumulative distributions for consumption and concentration data; Cumulative, LogLogistic, LogNormal or Weibull distributions for subjective symptoms (results of the fitted distribution that gave the highest risk shown); LogLogistic, LogNormal or Weibull distributions for objective symptoms (results of the fitted distribution that gave the highest risk shown);

The outcome p (the chance on an allergic reaction) is the success probability for a Bernoulli distribution; Pooled standard deviations were too low to have impact on the outcome.

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Food Additives and Contaminants

  gelatin (Sigma G7765) to the extraction solution (10% w/w) prior to the sample extraction as well as heating the mixture to 50-60 °C during extraction (according to the instructions of Tepnel). Commercially available ELISA kits were used to determine the concentrations of casein (Tepnel 902062W), β-lactoglobulin (Tepnel 902060Y) and egg protein (Tepnel 902072T). The procedure was performed according to the directions of use included by

  , Van Hoffen E, Michelsen A, Guikers K, Van der Tas CHW, Bruijnzeel-Koomen CAFM, Knulst AC. Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved. Clinical and Experimental Allergy 2008; 38: 995-1002 -Madsen CB, Hattersley S, Buck J, Gendel SM, Houben GF, Hourihane JOB, Mackie A, Mills ENC, Nørhede P, Taylor SL, Crevel RWR. Approaches to Risk Assessment in Food Allergy: Report from a Workshop "Developing a framework for assessing the risk from allergenic foods". Food and Chemical Toxicology 2009; 47(2): 480-489 -Pele M, Brohée M, Anklam E, Van Hengel AJ. Peanut and hazelnut traces in cookies and chocolates: relationship between analytical results and declaration of food allergens on product labels. Food Additives and Contaminants 2007 24(12): 1334-1344 -Spanjersberg MQI, Kruizinga AG, Rennen MAJ, Houben GF. Risk assessment and food allergy: the probabilistic model applied to allergens. Food and Chemical Toxicology 2007; 45(1): 49-54 -Taylor SL, Crevel RWR, Sheffield D, Kabourek J, Baumert J. Threshold dose for peanut: risk characterization based upon published results from challenges of peanut-allergic individuals. Food and Chemical Toxicology 47(6

Figure 1

 1 Figure 1 Percentages of predicted responders in the milk allergic population to the different contaminated samples

  and table 2. The threshold data were fitted in different statistical distributions each resulting in a calculated risk. The worst-case outcomes are reported in this publication.

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

Table 1a

 1a Concentrations of casein and total milk protein in dark chocolate sprinkles not labeled precautionarily for the presence of milk protein

	Dark chocolate sprinkles	Casein concentration	Total milk protein
	-not labeled for milk allergens -	(mg/kg)	concentration*
				(mg/kg)
	Brand 1 (production code a) **	1500	1765
	Brand 1 (production code b)	85	100
	Brand 1 (production code c)	3015	3547
	Brand 2 (production code a)	459	540
	Brand 2 (production code b)	1568	1845
	Brand 3		15	18
	Brand 4		2	2
	Brand 5		775	912
	Brand 6		222	261
	Brand 7		213	251
	Table 1b
	Concentrations of casein and total milk protein in dark chocolate sprinkles with a precautionary labelling for milk
	allergens
	Dark chocolate sprinkles	Casein concentration	Total milk protein
	-labeled for milk allergens -	(mg/kg)	concentration*
				(mg/kg)
	Brand 8		3	4
	Brand 9 (production code a)	88	104
	Brand 9 (production code b)	156	184
	Table 1c
	Concentrations of casein and total milk protein in milk chocolate sprinkles
	Milk chocolate sprinkles	Casein concentration	Total milk protein
			(mg/kg)	concentration*
				(mg/kg)
	Brand 9		73000	85882
	Brand 2		53000	62353
	Brand 8		63000	74118
	*	calculated from casein concentration (casein concentration x 100 ÷ 85)
	**	complaint sample

Table 2

 2 Percentages of the milk protein allergic population expected to experience an allergic reaction due to unlabeled dark chocolate sprinkles

	Concentration of	Expected responders among milk allergic	Expected responders within the milk
	the milk protein	consumers of the product*		allergic population **
	contamination	(%)				(%)
	(mg/kg)	Subjective allergic	Objective allergic	Subjective allergic	Objective allergic
		reactions		reactions		reactions		reactions
		M	F	M	F	M	F	M	F
	Brand 1a 1765	57.5	50.3	7.7	5.8	7.2	5.0	1.0	0.6
	Brand 1b 100	21.6	17.1	1.1	0.8	2.7	1.7	0.1	0.1
	Brand 1c 3547	67.9	61.3	12.2	9.2	8.6	6.1	1.5	0.9
	Brand 2a 540	38.7	32.9	3.5	2.6	4.9	3.3	0.4	0.3

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Acknowledgement

The authors would like to thank Frans van der Kloet, Carina Rubingh, Linda van den Bosch and Karin Hulshof for their statistical support and their contribution to the automation of the probabilistic calculation process in allergen risk assessment.


Dr Henrike Westekemper

Sara Karimi

Dr Daniela Süsskind

Gerassimos Anastassiou

Dr Michael Freistühler

Dr Daniel Meller

Dr Michael Zeschnigk

Prof. Dr Klaus-Peter Steuhl

Prof. Dr Norbert Bornfeld

Kurt-Werner Schmid

Dr Gerasimos Anastassiou

Prof. Dr Kurt- Werner Schmid

Dr Florian Grabellus

EXPRESSION OF MCSP AND PRAME IN CONJUNCTIVAL MELANOMA

Keywords: MCSP, PRAME, Conjunctiva, melanoma, immunohistochemistry

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

Mail: Henrike.Westekemper@uk-essen.de Total word count: 3161 words (including text).

ABSTRACT:

Background: To analyse the expression of melanoma chondroitin sulphate proteoglycan (MCSP) and the preferentially expressed antigen of melanoma (PRAME) in conjunctival melanoma (CoM), lymph-node metastases of cutaneous melanoma (LN-met of CM) and conjunctival nevi (CoN) by immunohistology.

Methods: Immunohistology was performed in 70 samples of CoM, 25 of LN-met. of CM and 12 of CoN. and assessed by an immuno-reactive-score (0-12 points).

Statistical analysis was performed to disclose relevant differences in the expressionpattern. The diagnostic value of the markers was tested by receiver operating characteristics (ROC-analysis).

Results: MCSP and PRAME were expressed significantly higher in CoM and LNmetastases of CM than in CoN (p=<0.0001). Within CoM, a MCSP-expression <9.0 points meant higher risk for recurrences (Cox HR=3.1) and a shorter recurrence-free survival (p=0.002) than a MCSP-expression >9.0 points. ROC-analysis showed an area under the curve of 91.3% for MCSP (p=0.0002) and 93.8% for PRAME (p<0.0001).

Conclusions: MCSP and PRAME are differentially expressed in conjunctival melanomas and nevi. MCSP might have an impact on the risk for recurrence in being inversely correlated to the event. Both markers have high potential to discriminate CoM from CoN. Our results indicate that immunohistological characteristics gain relevance in the assessment of conjunctival melanoma.

INTRODUCTION:

In conjunctival melanoma (CoM) most predictable factors are based on clinical or histopathological characteristics [START_REF] Seregard | Conjunctival malignant melanoma in Sweden 1969-1991[END_REF][START_REF] Missotten | Conjunctival Melanoma in The Netherlands: A Nationwide Study[END_REF][START_REF] Shields | Primary acquired melanosis of the conjunctiva: risks for progression to melanoma in 311 eyes[END_REF][START_REF] Paridaens | Prognostic factors in primary malignant melanoma of the conjunctiva: a clinicopathological study of 256 cases[END_REF][START_REF] Anastassiou | Prognostic value of clinical and histopathological parameters in conjunctival melanomas: a retrospective study[END_REF][START_REF] Damato | An audit of conjunctival melanoma treatment in Liverpool[END_REF] but little is known about the tumour's biology and protein expression profile. [START_REF] Keijser | Immunophenotypic markers to differentiate between benign and malignant melanocytic lesions[END_REF] Concerning growth pattern and metastatic pathways, CoM resembles its cutaneous counterpart rather than other ocular melanoma (i.e. uveal). [START_REF] Esmaeli | Patterns of regional and distant metastasis in patients with conjunctival melanoma: experience at a cancer center over four decades[END_REF][START_REF] Iwamoto | Immunophenotype of conjunctival melanomas: comparisons with uveal and cutaneous melanomas[END_REF][START_REF] Hurst | Ocular Melanoma. A Review and the Relationship to Cutaneous Melanoma[END_REF][START_REF] Hu | Population-based incidence of conjunctival melanoma in various races and ethnic groups and comparison with other melanomas[END_REF] The data about the origin of conjunctival melanomas and their rates for local recurrence and metastatic disease show that the tumour's biology needs to be further illuminated in order to understand the underlying mechanisms. New therapeutic strategies are required to reduce recurrence rates and improve survival. [START_REF] Folberg | Malignant melanoma of the conjunctiva[END_REF][START_REF] Tuomaala | Population-Based Assessment of Clinical Characteristics Predicting Outcome of Conjunctival Melanoma in Whites[END_REF][START_REF] Wuestemeyer | Proton radiotherapy as an alternative to exenteration in the management of extended conjunctival melanoma[END_REF] Melanoma chondroitin sulphate proteoglycan (MCSP) has become a target in melanoma research when immunotherapy became relevant in cutaneous melanoma.

It was identified in the late 1970s as a transmembrane proteoglycan with two components. [START_REF] Wilson | Immunochemical characterization of a human high molecular weight melanoma associated antigen identified with monoclonal antibodies[END_REF] As a cell surface proteoglycan, MCSP serves as an adhesion receptor influencing cell adhesion and migration. [START_REF] Bemfield | Functions of cell surface heparan sulfate proteoglycans[END_REF] Additionally, it influences cell invasion [START_REF] Iida | Melanome Chondroitin Sulfate Proteoglycan Regulates Matrix Metalloproteinase-dependent Human Melanoma Invasion into Type I Collagen[END_REF] and proliferation. [START_REF] Burg | Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells[END_REF][START_REF] Chekenya | The NG2 chondroitin sulfate proteoglycan: role in malignant progression of human brain tumours[END_REF] It has been found that the MCSP-expression correlates to the metastatic potential of a soft tissue sarcoma and melanoma. [START_REF] Benassi | NG2 expression predicts the metastasis formation in soft-tissue sarcoma patients[END_REF][START_REF] Burg | Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells[END_REF][START_REF] Iida | Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin[END_REF][START_REF] Iida | Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A)[END_REF] A limited expression of MCSP is shown in normal tissues but is expressed in a high percentage of melanoma lesions. [START_REF] Ferrone | Selection and utilization of monoclonal antibody defined melanoma associated antigens for immunoscintigraphy in patients with melanoma[END_REF]; reviewed by Campoli et al. [START_REF] Campoli | Human High Molecular Weight-Melanoma-Associated Antigen (HMW-MAA): A Melanoma Cell Surface Chondroitin Sulfate Proteoglycan (MSCP) with Biological and Clinical significance[END_REF] The tumour antigen "preferentially expressed antigen of melanoma" (PRAME) is a non-mutated cancer-testis antigen that is expressed in fetal tissue, testis and in cancer, mainly in cutaneous melanoma. PRAME is expressed in 88% of primary cutaneous melanomas and 95% of melanoma metastases. [START_REF] Ikeda | Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor[END_REF][START_REF] Watari | Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene[END_REF] As it is neither expressed in benign cutaneous nevi nor normal skin, it is likely that PRAME expression occurs under melanocyte transformation. [START_REF] Haqq | The gene expression signatures of melanoma progression[END_REF] It supports cell survival and suppresses differentiation by interfering with retinoic acid signalling. [START_REF] Epping | The Human Tumor Antigen PRAME Is a Dominant Repressor of Retinoic Acid Receptor Signaling[END_REF] No data exist about PRAME expression in conjunctival melanoma.

In this study we analysed the expression of MCSP and PRAME in 70 cases of conjunctival melanoma (CoM) by immunohistochemistry and compared the results with lymph node (LN) metastases of cutaneous melanoma (CM) and conjunctival nevi (CoN). The differential diagnosis between conjunctival melanoma and nevi often gives rise to controversy between pathologists or pathologists and clinicians and has an important impact on the therapeutic approach. The established antigens in melanoma diagnostics cannot clarify the diagnosis in all cases. So we examined the potential of MCSP and PRAME to differentiate between these two conjunctival entities. Both antigens have an impact on melanoma progression and may represent therapeutic targets. While MCSP is of special interest as a melanoma specific antigen, PRAME is connected to a very important pathway being a target for modern therapies (RA-signalling pathway). We chose these two antigens to cover these issues for conjunctival melanoma. There does not exist any information about the two antigens in conjunctival melanoma research so far.

MATERIAL AND METHODS:

The study was funded by the Medical Faculty of the University Duisburg-Essen.

Demographic data:

In this study we analysed the clinical data of 70 patients with histologically confirmed malignant melanoma of the conjunctiva (CoM) and performed immunohistology of their tumour samples. The patients were treated between 1974 and 2006 in the Department of Ophthalmology of the University Hospital Essen or Tübingen. The samples were acquired from these departments and the Department of Pathology and Neuropathology of the University Hospital Essen. The mean age was 63.7 ± 16.0 years at the time of diagnosis. The mean follow-up was 69.6 months (min. 0.33, max 317.16 months, median 38.83). Forty cases (57.1%) were presented at the time of first diagnosis and then treated primarily in one of the participating centres. Twentytwo cases (31.4%) had already had a local recurrence at the time of presentation; in 8 cases (11.5%) we had no information about the earlier course of the disease. The tumours were staged following the 6 th edition of the TNM-staging-system for conjunctival melanoma (American Joint Committee on Cancer 2002). For demographic-and TNM-data see table 1. Lymph node metastases of cutaneous melanoma (LN-met. of CM) (n=25) and benign conjunctival nevi (CoN) (n=12) served as control groups. Clinical data of these control groups were not available. Primary cutaneous melanoma-samples served as positive controls for MCSP and PRAME. Therefore, we did not use primary cutaneous melanoma as a separate group in this study. We were interested on the expression of the antigens in metastases.

Metastases of conjunctival melanoma are numerously rare and not usually excised.

So we decided to include lymph node metastases of cutaneous melanoma as control group.

All experiments and procedures were conducted in accordance with the Declaration of Helsinki. An informed consent was obtained from the patients for the analysis of collected samples. The local Ethics Committee of the Medical Faculty of the University Hospital Essen, University of Duisburg-Essen, Germany approved the study.

Immunohistochemistry:

Diagnosis of melanoma was confirmed by hematoxylin-eosin (HE) slides and S-100-, HMB-45-and Melan-A immunostaining. Tissue microarrays (TMA) of conjunctival melanomas (n=70), lymph node metastases of cutaneous melanomas (n=25) and conjunctival nevi (n=12) were assembled with a core-size of 600µm and cut into 5µm thick slices. Suitable donor tissue for the construction of TMAs was identified and marked on HE sections. TMA blocks were constructed with a manual tissue arrayer (Beecher Instruments, Inc, Sun Prairie, WI, USA) with three punch specimens (diameter 0.6 mm) per tumor from corresponding blocks of formalin-fixed and paraffin-embedded tissue. The area of trephination was defined under microscopic control. In small samples 5µm slices were cut and placed directly on object holders.

Afterwards, the samples were allowed to dry at 52°Celsius (°C) overnight.

For antigen demasking and deparaffinisation sections were incubated in xylol and rehydrated through graded decreasing concentrations of alcohol (ethanol 100%, 90%, 70%). Afterwards, sections were rinsed with aqueous solution and washing buffer (DCS: WL583CO500). As pre-treatment citrate buffer (pH 6.0) (Zytomed Systems ZUC028-500) was applied to a water bath at 98°C for 20 minutes. Cooling had been allowed for 10 minutes before the samples were transferred to the washing buffer. Afterwards, Peroxidase block was performed for 5 minutes (Dako: S2001), and then the specific antibodies were applied for 30 minutes (PRAME: rabbit, polyclonal, dilution 1:200; MCSP: Mouse IgG 1 , LHM2, dilution 1:400, both Biozol Germany, both dilution-media Zytomed Systems ZUC025-500). A detection system of Zytomed Systems (POLHRP-100) was used. DAB (3,3'-Diaminobenzidin, Zytomed Systems DAB 530) provides brown staining to visualise the complexes. The sections were counterstained with haematoxylin 1:8. Endogenous melanin-pigment was identified in HE-stained samples and was appreciated for the estimation of the immuno-staining. A primary cutaneous melanoma (PRAME+, MCSP+) served as positive control for the TMA investigation. For negative controls specific primary antibodies were replaced by normal serum of the same species. The stained samples were analysed and graded by two independent examiners (HW and FG) using the immuno-reactive score (IRS) (also: Remmele score). [START_REF] Remmele | Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue[END_REF] Interobserver agreement was assessed by the Kappa-test. [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] In cases of interobserver differences, a third examiner re-estimated the sections and the results were discussed between all three examiners to find a congruent result. The Remmele-score is used to estimate the grade of immunoreaction. The product of the points for staining intensity (no staining: 0; weak staining: 1; moderate: 2; strong: 3) and percentage of stained cells (0%: 0; <10%: 1; 11%-50%: 2; 51%-80%: 3; 81%-100%: 4) results in a score from 0-12. [START_REF] Kohlberger | Modified true-color computer-assisted image analysis versus subjective scoring of estrogen receptor expression in breast cancer: a comparison[END_REF][START_REF] Remmele | Immunohistochemical determination of estrogen and progesterone receptor content in human breast cancer. Computer-assisted image analysis (QIC score) vs. subjective grading (IRS)[END_REF] A p-value of p≤0.05 was considered statistically significant. Results were presented in arithmetic means, standard deviation, medians, ranges, and percentage rates.

Statistical analysis

Statistical analysis was performed using

Survival analyses to test the relationship of antigen expression, clinical features and recurrences or metastatic disease were performed by the Kaplan-Meier analysis.

Risks were assessed using univariate Cox-regression analysis.

Mann-Whitney-U-test was used for the comparison of antigen expression and nominal variables (2-variables); the Kruskall-Wallis-test was performed for nominal variables with 3 and more variables. Bonferroni-correction was applied to multiple testing with an alpha level set to 0.05. For correlation of continuous variables the Spearman correlation was performed, the results visualised by linear regression.

The unknown cases of the clinical and histopathological parameters were not included in the calculations and were treated as missing values. The cut-off points for MCSP and PRAME of 9.0 points IRS were generated using the mean and median of all cases of CoM (MCSP: mean 8.9; median 9.0 / PRAME mean 9.4).

ROC-analysis (Receiver Operating Characteristic) was performed to analyse the diagnostic value of the antigen expression (sensitivity and specificity) in the distinction of CoM and CoN. In general, the area under the curve is given as an indicator for the accuracy of a tool (here antigen). All points above the diagonal line are better than a random guess. The asymptomatic significance (asymptomatic pvalue) of <0.05 indicates that a tool is better than a guess in predicting an event (nullhypothesis: true area=0.5). The cut-off points for each antigen are suggested by the statistical program and they include values from the minimum observed test-value minus 1 to the maximum observed test-value plus 1.

RESULTS:

Demographic data and therapy

Clinical and histopathological characteristics of the conjunctival melanoma-group are summarised in table 1. In our cohort, we could not identify clinical or histopathological 95% confidence interval (CI): 1.1-30.1). In Cox regression analysis, tumours originating from primary acquired melanosis with atypia (PAM) had a 2.4fold increased risk for recurrence compared to de novo tumours, (HR for PAM with atypia=1.3 p=0.58, 95% CI: 0.52-3.2; HR for de novo tumours=0.5; p=0.46, 95% CI: 0.11-2.7).

Expression of MCSP and PRAME in CoM, CoN and LN-metastases of CM

The Kappa-test revealed an overall interobserver reproducibility of 64.6%, which is a substantial agreement. [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] Both antibodies showed a cytoplasmic and nuclear immunoreaction. For MCSP the cytoplasmic staining exceeded nuclear staining. For PRAME nuclear staining was much more intense than cytoplasmic staining. This pattern was observed in conjunctival melanomas, conjunctival nevi, and lymph-node metastases of cutaneous melanoma in equal measure. The highest MCSP-expression with a mean IRS of 11.1 points and a very homogenous expression profile (SD ± 1.69) was found in LNmetastases of CM. CoM expressed MCSP on a high level, as well, but the mean IRS was significantly lower (8.9 points ± 2.96). CoN showed a minor MCSP-expression with a mean IRS of 3.8 points (SD ± 2.15; figure 1, A-C). The difference in MCSPexpression was statistically significant between each of the groups (p=0.0005 CoM to LN metastases; p<0.0001 CoM to CoN and LN metastases to CoN; figure 2A). PRAME-expression was highest in LN-metastases of CM (mean IRS 9.84 ± 2.4) and CoM (mean IRS 9.41 ± 3.4). Both groups expressed PRAME on a similar high level (figure 1B). CoN showed a significantly lower PRAME-expression with a mean IRSscore of 2.6 points (IRS score min. 0; max. 6; figure 1, D-F). The difference in PRAME-expression was statistically significant between CoM and CoN (p<0.0001) and between LN-metastases of CM and CoN (p<0.0001) (figure 2B).

A highly significant correlation was found between the expression of MCSP and PRAME in CoM (R 2 =0.4, p=<0.0001).

Expression of MCSP and PRAME in conjunctival melanoma

Statistically, we examined a possible correlation to gender, tumour origin, location of primary tumour (cTNM), pigmentation, infiltration depths (pTNM), predominant cell type, recurrence rate and time of first recurrence, metastatic disease, and occurrence of a second recurrence. We found no statistically significant correlation to these parameters and MCSP expression (data not shown). Tumours with a MCSPexpression <9.0 points IRS had a higher risk for local recurrence than tumours with an IRS-score ≥9.0 (Cox-regression analysis, HR=3.1; p=0.003; 95%-CI: 1.5-6.6; Wald 2 =8.6). Additionally, tumours with a MCSP-expression <9.0 points IRS had a shorter recurrence-free interval than tumours with a MCSP-expression ≥9.0 IRS (Kaplan-Meier analysis; log-rang p=0.002; Chi 2 =9.7) (figure 3). Analysing the subgroups, we found that tumours with a lower MCSP-expression had a higher risk to develop local recurrence and a shorter recurrence-free survival independent from their clinical characteristics (example for epibulbar / nonepibulbar tumours, p=0.005, Chi 2 =7.8). The same trend was observed in other characteristics as pigmentation (pigmented/amelanotic; p=0.002; Chi 2 =9.8), predominant cell type epithelioid (p=0.02; Chi 2 =5.1), or occurrence of a second recurrence (p=0.03; Chi 2 =4.9). We also observed a trend that tumours with the origin PAM with atypia (mean IRS 7.9 ± 2.7) had a lower mean MCSP expression than tumours appearing de novo (mean IRS 9.5 ± 2.6). This did not reach statistical significance (p=0.18) but gave us a link to a clinical explanation for our findings.

For PRAME, we did not find significant correlations to most parameters (data not shown). In tumours that were confined to the epithelium (pT1, Melanoma in situ), PRAME-expression was significantly lower than in tumours with subepithelial or deeper invasion (pT2-pT4) (p=0.04). Tumours classified as pT2, pT3 and pT4 had no significant difference in PRAME expression between the groups. We observed a trend that tumours with an IRS-score for PRAME <9.0 points had a lower risk for metastatic disease (Cox regression, HR=0.44, p=0.3; 95% CI: 0.1-2.1) and tended to develop metastases later than tumours with an IRS-score ≥9.0 points (log-rang p=0.3, Chi 2 =1.2). This reached to no statistical significance, possibly due to the limited number of cases with metastatic disease in this cohort.

ROC-Analysis and predictive value of MCSP and PRAME

We performed a ROC-analysis in order to assess the power of MCSP and PRAME to discriminate conjunctival nevi from conjunctival melanoma. For MCSP, the area under the curve was 91.3% (asymptomatic p-value=0.0002; 95% CI: 0.84-0.99, figure 4). For PRAME, the area under the curve was 93.8% (asymptomatic p-value<0.0001; 95% CI: 0.87-1.0; figure 4). These findings make both parameters suitable to differentiate conjunctival nevi from melanoma. In our cohort MCSP had the surprising effect, that CoM-tumours with lower MCSPexpression had a higher risk for local recurrence and developed their first recurrence earlier than tumours with a high MCSP-expression. Factors that had turned out to be prognostically relevant to the outcome of CoM in clinical studies before did not turn this trend in our study and might therefore be of subordinate predictive relevance compared to immunohistochemical characteristics. But despite our results, this can only be stated as a hypothesis and has to be confirmed by further studies. For explanation, one has to recall the mechanisms associated with MCSP. MCSP is able to influence intracellular signal cascades that have an impact on cell adhesion, motility and invasion. [START_REF] Yang | Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms[END_REF] One relevant mechanism in the communication of melanoma cells to extracellular matrix (ECM) is provided by the α4β1-integrin and its binding to fibronectin and to the vascular endothelial cell adhesion molecule VCAM [START_REF] Mould | Integrin alpha4beta1-mediated melanoma cell adhesion and migration on vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced IIICS region of fibronectin[END_REF] which is also expressed in conjunctival melanoma. [START_REF] Anastassiou | Expression of cell adhesion molecules and tumour infiltration leucocytes in conjunctival melanoma[END_REF] It is known that the MCSP rat-homologue NG2 collaborates with α4β1-integrin for stimulating focal contacts and stress fibre formation in melanoma cells. [START_REF] Iida | Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin[END_REF] This may contribute to the metastatic potential of a tumour. On the other hand, it may also be possible, that in cases of a low MCSP expression, the reduced communication of a tumour cell with the ECM and the immune-cells helps to establish a tumour recurrence in the local environment without being "recognised". This would explain the trend that a lower MCSPexpression is linked to a higher risk for local recurrence in our cohort of CoM.

Probably, our results could also be explained clinically, as follows: Tumours originating from PAM with atypia had a lower mean MCSP expression and a 2.4fold increased risk for recurrence compared to primary tumours in our cohort. The recurrence-rate may be higher because clinical assessment of PAM with atypia holds the risk of missing a recurrent transformation to melanoma due to their diffuse and variable appearance on the ocular surface. It can be assumed, that the lower mean expression of MCSP in this group leads to our finding that such tumours tend to have a higher risk for recurrence in the whole cohort.

In conjunctival melanoma PRAME is expressed on a high level. Together with the very low expression of PRAME in conjunctival nevi, our results correspond to the reported PRAME-expression in cutaneous melanoma and nevi. [START_REF] Ikeda | Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor[END_REF][START_REF] Haqq | The gene expression signatures of melanoma progression[END_REF] Our results indicate that invasive tumours express higher levels of PRAME than intraepithelial tumours. This suggests that PRAME-expression increases under melanoma progression. It is, however, no linear progression but rather seems to divide early (intraepithelial) from advanced (invasive) tumours. PRAME acts by interfering with retinoic-acid--signalling as a dominant inhibitor of the retinoic acid receptor (RAR). [START_REF] Epping | The Human Tumor Antigen PRAME Is a Dominant Repressor of Retinoic Acid Receptor Signaling[END_REF] Binding of retinoic acid to its receptor initiates the activation of target genes that induce proliferation arrest, differentiation and apoptosis. This is the basis of the antitumoral activity of retinoic acid. [START_REF] Altucci | The promise of retinoids to fight against cancer[END_REF][START_REF] Freemantle | Retinoids in cancer therapy and chemoprevention: promise meets resistance[END_REF] Subsequently, the change to, and increase of, PRAME-expression may support tumour progression as the suppression of RAR-signalling promotes tumour-growth and inhibits differentiation. The impact of PRAME on the prognosis of a tumour entity is variable. [START_REF] Steinbach | Clinical implications of PRAME gene expression in childhood acute myeloid leukemia[END_REF][START_REF] Doolan | Prevalence and prognostic and predictive relevance of PRAME in breast cancer[END_REF] Our samples showed a trend towards earlier occurrence of metastases in cases with high PRAME-expression combined with an increased risk for metastases. The statistical value was limited due to the limited number of cases in that subset.

In order to understand the role of PRAME and MCSP in conjunctival melanoma more completely, further genetic studies are required to elucidate the source and mechanism of the increased PRAME-and MCSP-expression in conjunctival melanoma.

MCSP and PRAME have a good diagnostic value to distinguish between conjunctival nevi and conjunctival melanoma. Whether the antigens are able to improve the immunohistological diagnosis in difficult cases has to be evaluated further.

In conclusion, in this study we reported on the expression of melanoma chondroitin sulphate proteoglycan (MCSP) and the preferentially expressed antigen of melanoma (PRAME) in conjunctival melanoma and benign conjunctival nevi. We showed that MCSP and PRAME were differentially expressed in conjunctival melanoma and nevi and can help to differentiate the lesions diagnostically.

The authors have no financial / conflicting interests to disclose.

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its licencees, to permit this article (if accepted) to be published in BJO and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence (http://bjo.bmjjournals.com//ifora/licence.pdf).

Legends to figures:

  Microsoft Excel 2000, SPSS (SPSS for Windows, version 17.0; SPSS Chicago, IL, USA) and StatView for Windows (Abacus Concepts Inc. Version 4.55).

  risk factors for local recurrence or metastatic disease (data not shown). Analysing therapeutic approaches, 12 patients had only been treated by tumour exzision and had not received adjuvant therapy. Five patients underwent adjuvant Mitomycin C local therapy, 8 cryo coagulation, 12 proton radiotherapy and 18 brachytherapy with ruthenium-106 plaques. Fifteen patients were given other adjuvant therapy. Cox regression analysis revealed an increased risk for local recurrence in patients who got local chemotherapy with Mitomycin C eye drops (p=0.04; Hazard ratio (HR) 5.7;

Figure 1 :

 1 Figure 1: Immunohistology of MCSP (A, B, C) and PRAME (D, E, F). Magnification

Figure 2 :

 2 Figure 2: A. Boxplot of results of MCSP-expression in the three diagnosis groups. B:

Figure 3 :

 3 Figure 3: Kaplan-Meier analysis for recurrence-free survival in patients with CoM.

Figure 4 :

 4 Figure 4: ROC-analysis of MCSP (continuous line) and PRAME (dotted line).

  Table 2 shows the cut-off points within the IRS score revealing that MCSP and PRAME were diagnostically valuable in the

	DISCUSSION:
	Our results indicate that MCSP and PRAME are differentially expressed in benign
	conjunctival nevi and malignant conjunctival melanomas. Conjunctival melanoma
	(CoM) and lymph-node (LN)-metastases of cutaneous melanoma (CM) express both,
	MCSP and PRAME, at a similar level, whereas conjunctival nevi (CoN) express both
	antigens on a significantly lower level. Both markers can discriminate conjunctival
	nevi from melanoma with high significance.
	intermediate	and	high	scores	(2.5-8.5	pts	IRS).

Table 1 :

 1 Demographic and pathological data of the CoM-group with the number of tumours (n=) of the whole group. CoM: conjunctival melanoma.

	22

Table 2 :

 2 Results of the ROC analysis. Presented are parts of the coordinates of the curve to illustrate the possible cut-off values of the IRS of the specific antigens.