Jérémy Pinte

Catherine Joly

Patrice Dole

Alexandre Feigenbaum

MASS DEPENDENCE AND ACTIVATION ENERGY OF DIFFUSION

Keywords: Packaging overall migration, Packaging plasticisers, Plasticisers, Simulants Food Types: Diffusion coefficient, FRAP, molecular weight, activation energy, polystyrene, 22 model probes 23

The published diffusion prediction models devoted to the diffusion of additives in food 10 packaging simplify reality by having only a few parameters. Therefore, extrapolation of such 11 models to barrier polymers, larger ranges of temperature and/or additive molecular weight 12 (M W ) are still questionable. Extra data is still required to generalize these existing prediction 13 models. In this paper, diffusion of a specifically designed homologous set of model additives 14 (from 236 to 1120 g.mol -1 ) was monitored in two polystyrenes at rubbery state (from 100 to 15 180°C): syndiotactic semi-crystalline polystyrene and its amorphous equivalent. Variations of 16 D and Ea with migrant M W and temperature were found to be surprisingly low. Comparison 17 of experimental behaviour with model predictions was performed. In their actual form, none 18 of the models is clearly able to describe all experimental data, but clues can be given, 19

showing converging of the different approaches. 20 21

Introduction 1 2

To ensure consumer health, food packaging materials must be inert to packaged foodstuffs, in 3 order to avoid contamination from harmful substances which can migrate from packaging 4 materials. When dealing with plastic, regulations reinforce the concept of an Overall 5 Migration Limit (OML) with a Specific Migration Limit (SML) for potentially toxic 6 substances filed in a positive list. Petitioners for a new packaging material and control 7 authorities have to test polymeric packaging to assess their compliance for food contact (e.g. 8 US Food Law Act and EU regulation 2002/72/CE). 9

Migration is kinetically limited by the diffusion step within the polymer matrix, and is 10 characterized by the associated diffusion coefficient D. The large amount of diffusion data in 11 polyolefins has made the drawing of good prediction models possible. These are useful in [START_REF] Al-Malaika | Migration of 4-substituted 2-hydroxy 3 benzophenones in low density polyethylene: Part I-Diffusion characteristics[END_REF] predicting migration rate into foodstuffs or food simulants. Recently, food safety authorities 13 have allowed their use in food packaging material assessments (e.g. art. 4 in EU regulation 14 2002/72/CE). 15 16

Prediction of diffusion coefficients 17

Different approaches have been developed to model diffusion in polymeric matrixes. From 18 Piringer's work, which set up a worst case model (eq. 1) to Limm and Hollifield's model (eq. 19

2), the diffusion processes can be simplified and implemented in ready-to-use models. , .exp .

W W W M D T M D M K T α     = -    
(2) 1 where D is the predicted diffusion coefficient (cm 2 .s -1 ), 2 D' 0 is the diffusion coefficient at infinite temperature (cm 2 .s -1 ), 3 α and K are tabulated polymer parameters (dimensionless) ,

4

T is the absolute temperature (K),

5

M W is the molecular weight of the diffusion species (g.mol -1 ).

6 7

The reliability of such empirical models has been tested [START_REF] Reynier | Prediction of worst case migration: 22 presentation of a rigorous methodology[END_REF]Helmroth 8 et al. 2002;Rosca et Vergnaud 2006). Both models are able to describe the diffusion of 9 additives in polyolefins, but prediction errors become significant when dealing with less 10 mobile polymers, or when enlarging the extrapolation range to high temperatures or M W s 11 [START_REF] Feigenbaum | Functional barriers: Properties and 26 evaluation[END_REF]; [START_REF] Dole | Typical diffusion 20 behaviour in packaging polymers: application to functional barriers[END_REF]. 12 13 With the goal of creating a more general model, Dole et al. proposed a different 14 approach to predict D with additive molecular weights that try to encompass both mobile and 15 barrier polymers (Figure 1) [START_REF] Dole | Typical diffusion 20 behaviour in packaging polymers: application to functional barriers[END_REF]). The D=f(M W ) correlations for a broad range of 16 polymers were measured. They then showed that the negative slope of these [log 10 D=f(M W )] 17 correlations decreased from high barrier polymers to lower barrier ones, while the Piringer 18 model uses the same slope for all polymers and the Limm model integrates a weaker 19 dependence. Consequently, as temperature directly affects polymer barrier properties (i.e. 20 matrix mobility), Dole et The Dole approach provides a more complex view of diffusion: the D=f(M W ) and Ea=f(M W ) 1 slopes are functions of additive M W but first strongly depend on polymer mobility (Table 1). 2

It is worth noting that both plasticization and T increase allow a barrier polymer to turn into a 3 more mobile one (plasticized vs. non-plasticized PVC, dry vs. 60%RH PA in Figure 1) 4 Piringer's model was built using diffusion coefficients of additives whose M W was 5 smaller than 500 g.mol -1 . Dole et al. studied monitored diffusion of additives whose M W was 6 up to 807 g.mol -1 but with very few data between 430 and 807 g.mol -1 . Extrapolation for high 7 M W additives still needs to be evaluated. In contrast, tabulated parameters in Limm's model 8 were determined by fitting with D of Irganox 1076 (531 g.mol -1 ) and Irganox 1010 (1178 9 g.mol -1 ). Therefore, Limm's model may not accurately predict the diffusion of small 10 additives. 11

When dealing with additives of increasing M W , the overall size, shape, and chemical 12 functionality vary. Therefore, diffusion data of commercial additives are quite scattered. 13 However, the models mentioned above average the molecular size effect by considering only 14 M W . Up to now, only the Piringer model was validated experimentally against a homologous 15 series of alkanes and alcohols (Reynier et al. 2001;Reynier et al. 2001). 16 17The same parallel can be drawn for the temperature range . The three models were all 18 built for a specific temperature range. The Piringer model has to be used at a given 19 temperature range depending on the studied polymer (175°C for PET, 70 °C maximum for 20 PS). Limm and Hollifield fits α and K parameters between 40 and 70 °C. The Dole approach 21 describes diffusion data at 40 °C but encompasses a great range of polymer mobility. This 22 model is therefore able to describe diffusion over an equivalent range of temperature. Reynier In order to determine the extent of D dependance on M W -, T-and polymer mobility, it is 1 still necessary to accumulate D data from homologous systems. This could then help to 2 determine proper fitting model parameters for barrier polymers and could lead to a general 3 model. 4 5 Therefore, this study was set up to follow the diffusion of a specifically designed 6 homologous set of high M W probes [START_REF] Pinte | Proposal of a Set of Model 18 Polymer Additives Designed for Confocal FRAP Diffusion Experiments[END_REF]). These probes were chosen to be 7 probably more accurately model commercial additives than alkanes used by Reynier et The series of fluorescent probes was synthesized as previously described (Pinte et al. 12 2008). This set of five homologous molecules, as shown in table 2, was made by grafting a 13 fluorescent head (7-nitrobenzofurazan) onto an amino acid-like tail. This "tail" was 14 synthesized by oligomerization of the same repeating unit. Tail molecular weight was 15 increased by repeating the same 'monomer-like' unit. For more detailed synthetic procedures, 16 please refer to the above mentioned article and its Supporting Information. 17 Instruments). The heating rate was 10K.min -1 . Glass transition temperatures were determined 21 as the inflection point of heat flow from the second run, whereas melting temperature and 22 melting enthalpy were determined from the first run. For sPS, crystallinity was found to be 23 70%, assuming a heat of fusion of 53 J.mol -1 [START_REF] Bruzaud | Syndiotactic 7 Polystyrene/Organoclay Nanocomposites: Synthesis via In Situ Coordination-Insertion 8 Polymerization and Preliminary Characterization[END_REF] [START_REF] Pinte | Proposal of a Set of Model 18 Polymer Additives Designed for Confocal FRAP Diffusion Experiments[END_REF]) by casting from an ethyl acetate solution onto a cover slide, followed by 3 solvent evaporation under dynamic vacuum at 90 °C, The cover slides were then mounted onto a 4 microscope slide by slight pressure and heating at 120_C for several seconds. sPS is non soluble in 5 the same systems as aPS. Therefore, samples were made using the following method: 6 i dissolution in a refluxed toluene solution containing the proper amount of the 7 fluorescent probe, 8

ii casting on a hot plate (100 °C) to allow quick evaporation of toluene and to 9 avoid sPS precipitation, 10 iii removal of toluene traces at 90 °C in a dynamic vacuum oven for two days, 11 iv pressing a small piece of doped sPS between a glass microscope slide and a 12 glass cover slide at 270 °C. 13

In order to have fixed crystallinity sPS samples, sPS slides were annealed at 270 °C for 14 2 minutes and cooled at constant rate of 10 °C.min -1 to allow crystallization to occur. 15 DSC showed no cold crystallization peak when heating these samples above Tg, up to 16 melting point. 17 18

Fluorescent probe diffusion was monitored by confocal Fluorescence Recovery After 19

Photobleaching (FRAP) as described elsewhere [START_REF] Pinte | Proposal of a Set of Model 18 Polymer Additives Designed for Confocal FRAP Diffusion Experiments[END_REF]). This microscopic 20 technique has been previously used as a relevant method to collect D data in polymers (Smith 21 1986). FRAP creates an in situ concentration gradient in the bulk of small polymer samples. 22

The flux of the fluorescent species then induces an intensity fluctuation over the Region of 23 interest (ROI). From a practical point of view, FRAP experiments are conducted in three 24 steps: (i) reading the initial intensity with low laser power, (ii) bleaching a specified area by 25 scanning a selected region of the sample (the ROI) with very high laser power, thus 26 The Shape ratio of the different probes used for FRAP experiment, denoting the ratio of a 6 migrant's major dimension to its minor dimension was determined from NBDNEt2 to 7 NBDNpip4. By 3D-drawing the molecules using MarvinSpace (software from ChemAxon), 8 both dimension of the molecules then the shape ratio were easily determined. 9

Experiments were set at a higher temperature than allowed on the microscope sample 10 holder. Therefore, sample slides were mounted once for reading and bleaching, then 11 demounted to be heated between two hot plates of a heating press, cooled down as quickly as 12 possible and remounted to read the fluorescence profile evolution. The last steps were 13 repeated until ROI intensity had varied enough to calculate D. Since the sample requires time 14 to reach thermal equilibrium under the press, diffusion time (i.e. time spent in the heating 15 press) was corrected to take this into account, assuming diffusion at lower temperatures to be 16 negligible. 17 FRAP kinetics were processed as described elsewhere [START_REF] Pinte | Proposal of a Set of Model 18 Polymer Additives Designed for Confocal FRAP Diffusion Experiments[END_REF]). Due to quick 1 diffusion (as high temperatures were explored), bleached ROIs were enlarged to extend 2 recovery timescales. Unfortunately, bleaching was not efficient enough in the depth of the 3 sample and apparent anomalous fluorescence recovery had to be corrected. As a consequence, 4 the mathematical analysis implemented by Pinte et al. was slightly modified to eliminate this 5 unwanted diffusion from planes below and above the focal plane and to analyze fluorescence 6 recovery due to x-axis 1D diffusion. 7

Several authors using the FRAP method have reported immobile fractions when 8 monitoring diffusion in complex systems [START_REF] Miura | What do FRAP curves tell us ?[END_REF][START_REF] Karboviak | Effect of plasticisers (water and glycerol) on the diffusion 9 of a small molecule in Iota-carrageenan biopolymer films for edible coating application[END_REF]. 9

Consequentially, equilibrium intensity in the bleached area may not be the same as in the 10 initial one. The ratio of the final intensity to the initial one represents the percentage of 11 mobile probes. The difference between this value and 100% then gives the amount of non 12 mobile probes. Our diffusion experiments in syndiotactic polystyrene were therefore first 13 conducted for a much longer time (6 months) than in the usual procedure in order to estimate 14 the equilibrium intensity. 15 16

Results and discussion 17

PS samples containing the probes (5 × 10 -10 mol.mg -1 ) were obtained. All probes were 18 correctly dispersed in the polymer matrixes, notwithstanding whether these were amorphous 19 or crystalline. However, some patterns were observed in sPS, revealing the partial exclusion 20 Literature on the FRAP technique mentions that in the case of complex polymer 1 systems, fluorescence recovery may not be total which is to be taken into account in the D 2 calculation. For example, Equilibrium intensity is lower, denoting non mobile probes. This is 3 supposed to be the case in semi-crystalline polymers where some of the probes could be 4 entrapped and not available for diffusion. This was checked in this study but no immobile 5 fraction was observed, even if some patterns could be observed in sPS samples (figure 3-6 sPS). Diffusion was found to be very similar in PS2M and in sPS. Equilibrium intensities 7

were not different and close to the initial ones (less than a 10% decrease). Crystallinity did 8 not induce a bigger change in mobile/non mobile probe ratios. Therefore, initial intensities 9 were used as equilibrium intensities. 10 11 Diffusion coefficients were measured for the five homologous probes, whose M W 12 ranged from 236 to 1120 g.mol -1 , in both amorphous and semi-crystalline PS, with an 13 experimental temperature increases from 100 °C to 180 °C. Those for PS2M are reported in 14 table 3. 15 16

[log 10 D=f(M W )] correlations 17

Diffusion coefficients as a function of M W for different temperatures are plotted on Figure 4. 18

The total decrease of log 10 D with M W for different temperatures is shown in table 3. 19

Increasing M W from 236 to 1120 g.mol -1 lead to a decrease of about two units of log 10 D for 20 all the tested temperatures. Indeed, as shown in figure 4 The constancy of the log 10 D = f(M W ) slope correlations relates the PS2M data to the 1 Piringer model, where influence of M W and T on D are independent. The D decrease is quite 2 small (roughly from 1 to 2 orders of magnitude) for an 890 g.mol -1 increase of the migrant 3 molecular weight. For example, Reynier et al. reported a 4orderofmagnitude decrease for 4 diffusion in polyolefins over the same range but for lower M W (from 156 to 807 g.mol -1 ) 5 (Reynier et al. 2001). But , the first molecule of the homologous series used in this study has 6 a higher M W compared to Reynier et al.'s one which was made for a migrant molecular 7 weight between Npip2 and Npip3 . As reported by Dole et al., the sharp decrease observed 8 forD mainly occurs for rather low M W that is to say between gases and small additives (M W 9 less than 200 g.mol -1 ). For higher molecular weight additives, extrapolation of such D = 10 f(Mw) curves could leads, in the present study, to a flatter slope of D=f(M W ) (Dole et al. 11 2006). 12 Migrant shape (steric hindrance) is known to be a limiting parameter for diffusion, 13 whereas increasing migrant degrees of freedom counterbalances this by increasing diffusion 14 (Al-Malaika et al. 1991). Reynier has reported a high decrease as predicted by the Piringer 15 model when taking into account homologous series of both alkanes and alcohols as well as 16 commercial additives for higher M W (Reynier et al. 2001). They observed that for high M W 17 migrants, increasing the degrees of freedom of the arms lead to a relative increase of D 18 (trilaurine and tripalmitine with long alkyl arms compared to Irgafos 168 and Irganox 1330, 19 with highly hindered arms). In fact, in our study, D decrease with migrant Mw is rather low 20 as reported above. This can be explained by the model additives series which are not shape-21 homologous over the whole range of M W (as initially expected) . Although they all have a 22 worm-like shape (shape ratio <1 as shown in figure 5), and due to the repet unit (table 3), this improved. This provides extra degrees of freedom when increasing migrant M W and thus, the 2 influence of shape through M W on D seems to be minimized. 3

The Piringer model implies a much higher dependence of D on M W (between NBDNet 2 4 and NBDNpip 4 , log 10 D=6.75 instead of about 2 in this study). Extrapolation of the Limm 5 model and the Dole approach to higher temperatures predicts variations closer to those 6 observed in this study (respective predicted decrease log 10 D of 2.75 and 3). D values have 7 been reported to be less dependent on M W in rubbery polymers than in more barrier ones 8

D=f(1/T) correlations 12

In consistency with any migration model, D was found to increase when increasing 13 temperature as shown in figure 6. The different D values between 120 °C and 180 °C ranged 14 from 3.2 to 3.9 log 10 D units (table 3). 15

Close to glass transition, D variations are emphasized. Although requiring further 16 confirmation, the higher the M W , the stronger the dependence with T. For NBDNet 2 to 17 NBDNpip 3 , the D jump temperature is between 100 and 120 °C whereas for NBDNpip 4 , it 18 occurs at a higher temperature (the jump is related to the D data which leave the D= f(Mw) 19 line observed at high temperature). The Tg jump seems to be a function of M W , as it is of 20 frequencies or heating speed in thermo-mechanical analysis (Sauerbrunn et al. Ea are between 140 and 180 °C slightly increased with molecular weight (table 4), 3 whereas between 120 and 180 °C, it could be considered as constant, considering the data 4 scattering . Results on a reduced range limit comparison with the Piringer model, which takes 5 a constant Ea into account, and make the PS2M data closer to the Limm and Hollifield model 6 (respective increase of Ea from Net 2 to Npip 4 is 1.5 vs 1.7). The effect of M W seems to prevail 7 over the shape factor ratio, in contrast to diffusion coefficients. This odd behavior, however, 8 makes one relationship clear: the weaker the dependence of the D=f(M W ) slope with 9 temperature, the smaller Ea variations. 10

Increasing linearity for higher M W leads to a weaker shape factor influence and 11 therefore decreasing dependence of Ea with M W . 12 13 Ea were then compared to data in rubbery PS and LDPE. Hall et al. studied the diffusion 14 of lophine in rubbery PS between 100 and 145 °C. [START_REF] Hall | Translational and 29 Rotational Diffusion of Probe Molecules in Polymer Films near Tg: Effect of Hydrogen 30 Bonding[END_REF]. From their data, the 15 calculated Ea (272 kJ.mol -1 ) is one and a half times larger than Hall et al.'s equivalent Ea 16 (M W of lophine is between that of NBDNet 2 and NBDNpip 1 ). This difference may lie in the 17 different shape and degree of freedom. As previously mentioned, the homologous set is worm 18 shaped with a propyl spacer between rigid cores providing degrees of freedom, whereas 19 lophine is more spherical with three short and rigid phenyl arms. This difference in Eas is 20 logical because the Ea represents the energy required to open a sufficient hole between 21 polymer chains for the migrant to diffuse: thus, a greater probe diameter means a higher Ea 22 Diffusion in two different PS, one amorphous and one semi-crystalline, was monitored at the 2 rubbery state for five homologous probes, offering a M W range of almost 900 g.mol -1 . 3 Diffusion was found to be less dependent on temperature or migrant molecular weight than 4 calculated from prediction models.. However, D decrease for M W range was found close to 5 the Limm model. This low dependence might be explained by the higher M W of the first 6 molecule of the model migrant series. Another hypothesis may be offered to explain the low 7 correlation between D and M W . Even if the probes are chemically homologous, they are 8 unfortunately not ideally homologous; their shape factor varies along the series from 9 relatively square to worm-like. This variation may counteract the decrease of D as M W gets 10 higher. Bearing this fact in mind, none of the models can be excluded, based on the study's 11 data. Moreover, all the three models can partially explain the observed effects of M W and T 12 on D. 13 Whatever hypothesis is taken into account, the following general relationship is 14 accurate: when D is not strongly affected by Mw or T, neither is Ea. Further experiments are 15 required to develop a general model for: (i) a more shape-homologous series of designed 16 migrants, and (ii) diffusion in glassy matrices, where very little data are still available because 17 of very long diffusion time The Frap technique could help to acquire information about that 18 issue. 19

  characterized by differential scanning calorimetry (MDSC 2940, TA 20

  photolysing the illuminated fluorescent probe, (iii) reading the recovery of the fluorescence 1

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  To help re-localization in the sample when re-mounting under the confocal microscope, 18 thin copper grids (Oxford Instruments, Saclay, France) were immersed in the PS in the casted 19 drop of PS2M or in the molten piece of sPS. The hexagonal pattern (figure3) provides 20 valuable information when saved coordinates failed to precisely point to the region of 21 interest. Due to the high temperatures required for the experiments, sandwiches of slides/PS 22 sample/coverslide were sealed with epoxy glue instead of nail polish. 23 24 Page 8 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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  of fluorescent probes from more crystalline regions. This did not disturb FRAP experiments 21 and diffusion of the different items of the homologous set was monitored in PS2M and in sPS 22 for different temperatures ranging from 120 to 180 °C. 23 24 25 Page 9 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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  , an arbitrary line can fit all data at 21 rubbery state, by only vertical shifts as lines are parallel. As reported in Table3, the slope can 22 be considered as almost constant, when not taking into account data at 100°C. The slope 23 seemed smaller at 100°C, when PS2M is at its DSC glass transition temperature. 24 25 Page 10 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  the change from NBDNpip n to NBDNpip n+1 . where the migrant linearity is 1

(

  [START_REF] Dole | Typical diffusion 20 behaviour in packaging polymers: application to functional barriers[END_REF]). But the flatter slope, (figure4), at 100 °C gives the opposite indication as 9 predicted byDole et al. 10 11

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  2003). 21Activation energies (Ea) are calculated from the Arrhenius law as usually describes by others 22 authors(Hayashi et al.1994, Roe et al.1998). Ea is then proportional to the slope of the curves 23 log D = f(1/T) and determine from the least squares approach. For homogeneity, activation 24 Page 12 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk between 120 and 180 °C, assuming they are constant over this range 1 of temperature. 2

(Food6Food

  Al-Malaika et al. 1991; Reynier et al. 2001). However, these reported Eas are very high 23 compared to those calculated by Al-Malaika et al. (Al-Malaika et al. 1991) for homologous 24 linear 2-hydroxy benzophenones in LDPE (between 69 and 86 kJ.mol -1 from 5 to 100 °C). 25 Page 13 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukThis could be explain by the mobility of the matrix: the temperature gap to the Tg is far 1 bigger in Al-Malaika's experiment (for LDPE: T-Tg ~ 150 °C) than in ours (0 < T-Tg < 80 2 °C) or in Hall's (0 < T-Tg < 45 °C). This corroborates the suggestion of Dole et al.(Dole et 3 al. 2006) that Ea may decrease with increasing temperature, but over a larger range and 4 farther from the Tg than studied here. 5D=f(crystallinity) 7Crystallinity is reported to decrease D by hampering migrant diffusion as described by 8Hedenqvist et al.[START_REF] Hedenqvist | Diffusion of small-molecule penetrants in 2 polyethylene: free volume and morphology[END_REF]. Some authors even describe an exponential 9 relationship between D and cristallinity[START_REF] Klopffer | Transport Properdines of Gases in Polymers: 12 Bibliographic Review[END_REF]. 10 11 Hedenqvist et al. report a five-fold reduction of D in linear PE and a two-fold one in 12 branched PE when increasing crystallinity by 50% (Hedenqvist et al. 1996). The same 13 observation was made by Chiang et al. when comparing quenched pseudo amorphous sPS 14 with isothermally crystallized sPS (Chiang et al. 2002). Such reductions are of the same 15 magnitude of what was found in our system (Figure 7). Nevertheless, larger Ds in sPS than in 16 PS2M at low temperature are odd. (from Figure 7 and other results not shown). In addition, 17 no increasing difference between D amorphous and D crystalline with the additive M W was found, in 18 contrast to what Hedenqvist et al. reported. The torturous diffusion path in sPS might be 19 counterbalanced by the increase of free volume due to increased tacticity and Tg heights. 20 Highly tactic polymers have been reported to have higher free volume than atactic ones due to 21 special chain conformation (Dammert et al. 1999; Soldera et Grohens 2002). When getting 22 close enough to Tg, tortuosity effect may become less important than free volume size and 23 chain mobility. 24 25 Page 14 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  Photophysical and photochemical Tools in Polymer Science. M. A. Winnik, D. Reidel 12 Publishing Company, pp 397-406. 13 14 Soldera, A. and Y. Grohens (2002). Molecular modeling of the glass transition of 15 stereoregular PMMAs. Polymer-Plastics Technology and Engineering 41(3), pp561 -571. 16 17 Tseng, K. C., Turro N. J., , Durning, C. J. (2000). Tracer diffusion in thin polystyrene films. 18 Polymer 41(12), pp 4751-4755. 19 20 21

Figure 1 . 11 Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Food 3 Figure 7 .Figure 1 .

 11123456371 Figure captions 2

Figure 2 .polystyreneFigure 3 .Figure 4 .Figure 5 .

 2345 Figure 2. Thermal characterization of virgin amorphous polystyrene (PS2M) and semi ccristaline

Figure 6 .FoodFigure 7 .

 67 Figure 6. Arrhenius plot of amorphous polystyrene (PS2M) diffusion coefficients.Probes legend is given in table2.Lines are drawn only as guides. The dotted line (Npip4), however, has a lower slope than other

general predictive model for additive diffusion in polymers? 26

  al. suggested that this high slope variation leads to a variation of 21 apparent Ea with temperature and M W . The empirical models above can still predict worst 22 case values, but the safety margins may be drastically reduced from polyolefins to other 23

	24 25	extrapolated systems. o r P e e r R e v i e w O n l Toward a F y

  al. 8(Reynier et al. 2001). Moreover, they are dedicated to the use of the FRAP technique. The 9

	1	To resume, the relationships D=f(M W ) and D=f(1/T) are established and discussed in
	2	this paper. Effects of crystallinity are also discussed. Comparison of experimental behavior
	3	with model predictions is made.
	4
	5	Material and methods
	6	Material
	10 11 12 13 14 15 16 17 18 19 20 21 22 7 8 9 10 11	FRAP technique (Fluorescence Recovery After Photobleaching) is a microscopic technique used to monitor diffusion in both biological samples (Seiffert et Oppermann 2005), and material ones (Smith 1986; Tseng et al. 2000). It allows the observation of a micro-domain of the packaging material, without simulant (or food) interactions. This offers a quick determination of intrinsic apparent diffusion coefficients of model additives, especially when dealing with low mobility matrixesfrom. The chosen matrix is PS, for which little modelling validation has been obtained up to now for the following reasons: i PS can be either totally amorphous or semi crystalline, depending on its tacticity, ii It's relatively high glass transition temperature (Tg) facilitates measurements in the vicinity of Tg, even at a glassy state, iii PS displays a large rubbery plateau without any important structural change (cold crystallization, melting). As a crystalline PS, syndiotactic PS (sPS) was preferred to isotactic PS because of its higher cold-crystallization and melting o r P e R e v i e w O n l y Amorphous atactic model polystyrene PS2M was obtained as a SEC (steric exclusion chromatography) standard from Sigma Aldrich (Saint Quentin Fallavier, France): W M = 1 850 000 g.mol -1 , n M = 1 760 000 g.mo l-1 , W M / n M =1.05. Syndiotactic polystyrene was purchased from Scientific Polymer Products (Ontario, USA): approx. temperatures. When correctly annealed, sPS shows no post-crystallization or F e r W M = 300 000 g.mol -1 , density=1.05.

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	1
	2	Films of amorphous polystyrene for FRAP experiments (PS2M) were made as described in
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
	24	). Both thermograms are
	25	shown in figure 2.

Table 1 . Factors influencing diffusion coefficients and activation energies according to the three models

 1

	1
	2
	Model	D influencing factors	Ea influencing factors
	Piringer's model	M W	T	polymer	polymer
	Limm and Hollifield's model	M W	T	polymer	polymer	M W
	Dole's model	polymer	M W	T	M W	polymer	T
	strong dependence (bold),

average dependence (normal), weak dependance (italic) 3 Page 19 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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

Table 2 . Characteristics and structures of the homologous fluorescent set of model additives.

 2

			Food Additives and Contaminants		Page 20 of 32
	1
		Name	NBDNEt 2	NBDNpip 1	NBDNpip 2	NBDNpip 3	NBDNpip 4
		n (number of 'monomer' units)	0	1	2	3	4
		Structure
		M W (g.mol -1 )	236	426	658	889	1120
		NBDNEt 2
		NBDpip 1
		NBDpip( 2 to 4)
	2	The filled elliptical head and open rectangles represent the NBD chromophore (in blue) and repeating units (pip)
	3			respectively.

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Table 3 . Diffusion coefficients measured in amorphous polystyrene (PS2M) at various temperatures (D 1 are in 10 -12 cm 2 .s -1 ). Probes legend are given in table 2.

 3

	Page 21 of 32		Food Additives and Contaminants
	2
		Probes			Temperature (°C)
		M W (g.mol -1 )	180	160	140	120	100	∆log 10 D b
	NEt 2	236	17000	1400	48	9,5	0,026	3.2
	Npip 1	426	12000	1000	60	7,0	0,0081	3.2
	Npip 2	658	5100	480	4	0,64	nd	3.9
	Npip 3	889	600	140	10	0, 29	0,0035	3.3
	Npip 4	1120	500	84	0,13	0.07	0,0008	3.8
		∆log 10 D a	-1.5	-1.2	-2.6	-2.1	-1.2
	3 4	a ∆log 10 D were calculated from NBDNEt 2 to NBDNpip 4 b ∆log 10 D were calculated between 120°C and 180°C

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Table 4 . Calculated activation energies (Ea) for the model additives in actactic polystyrene (PS2M).

 4

	1
	2	Arrhenius plot of PS2M diffusion coefficients.
	Model additive	M W (g.mol -1 )	Ea (kJ.mol -1 ) between 120 and 180 °C between 140 and 180 °C
	Net 2	236	187	170
	Npip 1	436	189	179
	Npip 2	658	219	237
	Npip 3	889	188	212
	Npip 4	1120	239	249
	Ea were calculated assuming a Arrhenian behavior and a constant Ea between 120 and 180 °C.

). Probes structures are given in table 2. 3 4 Page 31 of 32 http

			Food Additives and Contaminants
	1	List of abbreviations
	2	M W Molecular weight
	3	D	diffusion coefficient (
	4	Ea	Activation energy
	5	FRAP Fluorescence recovery after photo bleaching
	6	A' P tabulated polymer parameters (from the Piringer diffusion model)
	7	C tabulated polymer parameters (from the Piringer diffusion model)
	8	T temperature
	9	D* overestimated diffusion coefficient (obtained from the Piringer model)
	10 11 12 13 14	D'0 diffusion coefficient at infinite temperature (from the Limm model) F α tabulated polymer parameters (from the Limm model) K tabulated polymer parameters (from the Limm model) o PVC, polyvinylchloride r PA polyamide
	15 16	RH relative humidity PET polyethylene terephtalate P
	17 18 19 20 21	PS2M Polystyrene with High molecular weight (amorphous PS) e sPS syndiotactique polystyrene (semi-crystalline) ROI region of interest e NBD : NBD is the fluorescent kernel of the probes (nitrobenzoxadiazol) r
	22 23		R
	24 25 26 27 28 29 30 31		e v i e w
	32		l n O
			y

://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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et al. have shown that diffusion activation by a temperature increase or plasticization fits the

general relationship drawn in Figure1(Reynier et al. 2001).

melting over the studied range of temperatures (100 to 180 °C).

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ratio decreases along the series: NBDNet 2 has a more spherical shape than NBDNpip n .

Moreover, when jumping from NBDNet 2 to NBDNpip 1 , no extra degree of freedom is added,

Page 26 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Sylvie Sacher-Huvelin

Emmanuel Coron

Marianne Gaudric

Lucie Planche

Robert Benamouzig

Vincent Maunoury

Bernard Filoche

Muriel Frederic

Jean-Christophe Saurin

Clement Subtil

Jean-Christophe; Chu Edouard Saurin

Gastroenterology Herriot

Clement ; Subtil

Gastroenterology Chu Bordeaux

Stéphane Lecleire

Chu Rouen

Gastroenterology Cellier

Christopher ; Hegp

Gastroenterology Coumaros

Muriel Frédéric

Jean- Christophe Saurin

Clément Subtil

Christophe Cellier

Dimitri Coumaros

Denis Heresbach

Prof Jean Paul Galmiche
email: jeanpaul.galmiche@chu-nantes.fr

COLON CAPSULE ENDOSCOPY VERSUS COLONOSCOPY IN PATIENTS AT AVERAGE OR INCREASED RISK OF COLORECTAL CANCER

Keywords: Colorectal cancer < Disease-based, Capsule endoscopy < Topics, Colonoscopy < Topics, Diagnostic tests < Topics, Screening < colon capsule endoscopy, colonoscopy, screening, colonic neoplasia, accuracy

published or not. The documents may come

INTRODUCTION

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. The screening and surveillance of patients with average (asymptomatic, 50-74 years old), or increased (asymptomatic with a personal or family history of polyps and/or CRC), risk is based on the detection and removal of adenomatous polyps. In many countries, colonoscopy is considered to be the standard procedure for screening and surveillance. However, colonoscopy has some limitations including invasiveness, discomfort and embarrassment for the patient, the need for short-term hospitalisation and, finally, a relatively high cost. These inconveniences may limit the utility of colonoscopy, especially in screening strategies where acceptance of the test is of the utmost importance. Similarly, such disadvantages can impact on compliance in patients who require surveillance because of a personal or family history of colonic neoplasia. Capsule endoscopy is a new technology for the investigation of the small bowel, which has been developed very successfully during the last decade. Recently, a specific capsule device has been developed for colon endoscopy and proof-of-concept studies have shown encouraging results [START_REF] Eliakim | Evaluation of the PillCam colon capsule in the detection of colonic pathology: results of the first multicenter, prospective, comparative study[END_REF][START_REF] Schoofs | PillCam colon capsule endoscopy compared with colonoscopy for colorectal tumor diagnosis: a prospective pilot study[END_REF][START_REF] Van Gossum | Capsule endoscopy versus colonoscopy for the detection of polyps and cancer[END_REF][START_REF] Gay | Could the colonic capsule PillCam colon be clinically useful for selecting patients who deserve a complete colonoscopy?: Results of clinical comparison with colonoscopy in the perspective of colorectal cancer screening[END_REF] . In fact, none of these studies were designed for the assessment of colon capsule endoscopy (CCE) as a screening test, as most of the enrolled patients were symptomatic and/or hospitalised for known or suspected colonic diseases.

We, therefore, conducted a prospective, multicentre trial, in order to assess the diagnostic yield of CCE compared with colonoscopy in carefully-selected, asymptomatic patients at average or increased risk of CRC. Because the likelihood of cancer is higher in polyps ≥ 6 mm in size, our main criterion of judgement was the proportion of individuals with polyps of this size (or with CRC) detected by CCE, as compared with colonoscopy.

PATIENTS AND METHODS

Study group

The protocol was approved by the institutional review board of the Pays de la Loire and the study was registered in the EudraCT database (n° ID RCB 2007-A00056-47) and in the database Clinicaltrial.gov (n° : NCT00436514). Written, informed consent was obtained from all patients. Adults patients were enrolled prospectively from April 2007 to July 2009 in each of the 16 French academic centres if they fulfilled one of the following two criteria: (i) healthy, asymptomatic individuals 50 to 74 years old who accept colonoscopy in the context of a screening programme (average risk group); (ii) asymptomatic patients with a personal or family history of CRC or polyps, but without colonoscopy during the preceding three years (increased risk group). The main exclusion criteria were ; the presence of dysphagia; symptoms suggestive of intestinal obstruction; recently-complicated colonic diverticulosis; advanced heart or kidney failure; the presence of a cardiac pace-maker or other implanted electro-medical device; pregnancy.

Colon capsule endoscopy and colonoscopy

The characteristics of PillCam colon (Given Imaging Ltd) have been described previously [START_REF] Galmiche | Recent developments in capsule endoscopy[END_REF] .

Briefly, the colon capsule uses the same technology as the small bowel corresponding devices, but is slightly longer. It measures 11 x 32 mm and has dual cameras, located at both ends, allowing image acquisition with a frame rate of 4 frames per second.

The colon preparation procedure has been adapted from the conventional PEG preparation used for colonoscopy in order to ensure cleanliness, but also propulsion of the capsule through the GI tract. Patients underwent colon preparation as previously reported by others [START_REF] Eliakim | Evaluation of the PillCam colon capsule in the detection of colonic pathology: results of the first multicenter, prospective, comparative study[END_REF] . However, during the first part of this trial, we did not recommend a clear liquid diet the day before CCE. This recommendation was introduced after the first interim analysis planned in the study protocol (see results). Colon cleanliness was assessed using a fourgrade scale, as in previous studies, and results were expressed as excellent, good, fair or poor. The same classification was applied to assess colonic preparation during colonoscopy (after flushing of the colon, if required).

Colonoscopy was performed under general anaesthesia (as is standard practice in France). All detected polyps were removed and sent to the local Pathology Department for routine histology.

Study design (Table 1)

The study was conducted prospectively and its design followed the STARD recommendations [START_REF] Bossuyt | The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration[END_REF] . Patients at average or increased risk of CRC were enrolled in each centre and informed consent was obtained after explaining the objectives and modalities of the protocol. During this first visit, patients were instructed about the three-day, low-residue diet and the colon preparation procedure (four litres of PEG) required prior to CCE. Patients were hospitalised for approximately 36 hours. CCE was performed at approximately 10:00 a.m., one hour after the ingestion of the fourth litre of PEG. Domperidone, sodium phosphate and bisacodyl suppository were administered as previously described (Table 1). After completion of CCE (excretion of the capsule or at least 10 hours after capsule ingestion) patients were allowed to eat a light, low-residue snack in the evening of this first day.

Colonoscopy was performed the morning of the second day, approximately three hours after administration of an additional litre of PEG to ensure optimal cleanliness.

In each centre, one to three experienced endoscopists performed all colonoscopies, while capsule videos were interpreted separately by one single independent endoscopist per centre. CCEs and colonoscopies were performed by endoscopists, unaware of each others camera and then from the distal one. The sizes of polyps detected were estimated visually, according to the distance separating the colonic wall and the camera, as in previous trials.

However, contrary to Van Gossum et al, no correction of this rough estimate was applied.

Tolerability and safety were recorded during hospitalisation and, finally, at a follow-up visit approximately 30 days after the patient was discharged from the clinic. During this visit, patients were invited to express their opinions concerning CCE and colonoscopy using a Visual Analogic Scale (VAS) for both procedures.

Statistical analysis

As in previous trials, colonoscopy was considered as the gold standard and per-patient comparisons were made considering different primary and secondary criteria. However, because colonoscopy is not a perfect gold standard, we planned an additional analysis using a modified gold standard, taking into consideration the false positive results of CCE. Indeed, so-called "false positives" are in fact "true positives" if the presence of a polyp is confirmed by a second colonoscopy. We also made the same assumption when a second independent video reading of the discrepant findings between colonoscopy and CCE, performed by an expert panel, clearly confirmed the presence of the image of a polyp ≥ 6 mm.

According to the STARD recommendations, the analysis was performed on an intention-to-diagnose basis (with technical failures considered to be negative results of CCE).

The per-protocol cohort represents the patients who completed the study successfully and who underwent both CCE and colonoscopy with complete examination. Contrary to previous studies, which were based only on exploratory statistics, the aim of this trial was to test the non-inferiority of CCE as compared with colonoscopy. Considering the perspective of screening and surveillance, we assumed that an excellent negative predictive value (no more than 5% difference with colonoscopy) would be indispensable for such a test to be useful in practice. In addition, even if CCE was inferior to colonoscopy, we assumed that the difference in terms of sensitivity between CCE and colonoscopy should not exceed 20%. Using an α risk = 5%, a β risk = 10% and an expected prevalence of polyps ≥ 6 mm of 15% (primary criterion of judgement), we calculated that a sample size of 514 (550 with losses) would be necessary to test our non-inferiority hypothesis. An interim analysis was scheduled to be performed when 1/5 of the recruitment had been achieved, in order to assess colon cleanliness and safety. Finally, the following tests were used for the final analysis : the Mac Nemar test, the chi-square test and the Student's t-test on paired data. A p value < 0.05 was deemed to be statistically significant.

RESULTS

Patients

The flow distribution of the 545 patients enrolled in the trial is shown in Fig. 1. The demographic characteristics of the intention-to-diagnose cohort are shown in Table 2.

Propulsion of the capsule and colon cleanliness

Interim analysis of colon cleanliness was performed after enrolment of the first 105 patients [START_REF] Sacher-Huvelin | Wireless Capsule Colonoscopy Compared to Conventional Colonoscopy in Patients At Moderate or Increased Risk for Colorectal Cancer. Interim Analysis of a Prospective Multicenter Study[END_REF] .

Although there were no safety concerns, the proportion of patients with good or excellent preparation was only 55%. Thus, the protocol was amended and the recommendation [START_REF] Van Gossum | Capsule endoscopy versus colonoscopy for the detection of polyps and cancer[END_REF] concerning the use of a completely clear liquid diet the day before CCE was adopted.

Despite this change in the preparation, there was no improvement in the quality of preparation (data not shown) and results concerning cleanliness were pooled in the final analysis. As indicated by Fig. 2, colon cleanliness was considered to be excellent or good in 52% of patients at CCE and in 83% of cases at colonoscopy. In most patients (91%), the capsule was excreted within 10 hours of ingestion. Five patients with CRC were detected by colonoscopy compared with only three detected by CCE. The two missed cancers were located in the sigmoid colon and rectum, and both were relatively large tumours (35 mm and 15 mm, respectively). Re-reading of the videos of these two cancer cases failed to detect any abnormality that could have been missed by previous readers. The quality of preparation was good in one case and fair in the other case.

Prevalence and accuracy of detection of polyps

For the 545 patients, the CCE accuracy of detection of polyps ≥ 6 mm or CRC was 39% (95% CI 30-48) for sensitivity, 88% (95% CI 85-91) for specificity, 47% (95% CI 37-57) for the positive predictive value (PPV) and 85% (95% CI 82-88) for the negative predictive value (NPV). The non-inferiority between CCE and colonoscopy for the detection of polyps ≥ 6 mm was not acceptable for sensitivity (absolute difference -51% (95% CI -58;-43) nor for NPV (absolute difference -13% (95% CI -16;-10).

For 118 patients, the results of CCE and colonoscopy were discordant concerning the primary criterion of judgment. All of the CCE videos of these discordant cases were reviewed by the expert panel. This reinterpretation of the capsule videos improved the diagnostic yield of CCE, with sensitivity increasing to 57% (95% CI 48-66), specificity to 95% (95% CI 93-97), PPV to 73% (95% CI 63-82) and NPV to 90% (95% CI 87-92).

With respect to advanced adenomas (i.e. adenomas ≥ 10 mm and/or with a villous contingent and/or high grade dysplasia), the sensitivity of CCE was better, at 72%, and an NPV of 94%.

Overall, the results were almost the same in the screening and surveillance cohorts (data not shown) and in the intention-to-diagnose and per-protocol cohorts (data not shown).

Effect of cleanliness on the accuracy of CCE

As shown in Fig. 3, CCE accuracy was better in the group of patients with good or excellent cleanliness compared with those with poor or fair preparation. In the well-prepared patients, the NPV increased to 88% (95% CI 83-92) for polyps ≥ 6 mm and 98% (95% CI 96-99) for polyps ≥ 10 mm, but sensitivity remained low (53% [95% CI 39-67]) for polyps ≥ 6 mm).

Effect of modifying the gold standard on the accuracy of CCE and colonoscopy

The consideration of false positives of the CCE procedure as true positives, if confirmed by the expert panel (or, in four cases, by a second colonoscopy), minimised the differences in accuracy between CCE and colonoscopy for the diagnosis of polyps ≥ 6 mm. Indeed, the sensitivity and specificity of CCE increased to 51% (95% CI 42-59) and 94% (95% CI 91-96), respectively, compared with 83% (95% CI 76-89) and 100% for colonoscopy. Similarly, PPV and NPV were 72% (95% CI 63-81) and 85% (95% CI 82-89), respectively, for CCE versus 100% and 95% (95% CI 93-97) for colonoscopy. Thus, even in the best-case scenario, CCE was unable to satisfy the conditions of non-inferiority, as compared with colonoscopy.

Safety and tolerability

Nineteen adverse events were reported. Most of these were of mild or moderate severity (Table 4). Only three severe adverse events occurred, which were either potentially related to bowel preparation. No severe adverse event was related to the capsule itself.

The comparison of VAS scores showed a slight (probably not clinically relevant) statistical difference in favour of CCE compared with colonoscopy (8.74 ± 1.56 versus 8.25 ± 2.00; p < 0.0001).

DISCUSSION

This study, the largest trial of CCE reported to date, confirms the feasibility, safety and tolerability of this method for the screening and surveillance of patients at average or increased risk of CRC. Despite an excellent NPV, the sensitivity of CCE was lower than in previous studies conducted in patients with already-known, or suspected, disease.

Therefore, the hypothesis of non-inferiority of CCE compared with colonoscopy is ruled out by the present data. One strength of this study was the enrolment of a representative population of patients referred for screening or surveillance. The demographic characteristics of our cohort are indeed those expected in this context [START_REF] Rhun | High resolution with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study[END_REF][START_REF] Johnson | Accuracy of CT colonography for detection of large adenomas and cancers[END_REF][START_REF] Pickhardt | Computed tomographic virtual colonoscopy to screen for colorectal neoplasia in asymptomatic adults[END_REF][START_REF] Harewood | Colonoscopy practice patterns since introduction of medicare coverage for average-risk screening[END_REF] , although we enrolled fewer average-risk than increased-risk subjects. The proportion of polyps detected was slightly higher than initially expected, which may, ultimately, have resulted in a minor underestimation of the NPV of CCE. However, the issue concerning the results of this study is not NPV, but rather sensitivity. There are several reasons that may have contributed to a lower sensitivity of CCE as compared with previous trials. The poor quality of bowel preparation in nearly half of the subjects is a plausible explanation, because colon cleanliness directly influences the diagnostic performance of CCE, as shown by Van Gossum et al [START_REF] Van Gossum | Capsule endoscopy versus colonoscopy for the detection of polyps and cancer[END_REF] and confirmed by the present study. However, our findings are in agreement with those recently reported (in an abstract form) by Spada et al [START_REF] Spada | Pillcam colon capsule endoscopy for colon exploration : a single centre Italian experience[END_REF] ; indeed, in their series of 92 patients, only 43.5% of them had an adequate (good or excellent) bowel preparation. Accordingly the sensitivity of CCE in this study was somewhat lower (56%) that in previous series [START_REF] Eliakim | Evaluation of the PillCam colon capsule in the detection of colonic pathology: results of the first multicenter, prospective, comparative study[END_REF][START_REF] Schoofs | PillCam colon capsule endoscopy compared with colonoscopy for colorectal tumor diagnosis: a prospective pilot study[END_REF][START_REF] Van Gossum | Capsule endoscopy versus colonoscopy for the detection of polyps and cancer[END_REF][START_REF] Gay | Could the colonic capsule PillCam colon be clinically useful for selecting patients who deserve a complete colonoscopy?: Results of clinical comparison with colonoscopy in the perspective of colorectal cancer screening[END_REF] . In our study, the type of population enrolled (asymptomatic subjects) could be one of the factors that negatively influenced colon cleanliness. This does, however, reflect real-life conditions, where patients are prepared out of the clinic. In contrast, colonoscopy was performed on the second day in the clinic and in more stringent conditions; it is, therefore, not surprising that the preparation was considered good or excellent at colonoscopy in 83% of the cases. Moreover, the flushing of residual amounts of faeces may occur during colonoscopy (but not during CCE), which may further improve the quality of exploration by colonoscopy. In the group of patients with excellent or good colon cleanliness the sensitivity of CCE, although improved, remains clearly insufficient. In this respect it is important to underline that, in the first published metaanalysis [START_REF] Rokkas | Meta-analysis evaluating the accuracy of colon capsule endoscopy in detecting colon polyps[END_REF] of CCE trials, only data (including our preliminary results) referring to the best level of bowel preparation were included. The last meta-analysis published by Spada et al [START_REF] Spada | Meta-analysis shows colon capsule endoscopy is effective in detecting colorectal polyps[END_REF] found a sensitivity of 68% for significant findings (polyps ≥ 6mm and/or ≥ 3 polyps). In fact, after excluding one study for heterogeneity, the sensitivity falls to 62%. To the best of our knowledge there has been no validation study of the scale used for CCE colonic preparation assessment. We used the same scale as in previous CCE studies 1- 4, 12 . We cannot completely exclude that, apart from true differences in the quality of preparation between centres, some part of the variability could be related to inter-observer variations in the assessment method employed. This may also contribute to an explanation of the wide variability in colonic preparation quality scores reported in the meta-analysis [START_REF] Spada | Meta-analysis shows colon capsule endoscopy is effective in detecting colorectal polyps[END_REF] , as the proportion of patients who were well prepared ranged from 27% up to 89%.

Another reason for the lower performance of CCE in our study might be insufficient experience of the endoscopists in reading the CCE videos. This hypothesis is partly supported by the fact that the diagnostic yield improved upon re-reading of the videos of discordant cases by an expert panel, but again it did not reach the performance characteristics required for a screening or a surveillance procedure. However, we did not detect, in the context of this trial, any evidence of a learning curve effect. Indeed, no difference was observed in terms of diagnostic performance when we compared the group of 70 patients initially enrolled with the group of patients recruited later (data not shown). In addition, there was no difference between the low-volume recruitment centres and the largest ones. The differences between centres were actually more directly related to the overall quality of colonic preparation achieved in different hospital settings.

Finally, even after changing the gold standard definition to test the best possible scenario, the difference between CCE and colonoscopy remained outside the maximal interval accepted for the non-inferiority hypothesis. Hence, our negative conclusion concerning CCE sensitivity would seem to be robust and was unchanged by the several post-hoc analyses performed in selected subgroups. Similarly, the results were not better in the per-protocol cohort or in the average-risk compared with increased-risk patients.

The main criterion of judgement adopted in this trial (i.e. the proportion of patients with polyps of at least 6 mm or CCR) is the same as in other studies. It is justified by the fact that the risk of cancer is very low (but not zero) below this size threshold. As with other imaging technologies, such as CT-scanning (virtual colonoscopy), CCE does not allow the removal of polyps and, thus, it has been proposed that small, diminutive polyps do not need to be removed, due to the very low risk of malignancy [START_REF] Pickhardt | Computed tomographic virtual colonoscopy to screen for colorectal neoplasia in asymptomatic adults[END_REF][START_REF] Bond | Clinical relevance of the small colorectal polyp[END_REF][START_REF] Külling | Is histological investigation of polyps always necessary?[END_REF] . In contrast, the detection and removal of advanced adenomas is of crucial importance. In this context, it is important to stress the excellent NPV and the better sensitivity of CCE, reaching 72% for the whole cohort (and 78% in the well-prepared patients). CCE might, at least, be an option when colonoscopy is contra-indicated or incomplete for technical reasons and further trials are in progress to test this potential "second-line" indication of CCE.

All diagnostic parameters were calculated on a per-patient analysis. Therefore it is not surprising to see that sensitivity is better for small than large polyps. Indeed, the number of small polyps was higher than that of large polyps (for example, 177 out of 857 polyps had a diameter of at least 6 mm compared to 54 for those of at least 10 mm). Therefore, the likelihood of detecting a polyp in a patient is greater for small polyps, because their mean number per patient is higher (0.32) than for large polyps (0.10).

One limitation inherent to all current capsule endoscopy technologies is the difficulty of assessing polyp size accurately. Although we applied the same principles as in former studies, it must be recognised that the method used in all CCE studies provides an imperfect estimate, which clearly may have affected the validity of our primary criteria of judgement.

The second generation of capsule endoscopes will, hopefully, integrate a better system with respect to the measurement of polyp size. Our study was not designed to assess inter/intraobserver variability but within the panel of experts who reviewed the discordant cases the inter-observer agreement was 71% (data not shown) Finally, it must be acknowledged that we used a first generation of colon capsule.

Very-recently, a new device has been developed, which presents several technical improvements, including a larger field-of-view, a better sampling rate and, of greater importance, a grid for more objective measurement of polyp size. A recent trial of this new device [START_REF] Eliakim | Prospective multicenter performance evaluation of the second-generation colon capsule compared with colonoscopy[END_REF] conducted in a population with various colonic pathologies, reported higher sensitivity than in our study but, again, the population was not representative of a screening population, as in our trial. In summary, in this trial, which involved the largest population enrolled for colon capsule evaluation to date, and which was conducted in average-and increased-risk, asymptomatic patients, we did not establish the non-inferiority of CCE as compared with colonoscopy. Further studies should pay particular attention to colonic preparation in conditions compatible with real life. Quality control of video reading is probably required after an initial training period. Improving the technology of the device is, on its own, probably insufficient to fulfil the requirements of a test which is useful for screening and surveillance in patients at risk of CRC.

Financial support and Statement of interest

Author's declaration of personal interest

Robert Benamouzig is a board membership of Given Imaging.

Jean Paul Galmiche is a consultant of Given Imaging.

Declaration of funding interest

This study was funded by a national grant from the French Ministry of Health and sponsored by the Délégation à la Recherche Clinique (DRC) of the university hospital of Nantes.

The study benefited from some limited logistic support from Given Imaging, including the use of an already-available eCRF and a discount price for capsules. The design of the trial and the monitoring and analysis of data were, however, conducted independently by the sponsor (DRC) and the authors. 7 and table 2 16

F

The number of participants satisfying the criteria for inclusion who did or did not undergo the index tests and/or the reference standard; describe why participants failed to undergo either test (a flow diagram is strongly recommended).

7 and figure 1 Test results

17 Time-interval between the index tests and the reference standard, and any treatment administered in between.

  findings. All capsule endoscopists received specific training using CCE videos before the beginning of the trial. The training course (one full day) consisted of showing some characteristic videos in the morning (with explanations of the method of CCE video interpretation, especially polyp size estimation) followed by a validation of the training by three test-video sequences in the afternoon. The first author certified the validation of the training session by the following criteria; (i) adequate knowledge of the software capabilities, (ii) ability to detect more than 90% of lesions ≥6 mm, and (iii) estimation of polyp size. Polyps and lesions seen during colonoscopy were recorded according to their location and size, eventually, with the use of open-biopsy forceps. CCE videos were interpreted separately at a reading speed of approximately eight frames per second. Investigators were instructed to read the images taken from the proximal

Figure 1 :

 1 Figure 1: Flow of patients. Intention-to-diagnose and per-protocol cohorts.

Figure 2 :

 2 Figure 2: Proportion of patients (%) with excellent or good colonic preparation at colon capsule endoscopy and colonoscopy.

Figure 3 :

 3 Figure 3: Capsule accuracy of capsule endoscopy in patients with good or excellent cleanliness.

4 8

 4 Technical specifications of material and methods involved including how and when measurements were taken, and/or cite references for index tests and reference standard.

5 9

 5 Definition of and rationale for the units, cut-offs and/or categories of the results of the index tests and the reference standard.

3 10

 3 The number, training and expertise of the persons executing and reading the index tests and the reference standard.

5 11 5

 55 Whether or not the readers of the index tests and reference standard were blind (masked) to the results of the other test and describe any other clinical information available to the readers.Statistical methods12 Methods for calculating or comparing measures of diagnostic accuracy, and the statistical methods used to quantify uncertainty (e.g. 95% confidence intervals).

6 13 4 15

 64 Methods for calculating test reproducibility, if done. NA RESULTS Participants 14 When study was performed, including beginning and end dates of recruitment. Clinical and demographic characteristics of the study population (at least information on age, gender, spectrum of presenting symptoms).

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Flow of patients. Intention-to-diagnose and per-protocol cohorts. 85x51mm (400 x 400 DPI)

Table 3

 3 shows the per-patient prevalence of polyps and CRC detected by colonoscopy and the diagnostic performance of CCE. Overall, colonoscopy detected more patients with polyps than did CCE. Hence, 311 (57%) patients had polyps of any size detected by colonoscopy compared with 249 seen at CCE (46%; p < 0.0001). Regarding polyps ≥ 6 mm and ≥ 10 mm,

	the corresponding figures were 112 (21%) versus 94 (17%) (p = 0.097) and 43 (8%) versus
	29 (5%) (p = 0.03), respectively.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 : Study protocol: colonic preparation and drug administered (prokinetic and boosters)

 1

		Regimen I (n = 232)	Regimen II (n = 313)
	Day -3 to -2	Low residue diet		Idem
	Day -1	Low residue diet		Liquid diet
		18:00 -21:00	3 litres Colopeg®	Idem
		8:00 -9:00	1 litre Colopeg®
		9:45 -10:00	20 mg Motilium® & PillCam
	Day 0	12:00	Booster I (45 ml NaP)*	Idem
		15:30	Booster II (30 ml NaP)**
		18:00	10 mg Bisacodyl suppository**
		18:30	Low-fibre snack
	Day +1	6:00 -7:00	1 litre Colopeg®
		After 10:00	Traditional colonoscopy	Idem
	* Pending verification that PillCam left stomach with RAPID real-time viewer
	** If PillCam was not expelled from anus

Table 3 : The prevalence of lesions detected by colonoscopy in the 545 patients in the accuracy analysis and the sensitivity, specificity, positive predictive value and negative predictive value of CCE for the detection of these lesions.

 3

		Colonoscopy		Colon Capsule Endoscopy
		Prevalence	Sensitivity	Specificity	Positive	Negative
					Predictive	Predictive
					Value	Value
		N of patients (%)		% (95 % CI)
	Polyp
	Any size	311 (57.1)	58 (53-64)	71 (65-77)	73 (67-78)	56 (50-62)
	≥ 6 mm	112 (20.6)	39 (30-48)	88 (85-91)	47 (37-57)	85 (82-88)
	≥ 10 mm	43 (7.9)	35 (21-49)	97 (96-99)	52 (34-70)	95 (93-97)
	Adenoma
	Any size	192 (35.2)	61 (54-68)	63 (58-68)	47 (41-53)	75 (70-80)
	≥ 6 mm	80 (14.7)	44 (33-55)	87 (84-90)	37 (27-47)	90 (87-93)
	≥ 10 mm	36 (6.6)	39 (23-55)	97 (96-99)	48 (30-66)	96 (94-97)
	Advanced adenoma*
	Any size	54 (9.9)	72 (60-84)	57 (53-62)	16 (11-20)	94 (92-97)
	≥ 6 mm	45 (8.3)	49 (34-63)	86 (83-89)	23 (15-32)	95 (93-97)
	≥ 10 mm	36 (6.6)	39 (23-55)	97 (96-99)	48 (30-66)	96 (94-97)
	Colorectal Cancer**	5 (0.9)	60 (17-100) 100 (99-100) 60 (17-100) 100 (99-100)
	* Advanced adenoma was defined as an adenoma 10 mm or larger or an adenoma with
	villous features or high grade dysplasia.
	** All colorectal cancers were larger than 10 mm and localised, one in the caecum, one in the
	rectum and three in the sigmoid colon.

Table 4 : Adverse events

 4

	Adverse event	Number of	Severity	Relation	Evolution
		patients
	Haemorrhoid proctitis	1	Moderate	Probable	Resolved
	Vomiting	7	Mild	Probable	Resolved
	Hypokalaemia	1	Mild	Probable	Resolved
	Cervical pain	1	Mild	Probable	Resolved
	Dizzyness	1	Moderate	Probable	Resolved
	Bleeding after	1	Severe	Probable	Resolved
	mucosectomy
	Fever	2	Mild	None	Resolved
	Headache	1	Mild	None	Resolved
	Abdominal pain	1	Mild	Probable	Resolved
	Bronchospasm	1	Mild	Probable	Resolved
	Colonic perforation	1	Severe	Probable	Resolved
	Cardiac failure	1	Severe	Probable	Death

Table 1 18

 1 Distribution of severity of disease (define criteria) in those with the target condition; other diagnoses in participants without the target condition. of the results of the index tests (including indeterminate and missing results) by the results of the reference standard; for continuous results, the distribution of the test results by the results of the reference standard.

	7 and
	table 2

Table 3 20

 3 Any adverse events from performing the index tests or the reference standard.

	9 and
	Table 4

Alimentary Pharmacology & Therapeutic

Acknowledgments

The authors thank the following people for their contribution in this study:

The expert panel for the re-reading of CCE:

Bernard Filoche, Gérard Gay, Vincent Maunoury, Sylvie Sacher-Huvelin

The investigators, listed from highest to lowest number of patients enrolled in the study:

Marc Le Rhun, Emmanuel Coron, Mathurin Flamant, Marianne Gaudric, Stanislas Chaussade, Romain Coriat, Robert Benamouzig, Bakhtiar Bejou, Jean-Christophe Saurin, Thierry Ponchon, Jérôme Dumortier, Marie George Lapalus, Jeanne Boitard, Clément Subtil, Patrice Couzigou, Elise Chanteloup, Eric Terreborne, Michel Delvaux, Muriel Frédéric, Philippe Ducrotté, Stéphane Lecleire,Christophe Cellier, Joel Edery, Camille Savale, Dimitri Coumaros, Dimitri Tzilves, Denis Heresbach, Pierre Nicolas d'Halluin, Denis Sautereau, Anne Lesidaner, Franck Cholet, Thierry Barrioz. The following people participated in the coordination of the study: Stéphanie Bardot, Kafia Belhocine, Kevin Galery, Eliane Hivernaud, Anne Omnes, Véronique Sébille, Fabienne Vavasseur