Najet Yagoubi

Volatiles François Caire-Maurisier

Caroline Aymes-Chodur
email: caroline.aymes-chodur@u-psud.fr

Vincent Jandard

Annick Bourrel

Effects of electron beam sterilization on polyethylene terephthalate: Physico-chemical modifications and formation of non-volatile organic extractables

selon sa nature chimique, le type de rayonnements et d'atmosphère d'irradiation, va ensuite soit subir une dégradation soit une réticulation de ses chaînes macromoléculaires [START_REF] Charlesby | Radiation Effects in Solid Polymers[END_REF][START_REF] Dole | The radiation chemistry of macromolecules -Volume I[END_REF][START_REF] Dole | The radiation chemistry of macromolecules[END_REF]. Il est alors important d'évaluer la modification du matériau de conditionnement sous stérilisation ionisante et l'objectif de ce travail est d'étudier à la fois les modifications dans le volume du PET ainsi que d'analyser les extractibles issus de la stérilisation par électrons accélérés. En ce qui concerne la matrice polymérique et après stérilisation, nous n'avons pas observé de modification chimique par analyse spectrale, en revanche, nous avons pu mettre en évidence la coupure de chaînes par analyse thermique. De même, pour les composés extractibles organiques non volatiles, nous confirmons la présence de nombreux composés, en particulier des oligomères et des trimères, avec en particulier les acides benzoïque et téréphtalique. En ce qui concerne les recommandations pharmaceutiques, nous prétendons ici que la dose de stérilisation de 25 kGy utilisée, engendre la formation d'oligomères et de substances extractibles, un résultat qui semble prometteur pour une future analyse de risque.

KEY WORDS -MOTS CLÉ

Extractables; PET; oligomers; electron beam sterilization; thermal analysis; separation techniques. Extractibles; PET ; oligomères ; stérilisation aux électrons accélérés ; analyse thermique ; techniques séparatives.

INTRODUCTION

Polyethylene Terephthalate (PET) is widely used in the food packaging industry thanks to its high-strength barrier and easy thermoforming (e.g.: bottles, frozen products as well as packaged goods intended for microwave and convection oven use). In the field of the medical and pharmaceutical industry, because of its distinctive physical assets such as resistance, crystallinity, heat and solvent resilience as well as its biological characteristics such as biostability and promotion of tissue ingrowth, PET's applications include bottles, primary packaging material as well as implantable sutures, surgical mesh, vascular grafts, sewing cuffs for heart valves and components for percutaneous access devices.

For these purposes, PET can be submitted to ionizing radiation sterilization. The evaluation of packaging material modifications under radiation sterilization is of great interest. The objective of our paper is to study the electron beam effects on the physico-chemical properties of PET as well on the formation of extractables.

This PET material comes from of a multi-layer laminated film with amorphous polyethylene terephthalate (PET) combined with linear low density polyethylene.

A primer is applied to the PET film during the lamination process thus helping the bonding between both films. This complex allows reservoir manufacturing for BD-Uniject™ medical device. BD-Uniject™ prefillable injection device is for packaging and administering parenteral drugs through intra-muscular or sub cutaneous injections. It is aseptically filled, sealed and packaged by a pharmaceutical manufacturer. It consists in the following components assembled in open and ready to fill form ; reservoir which contains parenteral solution and needle assembly with a port, a valve disc combined with valve seat, a hub and cannula assembly, and finally the needle shield. The ready to fill form implies that the product is radiosterilised. To anticipate the likely interaction between container and content, we have to understand the effect of radiation among other topics. We focused on the reservoir because of an immediately contact with parenteral drug. Indeed, the effect of radiation can induce physical modifications in PET such as change in the mechanical properties due to scission/cross-linking of the macromolecular chains, oxidation, change of crystallinity and as a consequence of permeability; but radiation can also induce degradation of the additives into smaller molecules that have higher mobility through the macromolecular chains and as a consequence become potential leachables. Moreover, it is noteworthy that in the context of pharmaceutical packaging sterilization processes, degradation products and oligomers are likely to appear [START_REF] Jenke | Linking extractables and leachables in container/closure applications[END_REF][START_REF] Dennis | Evaluation of the chemical compatibility of plastic contact materials and pharmaceutical products; safety considerations related to extractables and leachables[END_REF][START_REF] Welle | Migration and sensory changes of packaging materials caused by ionising radiation[END_REF][START_REF] Welle | Migration of radiolysis products from radiation-sterilized plastics[END_REF] and further, they may spread from the container/closure system into the drug solution itself. Those compounds might have a bad effect on the stability of the formulation and the adsorption of the formulation on the plastic container can be modified. Hence, any thorough compatibility assessment needs to consider the sterilization impact on physical properties and potential leaching impurities [START_REF] Dennis | Evaluation of the chemical compatibility of plastic contact materials and pharmaceutical products; safety considerations related to extractables and leachables[END_REF][START_REF] Welle | Migration and sensory changes of packaging materials caused by ionising radiation[END_REF][START_REF] Welle | Migration of radiolysis products from radiation-sterilized plastics[END_REF][START_REF] Norwood | Best Practices for Extractables and Leachables in Orally Inhaled and Nasal Drug Products: An Overview of the PQRI Recommendations[END_REF][START_REF] Pan | Strategy for identification of leachables in packaged pharmaceutical liquid formulations[END_REF].

Polyethylene Terephthalate (PET) is a thermoplastic polyester synthesized by polycondensation of ethylene glycol and dimethyl terephthalate [START_REF] Yin | Novel catalysts based on titanium dioxide/silicon dioxide for poly(ethylene terephthalate)[END_REF][START_REF] Duh | Effects of the carboxyl concentration on the solid-state polymerization of poly(ethylene terephthalate)[END_REF][START_REF] Ignatov | PET synthesis in the presence of lanthanide catalysts[END_REF].

Numerous publications to date provide extensive information as regards PET oligomers identification, quantification [START_REF] Begley | Evaluation of polyethylene terephthalate cyclic trimer migration from microwave food packaging using temperature-time profiles[END_REF][START_REF] Barnes | Qualitative liquid chromatographic-atmospheric-pressure chemical-ionization mass spectrometric analysis of polyethylene terephthalate oligomers[END_REF][START_REF] Barth | Size exclusion chromatography and related separation techniques[END_REF][START_REF] Begley | High-performance liquid chromatographic determination of migrating poly(ethylene terephthalate) oligomers in corn oil[END_REF][START_REF] Costley | Extraction of oligomers from Poly(ethylene terephthalate) by microwave-assisted extraction[END_REF][START_REF] Küppers | The use of temperature variation in supercritical fluid extraction of polymers for the selective extraction of low molecular weight components from poly(ethylene terephthalate)[END_REF][START_REF] López-Cervantes | Migration of Ethylene Terephthalate Oligomers from Roasting Bags into Olive Oil[END_REF][START_REF] Nasser | Identification of oligomers in polyethyleneterephthalate bottles for mineral water and fruit juice: Development and validation of a high-performance liquid chromatographic method for the determination of first series cyclic trimer[END_REF][START_REF] Shiono | Determination of poly(ethylene terephthalate) oligomers in refrigeration oils by adsorption column chromatography-gel permeation chromatography[END_REF][START_REF] Shiono | Separation and identification of poly(ethylene terephthalate) oligomers by gel permeation chromatography[END_REF][START_REF] Youk | Polymerization of Ethylene Terephtalate Cyclic Oligomers with Antimony Trioxide[END_REF] and migration in food simulants [START_REF] Ashby | Migration from polythylene terephtalate under all conditions of use[END_REF]. However, few research papers have probed as yet the effects of sterilization on PET used in medical or pharmaceutical applications. Within the realm of pharmaceutical implementation, PET is used in containers intended for a wide range of preparations but not in direct contact with parenteral drugs, where acetaldehyde is solely referenced as a manufacturing residue: therefore its content must never exceed 10 ppm in granules as a rule [START_REF]Directorate for the Quality of Medicines and HealthCare of the Council of Europe[END_REF]. Whenever containers or delivery-systems require proper sterilization before use, the above-mentioned critical pre-requisite must be observed unequivocally.

Consequently, our goal is to evaluate the effects of electron-beam sterilization on Polyethylene Terephthalate (PET) used as a medical device for drug delivery, and subsequently to further lay the ground for compatibility studies involving a pharmaceutical and/or a biological medium. Insofar as sterilization induces modifications affecting all at once the polymeric bulk as well as the additives and oligomers, an integrative two-fold approach had to be implemented (one slant of approach focused on the matrix and the other slant concentrated on the additives, the degradation products and oligomers).

MATERIAL and METHODS

Materials and reagents

Medical grade PET films, about 130 µm thick, 15 cm width were supplied by Becton Dickinson Medical (France).

Tetrahydrofuran (THF) and Methanol (both HPLC grade) as well as benzoic and orthophosphoric acids were provided by Prolabo, terephthalic acid was supplied by Acros Organics.

Irradiations

The samples were irradiated at room temperature under air by an industrial electron beam accelerator (Energy of 10 MeV, power 10 kW, speed of the conveyor belt under the electron beam of 0.85 m/min) (Ionisos -Chaumesnil, France).

The PET films were placed between paper sheets in plastic bags before irradiation. The mean incident doses on the sample packages amounted to 25, 50 and 150 kGy. Around 140 g of PET films is dedicated for each dose. Chromic dosimeters were used to measure the dose. 25 kGy is the sterilization dose that is recommended by the Pharmaceutical authorities [START_REF]Directorate for the Quality of Medicines and HealthCare of the Council of Europe[END_REF][START_REF]Sterilization of health care products-Requirements for validation and routine control-Radiation sterilization[END_REF]. Once irradiated and before being studied, samples were maintained 3-4 weeks at 5°C, so as to slow down at best the decay of the radicals formed during irradiation, and to further restrict potential component migration.

In order to check radiation effects on PET, we used specific analytical techniques.

Hence, we chose to further elicit in a first step, the effects of irradiation on the polymer, subject to both chain scissions and cross-linking: such phenomena were singled out by means of analytical techniques such as spectral (Fourier Transform InfraRed spectroscopy (FTIR)) and thermal analyses (Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA)). In a second step, we concentrate on the additives, the degradation products and oligomers, known as extractables: they can be analyzed by means of weighing measurements along with chromatographic methods, namely Size Exclusion Chromatography (SEC) (providing some idea as regards oligomers molecular masses, albeit unspecific), and Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) (more specific). In terms of compatibility studies involving the polymer combined with pharmaceutical or biological media, toxicity tests still require further developments to date.

Characterization of the polymeric bulk Fourier Transform InfraRed (FTIR) analysis

FTIR spectra were recorded with a Perkin Elmer 2000 spectrometer (16 scans, 4 cm -1 resolution) equipped with a deuteriated triglycerine sulphate (DTGS) detector. In transmission mode, the sample holder was tilted at the Brewster's angle (also called the polarization angle) in order to eliminate interference fringes.

Ten different samples were analyzed for statistical reasons.

Differential Scanning Calorimetry (DSC) analysis

The DSC curves were recorded with a TA Q1000 calorimeter, under nitrogen blanketing, for a temperature ranging between 35 and 280°C. The scanning rate was optimized and finally set at 10°C/min. The calorimeter was calibrated before use with indium and tin standards. For all PET samples a sequence was set as follows: the temperature was initially set at 300°C over a 5 minute period in order to melt the polymer and improve its contact with the crucible; subsequently a first heating and cooling cycle was performed and the second heating scan was chosen, since further heating scans gave the same DSC curve. For each dose, three samples were analyzed.

Thermogravimetric analysis (TGA)

The TGA curves were recorded with a TA Q500 calorimeter, under nitrogen blanketing, for a temperature ranging between 30 and 600°C at a scanning rate optimized for 20°C/min. For each dose, three samples were analyzed.

Characterization of the non-volatile extractables

Soxhlet extractions

The soxhlet extraction was carried out on 20 g of PET accurately weighted, placed in cellulose extraction thimbles, over a 2-hour period in methanol under reflux.

The extraction solution evaporated until dry. The 250 mL glass flask used for collecting extracts were weighted before extraction and after evaporation, once cooled. When ready for HPLC measurements, dry extracts collected in the glass flask, were dissolved in 1 mL methanol (40°C) and 1 mL mobile phase. For each dose, three extractions were performed and each extraction solution was injected (20 µL) three times in the HPLC apparatus. In the case of SEC analysis, extractables were solubilized in methanol (40°C) prior to SEC analysis.

Weighing

An analytical Denver Instrument M-220D scale, capable of weighing from 1 mg up to 31 g with a 0.01 mg sensitivity level was used to evaluate the quantity of oligomers, additives and degradation products extracted from the polymer, by calculating the difference of weight of the glass ampoules used before and after extraction.

Size Exclusion Chromatography (SEC) analysis

Chromatograms were recorded with a PL-GPC 220 high temperature chromatograph (Polymer Laboratories, UK), equipped with a refractometer (RI) (Polymer Laboratories, UK) and an UV detector whose wavelength was set at 240 nm and 280 nm.

The chromatographic system had its temperature set at 35°C. Flow rate reached 1 mL/min in THF. The SEC system was controlled by the PL-GPC 220 Control software (2.01 version). Acquisition and analysis were controlled by a Viscotek software (Omnisec 4.0 version).

Two Polymer Laboratories (PL) 5 µm-mixed C and 100 Å-3 µm SEC columns were set up in series. Their molecular weights linear range ranked between 200 -2 x10 6 for the first one, and 4000 -2 x10 5 for the second one, their length reached 300 mm and their internal diameter, 7.5 mm (supplier's data).

Six polystyrene (PS) standards (Polymer Laboratories, UK) of narrow molecular weights (Mp) were used to perform a universal calibration.

Non irradiated PET extraction samples were obtained as described earlier and extracts, containing oligomers and small weight molecules, was solubilized in hot methanol prior to SEC analysis. Three different samples were analyzed such a way for statistical reasons.

Reversed Phase High Performance Liquid Chromatography (RP-HPLC) analysis

The liquid chromatograph was a Thermo Spectra Physics (TSP) system, involving a P4000 pump, a Rheodyne sampling valve with a 20 µL loop, an AS 1000 automatic sampler, and was coupled with a photodiode array UV 6000 detector, with pre-selected wavelength set at 240 nm. The software used was ChromQuest (3.0 version) from Thermo Finnigan. The separation was achieved on a 250 by 4.6 mm inner diameter, 5 µm Lichrosphere, 60 RP Select B column (C8) (Merck), heated at 30°C. The elution gradient was a solvent mixture (phosphoric acid buffer / methanol) with a 50/50 volume ratio. The phosphoric acid buffer was obtained from an orthophosphoric acid solution prepared at 0.1 M and added to a 0.1 M NaOH solution. The mobile phase had a pH adjusted to 2.8 with a 1 N HCl solution. The flow rate of the mobile phase was set at 1 mL/min.

RESULTS and DISCUSSION

There is substantial existing evidence to the effect that irradiation induces physical and chemical modifications in the polymeric bulk (e.g. chain scissions and cross-linking), as well as oxidation groupment formation [START_REF] Charlesby | Radiation Effects in Solid Polymers[END_REF][START_REF] Dole | The radiation chemistry of macromolecules -Volume I[END_REF][START_REF] Dole | The radiation chemistry of macromolecules[END_REF][START_REF] Buttafava | Effects induced by gamma irradiation of different polyesters studied by viscometry, thermal analysis and positron annihilation spectroscopy[END_REF][START_REF] Jeon | The effects of irradiation on physicochemical characteristics of PET packaging film[END_REF].

Irradiation also endorses a critical role in terms of extractable (oligomers and additives) degradation. The entities formed thereby, display high mobility levels in keeping with their low molecular weight, thus allowing for swift migration capacity into the pharmaceutical and biological media. Both the effect of the electron-beam radiation on PET bulk and non-volatile extractables are here presented. It is worse notice here that the effect of radiation on polymer might be different depending on the radiation type (electron-beam, gamma-ray, swift heavy ions, X-rays), especially because of the dose rate and the depth of induced radiation.

1-Effect of the sterilization on PET bulk -Physico-chemical methods.

Several papers have been published on radiation effects on PET by means of high energy ions beam [START_REF] Liu | Molecular conformation changes of PET films under high-energy Ar ion bombardment[END_REF][START_REF] Liu | Study of effects in polyethylene terephthalate films induced by high energy Ar ion irradiation[END_REF][START_REF] Steckenreiter | Chemical modifications of PET induced by swift heavy ions[END_REF][START_REF] Singh | Microstructural modifications in swift ion irradiated PET[END_REF][START_REF] Zhu | Modification of polyethylene terephthalate under high-energy heavy ion irradiation[END_REF][START_REF] Liu | Chemical modifications in polyethylene terephthalate films induced by 35 MeV/u Ar ions[END_REF][START_REF] Apel | Track structure in polyethylene terephthalate irradiated by heavy ions: Let dependence of track diameter[END_REF] and gamma rays [START_REF] Buttafava | Effects induced by gamma irradiation of different polyesters studied by viscometry, thermal analysis and positron annihilation spectroscopy[END_REF][START_REF] Jeon | The effects of irradiation on physicochemical characteristics of PET packaging film[END_REF][START_REF] Buchalla | Characterization of gamma-irradiated polyethylene terephthalate by liquid-chromatography-mass-spectrometry (LC-MS) with atmospheric-pressure chemical ionization (APCI)[END_REF][START_REF] Aytaç | Effect of gamma irradiation on the properties of tyre cords[END_REF][START_REF] Goulas | Effect of ionizing radiation on physicochemical and mechanical properties of commercial monolayer and multilayer semirigid plastics packaging materials[END_REF][START_REF] Kattan | Thermal behavior of gamma-irradiated amorphous poly(ethylene terephtalate) films[END_REF][START_REF] Komolprasert | Volatile and non volatile compounds in irradiated semi-rigid crystalline poly (ethylene terephtalate) polymers[END_REF], but electron-beam sterilization on PET is less described in the literature. Apel et al. [START_REF] Apel | Track structure in polyethylene terephthalate irradiated by heavy ions: Let dependence of track diameter[END_REF] and Buchalla et al. [START_REF] Buchalla | Characterization of gamma-irradiated polyethylene terephthalate by liquid-chromatography-mass-spectrometry (LC-MS) with atmospheric-pressure chemical ionization (APCI)[END_REF] mentioned the formation of a large amount of polar lowmolecular weight compounds. Buchalla et al. studied in depth the non-volatile radiolysis products of PET by means of Liquid Chromatography -Mass spectrometry (LC-MS). In their case, PET was irradiated by gamma rays at doses of 25 and 50 kGy. They evidenced that every radiation resistant polymer can lead to the formation of low-molecular weight degradation products.

1-FTIR analysis

FTIR measurements enabled film structural analysis as a function of the radiation dose. As shown on Figure 1, the transmission mode results revealed identical spectra within the dose. Such a result underscored the lack of any volume-based modification of PET chemical functions following irradiation (namely oxidation), even at the highest dose. Likewise, authors such as Aytac et al. [START_REF] Aytaç | Effect of gamma irradiation on the properties of tyre cords[END_REF] evidenced no change in the FTIR spectra of 150 kGy gamma irradiated cords used as tyre reinforcing material.

PET is known to be radiation resistant, owing to the presence of aromatic groups along with ester linkages in its backbone [START_REF] Goulas | Effect of ionizing radiation on physicochemical and mechanical properties of commercial monolayer and multilayer semirigid plastics packaging materials[END_REF][START_REF] Dole | The radiation chemistry of polymer composites[END_REF]. In keeping with findings released by Liu et al. for doses above 6 MGy [START_REF] Liu | Molecular conformation changes of PET films under high-energy Ar ion bombardment[END_REF][START_REF] Liu | Study of effects in polyethylene terephthalate films induced by high energy Ar ion irradiation[END_REF], we found that the 1579 cm -1 band intensity assigned to the aromatic skeletal stretching band of benzene rings, remained constant during irradiation. Several papers deal with the modification of PET under high energy heavy ions irradiations: Steckenreiter et al. [START_REF] Steckenreiter | Chemical modifications of PET induced by swift heavy ions[END_REF] as well as Singh et al. [START_REF] Singh | Microstructural modifications in swift ion irradiated PET[END_REF] studied swift heavy ion effects over PET by FTIR and showed alkyne formation near the track core and evidenced an enhanced degradation due to the presence of oxygen. Zhu et al. [START_REF] Zhu | Modification of polyethylene terephthalate under high-energy heavy ion irradiation[END_REF] showed that a single ion bombardment damaged the phenyl ring and the crystallinity of PET. As previously reported by Dole [START_REF] Dole | The radiation chemistry of macromolecules[END_REF], carbonyl groups are formed under radiation in oxygen atmosphere. In our case, we observed no alkyne end group formation nor any oxidation functions.

It is noteworthy however, that we used a different radiation framework. Moreover, we resorted to very small doses compared to the cited authors. Indeed, our goal was to be the closest as possible to the sterilization dose.

Finally, under irradiation conditions used in our study, no chemical modification was observed with reference to PET films.

2-Thermal analysis

Thermal characteristics and especially crystallinity yield is an important parameter to check after irradiation because a highly crystalline polymer will be more efficient against the molecular diffusion than an amorphous one. Figure 2 gives the DSC curve overlay obtained at the second heating stage for both nonirradiated and irradiated samples. Glass transition was readily observed and measured at a glass temperature (T g ) of 81°C, regardless of the dose. Radiation effects were displayed with both subsequent thermal events, namely: the endothermic transition emerging at about 176°C, and the shape of the melting peak at 242°C approximately. Following irradiation, the endothermic transition increases as well as the shoulder in the melting peak at a temperature below the maximum level (about 228°C). Chain scissions may account for such a phenomenon, insofar as they induce the formation of smaller molecular chains involving a lower melting point compared to initial chains.

Jeon et al. [START_REF] Jeon | The effects of irradiation on physicochemical characteristics of PET packaging film[END_REF] have referenced gamma irradiation PET degradation. Through Size Exclusion Chromatography (SEC), they evidenced the molecular weights decrease. Findings are congruent with results released by Buttafava et al. [START_REF] Buttafava | Effects induced by gamma irradiation of different polyesters studied by viscometry, thermal analysis and positron annihilation spectroscopy[END_REF].

The latter carried out viscosity measurements, thus disclosing the following trends: molecular weights decrease at 50-150 kGy gamma-rays doses, followed by an increase up to 300 kGy, prior to a subsequent decrease recorded at higher doses within the reach of 1000 kGy. In the course of our investigations, however, we observed no single T g value variation within our dose range, whereas Buttafava et al. [START_REF] Buttafava | Effects induced by gamma irradiation of different polyesters studied by viscometry, thermal analysis and positron annihilation spectroscopy[END_REF] and Kattan [START_REF] Kattan | Thermal behavior of gamma-irradiated amorphous poly(ethylene terephtalate) films[END_REF] reported a T g decrease at higher gamma-rays doses (up to 3500 kGy): such a finding points out to a chain scission-induced macromolecules mobility increase. Kattan took his research a step further: He calculated the number of chain scissions by evaluating T g decrease, a significant point when considering the dose range used then.

Many semi-crystalline polymers exhibit multiple melting behaviors from different sources depending on authors. Regarding the issue of endothermic transition at about 176°C, Kong [START_REF] Kong | The enthalpy of fusion and degree of crystallinity of polymers as measured by DSC[END_REF] and Wang alike [START_REF] Wang | The nature of secondary crystallization in poly(ethylene terephthalate)[END_REF] argued that depending on PET thermal treatment, a minor endotherm is likely to emerge due to secondary lamellae melting processes. Authors evidenced the formation of two distinctive lamellar thickness populations during the isothermal crystallization process at long times: the thicker lamellae formed first, and the thinner lamellae formed both during and after primary crystallization. Using isothermally crystallized PET, Woo et al. [START_REF] Woo | A differential scanning calorimetry study on poly(ethylene terephthalate) isothermally crystallized at stepwise temperatures: multiple melting behavior re-investigated[END_REF] demonstrated the existence of doublet melting peaks in addition to a series of multiple minor peaks. These results were related to the presence of multiple lamellae population, a distinctive semi-crystalline polymer feature. Liu et al. [START_REF] Liu | DSC and TEM investigations on multiple melting phenomena in isotactic polystyrene[END_REF] used both morphological and calorimetric analyses to evidence such multiple melting processes: hence, the lowest endotherm was attributed to the melting of lesser quality subsidiary crystals and the highest endotherm to the major crystallites. to our research, as crystallinity increased permeability decreased, a double-fold trend which entailed a valuable consequence, namely a lesser migration of low molecular weight molecules. Small molecules are less likely to spread through crystalline areas than through amorphous ones. Indeed, crystalline areas act as barriers fending off any risk of spreading.

From TGA measurements, we obtained the sample degradation temperature from the derivative curves. Results are given in Table 2. As dose increased, we observed no single temperature variation or any degradation percentage alteration, a fact which implies that even 150 kGy irradiation had no impact whatsoever over PET temperature stability. Degradation temperature was akin to the one claimed by Kinoshita et al. [START_REF] Kinoshita | Thermal degradation of polyesters by simultaneaous TG-DTA/FT-IR analysis[END_REF]: using TGA/FTIR simultaneous analysis, authors evidenced the main gases from PET, i.e. benzoic acid and carbon dioxide.

Finally, in the first part, dedicated to the matrix, we have shown that for the dose range used here, no chemical modifications were observed, whereas thermal analysis evidenced physical modifications attributed to chains scissions.

Then, we chose to further extend our investigations on irradiation effects over non-volatile organic extractables.

2-Effect of the sterilization on PET's non-volatile extractablesglobal analysis.

First, we aimed at achieving a global analysis of extractables in PET films by means of weight measurements. Extractables emerged following irradiation and were chain scission-related, as stated earlier. Hence, we resorted to SEC in order to determine extractable molecular weights. RP-HPLC was also used on account of its enhanced separating efficiency.

1-Weight measurements

PET is known to be radiation-resistant. Regarding mechanical features, authors showed that PET and (High Density Polyethylene /Polyamide) HDPE/PA were the most radiation-resistant and displayed moderate concentration of leachables.

Goulas et al. [START_REF] Goulas | Effect of ionizing radiation on physicochemical and mechanical properties of commercial monolayer and multilayer semirigid plastics packaging materials[END_REF] have studied the overall migration of several gamma-irradiated packaging materials in aqueous simulants and isooctane by weighing the samples.

We carried out three weight measures. As a result, the trend evidenced a significant weight decrease ranging between 0 and 25 kGy (i.e. from 0.040 ± 0.006 g down to 0.018 ± 0.010 g). The loss of volatile compounds involving low molecular weight molecules may indeed account for such a fact. Villain et al. [START_REF] Villain | Thermal degradation of poly(ethylene terephthalate) and the estimation of volatile degradation products[END_REF] reported aldehyde formation following PET thermal degradation. Komolprasert et al. [START_REF] Komolprasert | Volatile and non volatile compounds in irradiated semi-rigid crystalline poly (ethylene terephtalate) polymers[END_REF] argued that a 25 kGy gamma irradiation on PET triggered a significant volatile compound increase, as opposed to non-volatile compounds. 50 and 150 kGy doses used in our investigations induced weight increase up to 0.056 ± 0.003 g. Chain scission-related oligomers formation may indeed account for such an upward trend.

2-SEC analysis

After weight analysis, we turned to SEC analysis. Although not specific, SEC analysis stands as a semi-separative technique suited to releasing further extractable-related information. Figure 3 shows the chromatograms obtained with the RI and UV detectors set at 240 nm which is PET's most specific wavelength, and at 280 nm specific of phenolic groupments. Several peaks are presented in Figure 3. We determined molecular masses (Mp) based on calibration. Table 3 displays the masses of the different peaks. Peak n°1 was evidenced by refractometry only, and corresponds to high weight extractables with a 34390 g.mol -1 approximately Mp. As regards retention times exceeding 15 minutes, several peaks exhibit molecular weight variations within the 1275 to 221 g.mol -1 Mp range. Regardless of the detector used, the top ranking peak corresponds to peak number 3 with a 657 g.mol -1 Mp (Figure 3). Chromatographic profiles are similar for both wavelengths even though at 240 nm peaks are far more distinctive.

Considering that the monomer molecular weight is of 192 g.mol -1 , we can propose that oligomers observed have a polymerization degree varying from 2 to 6 approx.

(i.e. peaks 5 to 2, respectively). Shiono et al. [START_REF] Shiono | Determination of poly(ethylene terephthalate) oligomers in refrigeration oils by adsorption column chromatography-gel permeation chromatography[END_REF][START_REF] Shiono | Separation and identification of poly(ethylene terephthalate) oligomers by gel permeation chromatography[END_REF] used preparative SEC and mass spectroscopy on PET films to demonstrate the presence of oligomers, as well as cyclic dimers and trimers in particular. Molar masses reach 384 and 576 g.mol -1 , respectively. Cyclic trimer is predominant because of ring tension [START_REF] Nasser | Identification of oligomers in polyethyleneterephthalate bottles for mineral water and fruit juice: Development and validation of a high-performance liquid chromatographic method for the determination of first series cyclic trimer[END_REF][START_REF] Besnoin | Identification and characterization of reaction byproducts in the polymerization of polyethylene terephtalate[END_REF][START_REF] Harrison | Analysis of cyclic oligomers of poly(ethylene terephthalate) by liquid chromatography/mass spectrometry[END_REF]. Insofar as mass spectroscopy is more acute, weight determination is therefore much more accurate. We may hence consider that the top ranking peak in our study (peak number 3) corresponds to the cyclic trimer, that has the highest concentration, and that the accuracy of the calculation method accounts for the molecular mass difference (i.e. Mp= 576 g.mol -1 instead of 657 g.mol -1 ).

Ultimately, SEC used on non-irradiated PET enabled us to evidence the presence of oligomers obtained after extraction. Indeed, the medical grade PET used in our research remains as such, additive-free, according to the European Pharmacopoeia's specifications.

To extend further probing of extractables, we performed RP-HPLC analyses. The latter prove far more acute than SEC. Hence, they stand as a relevant preparatory task towards proper mass spectrometry coupling.

3-HPLC analysis

To date, numerous researchers have granted special attention to PET oligomers on the following grounds: highly radiation-resistant polymers such as PET, yield measurable amounts of low molecular weight products. Cyclic n-mers ( n=1 to 8) have thus been identified. Among them, the cyclic trimer has been referenced as the most concentrated form. Different methods have been developed and catered to effective PET oligomer identification. As a case in point: Shiono et al. [START_REF] Shiono | Determination of poly(ethylene terephthalate) oligomers in refrigeration oils by adsorption column chromatography-gel permeation chromatography[END_REF][START_REF] Shiono | Separation and identification of poly(ethylene terephthalate) oligomers by gel permeation chromatography[END_REF] as well as Nasser et al. [START_REF] Nasser | Identification of oligomers in polyethyleneterephthalate bottles for mineral water and fruit juice: Development and validation of a high-performance liquid chromatographic method for the determination of first series cyclic trimer[END_REF] carried out preparative SEC. They singled out oligomers from dimer to pentamer (by 1 H and 13 C NMR). Küppers et al. [START_REF] Küppers | The use of temperature variation in supercritical fluid extraction of polymers for the selective extraction of low molecular weight components from poly(ethylene terephthalate)[END_REF] implemented the Supercritical Fluid Extraction (SFE) technique towards selective oligomers extraction in PET. Bartle et al. [53] demonstrated that cyclic trimer molecules are not SFE accessible and that soxhlet extraction allows for enhanced extraction yields. Others [START_REF] Costley | Extraction of oligomers from Poly(ethylene terephthalate) by microwave-assisted extraction[END_REF] studied oligomer extracts through Microwave Assisted Extraction given its distinctive time-efficient and solvent saving features.

Mostly cyclic trimers and heptamers were thus extracted. HPLC coupled with UV has also been used [START_REF] Begley | High-performance liquid chromatographic determination of migrating poly(ethylene terephthalate) oligomers in corn oil[END_REF][START_REF] López-Cervantes | Migration of Ethylene Terephthalate Oligomers from Roasting Bags into Olive Oil[END_REF]. Lately, mass spectrometry involving thermospray [START_REF] Milon | Identification of poly(ethylene terephtalate) cyclic oligomers by liquid chromatography-mass spectrometry[END_REF] and Positive Ion Atmospheric Pressure Chemical Ionization has been implemented to study PET oligomers [START_REF] Barnes | Qualitative liquid chromatographic-atmospheric-pressure chemical-ionization mass spectrometric analysis of polyethylene terephthalate oligomers[END_REF][START_REF] De | Thermal stability of polyethylene terephthalate (PET): oligomer distribution and formation of volatiles[END_REF]. Such soft ionization, coupled with an HPLC/MS method enables the identification of oligomers from n=3 to 8.

It is noteworthy however, that few researchers have issued to date studies dedicated to low molecular weight compounds in irradiated PET. As a rare example, Buchalla et al. [START_REF] Buchalla | Characterization of gamma-irradiated polyethylene terephthalate by liquid-chromatography-mass-spectrometry (LC-MS) with atmospheric-pressure chemical ionization (APCI)[END_REF] evidenced non-volatile PET radiolysis products such as terephthalic acid methyl and ethylesters such as benzoïc acid. They showed that the concentration of terephthalic acid ethylester increased by a factor 2 after a 50 kGy gamma-irradiation (to ca. 2mg/kg) with a limit of detection of their method of 10 µg/L. Moreover, pure ethylene terephthalate oligomers are neither commercially available nor easy to prepare. In the framework of our investigations, we focused therefore on terephthalic and benzoic acids exhibiting close molecular structures (Figure 4): indeed, they stem from PET synthesis and degradation respectively. Figures 5a and 5b display the chromatographic profiles of both the acid standard solutions and the extraction solutions at the 240 nm wavelength (wavelength chosen from the maximum absorbance at 240 nm for the major peaks). Regarding the chromatographic profiles of PET extraction solutions, the overall profile remains complex and exhibits several major peaks. From the chromatographic system characteristics, firstly held compounds disclose polar properties and/or small molecular masses. As retention time increases, more apolar or larger compounds are separated. Whenever retention times exceed 25 minutes, the major peaks appear with regular 7-8 minutes approximative time intervals [START_REF]Directorate for the Quality of Medicines and HealthCare of the Council of Europe[END_REF][START_REF] Steckenreiter | Chemical modifications of PET induced by swift heavy ions[END_REF][START_REF] Komolprasert | Volatile and non volatile compounds in irradiated semi-rigid crystalline poly (ethylene terephtalate) polymers[END_REF] and 48 min), while their dose-dependant intensities increase markedly. Such peaks result most likely from the presence of oligomers with increasing polymerization order as their 3D chromatograms display the same UV spectrum.

Retention times under 25 minutes yield numerous peaks that correspond to the most polar compounds. We can observe in Figures 5a and5b that very intense peaks stand at about 4.5 minutes and 9 minutes. Those quickly eluted peaks certainly correspond to dimer and trimer oligomers observed earlier by SEC.

Although low retention times generate numerous peaks, only terephthalic and benzoic acids have been identified at 5.5 and 7.3 minutes retention times, respectively. We performed a calibration with a linear regression on both acids (n=3). Quantification results are presented in Figure 6. Acid concentrations were calculated on the basis of calibration equation parameters. Concentrations were further rationalized to the mass of PET extracted and to the volume of methanol used to dissolve extraction products. Since extraction was performed in methanol, we hypothesized therefore that trans-esterification could possibly affect acids [START_REF] Begley | High-performance liquid chromatographic determination of migrating poly(ethylene terephthalate) oligomers in corn oil[END_REF]. Hence, we chose to test such an assumption as follows: we let them boil separately in methanol for two hours; subsequent analysis revealed no difference whatsoever in HPLC with reference to native standards. To the extent that terephthalic acid is a synthesis residue, concentration increase stems therefore from the fact that extractables are easily forced out of the bulk whenever macromolecular chains are degraded. Once should notice that regulation n°10/2011 [START_REF] Commission Regulation | No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food[END_REF], devoted to plastic materials and articles intended to come into contact with food, specifies a specific migration limit of 7.5 ppm for terephthalic acid.

CONCLUSION

Our investigations performed on electron beam irradiated PET films, demonstrate that chain scissions can be evidenced. Thermal analyses reveal that the latter reduce crystallite size without altering either global crystallinity yield, or stability temperature. Findings are congruent with the study performed on PET extractables which exhibit a dose-dependant extraction yield increase. Indeed, as chain scissions occur, extractables prove more numerous; moreover, they are likely to migrate swiftly towards the polymer matrix and may be easily extracted.

Further, we substantiated the presence of terephthalic and benzoic acids. Even though concentration increase proves dose-dependant; the latter remain however far below official pharmaceutical recommendations.

Within the framework of our experiments, we evidenced two kinds of organic non-volatile extractables: low molecular weight extractables and oligomers, both obtained under low irradiation doses. Concentrations increase, while mobility becomes easier as crystallite size tends to decrease. SEC analysis singled out the trimer as the predominant oligomer. HPLC further substantiated the presence of oligomers. Any acute identification of extractable composition will however require in the future, extended probing through mass spectroscopy coupled with HPLC.

With respect to extractables, leachables do also require further scrutiny. Hence, ageing of materials under accelerated conditions needs to be experimented and mathematical migration models developed accordingly. Ultimately, such results provide a valuable contribution towards pharmaceutical and biological applications: indeed, low molecular weight compounds can migrate out of the polymer bulk and thus interact with the contact medium and we can wonder if they are not able to induce potential toxicity-related risks.

ACKNOWLEGMENTS

Authors wish to thank J. B. Hamel from Becton Dickinson, for his valuable help in developing this device.

Conflicts of interest: none

Figure 6

 6 Figure 6 presents data pertaining to terephthalic acid concentrations: a slight

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: FTIR in transmission mode of PET irradiated at different doses (0, 25, 50 and 150 kGy). / Spectres IRTF en mode transmission du PET irradié à différentes doses (0, 25, 50 et 150 kGy).

Figure 6 :

 6 Figure 6 : Terephthalic and benzoic acids mean concentrations (n=3) in PET irradiated with different doses. / Concentrations moyennes en acides téréphtalique et benzoïque (n=3) dans le PET irradié à différentes doses.

Table 1 :

 1 Crystallinity yields, χc, as a function of the dose, considering both endotherms in the temperature ranges of (140-259 °C) and (194-259 °C). / Taux de cristallinité, χc, en fonction de la dose, selon les deux endothermes entre (140-259 °C) et (194-259 °C).

	Dose (kGy)	Mean degradation temperature (°C) (n=3)	Mean degradation percentage (%) (n=3)
	0	446 ± 1	87 ± 6
	150	447 ± 2	87 ± 4

Table 2 :

 2 Temperatures and percentages of degradation as a function of the dose. / Températures et pourcentages de dégradation en fonction de la dose.

	Peak number	1		2		3		4		5
	Retention volume	12.0		14.9		15.7		16.2		16.6
	(min)
		Mean (n=3)	CV	Mean (n=3)	CV	Mean (n=3)	CV	Mean (n=3)	CV	Mean (n=3)	CV
	Mn (g/mol)	31401 3016 1230 148 702	8	434	4	307	15
	Mw (g/

mol) 38380 9335 1271 142 715 18 441 6 309 15 Mz (g/mol) 47896 19309 1320 134 730 30 448 9 311 15 Mp (g/mol) 34390 1440 1275 234 657 35 443 1 341 27

Table 3 :

 3 Molecular masses of extractables observed in non-irradiated PET. / Masses molaires des extractibles observés dans le PET non irradié.

Jeon et al. [START_REF] Jeon | The effects of irradiation on physicochemical characteristics of PET packaging film[END_REF] proposed, from Nuclear Magnetic Resonance (NMR) results on a 10 kGy gamma-irradiated PET, a mechanism of formation of diethylene glycol segments whereas Yoo et al. [START_REF] Yoo | Double melting behavior of cyclic tris(ethylene terephthalate)[END_REF] worked on the cyclic tris (ethylene terephthalate) (CTET) , a widely spread PET oligomer. Authors showed that the 316 °C melting endotherm, a distinctive CTET feature, would evolve to a double melting behavior (252 and 313°C) depending on heat treatment. Insofar as melting temperatures were substantially different from the endotherm observed in our research (i.e.176 °C), we attributed it to the emergence of a new lamellar population due to irradiation-induced polymer chain scissions. The formation of small molecules did not seem to influence the Tg, presumably owing to their scarcity. The high-temperature melting peak was construed as resulting from the melting of thick lamellae generated during the industrial film process, whereas the low-temperature melting peak was attributed to thin lamellae formed during irradiation.

Crystallinity yields were calculated from the ratio of the melting enthalpy (∆H) measured by the total area under the melting peak to the melting enthalpy of a fully crystalline PET, i.e. ∆H 0 =138 J.g -1 [START_REF] Wunderlich | Macromolecular physics. Crystal nucleation, growth, annealing[END_REF]. Integrations were conducted with a linear function. Results are displayed in Table 1.

Considering the crystallinity yield calculated from both endotherms (between 140 up to 259 °C), a slight increase was observed over 50 kGy, due to secondary lamellae formation. Congruent with our earlier statements, as irradiation dose increased chain scissions developed, fostering thereby secondary lamellae formation. Liu et al. [START_REF] Liu | Chemical modifications in polyethylene terephthalate films induced by 35 MeV/u Ar ions[END_REF] have reported crystallinity stability up to 4.0 MGy absorbed doses delivered by swift heavy ions. On the contrary, Zhu et al. [START_REF] Zhu | Modification of polyethylene terephthalate under high-energy heavy ion irradiation[END_REF] showed by both FTIR and X-rays Diffraction (XRD) that high energy heavy ions could damage the benzyl ring of PET and induce a loss of crystallinity. According