C Mnekbi

M Vincent

J F Agassant

POLYMER RHEOLOGY AT HIGH SHEAR RATE FOR MICROINJECTION MOULDING

Keywords: Microinjection moulding, high shear rate, HDPE, numerical simulation, Rem3D

In this paper, we present the rheology of a high-density polyethylene for microinjection moulding. At high flow rate, pressure becomes high enough so that shear heating and pressure-dependence of the viscosity cannot be neglected. Moreover, spurt and chaotic defects related to wall slip were encountered. Nevertheless, we used classical data processing. We obtained viscosity at high shear rate which is lower than the viscosity obtained with the Carreau-Yasuda extrapolation of the moderate shear rate data. In order to discuss the origin of this difference, we developed a flow numerical simulation in the capillary where we take into account pressure and temperature dependent viscosity. The relative importance of these phenomena and their coupling is presented. Then, injection moulding experiments were carried out in a plaque mould. Different moulding parameters were tested. A three dimensional injection moulding numerical simulation, using Rem3D software, was carried out. The quality of the different sets of rheological data is assessed by comparing computed and measured pressure.

INTRODUCTION

In microinjection moulding, high velocity combined to small flow gaps lead to different flow behaviour compared to classical injection moulding [START_REF] Whiteside | Micromolding: extreme process monitoring and inline product assessment[END_REF]. One of the questions is the viscosity data at high shear rate [START_REF] Takahashi | Rheological behavior of SAN PC blends under extremely high shear rate[END_REF][START_REF] Cardinaels | Evaluation and comparison of routes to obtain pressure coefficients from high-pressure capillary rheometry data[END_REF], as numerical simulation of mould filling requires precise rheological data. The aim of this work is to identify what physics is involved in such conditions, to obtain viscous rheological data, and to test them by comparing the results of numerical simulation to measurements in an instrumented mould.

HDPE RHEOLOGY

RHEOLOGY EXPERIMENTS

Material

A commercial semi-crystalline thermoplastic, HDPE (high-density polyethylene) with the grade ELTEX HD 6070, is supplied by Ineos. As the HDPE was not stabilised by the supplier, we added 0.1% of a stabilizer (IRGANOX).

Experimental protocol

A rotational rheometer (ARES) is used in dynamic oscillatory mode to determine polymer linear viscoelastic properties at imposed strain. Measurements are performed in a frequency sweep mode (0.1 to 100 rad/s), at an imposed strain of 10 %, for temperatures between 140 and 190 °C. The viscosity, the elastic modulus G' and the viscous modulus G" are determined.

Then according to the time-temperature superposition principle, the experimental results are superposed on a master curve at a reference temperature, by means of a shift factor a T . A capillary rheometer, the Rheoplast, has a conical Couette plastication zone, in which the polymer is melted by shear-heating and by conduction, then relaxed to homogenize the temperature and finally extruded into a capillary. A pressure transducer is mounted immediately above the capillary to record the pressure drop. The capillary rheometer is used with six dies with diameter D equal to 1 mm and 0.5 mm, and length over diameter L/D ratio equal to 4, 8 and 16 respectively, at 190°C, for injection volume flow rates between 2 and 1018 mm 3 ⋅s -1 . The maximum recorded pressure is 60 MPa.

Flow hypothesis

We suppose a certain number of assumptions for the evaluation of capillary data: -flow is incompressible, isothermal and established; -melt sticks to the wall; -viscosity is not pressure dependent. These assumptions permit to use Bagley and Rabinowitsch corrections.

Experimental results

First, we selected the large diameter 1mm. We limited apparent shear rate value to a maximum of 10 4 s -1 . We did not observe extrusion defects. In those conditions the assumptions set out above are true and classical Bagley and Rabinowitsch corrections can be used [START_REF] Agassant | La mise en forme des matières plastiques[END_REF]. Time-temperature superposition was done according to a shift factor to a reference temperature of 190°C. The viscosity versus the frequency given by the ARES and the viscosity versus the shear rate given by the Rheoplast are shown in Figure 1. We can see that the Cox-Merz transformation is satisfied. In a second step, we imposed high injection speed in the capillary rheometer using the 0.5 mm die diameter. We reach high shear rates values up to 10 5 s -1 . Around a shear rate value of 10 4 s -1 , we observe polymer spurt, then chaotic defects. However, the evolution of measured pressure for a constant shear rate still gives a plateau, which permits the determination of a flow curve. HDPE measurements for high shear rate leads to high pressure values up to 60 MPa.

At such a pressure level, dissipative heating and pressure dependent viscosity are no longer negligible [START_REF] Couch | High pressure capillary rheometry of polymeric fluids[END_REF].

Dissipative heating decreases the viscosity, whereas the rise of pressure enhances the viscosity. Both of these dependencies are estimated afterwards in this paper. We also choose to neglect the extrusion defects for data processing. We employ Bagley and Rabinowitsch corrections to draw viscosity behaviour. An extended viscosity curve given by the capillary rheometry is superimposed on the results of Figure 1.

The shear rate reaches 10 5 s -1 (Figure 2). The viscosity at high shear rate is lower than the viscosity with the Carreau-Yasuda extrapolation of the moderate shear rate data.

CAPILLARY FLOW SIMULATION WITH TEMPERATURE AND PRESSURE DEPENDENCE OF VISCOSITY

Theoretical issues

At high shear rate, the dissipative heating and pressure dependence of viscosity have to be taken into account [START_REF] Laun | Capillary rheometry for polymer melts revisited[END_REF]. A capillary flow simulation take into account the dissipative and pressure dependent viscosity effect on the pressure. The viscosity function is expressed by a Carreau-Yasuda law (equation 1).

( ) [ ] ) 1 ( 0 1 ) ( a m a - + = γ λ η γ η 1 1 (1)
1 0 is zero shear rate viscosity (at T 0 reference temperature and P 0 atmospheric pressure), 2 is a characteristic time, m is the power law exponent and a transition width parameter. The temperature shift factor follows Arrhenius law, (equation 2), as we work with a semi-crystalline thermoplastic:

1 2 3 4 5 6 7 7 8 9 A A B C - = 0 1 1 exp T T R Ea a T (2)
Ea is the activation energy. Shear rate and temperature dependent viscosity is calculated according to equation 3.

) , ( ) , ( 0 T a a T T T γ η γ η 1 1 = (3)
Shear rate and pressure dependent viscosity (equation 4)

) ). (exp( ). exp( ) , ( γ χ η χ γ η 1 1 P P P = (4)
We can combine both the pressure and temperature dependency of viscosity (equation 5). ) , .

). exp( (. ). exp( ) , , ( 0

T a P a P P T T T γ χ η χ γ η 1 1 = (5)
We assume that the velocity vector is parallel to the tube axis, and the component parallel of the axis z, w, is only a function of the radius r. We assume a generalized Newtonian behaviour law. Neglecting gravity and inertia, the force balance equation leads to:

dz dp dr r r w r R 1 2 3 4 5 6 = D η 2 ) ( (6)
The integration of the flow velocity leads to volume flow rate Q:

D D D 7 7 8 9 A A B C = = R r R R rdr rdr dz dp rdr r w Q 0 0 2 2 ) ( 2 η π π (7)
We use a slab method to define a bi-dimensional meshing of the capillary. The pressure variation in slab n of length ∆z is given by:

D D 7 7 8 9 A A B C ∆ = ∆ - - - R r R n n n n rdr P T rdr Q z P 0 1 1 1 , , ( 2 2 γ η π 1 (8)
To determine the temperature regime in the capillary, we calculate the Cameron number for the dissipative heating characterisation. In our case, Ca 1 10 -2 , the temperature regime is adiabatic. Dissipative heating for an adiabatic temperature regime in a slab is expressed by (9).

c P T n n . ρ ∆ = ∆ ( 9
)
Where 3 is the density and c is the specific heat capacity. The simulation is run with a power law initialisation in the first slab. Then, we calculate 2P i and 2T i in each slab i successively. At the end of calculi, we deduce the P i values.

Modelling results

We simulate the HDPE flow into a die with a ratio L/D of 8 at 200°C. The geometry of the slab method simulation is given by Figure 3. The material parameters are listed in Table 1. . Figure 4 shows the four curves of pressure profile for an imposed shear rate of 10 5 s -1 . Activation energy As predicted with experimental observations, the viscosity pressure dependency increases the pressure value in the die, while the dissipative heating decreases this pressure value. But when we combine both the pressure and temperature dependency of viscosity, we return to a pressure value at the die inlet which is the same as the pressure value when both the dependency parameters are not taken into account. As a first approximation of the modelling results, we can suppose that the experimental results at high shear rate are still valid.

INJECTION MOULDING

INJECTION MOULDING EXPERIMENTS

Injection moulding experiments were carried out in a plaque mould 0.5 mm thick, 25 mm long and 25 mm wide, equiped with three pressure sensors P 1 in the channel, P 2 near the entrance of the plaque, and P 3 near the end. A temperature sensor T m was placed in the upper part of the mould. A light-absorption sensor was used to detect the polymer crystallization during the process. P2 and P3 transducers also gave temperatures T2 and T3 (Figure 5). The screw displacement was also recorded. The mould was mounted on a pneumatic Getelec injection moulding machine. Different moulding parameters were tested. Figure 6 shows the pressures and temperatures recorded during an injection moulding test, with a pneumatic injection pressure of 0.5 MPa. The melt temperature is 240°C and the mould temperature is 75°C. The average shear rate reached 5.10 4 s -1 . We can see that the displacement of the screw vs. time is not linear. Therefore, the entrance flow rate is not constant. At the end of mould filling, at 0.29 s, we observe a pressure increase due to the material compressibility.

INJECTION MOULDING SIMULATION

A three dimensional injection moulding numerical simulation, using Rem3D software, was carried out. This simulation was based on finite element equation solvers (a flow Stokes solver, a flow front transport equation solver and a heat equation solver). The entire cavity to be filled was meshed with tetrahedral elements using a fully automatic 3D mesh generator. The finite element method was used to solve the thermo-mechanical equilibrium equations at each time step of the process. We used a thermo dependent Carreau-Yassuda law taking into account the material compressibility. The filled part is shown in Figure 6. The quality of the different sets of rheological data is assessed by comparing computed and measured pressure Figure 7. The starts of each pressure P1, P2 and P3 are well reproduced by the computation. The filling end coincides in both cases. Nevertheless, the computed pressure values P1 and P2 are more important than the experimental ones. This can be due to the incertitude of the introduced flow rates in the simulation. Another hypothesis for this difference is the material data. We consider a rheolgy neglecting the wall sleep related to the extrusion defects and observed in the flow curves. A wall sleep causes a pressure decrease in the measured case.

CONCLUSIONS

We presented the rheology of a high-density polyethylene. When we used a capillary rheometer with conventional diameter (1 mm) and injection speed, we obtained viscosity data up to an apparent shear rate of 10 3 s -1 . When using smaller diameter (0.5 mm) at high injection rate, the apparent shear rate reached 10 5 s -1 . Pressure became high enough so that shear heating and pressure-dependence of the viscosity could not be neglected. Moreover, spurt and chaotic defects related to wall slip were encountered. Nevertheless, we again used classical data processing. We obtained viscosity at high shear rate which is lower than the viscosity obtained with the Carreau-Yasuda extrapolation of the moderate shear rate data. In order to discuss the origin of this difference, we developed a flow numerical simulation in the capillary where we took into account pressure and temperature dependent viscosity. We deduced that at shear rate until 10 5 s -1 , the decrease of the pressure due to the heat dissipation and its growth due to the piezodependency cancelled out mutually. In other words, the experimental results at high shear rate should still be valid.

We exploited the experimental rheological data and we tested them by comparing the results of injection moulding numerical simulation to measurements in an instrumented mould. In the simulation, we overestimated the measured pressure.

Figure 2 :

 2 Figure 2: Rotational and high shear rate capillary rheometries results superimposed for HDPE AT 190°C

Figure 3 :

 3 Figure 3: Slab method simulation geometry

Figure 4 :

 4 Figure 4: Capillary flow simulation for HDPE at 190°C, shear rate 10 5 s -1

Figure 5 :

 5 Figure 5: Mould. Only half is represented

Figure 6 :

 6 Figure 6: HDPE injection moulding test acquisition

Figure 7 :

 7 Figure 7: Measured and computed pressures comparisons

Table 1 :

 1 HDPE parameters at 190°C

	Zero shear viscosity	0 [Pa.s]	1200
	Zero characteristic time	2 [s]	0.01
	Transition width parameter	a	1
	Power law exponent	m	0.27
	Piezodependency coefficient	[Pa -1 ]	2.10 -8
	Heat capacity	3.c	2.10 -6
		[J/°Cm 3 ]

ACKNOWLEDGEMENT

The authors gratefully acknowledge financial support from the French Research National Agency ANR for Microconnect project. The injection moulding tests were carried out thanks to G. Regnier and N. Bou Malhab from LIM of Ensam Paris. http://www.microinjectionmolding.eu/


Areski Cousin
email: areski.cousin@univ-lyon1.fr

Diana Dorobantu
email: diana.dorobantu@adm.univ-lyon1.fr

Didier Rullière
email: didier.rulliere@univ-lyon1.fr.

A note on the computation of an actuarial Waring formula in the finite-exchangeable case *

We present in this paper the actuarial Waring formula, which is used in several fields, like life-insurance or credit risk. In a particular framework where considered random variables are exchangeable, we show that some problems can occur when using this formula. We propose alternative recursions in order to improve the complexity of the calculations, and to cope with the numerical instability of the formula.

Introduction

The considered actuarial Waring formula

Let n ∈ N and Ω = {1, . . . , n}. Consider a sequence of Bernoulli random variables {X i } i∈Ω , X i ∈ {0, 1}, i ∈ Ω, non necessarily an iid sequence. The actuarial Waring formula gives the law of X 1 + • • • + X m as a linear combination of some coefficients µ k , which are sums of probabilities of kind P [X i 1 = 1 ∩ • • • ∩ X i k = 1], i 1 , . . . , i k ∈ Ω. These coefficients are quite easy to express in several cases, such as the independent case (not necessarily identically distributed), or such as the exchangeable case.

The Waring formula is based on the inclusion-exclusion principle (see Feller, 1968, chapter IV.3, page 106). The reference of this formula as "Waring formula" is given in some actuarial writing, like in [START_REF] Gerber | Life Insurance Mathematics[END_REF]), ch.8.6, in Tupper (1981) or in some probability books, like in [START_REF] Bizley | Probability, an intermediate text book[END_REF], p74. A reference of this formula as "Waring's Theorem" is also given in [START_REF] Macdonald | Waring's Theorem[END_REF].

This formula is known in the life-insurance field under the name of "Zmethod"(cf. [START_REF] Scott | The Z-method for assurances[END_REF]Neill, 1977, section 7.4). It appears in some early references, like in a book written by George King and published in 1902 by the U.K. Institute of Actuaries (see [START_REF] King | Life contingencies, Text book of the principle of interest, life annuities and assurances, and their practical application[END_REF]. We found an earlier mention of this formula in [START_REF] Baily | The doctrine of life-annuities and assurances, analytically investigated and explained[END_REF], p.48, §65, which refers to the original writing of Waring (1792) at the end of the 18th century. In order to distinguish this formula with other famous formulas from Edward Waring, we will call it the "actuarial" Waring formula. This actuarial Waring formula corresponds to a particular case of the more recent Schuette-Nesbitt formula (see Gerber, 1995, ch 8.6, p.89).

First, let us define first some coefficients that will be useful to write the Waring formula. Let m ∈ N, m n. We introduce the coefficients of order k, k m, for the set

{X i , i ∈ Γ m } where Γ m ⊂ Ω is such as card(Γ m ) = m: Definition 1.1 (µ k coefficients) Let Γ m ⊂ Ω, where m = card(Γ m ). The coefficient of order k (k ≤ m) for the set {X i , i ∈ Γ m }, denoted µ k (Γ m ), is defined as µ k (Γ m ) = 1 C k m j 1 <j 2 <..<j k j 1 ,...,j k ∈Γm P [X j 1 = 1 ∩ ... ∩ X j k = 1], 1 ≤ k ≤ m, µ 0 (Γ m ) = 1 (including if Γ m = ∅).
. C k n = n! k!(n-k)! denotes the binomial coefficient, 0 ≤ k ≤ n, n ∈ N.
Theorem 1.1 (The actuarial Waring formula) Let X 1 , ..., X n be n dependent Bernoulli random variables and let Γ m be a subset of

Ω such that card(Γ m ) = m ≤ n. Then W µ (k, Γ m ) = P   i∈Γm X i = k   = 1 k≤m C k m m-k j=0 C j m-k (-1) j µ j+k (Γ m ).
Proof: A proof can be found in Feller (1968) (chapter IV.3, page 106). This formula can also be seen as a particular case of the Schuette-Nesbitt Formula often used in actuarial science (see White and Greville, 1959;[START_REF] Schuette | Discussion of the preceding paper by Robert P. White and T[END_REF][START_REF] Gerber | A Proof of the Schuette-Nesbitt Formula for Dependent Events[END_REF][START_REF] Buchta | An Elementary Proof of the Schuette-Nesbitt Formula[END_REF]Gerber, 1995, chapter 8.6, page 89 for some proofs). 2

This actuarial Waring formula was originally used in the life-insurance field, where random lifetimes and death indicators are often considered to be independent but not identically distributed (due to the difference of ages among considered people): in these circumstances, coefficients µ k are easy to compute, and the law of a number of death among a group of independent but heterogeneous people is easy to obtain. This formula is also useful when we consider finite-exchangeable random variables, even without an underlying random factor. We previously used it in order to compute a number of defaults into a credit-risk model (see [START_REF] Cousin | An extension of Davis and Lo's contagion model[END_REF].

Remark 1.1 (Exchangeable case) If the random variables {X i , i ∈ Ω} are exchangeable, then for all Γ m ⊂ Ω we have

µ k (Γ m ) = µ k = P [X 1 = 1 ∩ ... ∩ X k = 1], 1 ≤ k ≤ m.
Remark 1.2 (Independent case, not i.d.) If the random variables {X i , i ∈ Ω} are independent, but not identically distributed, then for all Γ m ⊂ Ω we have

µ k (Γ m ) = 1 C k m j 1 <j 2 <..<j k j 1 ,...,j k ∈Γm P [X j 1 = 1] . . . P [X j k = 1], 1 ≤ k ≤ m,
Remark 1.3 (Infinite-exchangeable case) If the random variables {X i , i ∈ Ω} are infinite exchangeable, then due to De Finetti's Theorem, there exists an underlying random factor Θ such that {X i , i ∈ Ω} are independent given the value of a common probability Θ (see [START_REF] De Finetti | Funzione caratteristica di un fenomeno aleatorio[END_REF]. In this case,

P   i∈Γm X i t = k   = C k m E[Θ k (1 -Θ) m-k ],
and Waring formula correspond to the power development of this expression, with µ k = E Θ k . In this last case, these probabilities may also be computed by numerical integration, given the distribution of the underlying factor Θ.

Nevertheless, when Ω is finite, stating that the sequence X 1 , X 2 , . . . is finiteexchangeable does not involve the existence of an underlying common factor Θ, since De Finetti's theorem does not apply in this case.

Remark 1.4 (Analytic solutions)

In some cases, W µ (k, Γ m ) has an analytical simple expression. As an example, with a beta-distributed underlying factor Θ, W µ (k, Γ m ) can be expressed easily using Euler Gamma function (see [START_REF] Cousin | Valuation of portfolio loss derivatives in an infectious model, book chapter[END_REF]

The problem

We consider here the exploitation of the Waring formula in the finite-exchangeable case. We used such an assumption in a previous work modeling dependencies and infections in a credit-risk model (see [START_REF] Cousin | An extension of Davis and Lo's contagion model[END_REF].

Suppose that {X i }, i ∈ Ω are exchangeable random variables, so that for all Γ m ⊂ Ω, µ k (Γ m ) = µ k . In this case, all W µ (k, Γ m ) are equals for any subset of cardinal m, so that we will simply write

W µ (k, m) instead of W µ (k, Γ m ).
In this case,

W µ (k, m) = P [X 1 + • • • + X m = k] = 1 k≤m C k m m-k j=0 C j m-k (-1) j µ j+k ,
where

µ k = P [X 1 = 1 ∩ ... ∩ X k = 1], 1 ≤ k ≤ m, µ 0 = 1 .
Waring formula is useful when

• Coefficients {µ k } k∈Ω are given.
• We want to compute the law of m i=1 X i , for each m ∈ Ω. Suppose that coefficients µ k are given, and that we want to compute all

W µ (k, m), k, m ∈ Ω. W µ (k, m) = 1 k≤m C k m m-k j=0 C j m-k (-1) j µ j+k .
One can then make two remarks:

• First, the loops number for a given couple (k, m) ∈ Ω 2 , k m, is of order m -k, so that computing this formula for any couple (k, m) involves approximatively n 3 /6 loops, which may be important when n is large.

• Second, binomial coefficients C k m and C j m-k may become extremely large when n is large, so that numerically, we are then adding some huge quantities to get a final probability belonging to [0, 1]. Even when writing binomial coefficients as exponentials of log-binomial coefficients, due to computer arithmetic, we can not ensure that numerically (exp(γ) + 1) -(exp(γ)) equals one for large γ. We will investigate more precisely these issues in section 3.2.

In the following, we will try to propose some solutions for these two problems, by using some recursions.

Proposed recursions in the exchangeable case

Basic recursion

Theorem 2.1 (First recursion) Coefficients W µ (k, m), can be computed recursively, for m varying from 1 to n, for k varying from m -1 to 0, using:

W µ (m, m) = µ m , W µ (k, m) = m m -k W µ (k, m -1) - k + 1 m -k W µ (k + 1, m) .
with the value when m = 0, W µ (0, 0) = 1.

Proof: Consider k, m ∈ N, m 1, m -k 2. First write W µ (k, m)/C k m = m-k j=0 C j m-k (-1) j µ j+k = µ k + (-1) m-k µ m + m-k-1 j=1 C j m-k (-1) j µ j+k Using C j m-k = C j m-k-1 + C j-1 m-k-1 , W µ (k, m)/C k m = m-k-1 j=0 C j m-k-1 (-1) j µ j+k + m-k j=1 C j-1 m-k-1 (-1) j µ j+k = W µ (k, m)/C k m-1 + (-1) m-k-1 i=0 C i m-k-1 (-1) i µ i+1+k = W µ (k, m)/C k m-1 -W µ (k + 1, m)/C k+1 m .
and checking that the result is also true for m -k = 1, the recursion holds. 2

Remark 2.1 (Symmetrical recursion) By symmetry, working on X = 1 -X and applying the same recursion, we also get for k ∈ {1, . . . , n}:

W µ (k, n) = n k W µ (k -1, n -1) - n -k + 1 k W µ (k -1, n) , Remark 2.2 (Local numerical problem) Suppose µ n = p n , correspond- ing to the iid case. Since for k = n, W µ (n -1, n) = n(µ n-1 -µ n ), applying this recursion with p = 1 -leads to (1 -) n-1 = (1 -) n-1 -(1 -) n
, which can cause local numerical problems when is small. These problems will be studied in Section 3.1 and 3.2.

Normalized recursions

We can remark that if the algorithm complexity has been reduced, there remain some numerical problems. We can first imagine changing the recursion, since this recursion is simplified in some case. A first investigation consists in looking for another recursion that would have a better stability.

A way to propose other recursions is to express each W µ (k, n) as a function of a given value W 0 (k, n) and some residual terms, obtained by recursion, that would have a better stability.

Proposition 2.2 (Additive normalization) Suppose we have a quantity

W 0 (k, n) such that for n 1, k n: W 0 (k, n) = n n -k W 0 (k, n -1) - k + 1 n -k W 0 (k + 1, n) ,
And suppose furthermore that this quantity is numerically easy to obtain. If we write

W (k, n) = W µ (k, n) -W 0 (k, n), then W (k, n) = n n -k W (k, n -1) - k + 1 n -k W (k + 1, n) ,
so that W (k, n) may be computed by recurrence, and then W µ (k, n) deduced from W (k, n).

As an example, following quantities may be used for additive normalization:

• W 0 (k, n) = C k n p k (1 -p) n-k (iid case) • W 0 (k, n) = P [N n = k],
where N n is a beta-mixed binomial law

In the particular iid case or beta-mixed binomial case, one can thus find W 0 such that all W (k, n) are equal to zero, so that the recursion remains stable.

Proposition 2.3 (Multiplicative normalization) Suppose we have a quantity

W 0 (k, n) such that for n 1, k n, W 0 (k, n) = 0. If we write W (k, n) = W µ (k, n)/W 0 (k, n), then W (k, n) = α n k W (k, n -1) + β n k W (k + 1, n) , with α n k = n n-k W 0 (k,n-1) W 0 (k,n) and β n k = -k+1 n-k W 0 (k+1,n) W 0 (k,n) .
so that W (k, n) may be computed by recurrence, and then

W µ (k, n) deduced from W (k, n).
We can also show that

β n k = 1 -α n k when W 0 (k, n) is such that W 0 (k, n) = n n -k W 0 (k, n -1) - k + 1 n -k W 0 (k + 1, n) .

Remark 2.3 (Normalization example)

One can think about finding one W 0 that would lead to a greater stability of the recursion. Using for example

W (k, n) = 1 1 -p W (k, n -1) - p 1 -p W (k + 1, n) , when W 0 (k, n) = C k n p k (1 -p) n-k .
In the particular iid case, all W are here equal to one, so that the recursion 1 = 1/(1 -p) -p/(1 -p) remains very stable (except for p close to 1 ). In the general case, we will see that there remain some numerical problems (see Section 3.1).

Theorem 2.4 (Simpliest proposed recursion) Setting

W (k, n) = W (k, n)/C k n , one have for n 1 and k < n: W (n, n) = µ n , W (k, n) = W (k, n -1) -W (k + 1, n) ,
So that we can build W recursively, with W (0, 0) = 1. Waring coefficients are then given by W

(k, n) = C k n W (k, n). Proof: This result is a direct consequence of Proposition 2.3 with W 0 (k, n) = C k n .
Once written, we can check that in the particular finite-exchangeable case, it is also deriving directly from the fact that

W (k, n) = x 1 +•••+xn=k P [X 1 = x 1 , . . . , X n = x n ] = C k n P [X 1 = 1, . . . , X k = 1, X k+1 = 0, . . . , X n = 0]. If W (k, n) = P [X 1 = 1, . . . , X k = 1, X k+1 = 0, . . . , X n = 0],
then by the same kind of arguments as in [START_REF] Kendall | On finite and infinite sequences of exchangeable events[END_REF],

W (k, n) = P [X 1 = 1, . . . , X k = 1, X k+1 = 0, . . . , X n-1 = 0] -P [X 1 = 1, . . . , X k = 1, X k+1 = 0, . . . , X n-1 = 0, X n = 1] ,
And using exchangeability we get:

W (k, n) = W (k, n -1) -W (k + 1, n) ,
2 Considering the complexity problem, computing all W µ (k, n) has now a complexity of order n 2 rather than n 3 , meaning the calculation of order n times faster, which may be important when n is large. Furthermore, all involved operations are very basic operations, directly available in the arithmetic coprocessor Floating Point Unit, allowing very low level programming of the recurrence.

Considering the numerical stability problem, all W are probabilities, which is likely to help controlling the local errors. Nevertheless, we will see that numerical stability is not ensured, so that we may have to use a greater arithmetic precision than the 64-bit IEEE754 standard. One advantage of such a recurrence is that it involves only basic operations for which many arbitrary precision floating point libraries exist.

The residual problem

Local errors and propagation errors

In proposed recursions, there are essentially two kind of errors involving the floating point arithmetic precision:

• Local calculation errors, for a fixed couple (k, n).

• Propagation errors, linked to the propagation (and emphasis) of previous errors.

We present here some situations where local errors or where propagation errors may be involved. These errors will not be here precisely quantified, since we will see in a further section that it is not possible to eliminate these errors due to the sensitivity of the formula on initial parameters.

Local errors

Local errors are those for which, starting with slightly modified values in the recurrence, for a given couple (k, n), one may get large errors on W µ (k, n). Consider for example (see Remark 2.3)

W (k, n) = 1 1 -p W (k, n -1) - p 1 -p W (k + 1, n) ,
due to the normalization method, the W are here a ratio of two probabilities and may be huge. We can then obtain situations where a small quantity is equal to the difference of two huge quantities, causing a large local error. Another example of local error is given in Remark 2.2.

Propagation errors Propagation errors are due to the iteration process: starting with slightly modified values in the recurrence, for a given couple (k, n), one get relatively small errors on W µ (k, n). But the error is due to the iteration process, which emphasis at each step the small error on W µ (k, n).

Consider for example

W (k, n) = n n -k W (k, n -1) - k + 1 n -k W (k + 1, n) , If W (k + 1, n) is replaced by W (k + 1, n) + , then W (k, n)
will be translated by a quantity k+1 n-k , that is for example n in the case k = n-1. If is small, n will be locally an acceptable error. Nevertheless, during the first steps the error will be multiplied at each step by a factor near n. For n = 100, an initial error of 10 -16 becomes too large after only ten iterations for example. The initial error may be smaller if µ n is very small, but in the case where µ n is too large, propagation errors may occur.

Numerical sensitivity

Let us start with the Waring formula,

W µ (k, m) = 1 k≤m C k m m-k j=0 C j m-k (-1) j µ j+k ,
and consider two set of possible initial coefficients {µ k } and {μ k }, differing slightly (due to the computer limited arithmetic precision for example). Will the quantities W µ (k, m) and W μ(k, m) be very different ? Consider the particular value

W µ (m, 2m) = C m 2m m j=0
C j m (-1) j µ j+m .

Consider for example that {µ k } and {μ k } are differing only on one value:

μk = µ k , k < 2m , μ2m = µ 2m + .
After some basic calculations, and even supposing all calculations are done with an infinite precision, we can easily show that

W μ(m, 2m) = W µ (m, 2m) + (-1) m C m 2m .
Suppose for example that = µ 2m 10 -16 , due to the numerical uncertainty on the µ 2m quantity. The absolute error ∆ = |W μ(m, 2m) -W µ (m, 2m)| is in this case: ∆ = C m 2m µ 2m 10 -16 . This error can be huge, and obviously greater than one, when m is large and µ 2m not enough close to zero. When µ 2m = p 2m (iid case), this can occur only when p is close to one, but in the general exchangeable case, this can occur when an important dependency structure among indicators X 1 , X 2 , . . . cause the higher moments µ k to be important. Since all W µ (k, m) are probabilities and should belong to [0, 1], situations where ∆ is greater than one are not acceptable. In the exchangeable case, this reveals that in some particular cases, the final law of X 1 + • • • + X m may be too highly sensitive on the input moments µ k = E Θ k of the underlying factor Θ.

Conclusion

We have investigated the calculation of the sum of indicators random variables X 1 +• • •+X m , when they were not necessarily iid, based on the Waring formula.

We have seen some basic recursions which allow to compute very simply the Waring formula in the exchangeable case, and to reduce the complexity of the formula when computing the whole law of

X 1 + • • • + X m .
Despite their simplicity we have seen that all proposed recursion were facing either local arithmetic errors, either propagation errors, and that it was not possible to reduce these errors in all cases, since the underlying formula was in some cases too sensitive on some input parameters.

As a conclusion three solutions seem to us possible:

• Checking that input parameters are not leading to unacceptable errors.

In particular, initial coefficients µ n should be small enough for large n. Since initial moments of X 1 + • • • + X m are usually easy to get, one can a posteriori check that the law of X 1 + • • • + X m leads to the correct moments, including the sum of probability equals to one.

• Using a greater computer arithmetic floating point precision, which might be easy since all operations involved in the recursions are very basic operations, for which many arbitrary precision arithmetic exists. Investigations could also be developed in order to bound each quantity in the recursion, using interval arithmetic for example.

• Changing input parameters {µ k }, and using another formula in problematic cases. As an example, in the infinite-exchangeable framework with an underlying factor Θ, according to remark 1.3, numerical integration can be used to compute W µ (k, m), without starting from {µ k } coefficients. Another example is getting coefficients ln W µ (k, m) as a function of coefficients ln µ n , which might also reduce the required floating point precision in some cases.

2

  considered actuarial Waring formula . . . . . . . . . . . . 1 1.2 The problem . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Proposed recursions in the exchangeable case 5 2.1 Basic recursion . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 Normalized recursions . . . . . . . . . . . . . . . . . . . . errors and propagation errors . . . . . . . . . . . . . . . 8 3.2 Numerical sensitivity . . . . . . . . . . . . . . . . . . . . .

  Tupper, J.L., (1981)King's Z-Method and Compound Statuses: An Algebraic Approach, Actuarial Research Clearing House, Vol. 2.Waring, E.(1792) On the principles of translating algebraic quantities into probable relations and annuities, printed by J. Archdeacon, for J. Nicholson.White, R.P., Greville, T.N.E.,(1959) On computing the probability that exactly k of n independent events will occur. Transactions of The Society of Actuaries, Vol. 11., No 29AB.

* This work has been funded by ANR Research Project ANR-08-BLAN-0314-01.


Marco Arlorio
email: arlorio@pharm.unipmn.it

Jean Daniel Coisson

Matteo Bordiga

Fabiano Travaglia

Cristiano Garino

Laurian Zuidmeer

Ronald Van Ree

Maria Gabriella Giuffrida

Amedeo Conti

Aldo Martelli

Matteo ; Daniel Bordiga

Discaff Travaglia

Fabiano Garino

Cristiano Zuidmeer

Laurian Van Ree

Ronald Giuffrida

Maria Conti

Amedeo Martelli

A Martelli

Olive oil adulterated with hazelnut oils: simulation to identify

Keywords: Chromatography -electrophoresis, Immunoassays Additives/Contaminants: Allergens Hidden allergens, olive oil, hazelnut oil, oleosins

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

Introduction

Due to its nutritional and commercial value, the consumption of olive oil has increased considerably over recent years, representing 15.2% of the total oil utilization in the European Union [START_REF] Firestone | Detection of adulterated and misbranded olive oil products[END_REF]. Production of olive oil represents about 3% of the world oils and fat markets, with 78% of global production taking place in Mediterranean countries, where oil industry is important in sustaining viability of agricultural economy. The cultivation, harvesting and extraction of olive oil is a hard and time-consuming task that adds to its relatively high commercial price. Olive oil is either classified as "virgin olive oil" (i.e. cold-pressed olive oil produced by the use of physical means without chemical treatment), as "olive oil" (i.e. a mixture of virgin and refined oil) or "refined oil" (i.e. olive oil chemically treated to neutralize strong taste and in particular acidity) (Reg. EC 1531/2001).

Oils labelled as "extra-virgin olive oil" (EVOO) must not contain any refined olive oil; oils labelled as "olive oil" must not contain oil from other oleaginous nuts or seeds. To increase profits, unscrupulous dealers may be tempted to add lower-priced vegetable or hazelnut oils to fresh extra virgin olive oils. This fraudulent practice, in addition to being unfair to the consumer in terms of the cost (Chiavaro 2008), may also cause severe health and safety problems such as the occurrence of Spanish toxic oil syndrome in 1981 [START_REF] Gelpi | The Spanish toxic oil syndrome 20 years after its onset: A multidisciplinary review of scientific knowledge[END_REF], Blanch 1998).

Hazelnut oil (HAO), obtained from Corylus avellana L. seeds, is often used in extra-virgin olive oil (EVOO) adulteration, because of the similar chemical composition with respect to both major and minor compounds [START_REF] Vichi | Detection of hazelnut oil in virgin olive oil by assessment of free sterols and triacylglycerols[END_REF]. The high degree of similarity complicates the detection of HAO in EVOO, especially at concentrations below 20% [START_REF] Parcerisa | Fatty acid, tocopherol, and sterol content of some hazelnut varieties (Corylus avellana L.) harvested in Oregon (USA)[END_REF][START_REF] Ozen | Detection of Hazelnut Oil Adulteration Using FT-IR Spectroscopy[END_REF] . The seriousness of the problem has been aggravated during the last twenty years. In 1993/94 1,829,000 tonnes of olive oil were consumed world wide. In 1995 it has been reported that quantities of Turkish hazelnut oil was illegally introduced into the European community, without proper declaration to customs and excise, in order to adulterate olive oil [START_REF] Aparicio | Development and Assessment of Methods for the Detection of Adulteration of OliVe Oil with Hazelnut Oil; Final Report[END_REF]). The loss to European consumers was estimated at 40 million euros due to hazelnut oil being three times as cheap as olive oil. In 1997 the European Commission, Unit for the Coordination of Fraud Prevention Many analytical approaches have been developed during last years in order to detect HAO in EVOO, principally based either on chromatographic analyses of free and esterified sterols, or on the detection of other minor secondary compounds, like filbertone ((E)-5 methylhept-2-en-4-one) [START_REF] Parcerisa | Analysis of olive and hazelnut oil mixtures by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry of triacylglycerols and gas-liquid chromatography of non-saponifiable compounds (tocopherols and sterols)[END_REF]Flores 2006a;Flores 2006b). Other advanced approaches such as differential scanning calorimetry, nuclear magnetic resonance and isotopic ratio coupled with mass spectrometry were also described (Chiavaro 2008;[START_REF] Sacchi | 1 H and 13 C NMR of virgin olive oil. An overview[END_REF][START_REF] Remaud | Authentication of Mustard Oils by Combined Stable Isotope Analysis (SNIF-NMR and IRMS)[END_REF]. Until today, no validated PCR-based methods for identifying fraudulently spiked HAO in EVOO have been reported.

HAO, like other edible oils, is largely considered a "protein-free" food but solvent-assisted extraction for "industrial" oil can potentially (and probably) influence the presence of residual hazelnut proteins, as has been shown for other nuts and legume oil preparations [START_REF] Crevel | Allergenicity of refined vegetable oils[END_REF]. Some evidence highlighted that peanut oil (as well as the soybean oil) can not always be considered as a safe product for peanut-allergic patients [START_REF] Hourihane | Randomised, double-blind, cross-over challenge study of allergenicity of peanut oils in subjects allergic to peanuts[END_REF][START_REF] Crevel | Allergenicity of refined vegetable oils[END_REF][START_REF] Ring | Allergy to peanut oil--clinically relevant[END_REF]. Apart from a recent paper focused on the use of a biosensor [START_REF] Bremer | Biosensor immunoassay for traces of hazelnut protein in olive oil[END_REF], no specific data are available for protein content of either raw or refined HAO.

Hazelnut allergy is often the result of primary sensitization to birch pollen, more in particular to the major birch pollen allergens Bet v 1 and Bet v 2. Cross-reactivity of IgE antibodies to their homologues Cor a 1 and Cor a 2 in hazelnut is at the basis of hazelnut allergy [START_REF] Hirschwehr | Identification of common allergenic structures in hazel pollen and hazelnuts: a possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen[END_REF].

Other hazelnut proteins that can elicit an allergic response are non-pollen related seed storage proteins, such as Cor a 11 (7S globulin) [START_REF] Lauer | Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity[END_REF]) and Cor a 9 (11S globulin) [START_REF] Beyer | Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions[END_REF] and the non-specific lipid transfer protein (nsLTP) Cor a 8 [START_REF] Schocker | Recombinant lipid transfer protein Cor a 8 from hazelnut: A new tool for in vitro diagnosis of potentially severe hazelnut allergy[END_REF]. More recently two oleosin isoforms, now designated Cor a 12 and Cor a 13, were identified as new allergens in hazelnut [START_REF] Akkerdaas | Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library[END_REF]. Moreover, we very recently isolated and cloned 2S albumin from hazelnut [START_REF] Zuidmeer | Isolation, cloning and confirmation as an allergen of the 2S Albumin from hazelnut[END_REF]GenBank FJ358504;release The problem of so-called "hidden allergens" in food is an important issue for the assessment of food safety. Hidden ingredients in food can either originate from cross-contamination during food processing, or from fraudulent use of non-declared ingredients in food [START_REF] Steinman | Hidden allergens in foods[END_REF].

Fraudulent introduction of "hidden allergens" is a more serious risk for sensitized consumers because the producer wants the adulteration to go unnoticed. Cases of death for anaphylactic shock in children, adolescents, and adults after ingestion of foods able to trigger an allergic reaction in sensitive individuals have been reported. These deaths are often caused by the presence of "hidden" ingredients, sometimes not even declared. Peanuts and nuts are particularly implicated in this kind of severe responses, provoking asthma crisis in sensitive patients [START_REF] Sampson | Fatal and near fatal-induced anaphalaxis[END_REF]).

The lipophylic protein fraction containing oleosins and non-specific lipid transfer proteins (nsLTPs), considered allergens in several foods [START_REF] Ballmer-Weber | Lipid transfer protein as a potential panallergen?[END_REF][START_REF] Fernandez-Rivas | Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population[END_REF][START_REF] Pons | The 18 kDa peanut oleosin is a candidate allergen for IgE-mediated reactions to peanuts[END_REF][START_REF] Schocker | Recombinant lipid transfer protein Cor a 8 from hazelnut: A new tool for in vitro diagnosis of potentially severe hazelnut allergy[END_REF][START_REF] Fernandez-Rivas | Apple allergy across Europe: how allergen sensitization profiles determine the clinical expression of allergies to plant foods[END_REF], could be present as hidden residual ingredient either in HAO, and, consequently, in adulterated EVOO. The potentially severe risk that such products entail for hazelnut-allergic people has thus far been underexposed.

The first aim of the present study was to evaluate by SDS-PAGE the presence of residual protein in EVOO spiked with hazelnut oil obtained by cold-pressing or solvent-extraction and subsequently to prove through the use of mass spectrometry the eventual existence of relevant hazelnut allergens. Secondly our goal was, using immunoblotting, to establish if serum IgE of some hazelnut allergic patients with known reactivity both to Cor a 1 and oleosin confirm the presence of allergens in order to understand whether there is actually a real concern for sensitized people.

Materials & Methods

Samples

Five commercial samples of raw HAO (cold-pressed) and six commercial ones of olive oil (three labelled "extra virgin olive oil" EVOO (cold-pressed) and three "olive oil" OO) were purchased in local stores (Piedmont, Italy). Soremartec (Alba, Italy) kindly provided a series of six cold-pressed HAO (both three from un-roasted and three from roasted hazelnut seeds, Corylus avellana L.).

Solvent-extracted HAO was obtained from raw hazelnuts ("Nocciola Piemonte" PGI certified from Piedmont, Italy) using the following procedure. Before oil extraction, 20 g of hazelnuts were crushed in mortar using liquid nitrogen and subsequently transferred into the extractor paper vessel. Extraction was carried out in a semi-automated extractor (Büchi Extraction System B.811) for 9 hours using dichloromethane as solvent. Oil samples were filtered using a 0,4 µm syringe filter.

Preparation of reference olive oil sample spiked with different percentage of hazelnut oil

Solvent-extracted hazelnut oil was added to olive oil aliquots (commercial sample of EVOO) at 0.5, 1, 2, 3, 4 and 5% (v/v).

Extraction of protein from oils

Diethylether (100 mL) was added to 100 g of oil (after gently mixing of the bottle, in order to obtain a homogeneous surfactant phase mixed with the oil dreg typical of EVOO) and the mixture was maintained at 18 °C for 30 min. 20% w/v trichloroacetic acid (100 mL), previously cooled at 4 °C, were added and the mixture was transferred in a separating funnel. The trichloroacetic acid extraction step was repeated three times. The three aqueous fractions were collected and washed twice with diethylether (2 x 100 ml) to remove all the lipid material. The aqueous fraction was partially concentrated using a rotary evaporator at < 30 °C and centrifuged at 20,800 g for 20 minutes at 4 °C. The supernatant containing trichloroacetic acid was then discarded. The pellet was washed twice with acetone (2 x 10 ml) to remove all trichloroacetic acid traces, and dried in a vacuum oven for 2 h at room temperature. The dried pellet, obtained as an average of three

SDS-PAGE

Extracted proteins were dissolved in Laemmli sample buffer without 2-mercaptoethanol [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]) and separated, 10 ug of protein per lane, by sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE (Mini-PROTEAN II Cell, Bio-Rad, Richmond, CA) with 12% acrylamide resolving and 4% acrylamide stacking gel. Following electrophoresis, the gel was fixed in 7% acetic acid and 40% methanol for 1 h, then stained with brilliant blue G-colloidal (Sigma Chemical Co., Milan, Italy) for 1 h, and destained with 25% methanol for 48 hours [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]). In the preliminary phase of work in addition to Coomassie Blue also Silver staining procedure was applied [START_REF] Switzer | A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels[END_REF].

Patient sera

For the immunoblotting, one in-house reference serum with known IgE-reactivity to Cor a 1 and profilin was used. In addition, 4 patient sera with a positive clinical history to hazelnut were used.

Known specific IgE (sIgE) are listed (IU/ml). 1: Hazelnut (8.8), Cor a 1 (7.2), Cor a 2 (0.3), oleosin (0.1) 2: Hazelnut (8.1), Cor a 1 (0.3), oleosin (21.9) 3: Hazelnut (15), Cor a 14 (15.6), oleosin (0.8) 4: Hazelnut (2.1), Cor a 1 (0.9), Cor a 8 (0.2) and oleosin (7.1). Total IgE content has not been determined.

SDS-PAGE/immunoblotting

Proteins were separated by SDS-PAGE (NuPAGE  4-12% Bis-Tris gel, protein: 10 µg/cm) and western blotting was performed by transferring the proteins semi-dry to nitrocellulose on a Novablot electrophoretic transfer apparatus, according to the protocol of the manufacturer (Invitrogen, Carlsbad, California, USA). After blocking with PBS/5% ELK/0.02% Tween-20 for a minimum of 10 minutes, the blots were cut into strips and incubated overnight with 150 µl human serum, 1:3.000 (v/v) in-house polyclonal rabbit antisera (to hazelnut extract (extracted at neutral and low pH), Cor a 8 or natural hazelnut oleosin) or 1:30.000 (v/v) in-house monoclonal mouse antiserum (to the major birch pollen allergen Bet v 1, shown to be cross-reactive to Cor a 1) in 3 ml of PBS/0.02% Tween-20/0.5% ELK. After washing 5x (PBS/0.02% Tween-20), blots were incubated (4hrs) with 1:30.000 (v/v) HRP-labelled goat anti-human IgE (KPL, Gaithersburg, MD, USA) and washed as before. Detection was performed with ECL according to the protocol of the manufacturer (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Blots were exposed to X-ray film (Fuji Super RX, Fujifilm coop., Tokyo, Japan).

MALDI-TOF/TOF MS analysis of protein extracts

For "in gel" reduction and trypsin digestion, seven bands, chosen for their intensity, were cut out (fig. 1), reduced with 100 mM DTT (56 °C, 1h), alkylated by adding iodoacetamide to a final concentration of 200 mM, and incubated in the dark at room temperature for 45 min. The proteins were then "in gel" digested with trypsin (Promega, USA), as previously described [START_REF] Lauer | Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity[END_REF]). For MALDI-TOF mass spectrometry, 0.5 µl of each peptide mixture was applied to a target disk and allowed to air-dry. Subsequently, 0.5 µl of matrix solution (1% w/v αcyano-4hydroxycinnamic acid in 30% acetonitrile, 0.1% TFA) were applied to the dried sample and again allowed to dry. Spectra were obtained using a Bruker Autoflex II MALDI-TOF/TOF spectrometer (Bruker Daltonik, Germany). The MS data were searched against UniProt database with Mascot search engine (http://www.matrixscience.com) following the peptide mass fingerprinting (PMF) method [START_REF] Pappin | Rapid identification of proteins by peptide-mass fingerprinting[END_REF]. Mass tolerance of 30 ppm, hydrolysis with trypsin and two missed cleavages, carbamidomethylation of cystein as fixed modification, oxidation of methionine as variable modification were specified, no post-traslational modification were included in the search. To confirm the protein identification, the most intense signal of each spectrum underwent to MS/MS analysis. The MS/MS spectra were automatically analysed by the Flex Analysis software (Bruker Daltonik, Germany).

Results and Discussion

Several studies reporting the authenticity assessment of olive oil based on analytical techniques In addition, samples of HAO were obtained by solvent extraction with dichloromethane. A method for isolating and concentrating protein based on diethylether and trichloroacetic acid was used on all these samples. Both in case of EVOO and cold-pressed HAO visible solid residue (dreg) at the bottom of the bottle was carefully mixed with the oil prior to protein isolation, in order to obtain a representative extraction of the "whole" protein residue from the oil sample. OO did not contain such residue. After the extraction, the oil was filtered avoiding any other kind of refining process.

We chose not to consider any additional refining steps following filtration (e.g. deacidification, deodorisation, etc.), to allow a clear comparison of residual protein using two extraction procedures, i.e. solvent and cold-pressed extraction. Moreover solvent-extracted HAO is very likely to be used in fraudulent adulteration of EVOO as it is a simple and cheap one step procedure. Any additional refining steps would make the process of adulteration financially less attractive. The extraction method used in our laboratory (accelerated solvent extraction) is comparable to the industrial-solvent extraction of seed oils, allowing us to use a good model-system to evaluate the residual presence of proteins secondary to the disruption of tissue.

Solvent-extracted hazelnut oil proved to be the only one of the analyzed samples, subjected to this procedure of extraction-concentration, to have made a protein precipitate (160 mg/albumin equivalent from 100 g of oil); cold-pressed HAO and olive oil did not showed the presence of protein. Only the solvent-extracted procedure is therefore able to extract proteins from hazelnut.

We have also increased by three times the initial amount of cold-pressed oil (300 g) and tried a SDS-PAGE separation followed by a very sensitive silver staining technique with a detection level down to the 0.3-10 ng level [START_REF] Switzer | A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels[END_REF]. Even using this technique we were unable to visualize any band. Considering these results, regard to the gel staining, we have chosen to apply the protocol of Colloidal Coomassie Brilliant Blue (G-250), more sensitive (ca. 30 ng per band) than Coomassie Brilliant Blue (R-250) (ca. 100 ng per band), and fully compatible with mass spectrometry analysis [START_REF] Neuhoff | Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis[END_REF]. We found that the sensitivity of this staining technique was suitable for the purpose of this study.

Protein isolation from EVOO, OO and cold-pressed HAO did not result in any detectable bands on SDS-PAGE (fig 1). The same analysis of solvent-extracted HAO clearly demonstrated the presence of different proteins with apparent molecular masses ranging from 10 to 60 kDa (fig. 1).

The visible solid residue in EVOO and cold-pressed HAO can most likely be explained by the presence of phenolics ad fibre compounds. Solvent-extracted HAO probably showed clear presence of residual proteins, because the solvent more efficiently extracted protein from hazelnut seeds as well as disaggregated residual protein-coated oil bodies. As already known, the use of different solvents in Soxhlet extracting method is currently chosen to lead the lipid extraction at inter-cellular or intra-cellular level. Hence, we justify the presence of proteins in solvent-extracted and micro-filtered HAO, according to the "technological" differences occurring among the different oils employed in our analysis.

Concerning the EVOO samples spiked with solvent-extracted HAO, a conventional SDS-PAGE was able to show two bands down to 1% (v/v) solvent-extracted HAO. Hazelnut oil was added to olive oil aliquots at 0.5, 1, 2, 3, 4 and 5% (v/v) corresponding to 0.8, 1.6, 3.2, 4.8, 8 mg of hazelnut protein respectively (fig. 2). This result highlights how this simple method could be considered a good tool for the discrimination of HAO adulterated EVOO's, even though more advanced analytical chemical techniques were previously suggested [START_REF] Del Castello | Rapid Recognition of Olive Oil Adulterated with Hazelnut Oil by Direct Analysis of the Enantiomeric Composition of Filbertone[END_REF][START_REF] Baeten | Detection of the presence of hazelnut oil in olive oil by FT-raman and FT-MIR spectroscopy[END_REF]Flores 2006a;Chiavaro 2008). In our case, the sensitivity was higher than those observed monitoring the presence of filbertone using an on-line hyphenated HPLC-GC technique, allowing the detection of adulterated olive oil with 5-10% of virgin and refined hazelnut oils, respectively (Flores 2006b). A restriction of our suggested technique could be that the presence of the hazelnut proteins should be correlated only with some categories of HAO (solvent-extracted), as clearly shown in this study. When we applied the method to some commercial samples of HAO (undeclared whether cold-pressed or refined), no protein contamination in this set of commercial samples was detected, pointing towards the need of a more accurate and enlarged study in this field.

The detection of protein in adulterated oils is a major concern for allergic consumers. The allergenicity of different oils processed by different methods was previously investigated, namely in peanut (Arachis hypogaea L.) and soybean (Glycine max L.) oils [START_REF] Crevel | Allergenicity of refined vegetable oils[END_REF]. The presence of hazelnut protein in HAO was demonstrated in several studies on nut oils; hazelnut gourmet oil was reported to contain up to 62.0 ± 1.1 µg of protein on 1 ml of oil [START_REF] Teuber | Allergenicity of gourmet nut oils processed by different methods[END_REF]. To date the possible allergenicity of nut oils is still under discussion (Peeters 2004); the understanding of this safety issue is clearly impacted by different oil extraction and refining processes. The detection of residual hazelnut protein in adulterated olive oil (as well as the qualitative description and identification) was never deeply investigated. Two isoforms of oleosin with molecular masses of 16.7 and 14.7 kDa have recently been identified as new hazelnut allergens [START_REF] Akkerdaas | Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library[END_REF].

Oleosins isolated from seed-or nut-derived oilbodies have been reported to be contaminated with small but significant quantities of albumins and globulins (Millichip 1996). Albumins and globulins in seeds and nuts are major sources of allergenicity. Taken together, oil extraction from hazelnuts may result in residual protein content with a high likelihood of including allergens.

Considering the presence of allergenic proteins in solvent-extracted HAO, we aimed at identifying proteins using MALDI-TOF MS. This analysis clearly demonstrated the presence of two oleosin isoforms (UniProt ref. Q84T91, Q84T21) (fig. 1, bands 1 and 2), newly described by Akkerdaas (http://expasy.org/uniprot/Q84T91_CORAV; http://expasy.org/uniprot/Q84T21_CORAV) and of two bands both containing 11S globulin-like protein (UniProt ref. Q8W1C2) (fig. 1, bands 3 and 4) [START_REF] Beyer | Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions[END_REF]. Fifteen peptides, belonged to the 11S globulin-like protein, in band 4 whereas only nine in band 3 probably because the different amount of the protein in the two bands. The presence of 11S globulin-like protein in two distinct bands is not surprising and it is probably due to the post-translational modifications occurring on the protein and responsible for the slight differences of the molecular weight compared to the nominal one reported in Table 1. No specific investigation on post-translational modifications was performed because the MS/MS data were obtained only for one signal and the MS data are unreliable without MS/MS confirmation. Evidence for the presence of other allergens like Cor a 1 or Cor a 8 was not found. A more accurate bidimensional analysis should probably be carried out to improve the selectivity of protein fingerprint separation, permitting the clear identification of unknown proteins.

As we detected oleosin and Cor a 9 in hazelnut oil, immunoblotting method was applied in order to confirm the presence of possible allergens testing sera from both patients and sensitized animal (rabbit and mouse). We analyzed sera from four patients with a positive clinical history to hazelnut, and an in-house serum with IgE reactivity to Cor a 1 and 2. The four patient sera (Fig. 3, lanes 12-15) did not show any IgE reactivity, however the in-house serum showed IgE reactivity to a protein with an apparent molecular mass of 17-18 kDa (Fig. 3, lane 1). The main difference between the reference serum and the other sera is that the former has IgE-reactivity to profilin, while the four others do not. Profilin was not one of the proteins recognized in the Maldi-TOF analysis, however, that may be due to the low amounts present in the extract.

Two rabbit antisera against hazelnut extracts confirmed the presence of hazelnut-derived protein (Fig. 3, lanes 4 and 6). According to these preliminary results, although only one in-house serum from an allergic patient has given a positive response in the immunoblot, the initial assumption of a potential risk for the consumer can be considered plausible. The in-house serum is from a patient with known inhalant allergies, however, since this a serum from a retrospective patient, we do not have data on the clinical reactivity to hazelnut. So, although we do know the IgE-reactivity to Cor a 1 and 2 on blot and in RAST/CAP experiments, we can't claim the reactivity will lead to clinical reactions on hazelnut. However, the purpose of the paper was to detect allergens in adulterated olive-oil, and for this reason this serum can be used. The fraudulent adulteration of olive oil with hazelnut oil, already a problem in terms of commercial costs for the consumer, it appears at this point exacerbating those health and food safety related problems with an additional risk associated to its allergenic component. This new argument, even if highlighted by a preliminary study, should certainly be taken into account for the most severe reported cases of allergy in future researches.

Conclusions

In conclusion, there is a potential risk for hazelnut-sensitized patients when exposed to fraudulently spiked virgin olive oil, especially in the case of solvent-extracted not-refined HAO. This preliminary observation confirms other previous highlights, concerning the putative presence of allergens in oils from different origin. We can conclude that the method used here represents a step toward the more difficult detection of hazelnut oil in other olive oils enlarging the problems associated with this fraudulent practice that, besides provide an economic damage, introduces a potential health risk for sensitized patients. Tab. 1: Identification by PMF and MS/MS of proteins selected from the total protein extract from solvent-extracted HAO (used to spike EVOO) Figure 1: SDS-PAGE of total proteins isolated from solvent-extracted HAO (1) and from EVOO (2) (Colloidal Coomassie Brilliant Blue staining). Numbers highlight the proteins as "putative allergens" and (*) the bands analyzed but not identified.

Figure 2: SDS-PAGE of total protein extract isolated from EVOO, artificially spiked with different percentage of solvent-extracted HAO (from 5 to 0,5%, V/V). The arrow highlights hazelnut oleosins identified by MS/MS analysis.

Figure 3: immunoblotting profiling performed on the total protein fraction isolated from hazelnut oil. Lane 1: Bet v 1/ Bet v 2 -and homologues -recognizing serum; lane 2: Rabbit anti-hazelnut oleosin; lane 4: Rabbit anti-hazelnut; lane 6: Rabbit anti-hazelnut (pH 2.5 performed extraction); lane 8: Rabbit anti-LTP; lane 10: Mouse anti-Bet v 1 (Cor a 1); lanes 3,5,7,9,11: Pre-immune sera (taken previously immunization with the allergen or extract, then before the animal develops specific antibodies); lanes 12-15: Hazelnut positive human sera; lanes LC: Label controls. Spots in lanes 2 and 5 are caused by a phenomenon of background.

F

  , estimated that more than 20,680 tonnes of hazelnut oil was used for producing 103,400 tonnes of adulterated olive oil[START_REF] Uclaf | The Fight against Fraud[END_REF] European Union Research Committee 2001).

  was quantified as mg/albumin equivalent according to Bradford 1976. The pellet was stored at -20 °C.

  out (Ruiz del Castello 1998;[START_REF] Crevel | Allergenicity of refined vegetable oils[END_REF][START_REF] Parcerisa | Analysis of olive and hazelnut oil mixtures by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry of triacylglycerols and gas-liquid chromatography of non-saponifiable compounds (tocopherols and sterols)[END_REF][START_REF] Vichi | Detection of hazelnut oil in virgin olive oil by assessment of free sterols and triacylglycerols[END_REF][START_REF] Baeten | Detection of the presence of hazelnut oil in olive oil by FT-raman and FT-MIR spectroscopy[END_REF] Flores 2006a). Our aim was to evaluate the isolation and concentration of putative residual protein from EVOO, OO and HAO; then estimating by SDS-PAGE the presence of residual protein in EVOO spiked with hazelnut oil. Retail samples of EVOO, OO and cold-pressed HAO were used.

  Ruiz Del Castillo ML, Herraiz M, Blanch GP. 2006b. Detection of the adulteration of olive oils by solid phase microextraction and multidimensional gas chromatography. Food chemistry

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: SDS-PAGE of total proteins isolated from solvent-extracted HAO (1) and from EVOO (2) (Colloidal Coomassie Brilliant Blue staining). Numbers highlight the proteins as "putative allergens" and (*) the bands analyzed but not identified. 179x155mm (150 x 150 DPI)

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Acknowledgements

We acknowledge Piet Schilte and Andre Knulst for the kind use of these sera and Soremartec (Alba, Italy) for kindly supplying the cold-pressed virgin HAO. This work was funded by Regione Piemonte (Ricerca Sanitaria Finalizzata 2008 and "RINTRACOR Project") and the international partnership (Nutrial Network project) was sponsored by Cariplo Foundation (Milan, Italy).


S M Barlow
email: suebarlow@mistral.co.uk

Risk assessment of food contact materials: past experience and future challenges

Keywords: Risk assessment, food contact materials, food packaging, European Union, tiered testing

The tiered approaches used in the European Union (EU) and the United States of America (USA) for the risk assessment of substances in food contact materials are based on the principle that the amounts of data required to establish safety-in-use depend on the extent of dietary exposure. Tiered approaches are increasingly recognised as sound approaches for chemical risk assessment, which not only offer consumers reasonable certainty of no harm but also avoid unnecessary toxicological testing and focus scarce risk assessment resources on substances of potential concern. Although the EU and USA approaches were developed separately, their tiered testing recommendations are similar. This paper indicates the savings they have brought in the use of animals and suggests areas where testing recommendations for higher dietary exposures might be further refined. It also discusses some of the future challenges in risk assessment of food contact materials.

Introduction

The first EU framework legislation on food contact materials, Council Directive 76/893/EEC, was introduced over 30 years ago [START_REF] Cec | Council of the European Communities[END_REF]. At the same time, the European Commission's Scientific Committee for Food (SCF) began the work of evaluating the many thousands of monomers, other starting substances, and additives that are used in the manufacture of food contact materials, a task that has been continued since 2003 by the European Food Safety Authority (EFSA). The SCF initially concentrated on evaluation of substances commonly used in plastics for which there was suspicion of toxicity, such as vinyl chloride, acrylonitrile, vinylidene chloride and styrene monomers (SCF, 1975[START_REF] Scf | Reports of the Scientific Committee for Food concerning Acrylonitrile Monomer (opinion expressed 15 January 1981), Vinylidene Chloride Monomer[END_REF], and the plasticisers, di-2-ethylhexylphthalate and di-2-ethylhexyladipate (SCF, 1984). The Committee also evaluated substances used in regenerated cellulose films [START_REF] Scf | list of substances to be authorized in the manufacture of regenerated cellulose films intended to come into contact with foodstuffs[END_REF]. In 1986 the Committee published its first consolidated report on monomers and other starting substances used in plastics [START_REF] Scf | Report of the Scientific Committee for Food on Certain Monomers and Other Starting Substances to be Used in the Manufacture of Plastic Materials and Articles Intended to Come Into Contact with Food[END_REF].

In 1989 the first framework Directive of 1976 was repealed and replaced by Council Directive 89/109/EEC [START_REF] Cec | Council of the European Communities[END_REF], which, in turn, has been replaced by Regulation (EC) [START_REF] Cec | /2004 of the European Parliament and of the Council of 27 October 2004[END_REF][START_REF] Cec | /2004 of the European Parliament and of the Council of 27 October 2004[END_REF][START_REF] Cec | /2004 of the European Parliament and of the Council of 27 October 2004[END_REF]. This legislation made it obligatory for the Commission to consult the SCF and subsequently EFSA on public health aspects of substances used in food contact materials. Accordingly, many opinions on the safety-in-use of such substances were adopted between 1986 and 2003 by the SCF and since then by the relevant EFSA Scientific Panel. These opinions form the basis for the positive lists of authorised substances incorporated into EU legislation. The main focus over the last three decades has been on substances used in plastics, with occasional opinions on other substances of potential concern, such as lead and cadmium (SCF, 1992a(SCF, , 1992b)), and the printing-ink component, 2-isopropyl-thioxanthone (ITX) (EFSA, 2005).

This paper is written from a European perspective, with reference to the similar safety assessment approach used in the United States of America (USA), and considers what has been learned from the past 30 years experience on the risk assessment of food contact materials and the nature of future challenges.

Use of tiered approaches for the safety assessment of food contact materials

The first EU guidelines on the toxicological evaluation of substances used in food contact materials were published by the SCF in 1977, followed by periodic updates [START_REF] Scf | Toxicological evaluation of a substance for materials and articles intended to come into contact with foodstuffs[END_REF](SCF, , 1992c[START_REF] Scf | Guidelines of the Scientific Committee on Food for the Presentation of an Application for Safety Assessment of a Substance to be Used in Food Contact Materials Prior to its Authorisation[END_REF]. The original guidelines were not prescriptive as to what toxicological tests should be conducted, but outlined the tests that might be required in certain cases as including acute toxicity, 90-day oral, longterm, reproduction, teratogenicity, and mutagenicity studies. The necessary tests depended on the physicochemical properties, the chemical structure, the toxicity of related compounds and the quantities migrating into food [START_REF] Scf | Toxicological evaluation of a substance for materials and articles intended to come into contact with foodstuffs[END_REF]. Thus, there was early adoption of the idea of a tiered approach to toxicity testing, in which the amount of toxicity data required should increase as the known or anticipated human exposure increases.

Tiered approaches to toxicity testing are based on the founding principle of toxicology, first articulated by Paracelsus in the 16 th century, which can be paraphrased as 'the dose makes the poison'. Tiered approaches are now widely recognised as sound, not only from a public health perspective, but also because of the need to focus scarce toxicology resources on the testing and evaluation of substances to which there may be greater human exposure and therefore a greater likelihood of causing toxicity.

Tiered approaches to toxicity testing also help to reduce the unnecessary use of animals. Such approaches are used not only in the field of food contact materials, but also in other chemical sectors, including the new EU REACH legislation covering industrial chemicals (ECHA, 2008), and the testing requirements for food additives in the USA (FDA, 2006). The EU has maintained a tiered approach for testing and evaluation of substances used in food contact materials in subsequent revisions and updates to the 1977 guidelines and these more recent guidelines set out clear requirements for the toxicity tests that are needed, depending on which of three tiers of estimated human exposure a substance may fall into (SCF, 1992c[START_REF] Scf | Guidelines of the Scientific Committee on Food for the Presentation of an Application for Safety Assessment of a Substance to be Used in Food Contact Materials Prior to its Authorisation[END_REF].

The key aim of the evaluation of substances used in food contact materials is to establish their safetyin-use, albeit recognising that this can never be a guarantee of absolute safety. In this regard an important concept was elaborated by the US Food and Drug Administration (FDA), referring to food (Sotomayor et al., 2007) A comparison of the tiered approaches to data requirements in the USA and the EU is shown in Table 1. The approaches were developed separately and are not identical, the USA essentially having 4 tiers and the EU 3 tiers. Nevertheless, the recommendations for testing are similar for similar dietary exposures.

<<Insert Table 1 here>> animals at US tier 3, while EU tier 1, US tier 1 and US tier 2 require no live animal testing. These tiered approaches appear to have served public health adequately, insofar as the occurrence of identifiable health problems in humans has been avoided.

Possible refinements and improvements to the current safety assessment approaches

Genotoxicity testing

Genotoxicity data form the core of the majority of food contact material data submissions and are normally required for all substances by the EU. In the USA, genotoxicity data are not required below the Threshold of Regulation value of 0.5 µg/kg diet, corresponding to an intake of 1.5 µg/person per day, though if such data exist they should be submitted [START_REF] Fda | Food Additives: Threshold of Regulation of Substances Used in Food-Contact Articles; Final Rule[END_REF]. The scientific basis for the waiving of a requirement for genotoxicity data in the USA comes from examination of numerous datasets on substances that are known to be carcinogenic, by mathematical modelling of their observed dose-response curves and extrapolation down to much lower exposures in humans. Such investigations have show that for carcinogen exposures below 1.5 µg/person per day the estimated lifetime risk of cancer would be very low (less than 1 in a million) [START_REF] Rulis | Establishing a threshold of regulation[END_REF][START_REF] Cheeseman | A tiered approach to threshold of regulation[END_REF].

This same value of 1.5 µg/person per day has been used in the development of the decision tree for evaluation of substances for which there are few or no toxicity data, based on the concept of Thresholds for Toxicological Concern (TTC). In the TTC decision tree, 1.5 µg/person per day is the human exposure threshold value below which the risk of cancer is considered to be very low [START_REF] Kroes | Structure-based thresholds of toxicological concern (TTC): Guidance for application to substances present at low levels in the diet[END_REF][START_REF] Munro | The threshold of toxicological concern (TTC) in risk assessment[END_REF]. If this TTC value were to be utilised in EU evaluations of substances used in food contact materials, it would likely make a small contribution to reduction of testing, since the FDA has estimated that human exposure falls below this threshold in about 15% of all petitions on food contact materials (Sotomayor et al., 2007).

In considering the likely protection that is offered by the TTC value of 1.5 µg/person per day, it is interesting to note that under EU legislation, a number of substances known to be both genotoxic and carcinogenic are allowed to be used in food contact materials, including some that are classified by , 1987). These well-known substances have been used in food contact materials worldwide for many years and are authorised for use under EU legislation on condition that they do not migrate into food in amounts that are detectable by an agreed sensitive method. In practice, this means that concentrations in food should be below 10 µg/kg of diet, which, interestingly, is 20 times higher than the TTC threshold of 1.5 µg/person per day, which equates to 0.5 µg/kg of diet (though it is appreciated that the genotoxic and carcinogenic substances mentioned above are actually likely to be present in food at concentrations that are far lower than 10 µg/kg of diet). Thus if the TTC value of 1.5 µg/person per day were to be used in the EU to waive the requirement for genotoxicity testing, it should not imply any reduction in the current levels of protection.

Possibilities for flexibility on other data recommendations

Scientific arguments in support of reductions in testing

It is clearly a shared responsibility between petitioners, as data providers, and risk assessors, as data evaluators, to approach the needs for risk assessment with a view to reducing the number animals used to the minimum necessary to reach a sound conclusion about the safety-in-use of a substance.

This requires application, on a case-by-case basis, of what have become known as 'intelligent' or 'integrated' testing strategies and risk assessment strategies. It recognises that guidelines are only for guidance and not a list of mandatory studies.

In that context, EU risk assessors consider scientific arguments that may reduce the amount of testing needed to reach a sound conclusion on the safety-in-use of a particular substance. For example:-

• If the chemical structure of a substance is considered to be innocuous, or the substance breaks down to innocuous substances in the gut, the EU tier 2/3 data may not be needed, even if migration is estimated to be above the tier 2 threshold of 50 µg/kg of diet.

• If a substance is closely structurally related to another substance, or substances, for which there are already toxicity data, then based on these similarities, it may be possible to make • If there are chronic toxicity/carcinogenicity studies, then a 90-day study in a second species may not be required at EU tier 3.

• If evidence of absence of bioaccumulation has been provided at EU tier 2, then a study of absorption, distribution, metabolism and excretion (ADME) may not be required at EU tier 3.

The above scenarios have been all encountered from time to time in EU evaluations and the flexibilities indicated above have often been applied by the risk assessors.

Substitution of 90-day oral studies by 28-day oral studies

For some substances falling into EU tier 2 data recommendations, there may be circumstances where it could be considered whether a well-conducted 28-day oral study might obviate the need for a 90day oral study. The OECD Test Guideline 407 for a 28-day oral study is now a more powerful indicator of toxicity than it was when first issued in the 1980s. In 1995 it was updated to include additional parameters that are better indicators of neurotoxicity and immunotoxicity and it has recently been further updated to include additional parameters for detection of endocrine effects (OECD, 2008a). For many newer industrial chemicals, depending on the tonnage marketed, a 28-day, but not a 90-day oral study may be available to fulfil requirements under the Notification of New Substances

Regulations, now superseded by the REACH legislation. There is a continuing debate among toxicologists about the circumstances in which a 28-day study might be an adequate substitute for a 90-day study. In the case of substances used in food contact materials, it could be considered whether an adequately designed 28-day oral study, say in combination with an ADME study to exclude the possibility of accumulation with repeated dosing, would be sufficient for conclusion of a safety evaluation at EU tier 2. Judgement of the sufficiency of such an approach might also depend on other factors, such as the absence of structural alerts for toxicity, or knowledge of the toxicity of related substances. replacing the two-generation study with an extended one-generation study, which would reduce the number of animals used from around 2600 to 1400 [START_REF] Cooper | A tiered approach to life stages testing for agricultural chemical safety assessment[END_REF]. The proposal has been designed to ensure that endocrine disrupters acting primarily during prenatal development or in early postnatal life would be detected. Although originally developed in the context of agricultural chemicals, the ILSI HESI proposal could be applied equally well to other types of chemical and the OECD has taken up the extended one-generation study as a draft guideline on which there has already been a public consultation (OECD, 2008b).

Evolving strategies for reproductive and developmental toxicity testing

In the area of developmental toxicity testing, the Veterinary International Conference on Harmonisation (VICH), ILSI HESI, and others, have proposed strategies that may obviate the need in certain cases for testing in a second species [START_REF] Hurtt | Proposal for a tiered approach to developmental toxicity testing for veterinary pharmaceutical products for food-producing animals[END_REF][START_REF] Cooper | A tiered approach to life stages testing for agricultural chemical safety assessment[END_REF][START_REF] Janer | A retrospective analysis of developmental toxicity studies in rat and rabbit: What is the added value of the rabbit as an additional test species?[END_REF]. It seems likely that these strategy proposals will eventually also impact on testing recommendations for reproductive and developmental toxicity testing of substances used in food contact materials.

Future challenges

Areas requiring new guidelines

Looking ahead to future challenges in risk assessment of substances used in food contact materials, there are a number of obvious and immediate challenges, such as what will be the data needs for active and intelligent packaging, or for food contact materials containing substances that are present in nanoscale form, or for recycled plastics. For recycled plastics, EFSA has now issued guidelines [START_REF] Efsa | Guidelines on submission of a dossier for safety evaluation by the EFSA of a recycling process to produce recycled plastics intended to be used for manufacture of materials and articles in contact with food. Adopted on 21 May 2008[END_REF] and the challenge will be to establish whether, over time, the data requirements in these guidelines adequately cover potential problems that may be anticipated with the presence of contaminants in recycled plastics. The impact of the increasing use of functional barriers in food contact materials also needs to be considered in relation to data needs for substances present in materials that are behind the barrier, which may include recycled plastics [START_REF] Dole | Additives, Flavourings, processing Aids and materials in Contact with Food on a request from the Commission related to 2-isoprpyl-thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenzoate(EHDAB) in food contact Materials (Adopted on 7 December 2005)[END_REF]. EFSA has also developed draft guidelines for the assessment of substances used in active and intelligent packaging, which were published for public consultation in 2009 (EFSA, 2009a). In these draft guidelines it is proposed to follow the same tiered approach for toxicity testing requirements as is used for conventional food contact materials (SCF, 2001).

In the rapidly expanding area of the application of nanoscience and nanotechnology to food and feed products, EFSA is in the process of developing advice on the potential risks from substances present in nanoscale form in food and feed and how they may be assessed for safety prior to marketing. In the food sector, it is apparent that food contact material applications of nanoscience represent the largest innovatory sector. The initial guidance from EFSA's Scientific Committee addressed generic issues in the risk assessment of chemicals present in food in nanoscale form (EFSA, 2009b). It can be anticipated that further guidance specific to the various applications in food, including food contact materials, will need to be developed. The several challenges here are not only ones for risk assessors, such as detection of unanticipated toxicities from nanomaterials, but also ones for petitioners generating the data needed for safety assessments, since some of the biggest challenges lie in the need to develop methodologies for the detection and quantitation of engineered nanomaterials in food.

Evaluation of substances hitherto unregulated

While all food contact materials should conform to general EU food law, in that they should not endanger human health, several sectors do not have EU specific regulations and hence no positive lists of authorised substances; these include paper and board, rubber and other natural materials, coatings, printing inks, adhesives, solvents, ceramics and metals. To date, these types of materials have received very little regulatory attention. Systematic evaluation of substances used in these types of food contact materials would challenge the resources available for testing and risk assessment, as the last 20 years. If these other types of food contact material were to come under regulatory scrutiny, it would make sense, at least from the public health viewpoint, to focus risk assessment efforts first on those substances which might be more likely to give rise to health risks. Prioritisation for early evaluation might include aspects such as migration above 5 mg/kg diet, fatty food contact, use in packaging for foods specifically made for infants and young children, and suspect chemical structures.

Making use of newer developments in risk assessment

The TTC concept has already been mentioned above in the context of estimating risks from carcinogens, but the TTC approach also encompasses evaluation of substances for the likelihood of toxicity of any type. EFSA's Scientific Committee is currently considering the wider applicability of the TTC concept to the work of all its Scientific Panels dealing with chemicals, including the Panel that evaluates food contact materials. The dietary exposures corresponding to EU tier 2 data recommendations in the EU scheme range from 50 µg/kg up to 5 mg/kg of diet. If the TTC concept [START_REF] Kroes | Structure-based thresholds of toxicological concern (TTC): Guidance for application to substances present at low levels in the diet[END_REF][START_REF] Munro | The threshold of toxicological concern (TTC) in risk assessment[END_REF] were to be applied within this EU tier 2 range, it could allow substances with simple chemical structures, that are efficiently metabolised and of low potential toxicity (TTC structural Class I, for which the human exposure threshold is 1800 µg/day) to be evaluated in the absence of toxicity data, other than genotoxicity data, provided migration did not exceed 600 µg/kg of diet. Similarly, substances with intermediate chemical structures, that are less innocuous than simple structures but do not have a positive indication of toxicity (TTC structural Class II, for which the human exposure threshold is 540 µg/day) could be evaluated in the absence of toxicity data, other than genotoxicity data, provided migration did not exceed 180 µg/kg of diet.

The TTC approach could also be used for initial screening of substances that are not intentionally added to food contact materials but nevertheless may migrate into food. These include impurities, breakdown products and transformation products originating from the use of an intentionally added substance. Any such not-intentionally-added substance present in food at concentrations below the relevant TTC value would not need to be further considered or at least considered as low priority for further evaluation, while those present in concentrations exceeding the relevant TTC value could be treated according to the existing EU tiered approach. Since genotoxicity data are the key information for the majority of evaluations in the area of food contact materials, it would also be worthwhile to explore the value of evolving computational toxicity methods that use chemical structure-activity relationships derived from test data to predict the likelihood of genotoxicity in structurally-related but untested substances (see, for example, Cariello et al., 2004;[START_REF] Kazius | Derivation and validation of toxicophores for mutagenicity prediction[END_REF][START_REF] Rothfuss | Computational prediction of the chromosome-damaging potential of chemicals[END_REF][START_REF] Mazzatorta | Integration of structure-activity relationship and artificial intelligence systems to improve in silico prediction of Ames test mutagenicity[END_REF].

Conclusions

In view of the very large number of substances used in food contact materials, risk assessment approaches need to be both firmly science-based and resource efficient, in order to deliver adequate and timely protection for public health. The tiered approaches to toxicity data needs for risk assessment, used both in the EU and the USA, appear to have served public health needs well, while at the same time allowing substances with large and complex databases, such as phthalates and bisphenol A, to be singled out for more detailed assessment. However, the numbers of substances coming forward for evaluation are unlikely to diminish. So continuing efforts will be needed to refine data needs and to incorporate new, validated risk assessment methods that allow risk assessors to focus on those substances that are more likely to pose a risk to human health.

F

  Risk assessment of food contact materials: past experience and future challenges

  toxicological properties of the substance under evaluation ('read across').

  and developmental toxicity testing have received a lot of attention in the last 5 years or so. Among repeated-dose tests, multi-generation studies such as the two-generation study, consume the largest numbers of animals. The Agricultural Chemical Safety Assessment Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) has proposed

  of plastics, which has consumed a major part of EU risk assessment resources for

Table 2

 2

	shows the approximate number of substances evaluated in the USA and the EU over recent
	years and the distribution of those substances between the various tiers. The dietary exposure levels
	referred to in Table 2 are essentially 'worst case' estimates, derived either from measurements of the
	migration of the substance under evaluation into food or food simulants, or, in cases where migration
	data have not been provided, from assuming that all of the substance present in a food contact
	material does migrate into food.
	<<Insert Table 2 here>>
	In the US FDA evaluations between 2000 and 2006, the estimates of dietary exposure were below 50
	µg/kg diet for 85% of substances and for these either US tier 1 (no data) or US tier 2 (in vitro
	genotoxicity tests only) would have been the data recommendations (Sotomayor et al., 2007). In the
	EU evaluations between 2000 and 2007, the corresponding figure for substances with estimates of

dietary exposure below 50 µg/kg diet was 60%, and for these EU tier 1 (in vitro genotoxicity tests only) would have been the data recommendations. Only 15% of substances evaluated in the USA fell into US tier 3 or US tier 4 categories for data recommendations, while 40% of substances evaluated in the EU fell into EU tier 2 or EU tier 3 categories for data, with only a few of these falling into the tier 3 category, in which a considerable increase in the amount of testing is recommended. Thus these tiered approaches make a substantial contribution to reducing the need for extensive testing in animals. For example, at EU tier 3, and likely also at US tier 4, testing requirements would involve the 6 use of at least 3800 animals, whereas this number reduces to 80 animals at EU tier 2 and 200

Table 1 : Comparison of tiered approaches in the USA and the EU Tiered data approaches Dietary exposure (µg/kg of diet) USA EU

 1 Arvidson, K., Mayer, J., McDougal, A., and Sheu, C. 2007. Regulatory Report: Assessing the Safety of Food Contact Substances. Food Safety Magazine August/September 2007.Available from: http://www.cfsan.fda.gov/~dms/fcnrpt2.html

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Table 2 : Numbers of substances evaluated at each tier in the USA and in the EU USA EU 383 submissions evaluated 2000-2006

 2

	Page 21 of 21
		193 submissions evaluated 2000-2007
	• 85% had exposure levels below 50µg/kg	• 60% had exposure levels below 50µg/kg
	diet (genotoxicity tests only) ~ tier 1 or 2	diet (genotoxicity tests only) ~ tier 1
	• 15% had exposure levels above 50µg/kg	• 27% had exposure levels of 50 -
	diet ~ tier 3 or 4	5000µg/kg diet ~ tier 2
		• 13% had exposure levels above
		5000µg/kg diet ~ tier 3

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