Fabien Candau

Michaël Goujon

Jean-François Hoarau

Serge Rey
email: serge.rey@univ-pau.fr

Goujon Michaël

Cerdi

Jean-François Hoarau Cemoi

REAL EXCHANGE RATE AND COMPETITIVENESS OF AN EU'S ULTRA-PERIPHERAL REGION: LA REUNION ISLAND

Keywords: Real exchange rate, Ultra-Peripheral Region, La Reunion, Competitiveness, Euro, Misalignment. JEL Classification: F14, F31, C22

This article aims at analyzing the problem of real exchange rate appreciation and competitiveness in the EU's ultra-peripheral regions by the case study of La Reunion Island. After describing economic characteristics of this French overseas department, such as the large deficit of the trade balance, this article looks for explanation by calculating and examining the statistical properties of the Real Effective Exchange Rate (REER). Our results show that this rate is stationary around a trend and provides evidence of i) an appreciation of the REER, but ii) no permanent overvaluation of the currency. These two results can be explained by the economic catch-up of La Reunion characterized by gains in relative productivity, and by the dynamics of the terms of trade and a permanent increase in the transfers from metropolis.

Introduction

The outermost regions of Europe, with more than 4 millions of inhabitants (Eurostat, 2011), represent a particular challenge for the European integration. Located in the Atlantic and Indian Oceans, in the Caribbean Sea and in South America, these regions are eloquently defined along remoteness, small size, economic dependency on a few products and insularity.1 These ultra-peripheral regions are the four French overseas departments (Reunion, Guadeloupe, French Guyana and Martinique), two Portuguese autonomous regions (Azores and Madeira) and one Spanish autonomous community (Canary Islands) 2 . The Euro is the official currency in these regions, which is problematic in term of competitiveness when exchange rate changes, driven by the overall situation of the Euro Zone, are disconnected from economic fundamentals in these islands. Actually, ultra-peripheral regions may face the same problem than some Southern European countries. Obviously, overseas departments receive transfers from the metropolitan country that represent a major difference, but this advantage could fade away in a near future. Austerity and budget constraints in Europe will inevitably lead to a reduction in transfers and many politicians in ultra-peripheral regions fear to lose an important instrument of adjustment. With less transfers, economic conditions of development can deteriorate with increasing risks of instability and poverty. In this context, a strong Euro is devastating for exports but brings purchasing power on imports that characterized these regions. The current article focuses on La Reunion Island that is representative of many problems encounter by an ultra-peripheral region. Conclusions that can be drawn from our study may contribute to the debate on specialization and suboptimal monetary union. Basic development indicators show that despite significant improvements, gaps between La Reunion and its metropolitan patron remain relatively large. Concerning the GDP per capita, according to INSEE [The French Statistical Office], the level of La Reunion represents only 37% and 59% of the national average in 1985 and 2008 respectively 3 . Furthermore these results are supported in all the three dimensions of the Human Development Index, health, education and standard of living [START_REF] Goujon | L'indice de développement humain: Une évaluation pour La Réunion[END_REF][START_REF] Goujon | Indice de développement humain: des progrès depuis 20 ans mais un retard persistant[END_REF][START_REF] Sudrie | Quel développement des départements et collectivités d'outre-mer ? Une approche par l'indice de développement humain[END_REF].The vulnerability of this small island is also pronounced in the external dimension [START_REF] Candau | L'impact de la distance et de l'intégration sur le commerce d'une région ultrapériphérique d'Europe: l'île de La Réunion[END_REF]. The export rate is particularly low, averaging 6% of GDP in 2005 and the ratio of exports to imports is stable around 6-7% only since the beginning of the 1990s. The current trade balance deficit increased continuously over the last three decades to exceed 25% of GDP in the late 2000s.

3 Such economic results would suggest a lack of competitiveness. In order to analyze such a possibility for La Reunion, we adopt in this article a medium run macroeconomic perspective. More precisely, we calculate a usual indicator in international economics, the Real Effective Exchange Rate (REER, Hinkle and Nsengiyumva, 1999) against the major trade partners over the last decades. We find that REER fluctuations result from changes in the nominal exchange rate but also from inflation rate differentials between La Reunion and its trade partners. Moreover, the REER shows an appreciation trend. This outcome should imply a loss in competitiveness if not caused by an improvement in fundamentals, as an increase in the relative productivity. So, we try to identify whether this appreciation trend is associated to an equilibrating or a disequilibrating movement.

In a first step, using quarterly data, we follow [START_REF] Kejriwal | Unit roots, level shifts, and trend breaks in per capita output: A robust evaluation[END_REF] and verify that there is no structural break in the REER series. Then, we can use a set of standard unit root tests [START_REF] Dickey | Likelihood ratio statistics for autoregressive time series with a unit root[END_REF][START_REF] Phillips | Testing for a unit root in time series regression[END_REF][START_REF] Kwiatkowski | Testing the null hypothesis of stationarity against the alternative of a unit root[END_REF][START_REF] Elliott | Efficient tests for an autoregressive unit root[END_REF][START_REF] Ng | Lag length selection and the construction of unit root tests with good size and power[END_REF],which show that La Reunion's REER is stationary around a deterministic trend. In a second step, using annual data, we try to explain the appreciation trend by estimating an equation for REER's fundamentals. Results show that REER is driven by gains in productivity, terms of trade and public transfers.

In a third step, REER misalignments are computed as the gap between observed REER and the equilibrium REER defined as a simple deterministic trend or as the predicted REER from the equation of REER's fundamentals. Both methods detect similar phases of misalignments, and estimated misalignments are found to be small. The rest of the paper is as follows. Section 2 gives several stylized facts about the structure of La Reunion's foreign trade and the catching up effect at work. Section 3 presents analytical elements of the REER. Section 4 reports computations of the bilateral and multilateral levels of the real exchange rates for La Reunion over the period 1990-2010. Section 5examines the statistical properties of the REER using the different unit root tests mentioned above. In this section, we also introduce a first estimation of the REER misalignment. Finally, Section 6 concludes.

Trade structure and trade performances of La Reunion

The weak economic performances of La Reunion Island has been studied both at microeconomic and long term macroeconomic perspectives [START_REF] Dimou | Economies insulaires et trajectoires de développement, une comparaison entre Maurice et La Reunion[END_REF][START_REF]Transferts et déséquilibres macroéconomiques des économies ultramarines[END_REF].

Here we adopt a medium run macroeconomic perspective by focusing on the international competitiveness4 . Indeed, as showed below, La Reunion's economy is characterized by a large trade balance deficit, which is a usual suspect for a deficiency in competitiveness.

La Reunion's trade balance (trade with the metropolitan France being included) has experienced a permanent deficit for four decades (Figure 1). Since the 1980's, this deficit has progressively widened and exceed four billion euros in 2008. In percentage of GDP, the deficit is globally stable around 28%. This situation is mainly explained by weak exports, which amount €0.259 billion only in 2008, compared to a level of imports of €4.53 billion.

Consequently the ratio of exports to imports is very low, falling below 10% at the end of the 1980's and stabilizing around 6-7% in the last two decades.5

Insert Figure 1 Figures 2a and 2b highlight the overwhelming weight of metropolitan France in both imports and exports. After France, La Reunion's imports originate from Asia, the European Union and South Africa, while exports are oriented towards Indian Ocean's partners, Asia, the European Union and the United States.

Insert Figures 2a and 2b

The last characteristic of La Reunion's trade concerns the structure of traded goods. Imported goods essentially consist in final consumption goods while exports include mainly agrofood products (sugar is still the first exported good). If France is not considered, the profile of exports does not change but imports are now largely dominate by industrial products (Figure 3).

Insert Figure 3

The main conclusion is that La Reunion's trade is mainly dominated by goods that are highly exposed to international competition. Domestic consumption significantly depends on imported goods as local firms only supply half of the domestic demand. Moreover, their local firms' market share stagnates since several years (Jourdan and Parain, 2009). This external dependence is associated to a structural weakness of exports resulting from an underdeveloped industrial sector6 , traditionally explained by structural handicaps linked to insularity or/and developmental strategies [START_REF] Dimou | Economies insulaires et trajectoires de développement, une comparaison entre Maurice et La Reunion[END_REF][START_REF] Bayon | Des économies vulnérables et dépendantes[END_REF]. Nevertheless, while it is often neglected in the context of regional economies, a real overvaluation of the local currency would be a usual suspect for an external competitiveness worsening (as discussed in [START_REF] Hoarau | Les exportations: un moteur possible pour la création d'emplois[END_REF].

Defining an indicator of macro-economic competitiveness for La Reunion Island

We give particular attention to the price/cost competitiveness, which can be defined as the ratio of production costs or prices between countries, expressed in a common currency. The usual indicator is the real exchange rate (RER) that is usually built on the basis of consumer 5 prices indices, instead of the prices of internationally traded goods, capturing a more extended notion of competitiveness.7

Measures of the RER

Many definitions of the RER exist, that can be gathered under two broad categories (Edwards, 1988). The first category is based on purchasing power parity, leading to the "external" RER (ERER) that aims at measuring the country's external competitiveness, i.e. the competitiveness of a country compared to its foreign partners. The external RER is computed as the ratio of the world prices to domestic prices, expressed in the same currency, N.P/P*, N being the nominal exchange rate (number of foreign currency units per unit of domestic currency), P* an index of world prices and P an index of domestic prices. Then, an increase in N (a nominal appreciation) or in P/P* (a faster increase in domestic prices compared to foreign prices) generate a real appreciation, and should cause a loss of competitiveness of the domestic economy.

The second category is based on the dependent economy or Scandinavian model (Salter 1959, Swan 1960[START_REF] Aukrust | Inflation on the open economy: a Norwegian model[END_REF]. It leads to the "internal" real exchange rate (IRER), which aims at capturing the country "internal competitiveness", i.e. the incentives to produce tradable goods (exportable and importable goods) compared to non-tradable goods. Formally, the IRER is computed as the relative price P NT /P T with P NT the prices of non-tradable goods and P T the prices of tradable goods8 . An increase in the IRER encourages production of non-tradable goods rather than traded goods, resulting in resources transfers to the non-tradable goods sector and therefore a lower capacity to export and to produce import-substitutable goods, resulting in a loss of competitiveness. Arguably, for a small developing country, tradable goods prices are determined on the world markets, so that external competitiveness depends only on internal incentives to produce tradable goods.

The analytical relationship between the measures of RER

For La Reunion Island, the dependent economy model seems rather appropriate. However, as in most of the empirical analysis, the measure we used is the ERER since data are more available than for the IRER. In the following, we examine the relationship between these two measures to conclude that the choice of the ERER is relevant in our case9 .

The ERER is equal to: 6

P N ERER = ) 1 *( ) 1 ( * , , . . λ θ - -         t t t T t T t IRER IRER P P N (4)
The ERER is therefore a combination of three components: (i) the relative price of tradable goods between the domestic and foreign countries, (ii) the IRER in the domestic country, (iii) the IRER in foreign countries. Then, the ERER and the domestic IRER would systematically follow the same dynamics under two conditions. The first condition is the law of one price for tradable goods which leads the prices of tradable goods between countries to equalize. In this case, * , , .

t T t T t P P N = (5)
The first term of equation ( 4) is then equal to 1, and the equation ( 4) reduces to:

) 1 ( * ) 1 ( λ θ - - = t t t IRER IRER ERER (6)
The second condition is that changes in the foreign IRER are negligible compared to changes in the domestic IRER. In the simplified case where there is no change in IRER*, the changes in the ERER are proportional to those of the domestic IRER. If the two conditions are met, the use of the ERER is relevant because it is sufficiently general to capture both external and internal price-competitiveness of the economy.

The real exchange rate of La Reunion Island

Although La Reunion Island is not an independent country, the small-economy model can be applied because of its remoteness from the metropolitan country. The only drawback might concern the currency since this overseas territory has the same than its main trading partner (France and the Euro-zone). Therefore, contrasting with other similar territories with independent currency (like Mauritius), La Reunion cannot pursue an autonomous monetary policy. The use of the French Franc and of the Euro since 1999 imposes that its competitiveness (against its other euro-zone partners) is impacted by the fluctuations of the exchange rate of the euro against the currencies of its partners. However, even in this field, La Reunion's case is not so different from the ones of countries that have abandoned their own currencies for an international currency (dollarization, euroization) or that have opted for an anchor to a "strong" currency. 10 The computation of the ERER for La Reunion finally follows a standard method. Against the euro-zone countries, competitiveness only depends on the inflation differential. Against the other partners, competitiveness is function of both the inflation differential and the change in the (bilateral) nominal exchange rates.

The real effective exchange rate

Country competitiveness can be studied with respect to each trading partner or to a group of partners, i.e. using bilateral RERs or a multilateral RER, the so-called real effective exchange rate (REER).

The REER is defined as the geometric average of the bilateral RERs:

REER = ∏ = n i 1 ωi i réu réu/i P P . N       with ∑ = n i 1 ωi = 1 (7)
Where N reu/i is the index of the bilateral nominal exchange rate between the currency of country i and the La Reunion euro (number of foreign currency units for 1 euro). P reu and P i are La Reunion's and foreign country's consumption price index respectively. The weights ω i reflect the trade structure of La Reunion, limited to the first n trade partners 11 . An increase (decrease) in the REER is a real appreciation (depreciation) of La Reunion's currency. The REER can be decomposed into two components, the nominal effective exchange rate (NEER, the geometric average of the bilateral nominal exchange rates), and the effective relative price index (ERP, the geometric average of the relative prices):

10 Few studies are dedicated to the competitiveness of small insular and possibly dependent territories. Edwards andMagendzo (2001, 2006) find that "dollarized" countries, including dependent territories or colonies, experienced a lower rate of inflation and a lower GDP growth than non-dollarized ones; but a similar macroeconomic volatility.

11 See appendix 1 for details.

8

REER = NEER.ERP with NEER = ∏ = n i 1 { } ωi N réu/i and ERP = ∏ = n i 1 ωi i réu P P       (8)
Data availability allows the REER to be computed against 20 trading partners gathering 87% of La Reunion's trade in 2000. Figure 4 reports changes in the computed NEER, ERP and REER for La Reunion.

The ERP shows a downward trend over the period, meaning that inflation in La Reunion is lower than foreign inflation, which contributes to the depreciation of the REER, while the NEER shows an upward trend. Altogether, the change in the NEER dominates, pushing the REER to appreciate, signaling a possible loss in competitiveness especially from the early 2000s.

Insert Figure 4

However the change in the REER may mask disparities with regard to partner countries, which can be revealed only through the examination of the bilateral RERs.

Bilateral real exchange rates

A logarithmic form of equation 7 ensures that the real effective exchange rate is a linear combination of bilateral rates. It may be useful to distinguish bilateral RERs, against developed countries, and against developing countries.

Consider equation ( 4), ( ) In the case of perfect competition, and assuming equality in average cost and marginal cost (constant returns), firms set prices at the level of unit costs (wages adjusted for labor productivity) in each sector: where T w and NT w are the wage rates in the sectors of traded and non-traded goods, e a and ne a the productivities of these two sectors. Combining equations ( 4) and ( 9) and assuming equality of traded goods prices (law of one price), the real exchange rate expressed in first difference becomes:

( ) λ θ - -         = 1 * , * , 1 , ,
[ ] [ ] * * * * ). 1 ( ). 1 ( NT T T NT NT T T NT t a a w w a a w w - + - - - - + - - = λ θ , (10)
) ( ) ( ). 1 ( * * NT T NT T t a a a a - - - - = θ (11)
An increase in the real exchange rate will result from higher productivity gains in the traded good sector of La Reunion. This is the so-called Balassa/Samuelson productivity bias, which can explain a continuous appreciation of the real effective exchange rate of La Reunion (see Figure 4).This conclusion may emerge from the observation of the limited changes in bilateral RER vis-à-vis Germany and France (see Figure5).Vis-à-vis France, only the inflation differential explains the changes in the RER because of the common currency (Franc and Euro). For the same reasons, bilateral RER changes are limited since 1999 with the adoption of the Euro. Inflation in La Reunion however is significantly lower than inflation in Belgium, Netherlands, Italy, and especially in Spain, leading La Reunion to benefit from a real depreciation vis-à-vis these countries (around 10% vis-à-vis Spain).

Insert Figure 5

Conversely, the trend observed since the early 2000s in bilateral RER vis-à-vis countries outside the euro area (see figure 6) has more chance to be due to changes in the nominal exchange rates.

Insert Figure 6

Insofar as these countries have relatively low levels of inflation, fluctuations are explained by changes in the euro against the currencies of these countries. Especially since 2001, the Euro appreciates against the British Pound, the Japanese Yen and the U.S. dollar (and thus against the Singapore Dollar and the Korean Won that are anchored to the U.S. dollar). The real appreciation is approximately 30% against the British pound.

Competition with developing countries

Assuming that in developing countries wages do not increase at the same rate in both sectors, equation 11 becomes:

[ ]

) ( ) ( ) ( ). 1 ( * * * * NT T NT T NT T t a a w w a a - + - - = θ , (12)
or [ ]

) ( ) ( ). 1 ( * * NT T NT T t c c a a - + - - = θ , ( 12'
)

cbeing the unit labor cost. If we consider 0 > -NT T a a & &
, it is possible that the RER of La Reunion appreciates against some competitors and depreciates against others, depending on

whether 0 * * > -NT T c c & &
or not.

Note that emerging countries can experience large changes in both their nominal exchange rates against the euro and their domestic prices. Vis-à-vis the three Asian emerging countries, the real appreciation reaches about 30% since these countries anchor their currencies to the U.S. dollar.

Insert Figure 7

The Argentine peso was sharply devalued in 2002 during the financial crisis and then gradually depreciates, offsetting higher inflation, resulting in a very strong appreciation of the RER of La Reunion. Against Turkey, however, La Reunion benefits from a real depreciation of its currency (Turkey experiencing higher inflation). Vis-à-vis the Indian Ocean's partners, the RER of La Reunion depreciates against Kenya, Madagascar since 2005 (after a very strong devaluation of the Malagasy Franc/Ariary in 2004), but appreciates against South Africa and Mauritius.

Insert Figure 8

In conclusion, while different trends in bilateral real exchange rates can be observed, the REER reveals a clear real appreciation. This trend, especially since the 2000s, mainly reflects the nominal appreciation of the euro against the yen, the sterling pound and against the U.S. dollar (as well as currencies anchored to these currencies12 ). Do these dynamics mean that La Reunion has an overvalued exchange rate?

Is the REER of La Reunion overvalued?

Usually a country experiencing large trade deficits is suspected to suffer from a currency overvaluation. This phenomenon has been observed for instance in emerging countries in the late1990s and in other countries linking their currencies to the US Dollar in the early 2000s.

One could diagnose the same disease for La Reunion because of the strong euro and the regular deterioration of trade deficit over the last decades. However, economic catch-up with no inflation that is observed in this territory does not advocate for a (internal) disequilibrium.

The concept of equilibrium exchange rate

The level of (and the change in) the REER is not enough to conclude that the currency is under or overvalued. This requires defining an equilibrium REER and assess misalignment of the REER from this equilibrium.

There are different notions of equilibrium RER (Hinckle and Montiel, 1999[START_REF] Egert | Equilibrium exchange rates in transition economies: Taking Stock of the Issues[END_REF][START_REF] Hoarau | Les régimes monétaires en Australie, 1977-2000: Une mise en perspective à l'aide d'un indicateur de distorsions du taux de change réel[END_REF]. In a simple definition, from the work of [START_REF] Cassel | Abnormal deviations in international exchanges[END_REF] on the Purchasing Power Parity (PPP), the equilibrium exchange rate is the one that comes from the equalization of domestic and foreign prices for similar baskets of goods. Then, the equilibrium RER is equal to unity (absolute PPP) or to a constant (relative PPP). This equilibrium would exist if the law of one price can be generalized to the entire economy. However, it is generally considered that price rigidities in the short runwould only allow the PPP defining a very long-term equilibrium rate. One of the first challenges of the PPP came from [START_REF] Balassa | The Purchasing Power Parity Doctrine: A Reappraisal[END_REF] and [START_REF] Samuelson | Theoretical Notes on Trade Problems[END_REF], which show that productivity differentials between tradable and non-tradable sectors can produce permanent deviations from the simple PPP(while the law of one price may remain valid for the tradable sector). A country with productivity gains mainly located in the tradable sector would then experience an appreciation of its RER. This case has been then generalized in the literature by considering an equilibrium exchange rate, consistent with both external balance (current account equilibrium) and internal balance (equilibrium in non-tradable goods market, i.e. non-inflationary growth).Changes in the equilibrium RER then follow changes in fundamental factors that affect the conditions of external and internal balances. Williamson (1994 p.2) synthesizes these approaches writing: "We all believe that equilibrium real exchange rates can change over time, by enough to matter, as a result of changes in productivity (Bela Balassa' productivity bias), the terms of trade, foreign asset positions, and underlying capital flows". But, when he compares his FEER approach with other models, he admits (p.13) that: "We seem to differ in two important dimensions. One concerns what we treat as fundamentals". Generally, according to the models and the definition of the real exchange rate, these factors may also include social spending or government spending, tariffs, real interest rates ... [START_REF] Hinkle | Exchange rate misalignment: Concepts and measurement for developing countries[END_REF][START_REF] Macdonald | Equilibrium exchange rates[END_REF][START_REF] Rey | L'apport du NATREX à la modélisation des taux de change d'équilibre: théorie et application au dollar canadien[END_REF]. But the estimation of the equilibrium RER, and the measurement of misalignments, depends on statistical properties of the REER series, which is explored in the next section.

Estimation of equilibrium real exchange rate

To calculate an equilibrium exchange rate for La Reunion, we need information on both statistical properties of the REER and on possible fundamental factors of the real exchange rate. We retain two complementary approaches. The first one uses quarterly series of the REER over the period 1990-2010. For this period, quarterly data are only available for the REER and not for usual fundamentals. We only test for the presence of a deterministic trend in quarterly data of REER. If the series of REER is stationary around the deterministic trend, the equilibrium REER may be defined by this trend. 13The second approach use annual data for the REER and its fundamentals over 1980-2011, allowing to test for the determinants of the REER appreciation.

Trend in the REER

Before assessing an equilibrium exchange rate for the La Reunion's REER, we need to know whether this latter could be identified as a unit root or a stationary process. Many standard unit root tests exist in the literature (see Darné and Diebolt, 2005 for an exhaustive review). However these ones are not robust for at least two reasons. Firstly, the possible presence of structural breaks is not taken into account, which can lead to wrongly accept the null hypothesis of a unit root (Perron, 1989[START_REF] Perron | Dealing with structural breaks[END_REF]. Since the seminal work of Perron (1989), many procedures including the presence of structural breaks have been developed (Perronand Vogelsang, 1992;[START_REF] Lumsdaine | Multiple trend breaks and the unit root hypothesis[END_REF][START_REF] Perron | Further evidence on breaking trend functions in macroeconomics variables[END_REF][START_REF] Papell | Additional evidence of long-run purchasing power parity with restricted structural changes[END_REF]). Nevertheless, one crucial problem remains: the commonly used unit root tests consider structural breaks only under the alternative hypothesis of stationarity. Then, they could suffer from serious power and size distortions [START_REF] Lee | Break point estimation and spurious rejections with endogenous unit root tests[END_REF]Strazicich, 2001, 2003;[START_REF] Kim | Unit root tests allowing for a break in the trend function at an unknown time under both the null and alternative hypotheses[END_REF]. Secondly, the presence of a deterministic trend can also bias the findings resulting from unit root tests [START_REF] Dejong | Integration versus Trend Stationarity in Time Series[END_REF][START_REF] Harvey | Simple, robust and powerful tests of breaking trend hypothesis[END_REF]. Indeed, conclusions based on a specification including a deterministic trend would be inappropriate if this one finally does not exist. Several tests for identifying the existence of a linear trend have been recently proposed irrespective of the integration order of the series, namely if the series are I(O) or I(1) (Harvey et al., 2007;Perron and Yabu, 2009a).

Accordingly, here we follow Kejrival and Lopez (2013) by proceeding in two steps: (i) testing for the presence of structural breaks both in the level and the slope of the linear trend, and (ii) implementing the reliable unit root tests according to the previous results.

• Detection of structural breaks in the linear trend Our econometric methodology is based on the strategy adopted by Kejrival and Lopez (2013).

Consider the general specification: The stochastic one { } t u is assumed to be stationary, but not necessarily identically and independently distributed thereby allowing for a general error structure for t u .

1 1 1 1 1 0 , ,..., 2 , ,..., 1 v u T t v u u T t with DT DU y t t t K i t it i K i it i t t = = + = = + + + + = - = = ∑ ∑ α µ β µ β µ Where ) ( i it T t I DU > = , ) ( ) ( i i it T t I T t DT > - = , i=1
In the first step, we test for the presence of one structural break (K=1) in the slope of the deterministic trend that is the null hypothesis 0 = i β against the alternative 0 ≠ i β . To this regard, we run the recent approach of Perron and Yabu (2009b). Following Perron and Yabu (2009a), these authors proposed a robust test on the slope of a linear trend function integrating potential structural breaks at unknown dates when the integration order of the series is not a priori identified, i.e. either I(0) or I(1). The stability of the trend function is tested by the Feasible Quasi Generalized Least Squares procedure. In the second step, we try to detect the presence of a break in the level of the linear trend by testing the null hypothesis 0 = i u against the alternative 0 ≠ i u

. Thus, we implement the [START_REF] Harvey | Robust methods for detecting multiple level breaks in autocorrelated time series[END_REF] procedure which built a test for detecting multiple level breaks that is robust to unit root or stationarity properties of the data. Finally, the results are given in Table (1). These ones clearly show that it is impossible to reject the null hypothesis, so that there is no evidence of structural breaks in the REER of La Reunion14 .

INSERT Table 1 • Testing for the presence of a unit root Given the rejection of a structural break, we can apply a battery of standard stationarity or unit root tests. Two sets of tests were used: (i) the classical tests of Dickey and Fuller in its expanded version (1981) [ADF], [START_REF] Phillips | Testing for a unit root in time series regression[END_REF] [PP] and [START_REF] Kwiatkowski | Testing the null hypothesis of stationarity against the alternative of a unit root[END_REF] [KPSS] and (ii) efficient tests of [START_REF] Elliott | Efficient tests for an autoregressive unit root[END_REF] [ERS] and [START_REF] Ng | Lag length selection and the construction of unit root tests with good size and power[END_REF] [MZA and MZT], which are largely used in the literature [START_REF] Taylor | Real exchange rates and purchasing power parity: mean-reversion in economic thought[END_REF]Taylor 2004, Taylor 2006). Two specifications were considered for each procedure: with a deterministic trend and a constant [model 1], which allows testing a PPP adjusted for productivity gains in the Balassa-Samuelson version (Papell andProdan, 2006, Hoarau, 2009), and with only a constant [model 2], that allows testing the simple version of the Cassel's PPP.

Results in Table 2 lead to the same conclusions. First, the specification that best fits the data includes a deterministic trend, which is still significant at5%, rejecting Cassel's PPP. Second, trend-stationarity is significant in model 1 at the 5% level (but not at the very restrictive threshold of 1%).

Insert table 2

These results reveal one of the predictions of the Balassa-Samuelson theory, with the change in La Reunion's REER showing an appreciation trend (section 2), associated with the productivity catch-up phenomenon that can be observed on the basis of GDP per capita (see section 1).

Equation of REER's fundamentals

Insofar La Reunion is a small economy, the dynamics of the real exchange rate can be explained by productivity gains (Balassa-Samuelson effect), terms of trade, the social consumption, etc...(cf. among others Edwards, 1994[START_REF] Stein | The natural real exchange rate of the US dollar and determinants of capital flows[END_REF]. Technological progress is proxied by the rate of growth in per capita real GDP ( LnGDPpc ∆ ). Imports and exports prices are unavailable for La Reunion. Since developed countries constitute the main partners of La Reunion, we suppose that imports and exports prices of La Reunion are respectively proxied by exports (hereafter PX*) and imports prices (hereafter PM*) of advanced economies. Finally, large public transfers from the metropolis are proxied by transfers-to-GDP ratio. The REER model is:

) , , ( * * GDP Transf PM PX GDPpc REER REER ∆ = (13)
Where GDPpc represents the real GDP per capita, (PX*/PM*) the relative price or inverse of terms of trade, and (Transf/GDP) the public transfers in terms of GDP.

To estimate this model it is necessary to study the statistical properties of the variables. Sources of data and results of unit root tests are provided in appendix 3. All variables (in logarithm) are stationary. GDP per capita is I(1) in (log) level, but I(0) in first difference. The previous result concerning REER is confirmed with annual data and on a longer period.

Assuming that fundamentals are exogenous, we use the OLS estimator. The equation to be estimated is: or by a trend, and the error terms, t ε , are i.i.d. This autoregressive form allows distinguishing short run effects, a1, a2, a3 and long-run effects a1/1-λ, a2/1-λ and a3/1-λ15 . Table 3 reports the estimated coefficients with LnGDPpc ∆ in column 1 and the trend in column 2. The estimated coefficient for LnGDPpc ∆ has the expected positive sign but is insignificant, while the coefficient of the trend is significant at the10 % level and the Durbin'h confirms the hypothesis of no autocorrelation in the residuals. So we retain the results with the trend. A rise in the relative prices, i.e. a deterioration of the La Reunion's terms of trade appreciates the REER. 16 An increase in transfers appreciates the REER as expected.

Insert table 3

The long-run relationship is

Transfers have a high impact on the REER in the long-run. A rise of the transfers-to-GDP ratio of 1% generates a real appreciation of 0.28%. The coefficient of the trend is small, but the transfer variable would partly capture the Balassa effect if GDP per capita depends on transfers 18 . Figure 9 shows increasing trends in these three variables over the entire period that clearly calls for an appreciation of the RER.

Insert Figure 9 5.3. Assessment of REER misalignments A misalignment (noted MIS) is defined as the gap (in percentage) between the current REER and the estimated equilibrium REER, or MIS=Ln REER-Ln REEReq. A positive (negative) gap implies a real overvaluation (undervaluation).The major difficulty is to define and calculate the equilibrium REER. Following our previous results and the approach of [START_REF] Baffes | Single-equation estimation of the equilibrium real exchange rate[END_REF], we retain two operational methods to calculate REER misalignments.

Firstly, trend stationarity allows defining the equilibrium REER as a deterministic trend. Figure 11 shows the REER deviations from the trend, the so-called misalignments (in percentage) 19 . Three main periods are detected: (i) an overvaluation over the 1990s, and from 2006 to 2008, (ii) an undervaluation over the period 1999-2002, and (iii) a relative balance over the period 2002-2006.

Insert figure 10

Insert figure 11 Second, considering now the equation of REER's fundamentals, the equilibrium REER is obtained by using equilibrium values of the fundamentals in equation ( 15). These equilibrium values are obtained by an exponential smoothing [START_REF] Bowerman | Time Series and Forecasting: An Applied Approach[END_REF].Figure 12 reports the observed REER and the estimated equilibrium value and Figure 13 shows estimated misalignments.

Insert figure 12

Insert figure 13 The REER appears to be overvalued in the early 1980s, during the second half of the 1990s and during the 2000s. The nominal appreciation of the euro has certainly contributed to the phase of overvaluation in 2002-2008. Overall, both methods detect similar phases of misalignments and show that estimated misalignments are small. Apart from the early 1980s when there is an overvaluation of 7%, thereafter, the over or under evaluations lie in a range of 2% to 3% only.

Conclusion and final remarks

The main results show that there is no permanent overvaluation of the exchange rate for La Reunion, despite a continuous appreciation of the REER. Indeed, the appreciation of the REER is consistent with the phenomenon of economic catch-up, characterized by relative gains in productivity (Balassa-Samuelson hypothesis). We also show that transfers have contributed to this appreciation, but also to the gains in productivity. The importance of transfers, particularly for the dependent territories, is not always discussed in studies on territories with no separate legal tender (see for instance Edwards, 2002[START_REF] Edwards | Strict Dollarization and Economic Performance: An Empirical Investigation[END_REF].

We do not detect an evident problem of competitiveness at the macro level, based on the usual measures of REER and misalignments. Permanent and high trade deficits of La Reunion are then not caused by an overvalued currency and should be explained by, for instance, structural factors, or socioeconomic and political factors. La Reunion and other countries that are trading partners today have in common to be the former colonies of France, which contributes to higher trade intensity. Finally, structural factors, such as geographical distance, should be decisive for trade in a small island economy (see [START_REF] Candau | L'impact de la distance et de l'intégration sur le commerce d'une région ultrapériphérique d'Europe: l'île de La Réunion[END_REF].

= = = θ θ θ

H

. A rejection of H 0 implies that we have a long-run relationship. The Fstatistic is 4.307. At the 5% level the test is inconclusive but the F-statistic exceeds the upper bound at the 10% level. So, we conclude that there is evidence of a long-run relationship between the real GDP per capita, the transfers and the global productivity. We can verify that the long-run elasticity between the GDP per capita and the productivity is (0.1957/0.2795) =0.700, and (0878/0.2795) = 0.314 between the GDP per capita and the transfers. Finally, these results confirm the role of productivity in the dynamic of the GDP but also the importance of transfers that contribute at the appreciation of the REER, notably across their impact on the real GDP per capita.

.

  ,...K. A break in the trend occurs at time The breaks date ( i T ), and the number of breaks (K) are supposed to be unknown. The error u can follow either a I(0

Appendix 2 :

 2 Trade by products and weights of trading partners Data on international trade of La Reunion come from La Direction Nationale des Statistiques du Commerce Extérieur (DNSCE).

Figure 1 :Figure

 1 Figure 1: Trade balance of La Reunion (million euros) Source: Authors' calculations, data DNSCE

Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :

 3456789 Figure 3: Trade by products of La Reunion -2006 Source: INSEE-Réunion

Table A2 :

 A2 Weights of trading partners used to construct the real effective exchange rate of La

	Reunion Year 2000
						EXP+IMP2000		EXP+IMP2000
		EXP 2000 Share IMP 2000	Share	(26 countries) Share	(20 countries) Share
	U.A.E.	23486	0%	6493222	0%	6516708	0%		0%
	Argentina		0%	19277269	1%	19277269	1%	19277269	1%
	Belgium	111355	0%	49659033	2%	49770388	2%	49770388	2%
	Bahrain		0%	44702082	2%	44702082	2%		0%
	Chine	5959882	3%	31279118	1%	37239000	1%	37239000	1%
	Germany	1508327	1%	106245650	4%	107753977	4%	107753977	4%
	Spain	2170956	1%	52991927	2%	55162883	2%	55162883	2%
	France	136752730	65% 1714733906	67%	1851486637 67%	1851486637 72%
	United Kingdom	4312607	2%	33048428	1%	37361035	1%	37361035	1%
	Italy	2468216	1%	83318035	3%	85786251	3%	85786251	3%
	Japan	25550930	12%	53750720	2%	79301650	3%	79301650	3%
	Kenya	14213	0%	14100415	1%	14114628	1%	14114628	1%
	Comoros	955132	0%	40980	0%	996112	0%		0%
	South Korea	24710	0%	21733601	1%	21758311	1%	21758311	1%
	Madagascar	9756678	5%	17103235	1%	26859913	1%	26859913	1%
	Maurice	4525095	2%	21657934	1%	26183029	1%	26183029	1%
	Malaysia	269513	0%	24285443	1%	24554956	1%	24554956	1%
	Pays Bas	375466	0%	27119570	1%	27495036	1%	27495036	1%
	Saudi Arabia		0%	124407741	5%	124407741	4%		0%
	Singapore	89218	0%	3081964	0%	3171182	0%	3171182	0%
	Thailand	214308	0%	34392560	1%	34606868	1%	34606868	1%
	Turkey		0%	5950981	0%	5950981	0%	5950981	0%
	United States	4061043	2%	16135634	1%	20196677	1%	20196677	1%
	South Africa	468368	0%	55424755	2%	55893123	2%	55893123	2%
	Zimbabwe		0%	178731	0%	178731	0%		0%
	Mayotte	9452851	5%	249794	0%	9702645	0%		0%
	Total of partners 209065084 100% 2561362728	100%	2770427813 100%	2583923795 100%
	Total trade
	(euros)	226774777		2730209160		2956983937		2956983937
	Export/import
	coverage	92%		94%		94%		87%

Table A4 :

 A4 ARDL model for the real GDP per capita (∆ Logarithm). The F-statistic for testing the existence of a long-run relationship between the GDP per capita, the global productivity and the transfers in terms of GDP is 4.307. Critical values bounds for the F-statistic are[3.17, 4.14], [3.79, 4.85] at the 10% and the 5% level (cf. Table CI(iii),Pesaran et al. (2001 p. 300)).We include euro dummy variable which takes value 1 for dates later than 1999.

	Regressor		Coefficient	Standard error	p-value
	∆Ln GDP t-1		0.2779	0.2207	0.222
	∆Ln (Transf/GDP)	-0.1727	0.1159	0.151
	∆Ln (Transf/GDP) t-1	0.1004	0.0880	0.267
	∆Ln Prod		0.3002	0.1115	0.014
	∆Ln Prod t-1		0.1256	0.1078	0.257
	Ln GDP t-1		-0.2795	0.1145	0.024
	Ln Prod t-1		0.1957	0.1171	0.578
	Ln (Transf/GDP) t-1	0.0878	0.1554	0.110
	Dummy			0.0374	0.0142	0.016
	Intercept		1.7842	0.6712	0.015
	R	2 =	0	.	732	, F-statistic = 6.080, DW=2.129
		0	:	1	2	3	0

Notes:

Table 1 :

 1 Misalignments of the real exchange rate of La Reunion, 1990-2010 Tests for the presence of structural breaks in the linear trend Series Structural breaks in the slope ExpW : the t-stat ExpW and U result from the Perron and Yabu (2009b) and Harvey et al. (2010) tests, respectively.

	4.66
	4.65
	4.64
	4.63
	4.62
	4.61
	4.60
	4.59
	4.58
	4.57
	90	92	94	96	98	00	02	04	06	08	10
					REER	Equilibrium REER

Note

Table 2 :

 2 Unit root tests for the real effective exchange rate (Log) of LaReunion, 1990Reunion, -2010

	Tests		Model 1			Model 2	Conclusion
		t-stat	Critical value	t-stat	Critical value
			1%	5%	10%		1%	5%	10%
	ADF -3,62** -4,07 -3,46 -3,16	-1,67	-3,51 -2,89	-2,58	Trend stationary
	PP	-3,61** -4,07 -3,46 -3,16	-1,84	-3,51 -2,89	-2,58	Trend stationary
	KPSS	0,10	0,21	0,14 0,11 1,07***	0,73	0,46	0,34	Trend stationary
	ERS	-3,51** -3,65 -3,09 -2,80	-0,35	-2,59 -1,94	-1,61	Trend stationary
	MZa -19,0** -23,8 -17,3 -14,2	-0,65	-13,8	-8,1	-5,7	Trend stationary
	MZt	-3,03** -3,42 -2,91 -2,62	-0,36	-2,58 -1,98	-1,62	Trend stationary

***,**, * indicate that the null hypothesis is rejected at the 1% level, at 5% level and at 10% level.

Table 3 :

 3 Equation of REER (Logarithm)

	Regressors			(1)					(2)
	Ln REER t-1			0.6821**				0,5895**
				(0.000)					(0.000)
	Ln (PX*/PM*) t-1			0.1006**				0,0838**
				(0.017)					(0.045)
	Ln (Transf/GDP) t			0.1774**				0,1161**
				(0.0002)					(0.008)
	∆ Ln GDPpc t			0.0961
				(0.203)
	Trend								0,0007*
									(0.109)
	Intercept			1.6308**				1,9858**
				(0.0002)					(0.001)
	R	2 =	. 0	923	R	2 =	0	926 .	;
	F-statistic = 91.524	F-statistic = 95.224
	Durbin's h = -2.791	Durbin's h = -0.615

See Article

of the European Community Treaty. 2 On 11July 2012,the European Council recommended that Mayotte (French department in the Indian Ocean) take the status of an "ultra-peripheral region" of the European Union as of1 January2014. 3 18200 € against 30700 € in 2008.

We focus on price competitiveness only, excluding non-price competitiveness dimensions (quality, marketing …).

Excluding metropolitan France, the deficit reaches €1.6 billion, with exports at 0,15 billion and imports at € 1.75 billion in 2008. The associated ratio of exports to imports was 8.6%.

Taking into account services does not change significantly this finding.

As noted by[START_REF] Chinn | A Primer on Real Effective Exchange Rates: Determinants, Overvaluation, Trade Flows and Competitive Devaluation[END_REF], "[...] there are numerous measures of effective exchange rates. There is no simple answer to the question of which one is the best. Rather, the selection of the effective exchange rate measure depends upon the economic issue being analyzed"(Chinn, 2006, pp. 126)We will discuss this further in the following for the case of La Reunion.

Defining a synthetic price index of tradable (exportable + importable) goods makes sense when terms of trade are not affected by significant changes.

This relationship is discussed in[START_REF] Hinkle | Exchange rate misalignment: Concepts and measurement for developing countries[END_REF].

& & & & & & & & &

& & & & &

& & & & & & & --

Cf. Levy-Yeyati and Sturzenegger, (2005).

See Baffes et al. (1999) for a discussion on the determination of the equilibrium value of the REER when it is stationary (example of Burkina-Faso).

We also implemented the outliers' detection method ofGomez and Maravall (1997). The resulting outcomes support the previous finding of no structural breaks in the series.

For a similar specification, seeEdwards (1994)..

A deterioration of the terms of trade has a negative income effect that induces a fall in the demand for nontradable goods and consequently a real depreciation of the RER. This income effect dominates here the substitution effect that appreciates the RER through a rise in imported good price.

[START_REF] Chinn | A Primer on Real Effective Exchange Rates: Determinants, Overvaluation, Trade Flows and Competitive Devaluation[END_REF] For the relative price, the long-run elasticity is (0.0838/(1-0.5895)) = 0.2041,

etc… 18 Indeed, we show the existence of a long-run relationship between the real GDP per capita, the transfers and the global productivity. Detailed results are presented in appendix 4.

Note that our econometric methodology used to estimate the equilibrium REER is very trivial. So we could anticipate that the misalignment periods should be more pronounced in reality. Therefore the resulting estimates must be taken with caution for operational exercises.

* Significant at 10%;**significant at 5%. P-value in parenthesis.

Appendix 1: Details on the computation of the real effective exchange rate REER Data sources Data on prices and exchange rates comes mainly from the international financial statistics of the IMF. Consumer price indices (CPI) and nominal exchange rates of the national currencies against the USD (NER) are used. For Bahrain, the CPI is missing for the period 2001-04: the data are therefore linearly interpolated. The NER of China is the swap exchange rate (and not the official) that is used in the bulk of the forex transactions between 1990 and 1993 (Source: CERDI). The China's CPI is generated on the basis of the change over the same period of the previous year reported in international statistics (source: CERDI, and Fry, 1995). For Euro-zone countries, the NER starting from 1999 is NER i/$ = NER i/€ . NER € /$. For Mayotte and La Reunion, the NER € /$ is the one of France (IMF-SFI) and the CPI comes from the national statistical office INSEE. Some countries accounting for a very marginal share in La Reunion's trade have been excluded. Some countries that experience hyperinflation and/or dramatic fall in the NER have been excluded since they can bias computations, despite their low weight in the trade (e.g. Zimbabwe in the 2000s).

Computations

CPI is the consumer price index, base 2000Q1 = 100. NERi is the NER i / $ , base 2000Q1 = 100. RCPIi is the relative prices index = CPI reunion / CPI partner i, multiplied by 100. An increase in the RCPIi is a real appreciation (La Reunion's prices increase faster than partner's one). BNERi is the index of the bilateral exchange rate = NER partner i / NER reunion , multiplied by 100. An increase in the BNERi is a nominal appreciation of the La Reunion's Euro against the currency of the partner. BRERi is the index of the bilateral real exchange rate = CPIRi x BNERi / 100. An increase in BREBi is an appreciation of the bilateral real exchange rate. Weighting pattern is the weight used to compute the effective index. NEERi is the index of nominal effective exchange rates, applying a weighted geometric mean of the partners BNERi. An increase is an appreciation. REPi is the index of the effective relative prices, following a weighted geometric mean of the partners RCPIi. REERi is the index of real effective exchange rate, following a weighted geometric mean of the partners BREBi. An increase of the REERi is a real appreciation.

Global productivity:

The global productivity is calculated as the ratio between Added Value of the economy of La Reunion and the total of salaried employees in the private sector and the public sector (INSEE).

Transfers:

Transfers data are the net transfers from the metropolis. They are extracted from various numbers of "Panorama de l'économie de La Réunion" for the 1980s and 1990s,INSEE, and IEDOM for the recent years.

Relative prices:

Data on imports and exports prices of advanced economies are extracted from IFS data, IMF.

A3.2 ADF unit root tests

We test the presence of a unit root in variables of the model using the ADF test, over the period 1980-2011, i.e. on 32 years.

Appendix 4: Relationship between GDP per capita, productivity and transfers

To explain GDP per capita we adopt a model where the explanatory variables are the transfers and the global productivity of economy. Preliminary unit root tests conclude that the GDP per capita and the global productivity are non stationary while the transfer ratio variable is trendstationary. Since the explanatory variables are I(0) and I(1) we adopt the ARDL approach [START_REF] Pesaran | Bounds testing approaches to the analysis of level relationships[END_REF] to estimate the level relationship between the real GDP, the transfers and the productivity. Considering the small size of the sample (32 observations), we retain one lag for each explanatory variable. The model is:


Frédéric Cosnier
email: frederic.cosnier@inrs.fr

Stéphane Grossmann

Hervé Nunge

Céline Brochard

Samuel Muller

Anne-Marie Lambert-Xolin

Sylvie Sébillaud

Benoît Rieger

Aurélie Thomas

Marie-Josèphe Décret

Sylvie Sebillaud

Manuella Burgart

Laurent Gaté

Benoît Cossec

Pierre Campo

Metabolism of inhaled methylethylketone in rats

Keywords: Methylethylketone, 2-butanone, metabolism, inhalation, rat, 2, 3-butanediol

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

Methylethylketone (MEK) (or 2-butanone) (CAS n° 78-93-3) is commonly used as a solvent in the manufacture of adhesives and coatings (especially vinyl, nitrocellulose and acrylic), dewaxing agents of mineral oils, and extraction agents for foodstuffs, and also as a synthesis intermediate (INRS, 2009). In these contexts, it is often associated with other substances [START_REF] Yoshikawa | Biological monitoring of occupational exposure to methyl ethyl ketone in japanese workers[END_REF]. MEK exposure alone shows low acute and subchronic toxicity but, when combined with other substances, it can make the other chemicals harmful [START_REF] Yang | The toxicology of methyl ethyl ketone[END_REF]ATSDR, 1992;INRS, 2009). Indeed, it has been demonstrated that MEK potentiates the hepatotoxic effects of carbon tetrachloride [START_REF] Dietz | Potentiation of ccl4 of hepatotoxicity in rats by a metabolite of 2-butanone: 2,3-butanediol[END_REF], and the neurotoxicity of ethanol, n-hexane, ethyl-n-butyl ketone (EnBK) and methyl-n-butyl ketone (MnBK) [START_REF] O'donoghue | Further studies on ketone neurotoxicity and interactions[END_REF][START_REF] Yang | The toxicology of methyl ethyl ketone[END_REF][START_REF] Noraberg | Neurotoxic interactions of industrially used ketones[END_REF].

Recently, it has been demonstrated that the presence of MEK, at concentrations consistent with those found in industrial environments, can modify the metabolism of toluene, an aromatic solvent known to be neurotoxic and ototoxic (ATSDR, 2015). The interaction between the two solvents was notably linked to an increase in the blood toluene concentration in rats co-exposed to the solvents by inhalation [START_REF] Cosnier | Impact of coexposure on toluene biomarkers in rats[END_REF]. However, the nature of this interaction and its impact on MEK metabolism or urinary elimination have yet to be fully elucidated. Occupational exposure to MEK is generally bio-monitored by measuring the MEK concentration in exposed workers' blood (at the end of a work shift), or in the urine (for high exposures) [START_REF] Miyasaka | Biological monitoring of occupational exposure to methyl ethyl ketone by means of urinalysis for methyl ethyl ketone itself[END_REF][START_REF] Ong | Biological monitoring of occupational exposure to methyl ethyl ketone[END_REF][START_REF] Sia | Environmental and biological monitoring of methyl ethyl ketone (mek)[END_REF][START_REF] Kawai | Methyl isobutyl ketone and methyl ethyl ketone in urine as biological markers of occupational exposure to these solvents at low levels[END_REF].

Before assessing the impact of the interaction between toluene and MEK on short and medium-term excretions of various biomarkers, thorough investigation on the metabolism of MEK was necessary.

Previous investigations established that MEK can be absorbed through the skin and by inhalation. In rodents, MEK is reduced by alcohol dehydrogenase to 2-butanol (2BuOH) and oxidised to 3-hydroxy-2-butanone (OHMEK), presumably by cytochrome P450-dependent monooxygenases. OHMEK is then reduced to 2,3-butanediol (23BD). These metabolites can be excreted in the urine, as O-glucuronides or O-sulfates, or may enter the intermediary metabolism to be eliminated as CO 2 or incorporated into tissues (ATSDR, 1992) (Figure 1). These pathways were described based on observations made after i.p. injection of guinea pigs with MEK (DiVincenzo et al., 1976) and its oral administration in rats [START_REF] Dietz | Pharmacokinetics of 2-butanol and its metabolites in the rat[END_REF]. To the best of our knowledge, no study has investigated the excretion of MEK metabolites in animals after inhalation exposure. In humans, few investigations have studied 2-butanone metabolism. However, OHMEK and 23BD have been detected in human urine after inhalation exposure [START_REF] Perbellini | Methyl ethyl ketone exposure in industrial-workers -uptake and kinetics[END_REF]Liira et al., 1988b;Liira et al., 1990a).

To complete what is known about MEK metabolism, this study investigated the fate of MEK in rats after inhalation exposure for 1 month by analysing blood, brain and urine samples. To simultaneously determine the concentrations of urinary metabolites of MEK (MEK, 2BuOH, OHMEK and 23BD), a specific method had to be develop. The hepatic metabolism of MEK by measuring cytochrome P450 (CYP) concentrations, CYP1A2, CYP2E1 and glutathione Stransferase (GST) activities, as well as the GSH/GSSG balance was also investigated. Among the various cytochromes, a special attention has been brought to CYP2E1 and CYP1A2 since they are mainly involved in the metabolism of carbon tetrachloride, ethanol, hexane and aromatics; solvents which could be found in mixtures with MEK.

Materials and methods

Animals

4-week-old male Brown Norway rats were purchased from Janvier Labs (Le Genest St Isle,St Berthevin,53941,France) and weighed between 70 and 90 g on arrival at the animal facility.

Before exposure, all animals were acclimatised for at least 2 months with a 12:12 h day/night cycle in polycarbonate cages containing irradiated cellulose BCell8 bedding (ANIBED, Route de Lude, 72510 Pontvallain. A04 irradiated pelleted food (SAFE, Augy, France) and tap water were available ad libitum. The temperature in the animal facility was 22 ± 2 °C and the relative humidity was 55 ± 10%. Animal experiments were performed in line with ethical regulations on animal experimentation (European Directive 2010/63/EU) in a French Ministry of Agriculture-accredited animal facility (Accreditation D54-547-10). The present study was approved by the local ethics committee and was registered as CELMEA-2012-0029.

Test material

Methylethylketone puriss. p.a., ACS reagent, ≥99.5% (GC) (Fluka ref # 04380) was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). All other chemicals used were of analytical grade.

Inhalation exposure

Groups of rats (~ 250 g at the beginning of the exposure) were exposed to MEK vapours using a whole-body exposure system. Animals were exposed to 20, 200 or 1400 ppm for 6 hours/day (from 09:00 am to 03:00 pm), 5 days/week for 4 weeks in 200 L glass/stainless steel inhalation chambers. The 20, 200 and 1400 ppm MEK concentrations were chosen because they correspond to 1/10 and to the French 8-hour occupational exposure limit and to the maximum level of exposure detected in French industries between 2000 and 2010 (based on the INRS Solvex database), respectively. Unexposed rats (controls) were maintained in similar chambers, but exposed to clean air only. During the 6-hour exposure period, the rats did not have access to food or water. All the exposure parameters: temperature (22 ± 2 °C), humidity (55 ± 10%), air flow in the inhalation chambers, test substance concentrations, etc.

were determined in accordance with OECD guideline No. 412 to test chemical toxicity (Subacute Inhalation Toxicity: 28-Day Study. Details of how test substance vapours were generated and concentrations controlled can be found in [START_REF] Cosnier | Impact of coexposure on toluene biomarkers in rats[END_REF].

For each exposure condition (MEK20, MEK200 and MEK1400), 3 groups of rats were investigated. Urine was collected from the first group exposed to MEK (n = 12); blood was collected and organs were removed (in particular liver, brain and kidneys) from the second group exposed to MEK (n = 2×8); blood and organs were also collected from the third group, control animals (n = 2×8).

Urine collection

Five time-points were considered for urine collection: the first sample was taken 3 days before the start of inhalation exposure (control time = d0) and subsequent samples were taken after the first (d1), the fifth (d5), the tenth (d10) and the twentieth (d20) days of exposure.

Animals were placed in individual metabolic-type stainless steel cages with free access to food and water from 3:00 pm to 9:00 am the following day. During this 18-h period, urine was collected and refrigerated (by precaution) by a cooling system surrounding the collection tubes. Urine samples were frozen immediately after collection and stored at -20 °C. They were thawed just before analysis.

Blood and organ collection

Blood samples were collected from eight rats each from the second and third groups, at d1 and d5, and from the remaining eight rats per group at d10 and d20. Rats were removed one by one from the inhalation chamber using an airlock while atmospheric concentrations were maintained constant. Blood collection was performed and completed on one rat before taking another animal from the chamber. Briefly, blood was collected from the tail vein of rats anaesthetised with isoflurane; sampling was completed within 5 min (3.5 min in mean). Blood samples ( 0.8 mL) were dispensed into 2-mL heparinised (30 µL/150 UI) vials and frozen at -20 °C.

At d5 and d20, once blood collection had been completed, rats were sacrificed following a deep anaesthesia induced by i.p. injection of a ketamine/xylazine mixture (45mg/kg and 5 mg/kg, respectively). Anaesthetised animals were then intracardially perfused with heparinised physiological saline, exsanguinated and dissected to retrieve organs, including the liver. Organs were frozen in liquid nitrogen immediately after collection prior to storage at -80 °C.

Urinary analysis

Creatinine concentrations in urine were determined by the Jaffe method using a creatinine test kit (Randox Laboratories, Roissy en France, France). For the purposes of this study, creatinine results were primarily used to identify or explain possible extreme values of MEK metabolites concentrations.

Urinary metabolites of MEK, whether in free or glucuroconjugated forms, were determined by a method specifically developed for this study. The method developed was based on the works of [START_REF] Kezic | Determination of methyl ethyl ketone and its metabolites in urine using gas-chromatography[END_REF][START_REF] Kezic | Gas-chromatographic determination of 2,3butanediol isomers in urine[END_REF] and can simultaneously measure the five metabolites: MEK, 2BuOH, OH-MEK, ±23BD and meso23BD.

Sample preparation

Frozen urine samples were allowed to thaw and equilibrate to room temperature. Samples were agitated and 300-μL aliquots were transferred into 2-mL Eppendorf Safe-Lock microcentrifuge tubes. Two internal standards: 1-butanol (50 µL, 0.54 g/L) and 1,2-butanediol (12BD) (50 µL, 0.06 g/L) were added, and the total volume was adjusted to 500 µL with ultrapure water. Derivation of 12BD and 23BDs in samples was performed in a few seconds by adding a solution of 4-bromophenylboronic acid in dichloromethane (DCM) (3.2 g/L). 900 µL of DCM was then added. The mixture was vortex-mixed for 5 sec and centrifuged at 10 000 rpm for 3 min before collecting the organic layer. The sample was then ready for GC-MS/MS analysis. All samples were analyzed in duplicate.

To determine total metabolites (free plus conjugated), an enzymatic hydrolysis step was added prior to the derivation step. For this, β-Glucuronidase from Helix pomatia (type HP-2, aqueous solution,  100 000 units/mL) (30 µL) and phosphate buffer pH = 4.7 (170 µL, solution of Na 2 HPO 4 /NaH 2 PO 4 , 0.18/13.7 g/L) were added to defrosted urine samples. The mixtures were incubated at 37 °C for 18 hours.

Gas chromatography

Gas chromatography (GC) was performed on a 450-GC system (Bruker, Wissembourg, France) equipped with a CP-8400 autosampler (Bruker). Compounds were separated on an Rxi-17 MS (Restek, France) capillary column (30 m length × 0.25 mm I.D. × 0.25 µm film thickness) using helium as the carrier gas at a constant flow of 1.0 mL/min. The sample (1 µL) was injected in splitless mode (injector type 1177). Injection temperature was 300 °C.

The split delay and split ratio were set at 0.2 min and 1/30, respectively. The oven temperature was held at 40 °C for 3.0 min, then increased to 300 °C at a rate of 20 °C/min (total time: 16 min). The transfer line was maintained at 250 °C throughout.

Tandem mass spectrometry

GC-MS/MS analyses were performed on a 320 triple quadrupole mass spectrometer (Bruker, Wissembourg, France) in electronic impact (EI) mode. The ion source temperature was set to 300 °C with ionization energy at 70 eV. Mass spectra were acquired in selected reaction monitoring (SRM) mode using argon 99.9999% (Air Liquide) as collision gas at a pressure of 2.0 mtorr in the collision cell. The source was flushed with helium gas for the first 1.6 min of the injection. To ensure selectivity, two SRM ion transitions were chosen per compound, when possible based on their abundance. Selecting two transitions maximised the signal-tonoise ratio in matrix extracts. One transition was used as quantifier and the second as a qualifier. Dwell time and collision energy were set at 0.1 s and 10 eV, respectively, for all transitions.

Details regarding which SRM transitions were used for each compound are provided in Table 1.

The main performance characteristics of the method (validated linearity ranges, limits of detection (LOD), limits of detection (LOQ), within-run and between-run precisions and recoveries) are summarised in Table 2.

Blood and brain analysis MEK and 2BuOH concentrations in blood and brain were determined using a similar protocol involving liquid/liquid extraction of the tissues (800 µL of blood or half a brain) in DCM (500 or 750 µL for blood or brain samples, respectively) using n-propylacetate as an internal standard (5 µL, 1.5 mg/L in DCM). For brain samples, a homogenate was first prepared by grinding the brain in tubes containing ceramic beads (Fast Prep instrument, MP Biomedicals, with Lysing Matrix D tubes). After 30 min agitation, samples were centrifuged at 1250 rpm, 4 °C for 10 min. The lower DCM layer contained the MEK and the 2BuOH. 0.3 mL of this layer was transferred to a vial containing a glass insert. The sample (1 µL) was then ready for GC-MS analysis using the chromatographic conditions described in [START_REF] Cosnier | Impact of coexposure on toluene biomarkers in rats[END_REF].

In these conditions, the LODs (and LOQs) for MEK were 0.018 (and 0.054) µg/g in blood and 0.060 (and 0.180) µg/g in brain. For 2BuOH, the LOD (and LOQ) was 0.024 (and 0.072) µg/g in blood (this compound was not investigated in brain).

Liver assays

Biochemical assays were carried out on liver homogenates (reduced (GSH) and oxidised glutathione (GSSG)), liver microsomes (total cytochrome P450 (CYP) level and CYP1A2 and CYP2E1 activities), and cytosolic extracts (activity of GSTs).

GSH was measured by a colorimetric method using 5,5'-dithiobis 2-nitrobenzoic acid. GSH was blocked with 2-vinylpyridine, then its oxidised form (GSSG) could be measured following reduction by glutathione reductase [START_REF] Griffith | Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine[END_REF].

The total cytochrome P450 level was determined by the [START_REF] Omura | The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature[END_REF] method; this method is based on the absorption difference at 450 nm between the reduced form of the pigment and the carbon monoxide-conjugated form in the presence of dithionite.

CYP2E1 activity was determined according to the method described by [START_REF] Koop | Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome p-450 isozyme 3a[END_REF], based on the (colorimetric) measurement at 456 nm of 4-nitrocatechol obtained from p-nitrophenol after oxidation (hydroxylation) by CYP2E1. CYP1A2 activity was measured in a similar manner by monitoring the O-deethylation of ethoxyresorufin.

GST was measured according to the method developed by [START_REF] Habig | Glutathione s-transferases. The first enzymatic step in mercapturic acid formation[END_REF] using 1-chloro-2,4 dinitrobenzene as substrate.

For assays of GSH, GSSG, CYP or GST, the amounts measured were normalised based on the total protein content measured in the different samples by the Lowry assay [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF]). Except for GSH and GSSG, all the assays discussed above were carried out on a fully automated random access bench-top clinical chemistry analyzer (Randox RX-Daytona).

Statistical analysis

All statistical analyses were performed using Statgraphics Centurion XVI Software (Version 16.2.04) (StatPoint Technologies, Inc.). The threshold for statistical significance was set at 5%. For the analysis of results, different statistical procedures were implemented. For blood and urine data, analyses were performed using a general linear model including fixed effects (the MEK exposure level and/or the collection day), and a random effect (subject) to take the non-independent nature of the data into account. The interaction between the two fixed effects was also tested. Post-hoc tests were performed using the Bonferroni method. Atmospheric, blood and urinary concentrations are presented in the tables in the form arithmetic mean ± standard deviation (unless indicated otherwise). Analyses of variance of two factors (group and day) were used to analyse brain and liver data (group = exposed or control, day = d5 or d20).

Results

Animal and organ weights

Exposure to MEK did not affect weight-gain in animals (~ 340 g at the end of the exposures); in addition, the weights of the brains, lungs and kidneys of animals exposed by inhalation were no different from control animals (data not shown). However, the relative weights of livers (as a ratio of body weight) were significantly lower in exposed animals compared to controls. This difference was stable (-10.5 ± 2.8%) and did not depend on the atmospheric concentration of MEK nor the day of sacrifice (d5 or d20).

Blood and brain concentrations

The blood concentration for MEK (MEK-B) increased with its atmospheric concentration (MEK-A), from ~ 0.20 µg/g for MEK20 to ~ 162 µg/g for MEK1400 (Table 3). Except for MEK1400 between d1 and d20, no significant day-effect was identified based on paired t-test and Bonferroni correction. MEK-B was thus quite stable during the inhalation period.

Classical linear (y = mx + c) and multiplicative (logy = mlogx + c) models failed to describe the relation between MEK-B and MEK-A.

In contrast to MEK, its metabolite 2BuOH was only quantifiable in blood with exposure to MEK1400 (Table 3). When detected, the 2BuOH concentration (~ 2.4 µg/g) was 68 times lower than the MEK concentration.

The MEK brain concentration (MEK-C) was found to increase proportionally to MEK-B (Table 3), but was quantifiable only for MEK200 at d5 and for MEK1400 at d5 and d20. The concentrations at these time-points were between 6 and 3 times lower that those measured in the blood. However, it should be noted that brain levels of MEK were significantly higher at d20, suggesting accumulation over time.

Metabolite assay in urine

The LODs of the analytical method for a 300-µL aliquot of urine were 0.70, 0.26, 0.50, 0.11 and 0.29 mg/L for 2BuOH, MEK, OHMEK, 23BD and meso23BD, respectively (Table 2). These limits were lower than those reported by [START_REF] Kezic | Determination of methyl ethyl ketone and its metabolites in urine using gas-chromatography[END_REF][START_REF] Kezic | Gas-chromatographic determination of 2,3butanediol isomers in urine[END_REF]; expressed in terms of minimum detectable quantities (taking into account the volume of urine used), the method developed for this study presented a 3.5-, 8-, 6-, 15-and 6fold gain in sensitivity for 2BuOH, MEK, OHMEK, 23BD and meso23BD, respectively.

Compared to methods described in the literature to assay MEK, OHMEK or 23BDs individually [START_REF] Van Doorn | Determination of methyl ethyl ketone in human urine after derivatization with o-nitrophenylhydrazine, using solidphase extraction and reversed-phase high-performance liquid chromatography and ultraviolet detection[END_REF]Otsuka andOhmori, 1992, 1994), in the worst case the current method was 5, 12 or 13 times less sensitive, respectively. This loss in sensitivity could be related to the fact that these individual assays used larger volumes of urine and more sophisticated techniques: headspace, micro-solid phase extraction, oxidation and derivatisation steps or used a specific detector [START_REF] Miyasaka | Biological monitoring of occupational exposure to methyl ethyl ketone by means of urinalysis for methyl ethyl ketone itself[END_REF][START_REF] Gori | Highperformance liquid-chromatographic determination of methyl ethyl ketone in urine as its 3-methyl-2-benzothiazolinone hydrazone derivative[END_REF][START_REF] Chou | Detection of methyl ethyl ketone in urine using headspace solid phase microextraction and gas chromatography[END_REF][START_REF] Antunes | Determination of methyl ethyl ketone in urine samples by headspace solid phase micro extraction (spme) sampling associated to gas chromatography with flame-ionisation detector (gc-fid)[END_REF][START_REF] Pacenti | New automated and high-throughput quantitative analysis of urinary ketones by multifiber exchangesolid phase microextraction coupled to fast gas chromatography/negative chemicalelectron ionization/mass spectrometry[END_REF].

In rats exposed to MEK20 and MEK200, free 2BuOH, MEK and OHMEK were not present in measurable amounts in their urine; in contrast, exposure to MEK1400 triggered a marked increase in the amount of these free metabolites (Table 4). For MEK itself, the increase was spectacular: lower than 0.26 mg/L at MEK200 to 31.9 mg/L (on average) at MEK1400. 23BDs were detectable from endogenous sources in urine from unexposed animals (Table 4).

At MEK20, the levels excreted by exposed animals were not statistically different from those excreted by controls, however, at MEK200 and at MEK1400 the difference was significant.

No day-effect was observed with MEK200 in terms of excretion, which remained stable over time; however, excretion of 23BDs after exposure to MEK1400 gradually increased between d1 and d20. 23BDs were excreted with a mean distribution ratio of 1:3 between 23BD and meso23BD forms (1:5 for endogenous excretion). At 1400 ppm, combined excretion for the two forms was 8 to 22 times higher (depending on the day) than MEK excretion (31.6 mg/L); OHMEK (~ 3.1 mg/L) and the 2BuOH (~ 1.5 mg/L) followed in order of abundance (Table 4).

Assays of the total (free + conjugated) metabolite concentrations (at d0, d1 and d5) after hydrolysis of urine revealed that the proportion of free form was metabolite-dependent. Indeed, most OHMEK (~ 78%) and half the 2BuOH were conjugated; in contrast, more than 85% of urinary MEK was unconjugated. With 23BDs, the endogenous fractions (measured at d0) increased when urine was hydrolysed and were not homogenous (between animals in the MEK20, MEK200 and MEK1400 groups). This effect complicated the analysis of results.

The proportion of conjugated forms of ±23BD related to exposure appeared to decrease with the exposure level. Thus, at MEK20, conjugated ±23BD was a major form, whereas at MEK1400, it was negligible compared to the free form (Figure 2). For meso23BD, this phenomenon was even more pronounced: more than half of the total amount excreted was in the free form at MEK20 and MEK200, and more than 90% at MEK1400.

Liver assays

No statistically significant difference in the total CYP concentration between exposed and controls groups was found for any MEK concentration, or day considered (520 pmol/mg protein on average). In contrast, CYP1A2 and CYP2E1 activities measured in the liver of exposed animals were almost always higher than those measured in control livers (16 pmol/min/mg protein for γCYP1A2 and 230 pmol/min/mg protein for γCYP2E1) (Figure 3).

For both CYP isoforms, a dose/effect relationship was observed; the day-effect was however not statistically significant.

In addition, GST activity was altered only with MEK200 and MEK1400 (data not shown), where the concentrations observed in exposed animals were less than those of controls.

Finally, with regard to the GSH concentrations, controls always had a higher concentration than treated animals. This phenomenon was less pronounced with GSSG.

Discussion

A limited number of studies describing the uptake, distribution, metabolism, and elimination of MEK in experimental animals and humans have been published. This study is the first to explore the absorption and metabolism of MEK in rats following inhalation exposure through the measurement of some hepatic parameters and its elimination in the urine.

As previously shown by [START_REF] Liira | Metabolic interaction and disposition of methyl ethyl ketone and meta-xylene in rats at single and repeated inhalation exposures[END_REF], after a 600-ppm inhalation for 6 hours over 1 day or for 6-10 hours/day over 8 days, MEK does not appear to accumulate in the blood based on the duration of exposure. Thus, blood concentrations were similar after 6-hour or 1-month inhalation periods. In addition, blood concentrations measured at 200 ppm (~ 1.8 µg/g) or 1400 ppm (~ 160 µg/g) were consistent (not equals but same order of magnitude) with those found in rats exposed to 600 ppm (~ 1090 µmol/L = 78 µg/g) or in humans exposed for 4 hours at the same concentration (3.5 -7.2 µg/mL  3.3 -6.8 µg/g) (Liira et al., 1988b). It should be mentioned that in our case the blood concentrations measured are not at "steadystate" but at the very beginning of the declining phase (around 3.5 min after the end of exposure) knowing the fact that half-life of MEK in blood is 30 min (Liira et al., 1988b).

As expected from solubility data for MEK in various tissues [START_REF] Perbellini | Methyl ethyl ketone exposure in industrial-workers -uptake and kinetics[END_REF][START_REF] Thrall | Development of a physiologically based pharmacokinetic model for methyl ethyl ketone in f344 rats[END_REF], [START_REF] Liira | Metabolic interaction and disposition of methyl ethyl ketone and meta-xylene in rats at single and repeated inhalation exposures[END_REF] reported a constant MEK concentration in perirenal fat (~ 0.70 µmol/g = 50 µg/g). In the present investigation, a similar concentration was found in brain after 20 days' exposure at 1400 ppm. However, we did observe a slight increase in brain concentrations between d5 and d20 at MEK200 and MEK1400; thus, a slight accumulation in this organ over time cannot be excluded.

The strong increase in MEK blood concentration between MEK200 and MEK1400 was probably the result of metabolic saturation, as reflected by the appearance of 2BuOH in urine after exposure to MEK1400. Indeed, using F344 male rats exposed to 100-2000 ppm MEK in a closed, recirculating gas uptake system, [START_REF] Thrall | Development of a physiologically based pharmacokinetic model for methyl ethyl ketone in f344 rats[END_REF] demonstrated that MEK is metabolised by a process which is both a saturable and first-order. Using physiologically relevant pharmacokinetic model simulations (PBPK) for 8-hour exposures, Liira et al. (1990a) estimated that this metabolic saturation would occur in humans at 100 ppm at rest and at 50 ppm during exercise.

As in humans (Liira et al., 1988b), 23BD is the major metabolite in rat urine following inhalation. It exists under two forms: ±23BD and meso23BD (with a mean distribution ratio of 1:3). 23BD are also produced endogenously and excreted in urine. This endogenous production is related to pyruvate (a product of glycolysis) metabolism in mammals [START_REF] Montgomery | Assay of physiological levels of 2,3butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry[END_REF]. Other metabolites: 2BuOH and OHMEK, but also MEK itself, were only detectable in urine at the highest concentration investigated (1400 ppm = 7 ×8h-OEL) when metabolic saturation occurred; therefore, these metabolites should not be present except in accidental conditions. This represents a difference between humans and rats, because MEK and OHMEK have been detected in urine from humans exposed to normal occupational levels [START_REF] Miyasaka | Biological monitoring of occupational exposure to methyl ethyl ketone by means of urinalysis for methyl ethyl ketone itself[END_REF][START_REF] Perbellini | Methyl ethyl ketone exposure in industrial-workers -uptake and kinetics[END_REF]Liira et al., 1988a;Ong et al., 1991;[START_REF] Sia | Environmental and biological monitoring of methyl ethyl ketone (mek)[END_REF]. Another difference between human and rat metabolism and excretion lies in the fact that the glucuronide or sulfate form of these metabolites does not appear to be a major metabolic pathway in rats.

If we consider the rat minute ventilation equal to 110 mL/min [START_REF] Strohl | Ventilation and metabolism among rat strains[END_REF], the amount of MEK inhaled during a 6-h exposure would correspond to 330 and 2307 µmol for MEK200 and MEK1400 exposure, respectively. The corresponding amounts of MEK and urinary metabolites excreted (free or total) during the 18-h post-exposure period (considering an average diuresis of 7.5 mL) were less than 1.5 and 56 µmol, respectively. Thus, the urinary excretion measured accounted for less than 0.45% (at MEK200) and 2.4% (at MEK1400) of the total amount inhaled, or 0.9% and 4.8% of the absorbed MEK if pulmonary uptake was equivalent to that measured in humans (~50%) (Liira et al., 1988a;Liira et al., 1988b;Liira et al., 1990b). These data are consistent with data from human studies [START_REF] Perbellini | Methyl ethyl ketone exposure in industrial-workers -uptake and kinetics[END_REF]Liira et al., 1988b;Liira et al., 1990b) and are clearly different from those obtained in rats after oral administration, where about 30% of the dose administered was converted to 2,3butanediol, 4% was converted to 2-butanol, and 4% was converted to 3-hydroxy-2-butanone [START_REF] Dietz | Pharmacokinetics of 2-butanol and its metabolites in the rat[END_REF].

In contrast to what was observed by [START_REF] Toftgard | Changes in rat liver microsomal cytochrome p-450 and enzymatic activities after the inhalation of n-hexane, xylene, methyl ethyl ketone and methylchloroform for four weeks[END_REF] and [START_REF] Cavender | A 90-day vapor inhalation toxicity study of methyl ethyl ketone[END_REF] after exposure of rats to 800-ppm MEK for 5 weeks and 5000 ppm for 12 weeks, respectively, in our hands, a slight decrease in absolute and relative liver weight was observed in exposed animals. This decrease in liver weight was associated with a slight induction of the enzymatic activity of the two CYP isoforms investigated (CYP1A2 and CYP2E1). However, MEK exposure (even at 1400 ppm) was not found to alter the total cytochrome P450 concentration.

Until now, no changes had been observed in CYP isozyme profiles after MEK inhalation [START_REF] Toftgard | Changes in rat liver microsomal cytochrome p-450 and enzymatic activities after the inhalation of n-hexane, xylene, methyl ethyl ketone and methylchloroform for four weeks[END_REF], and only few studies had shown that MEK could induce microsomal liver enzymes in rats after acute oral exposure [START_REF] Robertson | Effects of methyl ethyl ketone pretreatment on hepatic mixed-function oxidase activity and on in vivo metabolism of n-hexane[END_REF][START_REF] Raunio | Cytochrome p450 isozyme induction by methyl ethyl ketone and m-xylene in rat liver[END_REF]ATSDR, 1992) or after acute inhalation of high MEK concentrations (≥ 1500 ppm) [START_REF] Brondeau | Acetone compared to other ketones in modifying the hepatotoxicity of inhaled 1,2-dichlorobenzene in rats and mice[END_REF].

In addition to these enzymatic effects, MEK exposure induced a decrease in the liver glutathione concentration (both reduced and oxidised forms) as well as in GST activity (at exposure levels over 200 ppm). For [START_REF] Liira | Metabolic interaction and disposition of methyl ethyl ketone and meta-xylene in rats at single and repeated inhalation exposures[END_REF], this decrease could be caused by production of thioethers. However, our preliminary results have found no evidence for the formation of mercapturic acid.

These metabolic data suggest that MEK could influence the metabolism (and potentiate the toxicity) of other substances, such as, for example, aromatic hydrocarbons which are primarily metabolised through oxidation by CYP1A2 and CYP2E1, but can also be eliminated via the glutathione pathway (Liira et al., 1988a;[START_REF] Raunio | Cytochrome p450 isozyme induction by methyl ethyl ketone and m-xylene in rat liver[END_REF][START_REF] Liira | Metabolic interaction and disposition of methyl ethyl ketone and meta-xylene in rats at single and repeated inhalation exposures[END_REF][START_REF] Uaki | Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats[END_REF][START_REF] Cosnier | Impact of coexposure on toluene biomarkers in rats[END_REF]. This work provides the foundation for future studies related to interactions with MEK. Another benefit of this study is to provide blood and urine data that could be useful for pharmacokinetic model development/and or verification. *medians are given in brackets when multiple measurements were between the limits of detection and the limits of quantification (= 3 × LODs) = 2.1, 0.8, 1.5, 0.3 and 0.9 mg/L for 2BuOH, MEK, OHMEK, 23BD and meso23BD, respectively.

Figure 1 .Figure 3 .

 13 Figure 1. MEK metabolism, adapted from DiVincenzo et al.(1976) and[START_REF] Dietz | Pharmacokinetics of 2-butanol and its metabolites in the rat[END_REF]

Table 2 .

 2 Performance parameters for the analytical method developed to simultaneously measure urinary metabolites

		Linearity range* (mg/L)	LOD (mg/L)	Within-run precision (%RSD)	Between-run precision (%RSD)	Recovery (%)
	2BuOH	2.1 -30	0.70	≤ 12.0	≤ 12.6	87 ± 18
	MEK	0.8 -50	0.26	≤ 12.1	≤ 15.8	95 ± 12
	OHMEK	1.5 -25	0.50	≤ 11.5	≤ 15.6	87 ± 13
	23BD	0.3 -120	0.11	≤ 3.3	≤ 12.3	66 ± 11
	m23BD	0.9 -500	0.29	≤ 4.6	≤ 9.2	79 ± 9
	* The lower limit corresponds to the limit of quantification (LOQ) = 3 × LOD

Table 3 .

 3 Atmospheric (MEK-A), blood (MEK-B and 2BuOH-B) and brain (MEK-C) concentrations in µg/g (mean ± standard deviation or median*)

Table 4 .

 4 Concentrations of urinary metabolites (free or total) in mg/L (mean ± standard deviation or median*)

			Day	2BuOH	MEK	OHMEK	±23BD	meso23BD
			d0				[0.25]	1.75 ± 0.37
			d1				0.59  0.17	2.52  0.47
		MEK20	d5				[0.35]	1.89  0.37
			d10				[0.34]	1.81  0.27
			d20				0.42  0.10	2.44  0.63
			d0				[0.24]	1.83 ± 0.66
			d1				2.04  0.97	7.24  2.55
	Free	MEK200	d5				2.43  0.49	8.26  2.68
			d10				2.48  0.71	7.30  3.48
			d20				2.05  0.40	6.47  2.91
			d0				[0.21]	1.42 ± 0.33
			d1	[1.69]	34.7  15.8	[1.85]	59.0  22.4	223.8  71.2
		MEK1400	d5	[1.41]	33.6  8.37	2.76  0.85	93.2  29.6	335.5  87.4
			d10	[1.34]	30.7  6.80	4.30  1.38	146.5  43.8	447.8  93.6
			d20	[1.07]	27.7  14.2	3.25  0.88	153.1  32.7	465.9  27.4
			d0				1.62 ± 0.81	1.55 ± 0.64
		MEK20	d1				3.46 ± 1.36	2.76 ± 0.76
			d5				3.34 ± 0.73	1.84 ± 0.42
			d0			[0.75]	7.43 ± 3.06	6.37 ± 1.90
	Total	MEK200	d1			[0.97]	12.4  5.5	16.6  5.2
			d5			[1.17]	11.2  4.0	16.2  2.4
			d0			[1.16]	12.1 ± 1.97	8.37 ± 2.80
		MEK1400	d1	[3.15]	41.0  20.2	7.91  5.00	87.7  30.4	244.0  96.2
			d5	[3.53]	37.7  9.4	12.4  8.30	170.2  58.1	378.3  124.3

Acknowledgements

The authors gratefully acknowledge Élodie Bonfanti, Chantal Cour, Lionel Dussoul, Jean-Claude Gondrexon, Jean-Claude Micillino and Sylvie Michaux for their technical assistance.

Declaration of interest

This work was entirely supported by the French Research and Safety Institute for the Prevention of Occupational Accidents and Diseases (INRS, France). The authors report no declaration of interest. # based on three measurements during the 6-h exposure * medians are given in brackets when multiple measurements were between the limits of detection and the limits of quantification (=3 × LODs) = 0.054, 0.072 and 0.18 µg/g for MEK-B, 2BuOH-B and MEK-C, respectively.