INERIS Ghislaine Lacroix
email: ghislaine.lacroix@ineris.fr.

Sylvie Tissot

Françoise Rogerieux

Rémy Beaulieu

Line Cornu

Chantal Gillet

Franck Robidel

Jean-Pierre P Lefevre

Frédéric Y Bois

Decrease in ovalbumin-induced pulmonary allergic response by benzaldehyde but not acetaldehyde exposure in a guinea pig model

Keywords: Acetaldehyde, Benzaldehyde, Inhalation toxicology, Guinea pigs, Ovalbumin-sensitization, Respiratory functions, Allergy, Inflammation

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INTRODUCTION

Carbonyl compounds, particularly aldehydes, are receiving increasing attention as air pollutants because of their potential as irritants and carcinogens [START_REF] Grafstrom | In vitro studies of aldehyde effects related to human respiratory carcinogenesis[END_REF][START_REF] Leikauf | Mechanisms of aldehyde-induced bronchial reactivity: role of airway epithelium[END_REF][START_REF] Morris | Dosimetry, toxicity and carcinogenicity of inspired acetaldehyde in the rat[END_REF].

Aldehydes are fairly common primary compounds, which derive from stationary sources (incineration and home wood fire) or mobil sources (diesel, internal combustion and jet engine emissions), but they are more frequently secondary pollutants in so far as they are almost obligatory intermediates in the photo-oxidation of organics compounds in the atmosphere [START_REF] Carlier | The chemistry of carbonyl compounds in the atmosphere -A review[END_REF].

Aldehydes are present both outdoors and indoors with concentrations inside often higher than outside [START_REF] Reiss | Measurement of organic acids, aldehydes, and ketones in residential environments and their relation to ozone[END_REF]. Approximately 50 percent of the total aldehyde burden is formaldehyde, which can range from 10 to 30 ppb in heavily contaminated air masses [START_REF] Leikauf | Mechanisms of aldehyde-induced bronchial reactivity: role of airway epithelium[END_REF]. Estimates for other aldehydes indicate that another 5 to 10 percent of the total aldehyde burden is acrolein, which ranges from 5 to 30 ppb during peaks [START_REF] Leikauf | Mechanisms of aldehyde-induced bronchial reactivity: role of airway epithelium[END_REF]. The remaining 40 to 45 percent consists of other aldehydes such as acetaldehyde [START_REF] Leikauf | Mechanisms of aldehyde-induced bronchial reactivity: role of airway epithelium[END_REF][START_REF] Reiss | Measurement of organic acids, aldehydes, and ketones in residential environments and their relation to ozone[END_REF] and, to a lesser extent, propanal, butanal and benzaldehyde [START_REF] Reiss | Measurement of organic acids, aldehydes, and ketones in residential environments and their relation to ozone[END_REF].

The toxicity of inhaled formaldehyde [START_REF] Bardana | Formaldehyde: an analysis of its respiratory, cutaneous, and immunologic effects[END_REF]IARC, 1995b;[START_REF] Mclaughlin | Formaldehyde and cancer: a critical review[END_REF] and acrolein [START_REF] Ghilarducci | Fate and effects of acrolein[END_REF]IARC, 1995a;[START_REF] Kehrer | The molecular effects of acrolein[END_REF] is well documented, but less data are available on inhaled acetaldehyde and practically no studies on other aldehydes such as benzaldehyde. In humans, acute exposure to acetaldehyde vapors resulted in irritation of the eyes and mucous membranes, reddening of the skin, pulmonary edema and sore throat [START_REF] Iarc | Acetaldehyde[END_REF]. Repeated exposure causes dermatitis and conjunctivitis [START_REF] Iarc | Acetaldehyde[END_REF]. Acute, subchronic and chronic exposure to acetaldehyde produces respiratory tract injury in the rat, especially in the nasal cavities [START_REF] Morris | Dosimetry, toxicity and carcinogenicity of inspired acetaldehyde in the rat[END_REF]. Inhaled benzaldehyde also causes damage to the respiratory epithelium lining the nasal septum in rats and impairment of the central nervous system [START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. All of these studies, however, have been done with quite high levels of aldehydes (above 100 ppm) which are not representative of ambient concentrations.

In parallel, epidemiological studies indicate an increase in the number of asthma cases in urban population, especially children [START_REF] Beasley | Prevalence and etiology of asthma[END_REF]. Atmospheric pollutants are thought to be at least responsible for damage to the respiratory function in humans and are believed to increase susceptibility to airborne allergens [START_REF] Higgins | Environmental exposure to air pollution and allergens and peak flow changes[END_REF].

Our hypothesis is that acetaldehyde or benzaldehyde exposure induces increased pulmonary allergic responses in laboratory animals. To test it, the effects of exposure to low levels of acetaldehyde (200 ppb) or benzaldehyde (500 ppb) were assessed on the respiratory functions of guinea pigs using a model of ovalbumin-sensitized animals [START_REF] Lebrec | Effect of influenza virus infection on ovalbuminspecific IgE responses to inhaled antigen in the rat[END_REF][START_REF] Ormstad | The fungal cell wall component beta-1,3glucan has an adjuvant effect on the allergic response to ovalbumin in mice[END_REF]. Concentrations were chosen in order to get close to environmental levels. The concentrations used are about ten times higher than recorded peak levels in heavily polluted areas. Lower levels would have led to technical problems, in particular in terms of exposure concentration variability. These compounds were administered in such a way as to simulate human chronic exposures to low concentrations.

METHODS

Animal model

Four-week male Hartley guinea pigs (GPs) were obtained from Charles River France. They were individually identified with an ear tattoo. Clinical observations were performed at reception and the animals (weighing 414.4 24.6 g) were randomly assigned to six groups with a method that provided approximately equal initial average body weight for each group.

Pulmonary effects were investigated in ovalbumin (OA)-sensitized or non-sensitized (control) GPs. Half of the animals used in this study were intraperitoneally injected on the first day and 24h later with 0.5 ml of a NaCl solution containing 100 g OA and 100 mg Al(OH) 3 . Control GPs were injected only with NaCl solution. Sensitization was checked after 3 weeks by intradermal reaction to 25 l of a solution containing 200 µg OA/ml. Reaction results were observed 24 h and 48 h later. Animal provocation was performed 4 weeks after the first injection by aerosolized OA inhalation (0.1% in saline for 10 min). Non sensitized GPs were not challenged with OA except half of the control group, in order to check if aerosolized OA had an effect by itself.

Generation of aldehyde-containing air

Acetaldehyde (Rectapur, 98% in purity) and benzaldehyde (99% in purity) were supplied by Merck Eurolab. Acetaldehyde bottles were stored at 4°C. Benzaldehyde was stored in a ventilated room at ambient temperature.

Acetaldehyde atmospheres were generated by injection of acetaldehyde vapor in pure air with a gas syringe (SGE) and a perfusion pump (A99, Razel). To control acetaldehyde concentration in the exposure chambers, acetaldehyde vapor was injected with the proper flow in the main airflow passing through the exposure chamber.

Benzaldehyde vapor was generated by bubbling air in liquid benzaldehyde. The vapor obtained was then mixed with pure air circulating in the chambers. Bubbling airflow rate, height of the liquid benzaldehyde column and main airflow rate through the chambers determined the concentration in the atmosphere.

Exposure conditions

Groups of 8 animals (control or OA-sensitized) were exposed by whole body inhalation in 140 liters Plexiglas® chambers, either to acetaldehyde (200 ppb, 0.36 mg/m 3 ) or benzaldehyde (500 ppb, 2.2 mg/m 3 ). The total airflow through the chambers was 3000 L/h for benzaldehyde and 1000 to 2500 L/h for acetaldehyde. The exposure schedule to each aldehyde was six hours/day, five days/week, for four weeks.

Between exposures, four animals were housed per cage in suspended Plexiglas® cages fitted with dust-free bedding. Animals were not fed during exposure but had access to food and water ad libitum between each exposure period.

Analytical chemistry

Inhalation chambers atmosphere samples were collected, twice a day, every day (90 min and 270 min after the beginning of exposure), on dinitrophenylhydrazine silicate adsorbent tubes (Sep pack). After desorption with acetonitrile, analyses were performed by HPLC chromatography with UV detection (model 9050, Varian) under the following characteristics: Kromasil column (C18 3.5µ 150 mm), elution solutions: acetonitrile and water, flow: 0.6 l/min, detection: 365 nm.

Experimental design

Six groups of eight guinea pigs were used: Group 1: air-exposed control Group 2: air-exposed ovalbumin-sensitized Group 3: benzaldehyde-exposed Group 4: benzaldehyde-exposed ovalbumin-sensitized Group 5: acetaldehyde-exposed Group 6: acetaldehyde-exposed ovalbumin-sensitized Animals were acclimated for at least 7 days before the experiment started. At day 0 and day 1, intraperitoneal injections of OA (groups 2, 4 and 6) or NaCl (groups 1, 3, 5) were performed. At day 21, sensitization was determined (intradermal reaction). Aldehyde exposure began the same day as animal sensitization and lasted from day 0 to day 26. Assessment of respiratory function parameters took place both before and after OA provocation, three days after the end of exposure (day 29). The day after (day 30), animals were sacrificed by intraperitoneal injection of pentobarbital sodium in order to evaluate the inflammatory and atopic responses.

Respiratory function parameters

The parameters studied were the breathing rate or respiratory frequency (RF), the tidal volume (TV) which is the volume of gas inhaled per breathing cycle, the minute volume (MV=TV RF), the inspiratory (PIF) and expiratory (PEF) peak flows, the relaxation time (RT) and the enhanced pause (Penh) which represents the resistance of airways and the state of bronchoconstriction [START_REF] Hamelmann | Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography[END_REF]. They were measured by means of whole body plethysmographs connected to a data acquisition software package (BUXCO Electronics Inc., Sharon, CT, USA). Baseline measurements were made during 15 min individually on each animal. They were interrupted for 10 min during which the animals were stimulated with the allergen. Measurements were recorded continuously after that time for one hour.

Hematology and serum chemistry

The day after the assessment of respiratory function parameters, animals were anesthetized with intraperitoneal injection of 6% pentobarbital sodium (0.2 ml/100g). Blood samples were taken from the vena cava. The following hematological parameters were determined with a hematology automated instrument (Technicon H1, Bayer): red blood cell (RBC) and white blood cell (WBC) counts, hemoglobin (Hb), hematocrit (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin 7/27 (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets and leukocyte differential counts.

The following biochemical parameters were determined in plasma, using an automated instrument (Cobas Fara II, Roche): alanine aminotransferase (ALT), albumin (Alb), aspartate aminotransferase (AST), calcium (Ca), cholesterol (Chol), gamma-glutamyl transferase ( -GT), glucose (Gluc), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), inorganic phosphorus (Phos), total proteins (Prot) and triglycerides (Trig).

Histopathology

At necropsy, a gross pathological examination was conducted and the observations recorded.

Trachea, lungs and tracheo-bronchial lymph nods were collected from two animals per group. Nasal tissue was also collected from all animals. Tissues were fixed in 10% formaldehyde, processed and embedded in paraffin. They were sectioned at 5 µm, stained with hematoxylin-eosin-saffron and examined by light microscopy. Nasal tissues were first decalcified for 24 h (Decalcifier II solution, Surgipath) before processing and embedding.

Cellular and biochemical pulmonary parameters

From six animals per groups, lungs were lavaged in situ with 3 x 20 ml of a 0.9 % NaCl solution. The collected cells were isolated by centrifugation (350 g, 5 min), counted with an hematology automated analyzer (Technicon H1, Bayer) and cellularity determined microscopically. The remaining cells were resuspended in RPMI medium (Gibco) and seeded onto 6-well plates at a concentration of 3 x 10 6 cells/well. After 3 hours of attachment, the medium was changed and 2 ml of serum free RPMI medium, supplemented with 0.2% bovine serum albumin, was added. After 18 h in a 5% CO 2 humid atmosphere at 37°C, the conditioned culture media were collected and stored frozen at -80°C until used. The remaining bronchoalveolar lavage fluid (BALF) was concentrated about 10-fold using a centrifugal filter device (Ultrafree 15, Millipore) and kept frozen at -80°C for assessment of protein and eicosanoid concentrations. Total protein content in BALF was assessed by the method of [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Prostaglandin E 2 , LTB 4 and LTC 4 /D 4 /E 4 were measured in BALF and conditioned culture media using immunoenzymatic systems (PGE 2 EIA system, LTB 4 EIA system, LTC 4 /D 4 /E 4 EIA system, Amersham Pharmacia Biotech).

Data from respiratory function parameters were analyzed using a non-parametric test (Kruskal-Wallis ANOVA, SPSS). For cellular and biochemical data, interaction between sensitization and aldehyde exposure was assessed using a two-way analysis of variance (ANOVA). These data were further analyzed using a one-way ANOVA and the Scheffe test (SPSS) to compare: 1) control to sensitized GPs responses for a given treatment (air, acetaldehyde or benzaldehyde),

2) the effect of aldehydes in control versus sensitized GPs.

Significance was considered achieved for p<0.05.

RESULTS

Animal model characteristics

Sensitized guinea-pigs presented the following characteristics, related to inflammatory and allergic responses (Table 1):

-Increased Penh reflecting enhanced bronchoconstriction.

-Changes in pulmonary histology reflecting an irritation of the respiratory tract.

-Increased number of eosinophils in blood.

-Increased number of inflammatory cells (macrophages, neutrophils and eosinophils) in BAL.

-Increased levels of proteins in BALF reflecting an alteration of vascular and epithelial permeability of the respiratory tract.

-Increased levels of the bronchoconstrictor lipid mediator LTC 4 /D 4 /E 4 .

No allergic reaction was noted in non-sensitized GPs challenged with OA. Therefore, all results from control GPs have been pooled.

Exposure concentrations

The concentrations recorded in the chambers are reported in Table 2. The geometric mean was used to estimate the average exposure because atmospheric concentrations tend to be log-normally distributed [START_REF] Nikas | Environmental versus analytical variability in exposure measurements[END_REF][START_REF] Rappaport | Assessment of long-term exposures to toxic substances in air[END_REF]. In this case, the geometric mean gives a better estimate of the integrated exposure. For acetaldehyde, target values differed slightly from observed values and a quite high variability was observed. However, we estimated that the animals received approximately the target dose during the 4-week interval.

Mortality, clinical signs and body weights

Exposures to benzaldehyde or acetaldehyde did not induce any mortality. However, two sensitized airexposed GPs died during the OA provocation. These animals developed acute pulmonary edema due to bronchoconstriction and inflammatory response. No alteration of body weight gain or impairment of state of health was noted during exposures (data not shown).

Respiratory functions

In control GPs, ovalbumin (OA) provocation induced no modifications in respiratory functions. In contrast, sensitized air-exposed GPs showed a typical asthmatic response to OA challenge, i.e. bronchoconstriction (increased Penh), an increase of the breathing rate and clinical signs of dyspnea, cyanosis of the ears and ocular membranes. Other respiratory parameters evaluated were also modified by OA challenge in control sensitized GPs. A significant increase in MV, PEF, PIF, TV and a significant decrease in RT were observed (data not shown).

In non-sensitized GPs, no difference in bronchial reactivity (i.e. Penh) was observed between airexposed and aldehyde-exposed animals (fig. 1A). Other respiratory parameters evaluated in nonsensitized animals were also not modified by aldehyde exposure.

For Penh, sensitized acetaldehyde-exposed GPs show a narrowed peak response to OA stimulation compared to air-exposed animals (fig. 1B). Sensitized benzaldehyde-exposed GPs show no marked response to OA stimulation, except for one animal, for which an intense bronchoconstriction was observed after 50 minutes (bringing the average to a very high value at that time). In benzaldehydeexposed animals, a similar suppression of allergic response was observed for the other respiratory parameters (data not shown).

Histopathology

For benzaldehyde or acetaldehyde-exposed GPs, histological examination of nasal cavities revealed a slight irritation (metaplasia/hyperplasia) of the respiratory epithelium for non-sensitized GPs and a proliferative subacute allergic rhinitis for sensitized GPs. Rhinitis alterations were more important for benzaldehyde than for acetaldehyde in sensitized GPs.

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For the two aldehydes, histological examination of the trachea and lungs showed a slight irritation of respiratory epithelium for non-sensitized GPs and a moderate one for sensitized GPs. An increase of OA-induced typical allergic histological alterations was also noted for aldehyde-exposed sensitized GPs.

Blood parameters

The only modification was a significant increase in eosinophils in air-exposed sensitized GPs compared to air-exposed non-sensitized GPs (Fig. 2A). The other cells (monocytes, lymphocytes, neutrophils and basophils) were not modified by OA-sensitization. Exposure to acetaldehyde or benzaldehyde produced no significant effect on the number of monocytes in non-sensitized GPs compared to the corresponding control (Fig. 2B).

In air-exposed animals, sensitization and challenge induced a significant increase in total proteins but not albumin. Sensitization also induced a significant increase in -GT. In non-sensitized and sensitized GPs, benzaldehyde or acetaldehyde exposure induced significant reduction of triglycerides.

Acetaldehyde exposure also significantly decreased blood cholesterol in both sensitized and nonsensitized GPs (data not shown).

Bronchoalveolar lavage (BAL) cells

In non-sensitized GPs, acetaldehyde exposure (but not benzaldehyde exposure) induced a significant increase in the number of alveolar cells, reflecting a pulmonary inflammation (Fig. 3A).

OA-sensitization animals significantly increased the number of cells recovered by BAL (Fig. 3A). This difference between sensitized and non-sensitized GPs was not found in benzaldehyde-exposed GPs.

In sensitized benzaldehyde-exposed GPs, the increased number of alveolar cells was not significantly different from healthy benzaldehyde-exposed GPs, which in turn was not different from control airexposed GPs. In control air-exposed GPs, the predominant cells recovered from BAL were alveolar macrophages (AM) (Fig. 3). In air-exposed sensitized animals, an influx of alveolar macrophages (AM), eosinophils (PE) and neutrophils (PN) was observed (Fig. 3C and3D).

Benzaldehyde exposure did not modify the BAL cytology in non-sensitized GPs. In sensitized animals, benzaldehyde exposure induced a significant decrease in the number of PN in BAL (Fig. 3). not significantly modify the BAL cytology compared to sensitized air-exposed GPs (Fig. 3).

Bronchoalveolar lavage (BAL) proteins

In non-sensitized GPs, neither benzaldehyde nor acetaldehyde exposure modified total protein concentrations in BALF compared to air-exposed GPs (Fig. 4). OA sensitization significantly enhanced total protein concentration in BALF (Fig. 4). This protein increase was abolished in sensitized GPs exposed to benzaldehyde (i.e. not significantly different from non-sensitized air-exposed GPs). In contrast, the sensitization-induced increase in protein concentrations was still observable in acetaldehyde-exposed GPs (Fig. 4).

Eicosanoid levels

In non-sensitized GPs, neither benzaldehyde nor acetaldehyde exposure modified PGE 2 , LTB 4 and LTC 4 /D 4 /E 4 concentrations in BALF compared to air-exposed GPs (Fig. 5).

OA sensitization increased LTC 4 /D 4 /E 4 concentrations in BALF. Benzaldehyde exposure of sensitized GPs reversed this pattern and decreased LTC 4 /D 4 /E 4 concentrations in BALF. Acetaldehyde exposure had no significant effect on the lipid mediator concentrations in BALF of sensitized GPs.

The concentration of PGE 2 in alveolar macrophage conditioned media was not significantly modified by either the sensitization or the aldehyde exposure. LTB 4 and LTC 4 /D 4 /E 4 were not detected in alveolar macrophage conditioned media.

DISCUSSION

The OA-induced allergic reaction was associated with a pulmonary inflammatory response, characterized by increased levels of the proinflammatory lipid mediator LTB 4 , neutrophil influx and increased levels of proteins in BALF, reflecting an alteration of vascular and epithelial permeability of the respiratory tract. These findings, which have been previously described [START_REF] Karol | Characteristics of weekly pulmonary hypersensitivity responses elicited in the guinea pig by inhalation of ovalbumin aerosols[END_REF][START_REF] Morley | Acute airway hyperreactivity in the guinea-pig[END_REF][START_REF] Santing | Relationships among allergeninduced early and late phase airway obstructions, bronchial hyperreactivity, and inflammation in conscious, unrestrained guinea pigs[END_REF][START_REF] Underwood | Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness[END_REF], are relevant to some physiologic features of asthma [START_REF] Bice | Animal models of asthma: Potential usefulness for studying health effects of inhaled particles[END_REF]. Animal sensitization and challenge also induced an increase in blood total proteins but not albumin, which was probably due to enhanced synthesis of proteins related to inflammation and allergy (complement, orosomucoid, immunoglobulins) [START_REF] Lebrec | Effect of influenza virus infection on ovalbuminspecific IgE responses to inhaled antigen in the rat[END_REF][START_REF] Ormstad | The fungal cell wall component beta-1,3glucan has an adjuvant effect on the allergic response to ovalbumin in mice[END_REF]. The increase in blood -GT, which is a marker of hepatic damage, may be due to the use of the adjuvant Al(OH) 3 , a known hepatotoxic compound [START_REF] Klein | Effects of aluminum on the liver following high-dose enteral administration to rats[END_REF][START_REF] Stein | Aluminium induced damage of the lysosomes in the liver, spleen and kidneys of rats[END_REF].

Very few studies have explored the toxicity of inhaled benzaldehyde or acetaldehyde. Inhalation exposure of Sprague-Dawley rats to benzaldehyde concentrations ranging from 500 to 1000 ppm for 14 consecutive days produced a mild irritation of the mucosa and severe impairment of the central nervous system as evidenced by abnormal gait, tremors and a positive Straub sign [START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. These changes also included hypothermia and a reduction of the breathing rate.

Histopathologic examination of tissues from exposed rats showed a goblet cell metaplasia that was largely confined to the respiratory epithelium lining the nasal septum in male rats. Hematologic changes were also observed, mostly in female rats which appeared to be more susceptible than males to the toxic effects of benzaldehyde. Acute, subchronic and chronic inhalation exposure to acetaldehyde produces respiratory tract injury with the most severe damage being produced in the nasal cavity [START_REF] Morris | Dosimetry, toxicity and carcinogenicity of inspired acetaldehyde in the rat[END_REF]. The 4-h LC 50 in the rat is 13,300 ppm [START_REF] Appelman | Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies[END_REF]. Repeated exposures (6h/day, 5 days/week, 4 weeks) of rats to 400, 1 000, 2 200 or 5 000 ppm produced marked injury to the nose. Degeneration of olfactory nasal tissues was noted at all concentrations. Tracheal and laryngeal lesions were observed at the 2 200 and 5 000 ppm concentrations only, and mild injury to the lower respiratory tract was observed only at the highest exposure level [START_REF] Appelman | Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies[END_REF]. Subchronic (13, 26 or 52 weeks) and chronic (28 months) exposure of rats produced injury to the nasal mucosa and larynx without apparent injury to the tracheobronchial tree or lungs [START_REF] Woutersen | Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 months[END_REF]1986). Chronic exposure of rats to 750 ppm results in nasal adenocarcinomas, whereas exposure to 1 500 ppm produces both adenocarcinomas and squamous cell carcinomas [START_REF] Woutersen | Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 months[END_REF]1986). In our experimental conditions, which occurred at considerably lower levels than those used in the above studies, benzaldehyde or acetaldehyde exposure did not markedly modify blood parameters.

Decreased hemoglobin and hematocrit have been noted in rats exposed to 1 000 ppm benzaldehyde during 14 days [START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. At that dose, female rats had also a decrease in number of RBC whereas male showed a significant increase in number of WBC. Blood content was also modified by acetaldehyde or benzaldehyde at doses between 400 and 5 000 ppm during 2 to 4 weeks [START_REF] Appelman | Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies[END_REF][START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. Blood biochemical parameters were not modified except for a decrease in triglycerides in aldehyde-exposed animals and a decrease in cholesterol in acetaldehyde-exposed animals. A decrease in blood cholesterol (but not in triglycerides) has been noted in male rats exposed at 500 and 750 ppm benzaldehyde for 14 days [START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. However, no biological significance could be attributed to these changes because the values reported fall within the normal range usually found in control rats of that age range.

In non-sensitized animals the two aldehydes caused respiratory tract irritation, which is known for acetaldehyde [START_REF] Appelman | Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies[END_REF] and suspected for benzaldehyde [START_REF] Laham | Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat[END_REF]. This irritation was accompanied, in acetaldehyde-exposed animals, by pulmonary inflammation, characterized mainly by a macrophage influx in alveolar spaces. Such a response was not observed in benzaldehyde-exposed animals. Acetaldehyde-induced macrophagic response is similar to that obtained after exposure to ozone [START_REF] Hotchkiss | Response of rat alveolar macrophages to ozone: quantitative assessment of population size, morphology, and proliferation following acute exposure[END_REF] or coal dust [START_REF] Oberson | Modification of matrix metalloproteinase activities from alveolar macrophages during chronic coal mine dust exposure in rats[END_REF]. The macrophage increase, observed in our study, may be due to recruitment of blood monocytes as demonstrated for other pollutants such as ozone [START_REF] Hotchkiss | Response of rat alveolar macrophages to ozone: quantitative assessment of population size, morphology, and proliferation following acute exposure[END_REF][START_REF] Zhao | Chemokine regulation of ozoneinduced neutrophil and monocyte inflammation[END_REF]. This hypothesis is supported by the fact that the number of blood monocytes decreased in acetaldehydeexposed GPs. Histological results did not show allergic features, such as eosinophilic infiltration, in aldehyde-exposed non-sensitized GPs.

Numerous studies, including clinical observations, epidemiology and human or animal controlled exposures suggest a connection between air pollutants and an increase in the incidence of asthma and allergy [START_REF] Devalia | Pollution-allergen interactions: challenge studies in man[END_REF][START_REF] Gilmour | Interaction of air pollutants and pulmonary allergic responses in experimental animals[END_REF][START_REF] Koenig | Air pollution and asthma[END_REF][START_REF] Sandström | Air pollution-allergy interaction: experiences from animal studies[END_REF]. However, a decrease in allergic reaction has been recently observed in OA-sensitized mice exposed during the aerosol challenge phase to 0.7 or 5 ppm NO 2 (Morris et al., 2001b) or a mixture of nasal irritants (3 ppm acrolein + 60 ppm acetaldehyde + 40 ppm acetic acid) (Morris et al., 2001a). Male C57/B6J mice were sensitized by weekly ip OVA injection for 3 weeks followed by daily 1-hour OVA aerosol challenge for 3 or 10 days. Mice were exposed to air or pollutants for 2 hours immediately after each daily OVA exposure. BAL eosinophil levels were significantly lower in OVA-pollutant compared to OVA-air animals. OVA-air exposed mice demonstrated a markedly enhanced airway resistance to aerosolized metacholine, an effect that was absent in the OVA-NO 2 exposed mice. Mechanisms of decreased allergic response in exposed animal are unknown. According to Morris et al., the decrease in OVAinduced allergic airway disease may be due to pharmacokinetics alterations (alteration in regional deposition pattern of OVA aerosol) or dynamic alterations (change in cytokine levels for example).

properties at concentrations relevant to human exposures. In sensitized animals, exposure to acetaldehyde does not modify the inflammatory and allergic responses induced by sensitization. In contrast, exposure to benzaldehyde modifies several respiratory functional, cellular and biochemical parameters leading to a large decrease in the severity of asthmatic responses. In a mixture of pollutants, benzaldehyde might therefore be able to mitigate the effects of other compounds in the asthmatic response. Further investigations are needed to determine if the attenuated response observed in sensitized GPs exposed to benzaldehyde is due to an alteration of the mechanism of sensitization or to a more direct effect on various mechanisms of the allergic response.

AKNOWLEDGMENTS

This study has been supported by a grant from French Environment Ministry (Loi sur l'air, convention 13/98).

FIGURE CAPTIONS

FIGURE 1 .

 1 FIGURE 1. Bronchoconstriction in air and aldehyde-exposed guinea pigs for non-sensitized (A) and sensitized (B) guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. : Air-exposed animals, : benzaldehyde-exposed animals, : acetaldehyde-exposed animals. Data are means SEM for n=8 animals.

FIGURE 2 .

 2 FIGURE 2. Number of eosinophils (A) and monocytes (B) in blood of air and aldehyde-exposed guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. a: significantly different (p<0.05) from the corresponding non-sensitized GP group. Data are means SD for n=6 animals.

FIGURE 3 .

 3 FIGURE 3. Number of total cells (A), alveolar macrophages (B), eosinophils (C) and neutrophils (D) in BAL of air and aldehyde-exposed guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. a: significantly different (p<0.05) from the corresponding non-sensitized GP group. b: significantly different (p<0.05) from the control GP group. c: significantly different (p<0.05) from the air sensitized GP group. Data are means SD for n=6 animals.

FIGURE 4 .

 4 FIGURE 4. Total proteins in BALF of air and aldehyde-exposed guinea pigs. Acetaldehyde exposure:

FIGURE 5 .

 5 FIGURE 5. Eicosanoids in BALF of air and aldehyde-exposed guinea pigs. A: PGE 2 , B: LTB 4 , C: LTC 4 /D 4 /E 4 . Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. a: significantly different (p<0.05) from the corresponding non-sensitized GP group. c: significantly different (p<0.05) from the air sensitized GP group. Data are means SD for n=6 animals.

FIGURE 6 .

 6 FIGURE 6. PGE 2 in alveolar macrophage conditioned media of air and aldehyde-exposed guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. Data are means SD for n=6 animals.

FIGURE 1 .FIGURE 2 .FIGURE 3 .

 123 FIGURE 1. Bronchoconstriction in air and aldehyde-exposed guinea pigs for non-sensitized (A) and sensitized (B) guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. : air-exposed animals, : benzaldehyde-exposed animals, : acetaldehyde-exposed animals. Data are means SEM for n=8 animals.

FIGURE 4 .FIGURE 5 .FIGURE 6 .

 456 FIGURE 4. Total proteins in BALF of air and aldehyde-exposed guinea pigs. Acetaldehyde exposure: 200 ppb for 4 weeks. Benzaldehyde exposure: 500 ppb for 4 weeks. a: significantly different (p<0.05) from the corresponding non-sensitized GP group. c: significantly different (p<0.05) from the air sensitized GP group. Data are means SD for n=6 animals.

TABLE 1 .

 1 Baseline characteristics of sensitized and non-sensitized animal groups mean of values recorded before and after OA challenge in air-exposed non-sensitized GPs, b mean of the maximal value noted after OA challenge in air-exposed sensitized GPs. Other parameters: data are means SD for n=6 animals. *: significantly different from control (p<0.05). Control values are assumed, not measured. Data are geometric means geometric SEM (n = 40) for the duration of exposure (geometric SD or SEM correspond to multiplicative factors).

	Parameters		Non-sensitized GPs	Sensitized GPs
	Penh (bronchoconstriction)	0.5	a	* 5.5	b
	Eosinophils in blood (10	6 / ml)	0.03 0.02	* 0.27 0.12
	Histology				Eosinophilic and lymphocytic chorionic infiltration
	Alveolar cells (x 10 6 )		24.3 1.6	* 68.2 21.0
	Macrophages		21.9 1.6	* 34.3 6.9
	Eosinophils Neutrophils		2.0 0.9 0.08 0.06	* 31.1 14.4 * 2.0 1.2
	Proteins in BALF (µg/ml)	133.3 14.1	* 767.8 538.5
	Eicosanoids in BALF (ng/ml)
	PGE 2		16.7 9.3	7.7 5.1
	LTB 4		1.1 0.6	3.0 1.8
	LTC 4 /D 4 /E 4		8.6 2.4	* 16.4 6.4
	Penh:

a a


Dusserre G \ Salvi
email: gilles.dusserre@ema.fr

Review of sixty two risk analysis methodologies of industrial plants

Keywords: risk, analysis, methodology, industrial plant, hiérarchisation

Since a few ten or so years, many methodologies have been developed to lead a risk analysis on an industrial plant. In this paper, sixty two methodologies have been identified.

Methodologies have been separated into three different phases (identification, evaluation and hiérarchisation). In order to understand their functioning, it seemed interesting to look at input data, methods used, output data obtained and to rank them in several classes. All the input data have been grouped together into seven classes (plan or diagram, process and reaction, products, probability and frequency, policy, environment, text and historical). The methods have been ranked in six classes based on four usual criteria (qualitative, quantitative, deterministic, probabilistic). Finally, the output data have been classified in four classes (management, list, probabilistic, hiérarchisation). This classification allowed to detail risk analysis methodology. In the aim to understand the functioning of these methodologies, the connections between the three criteria previously defined have been brought to the fore. Then the paper deals with the application fields and the main limitations of these methodologies.

So, the hiérarchisation phase is tackled and the type of scale used too. This paper highlights the difficulty to take into account all risks of an industrial plant and suggests that there is not only one general method to deal with the problematic of industrial risks.

1-Introduction

The industrial risk problematic and the diversification of risk types have increased consequently with the industrial development. In the same time, the risk acceptability threshold of the population has decreased. In response to this preoccupation, competent authorities and industrialists have developed methodologies and tools for risk prevention and protection, as well as crisis management.

To face up to major accidents, a previous analyse should be done. The forward-looking risk analysis allows to do an exhaustive identification of potential hazardous sources to prevent accident scenarios and to assess potential impact on human, environmental and equipment targets in order to propose prevention or protection [START_REF] Lagadec | Politique, risque et processus de développement : Le risque technologique majeur[END_REF]. The risk analysis methodologies focus on the main hazard sources. Two principal sources of risk may be brought to the fore :

-industrial establishment; -dangerous goods transportation These two types of sources are quite different. At first sight, the quantities involved are really different, and the environment is not evolutionary for an industrial site at the opposite of the case of dangerous goods transportation.

So, to analyse and to manage safety aspects, various approaches had been proposed, they focus on organizational and technical sight. Sixty two risk analysis methodologies are set out in the following paper.

2-Risk analysis methodologies

The management of major industrial risk should be one of the most important preoccupation for operators. To deal with this problem many risk analysis methodologies were developed by industrialists and competent authorities.

2.1-Description of methodologies

This work allowed to find more than sixty risk analysis methodologies which may include three main phases :

-an identification phase based on a site description (hazardous activities, products and equipments). Those data are necessary to develop the processes of the methodologies, -an evaluation phase to realize a quantification of risk. There are two ways to lead this part, a deterministic approach and / or a probabilistic approach. This evaluation gives the consequences of scenarios which were previously found and allows to take into account their impacts on the industrial site or on its vicinity.

-a hiérarchisation phase which aims at ranking some results obtained in the two previous phases in order to put preponderant risks forward. Thanks to this hiérarchisation, the most important risks should be solved at first. The phase of risk identification is essential, because it puts the bases of the risk analysis. Indeed, the data of risk identification will be the input of the evaluation and / or hiérarchisation phases. Therefore it is necessary to make an identification phase in an exhaustive way, to obtain the best results.

The phase of risk evaluation should be realized according two different approaches :

-either by the evaluation of damages consequences (deterministic approach), -or by the evaluation of accident probability (probabilistic approach).

The phase of risk hiérarchisation ranks the risks obtained in the previous phase, in order to implement modifications or corrective actions on the most severe risk systems.

A risk analysis methodology does not necessary contain these three phases. It can be constituted of only the following combinations :

-an identification phase, -an identification and evaluation phases, -or an identification, evaluation and hiérarchisation phases.

However methodologies may be, to carry out a risk analysis three kinds of elements are required :

-the output data expected, -the input data available, -the selected method, Indeed, users propose some objectives to reach (output data expected), next users collect information concerning the studied system (input data available), at last users chose the method applied according to the two previous elements.

2.1.1-Types of method

The used methods can be described according to four properties :

-Deterministic -Probabilistic -Qualitative -Quantitative These methodologies may be classified in two principal groups, one qualitative and the other quantitative. Each group may be divided into three categories :

-only deterministic, -only probabilistic, -a combination of deterministic and probabilistic approach.

The deterministic methods are based on the products, equipments and on the consequence quantification for various targets like people, environment and equipments. The probabilistic methods are based on the probability or frequency of hazardous situation apparition or the occurrence of potential accident. The probabilistic methods are mainly focused on failure probability of equipment or their component. On the other hand, deterministic and combined deterministic and probabilistic methods are used to analyse the whole industrial establishment.

The classification of the methods are based on type of output data. In each category, methods can be ranked from the more simple which are constituted of one step only to the more complex which are based on the three steps. The complex methods are generally composed of modules issued from simple methods and other modules are added in order to realize a risk analysis more complete with results easier to analyse.

In the table n°l, sixty two methodologies are ranked according to the four criteria previously defined.

The great majority of methods are determinist, historically operators and public organisations had initially try to quantify damages and consequences of potential accidents, before to understand why and how it was occurred.

2.1.2-Types of input data

The input data may be technical like process characteristics or qualitative like safety policy.

The analysis of sixty two methods lead us to propose seven classes of input data. For each classes some information is given on the type of input data.

• Plans or diagrams are related to the description of the site, the installation, the units, the fluid networks, the safety barriers and the storages.

• Process and reactions are related to operations and tasks description, physical and chemical features of process, kinetic and calorimetrical parameters, operating conditions and normal functioning conditions.

• Substances are related to the type of substance, the physical and chemical properties, the quantities and the toxicological data.

a Probability and frequency are related to the type of failure, the probability and frequency of failure, the human failure, the failure rate and the exposure probability.

• Policy and Management are related to the maintenance, the organization, the safety policy, the Safety Management System, the transport management and equipment cost.

• Environment : is related to the site environment, the topographical data and the population density.

a Text and historical knowledge are related to the standard and regulations, and historical knowledge.

These input data are summarised in the table n°2. This table gives connections between input data and the methodologies reviewed.

Most methods are based on a general description of the site (Plans and Diagrams) and few methods take into account the Environment.

2.1.3-Types of output data

The output data may be qualitative like recommendations or quantitative like index of risk level for example. From the review of sixty two methods, four classes of output data can be proposed :

• Management is related to actions, recommendations, modifications, and formation or operation procedures.

• Lists are related to lists of errors, hazards, domino effects, causes/consequences, failures and damages, critical activities, failure mode, accident initiators, vulnerable place and major accident scenarios.

• Probabilistic is related to failure rate, reliability, scenarios or damages probability, accident frequency. a Hiérarchisation is related to level risk index, severity and criticity, fire, explosion, toxic leakage index, organizational index, classification according to type of risk.

The connections between output data and methodologies are presented in the table n°3.

Output data like Management and Lists are based on expert choice and give qualitative results while output data like Probabilistic and Hiérarchisation give quantitative results.

2.1.4-Links between input data, output data and methods

Now, it seems to be relevant to underline how links between input data, method and output data are functioning. The table n°4 can be used according to whether the user expects some results or has some available data :

-First, if industrialists need a certain type of results, then they read table n°4 by results (output data) columns. So different types of methods are proposed and finally necessary input data are identified.

Second, if only several input data are available, then the user reads the table n°4 by input data columns. The combining of available input data allows to show which methods are conceivable to lead risk analysis.

The table n°4 is a synthesis of the study and a tool for an identification of which methods could be used according objectives and available input data.

The analysis of precedent table n°4 brings to the fore that many input data are necessary to realize qualitative & deterministic risk analysis and for quantitative and deterministic or deterministic & probabilistic risk analysis.

However qualitative or quantitative methods are, results are the most complete when both deterministic & probabilistic methods are used.

Probabilistic methods need a few input data, but they don't take into account some specificities of the industrial establishment like Policy or Environment for example. Now, the functioning of methodologies have been brought to the fore, and it still has to tackle two important points :

-on the one hand, the application fields of those methodologies, -and on the other hand, their main limitations.

2.2-Application fields of methodologies

The application field of these different methodologies can be ranked into three categories (table n°5). The first, which is the most important in number of developed methodologies, concerns industrial site. Generally, some methodologies are developed for specific application or process and they aren't transposable on different type of industrial establishment.

The second application field is the transportation of dangerous goods.

The third one allows to take into account human factor in a specific environment. Some methodologies can be used for various purposes and several application fields (to illustrate, the most general methods are "What if and "Safety Analysis").

2.3-Limitations of methodologies

The main limitations of those methodologies may be summarised in the following points :

The more general the methodology is, the less it takes into account the specificities of the studied case.

-On the contrary, if the methodology is too specific it is less transposable to another case.

-Knowledge of the people who are participating in the risk analysis are quite important (different type of competences and people involvement).

-For probabilistic analysis, the validity of data is a decisive parameter.

-The taking into account of the updating of data consume a lot of timework.

-For some methodologies, the operational application is difficult to realize because of the lack of description. It seems to be useful to provide a guide book to explain how methodologies may be used.

-The complexity of methods requires specific training for their implementation.

-It can be noticed that there is a great disconnection between risk analysis methodologies and human factor.

A new trend shows a need to have results more quickly exploitable. So, the output data of evaluation phase must be completed by an additional step : the hiérarchisation phase.

3-The risk hiérarchisation

The recent evolution in risk analysis methodologies shows that easily applicable methods are proposed with a risk level index as result. The hiérarchisation constitutes the action to organize some elements, data, or events in increasing or decreasing order with the help of classification tabs in the aim to bring out the main points for analysis. These methodologies are simple and rapid to use, data tables are usually available for the hiérarchisation phase.

The association between risk level and geographical zones on site is primordial to have a good safety management. These different methodologies including hiérarchisation step are gathered in the table n°6.

Methodologies with hiérarchisation phase are almost all quantitative and deterministic. They are based on the development of a risk level index calculated for each element, unit or studied area in order to rank them. This ranking provides the way to identify clearly critical areas on industrial site. Thus it allows to realize priority actions in order to reduce the probability of occurrence (prevention) or to reduce the consequences of an accident (protection including emergency response).

To calculate this index, several parameters characterizing the site and processes are identified.

Then these parameters are ranked with the help of scales which are provided in a guide and which can be based on a deterministic or probabilistic approach.

The deterministic scales can be quantitative or qualitative for the following variables which are :

• Internal hazard of substances and equipments : reaction types (hydrolysis, oxidation, reduction, polymerisation,...) reaction parameters (stability, reactivity, exothermically, pressure or temperature of reaction,...) physical and chemical properties of substances, their toxicity with a correlation dose/effect and the study of their incompatibility. quantities of substances used and stored storage characteristics (pressure, temperature,...).

• severity of the consequences :

-human damage types ( over pressure blast, thermal flux, toxicity).

-equipment damage types.

-environmental damage types.

-financial loss on equipment or production.

• Layout and environment :

-distances between dangerous units of an industrial site.

-population density inside and outside the industrial site.

The probabilistic scales are quantitative and they are made of variables which are : occurrence frequency of hazardous event -incident or accident frequencies taking into account historical knowledge.

-consequences probabilities ( fatalities, structure damages, ground or water pollution,...).

The various scales have been developed for several types of application according to the particularities of certain industrial site in order to rank the risk in consistent manner. To generalize hiérarchisation scale, it seems to be necessary to elaborate an exhaustive list of influencing risk factors from many industrial sites and to take into account human, environmental and equipment damages.

In fact, the hierachisation phase allow a data processing more advanced in order to rank risks on the studied area (risk level index). This ranking provides a help decision for decisionmakers (industrialists and competent authorities).

4-Conclusion

The used of risk analysis methodologies contribute to prevent accidents and to be prepared for emergency response. This work based on the review of sixty two methodologies shows the difficulty to take into account all risks of an industrial site.. This paper highlights the different types of input data, methods, output data and their links. A risk analysis methodology can be simple and focusing only on identification of hazards or a combined risk analysis methodology. A combined risk analysis methodology may be composed of several simple risk analysis methodologies, with an identification, estimation and hiérarchisation phases in order to obtain a risk level index for example. The recent development of risk analysis methodologies strives towards easily applicable methods with a hiérarchisation phase which is based on specific scales depending on the studied installation. The application fields of methodologies are industrial site, hazardous goods transportation and human factors. The human factor risk analysis is often disconnected with classical risk analysis, that is due to the complexity of human risk analysis. The type of results are recommendations, lists, risk level index, event frequency and damage probability.

The sixty two methodologies identified show that there is not an uniqueness of methods to realise a risk analysis. On the contrary, there is a need to combine several methodologies. The application of these methodologies requires experience to obtain good results. In fact, these sixty two methodologies can constitute a starting point of a thought to elaborate a new methodology. To elaborate a methodology it seems interesting to propose an initial draft of methods. Then its application on real industrial sites will allow to improve it so therefore its transferability to other cases will be easier.

The whole remarks introduce in this study, carry to propose a draft of an ideal risk analysis methodology.

First, the studied area must be cut in four parts to lead the risk analysis :

-the term source (industrial establishment) -the flux (vector of propagation of accidents) -targets (human, environmental and equipments) -Control and management These four identified parts must be described in an exhaustive way and their interaction too. Second, the mainspring of risk analysis should allow an deterministic and probabilistic approach with a hiérarchisation phase.

And Finally, the output data will be of two different types :

-qualitative in order to provide recommendations -quantitative in order to evaluate the main consequences This methodology present a overall processes of risk analysis in order to provide some way of improvement and a decision help.

Qualitative

Action Errors Analysis AEA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Checklist [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Concept Hazard Analysis CHA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [3] Concept Safety Review CSR [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Failure Mode Effect Analysis FMEA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Goal Orinted Failure Analysis GOFA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Hazard and Operability HAZOP [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] [8] [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Human Hazard and Operability HumanHAZOP [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Insurers involvement in risk reduction process [START_REF] Sankey | An insurer's involvement in the risk reduction process[END_REF] Manager [START_REF] Pitblado | Quantitative assessment of process safety programs[END_REF] Optimal Hazard and Operability OptHAZOP [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [10] Plant Level Safety Analysis PLSA [START_REF] Toola | Plant level safety analysis[END_REF] Potential domino effects identification [START_REF] Delvosalle | Development of a methodology for the identification of potential domino effects in « Seveso » industries[END_REF] Preliminary Risks Analysis PRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [6] Process Risk Management Audit PRIMA [START_REF] Hurst | Measures of safety management performance and attitudes to safety at major hazard sites[END_REF] Profile Deviation Analysis PDA [START_REF] Korjusiommi | Hazard analysis for batch processes and for special operations[END_REF] Safety related questions for computer controlled plants [START_REF] Chung | Safety related questions for computercontrolled plants : derivation, organisation and application[END_REF] [13] Seqhaz Hazard Mapping SHM [START_REF] Korjusiommi | Hazard analysis for batch processes and for special operations[END_REF] Sneak Analysis [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Task Analysis TA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] What if? Analysis [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] World Health Organisation WHO [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Accident Sequences Precursor ASP [START_REF] Holmberg | Risk follow up by probabilistic safety assessment-experience from a finish pilot study[END_REF] Delphi Technique [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Earthquake safety of structures and installations in chemical industries [START_REF] Jezler | Earthquake safety of structures and installations in chemical industry in the context of risk analysis[END_REF] Maximum Credible Accident Analysis MCAA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Reliability Block Diagram RBD [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Safety Analysis SA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Safety Culture Hazard and Operability SCHAZOP [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Structural Reliability Analysis SRA [

sit ial • § X) Q. </J C CD i- L-

13

Accident Hazard Analysis AHI [17] [18] Accident Sequences Precursor ASP [START_REF] Holmberg | Risk follow up by probabilistic safety assessment-experience from a finish pilot study[END_REF] Annex 6 of SEVESO II Directive [START_REF]La directive Seveso II : Annexe 6[END_REF] AVRIM2 [START_REF] Ham | Experiences with a safety report according to Seveso II : a pilot project in the Netherlands[END_REF] Checklist [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Chemical Runaway Reaction Hazard Index RRHI [START_REF] Kao | Chemical runaway reaction hazard index and risk assessment[END_REF] Concept Hazard Analysis CHA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [START_REF] Rasmussen | Hazard identification on plant functional modelling[END_REF] Concept Safety Review CSR [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Defi method [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Delphi Technique [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Dow's Chemical Exposure Index CEI [START_REF]Dow's chemical exposure index guide[END_REF] Dow's Fire and Explosion Index FEI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] [START_REF]Dow's fire and explosion index guide[END_REF] Earthquake safety of structures and installations in chemical Event Tree Analysis ETA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [6] [33] [START_REF] Gadd | Transport Riskat : the HSE quantified risk assessment tool for toxic and flammable dangerous goods transport by road and rail in Great Britain[END_REF] Facility Risk Review [START_REF] Schlechter | Facility risk review as a mean to addressing existing risk during the life cycle of a process unit, operation or facility[END_REF] Failure Mode Effect Analysis FMEA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Failure Mode Effect Criticality Analysis FMECA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Fault Tree Analysis FTA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Fire and Explsion Damage Index FEDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Goal Orinted Failure Analysis GOFA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Hazard and Operability HAZOP [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] [8] [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Hazard Identification and Ranking HIRA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Khan | Accident hazard index : a multi-attribute method for process industry hazard rating[END_REF] IDEF3 [START_REF] Kusiak | Risk assessment of process models[END_REF] [START_REF] Larson | Managing design processes : a risk assessment approach[END_REF] Instantaneous fractionnai loss index IFAL [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Insurers involvement in risk reduction process [START_REF] Sankey | An insurer's involvement in the risk reduction process[END_REF] International Study Group on Risk Analysis ISGRA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Maintenance Analysis MA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Maximum Credible Accident Analysis MCAA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Checklist [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Event Tree Analysis ETA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [6] [33] [START_REF] Gadd | Transport Riskat : the HSE quantified risk assessment tool for toxic and flammable dangerous goods transport by road and rail in Great Britain[END_REF] Failure Mode Effect Analysis FMEA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Fault Tree Analysis FTA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Action Errors Analysis AEA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Human Hazard and Operability HumanHAZOP [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Manager [START_REF] Pitblado | Quantitative assessment of process safety programs[END_REF] Process Risk Management Audit PRIMA [START_REF] Hurst | Measures of safety management performance and attitudes to safety at major hazard sites[END_REF] Methodologies Method Organised Systematic Analysis of Risk MOSAR [2] [39] Methodology of domino effects analysis [START_REF] Dolladille | Proposition d'une méthode d'analyse d'effet domino[END_REF] Methods of potential risk determination and evaluation [START_REF] Jâger | Approach to a systematic determination and evaluation of risk potential[END_REF] Mond Fire Explosion and Toxicity Index FETI [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Optimal Hazard and Operability OptHAZOP [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Khan | OptHazop-an effective and optimum approach for Hazop study[END_REF] Optimal Risk Assessment ORA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Plant Level Safety Analysis PLSA [START_REF] Toola | Plant level safety analysis[END_REF] Potential domino effects identification [START_REF] Delvosalle | Development of a methodology for the identification of potential domino effects in « Seveso » industries[END_REF] Preliminary Risks Analysis PRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Probabilistic Safety Analysis PSA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Papazoglou | Probabilistic safety analysis in chemical installation[END_REF] Profile Deviation Analysis PDA [START_REF] Korjusiommi | Hazard analysis for batch processes and for special operations[END_REF] Quantitative Risk Assessment QRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Oien | Development of risk level indicators for petroleum production platform[END_REF] [34] [START_REF] Papazoglou | Probabilistic safety analysis in chemical installation[END_REF] [36] [START_REF] Leeming | Use of quantified risk assessment in evaluating the risks of transporting chlorine by road and rail[END_REF] industries [START_REF] Jezler | Earthquake safety of structures and installations in chemical industry in the context of risk analysis[END_REF] Rapid Ranking RR [8] [30] Rapid Risk Analysis Based Design RRABD [START_REF] Khan | Inherently safer design based on rapid risk analysis[END_REF] Reliability Block Diagram RBD [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Risk Level Indicators RLI [START_REF] Oien | Development of risk level indicators for petroleum production platform[END_REF] SAATY methodology [START_REF] Troutt | The potential value of SAATY's eigenvector scaling method for short-term forecasting of currency exchanges rates[END_REF] Safety Analysis SA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Safety Culture Hazard and Operability SCHAZOP [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Safety related questions for computer controlled plants [12] [13] Seqhaz Hazard Mapping SHM [START_REF] Korjusiommi | Hazard analysis for batch processes and for special operations[END_REF] Short Cut Risk Assessment SCRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Sneak Analysis [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Structural Reliability Analysis SRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Toxic Damage Index TDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] What if? Analysis [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] World Health Organisation WHO [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Hazard and Operability HAZOP [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Safety Culture Hazard and Operability SCHAZOP [START_REF] Kennedy | Development of a hazard and operability-based method for identifying safety management vulnerabilities in high risk systems[END_REF] Task Analysis TA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Work Process Analysis Model WPAM [START_REF] Davoudian | The work process analysis model (WPAM)[END_REF] Table n° 5 : Application fields of risks analysis methodologies.

Methodologies with hiérarchisation phase

Accident Hazard Analysis AHI [17] [18] Annex 6 of SEVESO II Directive [START_REF]La directive Seveso II : Annexe 6[END_REF] Chemical Runaway Reaction Hazard Index RRHI [START_REF] Kao | Chemical runaway reaction hazard index and risk assessment[END_REF] Dow's Chemical Exposure Index CEI [START_REF]Dow's chemical exposure index guide[END_REF] Dow's Fire and Explosion Index FEI [17] [21] Earthquake safety of structures and installations in chemical industries [START_REF] Jezler | Earthquake safety of structures and installations in chemical industry in the context of risk analysis[END_REF] Facility Risk Review [START_REF] Schlechter | Facility risk review as a mean to addressing existing risk during the life cycle of a process unit, operation or facility[END_REF] Failure Mode Effect Criticality Analysis FMECA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Fire and Explsion Damage Index FEDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Hazard Identification and Ranking HIRA [5] [18] Instantaneous fractionnai loss index IFAL [5] [17] Methodology of domino effects analysis [START_REF] Dolladille | Proposition d'une méthode d'analyse d'effet domino[END_REF] Methods of potential risk determination and evaluation [START_REF] Jâger | Approach to a systematic determination and evaluation of risk potential[END_REF] Mond Fire Explosion and Toxicity Index FETI [5] [17] Potential domino effects identification [START_REF] Delvosalle | Development of a methodology for the identification of potential domino effects in « Seveso » industries[END_REF] Probabilistic Safety Analysis PSA [5] [35] Risk Level Indicators RLI [START_REF] Oien | Development of risk level indicators for petroleum production platform[END_REF] SAATY methodology [START_REF] Troutt | The potential value of SAATY's eigenvector scaling method for short-term forecasting of currency exchanges rates[END_REF] Seqhaz Hazard Mapping SHM [START_REF] Korjusiommi | Hazard analysis for batch processes and for special operations[END_REF] Toxic Damage Index TDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Table n° 6 : Risks analysis methodologies with hiérarchisation step

Accident Hazard Analysis AHI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] [START_REF] Khan | Accident hazard index : a multi-attribute method for process industry hazard rating[END_REF] Annex 6 of SEVESO II Directive [START_REF]La directive Seveso II : Annexe 6[END_REF] Chemical Runaway Reaction Hazard Index RRHI [START_REF] Kao | Chemical runaway reaction hazard index and risk assessment[END_REF] Dow's Chemical Exposure Index CEI [START_REF]Dow's chemical exposure index guide[END_REF] Dow's Fire and Explosion Index FEI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] [START_REF]Dow's fire and explosion index guide[END_REF] Fire and Explosion Damage Index FEDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Hazard Identification and Ranking HIRA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [START_REF] Khan | Accident hazard index : a multi-attribute method for process industry hazard rating[END_REF] Instantaneous fractionnai loss index IFAL [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [17] Methodology of domino effects analysis [START_REF] Dolladille | Proposition d'une méthode d'analyse d'effet domino[END_REF] Methods of potential risk determination and evaluation [START_REF] Jâger | Approach to a systematic determination and evaluation of risk potential[END_REF] Mond Fire Explosion and Toxicity Index FETI [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] [17] SAATY methodology [START_REF] Troutt | The potential value of SAATY's eigenvector scaling method for short-term forecasting of currency exchanges rates[END_REF] Toxic Damage Index TDI [START_REF] Khan | Multivariate hazard identification and ranking system[END_REF] Defi method [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Event Tree Analysis ETA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [6] [START_REF] Tiemessen | risk assessment of the transport of hazardous materials[END_REF] [START_REF] Gadd | Transport Riskat : the HSE quantified risk assessment tool for toxic and flammable dangerous goods transport by road and rail in Great Britain[END_REF] Fault Tree Analysis FTA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [5] [START_REF] Nicolet-Monnier | Integrated regional risk assessment : the situation in Switzerland[END_REF] Maintenance Analysis MA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Short Cut Risk Assessment SCRA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] Work Process Analysis Model WPAM [START_REF] Davoudian | The work process analysis model (WPAM)[END_REF] AVRIM2 [START_REF] Ham | Experiences with a safety report according to Seveso II : a pilot project in the Netherlands[END_REF] Facility Risk Review [START_REF] Schlechter | Facility risk review as a mean to addressing existing risk during the life cycle of a process unit, operation or facility[END_REF] Failure Mode Effect Criticality Analysis FMECA [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] IDEF3 [START_REF] Kusiak | Risk assessment of process models[END_REF] [START_REF] Larson | Managing design processes : a risk assessment approach[END_REF] International Study Group on Risk Analysis ISGRA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] IPO Risico Berekening Methodiek IPORBM [START_REF] Tiemessen | risk assessment of the transport of hazardous materials[END_REF] Method Organised Systematic Analysis of Risk MOSAR [START_REF] Bgn | Draft methodology on risk assessment of unit operations and equipment[END_REF] [39] Optimal Risk Assessment ORA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF] Probabilistic Safety Analysis PSA [START_REF] Khan | Techniques and methodologies for risk analysis in chemical process industries[END_REF]

Rapid Risk Analysis Based Design RRABD [START_REF] Khan | Inherently safer design based on rapid risk analysis[END_REF] Risk Level Indicators RLI [START_REF] Oien | Development of risk level indicators for petroleum production platform[END_REF]