Tracey J Woodruff
email: woodrufft@obgyn.ucsf.edu

Lauren Zeise

Daniel A Axelrad

Kathryn Z Guyton

Sarah Janssen

Mark Miller

Gregory G Miller

Jackie M Schwartz

George Alexeeff

Henry Anderson

Linda Birnbaum

Frederic Bois

Vincent James Cogliano

Kevin Crofton

Susan Y Euling

Paul M D Foster

Dori R Germolec

Earl Gray

Dale B Hattis

Amy D Kyle

Robert W Luebke

Michael I Luster

Chris Portier

Deborah C Rice

Gina Solomon

John Vandenberg

R Thomas Zoeller

Meeting Report: Moving Upstream-Evaluating Adverse Upstream End Points for Improved Risk Assessment and Decision-Making

Keywords: adverse health effects, androgen antagonists, hazard identification, immunotoxicants, risk assessment, science policy, thyroid hormone, toxicologic assessments

To evaluate the potential of environmental chemicals to cause harm, to estimate the risks that chemical exposures pose to the population, and to identify opportunities for prevention and intervention, the type and extent of adverse effects associated with exposure to a chemical must be elucidated. To date, hazard and risk assessments have relied largely on data from traditional toxicologic studies, such as the 2-year, chronic toxicology and carcinogenesis studies or the two-generation reproductive toxicity assay. A primary goal of these studies is to identify whether chemical exposures cause overt disease outcomes, such as birth defects and neoplasia. These studies also provide data on biological events that precede these overt disease outcomes, often referred to as precursor effects. Adverse effects identified in existing hazard and risk assessments have often been the more overt diseases or defects, rather than events that occur earlier in the disease process.

. Identifying an adverse effect forms the basis for hazard identification and for defining the critical effect for quantitative risk assessment.

The evolution in toxicology testing from overt disease to elucidating toxicologic pathways has been recognized and endorsed by the

National Academy of Sciences and federal agencies [START_REF] Collins | Toxicology. Transforming environmental health protection[END_REF]National Research Council 2007). These organizations note that moving toward a focus on perturbations along the disease pathway should result in assays that can test more chemicals with reduced cost and time and fewer animals, and improve the scientific understanding of the relationships between exposures and health effects.

To move toward these goals, it is necessary to consider how data on early biological changes relate to the concept of an adverse effect and how these data might best be integrated into hazard and risk assessment. To this end, a workshop, Moving Upstream: A Workshop on Evaluating Adverse Upstream Endpoints for Improved Decision Making and Risk Assessment, was held 16-17 May 2007 in Berkeley, California.

Workshop Summary

Three case studies were presented at the workshop that described available data on toxicologic pathways connecting chemical exposures to early upstream biological events and then to subsequent overt effects, which are considered "downstream": a) thyroid hormone disruption and related toxicities; b) antiandrogen-mediated male reproductive effects; and c) upstream indicators of immunosuppression. To frame discussions in terms of implications for hazard identification and risk assessment processes, the case studies explored the following questions:

• What are the precursor effects or other early biological changes linked to the case study outcome? • Is there sufficient evidence to associate the upstream events with the overt downstream effects? • What related information would or does enhance our understanding of the relationship between the upstream event and downstream effects?

• Were the steps from the upstream event(s) to the overt downstream effect(s) identified? How does our understanding of these steps inform the use of the upstream event as a basis for risk assessment, particularly in situations when we only have data on upstream events? • How does an understanding of variability in background biological status (e.g., susceptibility or sensitivity, genetic or otherwise) affect our interpretation of an upstream event? • How might each end point be influenced by exposure to other chemicals? • In considering an upstream biological end point that is measured as a continuous variable [e.g., thyroid-stimulating hormone (TSH)], what approach should be taken in deciding whether a certain degree of upstream change will lead to the overt downstream outcomes (e.g., neurodevelopmental change)? How should changes in upstream indicators within "normal" ranges be considered in evaluating potential for overt downstream outcomes in susceptible individuals and populations? • Can the association between upstream events and downstream effects for chemicals considered in the case study be generalized to other chemicals that have the same upstream effects? • What are the obstacles to the use of the upstream event in risk assessment (hazard identification; dose-response assessment)? How can they be overcome?

Case Study Synopsis: Thyroid Hormone Disruption and Related Toxicities

Thyroid hormone overview. The thyroid hormones (TH) thyroxine (T 4 ) and triiodothyrine (T 3 ) are essential to neurologic development, skeletal growth, and normal function of the pulmonary system, metabolism, and cardiovascular system. This case study emphasized the effects of thyroid disruption on neurologic development.

Hypothyroidism is a condition of persistent deficits of TH and is diagnosed primarily by comparing an individual's level of TH and TSH to population reference ranges. Untreated congenital (neonatal) hypothyroidism can have severe consequences on neurologic development. Children diagnosed with congenital hypothyroidism are treated with T 4 , and even small doses (e.g., 2 µg/kg/day) significantly improve later cognitive performance, demonstrating the sensitivity of the developing brain to TH insufficiency [START_REF] Oerbeck | Congenital hypothyroidism: influence of disease severity and L-thyroxine treatment on intellectual, motor, and school-associated outcomes in young adults[END_REF][START_REF] Selva | Neurodevelopmental outcomes in congenital hypothyroidism: comparison of initial T 4 dose and time to reach target T 4 and TSH[END_REF].

Moderate maternal TH insufficiency during pregnancy can cause lasting developmental deficits in the child. Decrements in human maternal T 4 during the early fetal period are associated with adverse outcomes such as reduced IQ scores, even for small T 4 deficits that do not constitute maternal hypothyroidism [START_REF] Haddow | Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child[END_REF][START_REF] Morreale De Escobar | Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia?[END_REF][START_REF] Pop | Maternal hypothyroxinaemia during early pregnancy and subsequent child development: A 3-year follow-up study[END_REF].

Levels of TH in the body are determined by a complex interplay of dynamic processes, including dietary intake of iodine (necessary for synthesis of TH); the transport of iodine into the thyroid; its synthesis and storage in the thyroid gland; its release and transport through circulation; the deiodination of T 4 to T 3 in peripheral tissues; and the degradation of TH by hepatic enzymes. TH homeostasis is regulated through a negative feedback loop involving the hypothalamus and pituitary. When TH levels decline, the hypothalamus signals the pituitary gland to release TSH, which stimulates the thyroid to increase TH. When levels rebound, the hypothalamus signals the pituitary to decrease TSH. Finally, TH action is mediated through TH receptors throughout the body.

In hazard identification and risk assessment, TH levels could serve as an upstream indicator of adverse effects on neurologic development. classes, mechanisms of action, and effects for chemicals that disrupt TH homeostasis and action. For example, perchlorate inhibits the uptake of iodine, resulting in decreased synthesis of TH. Polychlorinated biphenyls (PCBs) and the pesticide acetochlor activate enzymes in the liver that increase excretion of TH, thus reducing circulating levels [START_REF] Crofton | Thyroid disrupting chemicals: mechanisms and mixtures[END_REF].

Factors influencing effects of thyroiddisrupting chemicals. Susceptibility varies markedly with life stage. The fetus, infant, and child appear the most susceptible to neurologic disruption. The permanent effects of TH insufficiency on neurologic development were discussed above. Infants are likely less resistant to fluctuations in TH synthesis than adults. The adult thyroid stores several months' supply of TH and the half-life of T 4 in serum is 7-10 days [START_REF] Greer | Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans[END_REF][START_REF] Vulsma | Maternal-fetal transfer of thyroxine in congenital hypothyroidism due to a total organification defect or thyroid agenesis[END_REF]. In contrast, the newborn thyroid stores a 1-day supply of TH and the half life of T 4 in serum is 3 days [START_REF] Vulsma | Maternal-fetal transfer of thyroxine in congenital hypothyroidism due to a total organification defect or thyroid agenesis[END_REF]). Therefore, effects on TH synthesis have more profound consequences for newborns than they do for adults.

Recognizing subpopulations at risk is a key consideration in evaluating effects on TH levels or action as adverse. Hypothyroidism is prevalent in the U.S. population. Between 1999 and2002, 7.3% of the U.S. population ≥ 12 years of age reported having thyroid disease or taking thyroid medication. Hypothyroidism is frequently undetected and therefore untreated. This condition often persists even in those who receive treatment. Among women receiving thyroid hormone replacement medication, 14% had TSH and T 4 measurements indicating continued hypothyroidism. Pregnancy causes an increased demand on the thyroid gland, and hypothyroidism is twice as common during pregnancy [START_REF] Aoki | Serum TSH and total T 4 in the United States population and their association with participant characteristics: National Health and Nutrition Examination Survey (NHANES 1999-2002)[END_REF].

The current method for assessing thyroid health may not be predictive of adverse downstream effects. Thyroid health is evaluated primarily by comparing an individual's level of TH and TSH to population reference ranges. However, there is a substantial variability among individuals in the level of TH that represents homeostasis, and the normal range of fluctuations for an individual is narrower than the normal range for a population (Figure 1). Consequently, a TH value within the population reference range is not necessarily normal or healthy for the individual [START_REF] Andersen | Narrow individual variations in serum T 4 and T 3 in normal subjects: a clue to the understanding of subclinical thyroid disease[END_REF], and TH changes within the normal population range may therefore be associated with adverse effects for some individuals.

Assessments considering thyroid-disrupting chemicals (TDCs) The mixture had a dose-additive effect on T 4 at environmentally relevant doses and a 2-to 3-fold greater than dose-additive effect on T 4 at higher doses [START_REF] Crofton | Thyroid-hormone-disrupting chemicals: evidence for dose-dependent additivity or synergism[END_REF], demonstrating that exposures to chemicals acting on different pathways can have cumulative effects on an upstream marker. Assessments considering single TDCs in isolation are therefore likely to underestimate the potential disruption of TH by real-world exposure to chemical mixtures. It is appropriate to presume cumulative effects unless there is evidence to the contrary, and it is important for risk assessments to consider real-life exposure mixtures.

Challenges for assessing risks of exposure to TDCs. Currently available mathematical models may not be able to accurately predict the effects of mixtures containing TDCs with multiple mechanisms of action. There are also numerous uncertainties in extrapolating thyroid data from laboratory animals to estimate effects in humans, including species differences in the expression or structure of specific functional proteins that may affect the toxicity of specific compounds.

TH levels influence a spectrum of health outcomes and symptoms, in addition to neurologic development. Lower T 4 and higher TSH levels are correlated with adverse effects on the pulmonary system [START_REF] Mendelson | Hormonal control of the surfactant system in fetal lung[END_REF] and cardiovascular system, including increased blood pressure and less favorable blood lipid profiles (Asvold et al. 2007a(Asvold et al. , 2007b)). A meta-analysis of 14 epidemiologic studies found an overall increase in risk of coronary heart disease of > 65% in those with subclinical hypothyroidism (elevated in TSH with normal T 4 ) [START_REF] Rodondi | Subclinical hypothyroidism and the risk of coronary heart disease: a meta-analysis[END_REF].

Conclusions regarding thyroid data. Many environmental chemicals are capable of disrupting thyroid hormone levels, and many of these have the effect of decreasing circulating levels of T 4 . Compensatory mechanisms may not be sufficient to counteract the potential downstream consequences of these T 4 decrements. First, findings in both animals and humans indicate that even small maternal T 4 decrements within the normal population range during pregnancy can have adverse neurodevelopmental consequences, such as reduction in IQ, in the developing child. Second, fetuses and infants do not have stored thyroid hormone, and thus have limited capacity to respond to thyroid hormone decrements during critical stages of development. Third, there is a substantial prevalence of thyroid hormone insufficiency in the population of U.S. women, indicating that compensatory processes are already compromised for many individuals.

Therefore, for hazard identification purposes, workshop participants agreed that TDC exposures that would result in reduced T 4 in a population should be considered an adverse effect. Further-upstream disruptions that result in lowered T 4 levels, such as inhibition of iodine uptake, should similarly be considered adverse when their potential consequences include alteration of T 4 levels. Because additivity or synergy of TDCs with different modes of action has been demonstrated, and background exposure to many TDCs is common, risk assessments should consider simultaneous exposures to multiple agents and account for these interactions.

Because pregnancy causes an increased demand on the thyroid gland, pregnant women are likely to have heightened sensitivity to thyroid toxicants, particularly if they have low dietary iodine or thyroid peroxidase antibodies. These sensitive subpopulations are likely to be sizable; for example, 38% of 126 pregnant women in the National Health and Nutrition Examination Survey (NHANES), 2001-2002, had low iodine intake (< 100 µg/L urinary iodine) [START_REF] Caldwell | Urinary iodine concentration: United States National Health And Nutrition Examination Survey 2001-2002[END_REF]. Because thyroid hormone levels affect cardiovascular risk factors, the potential impact of TDCs on the health of adults in the general population also is large. Therefore, effects on thyroid represent a risk to the general population and not just the developing organism.

Case Study Synopsis: Antiandrogen-Mediated Male Reproductive Effects

Overview of mammalian male reproductive development. Early in fetal development, male sexual differentiation is determined by expression of a Y chromosome gene, the sex-determining region Y (SRY). Appropriately timed SRY expression results in differentiation of Sertoli cells in the testes, initiating a cascade of events that result in development and differentiation of male reproductive structures and regression of female structures. The interstitial cells of the testes, Leydig cells, produce two important hormones for male reproductive development: the steroid sex hormone testosterone and the peptide hormone insulin-like-3 (INSL-3). Testosterone is necessary for differentiation of the Wolffian duct into the epididymis and secondary sex organs such as the seminal vesicles. Both testosterone and INSL-3 are necessary for testicular descent in mammals: Testosterone mediates transabdominal descent of the testes [START_REF] Klonisch | Molecular and genetic regulation of testis descent and external genitalia development[END_REF]), whereas INSL-3 is necessary for gubernacular ligament development. Contraction of the gubernaculums causes the inguino-scrotal descent of the testes into the scrotum [START_REF] Wilson | Phthalate ester-induced gubernacular lesions are associated with reduced INSL3 gene expression in the fetal rat testis[END_REF]). Disruption of testosterone and/or INSL-3 production or action can result in cryptorchidism (undescended testes), one of the most common birth defects in humans.

Exposure to antiandrogens during fetal life. During fetal life, there is a transient peak in testosterone levels necessary for proper development of male reproductive tissues. Disruption in androgen action during this critical time window results in a number of abnormalities that consistently develop in laboratory animals (e.g., rats and rabbits), including retained female structures, such as nipples in male rodents, and malformations of male reproductive structures, such as hypospadias (an abnormal location of the urethral opening) and cryptorchidism.

Multiple modes of action can disrupt androgen activity, including a decrease in testosterone production or interference with androgen receptor binding. Each of these modes of action has been well described in the literature for two different classes of chemicals with antiandrogenic effects.

First, carboximide pesticides such as linuron, vinclozolin, and procymidone are classic antiandrogens in that they bind antagonistically to the androgen receptor (AR), interfering with endogenous testosterone and dihydrotestosterone binding to the AR, and thus decreasing the expression of androgen-dependent genes. In fetal rat studies, exposure to these chemicals has been consistently associated with retained nipples, decreased sperm counts, decreased anogenital distance, hypospadias, and decreased size or agenesis of the accessory sex glands [START_REF] Gray | Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals[END_REF]. The severity of effects increases with the dose.

Second, there are certain phthalatesindustrial chemicals with widespread exposure in the general population-that do not bind to the AR but do interfere with androgen activity by inhibiting testosterone synthesis. Specifically, phthalates with four to six carbon side-chains interfere with testosterone production by inhibiting cholesterol uptake into the mitochondria by steroidogenic acute regulatory protein and by inhibiting some, but not all, of the enzymes in the steroidogenic pathway [START_REF] Barlow | Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate[END_REF]). These phthalates have been most consistently shown to reduce testosterone production in animal research to date, though other phthalates may be important [START_REF] Gray | Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat[END_REF][START_REF] Liu | Gene expression profiling following in utero exposure to phthalate esters reveals new gene targets in the etiology of testicular dysgenesis[END_REF].

Rat studies have demonstrated that a phthalate-induced decrease in testosterone results in a syndrome of anti-androgenic reproductive abnormalities, referred to as the "phthalate syndrome," which is characterized by malformations of the epididymis, vas deferens, seminal vesicles, and prostate; hypospadias, cryptorchidism, and testicular injury; permanent changes (feminization) in the retention of nipples and areolae (sexually dimorphic structures in rodents); and demasculinization of the growth of the perineum, resulting in a reduced anogenital distance [START_REF] Mylchreest | Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to di(n-butyl) phthalate during late gestation[END_REF]. As with other antiandrogenic chemicals, the severity of effects increases with dose.

The carboximide pesticides and four to six side-chain carbon phthalates have different modes of action resulting in the same spectrum of adverse outcomes in male reproductive development. In the developing male reproductive tissues, disruption of either of these two parts of the androgen action pathway converges into a final common pathway: a decrease in androgen-dependent gene expression.

The spectrum of effects seen in phthalate syndrome parallels a spectrum of human diseases called "testicular dysgenesis syndrome": infertility, cryptorchidism, hypospadias, and testicular cancer. Several epidemiologic studies have shown associations between certain phthalate metabolites and male developmental reproductive outcomes, including shortened anogenital distance in male infants prenatally exposed to phthalates [START_REF] Swan | Decrease in anogenital distance among male infants with prenatal phthalate exposure[END_REF], changes in reproductive hormone levels in male infants exposed to phthalates in breast milk [START_REF] Main | Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants 3 months of age[END_REF], and poor semen quality in adult males [START_REF] Hauser | Altered semen quality in relation to urinary concentrations of phthalate monoester and oxidative metabolites[END_REF]. The exposure levels in most subjects in these studies were comparable to the general population exposures.

Antiandrogen effects as adverse end points in hazard identification and risk assessment. An antiandrogenic agent, therefore-one that reduces androgen action by any mode of action (e.g., binding to the androgen receptor, or via interference with androgen synthesis)can be predictably associated with a series of adverse end points in animals and likely also in humans. Several additional scientific findings need also be considered when considering antiandrogen effects as upstream adverse end points.

Specific periods during development are uniquely susceptible to perturbations. Experiments in rats have demonstrated that both the dose and the timing of exposure are important for development of phthalate syndrome. Gestation days 12-19 have been identified as a sensitive period in rats, during which exposure to dibutyl phthalate induces phthalate syndrome, with some irreversible effects [START_REF] Carruthers | Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl phthalate[END_REF].

Hydrolytic phthalate monoesters are the bioactive metabolites responsible for phthalate toxicity. Excretion of phthalate monoesters in urine is enhanced by glucuronide conjugation. Experiments in rats have shown the glucuronidation pathway is immature and inefficient during the period of susceptibility for phthalate syndrome. This relatively low level of glucuronidation compared to adults could render the fetus much more susceptible to the biologically active phthalate metabolites during a critical period of sexual development. Exposure to rats during this period of gestation has demonstrated the proportion of free phthalate monoesters relative to glucuronidated phthalate monoesters in amniotic fluid is much higher than levels in the urine of the pregnant dam [START_REF] Calafat | Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate[END_REF].

Exposure to mixtures of AR antagonists and androgen synthesis disruptors. More than 95% of the population from 6 to > 65 years of age are exposed to at least five phthalates on a regular basis [START_REF] Silva | Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999-2000[END_REF]). Recent studies show that exposure to mixtures of chemicals that interfere with androgen action results in dose-additive effects. Rats exposed to a mixture of AR antagonists, vinclozolin, procymidone, and flutamide, all acting through the same mode of action, at doses that would not have caused hypospadias alone, resulted in > 50% of the animals having hypospadias [START_REF] Christiansen | Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat[END_REF]. [START_REF] Rider | A mixture of seven antiandrogens induces reproductive malformations in rats[END_REF] found that prenatal exposure to a mixture of seven phthalates and pesticides with differing modes of action (i.e., AR antagonist or inhibition of androgen synthesis) produced cumulative, dose-additive outcomes in the androgen-dependent tissues. Exposures to phthalates can result in adverse effects from modes of action other than disrupting testosterone synthesis. In addition to inhibiting the production of testosterone, phthalates also interfere with production of INSL-3, which is necessary for gubernaculum development [START_REF] Wilson | Phthalate ester-induced gubernacular lesions are associated with reduced INSL3 gene expression in the fetal rat testis[END_REF]) and subsequent descent of the testis into the scrotum. Unique to phthalate exposure is an absence of the gubernacular ligament in males exposed in utero. Thus, phthalates can act through two different modes of action to cause cryptorchidism.

Conclusion regarding antiandrogen data. The prenatal exposure of males to antiandrogenic chemicals illustrates several important points when considering upstream indicators of adverse effects: First, it is necessary for exposure to occur during the critical window, the period of reproductive organ development, in order for certain developmental effects to be observed. Second, perturbations early in the development of the male reproductive system predictably result in a wide array of adverse outcomes that are permanent and irreversible. Third, exposures to different chemicals with different modes of action can result in the same outcomes due to a deficiency in androgenmediated gene expression. Finally, exposure to one chemical can have multiple modes of action with effects on the same end point. In summary, exposures to antiandrogens can cause adverse effects in upstream indicators, including a reduction in fetal testosterone levels or androgen receptor binding, which can increase the risk of a constellation of downstream effects including cryptorchidism, hypospadias, and, later in life, infertility.

Case Study Synopsis: Immune Function, Immunotoxicity, and Resistance to Infection and Neoplasia: The Downstream Impacts of Unintended Immunosuppression

Immune system overview. The immune system consists of a complex system of tissues, cells, and soluble mediators that protects the body against foreign substances, including infectious agents and some types of tumor cells. Immune cells are located throughout the body, either in discrete organs, such as the spleen, thymus, and lymph nodes, or in diffuse accumulations of lymphoid and myeloid cells, as are found in association with the skin, lungs, and gastrointestinal tract. Destruction of infectious agents relies on both innate and adaptive immune responses. Innate responses are rapid, do not require clonal expansion, and are stimulated by recognition of pathogen-associated molecules by macrophages, natural killer cells, and granulocytes. Adaptive, or acquired antigen-specific responses, rely on antigen recognition and subsequent events that culminate in cell proliferation and maturation, mediator production, and generation of longlived memory cells that respond rapidly on subsequent exposures to the same or closely related antigens. The acquired immune response is mediated by two types of lymphocytes, T cells and B cells. T cells are a source of soluble mediators known as cytokines that stimulate other immune system cells, including B cells, and act as effector cells with cytotoxic activity. B cells mature into plasma cells, which serve to produce immunoglobulins (Ig), or antibodies, of various subclasses, including IgM, IgG, IgE, IgA, and IgD. Each of these antibody subclasses serves a unique function in the immune response.

Factors influencing immune function. Numerous factors influence the outcome of an encounter with an infectious agent. Innate defenses, critical in the early phase of resistance, may be overcome by large numbers of infectious agents. The virulence of the pathogen may also prevent effective innate and acquired responses from eliminating microorganisms before infection ensues. Host attributes such as age, sex, genotype, lifestyle, and disease status all influence immunocompetence, and each may influence the incidence or severity of infections.

Newborns are not immunologically mature at birth and are thus at increased risk of infection. Protective antibodies are transferred from mother to fetus across the placenta and to the newborn in breast milk, although passive protection decreases rapidly as these proteins are catabolized. Average IgM and IgG antibody levels do not reach 50% of adult levels until 7-12 months, and IgA reaches adult levels by 3-5 years. A preponderance of naive T cells (90% vs. 50% in adults) contributes to reduced cell-mediated immunity in the young. Immunocompetence also declines with advanced age; however, few studies have evaluated the effects of immunotoxicants in older laboratory animals and humans.

Studies in laboratory animals and humans have established that extrinsic factors, including environmental chemicals, drugs, physical agents, and psychological factors may influence the course of infection. Furthermore, unintended immunosuppression resulting from exposure to environmental chemicals alone or in combination with other intrinsic and extrinsic factors can shift the distribution of normal immune response, resulting in an increase in individuals classified as "immunosuppressed." Immunosuppressed individuals might express alterations of immune function such as reduced antibody production, decreased immune cell counts, or ineffective cell signaling. Recent data also suggest that, in addition to suppression, developmental exposure (i.e., exposure from birth through puberty) to certain chemicals may shift the pattern of cytokine production, leading to a greater incidence of allergy and asthma [START_REF] Luebke | The comparative immunotoxicity of five selected compounds following developmental or adult exposure[END_REF].

Effects on the immune system during periods of susceptibility.

In a review of select developmental immunotoxicology literature, [START_REF] Luebke | The comparative immunotoxicity of five selected compounds following developmental or adult exposure[END_REF] concluded that exposure to environmental chemicals during key developmental periods can affect immune function in laboratory animals. Developmental exposure suppresses T-cell function through adolescence in mice and throughout adulthood in rats. Gestational/neonatal exposure to diazepam (DZP) and diethylstilbestrol (DES) in rodents can cause long-lasting (up to lifetime) effects on the immune system, including suppression of IgM and IgG antibody responses and nonspecific lymphocyte proliferation (DES) at doses that cause only short-term immunotoxicity in adults. Developmental exposure to DZP, lead, or tributyltin oxide caused immunotoxicity at lower doses in young than in mature animals, and developmental effects were persistent. Clinical experience indicates that adult immune function typically recovers soon after therapeutic immunosuppressive treatment ends. However, the developmental data suggest that screening chemicals exclusively in adult animals may fail to detect persistent effects or those effects that occur at lower doses [START_REF] Luebke | Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin[END_REF].

Immune function also declines with age, as do the normal physiologic processes that limit microbial invasion. Although data are limited, this suggests that relatively small changes in immune function resulting from chemical exposure may have more severe consequences if combined with normal immunosenescence.

Several epidemiologic studies have described associations between early-life chemical exposure, altered immune end points, and frequency of infections. Increased incidence of otitis media and respiratory infections were reported in children exposed to PCBs [START_REF] Dallaire | Effect of prenatal exposure to polychlorinated biphenyls on incidence of acute respiratory infections in preschool Inuit children[END_REF][START_REF] Dewailly | Susceptibility to infections and immune status in Inuit infants exposed to organochlorines[END_REF][START_REF] Nakanishi | Respiratory involvement and immune status in yusho patients[END_REF][START_REF] Weisglas-Kuperus | Immunologic effects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children[END_REF][START_REF] Yu | The immunologic evaluation of the Yucheng children[END_REF]. For example, at 3 months of age, multiple upstream indicators of impaired immune function were detected in breast-fed infants with higher PCB exposure levels, including reduced numbers of white blood cells and lymphocytes, and lower serum IgA levels at 7 and 12 months of age [START_REF] Dewailly | Susceptibility to infections and immune status in Inuit infants exposed to organochlorines[END_REF]. These studies also reported changes in immune system biomarkers, such as changes in blood cell counts and T-cell subsets or decreased serum Ig levels with increasing PCB exposures [START_REF] Karmaus | Infections and atopic disorders in childhood and organochlorine exposure[END_REF][START_REF] Weisglas-Kuperus | Immunologic effects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children[END_REF]. Antibody responses to tetanus toxoid vaccine were significantly decreased after early postnatal PCB exposure [START_REF] Heilmann | Reduced antibody responses to vaccinations in children exposed to polychlorinated biphenyls[END_REF].

Individuals with very severe forms of immunosuppression, as occur in primary immunodeficiency diseases and AIDS, frequently develop opportunistic infections. Xenobiotic agents are likely to cause considerably more subtle immunologic effects. The interaction among host genetics, pathogen virulence, and pathogen dose plays a role in determining the frequency and severity of illness. For individuals exhibiting mild immunosuppression, as may be associated with xenobiotic chemical exposure, the infectious dose of a given pathogen may be lower than that which would cause disease in an individual whose immune system is functioning optimally. Limited studies in populations with mild forms of immunosuppression (e.g., under psychological stress or administered immunosuppressive therapies) have provided qualitative evidence that moderate changes in immune function can also lead to an increased incidence of infectious or neoplastic diseases [START_REF] Cohen | Psychological stress and susceptibility to the common cold[END_REF][START_REF] Kiecolt-Glaser | Psycho-oncology and cancer: psychoneuroimmunology and cancer[END_REF].

Conclusions regarding immunosuppression data. In summary, exposures to environmental contaminants have the potential to affect upstream immune indicators, including antibody synthesis, T-cell function, and other measures of immunocompetence, which can result in compromised downstream resistance to infection. However, the impact of background levels of xenobiotics on the burden of disease has not been clearly established. If a positive correlation between increasing exposure and disease is assumed, then even small changes in immune function will represent an increased risk for developing disease.

Common Themes

The case studies demonstrate that certain toxicity pathways are understood well enough that screening assays to identify early biological changes could be used for hazard identification and risk assessment. For example, if a chemical is found to suppress thyroid function, the associated data could be used to assess risk for developmental neurotoxicity, because the latter effect can be predicted on the basis of the mechanism of action alone. Similarly, when a chemical is found to be antiandrogenic, the current science would support using the data to assess increased risk of developmental effects. Data from whole-animal tests of the downstream outcomes would not be required for risk assessment of these outcomes. The findings of this workshop support the recent National Academy of Sciences recommendation that toxicity testing move toward the direct assessment of upstream events along toxicity pathways and gradually move away from the current whole-animal end point-based assays (National Research Council 2007).

The workshop also explored some of the challenges that arise when using upstream events to assess the adverse effects of chemical exposures during hazard identification or risk assessment. Several common themes emerged from the case study discussions. Some of these themes are not unique to the use of upstream indicators as the basis of hazard identification and risk assessment. Rather, they underscore issues that should be addressed in existing hazard identification and risk assessment practices.

Chemical and biological background. Each of the case studies illustrated the importance of considering preexisting or continuous exposure to environmental chemicals as well as preexisting biological or disease susceptibilities that contribute independently to risk of overt disease. Preexisting exposures or biological vulnerabilities can increase the baseline risk of the population or enhance already initiated disease processes (Figure 2). Consequently, slight perturbations in upstream biological indicators are more likely to increase risk of subsequent downstream events given an already more activated state among segments of the population.

Chemical background and exposure to mixtures. NHANES data indicate that the entire U.S. population has measurable levels of multiple environmental contaminants in their bodies (Centers for Disease Control and Prevention 2008). Other studies of exposure pathways show the population is in constant contact with xenobiotics in air, food, water, and consumer products (Environmental Working Group 2005[START_REF] Epa | Where You Live[END_REF]; National Library of Medicine 2008; U.S. EPA 2007b; U.S. Food and Drug Administration 2007). U.S. EPA guidelines for mixtures risk assessment recommend default assumptions of dose additivity for mixtures of chemicals with similar toxicologic activity and response additivity for mixtures of chemicals that act independently (U.S. EPA 2000a). The case studies showed that these preexisting and concurrent exposures can increase the effect a given chemical exposure has on disease risk, consistent with the dose-additivity default. For example, separate studies of antiandrogenic chemicals and thyroid-disrupting chemicals found that mixtures of chemicals acting on common systems via different modes of action had dose-additive effects. Although a less-than-additive response [referred to in the U.S. EPA mixtures guidelines as "antagonism" (U.S. EPA 2000a)], or a greater than additive response ("synergism") are possibilities, the case studies support application of dose additivity as a default, as recommended in the guidelines. Considering the influence of multiple chemical exposures on upstream events can facilitate a more realistic characterization of the risks of downstream effects when assessing a single chemical that is an additional increment to the background exposure mixture.

Biological background. Background health status, as influenced by age, preexisting disease, genetics, and other factors, can influence the effect of chemical exposure on upstream and downstream adverse end points. For example, the implications of a chemical's interference with iodine uptake into the thyroid are substantially greater for an infant than for an adult because of differences in thyroid hormone storage and circulating half life. Likewise, the implications are greater for the 38% of pregnant women in the United States who are deficient in iodine [START_REF] Aoki | Serum TSH and total T 4 in the United States population and their association with participant characteristics: National Health and Nutrition Examination Survey (NHANES 1999-2002)[END_REF]) and who simultaneously have greater thyroid hormone needs to support the neurologic development of a fetus. Individuals with preexisting immune suppression, such as organ transplant patients, those who are HIV positive, and those at early or late life stages, might experience disproportionately higher disease risk due to chemical exposure.

Periods of susceptibility. Exposure to environmental chemicals during periods of susceptibility can pose a unique risk of subsequent downstream effects, both in the short and long term, as well as diminished capacity for recovery from decrements to physiological systems. For example, exposure to thyroid-disrupting chemicals such as perchlorate during the fetal and early child development can produce adverse effects on neurologic development by decreasing TH levels; the same mild perturbations in TH levels would not present an adverse challenge to a healthy adult. Similarly, to impair male reproductive development in

Percent of population

Value of physiological parameter

Nonadverse Adverse

No co-exposures or biological susceptibility Co-exposures, no biological susceptibility Co-exposures and biological susceptibility rodents, exposure to phthalates must occur during gestation days 12-19. When exposure occurs during periods of developmental susceptibility, the dose required to produce an effect will typically be lower than the dose required to produce effects in fully developed, healthy adults.

Multiple or complex modes of action. Exposure to a chemical can influence disease processes through multiple modes of action and also increase the risk of more than one downstream, overt effect. Hence, measurements that focus on a particular pathway or individual downstream event may provide an incomplete picture of the range of effects produced by the chemical. Phthalates work through multiple pathways to decrease androgen production but also interfere with the production of INSL-3. The resulting deficits in these hormones during male fetal development can increase the risk of several different downstream effects, both manifested early in life (e.g., cryptorchidism) and later in life (e.g., effects on sperm). Data interpretations therefore should consider the possibility of a complex set of modes of action that produce adverse effects on the whole system. Focusing on one single aspect of these complex modes of action can result in inadequate characterization of system response.

Continuous versus discrete events. Overt downstream effects, such as cancer, birth defects, and infectious illnesses are typically discrete events. In contrast, upstream indicators of adverse effects, such as changes in hormone levels or cellular markers of immune function, are often measured on a continuous scale. Risk assessment practices recognize differences in frequency of discrete events as an indication of an adverse effect. Characterization of changes in continuous outcomes in risk assessment requires further consideration.

One key issue is assessing the level of change constituting adversity. Should any chemical-induced increase or decrease be considered adverse? Should there be a focus on particular cut points in the distribution of population or individual baseline levels? These questions should be considered in the context of the issues of background exposures and agerelated susceptibilities noted above. One possibility is to consider that deviations from expected baseline (e.g., for a given individual, with all its characteristics, susceptibilities, age, sex, physiologic state) will increase the probability (risk) of downstream adverse effects. For example, changes in biological function during susceptible periods, such as during development, can increase risk of subsequent downstream events compared to biological function changes of the same magnitude at other life stages. Whether the perturbation is reversible and whether it is possible to use population reference ranges to detect adversity are two key questions that may arise in the process of considering continuous upstream indicators of adverse effects.

Reversibility. Biological perturbations resulting from chemical exposure are often evaluated in regard to whether they are reversible. For example, exposure to chemicals can result in immune system suppression and increased opportunity for infections. A suppressed immune system may recover once chemical exposure is stopped, and the risk of infectious disease returns to original levels. However, for assessments of a constant chronic or lifetime exposure, recovery from shorter duration exposures should not be considered when evaluating whether the observed response is adverse. Whether a perturbation is reversible can depend on intrinsic factors, such as age or disease status, and extrinsic factors, such as preexisting or co-exposures to other contaminants or the timing of exposure. For example, gestational and neonatal exposure to DZP and DES in rodents can cause long-lasting effects on immune function; in contrast, adult rodents exposed to the same dose experience reversible effects [START_REF] Luebke | The comparative immunotoxicity of five selected compounds following developmental or adult exposure[END_REF].

Population variability and defining the normal range. Basing the definition of "normal" function for a biologic parameter on population reference ranges may not reflect an individual's normal range or variability and may fail to detect an adverse effect. For example, an individual may experience an adverse decline in thyroid hormone but still remain within the normal population reference range (Figure 1). For situations in which the interindividual variability in a continuous upstream indicator is high, alternative methods to detect adverse changes in the biologic parameter will need to be developed.

Conclusions and Recommendations

Workshop participants concluded that for the toxicity pathways represented in these case studies, it may be feasible to move toward using direct evaluation of upstream mechanistic indicators as the basis for risk assessment and decision-making.

The case studies illustrate the complexities of the biological changes influenced by exposures to environmental contaminants. Capturing and translating the complexities of the science is an ongoing challenge in the regulatory and policy arena. While science pursues new areas of inquiry, decision making requires timely answers to questions about risks and hazards to public health in order to mitigate future or current potential harm. The regulatory context also requires a different sufficiency of evidence whereby regulatory decisions can be made based on evidence that a chemical is likely to cause a particular outcome. The value of a parallel-tracks approach was noted, in which scientific investigation continues in one track while development of appropriate principles and practices for assessing the emerging science for timely public health-based decision-making continues in another.

Identifying classes of upstream events. The case studies of thyroid perturbation, antiandrogen activity, and certain types of immune changes each represent a class of perturbations whose links to downstream events were determined by workshop participants to be sufficiently characterized by the data to move toward considering issues of implementation. A suggested next step is to evaluate chemicals for their ability to cause the upstream perturbation identified in the case studies. For these chemicals, hazard and risk assessment could be initiated with data on the relationship between exposures and early perturbations. Fewer data are needed for subsequent downstream events, given what is already known on risks linking the pathway from early to late events, and with the presumption that downstream consequences are likely to occur when the upstream perturbation is observed.

Participants noted other types of early biological perturbations that were not discussed in the case studies but that are also likely sufficiently characterized as to be defined as a class. Early biological perturbations that appeared to be sufficiently characterized include certain high affinity and persistent interactions with the Ah receptor, changes in hormonal responses [which are identified in reproductive toxicity risk assessment guidelines as an adverse event (U.S. EPA 1996)], and cholinesterase inhibition [which has been identified as an indicator of adverse effects (U.S. EPA 2000b)]. It would also be beneficial to expand the list to include biological perturbations that appear common to many environmental chemicals, such as effects on birth weight or liver function.

Participants emphasized that additional information on upstream indicators does not necessarily reduce uncertainty, although it does add more information about the exposuredisease continuum and increases the predictive power of risk assessment and can demonstrate greater variability across study subjects than previously recognized. Hence, using upstream indicators does not necessarily mean reducing applicable uncertainty or adjustment factors.

Next steps. The case studies suggest chemical exposure-induced changes in upstream biological markers have utility for hazard identification and risk assessment. The approaches to using these upstream indicators should consider factors such as biological background, chemical background (and the potential for dose additivity or synergism), periods of susceptibility, and multiple and complex modes of action that can increase the risk of subsequent downstream events. The case studies also illustrated the need to better integrate the nonzero baseline concept into hazard and

Woodruff et al. 1570 VOLUME 116 |

 1570116 NUMBER 11 | November 2008 • Environmental Health Perspectives

Figure 1 .

 1 Figure 1. Distribution of 12 monthly measurements of total T 4 in 15 healthy men (white bars) and one individual (black bars). The distribution in one individual is about half the width of the distribution in the group. Frequency represents number of measurements. Adapted from Andersen et al. (2002) with permission.

Figure 2 .

 2 Figure 2. Distribution of a typical physiological parameter within the population and how that may vary depending on the influence of chemical and biologic background.

Table 1 .

 1 Table 1 provides an overview of Upstream adverse end points Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 20081569 Classes, mechanisms of action, and effects of thyroid-disrupting chemicals on thyroid hormone homeostasis.

	Class	Mechanism	Effects on thyroid hormones	Chemicals
	Iodine transport	Inhibit uptake of iodine	Decreased thyroidal synthesis of T 3 and T 4	Perchlorate, chlorate, bromate, nitrates, thiocyanate
	Synthesis inhibitors	Inhibition of thyroperoxidase	Decreased thyroidal synthesis of T 3 and T 4	Methimazole, propylthiourea, amitrole, mancozeb,
				soy isoflavones, benzophenone 2,
				1-methyl-3-propyl-imidazole-2-thione
	Transport disruption	Altered binding to serum transport	Unknown	Hydroxylated PCBs, EMD 49209;
		proteins		pentachlorophenol
	Enhanced hepatic catabolism	Up-regulation of	Increased biliary elimination of T 3 and T 4	Acetochlor, phenobarbital, 3-methylcholanthrene,
		glucuronylsyltransferases or		PCBs, 1-methyl-3-propyl-imidazole-2-thione
		sulfotransferases (via CAR/PXR
		or AhR)
	Enhanced cellular transport	Up-regulation of OATPs or MCT	Increased biliary elimination of T 3 and T 4	TCPOBOP, pregnenolone-16α-carbonitrile, TCDD,
		transporters via CAR/PXR or AhR		rifampicin, phenobarbital, oltipraz
	Sulfotransferases	Inhibition of sulfotransferases	Decrease sulfation of THs	Hydroxlyated PCBs, triclosan, pentachlorophenol
	Deiodinases	Inhibition or upregulation	Decreased peripheral synthesis of T 3	FD&C Red dye no. 3, propylthiouracil, PCBs,
		of deiodinases		octyl-methoxycinnamate
	TR agonists and antagonists	Direct or indirect alterations in	Altered activation of TH-dependent gene	Tetrabromobisphenol A, bisphenol A,
		TR-TRE binding	transcription	hydroxylated PCBs

in isolation are likely to underestimate the potential disruption of TH levels or action by real-world chemical mixtures.

	Studies of human body burdens indicate
	simultaneous human exposure to multiple
	TDCs, including dioxins, PCBs, perchlorate,
	brominated flame retardants, bisphenol A, and
	several pesticides (Centers for Disease Control
	and Prevention 2008). A mixture of 18 TDCs
	(dioxins, dibenzofurans and PCBs) was tested
	at doses comparable to human exposure levels
	for effects on serum T 4 in rats. Components of
	this mixture affect T 4 through two different
	mechanisms of action: The dioxins, dibenzo-
	furans, and dioxin-like PCBs in the mixture
	activate one set of liver enzymes, and the non-
	dioxin-like PCBs activate a separate set of liver
	enzymes.

  Upstream adverse end pointsEnvironmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008

Abbreviations: AhR, aryl hydrocarbon receptor; CAR: constitutive androstane receptor;FD&C, Federal Food, Drug, and Cosmetic Act (1938); MCT, monocarboxylate transporter; OATPs, organic anion-transporting polypeptides; PXR, pregnane X receptor;TCDD, 2,3,7, TCPOBOP, 1,]benzene; TH, thyroid hormone; TR, thyroid receptor; TRE, thyroid hormone response elements. Data from[START_REF] Crofton | Thyroid disrupting chemicals: mechanisms and mixtures[END_REF].

VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives

This work was supported by California Environmental Protection Agency, Office of Environmental and Health Hazard Assessment, contract OEHHA-06-S34; the U.S. Environmental Protection Agency, Office of Policy, Economics, and Innovation, National Center for Environmental Economics and National Center for Environmental Assessment, contract EP07H001060; the Intramural Research Program of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH); University of California, Berkeley, NIEHS Superfund Program at Berkeley, NIH grant P42 ES04705; and University of California, San Francisco.

This report has been reviewed by the National Institute of Occupational Safety and Health and the U.S. Environmental Protection Agency's Office of Research and Development, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of institutions affiliated with the authors.

Upstream adverse end points

Environmental Health Perspectives 1575 risk assessment-in other words, background exposures and processes can confer a greater potential for effects than when exposure to a chemical is considered in isolation. An important implication of this concept is that the current framework for addressing risks from noncancer health effects no longer fits with what we know about the science and needs of the decision-maker for current understanding of potential risk. Disease status, susceptibility, and chemical background are critical to consider when assessing the implications of exposure, as they can put a portion of the population in the dose range where small incremental exposures can increase risk of downstream events. This suggests that the assumption of a threshold dose level-below which no deleterious effects occur (U.S. EPA 2002)-may not apply, and incorporating concepts traditionally used for nonthreshold events should be considered.


Agnès Janes
email: agnes.janes@ineris.fr

Douglas Carson

Antoinette Accorsi

Jacques Chaineaux

Benoît Tribouilloy

Daniel Moranviller

A Janes -D

Carson -A

Accorsi -J

Chaineaux-B

Tribouilloy - D

Morainvillers

Correlation between self-ignition of a dust layer on a hot surface and in baskets in an oven

Keywords:

Nomenclature:

A: pre-exponential factor of Arrhenius equation [s -1 ] E: apparent activation energy [J.mol -1 ] h: global heat transfer on storage surface (convection and radiation) [W.m -2 .K -1 ] M: lumped parameter (which depends on parameters such as heat of combustion, heat capacity, density) [

INTRODUCTION

Self-ignition is a common source of ignition of porous storage of bulk materials such as powders and dusts. Evaluation of this ignition hazard, taking into account manufacturing or storage conditions, requires experimental determination of critical self-ignition temperatures as a function of volume.

One of the first standardized tests [START_REF] Cen | Electrical apparatus for use in the presence of combustible dust -Part 2-1: Test methods -Methods for determining the minimum ignition temperature of dust[END_REF] consists of measuring the minimum temperature of a flat hot surface which results in the ignition of a dust layer of a given thickness, which is generally 5 mm. This test is particularly relevant for industries where dust is handled and can accumulate on potentially hot surfaces. However, this test is not always easy to carry out when the dust is blown away due to air movement or when the layer cracks due to the hot surface. Thus, when testing a 5 mm layer is not possible, the same test can be carried out with a thicker dust layer.

Consequently, as we know [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF], the self-ignition temperature will be lower than that of a 5 mm layer. This vas more recently reported by EL SAYED [START_REF] El-Sayed | Smoldering combustion on dust layer on hot surface[END_REF].

A second test is used to determine critical self-ignition temperatures. Bulk materials are placed in several baskets of various sizes, which are placed in a heating oven. So far, according to a theoretical model, relation between self-ignition temperature and bulk storage size can be found. This second method is easier to conduct than the previous one. This second method is also used to classify self-ignition substances for the transport of dangerous goods [START_REF]Transport of Dangerous goods, Manual of Tests and Criteria[END_REF]. This classifying methodology was discussed in the past [START_REF] Chervin | Testing strategy for classifying self-ignition substances for transport of dangerous goods[END_REF] and [START_REF] Jones | A new and more reliable test for the propensity of coals and carbons to spontaneous heating[END_REF].

Phenomena highlighted in these two tests are similar and their results can be connected with theoretical relations.

The aim of this paper is to describe a correlation model between both methods.

Then, results of an experimental validation campaign are presented and discussed.

CONTEXT

Since application of European directive 1999/92/EC [7], several actions must be taken by employers. It includes: evaluation of explosive atmospheres1 hazards, classification of area where explosive atmospheres may occur, identification of technical and/or organisational explosion control measures, use of appropriate electrical and non-electrical apparatuses according to area classification, set up and regular update of the so called "explosion protection document".

Explosion hazards assessment shall take into account the likelihood that ignition sources will be present and become active and effective. Thus, substances used and their ignition probability shall be considered to select safe work equipment and safe working conditions.

As far as electrical apparatuses are concerned, EN 1127-1 standard [START_REF]CEN, Explosive atmospheres -Explosion prevention and protection -Part 1: Basic concepts and methodology[END_REF] sets two specifications for devices. One is about dust permeability and the other concerns limitation of surface temperature. Indeed, this standard considers that apparatuses can be safely operated when dust is present, if its maximum surface temperature is: lower than the two thirds of dust self-ignition temperature in cloud, and lower than the self-ignition temperature of a 5 mm layer minus 75 K.

SELF-IGNITION MODELING

Self-ignition is related to exothermic oxidation reactions within the mass of bulk particle storage.

When the storage conditions do not allow complete dissipation of heat generated by these slow going oxidation reactions by the means of a sufficient heat exchange between reactional system and external environment, a rise of temperature within the mass is observed.

Three parameters are predominant for self-ignition phenomenon: product temperature, in regard to ambient temperature, storage size, oxygen diffusion conditions through the storage.

Models of self-ignition were developed in the 1920's by SEMENOV. This work was then continued by FRANK-KAMENETSKII [START_REF] Franck-Kamenetskii | Diffusion and heat transfer in chemical kinetics[END_REF] and later by THOMAS [START_REF] Thomas | Ignition, heat release and non-combustibility of materials ASTM STP[END_REF].

A comparison of SEMENOV and FRANK-KAMENETSKII theories was recently proposed by Shouman [START_REF] Shouman | A review of one aspect of the thermal-explosion theory[END_REF]. SEMENOV's model considers an equal temperature throughout the volume, as shown on figure 1a. It is based on geometrical dimensions, surface and volume of storage. Since this model does not take into account heat gradients between centre and periphery, it is not valid for large size storage.

FRANK-KAMENETSKII's model takes into account a temperature gradient inside the sample, which depends on its geometrical dimensions and thermal conductivity. In this model, heat transfer is achieved by optimal conduction into the material, and by convection and radiation on the storage periphery. In this case, as shown on figure 1b, surface temperature is equal to ambient temperature. This last hypothesis implies a high and global coefficient of heat transfer on the storage surface. If not, the surface temperature remains higher than ambient temperature. Finally, THOMAS and BOWES'model [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF] takes into account both internal thermal resistances by conduction and external thermal resistance by convection and radiation. This theory considers thermal conductivity of material, geometrical dimensions of storage as well as parameters of convection and radiation at the interface between material and air. The relation between thermal conduction inside the storage and convection on the surface is given by equation 1 [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF], which uses the BIOT number (α). α = (h.r)/λ

(1)

BIOT number depends on storage size. It tends towards 0 when storage size tends towards 0 for equal values of global heat transfer coefficient on storage surface and thermal conductivity inside the sample.

When thermal conductivity of material is high compared to exchange coefficient on surface, BIOT number tends also towards 0. In this case, internal temperature of sample is homogeneous and higher than ambient temperature, that correspond to SEMENOV's theory.

On the contrary, if thermal conductivity of material is low compared to heat transfer on surface, BIOT number tends towards infinite, that corresponds to FRANK-KAMENETSKII's model.

If there is an equal repartition of heat transfer resistance between inside storage conduction and outside convection and radiation, BIOT number is equal to 1.

CORRELATION BETWEEN SELF-IGNITION OF A DUST LAYER AND SELF-IGNITION IN AN OVEN

Critical self-ignition temperature is determined for different sizes of cubic latticed baskets in isothermal ovens, which are filled by samples, as it is described in Pr EN 15188 standard [START_REF] Cen | Determination of the spontaneous ignition behaviour of dust accumulations[END_REF]. Experimental results take the form of couples of data (r, T c )

where r is the half-edge length of cubic basket and T c the critical self-ignition temperature, for each sample.

These values are introduced in the model written as shows equation 2 [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF].

Ln [( c .T c ²)/r²] = M -(P/T c ) (2)
where

M = Ln [(E.Q. .A)/(R. )] P = E/R Notes:
Ln is the Napierian logarithm M and P parameters can be calculated by linear regression (least square method).

To make the equations ( 2) and (3) be rigorous, it is necessary to add a multiplication coefficient equal to 1 [K -2 m 2 ] in the expression ( c .T c ²)/r².

In the particular case of a layer deposited on a hot surface, ambient temperature is not homogeneous and the heating is unsymmetrical.

The temperature of the layer in contact with the hot surface is equal to hot surface temperature itself, while the external layer surface temperature can be calculated with ambient temperature and BIOT number, according to equations 3 and 4 [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF].

Ln [( cp .T c ²)/r²] = M -(P/T c ) (3)
δ cp is calculated according to equation 5 below.

2 2 2 4 . 1 2 1 2 1 a p p cp T T RT E (4)
The validity conditions of equation 4 are [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF]:

(E/RTp 2 ) . (Tp -Ta) > 8, and α > 0.5.

In the case of self-ignition by a hot surface, the geometrical constant depends on several parameters, such as apparent activation energy (E), ambient temperature (T a ), hot surface temperature (T p ) and geometry of the deposit and efficiency of heat transfer between hot surface and dust layer. On the contrary, in the case of selfignition in an isothermal oven, the geometrical constant depends only on geometry of the basket. (5') Equation 5' gives the half-size of a cube tested in isothermal oven, which corresponds to the half-thickness of a layer deposited on a hot surface for which the self-ignition temperature is equal to the self-ignition temperature of the cube.

Prediction of the critical temperature of a dust layer deposited on a hot surface can be done by plotting this equation 5' on a graph. The intersection point with the curve obtained by isothermal oven test, for the same sample, gives the theoretical selfignition temperature of a layer on a hot surface.

APPARATUS AND PROCEDURE

IGNITION OF A DUST LAYER DEPOSITED ON A SURFACE HEATED AT A CONSTANT TEMPERATURE

The minimum ignition temperature of a dust layer of specified thickness is the lowest temperature of a hot surface at which ignition occurs. According to EN 50281-2-1 [START_REF] Cen | Electrical apparatus for use in the presence of combustible dust -Part 2-1: Test methods -Methods for determining the minimum ignition temperature of dust[END_REF] standard, determination of this temperature needs a heating electric resistance, with a metallic plate disposed on it. The maximum temperature of the plate used in this study is 450 °C.

Dust layer is prepared by filling the cavity formed by a metal ring of appropriate height, placed on the heated surface and by levelling the layer on the top of the ring. This ring, which internal diameter is equal to 100 mm, is left in place during the test.

A given dust is tested in a layer of 5.0 mm +/-0.1 mm depth. Two other depths are also studied: 12.5 mm +/-0.1 mm and 15.0 mm +/-0.1 mm. Several temperature measurements are carried out during a test (fig. 2): ambient temperature, with a thermocouple 2 placed at 1 m of the heated surface but shielded from heat convection and radiation from the surface, dust layer temperature, with a thermocouple stretched across the heated surface, parallel to it, 2 mm height from it, with the hot junction over the centre of the plate, heated surface temperature, with a thermocouple disposed inside the heated plate mass, parallel to it and with the hot junction on the centre of the plate.

The dust is put into the ring with a spatula and spread without compressing.

For the determination of the minimum ignition temperature of a layer of a given thickness, repeated trials are carried out, using a fresh layer of dust for each trial and with up-and-down adjustments to the temperature of the heated surface.

Ignition is considered to have occurred if: visible glowing or flaming is observed, or a temperature of 450 °C is reached, or a temperature rise of 250 K above the temperature of the heated plate is measured.

Figure 3 shows ignition of a 5 mm zinc dust on the heating surface.

On the contrary, if none of these three conditions is satisfied after a 30 min delay, ignition is not considered to have occurred. This non-ignition test is repeated two times.

Trials are made until the ignition temperature is framed by a temperature value for which ignition is observed and another for which no ignition has occurred. These two values must not be different by more than 10 K. The experimental ignition temperature of the dust layer is the lowest temperature.

We consider that these apparatus and procedure lead to an uncertainty of + 4.5 K / -14.5 K [START_REF] Janes | Application de la norme EN 15188 relative à l'auto-échauffement des solides[END_REF].

SELF-IGNITION OF DUST IN OVENS

Critical self-ignition temperatures for different size of cubic baskets are determined by successive trials in heating ovens.

According to the FRANK-KAMENETSKII's model, relation between self-ignition temperature and storage size can be set. Then, extrapolation from this relation can help, for different storage geometry to evaluate: the critical size (maximum size) of a storage for a specified temperature, which is particularly useful for low temperatures, 2 all temperature measurements in this study use chromel-alumel (K) thermocouples the critical storage temperature (maximum temperature) for specified size and geometry.

According to PrEn 15188 [START_REF] Cen | Determination of the spontaneous ignition behaviour of dust accumulations[END_REF], critical self-ignition temperatures are assessed using 125 L re-circulating air ovens, with a maximum temperature of 300 °C and equipped with a middle-height grid, as shown on figure 4. Ventilation is provided to exhaust any gases released from the dust.

The dust sample is filled into mesh wire baskets of different sizes with a spatula, without compressing and levelled on the top. Baskets are open at the top and closed at the bottom. The width of the mesh used, made of stainless steel, is equal to 10 µm, in order to avoid sifting but to allow oxygen diffusion. Volumes used in this study are: 8 cm 3 , 125 cm 3 , 343 cm 3 and 1000 cm 3 , so critical sizes of cubic baskets (half height) are: 1 cm, 2.5 cm, 3.5 cm and finally 5 cm. These baskets, being filled by the sample, are located on the centre of the oven preheated to the test temperature.

Two temperature measurements are made during a test (fig. 5): oven temperature, with a thermocouple freely installed in the air space at half distance between the sample surface and the inner wall of the oven. Tests have shown that maximum temperature difference between two points in an oven is about 2 K [START_REF] Janes | Application de la norme EN 15188 relative à l'auto-échauffement des solides[END_REF], sample temperature, with a thermocouple located with its hot junction directly at the centre of the sample.

If sample temperature, after an induction delay, grows abruptly and rises at least 60 K above the oven temperature, it is considered that self-ignition is occurring. On the contrary, we assume that self-ignition did not occur if sample temperature is remains close to oven temperature after an induction delay or exceed it of a few K.

A sufficient number of tests is carried out with a fresh dust sample for each trial, until the oven temperature is high enough to cause ignition of the sample but which is no more than 5 K higher than a temperature which fails to cause ignition.

Critical temperature, for a specified basket size, is equal to the middle value of the interval framed by these two previous temperatures. We consider that the undertaking of the procedure described lead to an uncertainty of +/-8.0 K [START_REF] Janes | Application de la norme EN 15188 relative à l'auto-échauffement des solides[END_REF].

When critical temperatures are obtained for the four baskets sizes, these experimental points can be correlated. For each of the four couple of data (r, T c ) obtained, two coefficients (A, B) are calculated, according to equations 6 and 7 below.

A = Ln (2.6 T c ²/r²) (6) B = 1/T c (7)
Notes:

Ln is the Naperian logarithm 2.6 is the value of the Franck-Kamenetskii parameter for a cubic storage (δ c ).

According to the previous equation 2 (see also section 4) below [START_REF] Bowes | Self heating : evaluating and controlling the hazards[END_REF], the plot of the four points (A, B) describes a line.

Ln

[( c .T c ²)/r²] = M -(P/T c ) (2)
M and P parameters are then calculated by linear regression (least square method).

PROTOCOL SELECTED FOR EXPERIMENTAL CORRELATION

In order to check experimentally the correlation between the results of self-ignition in ovens and self-ignition on heated surfaces, we proceeded as follow, for each of the 14 samples tested.

RESULTS AND DISCUSSION

Results of self-ignition tests in heating ovens are shown in table 1. Tables 2 to 5 show differences between experimental and theoretical results of self-ignition temperatures of dust layers.

INFLUENCE OF BIOT NUMBER (α) ON THEORETICAL SELF-IGNITION

TEMPERATURES

Comparison of theoretical values is shown on table 5. It shows that, for a given dust layer thickness, the calculated self-ignition temperature increases with α value.

Table 5 points out that the difference between theoretical values of self-ignition temperatures is influenced either by dust layer thickness or by the nature of the sample. One exception is noticed: in the case of the activated carbon dust, the variation is significantly higher when α value is modified. This could be explained by the high value of carbon dust specific surface, in comparison with the others. This influences the value of the M and P global parameters when calculating theoretical self-ignition temperatures.

DIFFERENCE BETWEEN EXPERIMENTAL AND THEORETICAL SELF-IGNITION TEMPERATURES

Differences between experimental and theoretical self-ignition temperatures are shown in tables 2 to 4. Accuracy of correlation obtained is illustrated on these tables by colouring: the darker, the less accurate correlation.

In a general basis, experimental results for 5 mm dust layers agree with theoretical previsions, excepted for two varieties of wheat dust (n°1 and 3) and for the zinc powder. Calculated self-ignition temperatures are higher than experimental values for two thirds of the tested samples. The best correlation (difference lower than 10 K) are obtained with a α value equal to 1 (5 cases out of 11).

Theoretical prevision under-estimates the experimental self-ignition temperature of 12.5 mm dust layers for every samples tested. Temperature differences are systematically higher are than 10 K. The closest calculated temperature to the experimental one is obtained when α value tends to infinite. The worse correlation is obtained for one variety of wheat dust (n°2).

Calculated self-ignition temperatures for 15 mm layers are lower than experimental values for 80 % of tested samples. The best correlation (difference lower than 10 K) are obtained when α value tends to infinite (3 cases of 7). The worse correlation is obtained for the coal dust.

SYNTHESIS

Principal lessons resulting from this study are summarised below.

For 5 mm dust layers, it is possible to determinate a theoretical self-ignition temperature based on self-ignition tests in heating ovens. The most adapted BIOT number (α) seems equal to 1, it corresponds to a repartition of heat transfer between conduction inside the layer and convection on the surface (THOMAS and BOWES's model). In these conditions, the uncertainty obtained for self-ignition temperature is less than 30 K, except zinc powder (150 K) and wheat dust n°1 (80 K). It must be pointed out that calculated values are generally higher than experimental ones.

For 12.5 mm and 15 mm dust layer thickness, determination of a theoretical selfignition temperature based on self-ignition tests in heating ovens is also possible, if the chosen BIOT number (α) tends to infinite. This condition corresponds to the limitation of heat transfer by conduction inside the dust layer (FRANK-KANENETSKII's model). For these conditions, the uncertainty on self-ignition temperature is about 10 to 30 K, except for wheat dust n°2 (98 K) and coal dust (70 K).

This study has also shown that for several tested samples (e.g. zinc, wheat or coal dust), there is a discrepancy between theoretical value of self-ignition temperature and the experimental ones. Different explanations can be found:

the most adapted α value can be lower than ½. This is probably true for zinc dust. In fact, a study carried out by INERIS [START_REF] Janes | Application de la norme EN 15188 relative à l'auto-échauffement des solides[END_REF] showed that FRANK-KAMENETSKIIs' model is not applicable to aluminum dust, due to good conduction efficiency. A complementary study is necessary to extend this hypothesis to other samples characterised by a high conduction coefficient, such as metal powders in specific granulometry conditions, samples chemical modification at low temperature, such as complex agroalimentary molecules. This could explain the poor correlation obtained for e.g. wheat dust, in the case of persulphate powder, a foaming was observed. This particular behaviour can also be one of the causes of a bad correlation.

In all these hypothesis, equations from FRANK-KAMENETSKII's model, which was used in the study, are not valid.

CONCLUSION

The correlation method describes in this paper can be considered valid to estimate self-ignition temperature of a dust layer for several samples from the results of selfignition tests in ovens, if the value of BIOT number (α) is judiciously chosen:

for 5 mm dust layers, the most adapted value is equal to 1, for 12.5 mm and 15 mm dust layers, it is preferable to choose a very high value for α.

Uncertainty on results is lower than 30 %, if temperature is calculated in K. It is in the same order than uncertainty induced by testing a 15 mm layer self-ignition temperature, which is less difficult to obtain but pessimistic, instead of a 5 mm layer self-ignition temperature.

Finally, when the sample behaviour (foaming...) does not allow to carry out the standardised procedure to measure self-ignition temperature of a 5 mm dust layer, we recommend:

to calculate the theoretical self-ignition temperature based on results of selfignition tests in heating ovens, within the validity domain of FRANK-KAMENETSKII's model, to consider the self-ignition temperature of a 15 mm thick sample, determined by the standardised method, in other situations.

In both cases, uncertainties on results must be taken into account.

Additional tests would help refine the knowledge of samples for which FRANK-KAMENETSKII's model is not valid, e.g. materials whose conduction coefficient is very high or samples which undergo chemical conversion at low temperatures.

AKNOWLEDGMENTS

This work was financially supported by the French Ministry of Environment and Sustainable Development.

Tables and figures

  dimensionless] P: lumped parameter [K] r: critical dimension (half-edge length of cubic basket at the self-ignition temperature) [m] R: universal gas constant. 8.314 [J.(mol.K) -1 ] Q: heat of reaction per unit mass [kJ.kg -1 ] T a : room temperature [K] T c : self-ignition temperature of a given sample volume [K] T e : isothermal oven temperature [K] T p : hot plate temperature [K] T s : upper surface temperature [K] T 0 : storage's central point temperature [K] z: critical dimension (half-thickness of the layer at the self-ignition temperature) [m] Greek symbols α: BIOT number [dimensionless] δ c : FRANK-KAMENETSKII parameter for heating oven tests. 2.6 for a cubic storage [dimensionless] δ cp : FRANK-KAMENETSKII parameter for hot plate tests [dimensionless] λ: thermal conductivity of material [W.m -1 .K -1 ]: density [kg.m-3 ]

  So far, reliable methods are needed to determine self-ignition temperatures in order to demonstrate the safe use of equipment. Standards applied in this study are: EN 50281-2-1 [1]: Electrical apparatus for use in the presence of combustible dust -Part 2-1: Test methods -Methods for determining the minimum ignition temperature of dust, PrEN 15188 [9]: Determination of the spontaneous ignition behaviour of dust accumulations.

By dividing equation 4

 4 by equation 2, for the condition T e (determination of selfignition temperature by isothermal oven method) = T p (determination of self-ignition temperature by hot surface method), equation 5 is obtained.

  Development of equation 5 leads to equation 5'.

1 .

 1 self-ignition tests in heating ovens, for the four sizes of cubic baskets and calculation of M and P parameters by correlation of experimental results, 2. calculation of geometrical constant (δ cp ) and self-ignition temperature of dust layers by graphical determination, for three different layer thickness (5 mm, 12.5 mm and 15 mm) and three different values of BIOT number (infinite, 1 and ½ ), 3. experimental determination of self-ignition temperature of dust layers, for the three different layer thickness (5 mm, 12.5 mm and 15 mm) of each sample, 4. comparison of experimental and theoretical results, for the three values of BIOT number. We worked with three values of BIOT number, as follows: α = infinite (heat transfers limited by conduction): conditions of FRANK-KAMENETSKII's model, α = 1 (heat transfers limited by conduction and convection): conditions of THOMAS and BOWES' model, α = ½: theoretical limit of THOMAS and BOWES' model.

Figure 1 :

 1 Figure 1: Allure of theoretical temperatures in SEMENOV (a) and FRANK-KAMENETSKII (b) models.

Figure 2 :Figure 3 :

 23 Figure 2 : Schematic view of the location of thermocouples while self-ignition tests on hot surface

Figure 4 :

 4 Figure 4: View of a filled basket inside a heating oven.

Figure 5 :

 5 Figure 5: Schematic view of the location of thermocouples while self-ignition tests in ovens

Table 1 :

 1 Results of self-ignition tests in heating ovens.

			Self-ignition temperatures in heating ovens (in
	Ref.	Samples	8 cm 3	125 cm 3	°C)	343 cm 3	1000 cm 3
			cubic	cubic		cubic	cubic
			basket	basket		basket	basket
	1	Purifying station mud dust	187	147		137	127
	2 Wheat dust n°1	222	187		147	142
	3 Potatoes powder	202	172		167	157
	4 Crushed waste dust	202	177		167	157
	5 Zinc powder	> 300	277		262	252
	6 Cacao powder	187	157		147	137
	7 Wood and flax dust	227	192		182	172
	8 Activated carbon dust	> 300	292		252	242
	9 Wheat dust n°2	222	182		172	162
	10 White wood dust	232	197		182	172
	11 Wood with asphalt dust	222	187		172	162
	12 Wheat dust n°3	222	187		177	162
	13 Persulphate powder	132	102		97	82
	14 Coal dust	222	167		147	132

Table 2 :

 2 Difference between theoretical and experimental self-ignition temperatures for 5 mm layers. Values are not calculable when experimental temperature is higher than 450 °C. Coloration represent the accuracy of correlation obtained: the more darkly, the less accurate correlation.

				Experimen	Difference between prevision and experimental
				tal results	values (in K) for 12.5 mm layers
	Ref .	Samples		(in °C) for 12.5 mm layers	between = 1 and experimental	between infinite and experimental	between = ½ and experimental
		Purifying station
	1	mud dust			Experimental value not determined
	2 Wheat dust n°1		Experimental value not determined
	3 Potatoes powder	280	-38	-22	-48
		Crushed waste
	4	dust			Experimental value not determined
	5 Zinc powder			Experimental value not determined
	6 Cacao powder	260	-34	-16	-46
	7	Wood and flax dust	320	-46	-26	-60
	8	Activated carbon dust	> 450	Values not calculable
	9 Wheat dust n°2	390	-122	-98	-136
	10 White wood dust		Experimental value not determined
		Wood	with
	11	asphalt dust			Experimental value not determined
	12 Wheat dust n°3		Experimental value not determined
		Persulphate
	13	powder			Experimental value not determined
	14 Coal dust			Experimental value not determined

Table 3 :

 3 Difference between theoretical and experimental self-ignition temperatures for 12.5 mm layers. Values are not calculable when experimental temperature is higher than 450 °C. Coloration represent the accuracy of correlation obtained: the more darkly, the less accurate correlation.

				Experiment	Difference between prevision and experimental
				al results	values (in K) for 15 mm layers
	Ref .	Samples	(in °C) for 15 mm layers	between = 1 and experimental	between infinite and experimental	between = ½ and experimental
	1	Purifying station mud dust	240	-22	0	-38
	2 Wheat dust n°1	270	-2	34	-26
	3 Potatoes powder	270	-34	-20	-44
		Crushed	waste
	4	dust			Experimental value not determined
	5 Zinc powder			Experimental value not determined
	6 Cacao powder	240	-20	-4	-34
	7	Wood and flax dust	300	-34	-14	-48
	8	Activated carbon dust	> 450	Values not calculable
	9 Wheat dust n°2	290	-30	-10	-46
	10 White wood dust		Experimental value not determined
		Wood with asphalt
	11	dust			Experimental value not determined
	12 Wheat dust n°3		Experimental value not determined
		Persulphate
	13	powder			Experimental value not determined
	14 Coal dust		362	-106	-70	-128

Table 4 :

 4 Difference between theoretical and experimental self-ignition temperatures for 15 mm layers. Values are not calculable when experimental temperature is higher than 450 °C. Coloration represent the accuracy of correlation obtained: the more darkly, the less accurate correlation.

« Explosive atmosphere » means a mixture with air, under atmospheric conditions, of flammable substances in the form of gases, vapours, mists or dusts in which, after ignition has occurred, combustion spreads to the entire unburned mixture[START_REF]Transport of Dangerous goods, Manual of Tests and Criteria[END_REF]