Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Health risk assessment to polychlorinated naphthalenes dioxin-like compounds in French sea food consumers.

There has been a recent revival of interest in some historical contaminants such as polychlorinated naphthalenes (PCNs). However, occurrence data are still lacking in some countries although industrial production of PCNs has been reported. This observation led to the first ever assessment of their presence in fish and seafood products in France in the present work. Their analysis was integrated in an already validated method applied for polychlorinated dibenzodioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), based on the structural similarity existing between these POPs. Performances of the method (LODs in the range 0.10-0.28 pg g-1 wet weight (ww), LOQs in the range 0.33-0.93 pg g-1 ww), enabled monitoring 69 di-to octachlorinated congeners in a large representative set of fish and seafood samples collected in 2005 in four coastal areas of the French mainland (n > 30). Their systematic presence was demonstrated in all the investigated seafood products, with levels (ΣPCNs in the range 2-440 pg g-1 wet weight) close to those already reported in other European fish and seafood sampled at a similar period. In addition, the robust measurement of almost all possible PCNs (69/75) allowed a fine interpretation of the observed profiles, highlighting in particular the specificities between species and fishing areas. Compared to the PCDD/Fs, PCBs, and polybrominated diphenylethers levels also measured for this set of samples, PCNs were observed as minor contributors to total concentrations (0.05-3.2%). The specific PCN related dietary dioxin-like exposure could be evaluated at 0.028-0.051 pg of toxic equivalent (TEQ) per kg of body weight per week for an adult, based on fish and seafood consumption only. Overall, this study provides the first baseline data on the occurrence of a large number of PCNs in France, which will allow future evaluation of temporal trends and associated risks.

Lifetime dietary exposure to bisphenol A in the general population and during pregnancy: Foetal exposure and health risk assessment.

Bisphenol A is a well-known chemical substance triggering reprotoxic and endocrine disruptor effects. Pregnancy is considered as a critical period of exposure to BPA because of the foetal sensitivity to endocrine disruption. Because of its wide use in food packaging, BPA is found in common foods and in infant formulae. We used a lifetime approach to simulate dietary exposure trajectories of a French population and to assess the associated health risk. Moreover, a semi-physiological based toxicokinetic model was used to simulate the maternal-foetal exchanges of BPA during pregnancy. Metabolism was taken into account by considering the glucuronidation of BPA by the foetal-placental unit, as well as the reactivation of BPA-glucuronide into BPA in the foetal compartment. From maternal critical daily exposures defined by ANSES based on effects for different endpoints of BPA in the unborn child (i.e. 0.083, 0.17, 0.29 and 0.33 µg/kg bw/d, respectively based on effects on mammary gland, brain and behaviour, metabolism and obesity and female reproductive system), resulting concentrations of BPA in the foetal compartment were estimated and health risk was assessed for the sub-population of unborn children. This work leads to the conclusion that while a health risk due to dietary exposures of the general population can be excluded, this is not the case for the sub-population of pregnant women, in view of the levels of foetal exposure to BPA.

Human dietary exposure to chemicals in sub-Saharan Africa: safety assessment through a total diet study.

BACKGROUND: Human dietary exposure to chemicals can result in a wide range of adverse health effects. Some substances might cause non-communicable diseases, including cancer and coronary heart diseases, and could be nephrotoxic. Food is the main human exposure route for many chemicals. We aimed to assess human dietary exposure to a wide range of food chemicals. METHODS: We did a total diet study in Benin, Cameroon, Mali, and Nigeria. We assessed 4020 representative samples of foods, prepared as consumed, which covered more than 90% of the diet of 7291 households from eight study centres. By combining representative dietary surveys of countries with findings for concentrations of 872 chemicals in foods, we characterised human dietary exposure. FINDINGS: Exposure to lead could result in increases in adult blood pressure up to 2·0 mm Hg, whereas children might lose 8·8-13·3 IQ points (95th percentile in Kano, Nigeria). Morbidity factors caused by coexposure to aflatoxin B1 and hepatitis B virus, and sterigmatocystin and fumonisins, suggest several thousands of additional liver cancer cases per year, and a substantial contribution to the burden of chronic malnutrition in childhood. Exposure to 13 polycyclic aromatic hydrocarbons from consumption of smoked fish and edible oils exceeded levels associated with possible carcinogenicity and genotoxicity health concerns in all study centres. Exposure to aluminium, ochratoxin A, and citrinin indicated a public health concern about nephropathies. From 470 pesticides tested across the four countries, only high concentrations of chlorpyrifos in smoked fish (unauthorised practice identified in Mali) could pose a human health risk. INTERPRETATION: Risks characterised by this total diet study underscore specific priorities in terms of food safety management in sub-Saharan Africa. Similar investigations specifically targeting children are crucially needed. FUNDING: Standards and Trade Development Facility.

Suspect and non-targeted screening of chemicals of emerging concern for human biomonitoring, environmental health studies and support to risk assessment: From promises to challenges and harmonisation issues.

Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017-2021) are described, with an emphasis on harmonisation measures.

Health risk assessment to dioxins, furans and PCBs in young children: The first French evaluation.

A total diet study (TDS) was conducted between 2010 and 2016 to characterize the health risk related to chemical residues in food of French not breastfed children under three years of age (infant TDS). Among the targeted substances, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) have been characterized as they accumulate through the food chain, especially in lipid-rich food items, and because they have been associated with a number of adverse effects in humans. Food samples (n = 180) were collected to be representative of the dioxins and PCB exposure through the whole diet of non-breastfed children from 1 to 36 months old and prepared as consumed (including cooking) prior to analysis. Dietary exposure was then assessed for 705 representative children under 3 years of age based on their food consumptions recorded through a 3-consecutive-days record. Levels of PCDD/Fs and PCBs in infant food were lower than those observed in common food, leading to significant differences in exposure according to age groups. Mean exposures to PCDD/Fs ranged from 0.22 to 0.44 pg TEQWHO05.kg bw-1.d-1 (0.40-0.65 at the 90th percentile), depending on the age group and the hypothesis considered to manage left-censored data. Mean exposure to non-dioxin-like PCBs ranged from 0.87 ng kg bw-1.d-1 (1.55 at the 90th percentile) in the 1-4 months old children to 3.53 ng kg bw-1.d-1 (5.44 at the 90th percentile) in the 13-36 months old children. For dioxins and NDL-PCBs, the tolerable daily intake (TDI) was exceeded for some age groups, in particular for older ones. Therefore, appropriate management measures must continue for reducing exposure; it concerns mainly common milk in youngest children, ultra-fresh dairy products and fish. For PCBs, recommendations on fish consumption should be reminded. Moreover, toxicity studies focusing on mixtures of dioxin-like compounds should be encouraged in order to take into account effect of mixtures.

Dietary risk assessment methodology: how to deal with changes through life.

Chemicals are present in food and numerous methods have been developed to assess dietary risk associated with these substances, but mainly by studying short periods of exposure. Usually, consumption data used to assess the risk are collected over a short period-of-time. The aim of this paper was to compile existing methods to assess dietary risk and identify their limitations in terms of lifetime risk assessment. To this end, we reviewed the different methods currently used at each step of risk assessment (i.e. methods to collect consumption data, to compute dietary exposures, and then to interpret these exposures in terms of risk for the consumer). Numerous methodologies exist to collect consumption data, contamination data collection, as well as for data treatment and interpretation. These methods consider different hypotheses and therefore lead to numerous uncertainties and discrepancies in the estimation of the exposure. Moreover, changes in eating habits through life, evolutions of food contamination, as well as health risk induced by fluctuating exposures, are rarely studied. A scientific consensus on the current risk assessment approaches is needed between national regulatory authorities. Moreover, additional research activities appear needed to take the evolution of consumption and contamination through time into account and to be able to assess the dietary health risk over the entire life.

Associations between persistent organic pollutants and endometriosis: A multipollutant assessment using machine learning algorithms.

Endometriosis is a gynaecological disease characterised by the presence of endometriotic tissue outside of the uterus impacting a significant fraction of women of childbearing age. Evidence from epidemiological studies suggests a relationship between risk of endometriosis and exposure to some organochlorine persistent organic pollutants (POPs). However, these chemicals are numerous and occur in complex and highly correlated mixtures, and to date, most studies have not accounted for this simultaneous exposure. Linear and logistic regression models are constrained to adjusting for multiple exposures when variables are highly intercorrelated, resulting in unstable coefficients and arbitrary findings. Advanced machine learning models, of emerging use in epidemiology, today appear as a promising option to address these limitations. In this study, different machine learning techniques were compared on a dataset from a case-control study conducted in France to explore associations between mixtures of POPs and deep endometriosis. The battery of models encompassed regularised logistic regression, artificial neural network, support vector machine, adaptive boosting, and partial least-squares discriminant analysis with some additional sparsity constraints. These techniques were applied to identify the biomarkers of internal exposure in adipose tissue most associated with endometriosis and to compare model classification performance. The five tested models revealed a consistent selection of most associated POPs with deep endometriosis, including octachlorodibenzofuran, cis-heptachlor epoxide, polychlorinated biphenyl 77 or trans-nonachlor, among others. The high classification performance of all five models confirmed that machine learning may be a promising complementary approach in modelling highly correlated exposure biomarkers and their associations with health outcomes. Regularised logistic regression provided a good compromise between the interpretability of traditional statistical approaches and the classification capacity of machine learning approaches. Applying a battery of complementary algorithms may be a strategic approach to decipher complex exposome-health associations when the underlying structure is unknown.

Assessment of Dechlorane Plus and related compounds in foodstuffs and estimates of daily intake from Lebanese population.

Dechlorane Related Compounds (DRCs), including Dechlorane Plus (syn/anti-DP or syn/anti-DDC-CO) and related compounds (Dec-601 or DDC-ID, Dec-602 or DDC-DBF, Dec-603 or DDC-Ant and Chlordene Plus or DDC-PDD), are a group of polychlorinated flame retardants of concern since they were first reported in various environmental and biota matrices about one decade ago. In this work, we investigated the dietary intake of the Lebanese population to these lipophilic environmental contaminants upon the evaluation of selected foodstuff contamination. Collected food samples (n = 58) were selected to be representative of the lipid fraction of the whole diet of the Beiruti population. The samples were analysed using pressurized liquid extraction, silica multilayer column followed by gel permeation chromatography for purification and GC-EI-HRMS for separation and detection. Detection frequency of at least one compound among Dechlorane Plus (syn-DP and anti-DP), Dechlorane 602, 603 and Chlordene Plus) was 91%. The mean concentrations of ∑6DRCs, by food group, ranged from 4.7 to 29.5 pg g-1 wet weight in lowerbound (LB) and from 6.7 to 76.9 pg g-1 wet weight in upperbound (UB). Based on food habits, the dietary intake of Beiruti adults was estimated to be between 3.71 (LB) and 5.61 (UB) ng day-1. Dechlorane Plus and Dechlorane 602 were the dominant compounds, contributing to 70 and 24% of the total intake (LB value), respectively. This study reports for the first time the occurrence of Dechloranes in Lebanese foods and proposes corresponding deterministic dietary exposure scenario.

Dietary exposure to perfluoroalkyl acids, brominated flame retardants and health risk assessment in the French infant total diet study.

Perfluoroalkyl acids (PFAAs) and brominated flame retardants (BFRs) are widely used and present in human food. Due to the increased susceptibility to pollutants of the young children, we conducted a total diet study focusing on this population. Around 200 baby and common food composite samples, prepared "as consumed", have been analysed for PFAAS, hexabromocyclododecanes, polybrominated biphenyls, polybrominated diphenyl ethers and tetrabromobisphenol A. The dietary exposure of 705 children aged 1-36 months was assessed. PFAAS were detected only in one fish sample. Detection rates varied from 4 to 93% for BFRs, depending on the congeners. Regarding the provisional health-based guidance values set by EFSA in 2018 for PFOA and PFOS at 0.8 and 1.8 ng kg bw-1.d-1, respectively, 20-100% of children exceeded them, depending on the age. Efforts should be made to decrease the PFAAs contamination of common foods. This study also highlighted that for other PFAAs, toxicological studies are needed to set dietary health-based guidance values, to assess their related health risk. Conversely, dietary exposures to BRFs were much lower than the respective health based guidance values or margins of safety were high enough, and consequently not considered at-risk due to very low contamination of the infant specific foods.

Human epidemiological evidence about the associations between exposure to organochlorine chemicals and endometriosis: Systematic review and meta-analysis.

BACKGROUND: Endometriosis is a gynaecological disease characterized by the presence of ectopic endometrial tissue that affects women during their reproductive years, having a strong impact on their lives, fertility and healthcare costs. The aetiology remains largely unknown, but current evidence suggests that it is multi-causal and oestrogen-dependent. Many epidemiologic studies have explored associations between organochlorine chemicals (OCCs) and endometriosis, but the findings are inconsistent. OBJECTIVES: A systematic review (SR) and meta-analysis were conducted to gather and synthesize all the available evidence from human epidemiological studies about the associations between OCCs and endometriosis. DATA SOURCES: The searches were conducted in PubMed and Web of Science in June 2016 with a final follow-up in August 2018. STUDY ELIGIBILITY CRITERIA: Only human epidemiological studies were considered, independent of participant age, body mass index or life-stage. Studies reporting individual measures of exposure to OCCs were included, considering but not limited to polychlorinated dibenzodioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), or organochlorine pesticides (OCPs). The primary health outcome was presence of endometriosis, including all sub-types. Eligibility criteria excluded articles not written in English, conference papers, reviews and studies with overlapping information. STUDY APPRAISAL AND SYNTHESIS METHODS: A SR protocol pre-registered at PROSPERO was applied in duplicate to gather and extract all eligible original papers from PUBMED and Web of Science databases. Odds ratios were pooled using the inverse variance method for random effects meta-analysis for each group of OCCs. Risk of bias was assessed using the National Toxicology Program/Office of Health Assessment and Translation (NTP/OHAT) Risk of Bias Rating Tool for Human and Animal Studies adapted to the review question. The confidence in the body of evidence and related level of evidence was measured by using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) based NTP/OHAT framework. The results were structured and presented in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Of the 51 studies retained for the full-text screening, 17 provided effect sizes and metrics sufficient for pooling estimates through meta-analysis. The overall odds ratios and 95% confidence intervals were 1.65 (1.14; 2.39) for dioxins (n = 10), 1.70 (1.20; 2.39) for PCBs (n = 9), and 1.23 (1.13; 1.36) for OCPs (n = 5). Despite being statistically significant, these estimates should be considered with caution given the notable heterogeneity and small estimated effect size. Misclassification of exposure, due to varying laboratory detection rate capabilities, and disease status, due to varying definitions of endometriosis, were identified as major sources of uncertainty. LIMITATIONS, CONCLUSIONS, AND IMPLICATIONS OF KEY FINDINGS: The level of evidence was considered to be "moderate" with "serious" risk of bias according the NTP/OHAT criteria, supporting the need for further well-designed epidemiological research to fill lingering data gaps. Given the complexity of endometriosis and lack of known biomarkers suitable for population-based research, carefully designed observational studies play an important role in better understanding the aetiology of endometriosis, as will evolving mixture modeling approaches capable of handling various environmental chemical exposures. Attention to critical windows of exposure will shed further light on the possible developmental origin of endometriosis. Considering the high economic and societal cost associated with endometriosis, further research on this field is urged. SYSTEMATIC REVIEW REGISTRATION NUMBER: CRD42018080956.

Development and Application of a Probabilistic Risk-Benefit Assessment Model for Infant Feeding Integrating Microbiological, Nutritional, and Chemical Components.

A probabilistic and interdisciplinary risk-benefit assessment (RBA) model integrating microbiological, nutritional, and chemical components was developed for infant milk, with the objective of predicting the health impact of different scenarios of consumption. Infant feeding is a particular concern of interest in RBA as breast milk and powder infant formula have both been associated with risks and benefits related to chemicals, bacteria, and nutrients, hence the model considers these three facets. Cronobacter sakazakii, dioxin-like polychlorinated biphenyls (dl-PCB), and docosahexaenoic acid (DHA) were three risk/benefit factors selected as key issues in microbiology, chemistry, and nutrition, respectively. The present model was probabilistic with variability and uncertainty separated using a second-order Monte Carlo simulation process. In this study, advantages and limitations of undertaking probabilistic and interdisciplinary RBA are discussed. In particular, the probabilistic technique was found to be powerful in dealing with missing data and to translate assumptions into quantitative inputs while taking uncertainty into account. In addition, separation of variability and uncertainty strengthened the interpretation of the model outputs by enabling better consideration and distinction of natural heterogeneity from lack of knowledge. Interdisciplinary RBA is necessary to give more structured conclusions and avoid contradictory messages to policymakers and also to consumers, leading to more decisive food recommendations. This assessment provides a conceptual development of the RBA methodology and is a robust basis on which to build upon.

Short-term effects of a perinatal exposure to the HBCDD α-isomer in rats: Assessment of early motor and sensory development, spontaneous locomotor activity and anxiety in pups.

The present study investigated the developmental neurotoxicity of an early exposure to α-HBCDD through the ingestion of contaminated hen's egg in pregnant and lactating Wistar female rats. Hens were given α-HBCDD-contaminated feed (40 ng/g fresh matter) for 5 and 10 days, which produced eggs with HBCDD content of 33 and 102 ng/glipid weight, respectively. Female rats were administered daily p.o. with an appropriate volume of the whole egg from the day of fertilization (GD0) to the weaning day for pups (PND21). Fetuses and pups were thus exposed continuously to α-HBCDD via the dam over a whole 42-day period that included both gestation and lactation. The administered egg volume was calculated on the basis of daily egg consumption in humans (0.7 egg/person/day) and duration of gestation and lactation in both species, which led animals to be exposed to α-HBCDD at levels of 22 and 66 ng/kg/day, respectively. Neurobehavioral development of pups was investigated from PND3 to PND25 using various tasks including the righting reflex (PND4), the grasping reflex (PND5), the negative geotaxis (PND9), the forelimb grip strength test (PND10) and the locomotor coordination test (PND20). Pup ultrasonic vocalizations were also recorded daily from PND4 to PND14. After weaning, behaviors related to spontaneous locomotor activity and anxiety were examined in the open-field (PND25) and in an elevated-plus maze (PND26), respectively. The results showed a significant decrease in body weight of pups exposed to the lower HBCDD level from PND3 to PND28, whereas the weight of rat pups given 66 ng/kg/day of HBCDD was not different from controls. During the first 3 weeks of life, impairments in motor maturation of pups were observed in a dose-dependent manner depending on the test, whereas no significant differences were reported between male and female pups. At PND26, the anxiety level of female rats exposed to the lowest dose of HBCDD (22 ng/kg/day) was significantly reduced whereas it remained unchanged in males. No significant variations were measured in rats exposed to the higher level of HBCDD (66 ng/kg/day). These results suggest the potent developmental neurotoxicity of an early chronic exposure to the HBCDD α-isomer through the ingestion of hen's eggs contaminated with this pollutant and question the long-lasting consequences of this exposure on behavior abilities and brain functioning in adulthood.

Chlorination of bisphenol A: non-targeted screening for the identification of transformation products and assessment of estrogenicity in generated water.

Besides the performance of water treatments on the removal of micropollutants, concern about the generation of potential biologically active transformation products has been growing. Thus, the detection and structural elucidation of micropollutants transformation products have turned out to be major issues to evaluate comprehensively the efficiency of the processes implemented for drinking water treatment. However, most of existing water treatment studies are carried out at the bench scale with high concentrations and simplified conditions and thus do not reflect realistic conditions. Conversely, this study describes a non-targeted profiling approach borrowed from metabolomic science, using liquid chromatography coupled to high-resolution mass spectrometry, in order to reveal potential chlorination products of bisphenol A (BPA) in real water samples spiked at 50µgL(-1). Targeted measurements first evidenced a fast removal of BPA (>99%) by chlorination with sodium hypochlorite (0.8mgL(-1)) within 10min. Then, the developed differential global profiling approach enabled to reveal 21 chlorination products of BPA. Among them, 17 were brominated compounds, described for the first time, demonstrating the potential interest of this innovative methodology applied to environmental sciences. In parallel to the significant removal of BPA, the estrogenic activity of water samples, evaluated by ER-CALUX assay, was found to significantly decrease after 10min of chlorination. These results confirm that chlorination is effective at removing BPA in drinking water and they may indicate that the generated compounds have significantly lower estrogenic activity.

Occurrence of perfluorinated alkylated substances in breast milk of French women and relation with socio-demographical and clinical parameters: results of the ELFE pilot study.

A previously developed and validated methodology based on liquid chromatography coupled to high resolution mass spectrometry was used for determine the concentration levels of 14 perfluoroalkylated substances (PFASs) in a set of 48 breast milk samples collected from French women in the frame of the ELFE pilot study. In accordance with other similar studies conducted at european and international levels, PFOS, PFOA, and PFHxS were detected and quantified in most of the analyzed samples (90%, 98% and 100%, respectively), and appeared as major contributors to the total PFAS exposure (38%, 37%, 25%, respectively), whereas the other targeted PFAS were very rarely, if not, found at the limits of detection of the method. Also in agreement with other published data, the concentration levels measured for the detected substances varied from <0.05 to 0.33µg/L for PFOS (median=0.079), from <0.05 to 0.22µg/L for PFOA (median=0.075), and from 0.04 to 0.07µg/L for PFHxS (median=0.050). On the basis of this relatively limited data set, no statistically significant relation was observed between these exposure levels and developmental outcomes, in particular the weight at birth. Similarly, no relation was observed between the measured PFAS levels and various socio-demographical parameters including the consumption of seafood, alcohol, smoking, or socio-economical level. These results suggest a need for further research and better knowledge regarding the sources, pharmacokinetics, and factors of exposure for other substances belonging to this class of emerging contaminants.

Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves.

Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based on mass spectrometry detection have been developed to ensure the control of such veterinary drug residues. Nevertheless, the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention. New analytical tools able to detect such abuse are today mandatory. In this context, metabolomics may represent new emerging strategies for investigating the global physiological effects associated to a family of substances and therefore, to suspect the administration of steroids. The purpose of the present study was to set up, assess and compare two complementary mass spectrometry-based metabolomic strategies as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such approaches. The protocols were developed in two European laboratories in charge of residues analysis in the field of food safety. Apart from sample preparation, the global process was different in both laboratories from LC-HRMS fingerprinting to multivariate data analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-MetAlign strategies. The reproducibility of both sample preparation and MS measurements were assessed in order to guarantee that any differences in the acquired fingerprints were not caused by analytical variability but reflect metabolome modifications upon steroids administration. The protocols were then applied to urine samples collected on a large group of animals consisting of 12 control calves and 12 calves administrated with a mixture of 17ß-estradiol 3-benzoate and 17ß-nandrolone laureate esters according to a protocol reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid administration have been evaluated in this context and proved the suitability of the approach for discriminating anabolic treated animals from control ones. Such an approach may therefore open a new way for the screening of anabolic steroid administration through targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study.

Development of a metabonomic approach based on LC-ESI-HRMS measurements for profiling of metabolic changes induced by recombinant equine growth hormone in horse urine.

Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter, it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH-treated horses by liquid chromatography-electrospray-high-resolution mass spectrometry (LC-ESI-HRMS) is described and assessed in this paper in order to develop a new screening tool for growth hormone abuse in horseracing. The strategy involves a limited sample preparation of the urine samples and the use of appropriate software for data processing and analysis. As preliminary work, reproducibility of both sample preparation and mass spectrometry (MS) measurements was evaluated in order to demonstrate the reliability of the method. Application of the developed protocol on two horses demonstrated the suitability of the developed strategy and preliminary results showed significant modifications of the metabolome after treatment with reGH.

Exposure assessment of French women and their newborn to brominated flame retardants: determination of tri- to deca- polybromodiphenylethers (PBDE) in maternal adipose tissue, serum, breast milk and cord serum.

In the frame of a French monitoring program, tri- to deca- polybromodiphenylethers (PBDE) have been measured in maternal and cord serum, adipose tissue, and breast milk samples, collected from 93 volunteer women during caesarean deliveries. The seven major tri- to heptaBDE (BDE-28, 47, 99, 100, 153, 154, and 183) were detected in adipose tissue and breast milk with cumulated median values of 2.59 and 2.51 ng g(-1) l w. Nine highly brominated octa- to decaBDE (BDE-196, 197, 201, 202, 203, 206, 207, 208 and 209) was performed in the same samples, with cumulated median values of 2.73 and 3.39 ng g(-1) l w in adipose tissue and breast milk, respectively. At this opposite, median levels of octa- to decaBDE in maternal and cord serum appeared significantly higher than the levels of tri- to heptaBDE in the same matrices, i.e. 8.85 and 12.34 versus 0.98 and 0.69 ng g(-1) l w, respectively.

Exposure assessment of French women and their newborns to tetrabromobisphenol-A: occurrence measurements in maternal adipose tissue, serum, breast milk and cord serum.

A French monitoring study was initiated to evaluate the exposure of fetus and newborn to brominated flame retardants (BFR). A previously developed multi-residue analytical method was used for measuring the main classes of BFR (tetrabromobisphenol-A, and tri- to decabomodiphenyl ethers) in various human biological matrices. Analyzed samples (maternal and cord serum, adipose tissue and breast milk) were collected from 93 volunteer women during caesarean deliveries. TBBPA was detected in 44% of the analyzed breast milk samples, at levels varying from 0.06 to 37.34 ng g(-1) lipid weight, but was not detected in adipose tissue. This compound was also detected in 30% of the analyzed serum samples, with similar average values in maternal and cord serum (154 pg g(-1) fresh weight versus 199 pg g(-1) fresh weight, respectively). The interpretation of the collected data permitted the demonstration of (1) a significant exposure to TBBPA both for mothers and fetuses and (2) a possible risk of overexposure of newborns through breastfeeding.

Exposure assessment of prepubertal children to steroid endocrine disruptors. 2. Determination of steroid hormones in milk, egg, and meat samples.

In the present study, the occurrence of the main sex steroid hormones in milk, egg, and meat was evaluated on the basis of a highly specific gas chromatography-tandem mass spectrometry measurement method. Globally, the results indicated that targeted estrogens and androgens occurred at similar levels (concentration levels in the 10-100 ng kg (-1) range) in the analyzed muscle and milk samples. The same compounds occurred at about 10-fold higher concentrations (i.e., in the 100-1000 ng kg (-1) range) in eggs and kidney samples. More precisely, egg and milk appeared as a non-negligible sources of estradiol (i.e., 2.2 +/- 0.8 and 3.1 +/- 2.0 ng day (-1), respectively), whereas testosterone exposure is caused by ingestion of meat and/or egg (i.e., 12.2 +/- 48.2 and 5.2 +/- 2.3 ng day (-1), respectively). The provided exposure data will be further exploited in the scope of a risk assessment study regarding endocrine disruption associated with these molecules.