Nanotechnology in food industry II: risk assessment and legislation

Currently, there is increasing use of nanomaterials in the food industry thanks to the many advantages offered and make the products that contain them more competitive in the market. Their physicochemical properties often differ from those of bulk materials, which require specialized risk assessment. This should cover the risks to the health of workers and consumers as well as possible environmental risks. The risk assessment methods must go updating due to more widespread use of nanomaterials, especially now that are making their way down to consumer products. Today there is no specific legislation for nanomaterials, but there are several european dispositions and regulations that include them. This review gives an overview of the risk assessment and the existing current legislation regarding the use of nanotechnology in the food industry.

Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in Portugal

There is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering several questions: 1) Are Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.

Is there a health risk concerning the children´s consumption of cereal-based products? A cumulative mycotoxin risk assessment approach

Historically, the health risk of mycotoxins had been evaluated on the basis of single-chemical and single-exposure pathway scenarios. However, the co-contamination of foodstuffs with these compounds is being reported at an increasing rate and a multiple-exposure scenario for humans and vulnerable population groups as children is urgently needed. Cereals are among the first solid foods eaten by child and thus constitute an important food group of their diet. Few data are available relatively to early stages child´s exposure to mycotoxins through consumption of cereal-based foods. The present study aims to perform the cumulative risk assessment of mycotoxins present in a set of cereal-based foods including breakfast cereals (BC), processed cereal-based foods (PCBF) and biscuits (BT), consumed by children (1 to 3 years old, n=75) from Lisbon region, Portugal. Children food consumption and occurrence of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) in cereal-based foods were combined to estimate the mycotoxin daily intake, using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) and aflatoxin daily exposure. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (HQ, ratio between exposure and a reference dose). The concentration addition (CA) concept was used for the cumulative risk assessment of multiple mycotoxins. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. Main results revealed a significant health concern related to aflatoxins and especially aflatoxin M1 exposure according to the MoET and MoE values (below 10000), respectively. HQ and HI values for the remaining mycotoxins were below 1, revealing a low concern from a public health point of view. These are the first results on cumulative risk assessment of multiple mycotoxins present in cereal-based foods consumed by children. Considering the present results, more research studies are needed to provide the governmental regulatory bodies with data to develop an approach that contemplate the human exposure and, particularly, children, to multiple mycotoxins in food. The last issue is particularly important considering the potential synergistic effects that could occur between mycotoxins and its potential impact on human and, mainly, children health.

Application of mathematical models to mycotoxins children risk assessment: a case study of Portuguese children exposure to co-occurring mycotoxins in processed cereal-based foods

People, animals and the environment can be exposed to multiple chemicals at once from a variety of sources, but current risk assessment is usually carried out based on one chemical substance at a time. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, a wide group of fungal secondary metabolites that are known to potentially cause toxicity and carcinogenic outcomes. Mycotoxins are commonly found in a variety of foods including those intended for consumption by infants and young children and have been found in processed cereal-based foods available in the Portuguese market. The use of mathematical models, including probabilistic approaches using Monte Carlo simulations, constitutes a prominent issue in human health risk assessment in general and in mycotoxins exposure assessment in particular. The present study aims to characterize, for the first time, the risk associated with the exposure of Portuguese children to single and multiple mycotoxins present in processed cereal-based foods (CBF). Portuguese children (0-3 years old) food consumption data (n=103) were collected using a 3 days food diary. Contamination data concerned the quantification of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) were evaluated in 20 CBF samples marketed in 2014 and 2015 in Lisbon; samples were analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Daily exposure of children to mycotoxins was performed using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) to the aflatoxin exposure. The magnitude of the MoE gives an indication of the risk level. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (ratio between exposure and a reference dose, HQ). For the cumulative risk assessment of multiple mycotoxins, the concentration addition (CA) concept was used. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. 71% of CBF analyzed samples were contaminated with mycotoxins (with values below the legal limits) and approximately 56% of the studied children consumed CBF at least once in these 3 days. Preliminary results showed that children exposure to single mycotoxins present in CBF were below the TDI. Aflatoxins MoE and MoET revealed a reduced potential risk by exposure through consumption of CBF (with values around 10000 or more). HQ and HI values for the remaining mycotoxins were below 1. Children are a particularly vulnerable population group to food contaminants and the present results point out an urgent need to establish legal limits and control strategies regarding the presence of multiple mycotoxins in children foods in order to protect their health. The development of packaging materials with antifungal properties is a possible solution to control the growth of moulds and consequently to reduce mycotoxin production, contributing to guarantee the quality and safety of foods intended for children consumption.

Toxicity and Contamination in Bear Creek Sediment: Spatial Analysis and Implications for Risk Assessment

The sediments of Bear Creek near Baltimore, Maryland demonstrate substantial toxicity to benthic organisms, and contain a complex mixture of organic and inorganic contaminants. The present study maps the spatial extent and depth profile of toxicity and contamination in Bear Creek, and explores correlations between heavy metals, organic contaminants, and toxic responses. Two novel analytical techniques – handheld XRF and an antibody-based PAH biosensor – were applied to samples from the site to quantify total metals and total PAHs in sediments. By comprehensively assessing toxicity in Bear Creek, the present study provides data to inform future risk assessments and management decisions relating for the site, while demonstrating the benefits of applying joint biological assays and chemical assessment methods to sediments with complex contaminant mixtures.

Exploring the potential role of allostatic load biomarkers in risk assessment of patients presenting with depressive symptoms

Background: Depression is a major health problem worldwide and the majority of patients presenting with depressive symptoms are managed in primary care. Current approaches for assessing depressive symptoms in primary care are not accurate in predicting future clinical outcomes, which may potentially lead to over or under treatment. The Allostatic Load (AL) theory suggests that by measuring multi-system biomarker levels as a proxy of measuring multi-system physiological dysregulation, it is possible to identify individuals at risk of having adverse health outcomes at a prodromal stage. Allostatic Index (AI) score, calculated by applying statistical formulations to different multi-system biomarkers, have been associated with depressive symptoms. Aims and Objectives: To test the hypothesis, that a combination of allostatic load (AL) biomarkers will form a predictive algorithm in defining clinically meaningful outcomes in a population of patients presenting with depressive symptoms. The key objectives were: 1. To explore the relationship between various allostatic load biomarkers and prevalence of depressive symptoms in patients, especially in patients diagnosed with three common cardiometabolic diseases (Coronary Heart Disease (CHD), Diabetes and Stroke). 2 To explore whether allostatic load biomarkers predict clinical outcomes in patients with depressive symptoms, especially in patients with three common cardiometabolic diseases (CHD, Diabetes and Stroke). 3 To develop a predictive tool to identify individuals with depressive symptoms at highest risk of adverse clinical outcomes. Methods: Datasets used: ‘DepChron’ was a dataset of 35,537 patients with existing cardiometabolic disease collected as a part of routine clinical practice. ‘Psobid’ was a research data source containing health related information from 666 participants recruited from the general population. The clinical outcomes for 3 both datasets were studied using electronic data linkage to hospital and mortality health records, undertaken by Information Services Division, Scotland. Cross-sectional associations between allostatic load biomarkers calculated at baseline, with clinical severity of depression assessed by a symptom score, were assessed using logistic and linear regression models in both datasets. Cox’s proportional hazards survival analysis models were used to assess the relationship of allostatic load biomarkers at baseline and the risk of adverse physical health outcomes at follow-up, in patients with depressive symptoms. The possibility of interaction between depressive symptoms and allostatic load biomarkers in risk prediction of adverse clinical outcomes was studied using the analysis of variance (ANOVA) test. Finally, the value of constructing a risk scoring scale using patient demographics and allostatic load biomarkers for predicting adverse outcomes in depressed patients was investigated using clinical risk prediction modelling and Area Under Curve (AUC) statistics. Key Results: Literature Review Findings. The literature review showed that twelve blood based peripheral biomarkers were statistically significant in predicting six different clinical outcomes in participants with depressive symptoms. Outcomes related to both mental health (depressive symptoms) and physical health were statistically associated with pre-treatment levels of peripheral biomarkers; however only two studies investigated outcomes related to physical health. Cross-sectional Analysis Findings: In DepChron, dysregulation of individual allostatic biomarkers (mainly cardiometabolic) were found to have a non-linear association with increased probability of co-morbid depressive symptoms (as assessed by Hospital Anxiety and Depression Score HADS-D≥8). A composite AI score constructed using five biomarkers did not lead to any improvement in the observed strength of the association. In Psobid, BMI was found to have a significant cross-sectional association with the probability of depressive symptoms (assessed by General Health Questionnaire GHQ-28≥5). BMI, triglycerides, highly sensitive C - reactive 4 protein (CRP) and High Density Lipoprotein-HDL cholesterol were found to have a significant cross-sectional relationship with the continuous measure of GHQ-28. A composite AI score constructed using 12 biomarkers did not show a significant association with depressive symptoms among Psobid participants. Longitudinal Analysis Findings: In DepChron, three clinical outcomes were studied over four years: all-cause death, all-cause hospital admissions and composite major adverse cardiovascular outcome-MACE (cardiovascular death or admission due to MI/stroke/HF). Presence of depressive symptoms and composite AI score calculated using mainly peripheral cardiometabolic biomarkers was found to have a significant association with all three clinical outcomes over the following four years in DepChron patients. There was no evidence of an interaction between AI score and presence of depressive symptoms in risk prediction of any of the three clinical outcomes. There was a statistically significant interaction noted between SBP and depressive symptoms in risk prediction of major adverse cardiovascular outcome, and also between HbA1c and depressive symptoms in risk prediction of all-cause mortality for patients with diabetes. In Psobid, depressive symptoms (assessed by GHQ-28≥5) did not have a statistically significant association with any of the four outcomes under study at seven years: all cause death, all cause hospital admission, MACE and incidence of new cancer. A composite AI score at baseline had a significant association with the risk of MACE at seven years, after adjusting for confounders. A continuous measure of IL-6 observed at baseline had a significant association with the risk of three clinical outcomes- all-cause mortality, all-cause hospital admissions and major adverse cardiovascular event. Raised total cholesterol at baseline was associated with lower risk of all-cause death at seven years while raised waist hip ratio- WHR at baseline was associated with higher risk of MACE at seven years among Psobid participants. There was no significant interaction between depressive symptoms and peripheral biomarkers (individual or combined) in risk prediction of any of the four clinical outcomes under consideration. Risk Scoring System Development: In the DepChron cohort, a scoring system was constructed based on eight baseline demographic and clinical variables to predict the risk of MACE over four years. The AUC value for the risk scoring system was modest at 56.7% (95% CI 55.6 to 57.5%). In Psobid, it was not possible to perform this analysis due to the low event rate observed for the clinical outcomes. Conclusion: Individual peripheral biomarkers were found to have a cross-sectional association with depressive symptoms both in patients with cardiometabolic disease and middle-aged participants recruited from the general population. AI score calculated with different statistical formulations was of no greater benefit in predicting concurrent depressive symptoms or clinical outcomes at follow-up, over and above its individual constituent biomarkers, in either patient cohort. SBP had a significant interaction with depressive symptoms in predicting cardiovascular events in patients with cardiometabolic disease; HbA1c had a significant interaction with depressive symptoms in predicting all-cause mortality in patients with diabetes. Peripheral biomarkers may have a role in predicting clinical outcomes in patients with depressive symptoms, especially for those with existing cardiometabolic disease, and this merits further investigation.

Harmonised framework for ecological risk assessment of sediments from ports and estuarine zones of North and South Atlantic

This paper presents a harmonised framework of sediment quality assessment and dredging material characterisation for estuaries and port zones of North and South Atlantic. This framework, based on the weight-of-evidence approach, provides a structure and a process for conducting sediment/dredging material assessment that leads to a decision. The main structure consists of step 1 (examination of available data); step 2 (chemical characterisation and toxicity assessment); decision 1 (any chemical level higher than reference values? are sediments toxic?); step 3 (assessment of benthic community structure); step 4 (integration of the results); decision 2 (are sediments toxic or benthic community impaired?); step 5 (construction of the decision matrix) and decision 3 (is there environmental risk?). The sequence of assessments may be interrupted when the information obtained is judged to be sufficient for a correct characterisation of the risk posed by the sediments/dredging material. This framework brought novel features compared to other sediment/dredging material risk assessment frameworks: data integration through multivariate analysis allows the identification of which samples are toxic and/or related to impaired benthic communities; it also discriminates the chemicals responsible for negative biological effects; and the framework dispenses the use of a reference area. We demonstrated the successful application of this framework in different port and estuarine zones of the North (Gulf of Cadiz) and South Atlantic (Santos and Paranagua Estuarine Systems).

Modeling Flood Stage-Duration-Frequency: A Risk Assessment of Critical Infrastructure in the Tidal Potomac

The service of a critical infrastructure, such as a municipal wastewater treatment plant (MWWTP), is taken for granted until a flood or another low frequency, high consequence crisis brings its fragility to attention. The unique aspects of the MWWTP call for a method to quantify the flood stage-duration-frequency relationship. By developing a bivariate joint distribution model of flood stage and duration, this study adds a second dimension, time, into flood risk studies. A new parameter, inter-event time, is developed to further illustrate the effect of event separation on the frequency assessment. The method is tested on riverine, estuary and tidal sites in the Mid-Atlantic region. Equipment damage functions are characterized by linear and step damage models. The Expected Annual Damage (EAD) of the underground equipment is further estimated by the parametric joint distribution model, which is a function of both flood stage and duration, demonstrating the application of the bivariate model in risk assessment. Flood likelihood may alter due to climate change. A sensitivity analysis method is developed to assess future flood risk by estimating flood frequency under conditions of higher sea level and stream flow response to increased precipitation intensity. Scenarios based on steady and unsteady flow analysis are generated for current climate, future climate within this century, and future climate beyond this century, consistent with the WWTP planning horizons. The spatial extent of flood risk is visualized by inundation mapping and GIS-Assisted Risk Register (GARR). This research will help the stakeholders of the critical infrastructure be aware of the flood risk, vulnerability, and the inherent uncertainty.

FLOOD RISK ASSESSMENT UNDER HISTORICAL AND PREDICTED LAND USE CHANGE USING CONTINUOUS HYDROLOGIC MODELING

Current procedures for flood risk estimation assume flood distributions are stationary over time, meaning annual maximum flood (AMF) series are not affected by climatic variation, land use/land cover (LULC) change, or management practices. Thus, changes in LULC and climate are generally not accounted for in policy and design related to flood risk/control, and historical flood events are deemed representative of future flood risk. These assumptions need to be re-evaluated, however, as climate change and anthropogenic activities have been observed to have large impacts on flood risk in many areas. In particular, understanding the effects of LULC change is essential to the study and understanding of global environmental change and the consequent hydrologic responses. The research presented herein provides possible causation for observed nonstationarity in AMF series with respect to changes in LULC, as well as a means to assess the degree to which future LULC change will impact flood risk. Four watersheds in the Midwest, Northeastern, and Central United States were studied to determine flood risk associated with historical and future projected LULC change. Historical single framed aerial images dating back to the mid-1950s were used along with Geographic Information Systems (GIS) and remote sensing models (SPRING and ERDAS) to create historical land use maps. The Forecasting Scenarios of Future Land Use Change (FORE-SCE) model was applied to generate future LULC maps annually from 2006 to 2100 for the conterminous U.S. based on the four IPCC-SRES future emission scenario conditions. These land use maps were input into previously calibrated Soil and Water Assessment Tool (SWAT) models for two case study watersheds. In order to isolate effects of LULC change, the only variable parameter was the Runoff Curve Number associated with the land use layer. All simulations were run with daily climate data from 1978-1999, consistent with the 'base' model which employed the 1992 NLCD to represent 'current' conditions. Output daily maximum flows were converted to instantaneous AMF series and were subsequently modeled using a Log-Pearson Type 3 (LP3) distribution to evaluate flood risk. Analysis of the progression of LULC change over the historic period and associated SWAT outputs revealed that AMF magnitudes tend to increase over time in response to increasing degrees of urbanization. This is consistent with positive trends in the AMF series identified in previous studies, although there are difficulties identifying correlations between LULC change and identified change points due to large time gaps in the generated historical LULC maps, mainly caused by unavailability of sufficient quality historic aerial imagery. Similarly, increases in the mean and median AMF magnitude were observed in response to future LULC change projections, with the tails of the distributions remaining reasonably constant. FORE-SCE scenario A2 was found to have the most dramatic impact on AMF series, consistent with more extreme projections of population growth, demands for growing energy sources, agricultural land, and urban expansion, while AMF outputs based on scenario B2 showed little changes for the future as the focus is on environmental conservation and regional solutions to environmental issues.

Structured professional judgement approach to risk assessment: generalisability across patient groups for the prediction of adverse outcomes in secure mental health care

This is a redacted version of the the final thesis. Copyright material has been removed to comply with UK Copyright Law.

Risk assessment of the herbicide clomazone in the aquatic life.

Clomazone (2-(2-chlorophenyl)methyl-4.4-dimethyl-3-isoxazolidinone) is a post emergence herbicide widely used in rice fields in Rio Grande do Sul (Brazil) with high activity against Gramineae at the recommended application rate(AR).of 700g/ha. The herbicide input into the aquatic ecosystem may occur by aerial application or water drainage. The presence of this chemical in the water may affect non-target organisms leading to impairments in the aquatic food chain. Studies were conducted in this work to evaluate the risk of Clomazone using the estimated mean affective concentration (EC50) for the microalgae Selenastrum capricornutum(96h), the duckweed Lemna valdiviana(96h) and the crustacean Daphnia similis(48h). The EC50 values were 11.2; 31.7 and 13.8 mg/l, respectively. According to the obtained data, and considering a direct input of the herbicide in a 10cm column water, the estimated maximum application rate that doesn't cause acute effects is 5.3 AR for S. capricornutum, 6.5 AR for D. similis and 15.0 AR for L. valdiviana. The estimated maximum application rate that doesn't cause chronic effects is 2.0 AR for D. similis, 1.6 AR for S. capricornutum and 4.5 AR for L. valviana.

Development of a patient-specific model for stroke risk assessment in atrial fibrillation patients

Atrial fibrillation is associated with a five-fold increase in the risk of cerebrovascular events,being responsible of 15-18% of all strokes.The morphological and functional remodelling of the left atrium caused by atrial fibrillation favours blood stasis and, consequently, stroke risk. In this context, several clinical studies suggest that stroke risk stratification could be improved by using haemodynamic information on the left atrium (LA) and the left atrial appendage (LAA). The goal of this study was to develop a personalized computational fluid-dynamics (CFD) model of the left atrium which could clarify the haemodynamic implications of atrial fibrillation on a patient specific basis. The developed CFD model was first applied to better understand the role of LAA in stroke risk. Infact, the interplay of the LAA geometric parameters such as LAA length, tortuosity, surface area and volume with the fluid-dynamics parameters and the effects of the LAA closure have not been investigated. Results demonstrated the capabilities of the CFD model to reproduce the real physiological behaviour of the blood flow dynamics inside the LA and the LAA. Finally, we determined that the fluid-dynamics parameters enhanced in this research project could be used as new quantitative indexes to describe the different types of AF and open new scenarios for the patient-specific stroke risk stratification.

Advancing of risk assessment of pesticides on insect pollinators: beyond honey bees (Apis mellifera L.)

The western honey bee, Apis mellifera L., is currently the model specie for pesticide risk assessment on pollinators with the assumption that the worst-case scenarios for this species are sufficiently conservative to protect other insect pollinators. However, recent studies have showed that wild species may be more sensitive to plant protection products, due to differences in biology and life cycles. Therefore, there is the need to extend the risk assessment within a more ecological approach, in order to ensure that there are no irreversible effects on non-target organisms and in the environment. My dissertation aims to expand the risk assessment to other insect pollinators (including wild and managed pollinators), in order to cover some of the gaps of the current schemes. In this thesis, it is presented three experiments that cover the early stages of a solitary bee (chapter 1), the development of molecular tools for early detection of sub-lethal effects (chapter 2) and the development of protocols to access lethal and sub-lethal effects on other pollinator taxa (Diptera; chapter 3).

Models and methods for damage and quantitative risk assessment of Natech scenarios

Natural events are a widely recognized hazard for industrial sites where relevant quantities of hazardous substances are handled, due to the possible generation of cascading events resulting in severe technological accidents (Natech scenarios). Natural events may damage storage and process equipment containing hazardous substances, that may be released leading to major accident scenarios called Natech events. The need to assess the risk associated with Natech scenarios is growing and methodologies were developed to allow the quantification of Natech risk, considering both point sources and linear sources as pipelines. A key element of these procedures is the use of vulnerability models providing an estimation of the damage probability of equipment or pipeline segment as a result of the impact of the natural event. Therefore, the first aim of the PhD project was to outline the state of the art of vulnerability models for equipment and pipelines subject to natural events such as floods, earthquakes, and wind. Moreover, the present PhD project also aimed at the development of new vulnerability models in order to fill some gaps in literature. In particular, a vulnerability model for vertical equipment subject to wind and to flood were developed. Finally, in order to improve the calculation of Natech risk for linear sources an original methodology was developed for Natech quantitative risk assessment methodology for pipelines subject to earthquakes. Overall, the results obtained are a step forward in the quantitative risk assessment of Natech accidents. The tools developed open the way to the inclusion of new equipment in the analysis of Natech events, and the methodology for the assessment of linear risk sources as pipelines provides an important tool for a more accurate and comprehensive assessment of Natech risk.

PAH biomarkers for human health risk assessment: a review of the state-of-the-art

Polycyclic aromatic hydrocarbons (PAH) are widely distributed in the environment, and some are carcinogenic to human beings. The study of biomarkers has helped clarify the nature and magnitude of the human health risks posed by such substances. This article provides a review of the state-of-the-art on PAH biomarkers for human health risk assessment and also discusses their applicability within the context of environmental management in Brazil. The article discusses the methodologies for determination of some biomarkers such as 1-hydroxypyrene and PAH-DNA adducts. Cytogenetic markers, frequency of chromosomal aberrations, and micronucleus induction were considered for the evaluation of cancer risk. The current stage of studies on validation of such biomarkers was also approached.