Overview on legislation and scientific approaches for risk assessment of combined exposure to multiple chemicals: the potential EuroMix contribution.

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on food-related chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.

Assessing the sustainability of egg production systems in The Netherlands.

Housing systems for laying hens have changed over the years due to increased public concern regarding animal welfare. In terms of sustainability, animal welfare is just one aspect that needs to be considered. Social aspects as well as environmental and economic factors need to be included as well. In this study, we assessed the sustainability of enriched cage, barn, free-range, and organic egg production systems following a predefined protocol. Indicators were selected within the social, environmental, and economic dimensions, after which parameter values and sustainability limits were set for the core indicators in order to quantify sustainability. Uncertainty in the parameter values as well as assigned weights and compensabilities of the indicators influenced the outcome of the sustainability assessment. Using equal weights for the indicators showed that, for the Dutch situation, enriched cage egg production was most sustainable, having the highest score on the environmental dimension, whereas free-range egg production gave the highest score in the social dimension (covering food safety, animal welfare, and human welfare). In the economic dimension both enriched cage egg and organic egg production had the highest sustainability score. When weights were attributed according to stakeholder outputs, individual differences were seen, but the overall scores were comparable to the sustainability scores based on equal weights. The provided method enabled a quantification of sustainability using input from stakeholders to include societal preferences in the overall assessment. Allowing for different weights and compensabilities helps policymakers in communicating with stakeholders involved and provides a weighted decision regarding future housing systems for laying hens.

Anal cancer survival: a socioeconomic analysis.

INTRODUCTION: Squamous cell carcinoma is the most common variant of anal malignancy. Certain disease-related factors have been established in determining survival. These include tumour size, differentiation and nodal involvement. Other factors such as HIV status, human papillomavirus infection, smoking and socioeconomic disparity may have important roles, however few data are available on the UK population. We aim to correlate social deprivation and survival of anal cancer patients at a tertiary centre. MATERIALS AND METHODS: All consecutive cases diagnosed with anal squamous cell carcinoma and treated as per local protocol between July 2010 and April 2017 were included. The pathological and demographical details were collected from a prospectively maintained database. Socioeconomic deprivation was defined for each postcode using the Index of Multiple Deprivation decile compiled by local governments in England. Survival was estimated using Kaplan-Meier analysis and Cox regression was used to investigate the effect of different factors on overall survival. RESULTS: A total of 129 patients with anal squamous cell carcinoma over a median follow-up of 43 months were included. Overall survival for the entire patient cohort was 87.7% (95% confidence interval, CI, 82.0-93.7%), 75.5% (95% CI 67.5-84.5%) and 68.9% (95% CI 59.7-79.6%) at one year, three years and five years, respectively. On multivariate analysis, Index of Multiple Deprivation and income do not significantly influence overall survival (p = 0.79, hazard ratio, HR, 1.07; 95% CI 0.61-1.63), (p = 0.99, HR=1.00; 95% CI 0.61-1.63), respectively. Increased risk of death was observed for male sex (p = 0.02, HR=2.80; 95% CI 1.02-5.50) and larger tumour size (p = 0.01, HR=1.64; 95% CI 1.12-2.41). CONCLUSION: In contrast to US studies, there is little difference in survival between the least deprived and most deprived groups. We attribute this to equal access to intensity-modulated radiation therapy-based chemoradiotherapy. Thus, a highly effective treatment made available to all mitigates any survival difference between socioeconomic groups.

Increased efficacy for in-house validation of real-time PCR GMO detection methods.

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD(r) and RSD(R)) were calculated. The results showed that not only the PCR reaction but also the factors 'DNA isolation' and 'PCR day' are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.

Toxicity and survival of anal cancer patients treated with intensity-modulated radiation therapy.

INTRODUCTION: The definitive treatment of anal cancer with chemoradiotherapy spares abdominoperineal resection for salvage treatment but carries a high burden of toxicity. Intensity-modulated radiation therapy has been implemented to reduce toxicity, reduce treatment breaks and improve survival. However, large and long-term studies are lacking. We aimed to investigate the toxicities and long-term survival of anal cancer patients treated with intensity-modulated radiation therapy at James Cook University Hospital, Middlesbrough. MATERIALS AND METHODS: We conducted a retrospective analysis of all patients with squamous cell anal cancer treated at James Cook University Hospital between July 2010 and April 2017. All patients were uniformly treated with intensity-modulated radiation therapy-based chemoradiation with curative intent. A subset of these patients was followed-up prospectively by an oncologist for acute and late toxicity. We calculated Kaplan-Meier estimates of survival statistics and compared our results with those of previous trials which used conventional radiotherapy. RESULTS: We studied 132 patients, including a toxicity subset of 64, for a median follow-up time of 43 months (range 3-84 months). Eleven patients (8.3%) underwent salvage abdominoperineal resection. Grade 3+ acute non-haematological, gastrointestinal, genitourinary and dermatological toxicity were found in 56.2%, 12.3%, 0% and 50.7% of the toxicity subset (n = 64). Median treatment duration was 37 days. Overall and colostomy-free survival at five years were 68.3% and 85.3%, respectively. Tumour size (P = 0.006) and age (P = 0.002) predicted shorter overall survival. CONCLUSIONS: Intensity-modulated radiation therapy probably reduces acute gastrointestinal and genitourinary toxicity compared with conventional radiotherapy, while resulting in similar overall and colostomy-free survival. We suggest that further dose escalation may improve survival in patients with T3/T4 tumours.

Cumulative risk assessment of the exposure to organophosphorus and carbamate insecticides in the Dutch diet.

We report the acute cumulative exposure to organophosphorus insecticides (OPs) and carbamates in the Dutch population and young children (1-6 years) via the diet. Residue data were derived from Dutch monitoring programmes performed during 2003-2005, and food consumption levels from the Dutch National Food Consumption Survey 1997/1998. The relative potency factor (RPF) approach was used to cumulate the exposure to OPs and carbamates using acephate and oxamyl as index compound respectively. The exposure was estimated using the probabilistic approach, including unit variability and processing effects. We demonstrate that about 3% of the composite samples analysed for OPs and 0.2% for carbamates contain combinations of these pesticides. The P99.9 of exposure to OPs and carbamates in the total Dutch population equals 23 and 0.64microg/kg BW/d respectively. For young children the corresponding exposure levels are 57 and 1.47microg/kg BW/d. When comparing the P99.9 of exposure with the ARfD, 50 and 9microg/kg BW/d for acephate and oxamyl respectively, there is only a possible health risk for young children. Spinach contributed most to the exposure to OPs in both age groups, followed by orange and mandarin. For carbamates apple (sauce) was the main product determining the exposure.

Prevalence of Salmonella in the broiler supply chain in The Netherlands.

This article presents detailed information on Salmonella prevalence throughout the broiler supply chain in The Netherlands, based on results from a national monitoring program. Data were collected during the period 2002 through 2005 and from six sampling points in the chain, covering hatchery up to and including processing. Trends in Salmonella prevalence over years and seasons were analyzed as well as the effect of slaughterhouse capacity on these trends. In addition, correlations between the occurrence of Salmonella at the various sampling points were calculated. The results showed a decreasing trend of Salmonella prevalence from 2002 through 2005 at all sampling points. A seasonal effect on the occurrence of Salmonella was found at the broiler farm, with a higher prevalence during the third and fourth quarter of the year (July through December). The higher the capacity of the slaughterhouse, the lower Salmonella prevalence on arrival at the slaughterhouse and the higher the prevalence at the end of slaughter and the end of processing. The detailed insights obtained in this study could be used to focus future field and experimental research on the prevention and control of Salmonella in the broiler supply chain. Results presented could also be used in risk assessment studies.

Campylobacter prevalence in the broiler supply chain in the Netherlands.

After a national control program, data on Campylobacter prevalence in the broiler supply chain in the Netherlands were gathered for 3 sampling points: departure of broiler farm and arrival and departure of the slaughterhouse. Monitoring data from this control program for 2002 to 2005 were analyzed to find correlations and possible trends in the data. As expected, the greatest correlations were found between adjacent sampling points. A high correlation was expected between number of animals slaughtered and Campylobacter prevalence, because it is assumed that larger companies will have greater hygiene standards due to better implication of food safety regulations. However, statistical analysis showed that there was no clear correlation between company size and Campylobacter prevalence. Data analysis further identified an increasing trend in Campylobacter prevalence at departure of slaughterhouse from 2002 to 2005 with strong seasonality at all 3 sampling points. Measures to control Campylobacter, therefore, need to be reconsidered and possibly intensified to achieve a reduction in Campylobacter positives.

Analysis of multivariate extreme intakes of food chemicals.

A recently published multivariate Extreme Value Theory (EVT) model is applied to the estimation of population risks associated with dietary intake of pesticides. The objective is to quantify the acute risk of pesticide intake above a threshold and relate it to the consumption of specific primary food products. As an example daily intakes of a pesticide from three foods are considered. The method models and extrapolates simultaneous intakes of pesticide, and estimates probability of exceeding unobserved large intakes. Multivariate analysis was helpful in identifying whether the avoidance of certain food combinations would reduce the likelihood of exceeding a threshold. We argue that the presented method can be an important contribution to exposure assessment studies.

Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset.

Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A large dataset of 42 hemp samples on which 3456 cDNA clones and 351 NIR wavelengths have been measured, was analyzed using graphical representations. For this purpose we propose clustered correlation and clustered discrimination images. Large, tissue-related differences are seen to dominate the cDNA-NIR correlation structure but smaller, more difficult to detect, variety-related differences can be found at specific cDNA clone/NIR wavelength combinations.

A feasibility study of accelerated fractionation in radiotherapy of carcinoma of the urinary bladder.

PURPOSE: To establish the shortest possible overall treatment time, with respect to acute and late toxicity, in which a radiation dose of 66 Gy in 2 Gy/fraction can be administered to the urinary bladder. METHODS AND MATERIALS: In a stepwise fashion the overall treatment time of our routinely used conventional treatment schedule (66 Gy in 2 Gy/fraction in 6.5 weeks) was reduced to 5 weeks (Step 1) and then 4 weeks (Step 2). Shortening of the overall treatment time was obtained by giving two daily fractions during the last part of the treatment. The interfraction interval was a minimum of 6 h. Toxicity was scored according to a modified scheme of the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer. Fifteen patients received the 5-week schedule and 25 patients received the 4-week schedule. RESULTS: Note that feasibility was very good for the 5-week schedule. No Grade > or = 3 acute bowel or bladder toxicity was seen in these patients. Severe late complications did not occur. In the 4-week schedule, acute bladder toxicity was unaffected but acute bowel toxicity was considerably increased. Of the 25 patients, 16 suffered from Grade 3 toxicity, 3 from Grade 4, and 1 from Grade 5 toxicity. Actuarial analysis at 3 years showed a 31% incidence of Grade 4 late bladder complications. CONCLUSION: It is concluded that the 5-week schedule is well tolerated. In contrast, the 4-week schedule shows unacceptable acute bowel toxicity and an increased incidence of late bladder complications (p = 0.043).

A risk-assessment model for toxic exposure of small mammalian carnivores to cadmium in contaminated natural environments.

A model is presented to assess the risk of cadmium toxicity in mammalian wildlife in contaminated environments. The target-organ load was used as a hazard indicator and animals were considered to be at risk when a critical value, i.e. the lowest-observed-adverse-effect level (LOAEL), was attained or exceeded. The model was developed for Sorex araneus, a small mammalian predator with a high potential of attaining the critical target-organ load. Model parameter values were estimated from both explicit analysis of original field data and from theoretical considerations. A submodel was derived from linear regression of data concerning 36 field sites to predict the bioconcentration of cadmium in earthworms, an important food item of small mammalian carnivores. In addition to soil cadmium concentration several soil factors affecting cadmium bioavailability were included in the submodel. A second submodel was developed to predict target-organ load from a non-linear age-dependent regression of data on 63 predators from two different field sites. A separate theoretical model was constructed for the prediction of target-organ load as a function of pollutant ingestion, assimilation rates, and internal partitioning. The risk of cadmium exposure was assessed by estimating the EC50 value, the environmental effect concentration of a toxic substance in soil at which 50% of an adult population of predators would attain a target-organ load equal to or higher than the LOAEL. The model allows one to establish availability-differentiated soil quality standards with regard to cadmium pollution in natural environments. It predicts an exponential increase of the risk of hazardous exposure of terrestrial wildlife in areas sensitive to increasing levels of soil acidification ('chemical time bomb effect').

Probabilistic modelling of exposure doses and implications for health risk characterization: glycoalkaloids from potatoes.

Potatoes are a source of glycoalkaloids (GAs) represented primarily by alpha-solanine and alpha-chaconine (about 95%). Content of GAs in tubers is usually 10-100 mg/kg and maximum levels do not exceed 200 mg/kg. GAs can be hazardous for human health. Poisoning involve gastrointestinal ailments and neurological symptoms. A single intake of >1-3 mg/kg b.w. is considered a critical effect dose (CED). Probabilistic modelling of acute and chronic (usual) exposure to GAs was performed in the Czech Republic, Sweden and The Netherlands. National databases on individual consumption of foods, data on concentration of GAs in tubers (439 Czech and Swedish results) and processing factors were used for modelling. Results concluded that potatoes currently available at the European market may lead to acute intakes >1 mg GAs/kg b.w./day for upper tail of the intake distribution (0.01% of population) in all three countries. 50 mg GAs/kg raw unpeeled tubers ensures that at least 99.99% of the population does not exceed the CED. Estimated chronic (usual) intake in participating countries was 0.25, 0.29 and 0.56 mg/kg b.w./day (97.5% upper confidence limit). It remains unclear if the incidence of GAs poisoning is underreported or if assumptions are the worst case for extremely sensitive persons.

A dose-effect relationship for the effect of deltamethrin on a linyphiid spider population in winter wheat.

The relationship between application rate and effect of deltamethrin (Decis) on a linyphiid spider population was studied in relation to ambient physical factors. A field experiment using five application rates and a control was conducted in winter wheat plots. The aim was to determine application rates that allowed for determination of negative as well as positive effects of physical factors on arthropod toxicity using pitfall traps. Several physical variables were measured in the crop canopy and deltamethrin deposition was determined at ground level. The use of Before-After-Control-Impact design (BACI) time series analysis for characterization of instantaneous pesticide effects and velocity of recovery, relative to the application rate sprayed was evaluated. An initial reduction in spider catches ranging from 58% to 85% percent for sprayed concentrations of 0.25 to 4 g active ingredient per hectare (ai ha-1) was observed, followed by exponential recovery with a half-life time of 2.8 days for all treatments. The effects observed in this study were relatively mild compared to other field-experiments. It was concluded that an application rate near 0.25 g ai ha-1 deltamethrin (i.e., one twentieth of the recommended rate) would be sufficient to study the relationship between physical factors and the effects of deltamethrin. BACI time series analysis was shown to be a useful technique for characterizing short-term pesticide effects.

Introduction to the Monte Carlo project and the approach to the validation of probabilistic models of dietary exposure to selected food chemicals.

The Monte Carlo project was established to allow an international collaborative effort to define conceptual models for food chemical and nutrient exposure, to define and validate the software code to govern these models, to provide new or reconstructed databases for validation studies, and to use the new software code to complete validation modelling. Models were considered valid when they provided exposure estimates (e(a)) that could be shown not to underestimate the true exposure (e(b)), but at the same time are more realistic than the currently used conservative estimates (e(c)). Thus, validation required e(b)

Validation of a probabilistic model of dietary exposure to selected pesticides in Dutch infants.

A probabilistic model for dietary exposure to pesticides was validated. For this, we evaluated the agreement of dietary exposure to six pesticides as estimated with the model with exposures measured in duplicate diet samples (='real intake') and those calculated with the point estimate. To calculate the exposure with the model and point estimate, consumption data of the duplicate diet survey and pesticide residue measurements from Dutch monitoring programmes in 2000 and 2001 were used. The model was considered validated when the outcome was both higher than the real intake and lower than the point estimate. Results showed that exposures estimated with the model were closer to the real intake than those of the point estimate, and that the model outcome was lower than the point estimate. Furthermore, it was shown that the probabilistic approach can address the exposure to a pesticide via the consumption of different food products, while the point estimate only estimates the exposure through the consumption of one product. The model validated is a valuable asset when estimating the dietary exposure to pesticides in both the authorization of new pesticides and the evaluation of exposures using monitoring data.

Patterns in clinical chemistry requests.

For each patient sample that is presented to the clinical chemistry laboratory a combination of various tests can be requested. This combination or profile will depend on the condition of the patient, and hence also on the requesting hospital department. Several techniques were applied to detect and describe patterns in tests requested by the cardiology, hepatology and nephrology sections of the out-patient's Department for Internal Medicine. Comparison of the frequencies of ordering the tests showed significant differences between these sections. Cluster analysis and multidimensional scaling were used to show similarities and differences in the test profiles that were used by the sections. These techniques are useful for generating hypotheses, but the statistical significance of the clustering found is difficult to assess.

Exploring multivariate clinical chemical routine data concerning three major disease groups.

In preparation for multivariate analysis, an exploratory study has been undertaken to investigate the relative position, separability, homogeneity and shape of three major disease groups, using data from a clinical chemical routine package.The data set consists of 46 hepatology patients, 50 nephrology patients and 46 cardiology patients, and the measured blood levels include 20 common clinical chemical routine assays. Missing value problems were avoided by deleting some of the variables and objects.A univariate analysis was used as the basis ofa rescaling of the data.Bivariate (pairwise) plots of some major assays each show limited separation. The set of three such plots of the three major principal components reveals more distinction between the groups than was offered by univariate analysis. Three-dimensional extensions of these techniques allow better insight than any of the two-dimensional plots, but these three-dimensional versions require more plots for complete interpretation.Non-linear mapping of the data is the best way of retaining the distances and a fairly good separation is achieved in the plot. The plot is less informative about shape and relative position of the classes.Representation of the data as pictures of faces does not offer additional information and visual clustering is worse than in any of the techniques mentioned.During the analysis many assumed properties of the data are confirmed and a good starting pointfor multivariate classification is attained. Easy visual detection of outliers is offered by all techniques. Unfortunately, valuable information is lost in this data set by deleting some incomplete variables.

On-line diode array UV-visible spectrometry in screening for drugs and drug metabolites by high-performance liquid chromatography.

The direct coupling of a multi-channel diode array UV-visible spectrophotometer to a powerful reversed-phase HPLC separation system is considered, especially for use in qualitative analysis, e.g., screening/identification of drugs and drug metabolites. The approach is illustrated by the screening for metabolites of butoprozine and ticlopidine directly in human and rat bile.