Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2ß, gap-43, neurofilament-h, tubulin-α and tubulin-ß. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals.

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys.

The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004-2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefitting from the capacity building set up by COPHES/DEMOCOPHES.

Lessons learnt on recruitment and fieldwork from a pilot European human biomonitoring survey.

Within the European Environment and Health Action Plan an initiative to establish a coherent human biomonitoring approach in Europe was started. The project COPHES (COnsortium to Perform Human biomonitoring on a European Scale ) developed recommendations for a harmonized conduct of a human biomonitoring (HBM) survey which came into action as the pilot study DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale). Seventeen European countries conducted a survey with harmonized instruments for, inter alia, recruitment, fieldwork and sampling, in autumn/winter 2011/2012. Based on the countries' experiences of conducting the pilot study, following lessons learnt were compiled: the harmonized fieldwork instruments (basic questionnaire, urine and hair sampling) turned out to be very valuable for future HBM surveys on the European scale. A school approach was favoured by most of the countries to recruit school-aged children according to the established guidelines and country specific experiences. To avoid a low participation rate, intensive communication with the involved institutions and possible participants proved to be necessary. The communication material should also include information on exclusion criteria and offered incentives. Telephone contact to the participants the day before fieldwork during the survey can prevent the forgetting of appointments and first morning urine samples. To achieve comparable results on the European scale, training of interviewers in all issues of recruitment, fieldwork and sampling through information material and training sessions is crucial. A survey involving many European countries needs time for preparation and conduct. Materials for quality control prepared for all steps of recruitment, fieldwork and sampling proved to be important to warrant reliable results.

Additive and synergistic antiandrogenic activities of mixtures of azol fungicides and vinclozolin.

OBJECTIVE: Many pesticides including pyrethroids and azole fungicides are suspected to have an endocrine disrupting property. At present, the joint activity of compound mixtures is only marginally known. Here we tested the hypothesis that the antiandrogenic activity of mixtures of azole fungicides can be predicted by the concentration addition (CA) model. METHODS: The antiandrogenic activity was assessed in MDA-kb2 cells. Following assessing single compounds activities mixtures of azole fungicides and vinclozolin were investigated. Interactions were analyzed by direct comparison between experimental and estimated dose-response curves assuming CA, followed by an analysis by the isobole method and the toxic unit approach. RESULTS: The antiandrogenic activity of pyrethroids deltamethrin, cypermethrin, fenvalerate and permethrin was weak, while the azole fungicides tebuconazole, propiconazole, epoxiconazole, econazole and vinclozolin exhibited strong antiandrogenic activity. Ten binary and one ternary mixture combinations of five antiandrogenic fungicides were assessed at equi-effective concentrations of EC25 and EC50. Isoboles indicated that about 50% of the binary mixtures were additive and 50% synergistic. Synergism was even more frequently indicated by the toxic unit approach. CONCLUSION: Our data lead to the conclusion that interactions in mixtures follow the CA model. However, a surprisingly high percentage of synergistic interactions occurred. Therefore, the mixture activity of antiandrogenic azole fungicides is at least additive. PRACTICE: Mixtures should also be considered for additive antiandrogenic activity in hazard and risk assessment. IMPLICATIONS: Our evaluation provides an appropriate "proof of concept", but whether it equally translates to in vivo effects should further be investigated.

The European COPHES/DEMOCOPHES project: towards transnational comparability and reliability of human biomonitoring results.

COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to "develop a coherent approach on human biomonitoring (HBM) in Europe". Within this twin-project it was targeted to collect specimens from 120 mother-child-pairs in each of the 17 participating European countries. These specimens were investigated for six biomarkers (mercury in hair; creatinine, cotinine, cadmium, phthalate metabolites and bisphenol A in urine). The results for mercury in hair are described in a separate paper. Each participating member state was requested to contract laboratories, for capacity building reasons ideally within its borders, carrying out the chemical analyses. To ensure comparability of analytical data a Quality Assurance Unit (QAU) was established which provided the participating laboratories with standard operating procedures (SOP) and with control material. This material was specially prepared from native, non-spiked, pooled urine samples and was tested for homogeneity and stability. Four external quality assessment exercises were carried out. Highly esteemed laboratories from all over the world served as reference laboratories. Web conferences after each external quality assessment exercise functioned as a new and effective tool to improve analytical performance, to build capacity and to educate less experienced laboratories. Of the 38 laboratories participating in the quality assurance exercises 14 laboratories qualified for cadmium, 14 for creatinine, 9 for cotinine, 7 for phthalate metabolites and 5 for bisphenol A in urine. In the last of the four external quality assessment exercises the laboratories that qualified for DEMOCOPHES performed the determinations in urine with relative standard deviations (low/high concentration) of 18.0/2.1% for cotinine, 14.8/5.1% for cadmium, 4.7/3.4% for creatinine. Relative standard deviations for the newly emerging biomarkers were higher, with values between 13.5 and 20.5% for bisphenol A and between 18.9 and 45.3% for the phthalate metabolites. Plausibility control of the HBM results of all participating countries disclosed analytical shortcomings in the determination of Cd when using certain ICP/MS methods. Results were corrected by reanalyzes. The COPHES/DEMOCOPHES project for the first time succeeded in performing a harmonized pan-European HBM project. All data raised have to be regarded as utmost reliable according to the highest international state of the art, since highly renowned laboratories functioned as reference laboratories. The procedure described here, that has shown its success, can be used as a blueprint for future transnational, multicentre HBM projects.

New perspectives in toxicological information management, and the role of ISSTOX databases in assessing chemical mutagenicity and carcinogenicity.

Currently, the public has access to a variety of databases containing mutagenicity and carcinogenicity data. These resources are crucial for the toxicologists and regulators involved in the risk assessment of chemicals, which necessitates access to all the relevant literature, and the capability to search across toxicity databases using both biological and chemical criteria. Towards the larger goal of screening chemicals for a wide range of toxicity end points of potential interest, publicly available resources across a large spectrum of biological and chemical data space must be effectively harnessed with current and evolving information technologies (i.e. systematised, integrated and mined), if long-term screening and prediction objectives are to be achieved. A key to rapid progress in the field of chemical toxicity databases is that of combining information technology with the chemical structure as identifier of the molecules. This permits an enormous range of operations (e.g. retrieving chemicals or chemical classes, describing the content of databases, finding similar chemicals, crossing biological and chemical interrogations, etc.) that other more classical databases cannot allow. This article describes the progress in the technology of toxicity databases, including the concepts of Chemical Relational Database and Toxicological Standardized Controlled Vocabularies (Ontology). Then it describes the ISSTOX cluster of toxicological databases at the Istituto Superiore di Sanitá. It consists of freely available databases characterised by the use of modern information technologies and by curation of the quality of the biological data. Finally, this article provides examples of analyses and results made possible by ISSTOX.

Economic benefits of methylmercury exposure control in Europe: monetary value of neurotoxicity prevention.

BACKGROUND: Due to global mercury pollution and the adverse health effects of prenatal exposure to methylmercury (MeHg), an assessment of the economic benefits of prevented developmental neurotoxicity is necessary for any cost-benefit analysis. METHODS: Distributions of hair-Hg concentrations among women of reproductive age were obtained from the DEMOCOPHES project (1,875 subjects in 17 countries) and literature data (6,820 subjects from 8 countries). The exposures were assumed to comply with log-normal distributions. Neurotoxicity effects were estimated from a linear dose-response function with a slope of 0.465 Intelligence Quotient (IQ) point reduction per µg/g increase in the maternal hair-Hg concentration during pregnancy, assuming no deficits below a hair-Hg limit of 0.58 µg/g thought to be safe. A logarithmic IQ response was used in sensitivity analyses. The estimated IQ benefit cost was based on lifetime income, adjusted for purchasing power parity. RESULTS: The hair-mercury concentrations were the highest in Southern Europe and lowest in Eastern Europe. The results suggest that, within the EU, more than 1.8 million children are born every year with MeHg exposures above the limit of 0.58 µg/g, and about 200,000 births exceed a higher limit of 2.5 µg/g proposed by the World Health Organization (WHO). The total annual benefits of exposure prevention within the EU were estimated at more than 600,000 IQ points per year, corresponding to a total economic benefit between €8,000 million and €9,000 million per year. About four-fold higher values were obtained when using the logarithmic response function, while adjustment for productivity resulted in slightly lower total benefits. These calculations do not include the less tangible advantages of protecting brain development against neurotoxicity or any other adverse effects. CONCLUSIONS: These estimates document that efforts to combat mercury pollution and to reduce MeHg exposures will have very substantial economic benefits in Europe, mainly in southern countries. Some data may not be entirely representative, some countries were not covered, and anticipated changes in mercury pollution all suggest a need for extended biomonitoring of human MeHg exposure.

Antiandrogenic activity of phthalate mixtures: validity of concentration addition.

Phthalates and bisphenol A have very widespread use leading to significant exposure of humans. They are suspected to interfere with the endocrine system, including the androgen, estrogen and the thyroid hormone system. Here we analyzed the antiandrogenic activity of six binary, and one ternary mixture of phthalates exhibiting complete antiandrogenic dose-response curves, and binary mixtures of phthalates and bisphenol A at equi-effective concentrations of EC(10), EC(25) and EC(50) in MDA-kb2 cells. Mixture activity followed the concentration addition (CA) model with a tendency to synergism at high and antagonism at low concentrations. Isoboles and the toxic unit approach (TUA) confirmed the additive to synergistic activity of the binary mixtures BBP+DBP, DBP+DEP and DEP+BPA at high concentrations. Both methods indicate a tendency to antagonism for the EC(10) mixtures BBP+DBP, BBP+DEP and DBP+DEP, and the EC(25) mixture of DBP+BPA. A ternary mixture revealed synergism at the EC(50), and weak antagonistic activity at the EC(25) level by the TUA. A mixture of five phthalates representing a human urine composition and reflecting exposure to corresponding parent compounds showed no antiandrogenic activity. Our study demonstrates that CA is an appropriate concept to account for mixture effects of antiandrogenic phthalates and bisphenol A. The interaction indicates a departure from additivity to antagonism at low concentrations, probably due to interaction with the androgen receptor and/or cofactors. This study emphasizes that a risk assessment of phthalates should account for mixture effects by applying the CA concept.

The new ISSMIC database on in vivo micronucleus and its role in assessing genotoxicity testing strategies.

This paper presents a new curated database on in vivo micronucleus mutagenicity results, called ISSMIC. It is freely available at: http://www.iss.it/ampp/dati/cont.php?id=233&lang=1&tipo=7. The experimental results were critically reviewed, and evidence on target cell exposure was considered as well. The inspection of ISSMIC demonstrates that a large proportion of reported negative results in the literature (231 out 566 ISSMIC chemicals) lack a clear-cut, direct demonstration of toxicity at the target cells. Using this updated database, the predictive value of a compilation of Structural Alerts (SA) for in vivo micronucleus recently implemented in the expert system Toxtree was investigated. Individually, most of the SA showed a high Positive Predictivity (∼80%), but the need for further expanding the list of alerts was pointed out as well. The role of in vivo micronucleus in strategies for carcinogenicity prediction was re-evaluated. In agreement with previous analyses, the data point to a low overall correlation with carcinogenicity. In addition, given the cost in animal lives and the time required for the experimentation, in many programs, the in vivo tests are used only to assess in vitro positive results. The ability of in vivo micronucleus to identify real positives (i.e. carcinogens) among chemicals positive in Salmonella or among chemicals inducing in vitro chromosomal aberrations was studied. It appears that the in vivo micronucleus test does not have added value and rather impairs the prediction ability of the in vitro tests alone. The overall evidence indicates that in vivo micronucleus--in its present form--cannot be considered an useful tool for routine genotoxicity testing but should be used in targeted mechanistic studies.

Some flame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro.

Contaminants including flame retardants, antimicrobial agents and phthalates, occurring as residues in human tissues were associated with altered endocrine function. In our study we analysed the flame retardants tetrabromobisphenol A (TBBPA), hexabromocyclodecane (HBCD), penta-bromodiphenylether (BDE-100) and hexa-BDE (BDE-155), the antimicrobial compounds triclosan (TCS) and triclocarban (TCC) and eight phthalates for their androgenic and antiandrogenic activity in vitro in the MDA-kb2 cell line. No or only weak androgenic activity was observed for all the tested compounds. TBBPA showed weak antiandrogenic activity, which was demonstrated for the first time. The flame retardants HBCD, BDE-100 and BDE-155 enhanced the dihydrotestosterone-dependent activation of androgen receptor-responsive gene expression but exhibited little or no agonistic activity. The enhancement reached 150%, which was similar to the antimicrobials (TCS up to 180%, and TCC up to 130%). This enhancement of androgenic activity represents a novel mode of action of the endocrine activity of flame retardants. In contrast, most phthalates showed antiandrogenic activity. Butylbenzyl phthalate (BBP), dibutyl phthalate (DBP) and diethyl phthalate (DEP) showed strong antiandrogenicity, whereas the action of diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), dimethyl phthalate (DMP), and the DEHP metabolite monoethylhexyl phthalate (MEHP) was lower. Our in vitro study demonstrates for the first time a weak antiandrogenic activity of TBBPA, and a significant enhancement of the androgenic activity of HBCD, BDE-100 and BDE-155, which represents a novel mechanism of hormonal activity of flame retardants.

Prediction of the rodent carcinogenicity of 60 pesticides by the DEREKfW expert system.

The two-year rodent bioassay represents the golden standard for evaluating the carcinogenicity of chemicals. Because of practical and ethical reasons, alternative approaches have been investigated for many years. Among these approaches, the (quantitative) structure-activity relationships [(Q)SARs] offer promising perspectives for quickly screening a large number of chemicals. To increase the acceptance of (Q)SARs among the regulators, their predictive power needs to be scientifically validated. In this article, we tested the capacity of the DEREKfW expert system to qualitatively predict the rodent carcinogenicity and the genotoxic potential of 60 pesticides recently registered in Switzerland. The percentage of false negatives was found to be 31% for carcinogenicity. The associated sensitivity of 69% indicates that most of the pesticides with positive rodent bioassay results were detected by DEREKfW. On the other hand, the low specificity of 47% indicates that many pesticides may be flagged as carcinogenic while rodent bioassays would not confirm this potential. This may lead to unnecessary testing or the unnecessary restriction of a chemical.

Assessing human health response in life cycle assessment using ED10s and DALYs: part 1--Cancer effects.

Life cycle assessment (LCA) is a framework for comparing products according to their total estimated environmental impact, summed over all chemical emissions and activities associated with a product at all stages in its life cycle (from raw material acquisition, manufacturing, use, to final disposal). For each chemical involved, the exposure associated with the mass released into the environment, integrated over time and space, is multiplied by a toxicological measure to estimate the likelihood of effects and their potential consequences. In this article, we explore the use of quantitative methods drawn from conventional single-chemical regulatory risk assessments to create a procedure for the estimation of the cancer effect measure in the impact phase of LCA. The approach is based on the maximum likelihood estimate of the effect dose inducing a 10% response over background, ED10, and default linear low-dose extrapolation using the slope betaED10 (0.1/ED10). The calculated effects may correspond to residual risks below current regulatory compliance requirements that occur over multiple generations and at multiple locations; but at the very least they represent a "using up" of some portion of the human population's ability to accommodate emissions. Preliminary comparisons are performed with existing measures, such as the U.S. Environmental Protection Agency's (U.S. EPA's) slope factor measure q1*. By analyzing bioassay data for 44 chemicals drawn from the EPA's Integrated Risk Information System (IRIS) database, we explore estimating ED10 from more readily available information such as the median tumor dose rate TD50 and the median single lethal dose LD50. Based on the TD50, we then estimate the ED10 for more than 600 chemicals. Differences in potential consequences, or severity, are addressed by combining betaED10 with the measure disability adjusted life years per affected person, DALYp. Most of the variation among chemicals for cancer effects is found to be due to differences in the slope factors (betaED10) ranging from 10(-4) up to 10(4) (risk of cancer/mg/kg-day).

Assessing human health response in life cycle assessment using ED10s and DALYs: part 2--Noncancer effects.

In Part 1 of this article we developed an approach for the calculation of cancer effect measures for life cycle assessment (LCA). In this article, we propose and evaluate the method for the screening of noncancer toxicological health effects. This approach draws on the noncancer health risk assessment concept of benchmark dose, while noting important differences with regulatory applications in the objectives of an LCA study. We adopt the centraltendency estimate of the toxicological effect dose inducing a 10% response over background, ED10, to provide a consistent point of departure for default linear low-dose response estimates (betaED10). This explicit estimation of low-dose risks, while necessary in LCA, is in marked contrast to many traditional procedures for noncancer assessments. For pragmatic reasons, mechanistic thresholds and nonlinear low-dose response curves were not implemented in the presented framework. In essence, for the comparative needs of LCA, we propose that one initially screens alternative activities or products on the degree to which the associated chemical emissions erode their margins of exposure, which may or may not be manifested as increases in disease incidence. We illustrate the method here by deriving the betaED10 slope factors from bioassay data for 12 chemicals and outline some of the possibilities for extrapolation from other more readily available measures, such as the no observable adverse effect levels (NOAEL), avoiding uncertainty factors that lead to inconsistent degrees of conservatism from chemical to chemical. These extrapolations facilitated the initial calculation of slope factors for an additional 403 compounds; ranging from 10(-6) to 10(3) (risk per mg/kg-day dose). The potential consequences of the effects are taken into account in a preliminary approach by combining the betaED10 with the severity measure disability adjusted life years (DALY), providing a screening-level estimate of the potential consequences associated with exposures, integrated over time and space, to a given mass of chemical released into the environment for use in LCA.