Combined exposure to lead, inorganic mercury and methylmercury shows deviation from additivity for cardiovascular toxicity in rats.

Environmental exposure to metal mixtures in the human population is common. Mixture risk assessments are often challenging because of a lack of suitable data on the relevant mixture. A growing number of studies show an association between lead or mercury exposure and cardiovascular effects. We investigated the cardiovascular effects of single metal exposure or co-exposure to methylmercury [MeHg(I)], inorganic mercury [Hg(II)] and lead [Pb(II)]. Male Wistar rats received four different metal mixtures for 28 days through the drinking water. The ratios of the metals were based on reference and environmental exposure values. Blood and pulse pressure, cardiac output and electrical activity of the heart were selected as end-points. While exposure to only MeHg(I) increased the systolic blood pressure and decreased cardiac output, the effects were reversed with combined exposures (antagonism). In contrast to these effects, combined exposures negatively affected the electrical activity of the heart (synergism). Thus, it appears that estimates of blood total Hg levels need to be paired with estimates of what species of mercury dominate exposure as well as whether lead co-exposure is present to link total blood Hg levels to cardiovascular effects. Based on current human exposure data and our results, there may be an increased risk of cardiac events as a result of combined exposures to Hg(II), MeHg(I) and Pb(II). This increased risk needs to be clarified by analyzing lead and Hg exposure data in relation to cardiac electrical activity in epidemiological studies.

Urinary metabolites of the UV filter 2-Ethylhexyl salicylate as biomarkers of exposure in humans.

The UV filter 2-ethylhexyl salicylate (EHS) is used in sunscreens and other personal care products worldwide and has been found in a variety of environmental media. We aimed to provide human toxicokinetic data on EHS as a tool for risk assessment. For that purpose, we investigated metabolism and urinary metabolite excretion after a single oral EHS dose (57.4-75.5 µg/(kg body weight)) in three male volunteers. In a suspect screening, we tentatively identified seven EHS metabolites. Three EHS specific metabolites were quantitatively investigated: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenzoate (5oxo-EHS), and 5-(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx-EPS). These metabolites were excreted with urinary excretion fractions of 0.28% (range: 0.13-0.54%), 0.11% (0.06-0.20%), and 0.24% (0.14-0.41%), respectively. The elimination was fast: peak urinary concentrations were found 1.6-2.6 h after dose and ≥95% of the total amounts were excreted within 24 h. Elimination kinetics were biphasic, with mean elimination half-lives of 0.8 h (first phase) and 6.6 h (second phase) for 5OH-EHS, 0.8 h and 6.3 h for 5oxo-EHS, and 1.1 h and 5.9 h for 5cx-EPS. After dermal exposure (sunscreen application), we found a considerably delayed EHS elimination. Based on urinary metabolite levels we calculated EHS exposure levels for a small pilot population.

The mechanisms associated with the development of hypertension after exposure to lead, mercury species or their mixtures differs with the metal and the mixture ratio.

Hypertension is considered to be the most important risk factor for the development of cardiovascular diseases. Beside life-style risk factors, exposure to lead and mercury species are increasingly discussed as potential risk factors. Although there are a few previous studies, the underlying mechanism by which exposure to lead and mercury disturb blood pressure regulation is not currently understood. Potential mechanisms are oxidative stress production, kidney damage and activation of the renin-angiotensin system (RAS), all of which can interact to cause dysregulation of blood pressure. Male rats (Wistar) were exposed to lead, inorganic mercury, methylmercury or two mixtures of all three metals for four weeks through the drinking water. The two mixture ratios were based on ratios of known reference values or environmental exposure from the literature. To investigate the potential mechanism of actions, blood pressure was measured after four weeks and compared to plasma nitrotyrosine or reduced/oxidized glutathione levels in liver as markers for oxidative stress. Plasma renin and angiotensin II levels were used as markers for RAS activation. Finally, kidney function and injury were assessed via urinary and plasma creatinine levels, creatinine clearance and urinary kidney-injury molecule (KIM-1). While exposure to lead by itself increased oxidative stress and kidney damage along with blood pressure, inorganic mercury did not affect blood pressure or any end-point examined. Conversely, methylmercury instead increased RAS activation along with blood pressure. Surprisingly, when administered as mixtures, lead no longer increased oxidative stress or altered kidney function. Moreover, the mixture based on an environmental ratio no longer had an effect on blood pressure, while the reference value ratio still retained an increase in blood pressure. Based on our results, the prominent mechanism of action associated with the development of hypertension seems to be oxidative stress and kidney damage for lead, while increased RAS activation links methylmercury to hypertension, but these mechanisms along with hypertension disappear when metals are present in some mixtures.

Cardiovascular responses to lead are biphasic, while methylmercury, but not inorganic mercury, monotonically increases blood pressure in rats.

Cardiovascular diseases, such as heart attack and stroke, are the major cause of death worldwide. It is well known that a high number of environmental and physiological risk factors contribute to the development of cardiovascular diseases. Although risk factors are additive, increased blood pressure (hypertension) is the greatest risk factor. Over the last two decades, a growing number of epidemiological studies associate environmental exposure to lead or mercury species with hypertension. However, cardiovascular effects beyond blood pressure are rarely studied and thresholds for effect are not yet clear. To explore effects of lead or mercury species on the cardiovascular system, normal male Wistar rats were exposed to a range of doses of lead, inorganic mercury or methylmercury through the drinking water for four weeks. High-resolution ultrasound was used to measure heart and vascular function (carotid artery blood flow) at baseline and at the end of the exposure, while blood pressure was measured directly in the femoral artery at the end of the 4-week exposure. After 4 weeks, blood pressure responses to lead were biphasic. Low lead levels decreased blood pressure, dilated the carotid artery and increased cardiac output. At higher lead doses, rats had increased blood pressure. In contrast, methylmercury-exposed rats had increased blood pressure at all doses despite dilated carotid arteries. Inorganic mercury did not show any significant cardiovascular effects. Based on the current study, the benchmark dose level 10% (BMDL10s) for systolic blood pressure for lead, inorganic mercury and methylmercury are 1.1, 1.3 and 1.0 µg/kg-bw/d, respectively. However, similar total mercury blood levels attributed to inorganic mercury or methylmercury produced strikingly different results with inorganic mercury having no observable effect on the cardiovascular system but methylmercury increasing systolic and pulse pressures. Therefore, adverse cardiovascular effects cannot be predicted by total blood mercury level alone and the mercury species of exposure must be taken into account.

Approaches to the safety assessment of engineered nanomaterials (ENM) in food.

A systematic, tiered approach to assess the safety of engineered nanomaterials (ENMs) in foods is presented. The ENM is first compared to its non-nano form counterpart to determine if ENM-specific assessment is required. Of highest concern from a toxicological perspective are ENMs which have potential for systemic translocation, are insoluble or only partially soluble over time or are particulate and bio-persistent. Where ENM-specific assessment is triggered, Tier 1 screening considers the potential for translocation across biological barriers, cytotoxicity, generation of reactive oxygen species, inflammatory response, genotoxicity and general toxicity. In silico and in vitro studies, together with a sub-acute repeat-dose rodent study, could be considered for this phase. Tier 2 hazard characterisation is based on a sentinel 90-day rodent study with an extended range of endpoints, additional parameters being investigated case-by-case. Physicochemical characterisation should be performed in a range of food and biological matrices. A default assumption of 100% bioavailability of the ENM provides a 'worst case' exposure scenario, which could be refined as additional data become available. The safety testing strategy is considered applicable to variations in ENM size within the nanoscale and to new generations of ENM.

Application of the TTC concept to unknown substances found in analysis of foods.

Unknown substances, not previously observed, are frequently detected in foods by quality control laboratories. In many cases, the assessment of these 'new' substances requires additional chemical analysis for their identification prior to assessing risk. This identification procedure can be time-consuming, expensive and in some instances difficult. Furthermore, in many cases, no toxicological information will be available for the substance. Therefore, there is a need to develop pragmatic tools for the assessment of the potential toxicity of substances with unknown identity to avoid delays in their risk assessment. Hence, the 'ILSI Europe expert group on the application of the threshold of toxicological concern (TTC) to unexpected peaks found in food' was established to explore whether the TTC concept may enable a more pragmatic risk assessment of unknown substances that were not previously detected in food. A step-wise approach is introduced that uses expert judgement on the source of the food, information on the analytical techniques, the dietary consumption of food sources containing the unknown substance and quantitative information of the unknown substance to assess the safety to the consumer using the TTC. By following this step-wise approach, it may be possible to apply a TTC threshold of 90 µg/day for an unknown substance in food.

Application of the Margin of Exposure (MOE) approach to substances in food that are genotoxic and carcinogenic.

This paper presents the work of an expert group established by the International Life Sciences Institute - European branch (ILSI Europe) to follow up the recommendations of an international conference on "Risk Assessment of Compounds that are both Genotoxic and Carcinogenic: New Approaches". Twelve genotoxic and carcinogenic chemicals that can be present in food were selected for calculation of a Margin of Exposure (MOE) between a point of departure on the dose-response for oral carcinogenicity in animal studies and estimates of human dietary exposure. The MOE can be used to support prioritisation of risk management action and, if the MOE is very large, on communication of a low level of human health concern. Depending on the approaches taken in determining the point of departure and the estimation of exposure, it is possible to derive very different values for the MOE. It is therefore essential that the selection of the cancer endpoint and mathematical treatment of the data are clearly described and justified if the results of the MOE approach are to be trusted and of value to risk managers. An outline framework for calculating an MOE is proposed in order to help to ensure transparency in the results.

Intake of selected nutrients from foods, from fortification and from supplements in various European countries.

BACKGROUND: Recent European Union regulation requires setting of maximum amount of micronutrients in dietary supplements or foods taking into account the tolerable upper intake level (ULs) established by scientific risk assessment and population reference intakes. OBJECTIVE: To collect and evaluate recently available data on intakes of selected vitamins and minerals from conventional foods, food supplements and fortified foods in adults and children. Intake of calcium, copper, iodine, iron, magnesium, phosphorus, selenium, zinc, folic acid, niacin and total vitamin A/retinol, B(6), D and E was derived from nationally representative surveys in Denmark, Germany, Finland, Ireland, Italy, the Netherlands, Poland, Spain and the United Kingdom. Intake of high consumers, defined as the 95th percentile of each nutrient, was compared to the UL. RESULTS: For most nutrients, adults and children generally consume considerably less than the UL with exceptions being retinol, zinc, iodine, copper and magnesium. The major contributor to intakes for all nutrients and in all countries is from foods in the base diet. The patterns of food supplements and voluntary fortification vary widely among countries with food supplements being responsible for the largest differences in total intakes. In the present study, for those countries with data on fortified foods, fortified foods do not significantly contribute to higher intakes for any nutrient. Total nutrient intake expressed as percentage of the UL is generally higher in children than in adults. CONCLUSION: The risk of excessive intakes is relatively low for the majority of nutrients with a few exceptions. Children are the most vulnerable group as they are more likely to exhibit high intakes relative to the UL. There is a need to develop improved methods for estimating intakes of micronutrients from fortified foods and food supplements in future dietary surveys.

Probabilistic risk assessment for linear alkylbenzene sulfonate (LAS) in sewage sludge used on agricultural soil.

Deterministic and probabilistic risk assessments were developed for commercial LAS in agricultural soil amended with sewage sludge. The procedure done according to ILSI Europe's Conceptual Framework [Schowanek, D., Carr, R., David, H., Douben, P., Hall, J., Kirchmann, H., Patria, L., Sequi, P., Smith, S., Webb, S.F., 2004. A risk-based methodology for deriving quality standards for organic contaminants in sewage sludge for use in agriculture-conceptual Framework. Regul. Toxicol. Pharmacol. 40 (3), 227-251], consists of three main steps. First, the most sensitive endpoint was determined. This was found to be the chronic ecotoxicity of LAS to soil invertebrates and plants. Additional endpoints, such as potential for plant uptake and transfer in the food chain, leaching to groundwater, surface erosion run-off, human health risk via drinking water, plant consumption and soil ingestion were also systematically evaluated but were all assessed to be of little toxicological significance. In the second step, a back-calculation was conducted from the Predicted No-Effect Concentration in soil (PNECsoil) to a safe level of LAS in sludge (here called 'Sludge Quality Standard'; SQS). The deterministic approach followed the default agricultural soil exposure scenario in the EU-Technical Guidance Document (TGD). The SQS for LAS was calculated as 49 g/kg sludge Dry Matter (DM). In order to assess the potential variability as a result of varying agricultural practices and local environmental conditions, two probabilistic exposure assessment scenarios were also developed. The mean SQS was estimated at 55 and 27.5 g/kg DM for the homogeneous soil mixing and soil injection scenarios, respectively. In the final step, the resulting SQS values were evaluated for consistency and relevance versus available information from agricultural experience and field tests. No build-up, adverse impact on soil fertility, agronomic performance, or animal/human health have been reported for agricultural fields which have received sludge with high LAS levels for up to 30 years. Distribution statistics of LAS concentrations in anaerobically digested sewage sludge measured across Europe were created (mean value: 5.56 g LAS/kg sludge DM). When compared to the above mean SQS values, adequate risk characterisation ratios of 0.08-0.2 were found. The 'ecological risk' parameter calculated for anaerobic sludge from the probabilistic approaches was below 3%. A regulatory Limit Value for LAS of 2.60 g/kg sludge DM was originally proposed in the 3rd Draft of the Working Document on Sludge [CEC, 2000b. Working Document on Sludge. Third Draft, Brussels 27 April 2000, DG. Environment, 18 p.]. The current assessment, based on an updated dataset and a refined assessment procedure, suggests that the need for a limit value for LAS in sewage sludge cannot be substantiated on a risk basis.