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Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio , as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

In vitro models of adipogenesis are phenotypic assays that most closely mimic the increase of adipose tissue in obesity. Current models, however, often lack throughput and sensitivity and even report conflicting data regarding adipogenic potencies of many chemicals. Here, we describe a ten-day long adipogenesis model using high content analysis readouts for adipocyte number, size, and lipid content on primary human mesenchymal stem cells (MSC) sensitive enough to compare bisphenol A derivatives quantitatively in a robust and high throughput manner. The number of adipocytes was the most sensitive endpoint capable of detecting changes of 20% and was used to develop a benchmark concentration model (BMC) to quantitatively compare eight bisphenols (tested at 0.1-100 μM). The model was applied to evaluate mixtures of bisphenols obtaining the first experimental evidence of their additive effect on human MSC adipogenesis. Using the relative potency factors (RPFs), we show how a mixture of bisphenols at their sub-active concentrations induces a significant adipogenic effect due to its additive nature. The final active concentrations of bisphenols in tested mixtures reached below 1 μM, which is within the concentration range observed in humans. These results point to the need to consider the toxicity of chemical mixtures.

Purpose. This study aimed to investigate chemical injuries caused by cleaning agents and disinfectants by reviewing poison control data. Methods. We performed a 5-year retrospective analysis of cal...

Hazard characterization during pharmaceutical development identifies the candidate drug's potential hazards and dose-response relationships. To date, the no-observed-adverse-effect-level (NOAEL) approach has been employed to identify the highest dose which results in no observed adverse effects. The benchmark dose (BMD) modeling approach describes potential dose-response relationships and has been used in diverse regulatory domains, but its applicability for pharmaceutical development has not previously been examined. Thus, we applied BMD-modeling to all endpoints in three sequential in vivo studies in a drug development setting, including biochemistry, hematology, organ pathology and clinical observations. In order to compare the results across such a broad range of effects, we needed to standardize the choice of the critical effect size (CES) for the different endpoints. A CES of 5%, previously suggested by the European Food Safety Authority, was compared with the study NOAEL and with the General Theory of Effect Size, which takes natural variability into account. Compared to the NOAEL approach, the BMD-modeling approach resulted in more informative estimates of the doses leading to effects. The BMD-modeling approach handled well situations where effects occurred below the lowest tested dose and the study's NOAEL, and seems advantageous to characterize the potential toxicity during safety assessment. The results imply a considerable step forward from the perspective of reducing and refining animal experiments, as more information is yielded from the same number of animals and at lower doses. Taken together, employing BMD-modeling as a substitute, or as a complement, to the NOAEL approach seems appropriate.

The toxicity of PCB-156 (2,3,3′,4,4′,5-hexachlorobiphenyl) was investigated in rats following subchronic dietary exposure. Groups of 10 male and female Sprague-Dawley rats were administered PCB-156 in the diet at 0, 0.01, 0.1, 1 or 10 ppm for 90 days. Dose-dependent increases were detected for the liver, lung and kidney weights, as well as for the liver EROD, PROD and UDPGT enzyme activities and liver uroporphyrin concentration. Dose-dependent decreases were observed in final body weight, body weight gain, and thymus weight. Apolar retinoid concentrations were decreased in the liver and lungs and increased in the kidneys. Histopathological examination of the liver, thyroid, and thymus showed mild to moderate dose-related changes. A LOAEL of 0.01 ppm was established, based on reduced apolar liver retinoid concentration. Benchmark dose-modelling corroborated the sensitivity of liver retinoid endpoints. The lower confidence limits (BMDL) for a 5% decrease in apolar liver retinoid concentrations were 0.0009 and 0.0007 ppm, respectively, in males and females, corresponding to a daily dose of 0.06 µg PCB-156 per kg body weight. Organizing dose-response data for the individual hepatic endpoints along the PCB-156 dosing scale revealed a sequence of events compatible with a causal link between depletion of apolar retinoids and the other liver biochemistry and pathology findings. Taken together, data suggest that the retinoid endpoints should be further evaluated for a causal relationship to PCB-induced liver toxicity and that retinoid system endpoints are identified and characterized to support health risk assessment in the emerging research fields of endocrine disruption and mixture toxicology.

Background: Extensive exposure to per- and polyfluoroalkyl substances (PFAS) have been observed in many countries. Current deterministic frameworks for risk assessment lack the ability to predict the likelihood of effects and to assess uncertainty. When exposure exceeds tolerable intake levels, these shortcomings hamper risk management and communication. Objective: The integrated probabilistic risk assessment (IPRA) combines dose-response and exposure data to estimate the likelihood of adverse effects. We evaluated the usefulness of the IPRA for risk characterization related to decreased levels of total triiodothyronine (T3) in humans following a real case of high exposure to PFAS via drinking water. Methods: PFAS exposure was defined as serum levels from residents of a contaminated area in Ronneby, Sweden. Median levels were 270 ng/mL [perfluorooctane sulfonic acid (PFOS)] and 229 ng/mL [perfluorohexane sulfonic acid (PFHxS)] for individuals who resided in Ronneby 1 y before the exposure termination. This data was integrated with data from a subchronic toxicity study in monkeys exposed daily to PFOS. Benchmark dose modeling was employed to describe separate dose–effect relationship for males and females, and extrapolation factor distributions were used to estimate the corresponding human benchmark dose. The critical effect level was defined as a 10% decrease in total T3. Results: The median probability of critical exposure, following a combined exposure to PFOS and PFHxS, was estimated to be [2.1% (90% CI: 0.4%–13.1%)]. Gender-based analysis showed that this risk was almost entirely distributed among women, namely [3.9% (90% CI: 0.8%–21.6%)]. Discussion: The IPRA was compared with the traditional deterministic Margin of Exposure (MoE) approach. We conclude that probabilistic risk characterization represents an important step forward in the ability to adequately analyze group-specific health risks. Moreover, quantifying the sources of uncertainty is desirable, as it improves the awareness among stakeholders and will guide future efforts to improve accuracy. https://doi.org/10.1289/EHP6654

Animal testing for toxicity assessment of chemicals and pharmaceuticals must take the 3R principles into consideration. During toxicity testing in vivo, clinical signs are used to monitor animal welfare and to inform about potential toxicity. This study investigated possible associations between clinical signs, body weight change and histopathological findings observed after necropsy. We hypothesized that clinical signs and body weight loss observed during experiments could be used as early markers of organ toxicity. This represents a potential for refinement in terms of improved study man­agement and decreasing of pain and distress experienced during animal experiments. Data from three sequential toxicity studies of an anti-cancer drug candidate in rats were analyzed using the multivariate partial least squares (PLS) regression method. Associations with a predictive value over 80% were found between the occurrence of mild to severe clinical signs and histopathological findings in the thymus, testes, epididymides and bone marrow. Piloerection, eyes half shut and slightly decreased motor activity were most strongly associated with the pathological findings. A 5% body weight loss was found to be a strong empirical predictor of pathological findings but could also be predicted accurately by clinical signs. Thus, we suggest using mild clinical signs and a 5% body weight loss as toxicity markers and as a non-invasive surveillance tool to monitor research animal welfare in toxicity testing. These clinical signs may also enable reduction of animal use due to their informative potential to support scientific decisions regarding drug candidate selection, dose setting, study design, and toxicity assessment.

The number of anthropogenic chemicals, manufactured, by-products, metabolites and abiotically formed transformation products, counts to hundreds of thousands, at present. Thus, humans and wildlife are exposed to complex mixtures, never one chemical at a time and rarely with only one dominating effect. Hence there is an urgent need to develop strategies on how exposure to multiple hazardous chemicals and the combination of their effects can be assessed. A workshop, “Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment” was organized in May 2018 together with Joint Research Center in Ispra, EU-funded research projects and Commission Services and relevant EU agencies. This forum for researchers and policy-makers was created to discuss and identify gaps in risk assessment and governance of chemical mixtures as well as to discuss state of the art science and future research needs. Based on the presentations and discussions at this workshop we want to bring forward the following Key Messages: • We are at a turning point: multiple exposures and their combined effects require better management to protect public health and the environment from hazardous chemical mixtures. • Regulatory initiatives should be launched to investigate the opportunities for all relevant regulatory frameworks to include prospective mixture risk assessment and consider combined exposures to (real-life) chemical mixtures to humans and wildlife, across sectors. • Precautionary approaches and intermediate measures (e.g. Mixture Assessment Factor) can already be applied, although, definitive mixture risk assessments cannot be routinely conducted due to significant knowledge and data gaps. • A European strategy needs to be set, through stakeholder engagement, for the governance of combined exposure to multiple chemicals and mixtures. The strategy would include research aimed at scientific advancement in mechanistic understanding and modelling techniques, as well as research to address regulatory and policy needs. Without such a clear strategy, specific objectives and common priorities, research, and policies to address mixtures will likely remain scattered and insufficient. Keywords: Chemical mixtures, Environmental chemicals, Combined exposure, Mixture risk assessment, Risk management

Chemical safety management is an iterative process where reviews of safety deficiencies (e.g., inspection notes) and failures (e.g., injuries) guide improvement efforts. The present work investigates safety in the school chemistry laboratory through two substudies. First, we interviewed 10 Swedish middle and high school chemistry teachers about how they work with safety and accident prevention in the chemistry laboratory. Second, we analyzed the call records of the Swedish Poisons Information Centre (PIC) concerning 10–19 olds and reports regarding severe injuries and accidents submitted to the Swedish Work Environment Authority (SWEA). The interviewed teachers encounter problems with chemical safety related to deficiencies in organizational support, such as lack of time and resources for preventive risk management and assignment to groups that are too large. The major safety challenge was reported to lie in students’ safety behavior. Although facilities were generally well-equipped we noted outdated safe...

Records of injuries and incidents provide an important basis for injury prevention related to hazardous substances at the workplace. The present study aimed to review available data on injuries and incidents involving hazardous substances and investigate how data from the Poisons Information Centre could complement the records of the Swedish Work Environment Authority. We found two major obstacles for using injury/incident data based on employers' mandatory reporting. First, it was not possible to quickly and reliably identify injuries caused by hazardous substances, and second, data identifying substances or products are not systematically included. For two out of five investigated injuries with lost working days likely due to chemical injuries, we could not identify substances and/or products involved. The records based on calls to the Poisons Information Centre allow better understanding of chemical hazards and products. Besides the large share of unidentified chemical hazards in the injury statistics, the most striking difference was found for cleaning agents. Cleaning agents were implicated in one-third of the occupational cases that the consulting Poisons Information Centre expert judged to pose a major risk and in need of immediate healthcare. Only one in 10 injuries with lost days reported by employers was related to this type of product. The identification of exposures and symptoms by the Poisons Information Centre allow recognition of chemicals with problematic occupational uses. Hence, these records may serve as an important complement to official injury statistics related to incidents with hazardous substances at work.

Increasingly, dose-response data are being evaluated with the benchmark dose (BMD) approach rather than by the less precise no-observed-adverse-effect-level (NOAEL) approach. However, the basis for designing animal experiments, using equally sized dose groups, is still primed for the NOAEL approach. The major objective here was to assess the impact of using dose groups of unequal size on both the quality of the BMD and overall animal distress. We examined study designs with a total number of 200 animals distributed in four dose groups employing quantal data generated by Monte Carlo simulations. Placing more animals at doses close to the targeted BMD provided an estimate of BMD that was slightly better than the standard design with equally sized dose groups. In situations involving a clear dose-response, this translates into fewer animals receiving high doses and thus less overall animal distress. Accordingly, in connection with risk and safety assessment, animal distress can potentially be reduced by distributing the animals appropriately between dose groups without decreasing the quality of the information obtained.

Asthma, a chronic respiratory disease, can be aggravated by exposure to certain chemical irritants. The objectives were first to investigate the extent to which experimental observations on asthmatic subjects are taken into consideration in connection with the registration process under the EU REACH regulation, and second, to determine whether asthmatics are provided adequate protection by the derived no‐effect levels (DNELs) for acute inhalation exposure. We identified substances for which experimental data on the pulmonary functions of asthmatics exposed to chemicals under controlled conditions are available. The effect concentrations were then compared with DNELs and other guideline and limit values. As of April 2015, only 2.6% of 269 classified irritants had available experimental data on asthmatics. Fourteen of the 22 identified substances with available data were fully registered under REACH and we retrieved 114 reliable studies related to these. Sixty‐three of these studies, involving nine of the 14 substances, were cited by the REACH registrants. However, only 17 of the 114 studies, involving four substances, were regarded as key studies. Furthermore, many of the DNELs for acute inhalation were higher than estimated effect levels for asthmatics, i.e., lowest observed adverse effect concentrations or no‐observed adverse effect concentrations, indicating low or no safety margin. We conclude that REACH registrants tend to disregard findings on asthmatics when deriving these DNELs. In addition, we found examples of DNELs, particularly among those derived for workers, which likely do not provide adequate protection for asthmatics. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd., We investigate the extent to which experimental observations on asthmatic subjects are taken into consideration by REACH registrants and determine whether asthmatics are provided adequate protection by the acute inhalation Derived No‐Effect Levels. Of 114 studies concerning 14 substances fulfilling our inclusion criteria, 63 studies, involving nine substances, were cited by the REACH registrants. However, only 17 of the 114 studies, involving four substances, were regarded as key studies. Furthermore, many of the Derived No‐Effect Levels were higher than Lowest‐ or No‐Observed Adverse Effect Concentrations values from these studies.

Larsson and coworkers recently presented a study in Food and Chemical Toxicology on the cumulative risks to the Danish population from dietary exposure to pesticide residues. They base their analysis on food monitoring data, spray journals, controlled field trials and food consumption data in the Danish population. A cumulative hazard-index (HI) approach is then used to estimate the overall risk from pesticide exposure, an approach well established in the literature. Based on an HI of 13-44%, the authors conclude that adverse health effects due to pesticide residues are “very unlikely” and equivalent to “1 glass of wine every seventh year”. Unfortunately, the paper fails to put the limitations of the underlying data and the applied methodology in context and it misinterprets the use of Assessment Factors in chemical safety assessment. The comparison of population-wide life-long involuntary exposure to pesticides with individual, time-limited, voluntary alcohol consumption is misleading, in particular in view that children are identified as the most vulnerable sub-population.

Convergent evidence associates endocrine disrupting chemicals (EDCs) with major, increasingly-prevalent human disorders. Regulation requires elucidation of EDC-triggered molecular events causally linked to adverse health outcomes, but two factors limit their identification. First, experiments frequently use individual chemicals, whereas real life entails simultaneous exposure to multiple EDCs. Second, population-based and experimental studies are seldom integrated. This drawback was exacerbated until recently by lack of physiopathologically meaningful human experimental systems that link epidemiological data with results from model organisms.We developed a novel approach, integrating epidemiological with experimental evidence. Starting from 1,874 mother-child pairs we identified mixtures of chemicals, measured during early pregnancy, associated with language delay or low-birth weight in offspring. These mixtures were then tested on multiple complementary in vitro and in vivo models. We demonstrate that each EDC mixture, at levels found in pregnant women, disrupts hormone-regulated and disease-relevant gene regulatory networks at both the cellular and organismal scale.

Objective: It has been suggested that asthmatics are more susceptible than healthy individuals to airborne irritating chemicals in general. However, there is limited human data available to support this hypothesis due to ethical and practical difficulties. We explored a murine model of ovalbumin (OVA)-induced airway inflammation to study susceptibility during acute exposure to chemicals with chlorine as a model substance. Methods: Naïve and OVA sensitized female BALB/c mice were exposed to chlorine at four different concentrations (0, 5, 30 and 80 ppm) for 15 minutes with online recording of the respiratory function by plethysmography. The specific effects on respiratory mechanics, inflammatory cells and inflammatory mediators (cytokines and chemokines) of the airways were measured 24 hours after the chlorine exposure as well as histopathological examination of the lungs. Results: Similar concentration-dependent reductions in respiratory frequency were seen in the two groups, with a 50% reduction (RD50) slightly above 5 ppm. Decreased body weight 24 hours after exposure to 80 ppm was also observed in both groups. Naïve, but not OVA-sensitized, mice showed increased bronchial reactivity and higher number of neutrophils in bronchoalveolar lavage fluid at 80 ppm. Conclusions: The results do not support an increased susceptibility to chlorine among OVA-sensitized mice. This animal model, which represents a phenotype of eosinophilic airway inflammation, seems unsuitable to study susceptibility to inhalation of irritants in relation to asthma.

IRET, CERCH Background: Mancozeb and its main metabolite ethylene thiourea (ETU) may alter thyroid function; thyroid hormones are essential for fetal brain development. In Costa Rica, mancozeb is aerially sprayed at large-scale banana plantations on a weekly basis. Objectives: Our goals were to evaluate urinary ETU concentrations in pregnant women living near large-scale banana plantations, compare their estimated daily intake (EDI) with established reference doses (RfDs), and identify factors that predict their urinary ETU concentrations. Methods: We enrolled 451 pregnant women from Matina County, Costa Rica, which has large-scale banana production. We visited 445 women up to three times during pregnancy to obtain urine samples (n = 872) and information on factors that possibly influence exposure. We determined urinary ETU concentrations using liquid chromatography mass spectrometry. Results: Pregnant women’s median urinary ETU concentrations were more than five times higher than those reported for other general populations. Seventy-two percent of the women had EDIs above the RfD. Women who lived closest (1st quartile, < 48m) to banana plantations on average had a 45% (95% CI: 23, 72%) higher urinary ETU compared with women who lived farthest away (4th quartile, ≥ 565m). Compared with the other women, ETU was also higher in women who washed agricultural work clothes on the day before sampling (11%; 95% CI: 4.9, 17%), women who worked in agriculture during pregnancy (19%; 95% CI: 9.3, 29%), and immigrant women (6.2%; 95% CI: 1.0, 13%). Conclusions: The pregnant women’s urinary ETU concentrations are of concern, and the principal source of exposure is likely to be aerial spraying of mancozeb. The factors predicting ETU provide insight into possibilities for exposure reduction. Antecedentes: el mancozeb y su principal metabolito etilen tiourea (ETU) pueden alterar la función tiroidea; Las hormonas tiroideas son esenciales para el desarrollo del cerebro fetal. En Costa Rica, mancozeb se fumiga por vía aérea en plantaciones de banano a gran escala semanalmente. Objetivos: Nuestros objetivos fueron evaluar las concentraciones urinarias de ETU en mujeres embarazadas que viven cerca de grandes plantaciones de banano, comparar su ingesta diaria estimada (EDI) con las dosis de referencia establecidas (RfD) e identificar factores que predicen sus concentraciones urinarias de ETU. Métodos: Inscribimos a 451 mujeres embarazadas del condado de Matina, Costa Rica, que tiene una producción bananera a gran escala. Visitamos a 445 mujeres hasta tres veces durante el embarazo para obtener muestras de orina (n = 872) e información sobre los factores que posiblemente influyen en la exposición. Determinamos las concentraciones urinarias de ETU mediante cromatografía líquida y espectrometría de masas. Resultados: Las concentraciones medias de ETU en orina de las mujeres embarazadas fueron más de cinco veces más altas que las informadas para otras poblaciones generales. Setenta y dos por ciento de las mujeres tenían EDI por encima de la RfD. Las mujeres que vivían más cerca (1er cuartil

Benchmark dose (BMD) modeling is increasingly used as the preferred approach to define the point-of-departure for health risk assessment of chemicals. As data are inherently variable, there is always a risk to select a model that defines a lower confidence bound of the BMD (BMDL) that, contrary to expected, exceeds the true BMD. The aim of this study was to investigate how often and under what circumstances such anomalies occur under current modeling practice. Continuous data were generated from a realistic dose–effect curve by Monte Carlo simulations using four dose groups and a set of five different dose placement scenarios, group sizes between 5 and 50 animals and coefficients of variations of 5–15%. The BMD calculations were conducted using nested exponential models, as most BMD software use nested approaches. “Non-protective” BMDLs (higher than true BMD) were frequently observed, in some scenarios reaching 80%. The phenomenon was mainly related to the selection of the non-sigmoidal exponential model (Effect = a · e b ·dose ). In conclusion, non-sigmoid models should be used with caution as it may underestimate the risk, illustrating that awareness of the model selection process and sound identification of the point-of-departure is vital for health risk assessment.

Different government agencies operating in the European Union regulate different types of chemical products but all require testing for carcinogenicity to support applications for product marketing and commercialisation. A conference was held in Brussels in 2013 where representatives of the pharmaceutical, animal health, chemical and plant protection industries, together with representatives of regulatory agencies, universities and other stakeholders, met under the auspices of The European Partnership for Alternative Approaches to Animal Testing (EPAA) to discuss the varying requirements for carcinogenicity testing, and how these studies might be refined to improve hazard evaluation and risk assessment while implementing principles of the 3Rs (replacement, refinement and reduction in animal studies). While there are some similarities, the regulatory approaches in pharmaceutical, animal health, chemical and plant protection sectors have varying degrees of flexibility in requirements for carcinogenicity testing, to an extent reflecting concerns over the magnitude and duration of human exposure, either directly as in therapeutic exposure to pharmaceuticals, or indirectly through the ingestion of residues of veterinary drugs or plant protection chemicals. The article discusses these differences and other considerations for modified carcinogenicity testing paradigms on the basis of scientific and 3Rs approaches.

The benchmark dose (BMD) approach is increasingly used as a preferred approach for dose-effect analysis, but standard experimental designs are generally not optimized for BMD analysis. The aim of this study was to evaluate how the use of unequally sized dose groups affects the quality of BMD estimates in toxicity testing, with special consideration of the total burden of animal distress. We generated continuous dose-effect data by Monte Carlo simulation using two dose-effect curves based on endpoints with different shape parameters. Eighty-five designs, each with four dose groups of unequal size, were examined in scenarios ranging from low- to high-dose placements and with a total number of animals set to 40, 80, or 200. For each simulation, a BMD value was estimated and compared with the "true" BMD. In general, redistribution of animals from higher to lower dose groups resulted in an improved precision of the calculated BMD value as long as dose placements were high enough to detect a significant trend in the dose-effect data with sufficient power. The improved BMD precision and the associated reduction of the number of animals exposed to the highest dose, where chemically induced distress is most likely to occur, are favorable for the reduction and refinement principles. The result thereby strengthen BMD-aligned design of experiments as a means for more accurate hazard characterization along with animal welfare improvements.

International audience; From the lectures presented at the 2nd International Workshop on Obesity and Environmental Contaminants, which was held in Uppsala, Sweden, on 8-9 October 2015, it became evident that the findings from numerous animal and epidemiological studies are consistent with the hypothesis that environmental contaminants could contribute to the global obesity epidemic. To increase awareness of this important issue among scientists, regulatory agencies, politicians, chemical industry management, and the general public, the authors summarize compelling scientific evidence that supports the hypothesis and discuss actions that could restrict the possible harmful effects of environmental contaminants on obesity.

Dose-effect evaluation is an increasingly important step of health risk assessment. The foreseen increase of in vitro methods argues for the development and evaluation of a clearly defined reference points for dose-effect modelling of in vitro data. In the present study, the traditional use of a concentration corresponding to 10% or 50% of the maximal effect (EC10 or EC50) is compared with a strategy, under which, a reference point (Benchmark dose, BMDT) is calculated that represents the dose where the slope of the dose-effect curve changes the most (per unit log-dose) in the low dose region. To illustrate the importance of the reference point, dose-effect data on CYP1A1 enzyme activity for 30 polychlorinated biphenyl (PCB) congeners were evaluated in order to calculate relative potencies, in relation to 2,3,7,8-TCDD, with confidence intervals (CIs). The present study shows that the interpretation of the results as potency and rank orders potentially depends on the choice and definition of the reference point (BMDT, EC10 or EC50). This is important as potency ranking may be used as a method for screening and prioritization, in research, in policy development or in pharmaceutical development. The use of the BMDT implies a focus on the change of structure in the parameter's dose–response rather than a particular percentage change in the response in such a parameter. In conclusion, the BMDT may be used as an alternative base for evaluation of dose-effect relationships in vitro. It offers an objective geometrical definition of a reference point in the low-dose region of the dose-effect curve. Copyright © 2012 John Wiley & Sons, Ltd.

Acute guidance values are tools for public health risk assessment and management during planning, preparedness and response related to sudden airborne release of hazardous chemicals. The two most frequently used values, i.e. Acute Exposure Guidance Levels (AEGL) and Emergency Response Planning Guideline (ERPG), were compared in qualitative and quantitative terms. There was no significant difference between the general level of AEGL and ERPG values, suggesting the two systems are equally precautious. However, the guidance values diverged by a factor of 3 or more for almost 40% of the substances, including many of high production volume. These deviations could be explained by differences in selection of critical effect or critical study and in a few cases differences in interpretation of the same critical study. Diverging guidance values may hamper proper risk communication and risk management. Key factors for broad international acceptance of harmonized values include transparency of the decision process, agreement on definition of toxicological tiers, and a target population including sensitive groups of the general population. In addition, development of purely health based values is encouraged. Risk management issues, such as land use and emergency response planning should be treated separately, as these rely on national legislation and considerations.

Objective International comparability of environmental tobacco smoke (ETS) exposure levels is difficult. This study assesses whether estimating children's exposure from information on adult smoking and exposure to ETS makes international comparisons more reliable. Methods The exposure among children was estimated using three different combinations (models) based on different sets of information on adult smoking, household composition or adult exposure to ETS at home in three cross-sectional nationally representative samples drawn from data sets from Estonia ( n = 2650), Finland ( n = 2829) and Latvia ( n = 5440) in the years 2002 and 2004. The first two models were based on adult smoking and the third also included ETS exposure. Results The parental smoking rate was similar to the general smoking prevalence. ETS exposure in non-smoking parents ranged from 22% in Finland to 60% in Latvia. All models gave rather comparative ranges except in Latvia, where the proportion of children with exposure varied from 67% with the simplest model to 81% with the most complex one. Conclusions Adult exposure at home or adult smoking prevalence, preferably among people with children, could be used as a proxy for children's exposure to ETS. It is recommended that population questionnaires include detailed information on exposure and household composition.

The dietary intake of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) in terms of toxic equivalents (TEQs) was investigated in Swedish children and young adults. Exposure was estimated from concentration data of six groups of individual food commodities (meat, fish, dairy products, egg, edible fats and other foodstuff) combined with food intake data from a 7-day record book obtained from 670 individuals aged 1-24 years. The results showed that Swedish boys and girls, up to the age of ten, had a median TEQ intake that exceeded the tolerable daily intake (TDI) of 2 pg TEQ/kg body weight. Children exceeding the TDI varied from almost all individuals among the youngest children to about 20% among young men and women. Dairy and fish products were the main sources of exposure for the average child, accounting for 59% of the total TEQ intake. The individuals most highly exposed were, on the other hand, characterized by a high consumption of fish. Since children constitute a vulnerable group, results obtained from the present study show that it is essential to perform age specific dietary intake assessments of pollutants and more carefully consider sensitive and/or highly exposed groups in the population in the risk management processes.

Reducing the number of laboratory animals used and refining experimental procedures to enhance animal welfare are fundamental questions to be considered in connection with animal experimentation. Here, we explored the use of cardinal ethical weights for clinical signs and symptoms in rodents by conducting trade-off interviews with members of Swedish Animal Ethics Committees in order to derive such weights for nine typical clinical signs of toxicity. The participants interviewed represent researchers, politically nominated political nominees and representatives of animal welfare organizations. We observed no statistically significant differences between these groups with respect to the magnitude of the ethical weights assigned, though the political nominees tended to assign lower weights. Overall, hunched posture was considered the most severe clinical sign and body weight loss the least severe. The ethical weights assigned varied considerably between individuals, from zero to infinite value, indicating discrepancies in prioritization of reduction and refinement. Cardinal ethical weights may be utilized to include both animal welfare refinement and reduction of animal use in designing as well as in retrospective assessment of animal experiments. Such weights may also be used to estimate ethical costs of animal experiments.

Background: Asthmatic individuals constitute a large sub-population that is often considered particularly susceptible to the deleterious effects of inhalation of airborne chemicals. However, for most such chemicals information on asthmatics is lacking and inter-individual assessment factors (AFs) of 3–25 have been proposed for use in the derivation of health-based guideline values. Objective: To evaluate available information in attempt to determine whether a general difference in airway response during short-term exposure between healthy and asthmatic individuals can be identified, and whether current AFs for inter-individual variability provide sufficient protection for asthmatics. Methods: After performing systematic review of relevant documents and the scientific literature estimated differential response factors (EDRF) were derived as the ratio between the lowest observed adverse effect levels for healthy and asthmatic subjects based on studies in which both groups were tested under the same conditions. Thereafter, the concentration–response relationships for healthy and asthmatic subjects exposed separately to four extensively tested chemicals (nitrogen dioxide, ozone, sulfuric acid, sulfur dioxide) were compared on the basis of combined data. Finally, a Benchmark Concentration (BMC) analysis was performed for sulfur dioxide. Results: We found evidence of higher sensitivity among asthmatics (EDRF > 1) to 8 of 19 tested chemicals, and to 3 of 11 mixtures. Thereafter, we confirmed the higher sensitivity of asthmatics to sulfuric acid and sulfur dioxide. No difference was observed in the case of ozone and nitrogen dioxide. Finally, our BMC analysis of sulfur dioxide indicated a ninefold higher sensitivity among asthmatics. Conclusion: Although experimental data are often inconclusive, our analyses suggest that an AF of 10 is adequate to protect asthmatics from the deleterious respiratory effects of airborne chemicals.

Baltic herring (Clupea harengus) oil was extracted and fractionated. To examine the contribution to toxicity and biological effects of different halogenated organic pollutants, the herring oil and the fractions were mixed into pelleted food and given to Sprague-Dawley female rats at three levels, corresponding to a human intake of 1.6, 8.2 and 34.4 kg fish per week. Herring oil, its fractions, as well as liver tissues from exposed rats, were analyzed for: eight chlorinated biphenyls, all 2,3,7,8-substituted chlorinated dibenzo-p-dioxins and dibenzofurans, hexachlorocyclohexanes, hexachlorobenzene, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), DDT-metabolites, three brominated diphenylethers as well as extractable organically bound chlorine and halogenated fatty acids. A bioassay (EROD) was used for measuring the dioxin-like enzyme induction activity. Nordic Sea lodda (Mallotus villosus) oil was used as a nutritionally equivalent control, with much lower levels of halogenated organic pollutants. A full toxicological subchronic examination is reported in the following paper (Stern et al. 2002). In this study, we report that the fractionation procedure resulted in a substantial reduction of most of the pollutants in the triacylglycerol fraction, and a pronounced enrichment of most of the pollutants into the two other fractions. However, all contaminants were present at some levels in all of the fractions. The concentrations of organohalogens found in this study were representative for Baltic herring during the mid-1990s. Rat liver tissue showed similar residue patterns as the diet, with the exception of chlorinated dibenzo-p-dioxin and dibenzofuran congeners that had a higher liver retention than pesticides, chlorinated biphenyls and brominated diphenylethers.

This study aimed to increase the knowledge about the toxicity of fish-derived organohalogen pollutants in mammals. The strategy chosen was to separate organohalogen pollutants derived from Baltic herring (Clupea harengus) fillet, in order to obtain fractions with differing proportions of identified and unidentified halogenated pollutants, and to perform a subchronic toxicity study in rats, essentially according to the OECD guidelines, at three dose levels. Nordic Sea lodda (Mallotus villosus) oil, with low levels of persistent organohalogen pollutants, was used as an additional control diet. The toxicological examination showed that exposure to Baltic herring oil and its fractions at dose levels corresponding to a human intake in the range of 1.6 to 34.4 kg Baltic herring per week resulted in minimal effects. The spectrum of effects was similar to that, which is observed after low-level exposure to pollutants such as chlorinated dibenzo-p-dioxins and dibenzofurans (CDD/F) and chlorinated biphenyls, despite the fact that these contaminants contribute to a minor part of the extractable organically bound chlorine (EOCl). The study confirmed previous findings that induction of hepatic ethoxyresorufin deethylase (EROD) activity takes place at daily intake levels 0.15 ng fish-derived CDD/F-TEQs/kg body weight. The study also demonstrated that hepatic vitamin A reduction takes place at somewhat higher daily exposure levels, i.e. 0.16–0.30 ng fish-derived CDD/F-TEQs/kg body weight. Halogenated fatty acids, the major component of EOCl, could not be linked to any of the measured effects. From a risk management point of view, the study provides important new information of effect levels for Ah-receptor mediated responses following low level exposure to organohalogen compounds from a matrix relevant for human exposure.

Increasingly, dose-response data are being evaluated with the benchmark dose (BMD) approach rather than by the less precise no-observed-adverse-effect-level (NOAEL) approach. However, the basis for designing animal experiments, using equally sized dose groups, is still primed for the NOAEL approach. The major objective here was to assess the impact of using dose groups of unequal size on both the quality of the BMD and overall animal distress. We examined study designs with a total number of 200 animals distributed in four dose groups employing quantal data generated by Monte Carlo simulations. Placing more animals at doses close to the targeted BMD provided an estimate of BMD that was slightly better than the standard design with equally sized dose groups. In situations involving a clear dose-response, this translates into fewer animals receiving high doses and thus less overall animal distress. Accordingly, in connection with risk and safety assessment, animal distress can potentially be reduced by distributing the animals appropriately between dose groups without decreasing the quality of the information obtained.

The polychlorinated biphenyl (PCB) mixture Aroclor 1254 alters bone tissue properties. However, the mechanisms responsible for the observed effects have not yet been clarified. This study compared the effect of Aroclor 1254 on the expression of osteoblast differentiation markers in MC3T3-E1 cells with the corresponding effect of the dioxin reference compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and two PCB congeners belonging to the category of non-dioxin-like PCBs. The aim of the study was to quantify the relative influence of dioxin-like and non-dioxin-like PCB-components on osteoblast differentiation. Expression of marker genes for AhR activity and osteoblast differentiation were analyzed, and relative potency (REP) values were derived from Benchmark concentration-effect curves. Expression of alkaline phosphatase and osteocalcin were decreased by both Aroclor 1254 and TCDD exposure, while the PCB-congeners PCB19 and PCB52 slightly induced the expression. The relative potency of Aroclor 1254 for inhibitory effects on osteoblast differentiation marker genes was within the expected range as estimated from the chemical composition of Aroclor 1254. These results are consistent with previously observed bone modulations following in vivo exposure to Aroclor 1254 and TCDD, and demonstrate that the inhibitory effects of Aroclor 1254 on osteoblast differentiation by the dioxin-like constituents are over-riding the contribution of non-dioxin-like PCBs.

Guideline values for emergency planning and response are aimed to protect the general public, including asthmatics and other susceptible groups, during sudden airborne releases of chemicals. A precondition of asthma may increase the individual susceptibility to acute exposures. This paper studies to what extent experimental data on asthmatics are included in the rationale and derivation of guideline values. An analysis of the Technical Support Documents (TSDs) of the Acute Exposure Guideline Levels (AEGLs) shows that only 23 of the 176 TSDs include references to experimental studies on asthmatics, 30 include a statement on asthmatics but no reference to experimental data, and 123 lack any explicit statement on asthmatics. The TSDs were further compared with the support documents of nine other programs for acute or occupational short-term values. All programs were incomplete with respect to experimental data on asthmatics. Omission of asthmatics may interfere with trustful and efficient health protective actions. We suggest that the availability of data on asthmatics should be carefully examined in the development of guideline values, and that the lack of such data should be explicitly noted. In the latter case, available data for other irritants may be used to justify an appropriate assessment factor.

The US Environmental Protection Agency voluntary phased-out residential use of chlorpyrifos in 2001. In contrast, in Costa Rica, chlorpyrifos-treated bags are increasingly used to protect banana and plantain fruits from insects and to fulfill product standards, even in populated areas. Objectives: To evaluate children’s exposure to chlorpyrifos in villages situated nearby banana plantations and plantain farms in Costa Rica. Methods: The study targeted two villages with use of chlorpyrifos-treated bags in nearby banana plantations and plantain farms and one village with mainly organic production. For 140 children from these villages, mostly indigenous Ngabe and Bribri, parent-interviews and urine samples ( ¨ n¼207) were obtained. Urinary 3,5,6-trichloro-2-pyridinol (TCPy) levels were measured as a biomarker for chlorpyrifos exposure. In the banana and plantain village also environmental contamination to chlorpyrifos was explored. Results: Children from the banana and plantain villages had statistically significant higher urinary TCPy concentrations than children from the referent village; 2.6 and 2.2 versus 1.3 mg/g creatinine, respectively. Chlorpyrifos was detected in 30% of the environmental samples as well as in 92% of the hand/foot wash samples. For more than half of the children their estimated intake exceeded the US EPA chronic population adjusted dose. For some, the acute population adjusted dose and the chronic reference dose were also exceeded. Conclusions: Our results suggest that children living nearby plantations with chlorpyrifos-treated bags are exposed to chlorpyrifos levels that may affect their health. Interventions to reduce chlorpyrifos exposure Antecedentes La Agencia de Protección Ambiental de los Estados Unidos suprimió voluntariamente el uso residencial del clorpirifos en 2001. En cambio, en Costa Rica, las bolsas tratadas con clorpirifos se utilizan cada vez más para proteger las frutas de banano y plátano de los insectos y para cumplir las normas de producto, incluso en zonas pobladas. Objetivos Evaluar la exposición de los niños al clorpirifos en las aldeas situadas cerca de las plantaciones de bananas y de plátanos en Costa Rica. Métodos El estudio se centró en dos aldeas que utilizaban bolsas tratadas con clorpirifos en plantaciones de bananas y plátanos cercanas y una aldea con producción principalmente orgánica. Para 140 niños de estas aldeas, en su mayoría indígenas Ngäbe y Bribri, se obtuvieron entrevistas con los padres y muestras de orina (n=207). Se midieron los niveles urinarios de 3,5,6-tricloro-2-piridinol (TCPy) como biomarcador de la exposición al clorpirifos. En la aldea de bananas y plátanos también se exploró la contaminación ambiental por clorpirifos. Resultados Los niños de las aldeas de bananas y plátanos tenían concentraciones de TCPy urinario más altas estadísticamente significativas que los niños de la aldea de referencia; 2,6 y 2,2 frente a 1,3 μg/g creatinina, respectivamente. Se detectó clorpirifos en el 30% de las muestras ambientales y en el 92% de las muestras de lavado de manos y pies. En más de la mitad de los niños su ingesta estimada superó la dosis ajustada de la población crónica de la EPA de los EE.UU. Para algunos, la dosis ajustada para la población aguda y la dosis de referencia crónica también fueron excedidas. Conclusiones Nuestros resultados sugieren que los niños que viven cerca de las plantaciones con bolsas tratadas con clorpirifos están expuestos a niveles de clorpirifos que pueden afectar a su salud. Las intervenciones para reducir la exposición al clorpirifos probablemente mejoren la salud de los niños y el medio ambiente en las regiones de cultivo de bananas y plátanos. Instituto Regional de Estudios en Sustancias Tóxicas, Universidad Nacional, Costa Rica Wageningen University, the Netherlands University of California at Berkeley, USA Karolinska Institute, Stockholm, Sweden University of Quebec at Montreal, Canada Lund University Hospital, Lund, Sweden Universidad Nacional, Costa Rica Technology and Agrarian Development Group, Wageningen University, the Netherlands University of California at Berkeley, USA Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden Center for Interdisciplinary Research in Biology, Health, Environment and Society (CINBIOSE), University of Quebec at Montreal, Canada Department of Occupational and Environmental Medicine, Lund University Hospital, Lund, Sweden

Dose-effect evaluation is an increasingly important step of health risk assessment. The foreseen increase of in vitro methods argues for the development and evaluation of a clearly defined reference points for dose-effect modelling of in vitro data. In the present study, the traditional use of a concentration corresponding to 10% or 50% of the maximal effect (EC₁₀ or EC₅₀) is compared with a strategy, under which, a reference point (Benchmark dose, BMD(T) ) is calculated that represents the dose where the slope of the dose-effect curve changes the most (per unit log-dose) in the low dose region. To illustrate the importance of the reference point, dose-effect data on CYP1A1 enzyme activity for 30 polychlorinated biphenyl (PCB) congeners were evaluated in order to calculate relative potencies, in relation to 2,3,7,8-TCDD, with confidence intervals (CIs). The present study shows that the interpretation of the results as potency and rank orders potentially depends on the choice and definition of the reference point (BMD(T) , EC₁₀ or EC₅₀). This is important as potency ranking may be used as a method for screening and prioritization, in research, in policy development or in pharmaceutical development. The use of the BMD(T) implies a focus on the change of structure in the parameter's dose-response rather than a particular percentage change in the response in such a parameter. In conclusion, the BMD(T) may be used as an alternative base for evaluation of dose-effect relationships in vitro. It offers an objective geometrical definition of a reference point in the low-dose region of the dose-effect curve.

Summary Background Exposure to second-hand smoke is common in many countries but the magnitude of the problem worldwide is poorly described. We aimed to estimate the worldwide exposure to second-hand smoke and its burden of disease in children and adult non-smokers in 2004. Methods The burden of disease from second-hand smoke was estimated as deaths and disability-adjusted life-years (DALYs) for children and adult non-smokers. The calculations were based on disease-specific relative risk estimates and area-specific estimates of the proportion of people exposed to second-hand smoke, by comparative risk assessment methods, with data from 192 countries during 2004. Findings Worldwide, 40% of children, 33% of male non-smokers, and 35% of female non-smokers were exposed to second-hand smoke in 2004. This exposure was estimated to have caused 379 000 deaths from ischaemic heart disease, 165 000 from lower respiratory infections, 36 900 from asthma, and 21 400 from lung cancer. 603 000 deaths were attributable to second-hand smoke in 2004, which was about 1·0% of worldwide mortality. 47% of deaths from second-hand smoke occurred in women, 28% in children, and 26% in men. DALYs lost because of exposure to second-hand smoke amounted to 10·9 million, which was about 0·7% of total worldwide burden of diseases in DALYs in 2004. 61% of DALYs were in children. The largest disease burdens were from lower respiratory infections in children younger than 5 years (5 939 000), ischaemic heart disease in adults (2 836 000), and asthma in adults (1 246 000) and children (651 000). Interpretation These estimates of worldwide burden of disease attributable to second-hand smoke suggest that substantial health gains could be made by extending effective public health and clinical interventions to reduce passive smoking worldwide. Funding Swedish National Board of Health and Welfare and Bloomberg Philanthropies.

The subacute toxicity of a commercial polybrominated diphenyl ether (PBDE) preparation, Bromkal 70-5DE, was investigated. In addition to a vehicle control, the mixture was given orally to male and female Sprague-Dawley rats for 28 d at three dose levels; 2.5, 25 and 250 mg kg(-1) b.w.d(-1). The observed effects include increased hepatic EROD activity (from 2.5 mg kg(-1)d(-1)); increased liver weight (males), increased PROD activity and depletion of hepatic retinoids (from 25 mg kg(-1)d(-1)); and increased liver weight (females), marked histological changes in the liver and lungs, as well as increased serum parameters such as total protein, cholesterol and albumin (from 250 mg kg(-1)d(-1)). Chemical analysis of the PBDE mixture with gas chromatography/mass spectrometry (GS/MS) showed impurities of polybrominated dibenzofurans and to a lesser extent dibenzodioxins, in total levels of about 7.0 microg g(-1) of Bromkal technical mixture. The animals were thereby exposed to an estimated dose of dioxin-like equivalents corresponding to 1.3-131 ng TEQ kg(-1) b.w.d(-1). It cannot be ruled out that this level of impurities can explain the hepatic EROD induction and hepatic retinoid depletion, which are considered typical markers of toxicity mediated via the aryl hydrocarbon receptor (AhR).

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.

This study was done to generate kinetic data on individual congeners of chlorinated biphenyls in the low dose range, which could be of value in the risk assessment procedure. Male Sprague-Dawley rats were given a single oral dose of a mixture of polychlorinated biphenyls (CBs) containing either CBs 105, 118, 138, 153, 156, 157, 170, and 180 (A-mix) or CBs 28, 52, 77, 87, and 101 (B-mix). Liver, serum, and adipose tissue were collected after 6 h up to 135 days, from rats given the A-mix, and after 6 h up to 4 days from rats given the B-mix. CB concentrations were measured in liver, serum, and adipose tissue. In addition, this study provides kinetic data of one of the major CB metabolites, 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). The low doses used resulted in serum CB concentrations similar to human background serum concentrations. In the A-mix experiment all CBs show high initial liver and serum concentrations followed by redistribution into adipose tissue. Differences between congeners were correlated to molecular weight. High molecular weight correlated to lower uptake and slower redistribution. During dynamic steady-state the tissue concentrations decreased with a calculated first order rate between 54-129 days for halving the concentrations (half-life). Most of the decrease in concentration was explained by the growth-related increase of tissue masses in general and adipose tissue in particular. In the B-mix experiment, the concentrations of CBs in adipose tissue decreased with between 25 and 59% from day 1 to day 4. These results show that the B-mix congeners, given at low dose, have longer half-lives than previously reported in high dose studies. Partition coefficients between body compartments are reported and for the first time a high and congener specific liver-to-serum ratio of CB 77 is observed.

Multivariate modelling of polychlorinated biphenyl-induced CYP1A activity in the MH1C1 rat hepatoma cell line