Christoph van Thriel

Translating neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts

The developing nervous system is particularly vulnerable to chemical insults. Exposure to chemicals can result in neurobehavioural alterations, and these have been used as sensitive readouts to assess neurotoxicity in animals and man. Deconstructing neurobehaviour into relevant cellular and molecular components may allow for detection of specific neurotoxic effects in cell-based systems, which in turn may allow an easier examination of neurotoxic pathways and modes of actions and eventually inform the regulatory assessment of chemicals with potential developmental neurotoxicity. Here, current developments towards these goals are reviewed. Imaging genetics (CB) provides new insights into the neurobiological correlates of cognitive function that are being used to delineate neurotoxic mechanisms. The gaps between in vivo neurobehaviour and real-time in vitro measurements of neuronal function are being bridged by ex vivo measurements of synaptic plasticity (RW). An example of solvent neurotoxicity demonstrates how an in vivo neurological defect can be linked via the N-methyl-d-aspartate (NMDA)-glutamate receptor as a common target to in vitro readouts (AB). Axonal and dendritic morphology in vitro proved to be good correlates of neuronal connectivity and neurobehaviour in animals exposed to polychlorinated biphenyls and organophosphorus pesticides (PJL). Similarly, chemically induced changes in neuronal morphology affected the formation of neuronal networks on structured surfaces. Such network formation may become an important readout for developmental neurotoxicity in vitro (CvT), especially when combined with human neurons derived from embryonic stem cells (ML). We envision that future in vitro test systems for developmental neurotoxicity will combine the above approaches with exposure information, and we suggest a strategy for test system development and cell-based risk assessment.

Sensory irritation as a basis for setting occupational exposure limits

There is a need of guidance on how local irritancy data should be incorporated into risk assessment procedures, particularly with respect to the derivation of occupational exposure limits (OELs). Therefore, a board of experts from German committees in charge of the derivation of OELs discussed the major challenges of this particular end point for regulatory toxicology. As a result, this overview deals with the question of integrating results of local toxicity at the eyes and the upper respiratory tract (URT). Part 1 describes the morphology and physiology of the relevant target sites, i.e., the outer eye, nasal cavity, and larynx/pharynx in humans. Special emphasis is placed on sensory innervation, species differences between humans and rodents, and possible effects of obnoxious odor in humans. Based on this physiological basis, Part 2 describes a conceptual model for the causation of adverse health effects at these targets that is composed of two pathways. The first, “sensory irritation” pathway is initiated by the interaction of local irritants with receptors of the nervous system (e.g., trigeminal nerve endings) and a downstream cascade of reflexes and defense mechanisms (e.g., eyeblinks, coughing). While the first stages of this pathway are thought to be completely reversible, high or prolonged exposure can lead to neurogenic inflammation and subsequently tissue damage. The second, “tissue irritation” pathway starts with the interaction of the local irritant with the epithelial cell layers of the eyes and the URT. Adaptive changes are the first response on that pathway followed by inflammation and irreversible damages. Regardless of these initial steps, at high concentrations and prolonged exposures, the two pathways converge to the adverse effect of morphologically and biochemically ascertainable changes. Experimental exposure studies with human volunteers provide the empirical basis for effects along the sensory irritation pathway and thus, “sensory NOAEChuman” can be derived. In contrast, inhalation studies with rodents investigate the second pathway that yields an “irritative NOAECanimal.” Usually the data for both pathways is not available and extrapolation across species is necessary. Part 3 comprises an empirical approach for the derivation of a default factor for interspecies differences. Therefore, from those substances under discussion in German scientific and regulatory bodies, 19 substances were identified known to be human irritants with available human and animal data. The evaluation started with three substances: ethyl acrylate, formaldehyde, and methyl methacrylate. For these substances, appropriate chronic animal and a controlled human exposure studies were available. The comparison of the sensory NOAEChuman with the irritative NOAECanimal (chronic) resulted in an interspecies extrapolation factor (iEF) of 3 for extrapolating animal data concerning local sensory irritating effects. The adequacy of this iEF was confirmed by its application to additional substances with lower data density (acetaldehyde, ammonia, n-butyl acetate, hydrogen sulfide, and 2-ethylhexanol). Thus, extrapolating from animal studies, an iEF of 3 should be applied for local sensory irritants without reliable human data, unless individual data argue for a substance-specific approach.

The impact of solvent mixtures on neurobehavioral performance: conclusions from epidemiological data.

The review of epidemiological studies investigating the neurobehavioral effects of occupational exposure to solvent mixtures sought to contribute to the following issues: (1) Identification of affected cognitive and motor functions. (2) Identification of sensitive neuropsychological tests. (3) Analysis of exposure-effect relationships. The approach was based on the meta-analytical method of effect size estimates. Fifty-three groups from occupational studies were included in the meta-analysis. Forty-eight neuropsychological performance variables could be analyzed as they were included in at least three studies. Seventeen articles provided detailed information on the constituents of mixtures, thereby enabling the computation of an exposure index that allowed the comparison of different mixtures. Significant negative effect sizes were obtained for 12 test variables measuring attention, memory, motor performance and constructional abilities. The greatest proportion of lower performance scores in the exposed groups was shown by different tests of attention: significant effect sizes between d=-0.16 and -0.46 were calculated. Tests of cognitive processing speed, response alternation and inhibition seemed to be sensitive tools for the detection of poorer performance. Exposure-effect relationships were mainly characterized by inconsistent patterns. Crude and inappropriately calculated exposure measures were blamed for this outcome. A healthy worker effect was suggested more consistently: studies examining groups with longer exposure duration obtained smaller effect sizes. Indications of confounding were observed; however, they did not seem sufficient to question consistent effect size patterns. Paying greater attention to the measurement of exposure and including measures of confounding is advisable for future studies and would enhance the explanatory power of cross-sectional studies and meta-analyses.

Putative adverse outcome pathways relevant to neurotoxicity

Abstract The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

Chronic solvent-induced encephalopathy: European consensus of neuropsychological characteristics, assessment, and guidelines for diagnostics

INTRODUCTION The presence of neuropsychological impairment is a hallmark of chronic solvent-induced encephalopathy (CSE), and using clinical neuropsychological procedures to generate a valid assessment of the condition is crucial for its diagnosis. The goals of this consensus document are to provide updated knowledge of the neuropsychological characteristics of CSE and to provide internationally acceptable guidelines for using neuropsychological assessments in the process of diagnosing patients who are suspected of having CSE. MATERIALS AND METHODS A European working group that was composed of experts in the field of the clinical diagnosis of CSE met at several round-table meetings and prepared this report. The first section of the consensus paper addresses a review of the relevant literature that was published between 1985 and March 2012. The second section addresses recommendations for the clinical neuropsychological assessment of patients who are suspected of having CSE. RESULTS The literature review indicates that the most common neuropsychological impairments in CSE patients are within the domains of attention, particularly the speed of information processing, memory, and motor performance. It appears that the influence of CSE on memory processes mainly involves immediate recall and generally involves verbal, visual and visuospatial material. In the second section, six recommendations are presented regarding important functional domains for the neuropsychological diagnostic process of CSE that relate to the evaluation of neuropsychological impairment, the assessment and evaluation of symptoms, differential diagnostic considerations, the reliability and validity of neuropsychological test results, and the retesting of patients. DISCUSSION AND CONCLUSIONS These recommendations will contribute to the improvement of the process for accurately diagnosing CSE, better counselling for CSE patients, the comparability of epidemiological data between countries, and finally, by raising awareness, these recommendations will contribute to combating the adverse health effects of occupational exposure to solvents.

Eye blinks as indicator for sensory irritation during constant and peak exposures to 2-ethylhexanol.

Two experiments were performed to re-evaluate the sensory irritating properties of 2-ethylhexanol in relation to dose and time and to examine the usability of electromyographic eye blink recordings as indicator of sensory irritation. Mean exposure levels of 1.5, 10 and 20ppm were realized in experimental models simulating either constant or variable 4h exposure. Each study was carried out with two subject samples, healthy young men with self-reported multiple chemical sensitivity (sMCS) and age matched controls. Although 2-ethylhexanol exposure was below the occupational threshold limit value of 50ppm, the study revealed strong dose-response relationships between airborne solvent concentrations and blink rates. During 40ppm peak exposures the blink rate increased threefold. In the course of 4h, exposure blink rates increased significantly showing no adaptation. Subjects with sMCS revealed, with one exception at start of exposure, no significantly higher blink rates than controls. The results indicate that the irritative potential of 2-ethylhexanol is higher than commonly expected. In both exposure scenarios with either constant or peak exposures, electromyographic eye blink recordings were an appropriate method for the examination of acute sensory irritations in time.

Performance alterations associated with occupational exposure to manganese—A meta-analysis

The review aimed at quantifying the evidence of performance effects resulting from occupational exposure to manganese. Epidemiological studies published between 1987 and 2008 were analyzed. The approach was based on the meta-analytical method of effect size estimates and sought to contribute to the following issues: (1) identification of the affected functions; (2) identification of sensitive neuropsychological tests; (3) analyses of exposure-effect relationships. Thirteen studies examining 958 exposed and 815 unexposed workers were included in the meta-analysis. Mean concentrations of inhalable manganese ranged from 0.05 to 1.59 mg/m(3), mean concentrations of manganese in whole blood ranged from 8.1 to 48.4 microg/L. Nineteen neuropsychological performance variables were analyzed as they were included in at least three of the identified studies. Apart from two outcomes, the overall effects displayed a negative impact of manganese on performance. Significant overall effects were obtained for six test variables; their size ranged from d=-0.23 to -0.36. Four of the variables measured motor speed and two of them speed of information processing. The analysis of exposure-effect relationships showed that larger effect sizes were more consistently associated with higher concentrations of inhalable manganese than with manganese in blood. The evidence of cognitive and motor performance effects is in accordance with the knowledge about accumulation of manganese in the basal ganglia and the effect of manganese on the neurotransmitter dopamine. Inconsistencies in the relationship between effect sizes and the biomarker manganese in blood were discussed in the context of results indicating that the biomarker might not be sufficiently meaningful for the neurobehavioral alterations. Simple motor performance tests with a distinct speed component seem to be highly recommendable for further studies, because they seem to be appropriate for measuring manganese-related changes, seem to provide homogenous results and their outcomes show consistent relations to exposure. The rigorous quantitative approach was especially appropriate for revealing exposure-effect relationships, but information about individual cumulative exposure would enhance the potential for risk assessment of manganese.

An integrative approach considering acute symptoms and intensity ratings of chemosensory sensations during experimental exposures.

Even at low concentrations airborne chemicals can excite olfactory and trigeminal receptors and inform the organism about the presence of airborne chemicals. Acutely, malodors or sensory irritations might trigger symptoms (e.g., bad air quality, eye irritations) and in the long-run functional impairment of chemoreception might occur. In humans, knowledge about short-term adaptational processes and effects of exposure peaks on these systems is limited. Therefore, two different experiments with human volunteers were conducted. In experiment A exposures (4h) with fluctuating concentrations of 2-ethylhexanol (1.5, 10, 20ppm C(TWA)) were investigated, experiment B used similar but constant vapor concentrations. Olfactory- and trigeminal-mediated symptoms and intensities of odor, eye, and nasal irritations were recorded. All measures showed a dose-dependent response and peak exposure effects. In the course of the 4h exposures solely olfactory symptoms decreased. Nasal irritations remained nearly unchanged across the 4h, eye irritations slightly increased. Inter-individual differences related to the personality trait of self-reported chemical sensitivity had only minor effects on chemosensory symptoms in experiment B and no effect on intensity ratings in both experiments. Chemosensory effects seem to be amplified by exposure peaks and less adaptive than assumed.

Neurobehavioural test results and exposure to inorganic mercury: in search of dose-response relations

The aim of the analysis was, in general, to find a way to summarise results of studies in search of exposure-response relationships and, in particular, to ask whether an exposure-response relationship can be ascertained for neurobehavioural studies on occupational mercury exposure. Eighteen studies dealing with human mercury exposure and examining 1,106 exposed and 1,105 control subjects were included in the analysis. Effect sizes were calculated for each of the single neuropsychological test results on cognitive and motor performance and were considered in relation to mean current concentrations of exposure. The total of effect sizes demonstrated a correlation to exposure in the range of r=0.50. Additional analyses showed that the influence of mercury on psychological functions was different and that results on motor performance compared with memory and attention revealed the greatest impairment in mercury-exposed workers. Implications for the reversibility of impairments could be discussed because three of the studies examined subjects whose exposure had ceased. Besides the usefulness of the approach, it became obvious that the available 18 studies are a small sample, even when non-dimensional effect-sizes are used; hence, not all psychological domains covered by tests could be analysed.

Adverse outcome pathways: opportunities, limitations and open questions

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.