Reprint of "CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals".

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritation is a regulatory requirement in safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as stand-alone assays. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed to assess the reliability of eight in vitro test methods and computational models as well as establishing an optimal tiered-testing strategy. For three computational models (Toxtree, and Case Ultra EYE_DRAIZE and EYE_IRR) performance parameters were calculated. Coverage ranged from 15 to 58%. Coverage was 2 to 3.4 times higher for liquids than for solids. The lowest number of false positives (5%) was reached with EYE_IRR; this model however also gave a high number of false negatives (46%). The lowest number of false negatives (25%) was seen with Toxtree; for liquids Toxtree predicted the lowest number of false negatives (11%), for solids EYE_DRAIZE did (17%). It can be concluded that the training sets should be enlarged with high quality data. The tested models are not yet sufficiently powerful for stand-alone evaluations, but that they can surely become of value in an integrated weight-of-evidence approach in hazard assessment.

CON4EI: Slug Mucosal Irritation (SMI) test method for hazard identification and labelling of serious eye damaging and eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement in the safety evaluation of industrial and consumer products. Although many in vitro ocular irritation assays exist, alone they are incapable of fully categorizing chemicals. The objective of CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for eye irritation assessment that can lead to complete replacement of the in vivo Draize rabbit eye test (OECD TG 405). A set of 80 reference chemicals was tested with seven test methods, one method was the Slug Mucosal Irritation (SMI) test method. The method measures the amount of mucus produced (MP) during a single 1-hour contact with a 1% and 10% dilution of the chemical. Based on the total MP, a classification (Cat 1, Cat 2, or No Cat) is predicted. The SMI test method correctly identified 65.8% of the Cat 1 chemicals with a specificity of 90.5% (low over-prediction rate for in vivo Cat 2 and No Cat chemicals). Mispredictions were predominantly unidirectional towards lower classifications with 26.7% of the liquids and 40% of the solids being underpredicted. In general, the performance was better for liquids than for solids with respectively 76.5% vs 57.1% (Cat 1), 61.5% vs 50% (Cat 2), and 87.5% vs 85.7% (No Cat) being identified correctly.

CON4EI: Short Time Exposure (STE) test method for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement in the safety evaluation of industrial and consumer products. Although many in vitro ocular irritation assays exist, alone they are incapable of fully categorizing chemicals. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium was developed to assess the reliability of eight in vitro test methods and establish an optimal tiered-testing strategy. One assay selected was the Short Time Exposure (STE) assay. This assay measures the viability of SIRC rabbit corneal cells after 5min exposure to 5% and 0.05% solutions of test material, and is capable of categorizing of Category 1 and No Category chemicals. The accuracy of the STE test method to identify Cat 1 chemicals was 61.3% with 23.7% sensitivity and 95.2% specificity. If non-soluble chemicals and unqualified results were excluded, the performance to identify Cat 1 chemicals remained similar (accuracy 62.2% with 22.7% sensitivity and 100% specificity). The accuracy of the STE test method to identify No Cat chemicals was 72.5% with 66.2% sensitivity and 100% specificity. Excluding highly volatile chemicals, non-surfactant solids and non-qualified results resulted in an important improvement of the performance of the STE test method (accuracy 96.2% with 81.8% sensitivity and 100% specificity). Furthermore, it seems that solids are more difficult to test in the STE, 71.4% of the solids resulted in unqualified results (solubility issues and/or high variation between independent runs) whereas for liquids 13.2% of the results were not qualified, supporting the restriction of the test method regarding the testing of solids.

CON4EI: EpiOcular™ Eye Irritation Test (EpiOcular™ EIT) for hazard identification and labelling of eye irritating chemicals.

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI project was to develop tiered testing strategies for eye irritation assessment. A set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different methods. Here, the results obtained with the EpiOcular™ Eye Irritation Test (EIT), adopted as OECD TG 492, are shown. The primary aim of this study was to evaluate of the performance of the test method to discriminate between chemicals not requiring classification for serious eye damage/eye irritancy (No Category) and chemicals requiring classification and labelling. In addition, the predictive capacity in terms of in vivo drivers of classification (i.e. corneal opacity, conjunctival redness and persistence at day 21) was investigated. EpiOcular™ EIT achieved a sensitivity of 97%, a specificity of 87% and accuracy of 95% and also confirmed its excellent reproducibility (100%) from the original validation. The assay was applicable to all chemical categories tested in this project and its performance was not limited to the particular driver of the classification. In addition to the existing prediction model for dichotomous categorization, a new prediction model for Cat 1 is suggested.

CON4EI: Development of testing strategies for hazard identification and labelling for serious eye damage and eye irritation of chemicals.

Assessment of acute eye irritation potential is part of the international regulatory requirements for safety testing of chemicals. In the last decades, many efforts have been made in the search for alternative methods to replace the regulatory in vivo Draize rabbit eye test (OECD TG 405). Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. The main objective of the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for serious eye damage and eye irritation assessment that can lead to complete replacement of OECD TG 405. A set of 80 reference chemicals (e.g. balanced by important driver of classification and physical state), was tested with seven test methods. Based on the results of this project, three different strategies were suggested. We have provided a standalone (EpiOcular ET-50), a two-tiered and three-tiered strategy, that can be used to distinguish between Cat 1 and Cat 2 chemicals and chemicals that do not require classification (No Cat). The two-tiered and three-tiered strategies use an RhCE test method (EpiOcular EIT or SkinEthic™ EIT) at the bottom (identification No Cat) in combination with the BCOP LLBO (two-tiered strategy) or BCOP OP-KIT and SMI (three-tiered strategy) at the top (identification Cat 1). For our proposed strategies, 71.1% - 82.9% Cat 1, 64.2% - 68.5% Cat 2 and ≥80% No Cat chemicals were correctly identified. Also, similar results were obtained for the Top-Down and Bottom-Up approach.

CON4EI: SkinEthic™ Human Corneal Epithelium Eye Irritation Test (SkinEthic™ HCE EIT) for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritancy is an international regulatory requirement and a necessary step in the safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as a stand-alone assay. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed with the goal of assessing the reliability of eight in vitro/alternative test methods as well as establishing an optimal tiered-testing strategy. One of the in vitro assays selected was the validated SkinEthic™ Human Corneal Epithelium Eye Irritation Test method (SkinEthic™ HCE EIT). The SkinEthic™ HCE EIT has already demonstrated its capacity to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage (No Category). The goal of this study was to evaluate the performance of the SkinEthic™ HCE EIT test method in terms of the important in vivo drivers of classification. For the performance with respect to the drivers all in vivo Cat 1 and No Cat chemicals were 100% correctly identified. For Cat 2 chemicals the liquids and the solids had a sensitivity of 100% and 85.7%, respectively. For the SkinEthic™ HCE EIT test method, 100% concordance in predictions (No Cat versus No prediction can be made) between the two participating laboratories was obtained. The accuracy of the SkinEthic™ HCE EIT was 97.5% with 100% sensitivity and 96.9% specificity. The SkinEthic™ HCE EIT confirms its excellent results of the validation studies.

Crohn's disease and sarcoidosis in siblings: follow-up of a published report with a new case and brief review of the literature.

Reported in 1971 were the cases of three brothers, two of whom had developed sarcoidosis and the third Crohn's disease. That now presented concerns one brother who, 50 years after the diagnosis and successful treatment of his sarcoidosis, was found incidentally, at colonoscopy performed to exclude malignancy, to have Crohn's colitis in the absence of any symptoms attributable to this. The report concludes with a brief review of the literature.

CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals.

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

Delineating the phenotypic spectrum of Bainbridge-Ropers syndrome: 12 new patients with de novo, heterozygous, loss-of-function mutations in ASXL3 and review of published literature.

BACKGROUND: Bainbridge-Ropers syndrome (BRPS) is a recently described developmental disorder caused by de novo truncating mutations in the additional sex combs like 3 (ASXL3) gene. To date, there have been fewer than 10 reported patients. OBJECTIVES: Here, we delineate the BRPS phenotype further by describing a series of 12 previously unreported patients identified by the Deciphering Developmental Disorders study. METHODS: Trio-based exome sequencing was performed on all 12 patients included in this study, which found a de novo truncating mutation in ASXL3. Detailed phenotypic information and patient images were collected and summarised as part of this study. RESULTS: By obtaining genotype:phenotype data, we have been able to demonstrate a second mutation cluster region within ASXL3. This report expands the phenotype of older patients with BRPS; common emerging features include severe intellectual disability (11/12), poor/ absent speech (12/12), autistic traits (9/12), distinct face (arched eyebrows, prominent forehead, high-arched palate, hypertelorism and downslanting palpebral fissures), (9/12), hypotonia (11/12) and significant feeding difficulties (9/12) when young. DISCUSSION: Similarities in the patients reported previously in comparison with this cohort included their distinctive craniofacial features, feeding problems, absent/limited speech and intellectual disability. Shared behavioural phenotypes include autistic traits, hand-flapping, rocking, aggressive behaviour and sleep disturbance. CONCLUSIONS: This series expands the phenotypic spectrum of this severe disorder and highlights its surprisingly high frequency. With the advent of advanced genomic screening, we are likely to identify more variants in this gene presenting with a variable phenotype, which this study will explore.

Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis.

OBJECTIVE: Validate independent component analysis (ICA) for removal of EMG contamination from EEG, and demonstrate a heuristic, based on the gradient of EEG spectra (slope of graph of log EEG power vs log frequency, 7-70 Hz) from paralysed awake humans, to automatically identify and remove components that are predominantly EMG. METHODS: We studied the gradient of EMG-free EEG spectra to quantitatively inform the choice of threshold. Then, pre-existing EEG from 3 disparate experimental groups was examined before and after applying the heuristic to validate that the heuristic preserved neurogenic activity (Berger effect, auditory odd ball, visual and auditory steady state responses). RESULTS: (1) ICA-based EMG removal diminished EMG contamination up to approximately 50 Hz, (2) residual EMG contamination using automatic selection was similar to manual selection, and (3) task-induced cortical activity remained, was enhanced, or was revealed using the ICA-based methodology. CONCLUSION: This study further validates ICA as a powerful technique for separating and removing myogenic signals from EEG. Automatic processing based on spectral gradients to exclude EMG-containing components is a conceptually simple and valid technique. SIGNIFICANCE: This study strengthens ICA as a technique to remove EMG contamination from EEG whilst preserving neurogenic activity to 50 Hz.

Measurement of neural signals from inexpensive, wireless and dry EEG systems.

Electroencephalography (EEG) is challenged by high cost, immobility of equipment and the use of inconvenient conductive gels. We compared EEG recordings obtained from three systems that are inexpensive, wireless, and/or dry (no gel), against recordings made with a traditional, research-grade EEG system, in order to investigate the ability of these 'non-traditional' systems to produce recordings of comparable quality to a research-grade system. The systems compared were: Emotiv EPOC (inexpensive and wireless), B-Alert (wireless), g.Sahara (dry) and g.HIamp (research-grade). We compared the ability of the systems to demonstrate five well-studied neural phenomena: (1) enhanced alpha activity with eyes closed versus open; (2) visual steady-state response (VSSR); (3) mismatch negativity; (4) P300; and (5) event-related desynchronization/synchronization. All systems measured significant alpha augmentation with eye closure, and were able to measure VSSRs (although these were smaller with g.Sahara). The B-Alert and g.Sahara were able to measure the three time-locked phenomena equivalently to the g.HIamp. The Emotiv EPOC did not have suitably located electrodes for two of the tasks and synchronization considerations meant that data from the time-locked tasks were not assessed. The results show that inexpensive, wireless, or dry systems may be suitable for experimental studies using EEG, depending on the research paradigm, and within the constraints imposed by their limited electrode placement and number.

Surface Laplacian of scalp electrical signals and independent component analysis resolve EMG contamination of electroencephalogram.

The serious impact of electromyogram (EMG) contamination of electroencephalogram (EEG) is well recognised. The objective of this research is to demonstrate that combining independent component analysis with the surface Laplacian can eliminate EMG contamination of the EEG, and to validate that this processing does not degrade expected neurogenic signals. The method involves sequential application of ICA, using a manual procedure to identify and discard EMG components, followed by the surface Laplacian. The extent of decontamination is quantified by comparing processed EEG with EMG-free data that was recorded during pharmacologically induced neuromuscular paralysis. The combination of the ICA procedure and the surface Laplacian, with a flexible spherical spline, results in a strong suppression of EMG contamination at all scalp sites and frequencies. Furthermore, the ICA and surface Laplacian procedure does not impair the detection of well-known, cerebral responses; alpha activity with eyes-closed; ERP components (N1, P2) in response to an auditory oddball task; and steady state responses to photic and auditory stimulation. Finally, more flexible spherical splines increase the suppression of EMG by the surface Laplacian. We postulate this is due to ICA enabling the removal of local muscle sources of EMG contamination and the Laplacian transform being insensitive to distant (postural) muscle EMG contamination.

In vitro human skin irritation test for evaluation of medical device extracts.

The aim of this study was to determine if the EpiDerm™ reconstructed human skin model (MatTek Corp.) could be an acceptable alternative to the ISO 10993-required rabbit skin irritation test for assessing medical device biocompatibility. Eleven medical device polymers were tested. Four extracts were prepared per polymer, two each with saline and sesame oil; half were spiked with two R-38 irritants, lactic acid for saline extracts and heptanoic acid for the sesame oil extracts. Tissue viability was assessed by MTT reduction and the proinflammatory response was assessed by IL-1α release. LOAELs of 2% for lactic acid in saline and 0.7% for heptanoic acid in sesame oil were determined. A cell viability reduction of >50% was indicative of skin irritation. Cells exposed to saline extracts spiked with 3.25% lactic acid had significantly reduced mean cell viabilities (12.6-17.2%). Cells exposed to sesame oil extracts spiked with 1.25% heptanoic acid also exhibited reduced mean cell viabilities (25.5%-41.7%). All spiked cells released substantial amounts of IL-1α (253.5-387.4pg/ml) signifying a proinflammatory response. These results indicate that the EpiDerm™ model may be a suitable in vitro replacement for the assessment of the irritation potential of medical device extracts.

Effects of anesthetic agents on seizure-induction with intra-cortical injection of convulsants.

RATIONALE: Studies of partial or generalized seizure pathophysiology often require the use of intact animals. Additionally, anesthesia may be required for ethical reasons or paralysis if instrumental measures require immobilization. We examined three commonly used injected anesthetic for their impact on seizures induced by three convulsant agents. METHODS: We prepared rats, under pentobarbitone anesthesia (65 mg/kg) with a catheter, electrodes and a dural window, for later non-noxious experimentation. Three to seven days later, kainic acid (1.25 µg), picrotoxin (225 ng) or fluorocitrate (0.8 nmol) were injected intra-cortically in animals paralysed with succinylcholine, or anesthetised with pentobarbitone, urethane or fentanyl plus droperidol. We recorded EEG activity, the latencies to seizure discharges, the occurrence of spreading depressions and the presence of movements in response to the convulsants. RESULTS: Fentanyl plus droperidol was the only anesthetic agent permissive for seizure-discharges and spreading depressions. No significant differences in the time for seizure onset for fentanyl plus droperidol compared to paralyzed unanesthetised rats were seen for any of the convulsants (Student's t-test p>0.20). Movements during seizures as well as other drug-induced behaviors continued to be expressed during anesthesia. CONCLUSION: Fentanyl plus droperidol has useful properties as an anesthetic agent in studies of seizure induction with different convulsants.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia.

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

Cell swelling, seizures and spreading depression: an impedance study.

The cellular processes that take place during the transition from pre-seizure state to seizure remain to be defined. In this study in awake, paralyzed rats, we used an electrical impedance measure of changes in extra-cellular intracranial volume to estimate changes in cell size in acute models of epilepsy. Animals were prepared with extradural electroencephalographic (EEG)/impedance electrodes and a venous catheter. On a subsequent day, animals were paralyzed, ventilated and treated with picrotoxin, kainic acid or fluorocitrate in doses that usually induce epileptiform discharges. We now report that increases in baseline impedance were induced by kainic acid and smaller increases by picrotoxin. We also demonstrated that epileptiform discharges were preceded by small, accelerated increases in impedance. Increases in baseline impedance were highly correlated with increases in power of non-ictal high frequency EEG activity. Seizures were accompanied by increases in impedance and all treatments induced transient, relatively large, increases in impedance often associated with unilateral reductions in low frequency EEG, likely periods of spreading depression. We conclude: cerebral cells swell in convulsant models of epilepsy, that there are pre-ictal accelerations in cell swelling, and that spreading depression-like events are frequently associated with seizures.

Gamma rhythms are not integral to EEG spindle phenomena.

OBJECTIVE: Gamma rhythms (30-100 Hz) have been shown to be associated with spindling activity induced by picrotoxin. To determine if gamma power is unique to picrotoxin spindles or is an integral part of physiological and pathological spindling activity we analysed and compared the strength and brain distribution of gamma EEG power during 4 spindling activities in the rat. METHODS: The electroencephalogram (EEG) was recorded from rats with chronically implanted electrodes during natural sleep, barbiturate anaesthesia, during naturally occurring absence epilepsy spike and wave discharges and following the systemic application of picrotoxin. Spectral analysis was applied off-line to compare the strength and brain distribution of gamma EEG power during the 4 spindling activities. RESULTS: Each spindle type contained significantly different levels of gamma power. Gamma power was significantly increased over background levels during picrotoxin spindles, slightly increased during absence spindles, slightly decreased during sleep spindles and significantly suppressed during barbiturate spindles CONCLUSIONS: Changes in the power of gamma frequencies during spindle types suggest that gamma frequencies are neither the cause of nor an integral part of a spindle. They appear to be correlated with levels of consciousness and may contribute to the process of epileptogenesis. SIGNIFICANCE: The findings are consistent with high frequency EEG activity being related to seizure-tendency.

Localised astroglial dysfunction disrupts high-frequency EEG rhythms.

We used cerebral cortex injections of fluorocitrate to determine if selective astrocytic disturbances affect the electroencephalogram (EEG). Rats were halothane-anaesthetized and 0.8 nmol of sodium fluorocitrate was injected into hindlimb (motor-sensory) cortex. Extra-dural EEG electrodes were implanted after which the anaesthesia was ceased. EEG was recorded at 1, 3, 5, 7, 24 and 48 hours. There was a broad-band reduction in frequencies in the EEG between 20 and 100 Hz commencing within 1 hour of injection and largely restricted to the side of injection and to frontal cortex, and maximal at 3 hours. Halothane had a suppressive effect on gamma power after citrate injection, but also prevented EEG suppression caused by fluorocitrate, consistent with the hypothesis that some of the action of fluorocitrate depended on gap-junctions. The findings are consistent with the hypothesis that primary astroglial dysfunction leads to reduced neuronal transmission and further supports gap-junctions as mediating fluorocitrate-induced astroglial effects.

Cognitive tasks augment gamma EEG power.

OBJECTIVE: Gamma EEG oscillations are low amplitude rhythms in the 30-100 Hz range that correlate with cognitive task execution. They are usually reported using time-locked averaging of EEG during repetitive tasks. We tested the hypothesis that continuous gamma EEG would be measurable during mental tasks. METHODS: We investigated sustained human gamma EEG oscillations induced by 8 cognitive tasks (Visual Checkerboard, Expectancy, Reading, Subtraction, Music, Expectancy, Word learning, Word recall, and a Video Segment) in 20 subjects using standard digital EEG recording and power spectral analysis. RESULTS: All of the cognitive tasks augmented gamma power relative to a control condition (eyes open watching a blank computer screen). This enhancement was statistically significant at more than one scalp site for all tasks except checkerboard. The Expectancy, Learning, Reading and Subtraction tasks expressed the most impressive gamma response, up to 5 fold above the control condition and there was some task-related specificity of the distribution of increased gamma power, especially in posterior cortex with visual tasks. CONCLUSIONS: Widespread gamma activation of cortical EEG can easily be demonstrated during mental activity. SIGNIFICANCE: These results establish the feasibility of measuring high frequency EEG rhythms with trans-cranial recordings, demonstrate that sustained gamma EEG activity correlates with mentation, and provides evidence consistent with the temporal binding model.