Identification of neurotoxicology (NT)/developmental neurotoxicology (DNT) adverse outcome pathways and key event linkages with in vitro DNT screening assays.

There is a need to assess compounds reliably and quickly for neurotoxicity (NT) and developmental neurotoxicity (DNT). Adverse outcome pathways (AOPs) enable the mapping of molecular events to an apical endpoint in a chemical agnostic manner and have begun to be applied in NT and DNT testing frameworks. We assessed the status of NT/DNT AOPs in the AOP-Wiki (ca. 2/1/23; https://aopwiki.org/), to characterize the state of AOP development, identify strengths and knowledge gaps, elucidate areas for improvement, and describe areas for future focus. AOPs in the Wiki database were assessed for inclusion of NT/DNT molecular events and endpoints, AOP development and endorsement, as well as the linkages of key neurodevelopmental processes with in vitro new approach methods (NAMs). This review found that 41 AOPs have been proposed detailing NT/DNT, of which eight were endorsed by working parties in OECD. Further, this review determined that learning and memory is included as an adverse outcome in eight NT/DNT AOPS, often without distinction regarding the varying forms of learning and memory, regional specification, temporal dynamics, or acquisition mechanisms involved. There is also an overlap with key events (KEs) and in vitro NAMs, which synaptogenesis appeared as a common process. Overall, progress on NT/DNT AOPs could be expanded, adding in modes of action that are missing, improvement in defining apical endpoints, as well as utilizing NAMs further to develop AOPs and identify gaps in current knowledge.

Proteome Profiling of Rat Brain Cortical Changes during Early Postnatal Brain Development.

Label-free quantitation (LFQ) was applied to proteome profiling of rat brain cortical development during the early postnatal period. Male and female rat brain extracts were prepared using a convenient, detergent-free sample preparation technique at postnatal days (PND) 2, 8, 15, and 22. The PND protein ratios were calculated using Proteome Discoverer, and the PND protein change profiles were constructed separately for male and female animals for key presynaptic, postsynaptic, and adhesion brain proteins. The profiles were compared to the analogous profiles assembled from the published mouse and rat cortex proteomic data, including the fractionated-synaptosome data. The PND protein-change trendlines, Pearson correlation coefficient (PCC), and linear regression analysis of the statistically significant PND protein changes were used in the comparative analysis of the datasets. The analysis identified similarities and differences between the datasets. Importantly, there were significant similarities in the comparison of the rat cortex PND (current work) vs mouse (previously published) PND profiles, although in general, a lower abundance of synaptic proteins in mice than in rats was found. The male and female rat cortex PND profiles were expectedly almost identical (98-99% correlation by PCC), which also substantiated this LFQ nanoflow liquid chromatography-high-resolution mass spectrometry approach.

Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Tests of untrained behaviors.

Manganese (Mn), an element that naturally occurs in the environment, has been shown to produce neurotoxic effects on the developing young when levels exceed physiological requirements. To evaluate the effects of this chemical in combination with non-chemical factors pregnant Long-Evans rats were treated with 0, 2, or 4 mg/mL Mn in their drinking water from gestational day (GD) 7 to postnatal day (PND) 22. Half of the dams received a variable stress protocol from GD13 to PND9, that included restraint, small cage with reduced bedding, exposure to predator odor, intermittent intervals of white noise, lights on for 24 h, intermittent intervals of lights on during dark cycle and cages with grid floors and reduced bedding. One male and one female offspring from each litter were tested to assess untrained behavior. Ultrasonic vocalizations (USV) were recorded from PND13 pups while they were isolated from the litter. Locomotor activity (MA) was measured in figure-eight mazes at PND 17, 29, and 79 (different set of rats at each time point). Social approach (SA) was tested at PND48. Acoustic startle response (ASR) and pre-pulse inhibition (PPI) were measured starting at PND58. At PND53 a sweetness preference for a chocolate flavored milk solution was assessed. There were sex related differences on several parameters for the USVs. There was also a Mn by stress by sex interaction with the females from the 4 mg/mL stressed dams having more frequency modulated (FM) call elements than the 4 mg/mL non-stressed group. There was an effect of Mn on motor activity but only at PND29 with the 2 mg/mL group having higher counts than the 0 mg/mL group. The social approach test showed sex differences for both the habituation and test phase. There was an effect of Mn, with the 4 mg/mL males having a greater preference for the stimulus rat than did the 0 mg/mL males. There was also a stress by sex interaction. The ASR and PPI had only a sex effect. Thus, with only the FM call elements having a Mn by stress effect, and the PND29 MA and SA preference index having a Mn effect but at different doses requires further investigation.

In vivo neurophysiological assessment of in silico predictions of neurotoxicity: Citronellal, 3,4-dichloro-1-butene, and benzyl bromoacetate.

Neurotoxicants may be widespread in the environment and can produce serious health impacts in the human population. Screening programs that use in vitro methods have generated data for thousands of chemicals. However, these methods often do not evaluate repeated or prolonged exposures, which are required for many neurotoxic outcomes. Additionally, the data produced by such screening methods may not include mechanisms which play critical biological roles necessary for in vivo neurotoxicity. The Hard and Soft Acids and Bases (HSAB) in silico model focuses on chemical structure and electrophilic properties which are important to the formation of protein adducts. A group of structurally diverse chemicals have been evaluated with an in silico screening approach incorporating HSAB parameters. However, the predictions from the expanded chemical space have not been evaluated using in vivo methods. Three chemicals predicted to be cumulative toxicants were selected for in vivo neurotoxicological testing. Adult male Long-Evans rats were treated orally with citronellal (CIT), 3,4-dichloro-1-butene (DCB), or benzyl bromoacetate (BBA) for 8 weeks. Behavioral observations were recorded weekly to assess motor function. Peripheral neurophysiological measurements were derived from nerve excitability (NE) tests which involved compound muscle action potentials (CMAPs) in the tail and foot, and mixed nerve action potentials (MNAPs) in the tail. Compound nerve action potentials (CNAPs) and nerve conduction velocity (NCV) in the tail were also quantified. Peripheral inputs into the central nervous system were examined using somatosensory evoked potentials recorded from the cortex (SEPCTX) and cerebellum (SEPCEREB). CIT or BBA did not result in significant alterations to peripheral nerve or somatosensory function. DCB reduced grip-strength and altered peripheral nerve function. The MNAPs required less current to reach 50% amplitude and had a lower calculated rheobase, suggesting increased excitability. Increased CNAP amplitudes and greater NCV were also observed. Novel changes were found in the SEPCTX with an abnormal peak forming in the early portion of the waveforms of treated rats, and decreased latencies and increased amplitudes were observed in SEPCEREB recordings. These data contribute to testing an expanded chemical space from an in silico HSAB model for predicting cumulative neurotoxicity and may assist with prioritizing chemicals to protect human health.

Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Maternal somatic measures and the postnatal growth and development of rat offspring.

Psychological stress experienced by the mother during pregnancy has been associated with emotional and cognitive disorders in children such as depression and anxiety. Socioeconomically disadvantaged populations are vulnerable to adverse life experiences and can also be disproportionally exposed to environmental contaminants. To better understand the neurodevelopmental impacts of an environmental toxicant coupled with elevated psychological stress, we exposed pregnant rats to a series of perinatal stressors. Manganese (Mn), a neurotoxicant at excessive concentrations was delivered through drinking water (0, 2, or 4 mg/mL) from gestational day (GD) 7 to postnatal day (PND) 22. A variable stress paradigm was applied to half of the animals from GD13 to PND9. Measurements of somatic development and behavior were examined in the offspring at different developmental stages. No evidence of overt maternal toxicity was observed although the 4 mg/mL Mn-exposed dams gained less body weight during gestation compared to the other dams. Stress also reduced gestational maternal weight gain. Daily fluid consumption normalized for body weight was decreased in the Mn-exposed dams in a dose-dependent manner but was not altered by the stress paradigm. Maternal stress and/or Mn exposure did not affect litter size or viability, but pup weight was significantly reduced in the 4 mg/mL Mn-exposed groups on PNDs 9 through 34 when compared to the other offspring groups. The efficacy of the manipulations to increase maternal stress levels was determined using serum corticosterone as a biomarker. The baseline concentration was established prior to treatment (GD7) and levels were low and similar in all treatment groups. Corticosterone levels were elevated in the perinatal-stress groups compared to the no-stress groups, regardless of Mn exposure, on subsequent time points (GD16, PND9), but were only significantly different on GD16. An analysis of tissue concentrations revealed Mn was elevated similarly in the brain and blood of offspring at PND2 and at PND22 in a significant dose-dependent pattern. Dams also showed a dose-dependent increase in Mn concentrations in the brain and blood; the addition of stress increased the Mn concentrations in the maternal blood but not the brain. Perinatal stress did not alter the effects of Mn on the maternal or offspring somatic endpoints described here.

The Future of Neurotoxicology: A Neuroelectrophysiological Viewpoint.

Neuroelectrophysiology is an old science, dating to the 18th century when electrical activity in nerves was discovered. Such discoveries have led to a variety of neurophysiological techniques, ranging from basic neuroscience to clinical applications. These clinical applications allow assessment of complex neurological functions such as (but not limited to) sensory perception (vision, hearing, somatosensory function), and muscle function. The ability to use similar techniques in both humans and animal models increases the ability to perform mechanistic research to investigate neurological problems. Good animal to human homology of many neurophysiological systems facilitates interpretation of data to provide cause-effect linkages to epidemiological findings. Mechanistic cellular research to screen for toxicity often includes gaps between cellular and whole animal/person neurophysiological changes, preventing understanding of the complete function of the nervous system. Building Adverse Outcome Pathways (AOPs) will allow us to begin to identify brain regions, timelines, neurotransmitters, etc. that may be Key Events (KE) in the Adverse Outcomes (AO). This requires an integrated strategy, from in vitro to in vivo (and hypothesis generation, testing, revision). Scientists need to determine intermediate levels of nervous system organization that are related to an AO and work both upstream and downstream using mechanistic approaches. Possibly more than any other organ, the brain will require networks of pathways/AOPs to allow sufficient predictive accuracy. Advancements in neurobiological techniques should be incorporated into these AOP-base neurotoxicological assessments, including interactions between many regions of the brain simultaneously. Coupled with advancements in optogenetic manipulation, complex functions of the nervous system (such as acquisition, attention, sensory perception, etc.) can be examined in real time. The integration of neurophysiological changes with changes in gene/protein expression can begin to provide the mechanistic underpinnings for biological changes. Establishment of linkages between changes in cellular physiology and those at the level of the AO will allow construction of biological pathways (AOPs) and allow development of higher throughput assays to test for changes to critical physiological circuits. To allow mechanistic/predictive toxicology of the nervous system to be protective of human populations, neuroelectrophysiology has a critical role in our future.

Exposure to Intermittent Noise Exacerbates the Cardiovascular Response of Wistar-Kyoto Rats to Ozone Inhalation and Arrhythmogenic Challenge.

Noise has become a prevalent public health problem across the world. Although there is a significant amount of data demonstrating the harmful effects of noise on the body, very little is known about how it impacts subsequent responses to other environmental stressors like air pollution, which tend to colocalize in urban centers. Therefore, this study was conducted to determine the effect of intermittent noise on cardiovascular function and subsequent responses to ozone (O3). Male Wistar-Kyoto rats implanted with radiotelemeters to non-invasively measure heart rate (HR) and blood pressure (BP), and assess heart rate variability (HRV) and baroreflex sensitivity (BRS) were kept in the quiet or exposed to intermittent white noise (85-90 dB) for one week and then exposed to either O3 (0.8 ppm) or filtered air. Left ventricular function and arrhythmia sensitivity were measured 24 h after exposure. Intermittent noise caused an initial increase in HR and BP, which decreased significantly later in the regimen and coincided with an increase in HRV and BRS. Noise caused HR and BP to be significantly elevated early during O3 and lower at the end when compared to animals kept in the quiet while the increased HRV and BRS persisted during the 24 h after. Lastly, noise increased arrhythmogenesis and may predispose the heart to mechanical function changes after O3. This is the first study to demonstrate that intermittent noise worsens the cardiovascular response to inhaled O3. These effects may occur due to autonomic changes and dysregulation of homeostatic controls, which persist one day after exposure to noise. Hence, co-exposure to noise should be taken into account when assessing the health effects of urban air pollution.

Acute in vitro effects on embryonic rat dorsal root ganglion (DRG) cultures by in silico predicted neurotoxic chemicals: Evaluations on cytotoxicity, neurite length, and neurophysiology.

The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1-100 µM) with test chemical; %LDH and NLPN were measured after 48 h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100 µM (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100 µM (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48 h; 50 and 100 µM for 4-C (28% and 60%), 100 µM Cit (52%), 100 µM 2,5- Hex (37%) 100 µM PAA (41%) and 100 µM for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100 µM: Acrylamide (-83%), 3,4-dichloro-1-butene (-93%), 4-C (-89%) and hexane (-79%, 50 µM). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity.

Application of the hard and soft, acids and bases (HSAB) theory as a method to predict cumulative neurotoxicity.

Xenobiotic electrophiles can form covalent adducts that may impair protein function, damage DNA, and may lead a range of adverse effects. Cumulative neurotoxicity is one adverse effect that has been linked to covalent protein binding as a Molecular Initiating Event (MIE). This paper describes a mechanistic in silico chemical screening approach for neurotoxicity based on Hard and Soft Acids and Bases (HSAB) theory. We evaluated the applicability of HSAB-based electrophilicity screening protocol for neurotoxicity on 19 positive and 19 negative reference chemicals. These reference chemicals were identified from the literature, using available information on mechanisms of neurotoxicity whenever possible. In silico screening was based on structural alerts for protein binding motifs and electrophilicity index in the range of known neurotoxicants. The approach demonstrated both a high positive prediction rate (82-90 %) and specificity (90 %). The overall sensitivity was relatively lower (47 %). However, when predicting the toxicity of chemicals known or suspected of acting via non-specific adduct formation mechanism, the HSAB approach identified 7/8 (sensitivity 88 %) of positive control chemicals correctly. Consequently, the HSAB-based screening is a promising approach of identifying possible neurotoxins with adduct formation molecular initiating events. While the approach must be expanded over time to capture a wider range of MIEs involved in neurotoxicity, the mechanistic nature of the screen allows users to flag chemicals for possible adduct formation MIEs. Thus, the HSAB based toxicity screening is a promising strategy for toxicity assessment and chemical prioritization in neurotoxicology and other health endpoints that involve adduct formation.

Commentary on topic: Should all tests of cognitive function - Learning, memory, attention - Be eliminated from the required protocols for developmental neurotoxicity testing?

The attached manuscript by Dr. Herr is a Commentary on Topic: Should All Tests of Cognitive Function - Learning, Memory, Attention - be Eliminated From the Required Protocols for Developmental Neurotoxicity Testing?

Taxonomic applicability of inflammatory cytokines in adverse outcome pathway (AOP) development.

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system function after developmental exposure to gasoline, E15, and E85 vapors.

The use of gasolines blended with a range of ethanol concentrations may result in inhalation of vapors containing a variable combination of ethanol with other volatile gasoline constituents. The possibility of exposure and potential interactions between vapor constituents suggests the need to evaluate the possible risks of this complex mixture. Previously we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Here we report an evaluation using the same battery of sensory function testing in offspring of pregnant dams exposed during gestation to condensed vapors of gasoline (E0), gasoline blended with 15% ethanol (E15) or gasoline blended with 85% ethanol (E85). Pregnant Long-Evans rats were exposed to target concentrations 0, 3000, 6000, or 9000 ppm total hydrocarbon vapors for 6.5h/day over GD9 - GD20. Sensory evaluations of male offspring began as adults. The electrophysiological testing battery included tests of: peripheral nerve (compound action potentials, nerve conduction velocity [NCV]), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual functions. Visual function assessment included pattern elicited visual evoked potentials (VEP), VEP contrast sensitivity, dark-adapted (scotopic) electroretinograms (ERGs), light-adapted (photopic) ERGs, and green flicker ERGs. The results included sporadic statistically significant effects, but the observations were not consistently concentration-related and appeared to be statistical Type 1 errors related to multiple dependent measures evaluated. The exposure concentrations were much higher than can be reasonably expected from typical exposures to the general population during refueling or other common exposure situations. Overall the results indicate that gestational exposure of male rats to ethanol/gasoline vapor combinations did not cause detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

Translational Biomarkers of Neurotoxicity: A Health and Environmental Sciences Institute Perspective on the Way Forward.

Neurotoxicity has been linked to a number of common drugs and chemicals, yet efficient and accurate methods to detect it are lacking. There is a need for more sensitive and specific biomarkers of neurotoxicity that can help diagnose and predict neurotoxicity that are relevant across animal models and translational from nonclinical to clinical data. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid (CSF) have great potential due to the relative ease of sampling compared with tissues. Increasing evidence supports the potential utility of fluid-based biomarkers of neurotoxicity such as microRNAs, F2-isoprostanes, translocator protein, glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1, myelin basic protein, microtubule-associated protein-2, and total tau. However, some of these biomarkers such as those in CSF require invasive sampling or are specific to one disease such as Alzheimer's, while others require further validation. Additionally, neuroimaging methodologies, including magnetic resonance imaging, magnetic resonance spectroscopy, and positron emission tomography, may also serve as potential biomarkers and have several advantages including being minimally invasive. The development of biomarkers of neurotoxicity is a goal shared by scientists across academia, government, and industry and is an ideal topic to be addressed via the Health and Environmental Sciences Institute (HESI) framework which provides a forum to collaborate on key challenging scientific topics. Here we utilize the HESI framework to propose a consensus on the relative potential of currently described biomarkers of neurotoxicity to assess utility of the selected biomarkers using a nonclinical model.

Expanding the test set: Chemicals with potential to disrupt mammalian brain development.

High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxicants. As new assays are developed, a "training set" of chemicals is used to evaluate the relevance of individual assays for specific endpoints. Different training sets are necessary for each assay that would comprise a developmental neurotoxicity test battery. In contrast, evaluation of the predictive ability of a comprehensive test battery requires a set of chemicals that have been shown to alter brain development after in vivo exposure ("test set"). Because only a small number of substances have been well documented to alter human neurodevelopment, we have proposed an expanded test set that includes chemicals demonstrated to adversely affect neurodevelopment in animals. To compile a list of potential developmental neurotoxicants, a literature review of compounds that have been examined for effects on the developing nervous system was conducted. The search was limited to mammalian studies published in the peer-reviewed literature and regulatory studies submitted to the U.S. EPA. The definition of developmental neurotoxicity encompassed changes in behavior, brain morphology, and neurochemistry after gestational or lactational exposure. Reports that indicated developmental neurotoxicity was observed only at doses that resulted in significant maternal toxicity or were lethal to the fetus or offspring were not considered. As a basic indication of reproducibility, we only included a chemical if data on its developmental neurotoxicity were available from more than one laboratory (defined as studies originating from laboratories with a different senior investigator). Evidence from human studies was included when available. Approximately 100 developmental neurotoxicity test set chemicals were identified, with 22% having evidence in humans.

Identification of fipronil metabolites by time-of-flight mass spectrometry for application in a human exposure study.

Fipronil is a phenylpyrazole insecticide commonly used in residential and agricultural applications. To understand more about the potential risks for human exposure associated with fipronil, urine and serum from dosed Long Evans adult rats (5 and 10mg/kg bw) were analyzed to identify metabolites as potential biomarkers for use in human biomonitoring studies. Urine from treated rats was found to contain seven unique metabolites, two of which had not been previously reported-M4 and M7 which were putatively identified as a nitroso compound and an imine, respectively. Fipronil sulfone was confirmed to be the primary metabolite in rat serum. The fipronil metabolites identified in the respective matrices were then evaluated in matched human urine (n=84) and serum (n=96) samples from volunteers with no known pesticide exposures. Although no fipronil or metabolites were detected in human urine, fipronil sulfone was present in the serum of approximately 25% of the individuals at concentrations ranging from 0.1 to 4ng/mL. These results indicate that many fipronil metabolites are produced following exposures in rats and that fipronil sulfone is a useful biomarker in human serum. Furthermore, human exposure to fipronil may occur regularly and require more extensive characterization.

Assessment of serum biomarkers in rats after exposure to pesticides of different chemical classes.

There is increasing emphasis on the use of biomarkers of adverse outcomes in safety assessment and translational research. We evaluated serum biomarkers and targeted metabolite profiles after exposure to pesticides (permethrin, deltamethrin, imidacloprid, carbaryl, triadimefon, fipronil) with different neurotoxic actions. Adult male Long-Evans rats were evaluated after single exposure to vehicle or one of two doses of each pesticide at the time of peak effect. The doses were selected to produce similar magnitude of behavioral effects across chemicals. Serum or plasma was analyzed using commercial cytokine/protein panels and targeted metabolomics. Additional studies of fipronil used lower doses (lacking behavioral effects), singly or for 14 days, and included additional markers of exposure and biological activity. Biomarker profiles varied in the number of altered analytes and patterns of change across pesticide classes, and discriminant analysis could separate treatment groups from control. Low doses of fipronil produced greater effects when given for 14 days compared to a single dose. Changes in thyroid hormones and relative amounts of fipronil and its sulfone metabolite also differed between the dosing regimens. Most cytokine changes reflected alterations in inflammatory responses, hormone levels, and products of phospholipid, fatty acid, and amino acid metabolism. These findings demonstrate distinct blood-based analyte profiles across pesticide classes, dose levels, and exposure duration. These results show promise for detailed analyses of these biomarkers and their linkages to biological pathways.

Use of electroencephalography (EEG) to assess CNS changes produced by pesticides with different modes of action: effects of permethrin, deltamethrin, fipronil, imidacloprid, carbaryl, and triadimefon.

The electroencephalogram (EEG) is an apical measure, capable of detecting changes in brain neuronal activity produced by internal or external stimuli. We assessed whether pesticides with different modes of action produced different changes in the EEG of adult male Long-Evans rats. The EEG was recorded using two montages (visual cortex referenced to the cerebellum and to the frontal cortex) in unrestrained rats at the time of peak behavioral effects. Pesticides included: permethrin and deltamethrin (Type I and Type II pyrethroids; 2 h), fipronil (single and repeated doses; phenylpyrazole; 6 h), imidacloprid (neonicotinoid; 2 h), carbaryl (carbamate; 0.5 h), and triadimefon (triazole; 1 h), using dosages that produced approximately an ED30 or an ED50-ED80 change in motor activity. Permethrin (43, 100 mg/kg) increased amplitudes or areas (delta, alpha, or gamma bands) in the EEG. Deltamethrin (2.5, 5.5 mg/kg) reduced the amplitudes or areas of the delta, theta, alpha, beta, and gamma bands, but the changes were not dose-related. A single treatment with fipronil (25, 50 mg/kg, but not 5, 10 mg/kg) decreased gamma band area. Additional changes in the delta, theta, and gamma bands were observed when fipronil (5, 10 mg/kg) was administered for 14 days. Imidacloprid (50, 100 mg/kg) did not alter the EEG. Carbaryl (10, 50 mg/kg) decreased theta area, and decreased delta and increased beta frequency. Triadimefon (75, 150 mg/kg) produced minimal changes in the EEG. The results show that the EEG is affected differently by approximately equipotent doses of pesticides with different modes of action.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system functions after developmental exposure to ethanol vapors.

Ethanol-blended gasoline entered the market in response to demand for domestic renewable energy sources, and may result in increased inhalation of ethanol vapors in combination with other volatile gasoline constituents. It is important to understand potential risks of inhalation of ethanol vapors by themselves, and also as a baseline for evaluating the risks of ethanol combined with a complex mixture of hydrocarbon vapors. Because sensory dysfunction has been reported after developmental exposure to ethanol, we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Pregnant Long-Evans rats were exposed to target concentrations 0, 5000, 10,000, or 21,000 ppm ethanol vapors for 6.5h/day over GD9-GD20. Sensory evaluations of male offspring began between PND106 and PND128. Peripheral nerve function (compound action potentials, nerve conduction velocity (NCV)), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual evoked responses were assessed. Visual function assessment included pattern elicited visual evoked potentials (VEPs), VEP contrast sensitivity, and electroretinograms recorded from dark-adapted (scotopic), light-adapted (photopic) flashes, and UV flicker and green flicker. No consistent concentration-related changes were observed for any of the physiological measures. The results show that gestational exposure to ethanol vapor did not result in detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

A four-step approach to evaluate mixtures for consistency with dose addition.

Mixture risk assessment is often hampered by the lack of dose-response information on the mixture being assessed, forcing reliance on component formulas such as dose addition. We present a four-step approach for evaluating chemical mixture data for consistency with dose addition for use in supporting a component based mixture risk assessment. Following the concepts in the U.S. EPA mixture risk guidance (U.S. EPA, 2000a,b), toxicological interaction for a defined mixture (all components known) is departure from a clearly articulated definition of component additivity. For the common approach of dose additivity, the EPA guidance identifies three desirable characteristics, foremost of which is that the component chemicals are toxicologically similar. The other two characteristics are empirical: the mixture components have toxic potencies that are fixed proportions of each other (throughout the dose range of interest), and the mixture dose term in the dose additive prediction formula, which we call the combined prediction model (CPM), can be represented by a linear combination of the component doses. A consequent property of the proportional toxic potencies is that the component chemicals must share a common dose-response model, where only the dose coefficients depend on the chemical components. A further consequence is that the mixture data must be described by the same mathematical function ("mixture model") as the components, but with a distinct coefficient for the total mixture dose. The mixture response is predicted from the component dose-response curves by using the dose additive CPM and the prediction is then compared with the observed mixture results. The four steps are to evaluate: (1) toxic proportionality by determining how well the CPM matches the single chemical models regarding mean and variance; (2) fit of the mixture model to the mixture data; (3) agreement between the mixture data and the CPM prediction; and (4) consistency between the CPM and the mixture model. Because there are four evaluations instead of one, some involving many parameters or dose groups, there are more opportunities to reject statistical hypotheses about dose addition, thus statistical adjustment for multiple comparisons is necessary. These four steps contribute different pieces of information about the consistency of the component and mixture data with the two empirical characteristics of dose additivity. We examine this four-step approach in how it can show empirical support for dose addition as a predictor for an untested mixture in a screening level risk assessment. The decision whether to apply dose addition should be based on all four of those evidentiary pieces as well as toxicological understanding of these chemicals and should include interpretations of the numerical and toxicological issues that arise during the evaluation. This approach is demonstrated with neurotoxicity data on carbamate mixtures.

Cholinesterase inhibition and depression of the photic after discharge of flash evoked potentials following acute or repeated exposures to a mixture of carbaryl and propoxur.

Previously, we reported that acute treatment with propoxur or carbaryl decreased the duration of the photic after discharge (PhAD) of flash evoked potentials (FEPs). In the current studies, we compared the effects of acute or repeated exposure to a mixture of carbaryl and propoxur (1:1.45 ratio; propoxur:carbaryl) on the duration of the PhAD and brain ChE activity in Long Evans rats. Animals were exposed (po) either to a single dose (0, 3, 10, 45 or 75 mg/kg), or 14 daily dosages (0, 3, 10, 30, 45 mg/kg), of the mixture. Acute and repeated treatment with 3mg/kg (or greater) of the mixture produced dose-related inhibition of brain ChE activity. Compared to controls, the PhAD duration decreased after acute administration of 75 mg/kg or repeated treatment with 30 mg/kg of the mixture. The linear relationship between the percent of control brain ChE activity and the PhAD duration was similar for both exposure paradigms. Dose-response models for the acute and repeated exposure data did not differ for brain ChE activity or the duration of the PhAD. Repeated treatment with the mixture resulted in slightly less (13-22%) erythrocyte ChE inhibition than acute exposure. Both acute and repeated treatment resulted in dose-additive results for the PhAD duration and less than dose-additive responses (6-16%) for brain ChE activity for the middle range of dosages. Acute treatment resulted in greater than dose-additive erythrocyte ChE inhibition (15-18%) at the highest dosages. In contrast, repeated treatment resulted in less than dose-additive erythrocyte ChE inhibition (16-22%) at the middle dosages. Brain and plasma levels of propoxur and carbaryl did not differ between the acute and repeated dosing paradigms. In summary, a physiological measure of central nervous system function and brain ChE activity had similar responses after acute or repeated treatment with the carbamate mixture, and brain ChE showed only small deviations from dose-additivity. Erythrocyte ChE activity had larger differences between the acute and repeated treatment paradigms, and showed slightly greater deviations from dose-additivity. Because these treatments utilized larger dosages than anticipated environmental exposures, concern for non-additive effects in humans is minimized. The small magnitude of the deviations from dose-additivity also suggest that in the absence of repeated exposure data, results from an acute study of readily reversible carbamate toxicity can be used to estimate the response to repeated daily exposures.