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The US EPA's Toxicity Forecaster (ToxCast) is a suite of high-throughput in vitro assays to screen environmental toxicants and predict potential toxicity of uncharacterized chemicals. This work examines the relevance of ToxCast assay intended gene targets to putative molecular initiating events (MIEs) of neurotoxicants. This effort is needed as there is growing interest in the regulatory and scientific communities about developing new approach methodologies (NAMs) to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity. Assay gene function (GeneCards, NCBI-PUBMED) was used to categorize gene target neural relevance (1 = neural, 2 = neural development, 3 = general cellular process, 3 A = cellular process critical during neural development, 4 = unlikely significance). Of 481 unique gene targets, 80 = category 1 (16.6 %); 16 = category 2 (3.3 %); 303 = category 3 (63.0 %); 97 = category 3 A (20.2 %); 82 = category 4 (17.0 %). A representative list of neurotoxicants (548) was researched (ex. PUBMED, PubChem) for neurotoxicity associated MIEs/Key Events (KEs). MIEs were identified for 375 compounds, whereas only KEs for 173. ToxCast gene targets associated with MIEs were primarily neurotransmitter (ex. dopaminergic, GABA)receptors and ion channels (calcium, sodium, potassium). Conversely, numerous MIEs associated with neurotoxicity were absent. Oxidative stress (OS) mechanisms were 79.1 % of KEs. In summary, 40 % of ToxCast assay gene targets are relevant to neurotoxicity mechanisms. Additional receptor and ion channel subtypes and increased OS pathway coverage are identified for potential future assay inclusion to provide more complete coverage of neural and developmental neural targets in assessing neurotoxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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.

The developmental effects of chemicals that co-occur in vulnerable populations with elevated psychological stress are of increasing concern to the public. To investigate these concerns, we developed a rodent model of co-occurring perinatal manipulations and conducted a series of cognitive assessments in male and female offspring. Manganese (Mn), a neurodevelopmental toxicant when exceeding physiological requirements, was delivered in the drinking water (0, 2, or 4 mg Mn/mL) of rats from gestational day (GD) 7 to postnatal day (PND) 22. A variable perinatal stress paradigm was applied to half of the animals from GD13 to PND9. Novel object recognition (NOR), Morris water maze (MWM), differential reinforcement of low-rates procedure (DRL) and cued and uncued choice reaction time (CRT) tests were used to assess cognitive functions in offspring. Mn (4 mg/mL) and stress impaired NOR in adolescent males but facilitated NOR performance in females. However, when stress and Mn were combined these effects were attenuated in both sexes. During training for the DRL, Mn (2 mg/mL) facilitated, while stress impaired, lever press learning in both sexes. Few effects related to the treatments were found on DRL or MWM. During cued CRT, Mn (2 and 4 mg/mL) and stress reduced accuracy in males, while stress and Mn (2 mg/mL) increased anticipatory responding and slowed decision time in both sexes. Stress combined with Mn (2 mg/mL) improved cued accuracy and decision time, and Mn attenuated the effect of stress on anticipatory responding in both sexes. Stress slowed female movement time but when combined with Mn (4 mg/mL) the effect of stress was attenuated. During uncued CRT, except for decision time (which replicated effects observed with the cued task), no other effects of Mn or its combination with stress occurred. Females remained negatively affected by stress in most uncued CRT performance measures, while stressed improved male uncued accuracy. Taken together these data do not support increased cognitive impairment produced by Mn when combined with stress. However, the effects of perinatal stress alone, on these cognitive functions may hinder the detection of effects due to chemical exposures and underscores the need to consider the psychological health and wellbeing of the mother and her environment in risk assessment for developmental neurotoxicity of chemicals., (Published by Elsevier Inc.)

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 (SEP CTX ) and cerebellum (SEP CEREB ). 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 SEP CTX with an abnormal peak forming in the early portion of the waveforms of treated rats, and decreased latencies and increased amplitudes were observed in SEP CEREB 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., (Published by Elsevier B.V.)

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

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., Competing Interests: Conflict of Interest: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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 (O 3 ). 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 O 3 (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 O 3 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 O 3 . This is the first study to demonstrate that intermittent noise worsens the cardiovascular response to inhaled O 3 . 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.

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., Competing Interests: Declaration of Competing Interest None., (Published by Elsevier Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., (Published by Elsevier Inc.)

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.

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., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Ltd.)

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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., (Published by Elsevier B.V.)

Polybrominated diphenyl ethers (PBDEs) are commonly used as commercial flame retardants in a variety of products, including plastics and textiles. Previous studies in our laboratory, and in the literature, showed that exposure to a specific PBDE congener (PBDE 47) during a critical period of brain development may lead to developmental delays and hyperactivity in adulthood. To date, the underlying causes of these behavioral alterations are unknown, although in vitro studies linked PBDEs with potential alterations in neurotransmitter levels, particularly acetylcholine (ACh) and dopamine (DA). Alterations in DA function have also been noted in cases of hyperactivity in rodents and humans. The current study examined monoamine levels in male mice acutely exposed to corn oil vehicle or PBDE 47 (1, 10, or 30 mg/kg) on postnatal day (PND) 10. Animals were sacrificed on PND 15, PND 20, and in adulthood (131-159 days old). The cortex, striatum, and cerebellum were isolated and analyzed by high-performance liquid chromatography to determine the concentration of monoamines within each brain region. A statistically significant increase in DA levels was seen within the cortex, regardless of age, but only in the 10-mg/kg PBDE treatment group. While these effects did not show a monotonic dose response, we previously reported hyperactivity in littermates in the same dose group, but not at the lower or higher dose. Thus, early developmental exposure to PBDE 47 alters the levels of cortical DA in male mice, which may correlate with behavioral observations in littermates.

Carbaryl is a N-methylcarbamate pesticide and, like others in this class, is a reversible inhibitor of cholinesterase (ChE) enzymes. Although studied for many years, there is a surprising lack of information relating tissue levels of carbaryl with ChE activity in the same animals. The present studies were undertaken to describe the dose-response relationship about 40 min (approximate time of maximal ChE inhibition) after oral treatment in adult, post-natal day (PND) 17, and PND11 rats. Additionally, the time-course of plasma ChE activity and carbaryl levels in adult rats was determined after a 30 mg/kg dosage of carbaryl. The time-course study found that carbaryl levels could be detected in plasma 1 h after dosing, but rapidly decreased below the level of quantitation by the 2 h time point. In the dose-response studies, treatment-related increases in plasma and brain carbaryl levels were observed 40 min after dosing. Plasma levels of carbaryl increased linearly, while brain levels appeared to asymptote after 75 mg/kg carbaryl. Plasma and brain levels of carbaryl appeared to be linearly related with a slope close to 1 after various dosages (range: 1-75 mg/kg) of carbaryl at the 40 min time point. Finally, the dose-related relationship between tissue levels of carbaryl and ChE activity was described using a first order exponential decay function with an asymptote. The parameters of this function did not appear to differ between adult, PND17, or PND11 rats. This indicates that age-related differences in brain ChE inhibition by carbaryl are unlikely to be the result of greater tissue levels of the pesticide in PND11 animals. These are the first studies to report the relationship between brain and plasma tissue levels of carbaryl and ChE activity on an individual animal basis. The results of these experiments will be useful to extend physiologically-based pharmacokinetic models for carbaryl and their application in risk assessment., (Published by Elsevier Ireland Ltd.)

Introduction: Exposure to irreversible cholinesterase (ChE)-inhibiting compounds, such as organophosphates may produce neuromuscular dysfunction. However, less is known about changes in neuromuscular transmission after treatment with reversible ChE-inhibitors. These studies adapted single fiber electromyography (SFEMG) techniques to quantify neuromuscular jitter in adult and juvenile rats after treatment with agents that alter cholinergic neurotransmission., Methods: Anesthetized adult and juvenile rats were tested using stimulation SFEMG, recorded in the gastrocnemius muscle, after stimulation in the sciatic notch region. The influence of electrode placement, treatment with decamethonium (to disrupt neuromuscular transmission) or physostigmine (reversible ChE inhibitor), and the impact of varied stimulation frequency were quantified., Results: No significant effects of needle placement or criterion amplitude were observed when calculating the mean consecutive difference (MCD). Treatment with decamethonium did not alter MCD values in adult or juvenile rats. However, decamethonium produced an increased blocking of muscle action potentials (MAP) in juveniles. Also, when stimulated at 9.09 Hz, both adult and juvenile animals had a greater decrease in MAP amplitude between the first and fourth responses (T(1)-T(4) decrement) after treatment with decamethonium. Prior to treatment with decamethonium, the T(1)-T(4) decrement averaged about 3 fold greater in juveniles than adults, and was larger at 3.03 and 9.09 Hz than with 0.91 Hz stimulation. Treatment with physostigmine resulted in at least 50% inhibition of muscle ChE activity, but produced minimal changes in the MCD values in adults or juveniles. Combined over treatments and stimulation frequencies, the median MCD for juveniles (11.6 micros) was less than that for of adults (18.8 micros). In juveniles, the median MCD increased from 9.3 micros to 13.9 micros as the stimulation rate was increased from 0.91 to 9.09 Hz. This stimulus-dependent change was more evident in juveniles than in adults., Discussion: A technique was developed to record stimulation SFEMG and neuromuscular jitter, in vivo, in adult and juvenile rats. The method was sufficiently sensitive to detect age-related differences, potentially allowing developmental processes to be examined. Based on the literature and the current data, the technique appears to be more sensitive to prolonged inhibition of ChE enzymes than the reversible inhibition produced by physostigmine.

The effects of N-methyl carbamate pesticides on the photic after discharge (PhAD) of flash evoked potentials (FEPs) and the relationship between inhibition of brain cholinesterase (ChE) activity and the PhAD were evaluated. FEPs were recorded in Long Evans rats treated with physostigmine (s.c.) 0, 0.05, 0.1, 0.2 or 0.3mg/kg (free base), in an ascorbic acid/saline vehicle, carbaryl (p.o.) 0, 1, 3, 10, 30, 50 or 75 mg/kg, or propoxur (p.o.) 0, 0.3, 3, 10, 20, 30, or 40 mg/kg in a corn oil vehicle. Physostigmine served as positive control based on literature data. Early (e.g. peak N(36)) and late FEP components (peak N(166) and PhAD) are related to the initial retino-geniculate afferent volley and higher cortical processing of visual information, respectively. Compared to controls, the PhAD duration decreased following treatment with 0.1 and 0.3mg/kg physostigmine, 7 5 mg/kg carbaryl or 30 mg/kg propoxur. Lesser changes were noted in FEP amplitudes or peak latencies. Treatment with 0.2 or 0.3 mg/kg physostigmine increased peak N(36) latency. Peak N(166) latency increased only following exposure to 40 mg/kg propoxur. None of the compounds altered peak N(36) or N(166) amplitudes. Hypothermia was observed at doses greater than 0.05 mg/kg physostigmine, at 30 or 50 mg/kg carbaryl, and after treatment with 10, 20 or 40 mg/kg propoxur. Inhibition of brain ChE activity occurred at dosages greater than 0.05 mg/kg physostigmine, 1mg/kg carbaryl, and 0.3 mg/kg propoxur. Linear regression analysis indicated that the decrease in PhAD duration correlated with decrease in brain ChE activity. The results indicate that at 30 min after treatment, inhibition of brain ChE activity did not affect cortical processing of the input from the retino-geniculate volley (evidenced by unaltered peak N(36) amplitude). However, the data suggest that disruption of cortical processing of visual signals related to FEP late components, as indicated by depression of the PhAD, was related to inhibition of brain ChE activity.

Carbonyl sulfide (COS), a chemical listed by the original Clean Air Act, was tested for neurotoxicity by a National Institute of Environmental Health Sciences/National Toxicology Program and U.S. Environmental Protection Agency collaborative investigation. Previous studies demonstrated that COS produced cortical and brainstem lesions and altered auditory neurophysiological responses to click stimuli. This paper reports the results of expanded neurophysiological examinations that were an integral part of the previously published experiments (Morgan et al., 2004, Toxicol. Appl. Pharmacol. 200, 131-145; Sills et al., 2004, Toxicol. Pathol. 32, 1-10). Fisher 334N rats were exposed to 0, 200, 300, or 400 ppm COS for 6 h/day, 5 days/week for 12 weeks, or to 0, 300, or 400 ppm COS for 2 weeks using whole-body inhalation chambers. After treatment, the animals were studied using neurophysiological tests to examine: peripheral nerve function, somatosensory-evoked potentials (SEPs) (tail/hindlimb and facial cortical regions), brainstem auditory-evoked responses (BAERs), and visual flash-evoked potentials (2-week study). Additionally, the animals exposed for 2 weeks were examined using a functional observational battery (FOB) and response modification audiometry (RMA). Peripheral nerve function was not altered for any exposure scenario. Likewise, amplitudes of SEPs recorded from the cerebellum were not altered by treatment with COS. In contrast, amplitudes and latencies of SEPs recorded from cortical areas were altered after 12-week exposure to 400 ppm COS. The SEP waveforms were changed to a greater extent after forelimb stimulation than tail stimulation in the 2-week study. The most consistent findings were decreased amplitudes of BAER peaks associated with brainstem regions after exposure to 400 ppm COS. Additional BAER peaks were affected after 12 weeks, compared to 2 weeks of treatment, indicating that additional regions of the brainstem were damaged with longer exposures. The changes in BAERs were observed in the absence of altered auditory responsiveness in FOB or RMA. This series of experiments demonstrates that COS produces changes in brainstem auditory and cortical somatosensory neurophysiological responses that correlate with previously described histopathological damage.

Exposure of pregnant Long-Evans rats to elemental mercury (Hg0) vapor resulted in a significant accumulation of Hg in tissues of neonates. Because elevated Hg in neonatal tissues may adversely affect growth and development, we were interested in how rapidly Hg was eliminated from neonatal tissues. Pregnant rats were exposed to 1, 2, or 4 mg Hg0 vapor/m3 or air (controls) for 2 hr/day from gestation day 6 (GD6) through GD15. Neonatal brain, liver, and kidney were analyzed for total Hg at various times between birth and postnatal day 90 (PND90). Milk was analyzed for Hg between birth and weaning (PND21). Before weaning, the Hg levels in neonatal tissues were proportional to maternal exposure concentrations and were highest in kidney followed by liver and then brain. There was no elimination of Hg between birth and weaning, indicating that neonates were exposed continuously to elevated levels of Hg during postpartum growth and development. Consumption of milk from exposed dams resulted in a slight increase in kidney Hg concentration during this period. Unexpectedly, neonatal Hg accumulation increased rapidly after weaning. Increased Hg was measured in both control and exposed neonates and was attributed to consumption of NIH-07 diet containing trace levels of Hg. By PND90, tissue Hg levels equilibrated at concentrations similar to those in unexposed adult Long-Evans rats fed the same diet. These data indicate that dietary exposure to trace amounts of Hg can result in a significantly greater accumulation of Hg in neonates than gestational exposure to high concentrations of Hg0 vapor.

Most toxicity data are based on studies using single compounds. This study assessed if there is an interaction between mixtures of the anticholinesterase insecticides chlorpyrifos (CHP) and carbaryl (CAR) using hypothermia and cholinesterase (ChE) inhibition as toxicological endpoints. Core temperature (T(c)) was continuously monitored by radiotelemetry in adult Long-Evans rats administered CHP at doses ranging from 0 to 50mg/kg and CAR doses of 0-150 mg/kg. The temperature index (TI), an integration of the change in T(c) over a 12h period, was quantified. Effects of mixtures of CHP and CAR in 2:1 and 1:1 ratios on the TI were examined and the data analyzed using a statistical model designed to assess significant departures from additivity for chemical mixtures. CHP and CAR elicited a marked hypothermia and dose-related decrease in the TI. The TI response to a 2:1 ratio of CHP:CAR was significantly less than that predicted by additivity. The TI response to a 1:1 ratio of CHP and CAR was not significantly different from the predicted additivity. Plasma and brain ChE activity were measured 4h after dosing with CHP, CAR, and mixtures in separate groups of rats. There was a dose-additive interaction for the inhibition of brain ChE for the 2:1 ratio, but an antagonistic effect for the 1:1 ratio. The 2:1 and 1:1 mixtures had an antagonistic interaction on plasma ChE. Overall, the departures from additivity for the physiological (i.e., temperature) and biochemical (i.e., ChE inhibition) endpoints for the 2:1 and 1:1 mixtures studies did not coincide as expected. An interaction between CHP and CAR appears to depend on the ratio of compounds in the mixture as well as the biological endpoint.

Traditional factorial designs for evaluating interactions among chemicals in a mixture may be prohibitive when the number of chemicals is large. Using a mixture of chemicals with a fixed ratio (mixture ray) results in an economical design that allows estimation of additivity or nonadditive interaction for a mixture of interest. This methodology is extended easily to a mixture with a large number of chemicals. Optimal experimental conditions can be chosen that result in increased power to detect departures from additivity. Although these designs are used widely for linear models, optimal designs for nonlinear threshold models are less well known. In the present work, the use of D-optimal designs is demonstrated for nonlinear threshold models applied to a fixed-ratio mixture ray. For a fixed sample size, this design criterion selects the experimental doses and number of subjects per dose level that result in minimum variance of the model parameters and thus increased power to detect departures from additivity. An optimal design is illustrated for a 2:1 ratio (chlorpyrifos:carbaryl) mixture experiment. For this example, and in general, the optimal designs for the nonlinear threshold model depend on prior specification of the slope and dose threshold parameters. Use of a D-optimal criterion produces experimental designs with increased power, whereas standard nonoptimal designs with equally spaced dose groups may result in low power if the active range or threshold is missed.

Mercury is known to alter neuronal function and has been shown to cross the placental barrier. These experiments were undertaken to examine if gestational exposure to mercury vapor (Hg(0)) would result in alterations in sensory neuronal function in adult offspring. Dams were exposed to 0 or 4 mg/m(3) Hg(0) for 2 h/day from gestational days 6-15. This exposure paradigm has been shown to approximate a maximal tolerated dose of Hg(0) for the dams. Between postnatal days 140-168, male and female offspring (one of each gender/dam) were examined using a battery of sensory evoked potentials. Peripheral nerve action potentials, nerve conduction velocity, somatosensory evoked responses (cortical and cerebellar), brainstem auditory evoked responses, pattern evoked potentials, and flash evoked potentials were quantified. Gestational exposure to 4 mg/m(3) Hg(0) did not significantly alter any of the evoked responses, although there was a suggestion of a decrease in compound nerve action potential (CNAP) amplitudes in male animals for the 3 mA stimulus condition. However, this possible change in CNAP amplitudes was not replicated in a second experiment. All evoked potentials exhibited predictable changes as the stimulus was modified. This shows conclusively that the evoked responses were under stimulus control, and that the study had sufficient statistical power to detect changes of these magnitudes. These results indicate that gestational exposure to 4 mg/m(3) Hg(0) did not result in changes in responses evoked from peripheral nerves, or the somatosensory, auditory, or visual modalities.

Carbonyl sulfide (COS), a high-priority Clean Air Act chemical, was evaluated for neurotoxicity in short-term studies. F344 rats were exposed to 75-600 ppm COS 6 h per day, 5 days per week for up to 12 weeks. In rats exposed to 500 or 600 ppm for up to 4 days, malacia and microgliosis were detected in numerous neuroanatomical regions of the brain by conventional optical microscopy and magnetic resonance microscopy (MRM). After a 2-week exposure to 400 ppm, rats were evaluated using a functional observational battery. Slight gait abnormality was detected in 50% of the rats and hypotonia was present in all rats exposed to COS. Decreases in motor activity, and forelimb and hindlimb grip strength were also detected. In rats exposed to 400 ppm for 12 weeks, predominant lesions were in the parietal cortex area 1 (necrosis) and posterior colliculus (neuronal loss, microgliosis, hemorrhage), and occasional necrosis was present in the putamen, thalamus, and anterior olivary nucleus. Carbonyl sulfide specifically targeted the auditory system including the olivary nucleus, nucleus of the lateral lemniscus, and posterior colliculus. Consistent with these findings were alterations in the amplitude of the brainstem auditory evoked responses (BAER) for peaks N3, P4, N4, and N5 that represented changes in auditory transmission between the anterior olivary nucleus to the medial geniculate nucleus in animals after exposure for 2 weeks to 400 ppm COS. A concentration-related decrease in cytochrome oxidase activity was detected in the posterior colliculus and parietal cortex of exposed rats as early as 3 weeks. Cytochrome oxidase activity was significantly decreased at COS concentrations that did not cause detectable lesions, suggesting that disruption of the mitochondrial respiratory chain may precede these brain lesions. Our studies demonstrate that this environmental air contaminant has the potential to cause a wide spectrum of brain lesions that are dependent on the degree and duration of exposure.

In this carbonyl sulfide (COS) study, magnetic resonance microscopy (MRM) and detailed light microscopic evaluation effectively functioned in parallel to assure that the distribution and degree of pathology in the brain was accurately represented. MRM is a powerful imaging modality that allows for excellent identification of neuroanatomical structures coupled with the ability to acquire 200 or more cross-sectional images of the brain, and the ability to display them in multiple planes. F344 rats were exposed to 200-600 ppm COS for up to 12 weeks. Prior to MRM, rats were anesthetized and cardiac perfused with McDowell Trump's fixative containing a gadolinium MR contrast medium. Fixed specimens were scanned at the Duke Center for In Vivo Microscopy on a 9.4 Tesla magnetic resonance system adapted explicitly for microscopic imaging. An advantage of MRM in this study was the ability to identify lesions in rats that appeared clinically normal prior to sacrifice and the opportunity to identify lesions in areas of the brain which would not be included in conventional studies. Other advantages include the ability to examine the brain in multiple planes (transverse, dorsal, sagittal) and obtain and save the MRM images in a digital format that allows for postexperimental data processing and manipulation. MRM images were correlated with neuroanatomical and neuropathological findings. All suspected MRM images were compared to corresponding H&E slides. An important aspect of this study was that MRM was critical in defining our strategy for sectioning the brain, and for designing mechanistic studies (cytochrome oxidase evaluations) and functional assessments (electrophysiology studies) on specifically targeted anatomical sites following COS exposure.

Background: Microtia is a reduction in pinna size, usually seen in humans in conjunction with other medical conditions. We report microtia in CD-1 mice after gestational exposure to ethane dimethanesulfonate (EDS), an alkylating agent and adult rat Leydig cell toxicant., Methods: Time-pregnant CD-1 mice were administered 0, 80, or 160 mg EDS/kg on gestation days (GD) 11-17, or 0 or 160 mg EDS/kg on GD 11-13, GD 13-15 or GD 15-17. Pinnae were measured on postnatal days (PND) 4, 8, 18, and 28; and were observed for detachment from birth through PND 8. Branchial-arch derived skeletal structures and histology of the pinna was examined on PND 4 and 24. Brainstem auditory evoked response (BAER) tests were carried out at approximately PND 160 to determine possible effects on hearing., Results: All offspring of EDS-treated dams exhibited bilateral, dose-related decreases in pinna size. Gestational exposure during GD 11-13 produced smaller ears than during GD 13-15 or 15-17, but not as small as the GD 11-17 regimen. Ossification of other pharyngeal arch derivatives was delayed whereas histology was unremarkable. BAER analysis showed a decrease in the proportion of adult offspring producing a quantifiable response to varied auditory stimuli among EDS-treated litters., Conclusions: Gestational exposure to EDS affects pinna development in the mouse, with a broad period of sensitivity during the second half of gestation. Microtia induced by EDS may be associated with hearing deficits, suggesting functional importance of pinna size or additional effects of EDS on ear development not detected by morphological examination.

Exposure to organic solvents frequently causes functional impairment of the central nervous system (CNS). One method to examine the effects of solvent exposure on visual function is flash-evoked potentials (FEPs). Greater knowledge of the role of various neurotransmitters in generating FEP peaks would be beneficial for understanding the basis of neurotoxicant-induced changes. FEP peak N166 is influenced by the psychological construct of arousal, which in turn is believed to be influenced by the function of neurons containing norepinephrine (NE). Because of its known effects on both NE and FEPs, we utilized carbon disulfide (CS2) as a means to examine the possible role of NE in modulating the amplitude of FEP peaks N36 and N166. Our hypothesis was that CS2-induced alterations in cortical NE levels would be correlated with changes in FEP peak N36 and N166 amplitudes. Adult male Long-Evans rats were implanted with electrodes over their visual cortex and allowed to recover. To develop peak N166, FEPs were recorded for two days prior to dosing. On the third day, FEPs were recorded prior to dosing, and one group of animals was sacrificed to serve as pretreatment controls. The remaining animals were dosed ip with 0 (corn oil vehicle; 2 ml/kg), 100, 200, or 400 mg/kg CS2. The treated animals were retested at 1, 4, 8, or 24 h after dosing, immediately sacrificed, and samples of the cortex, cerebellum, striatum, and brain stem were frozen for high performance liquid chromatography (HPLC) analysis of monoamine levels. Treatment with CS2 decreased peak N166 amplitude at 1 h, and peak N36 amplitude was depressed at 4 h, relative to the subject's pretreatment values. Peak latencies were increased, and colonic temperature was decreased by treatment with CS2. Exposure to CS2 depressed NE levels in the cortex, brain stem, and cerebellum 4 h after treatment. Conversely, at 4 h, levels of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid were increased in the brain stem and cerebellum, and levels of the DA metabolite homovanillic acid were increased in the brain stem. Levels of serotonin were unaffected by CS2 treatment. There was a slight increase in striatal levels of the serotonin metabolite 5-hydroxyindole acetic acid at all times after treatment with CS2. There was no apparent association between the decreases in NE levels and the reductions in amplitudes for peaks N36 and N166. The neurochemical mechanism for CS2-induced reductions in FEP peak amplitudes remains to be determined.

Pregnant Long-Evans rats were exposed to 0, 1 or 6 mg/kg/day of Aroclor 1254 (A1254; Lot no. 124-191), a commercial mixture of polychlorinated biphenyls (PCBs), from gestation day (GD) 6 through postnatal day (PND) 21. At 128-140 days of age, male and female offspring were tested for visual-, somatosensory- and peripheral nerve-evoked potentials. The evoked responses increased in amplitude with larger stimulus intensities, and gender differences were detected for some endpoints. In contrast, developmental exposure to A1254 failed to significantly affect the electrophysiological measures. A subset of the animals were tested for low-frequency hearing dysfunction using reflex modification audiometry (RMA). An elevated threshold for a 1-kHz tone was observed, replicating previous findings of A1254-induced auditory deficits [Hear. Res. 144 (2000) 196; Toxicol. Sci. 45(1) (1998) 94; Toxicol. Appl. Pharmacol. 135(1) (1995) 77.]. These findings indicate no statistically significant changes in visual-, somatosensory- or peripheral nerve-evoked potentials following developmental exposure to doses of A1254 that produce behavioral hearing deficits. However, subtle changes in the function of the visual or somatosensory systems cannot be disproved.

A system has been developed for collecting sensory evoked potentials simultaneously from multiple channels for multiple subjects at up to 80 kHz sample rate per channel. Sample rates up to 200 kHz are available for four or less chambers and a single channel per chamber. A variety of visual, somatosensory, and auditory stimuli may be presented singly or simultaneously. Collected waveforms are associated with searchable text (metadata) to allow convenient selection from a relational database. Multiple waveforms can then be easily grouped for analysis and processed. Results can be exported to other software for further graphics or statistical processing. Scripting and event logging are available to provide automation and improve data confidence. Sample data are presented from control animals for each of the sensory modalities for comparison with historical data collected from other systems.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long-Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+ buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+ buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels., (Copyright 1998 Academic Press.)

Trichloroethylene (TCE) is known to produce an unusual pattern of hearing impairment in laboratory animals marked by a preferential loss of threshold sensitivity at midfrequencies. The purpose of this research was to determine whether the TCE-induced auditory deficit results from cochlear dysfunction. Adult Long Evans hooded rats were exposed via inhalation to either 0 (clean air) or 4000 ppm TCE (6 h/day for 5 days). Auditory thresholds for 1-40 kHz tones were determined 3 weeks after exposure using reflex modification audiometry (RMA; n = 12/group). Cochlear electropotentials were measured during subsequent testing (n = 3-10/group) 5 to 7 weeks after exposure, including thresholds for cochlear action potentials (CAP) and the 1-microV cochlear microphonic for 2-40 kHz tones, and the N1 amplitude intensity function (40-90 dB SPL). Cochlear histopathology was assessed in midmodiolar preparations of a separate set of animals, exposed as before (n = 4/group). RMA testing confirmed a TCE-induced loss in midfrequency threshold sensitivity (8 and 16 kHz). CAP thresholds were elevated at midfrequencies (8 and 16 kHz) among TCE-treated subjects, along with a suppression of the N1 amplitude from 50 to 90 dB SPL. The cochlear microphonic, a nonpropagated ac potential generated largely by the outer hair cells, was not affected by the TCE treatment. Cochlear histopathology revealed a loss of spiral ganglion cells that was significant in the middle turn, but not in the basal turn. There was an inconsistent loss of hair cells among treated subjects. The data suggest strongly that the behaviorally determined loss in auditory function can be accounted for by a cochlear impairment and that the spiral ganglion cell may be a prominent target of TCE., (Copyright 1998 Academic Press.)

The effects of subchronic exposure to carbon disulfide (CS2) on ventral caudal tail nerve compound nerve action potential (CNAP) amplitudes and latencies, and nerve conduction velocity (NCV) in rats were examined. Male and female Fischer 344 rats were exposed to 0, 50, 500, or 800 ppm CS2 for 6 hrs/day, 5 days/week. Using separate groups, exposure duration was 2, 4, 8, or 13 weeks. Exposure to 500 or 800 ppm CS2 for 13 weeks decreased NCV compared to the 50 ppm CS2 group. CNAP amplitudes were increased, and peak P1P2 interpeak latency decreased, after exposure to 500 or 800 ppm CS2 for 13 weeks. Most of the changes in NCV and CNAPs were not attributable to differences in tail or colonic temperature. However, the increases in peak P1 amplitude may relate to the proximity of the electrodes to the tail nerves. Assessment of tail nerve morphology after 13 weeks exposure to 800 ppm CS2 revealed only minor changes compared to the extent of axonal swelling and degeneration observed in the muscular branch of the tibial nerve and axonal swelling in the spinal cord. As anticipated, in older animals the NCV increased, the CNAP amplitudes increased, and the CNAP latencies decreased. The biological basis for the changes in CNAPs produced by CS2 is under investigation. Future studies will focus on electrophysiological evaluation of spinal nerve function, to allow better correlation with pathological and behavioral endpoints.

Previous research showed that acute exposure to dichloromethane (DCM) produced a selective reduction in peak N30 of flash evoked potentials (FEPs) in rats. In contrast, acute exposures to p-xylene or toluene selectively reduced FEP peak N160. The present experiments compared the effects of DCM (log P = 1.25; oil:water partition coefficient), 1,3-dichloropropane (DCP; log P = 2.00), and 1,2-dichlorobenzene (DCB; log P = 3.38) on FEPs recorded from adult Long-Evans rats. Before administration of test compounds, FEPs were recorded for five daily sessions to develop FEP peak N160. Test compounds were dissolved in corn oil and administered i.p. at doses based on proportions of their LD50 values. The doses were: DCM, 0, 57.5, 115, 230, or 460 mg/kg; DCP, 0, 86, 172, 343, or 686 mg/kg; and DCB, 0, 53, 105, 210, or 420 mg/kg. Testing times after dosing varied among compounds and were based on pilot studies to measure both the times of peak effect and recovery. Each solvent produced significant changes in the latency and amplitude of multiple components of the FEP waveforms. However, the predominant effect of DCM was to reduce the amplitude of peak N30 (ED50 = 326.3 mg/kg), that of DCP was to reduce both peaks N30 (ED50 = 231.0 mg/kg) and N160 (ED50 = 136.8 mg/kg), and that of DCB was to reduce peak N160 (ED50 = 151.6 mg/kg). There was no consistent relationship between log P values and the potency of the compounds to alter FEP peaks N30 and N160. The results suggest that organic solvents have multiple acute effects on the function of the central nervous system, which are not predictable solely by the compound's lipid solubility.

Developmental exposure of Long-Evans rats to 0, 1, 4, or 8 mg/kg/day Aroclor 1254 (A1254) from Gestational Day 6 through Postnatal Day 21 produces an elevated behavioral threshold for a 1-kHz tone. Brainstem auditory evoked responses (BAERs) were assessed in a subset of these animals (about 1 year old) using filtered clicks at 1 (65 and 80 dB SPL), 4 (60 and 80 dB SPL), 16 (40 and 80 dB SPL), and 32 (40 and 80 dB SPL) kHz. Aroclor 1254 decreased BAER amplitudes at 1 and 4 kHz, but not at 16 or 32 kHz. A dose-related decrease in the baseline-to-peak P1A amplitude was observed for the 1-kHz (80-dB) stimulus. Doses of 1, 4, or 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of both P1AN1 and P1BN1 for a 1-kHz (80-dB) stimulus. Doses of 4 and 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of N1P2 and P2N2 for a 4-kHz (60-dB) or 1-kHz (80-dB) stimulus. At 8 mg/kg/day, A1254 also increased the latency of peak P4 at 1 kHz (65 dB). The decreases in peak P1A amplitudes are consistent with a dysfunction of the cochlea and/or auditory nerve. Together, the data confirm that developmental exposure of rats to A1254 produces a permanent low- to mid-frequency auditory dysfunction and suggest a cochlear and/or auditory nerve site of action.

Flash evoked potentials (FEPs) undergo within- and between-session changes and are modified by auditory white noise (26). We examined whether an auditory potential produced by the "click" associated with the strobe discharge could be recorded, and if alterations in an auditory response could explain the within- and between-session changes in FEPs. We also examined differences between a frontal cortex or a nasal reference electrode location on FEPs and auditory potentials. An auditory potential associated with the strobe discharge could be clearly recorded. This response was eliminated by the presence of 80 dB SPL masking white noise. However, the within- and between-session changes in FEPs could not be explained by modifications of the auditory potential. Animals whose ear drums were ruptured did not exhibit an auditory response, and their FEPs were similar to those of controls tested in the presence of masking white noise. A nasal reference electrode decreased the impact of auditory potentials on FEPs, but allow visual potentials (electroretinogram and optic tract activity) to influence FEPs. The data show that auditory potentials associated with the strobe discharge can be recorded from the visual cortex of rats, and that these auditory responses represent a possible confounding factor in the interpretation of toxicological studies employing FEPs.

3,3'-Iminodipropionitrile (IDPN) is a neurotoxicant that produces changes in flash evoked potentials (FEPs) 18 weeks after treatment. We examined dose- and time-related effects of IDPN on FEPs at earlier time points than previously studied (52). Adult male Long-Evans rats were given IDPN (0, 100, 200, 400 mg/kg/day x 3 days, i.p.) and FEPs were recorded 14 days later. IDPN (400 mg/kg/day) decreased the amplitudes of some of the "early" and "middle" FEP peaks (n30 and N56), and increased the latencies of some early peaks (P21 and P46). A separate group of rats was treated with IDPN (0 or 400 mg/kg/day x 3 days, i.p.) and FEPs were recorded 1, 3, 7, 14, and 35 days later. The latencies of of all portions of FEPs were increased by IDPN, with maximal changes occurring at 7 and/or 14 days. The amplitude of the middle portions of FEPs (peaks N56, P63, N70, P90) were altered as early as day 3, and some changes were observed up to day 14. In contrast, the "late" portion of FEPs (peak N160) was affected at later times (days 14 and 35). Corneal opacities were noted on days 3 and 7, but were largely reversible by day 14. In the time-course study, IDPN decreased colonic temperature on days 1, 3, 7, and 14. The present results suggest that IDPN alters both the early FEP peaks related to the initial afferent sensory volley, and cortical processing associated with the middle and later portions of FEPs.

Adult male Long-Evans rats received 3,3'-iminodipropionitrile (IDPN; 400 mg/kg i.p.) and were killed one day after one dose, or one, three, seven, thirty-five, or seventy day(s) following 3 consecutive daily doses for histological analysis of the eye. Histological alterations in visual structures were not observed before one day after the third dose of IDPN. Somato-dendritic swelling of cells in the inner nuclear (IN) layer was seen prior to retinal detachment (1 day after cessation of dosing) followed by progressive retinal degeneration (35 and 70 days). IDPN exposure resulted in opacification of the cornea and vascular hemorrhaging into the subretinal space (3 days) followed by complete detachment of the retina (7 days). The corneal opacification was transient and resolved by 14 days post-treatment. The retina underwent complete spontaneous reattachment between 35 and 70 days after IDPN administration. A subsequent experiment was performed to characterize the dose-response of IDPN on retinal histology, 2 weeks after the last dose (0, 100, 200, 400 mg/kg x 3 days). In the dose-response experiment, retinal detachment and degeneration in the IN layer were only apparent in the 400 mg/kg dose group. However, increased GFAP immunoreactivity in the retina was observed in the 200 mg/kg dose group without overt retinal pathology. Results indicate that the corneal opacification, vascular hemorrhaging, and detached retinae recovered in a time-dependant manner, while neurodegeneration of the visual retina was progressive, even after the retina had reattached. The present study indicates that this toxicant may have direct effects on both neural and non-neural structures, and characterizes the time-course and dose-response of histopathological changes in the retina.

Colchicine and ibotenic acid were compared for their ability to produce neurodegeneration and cognitive deficit after bilateral infusions into the nucleus basalis magnocellularis of male Long-Evans rats. Four weeks post-lesion, there was no difference in locomotor activity following infusion of either neurotoxicant or vehicle. In a passive avoidance task, both treated groups had significantly shorter step-through latencies compared with vehicle. Five weeks post-lesion, rats were killed for neurochemistry or histochemistry. Choline acetyltransferase (ChAT) activity in both the frontal and parietal cortex was significantly decreased (25-35%) in the colchicine- and ibotenic acid-infused rats when compared to control. There was no effect of either neurotoxicant on ChAT activity in the hippocampus or striatum. Both neurotoxicants produced damage in the general area of the ventromedial pallidum, although ibotenic acid infusion consistently produced a larger area of damage as assessed in Nissl-stained sections. Analysis of the number of ChAT-immunoreactive cells in the nucleus basalis magnocellularis (NBM) showed an average 60% cell loss following colchicine infusion and a 75% cell loss after ibotenic acid infusion. Area of glutamic acid decarboxylase (GAD) staining was significantly decreased in several regions surrounding the NBM for ibotenic acid (51% average decrease), and showed non-significant decreases (28%) following colchicine infusion. Colchicine infusion decreased dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum; ibotenic acid had no effect on brain catechol of indoleamine levels. The results indicate that although similar cholinergic hypofunction and behavioral deficits were achieved, several non-cholinergic differences between the neurotoxicants were detected.

The negative peak occurring approximately 160 ms after stimulation (peak N160) of flash evoked potentials (FEPs) of rats changes with repeated testing. Habituation, sensitization, and arousal have all been invoked to explain these changes, but few studies have directly tested these explanations. We examined within-session changes in peak N160 amplitude with repeated testing, and the modulatory effects of stimulus intensity and auditory white noise. Peak N160 amplitude increased with daily testing (between-session changes), and was larger at greater stimulus intensities. In contrast, peak N160 amplitude underwent within-session increases on early days and within-session decreases on later days. The within-session changes were not affected by stimulus intensity. In rats previously tested in a quiet environment, exposure to acoustic white noise increased motor activity and transiently decreased peak N160 amplitude, which then increased and subsequently decreased with continued photic and acoustic stimulation. Repeated testing in the presence of noise did not alter the within-session changes in peak N160 amplitude. Heart rate showed both within- and between-session decreases, but was unaffected by noise. The data suggest that the within-session changes in peak N160 amplitude may reflect a habituation-like response to the test environment.

Because solvents may selectively alter portions of visual evoked potentials, we examined the effects of carbon disulfide (CS2) on flash (FEPs) and pattern reversal (PREPs) evoked potentials. Long-Evans rats were administered ip carbon disulfide either acutely or for 30 days. FEPs or PREPs were recorded prior to and 1, 2, 4, 8, or 24 hr after a single dose of CS2 (0, 100, 200, 400, or 500 mg/kg). Flash evoked potentials were also recorded 1, 2, 6, and 24 hr after the last of 30 doses of 200 mg CS2/kg/day. Acute exposure to CS2 consistently decreased the amplitude of FEP peak N160 at 1 hr, depressed peak N30 amplitude over 2-4 hr, and increased the latency of peaks P21, N30, P46, N56, and N160 for up to 4 hr after treatment. Carbon disulfide decreased the amplitude of PREP peaks P65, N83, P88, and N122 4 hr after treatment. Colonic temperature was depressed up to 8 hr after treatment. Administration of 200 mg CS2/kg/day decreased the amplitude of FEP peak N30 and increased the latencies of peaks P21, N30, P46, N56, and N160 up to 24 hr after the last dose. The differential effects of CS2 on portions of FEPs indicate that FEP peaks can be independently modulated. Changes in PREPs were temporally correlated with alterations in early FEP peaks, but FEP peak N160 was depressed at an earlier time point. Repeated CS2 exposure affected FEPs at lower doses and for a longer time than an acute exposure, similar to the reported greater severity of neurological disturbances following repeated CS2 exposures in humans.

Tris(2-chloroethyl) phosphate (TRCP) is a flame retardant that has a wide variety of industrial applications. In subchronic studies, oral administration of TRCP to rats and mice has been reported to produce dose-, sex-, and species-dependent lesions in the hippocampal brain region. The present investigation has examined the metabolism, elimination, and regional brain distribution of [14C]TRCP in male and female rats. [14C]TRCP was administered by gavage (0, 175, 350, or 700 mg/kg) and urine, feces, exhaled volatiles, CO2, and selected tissues were collected. Regional brain distribution of 14C was determined 2 hr following single doses of TRCP to male and female rats, and 24 hr after a single dose and the last of 14 daily doses of TRCP to female rats. Results of these studies indicate that TRCP is readily absorbed from the gastrointestinal tract, distributed to all brain regions, and that metabolism and excretion are nearly complete in 72 hr. Most of the TRCP-derived radioactivity was excreted in urine (up to 85%), with feces, volatiles, and CO2 combined accounting for less than 10% of the dose. Predominant signs of toxicity associated with TRCP administration (350 and 700 mg/kg) were seizures within 2 hr of treatment, when most of the TRCP-derived radioactivity present in brain tissue was in the form of the parent compound. Traces of inextractable 14C were detected at later times, but this material was not concentrated in brain relative to other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Tris(2-chloroethyl) phosphate (TRCP), a flame retardant, produces a dose-, sex-, and species-dependent lesion in the hippocampal region of the brain following subchronic oral administration. This lesion is more common and more severe in female F344 rats than in male F344 rats, and is not observed in B6C3F1 mice. The present investigation of the metabolism of TRCP was designed to detect sex and species variations that might account for differences in toxicity. Elimination of TRCP-derived radioactivity was more rapid in mice, which excreted greater than 70% of an oral dose of 175 mg/kg in urine in 8 hr vs. approximately 40% for male or female rats. However, the metabolic profile of TRCP-derived radioactivity in urine was similar for both species. The major metabolite in female rat urine was identified as bis(2-chloroethyl) carboxymethyl phosphate. This metabolite co-chromatographed with the major metabolite found in both male rat and mouse urine. Two additional metabolites identified in female rat urine were bis(2-chloroethyl) hydrogen phosphate and the glucuronide of bis(2-chloroethyl) 2-hydroxyethyl phosphate. These metabolites also cochromatographed with metabolites found in male rat and mouse urine. TRCP metabolism in rats was not induced or inhibited by nine daily 175 mg/kg doses. Toxicity, as evidenced by seizures, was potentiated in male rats pretreated with inhibitors of aldehyde dehydrogenase.