Acute toxicologic and neurotoxic effects of inhaled 1,2-dichloroethane in adult Fischer 344 rats.

Acute toxicologic and neurotoxic effects were evaluated in Fischer 344 rats exposed to 0, 50, 200, 600, or 2000 ppm 1,2-dichloroethane (ethylene dichloride; EDC) for 4 h or 0, 50, 100 or 150 ppm for 8 h. Neurobehavioral and neuropathologic effects were assessed using a functional observational battery (FOB; baseline, days 1, 8, and 15), and by light microscopy, respectively. Acute toxicologic effects were assessed by bronchoalveolar lavage (BAL) and histopathology of the respiratory tract and selected target organs. Neurobehavioral effects consistent with central nervous system (CNS) depression were present at concentrations >200 ppm and were restricted to day 1. There were no neuropathologic changes in the CNS, however, olfactory epithelial regeneration 15 days after exposure to > or = 200 ppm was observed. The no-observed-effect concentration (NOEC) for behavioral neurotoxicity was 200 ppm EDC for 4 h. There were no effects on BAL parameters in any exposure group. Exposure to 2000 ppm EDC altered adrenal gland, kidney, and liver weights, and resulted in morphologic alterations in the kidney and liver. Degeneration/necrosis of the olfactory epithelium was observed at > or = 200 ppm for 4 h and > or = 100 ppm for 8 h. Based on olfactory epithelial degeneration/necrosis, the most sensitive indicator of toxicity in this study, the overall NOEC was 50 ppm EDC for up to 8 h in rats.

Diethylene glycol monobutyl ether (DGBE): two- and thirteen-week oral toxicity studies in Fischer 344 rats.

Standard toxicologic endpoints, supplemented by additional examinations, were studied for groups of 10 Fischer 344 rats/sex given drinking water formulated to supply 0, 50, 250, or 1000 mg diethylene glycol monobutyl ether (DGBE)/kg/day for 13 weeks. These dose levels were based upon initial investigations using drinking water formulated to supply 0, 1000, 1500 or 2000 mg DGBE/kg/day for two weeks. All rats survived the respective treatment intervals with no adverse treatment-related in-life effects, including no alterations in a functional observational battery. In both studies, rats given > or = 1000 mg/kg/day consumed less water and feed and weighed slightly less than controls. For rats given > or = 1000 mg DGBE/kg/day, the liver and red blood cells (RBC) were the primary target organs although the effects were slight. In the 13-week study, rats given 1000 mg/kg/day had statistically significant increased relative liver weight (7-10%) and hepatic cytochrome P450s (24-39%) and UGT (approximately 16%) levels along with slight, statistically significant, decreases in serum total protein, cholesterol and aspartate aminotransferase. Histopathologically, very slight hepatocyte hypertrophy and increased individual hepatocyte degeneration were found in females only. At 1000 mg/kg/day, the RBC count, hemoglobin (Hgb) and hematocrit (Hct) were minimally, but statistically significantly, decreased (5.1-8.7%) but RBC morphology, RBC indices, reticuloctye count and bone marrow and spleen histopathology were unaffected. Absolute and relative kidney weights statistically significantly increased (6-13%) with an equivocal increase in minor histopathologic changes typical of early spontaneous nephropathy. There were no adverse effects on urinalysis, clinical chemistry, sperm parameters or testis histopathology. At 250 mg/kg/day, there were equivocal decreases (approximately 2-3%) in RBC count, Hgb and Hct that were statistically significant for the RBC count and Hgb, but these changes were within the historical control range. This dose level was considered the no adverse effect level (NOAEL).

Lack of selective developmental neurotoxicity in rat pups from dams treated by gavage with chlorpyrifos.

Pregnant Sprague-Dawley rats were given chlorpyrifos (O:, O-diethyl-O:-[3,5,6-trichloro-2-pyridinyl] phosphorothioate; CPF) in corn oil by gavage from gestation day 6 (GD 6) through lactation day 10 (LD 10) at dosages of 0, 0.3, 1, or 5 mg/kg/day in a developmental neurotoxicity study that conformed to U.S. Environmental Protection Agency 1991 guidelines. GD 0 was the day when evidence of mating was observed and postnatal day 0 (PND 0) was the day of birth. Toxicity was limited to the highest dosage level (5 mg/kg/day) and, in the dams, consisted of muscle fasciculation, hyperpnea, and hyperreactivity. A nonsignificant overall trend toward weight gain and feed consumption was also observed in the high-dosage dams, with a statistically significant Group x Time interaction for reduced weight gain in the 5-mg/kg/day group near the end of gestation. Although many developmental indices were normal, pups from high-dosage dams had increased mortality soon after birth, gained weight more slowly than controls, and had several indications of slightly delayed maturation. The early deaths and delayed maturation were attributed to maternal toxicity, though a possible contributing role of direct pup toxicity in delayed development cannot be eliminated. In spite of the apparent delay in physical development, high-dosage pups tested just after weaning had normal learning and memory as tested on a T-maze spatial delayed-alternation task. Habituation, a primitive form of learning, was tested in 2 tasks (motor activity and auditory startle) and was not affected. No overt effects were noted in either dams or pups at 1 or 0.3 mg/kg/day. Based on these data, chlorpyrifos produced maternal and developmental toxicity in the 5-mg/kg/day-dosage group. There was no evidence of selective developmental neurotoxicity following exposure to chlorpyrifos.

Chlorpyrifos: lack of cognitive effects in adult Long-Evans rats.

Female Long-Evans rats were gavaged 5 days a week for 4 weeks with chlorpyrifos in oil at dosages of 0, 1, 3, and 10 mg/kg/day. Clinical observations were conducted, and memory was tested with a delayed matching-to-position task (DMTP). Before exposure started, the rats were divided into four groups of ten of comparable overall performance. Then, they were tested during four weeks of dosing and for another four weeks thereafter. The observer was not provided information about each rat's dose group identification. Miosis was a prominent sign observed in the 3 and 10 mg/kg/day groups. Rectal temperature was reduced in the 10 mg/kg/day group. Noncognitive performance measures in the DMTP test (e.g., actual total delay, void trials) were affected and consistent with decreased motor activity. There was a statistically significant difference in the intercept at the zero delay (i.e., a measure of encoding/motivation/attention), which was attributed to deviations from controls in the high-dosage group during dosing weeks 2 and 3 (in opposite directions). This difference was not considered treatment related. The slope of the retention gradient (i.e., a measure of forgetting rate) did not show any statistically significant difference between groups at dosages that inhibited brain cholinesterase by up to approximately 85%. In conclusion, chlorpyrifos decreased motor activity but had no effects on short-term memory (i.e., information retention capability) and on encoding/motivation/attention.

Lack of differential sensitivity to cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally with chlorpyrifos.

Pregnant Sprague-Dawley rats were exposed to chlorpyrifos (CPF; O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) by gavage (in corn oil) from gestation day (GD) 6 to postnatal day (PND) 10. Dosages to the dams were 0 (control), 0.3 (low), 1.0 (middle) or 5.0 mg/kg/day (high). On GD 20 (4 h post gavage), the blood CPF concentration in fetuses was about one half the level found in their dams (high-dose fetuses 46 ng/g; high-dose dams 109 ng/g). CPF-oxon was detected only once; high-dose fetuses had a blood level of about 1 ng/g. Although no blood CPF could be detected (limit of quantitation 0.7 ng/g) in dams given 0.3 mg/kg/ day, these dams had significant inhibition of plasma and red blood cell (RBC) ChE. In contrast, fetuses of dams given 1 mg/kg/day had a blood CPF level of about 1.1 ng/g, but had no inhibition of ChE of any tissue. Thus, based on blood CPF levels, fetuses had less cholinesterase (ChE) inhibition than dams. Inhibition of ChE occurred at all dosage levels in dams, but only at the high-dose level in pups. At the high dosage, ChE inhibition was greater in dams than in pups, and the relative degree of inhibition was RBC approximately plasma > or = heart > brain (least inhibited). Milk CPF concentrations were up to 200 times those in blood, and pup exposure via milk from dams given 5 mg/kg/day was estimated to be 0.12 mg/kg/day. Therefore, the dosage to nursing pups was much reduced compared to the dams exposure. In spite of exposure via milk, the ChE levels of all tissues of high-dosage pups rapidly returned to near control levels by PND 5.

Symposium overview: the use of delayed matching-to-sample procedures in studies of short-term memory in animals and humans.

Behavioral paradigms applicable for use in both human and nonhuman subjects for investigating aspects of working/short-term memory are presented with a view towards exploring their strengths, weaknesses, and utility in a variety of experimental situations. Such procedures can be useful in teasing out specific aspects of mnemonic processes including discrimination, encoding, and retention. Delayed matching-to-position, delayed matching-to-sample (DMTS), and titrating matching-to-sample procedures are highlighted. Additionally, the application of DMTS tasks in preclinical and clinical settings is presented: drug effects on memory processes can be explored preclinically in animal models; normative data have been developed in human populations where they have been used in adults to explore the relationships between mnemonic processes and specific clinical entities such as Parkinsonism, senile dementia of the Alzheimer's type, schizophrenia, and depression. Studies in children indicate that encoding and retention processes improve rapidly in the early years, plateauing prior to puberty. Noninvasive imaging techniques such as positron emission tomography (PET) indicate that activity in specific brain areas is associated with DMTS task performance and may serve to confirm roles for such structures in mnemonic processes.

4-Phenylcyclohexene: 2-week inhalation toxicity and neurotoxicity studies in Swiss-Webster mice.

4-Phenylcyclohexene (4-PCH) is a by-product formed during the polymerization of styrene-butadiene latex used in carpet backing. Limited reports suggest that exposure to very low levels of 4-PCH or other emission products following new carpet installation may result in health complaints. Significantly, it has been claimed that Swiss-Webster mice held in neck restraints and exposed head-only to approximately 0.4 ppm 4-PCH for a few hours suffered severe toxicity including death. A 2-wk inhalation and neurotoxicity study was therefore conducted in Swiss-Webster mice using standard methods of toxicity testing. Groups of 40 mice were exposed to 0, 7, 18 or 71 ppm (near-saturated atmosphere) 4-PCH vapour, 6 hr/day for 9 consecutive days. Data were collected on a wide variety of clinical, neurological and histopathological parameters including extensive neurohistopathology. All animals survived the exposures, and there were no treatment-related effects. Because of the occurrence of spontaneous lesions in two high-dose group mice, 40 additional males were exposed to 0 ppm or a near-saturated atmosphere of 4-PCH under the same exposure regimen. No treatment-related lesions were observed in the follow-up study, confirming the conclusions of the original study. These findings, consistent with the reported lack of toxicity of inhaled 4-PCH in rats, do not suggest a direct, organic, association between low-level 4-PCH exposure and human complaints. Further, the results of this study suggest that positive findings in mice may have been due to methodological problems and not to exposure to 4-PCH.

Single-dose and 13-week repeated-dose neurotoxicity screening studies of chlorpyrifos insecticide.

Chlorpyrifos (CPF), a widely used organophosphate insecticide, was screened for neurotoxic effects in Fischer 344 rats using United States Environmental Protection Agency 1991 guidelines for single-dose and 13-wk repeated dose studies. The studies emphasized a functional observational battery (which included grip performance and hindlimb splay tests), automated motor activity testing and comprehensive neurohistopathology of perfused tissues. Doses of up to 100 mg/kg body weight in corn oil by gavage in the single-dose study and up to 15 mg/kg body weight/day in diet for 13 wk in the repeated dose study were administered. It is known that CPF and other phosphorothionates can be activated to the oxon in local (extrahepatic) tissues. Local activation could possibly cause different effects in different tissues with cholinergic innervation, and thereby create syndromes unique to each phosphorothionate according to their structure. Consequently, the conduct of CPF neurotoxicity screening studies by contemporary guidelines offered opportunity to characterize the CPF over-exposure syndrome in rats. Single-dose high levels of oral exposure to CPF caused a range of clinical signs characteristic of cholinergic overstimulation. Although there was no clinical evidence of wide differences in sensitivity of one cholinergic response versus another, motor dysfunction (incoordination etc.) was more prominent than other signs, for example soiling. Effects were much more apparent in females and regressed over several days. Effects were minimal in the 13-wk study, and there was no evidence of accumulation of toxicity during the 13 wk of daily dietary exposure. Motor activity was decreased at the high dose in males and females at wk 4, but was not significantly different from controls in subsequent weeks. The 'normalization' of motor activity later in the study was interpreted as tolerance to repeated administration of CPF. Comprehensive neuropathological examination revealed no treatment-related lesions in either study.

A few considerations in the design and analysis of experiments in neurotoxicology.

To help minimize errors and confusion in the scientific literature, a few principles can be followed. The original intent and hypothesis of each study should be made clear, and deviations from the initial purpose should be stated. The hypothesis-testing portions of the research need to be clearly differentiated from the hypothesis generating sections. If data were selected and reanalyzed in unintended ways, the analyses should be clearly identified as hypothesis generating, and no conclusions should be drawn from such data. The data should be unassailable. The control group should be adequate. The data analysis should be consistent with the experimental design. Given that the larger the number of P-values, the greater the rate of false declarations, the total number of derived P-values should be reasonable and should be reported. The publication should unveil all details necessary to understand and replicate the research project, including the data analysis. Alternate etiologies should be seriously considered in light of potential confounding factors. It is important to be critical of the results of a study, especially when data confirm a preconceived hypothesis.

Vibration sensitivity recovery after a second course of acrylamide intoxication.

Five monkeys were trained to report detection of vibration or an electrical stimulus. Three of them had been dosed previously with acrylamide administered orally until toxic signs appeared, and were then allowed to recover. Approximately 30 weeks after the end of the first dosing course, they were dosed a second time with 10 mg/kg/day of acrylamide, 5 days a week, to mimic the dosage regimen of the first dosing period. Two monkeys received vehicle, as in the first dosing period. Vibration sensitivity was reduced in the exposed monkeys to an extent comparable to the first dosing period. Analysis of covariance showed that the rate of recovery from a second exposure to acrylamide was comparable to the rate of recovery from the first exposure. Vibration sensitivity appears useful as a monitoring tool to follow the time course of acrylamide-induced peripheral nerve dysfunction.

Uses of the coefficient of detection and sample size determination.

The coefficient of detection (CD) is said to represent the minimal difference statistically detectable with a t-test at some preselected alpha. Although the CD does not explicitly take statistical power into consideration, it is mathematically and empirically demonstrable that the CD operates at approximately 50% power. The CD, as reported in the Collaborative Behavioral Teratology Study (CBTS), also applies only to hypotheses about the difference between one sample mean and a selected standard value, and, consequently, does not apply to a situation where a difference between two sample means is considered. Therefore, the low one-sample CDs reported in the CBTS cannot be used to approximate sample sizes for purposes of meeting the requirement by the Environmental Protection Agency that a 20% difference between two sample means (e.g., control vs. experimental groups) should be detected with a power of 80% (or 90%) at a 5% certainty level.

One-year reliability of vibration sensitivity thresholds in human beings.

Vibration sensitivity of the 3rd and 5th digits was evaluated in 36 female volunteers (between 22 and 36 years of age at the beginning of the study) previously screened for the absence of neurological disorders. Both fingers were tested in the same subjects on weeks 1, 2 and 52 in a balanced crossover design. Twenty-nine volunteers completed all 3 sessions. All participants gave their informed consent prior to participation. The test session was divided into trials. Each trial consisted of 2 sequential intervals, identified by a signal, and vibration was applied to the fingertip during either the first or the second interval. The subject was instructed to press the push button corresponding to the interval that contained vibration (two-interval forced-choice procedure). Vibration amplitudes were varied according to the up-down transformed rule for the estimation of the amplitude corresponding to 75% correct detection. Vibration sensitivity thresholds were stable as reflected by the lack of a trend over time, and by the compound symmetry of the variance-covariance matrix, which reflects stability of threshold variances as well as of intersession correlations over time. Reliability of vibration sensitivity makes this test potentially suitable for longitudinal evaluation of somatosensory functions.

Critical assessment of motor activity as a screen for neurotoxicity.

Motor activity consists of a large number of motor behaviors not placed under stimulus control. The Environmental Protection Agency defines motor activity in terms of "activity counts," and has incorporated it in the guidelines for the assessment of neurotoxicity. While motor activity testing has some enticing operating characteristics, a dose-related change in activity counts can reflect an effect on the nervous system only in the absence of systemic toxicity. Furthermore, when a motor activity effect has been documented, ancillary data or further testing is needed to determine whether this effect is indeed adverse. In addition, the results of a motor activity test do not usually provide information about either the origin of the problem, or the follow-up tests that need to be conducted.

Quantitative sensory assessment in toxicology and occupational medicine: applications, theory, and critical appraisal.

Sensory systems are affected by a number of chemicals. Their functions can be quantitatively assessed with psychophysical methods. Psychophysics is the scientific study of the relationship between the physical dimension(s) of a stimulus and the behavioral response it generates. First, the unique contribution of psychophysics to toxicology is illustrated with a few examples taken from the fields of audition, vision, and somatosensory sensitivity. Then, a brief survey of the evolution of psychophysics is presented and followed by a review of its basic scientific foundations. Psychophysical evaluation is accomplished by delivering quantified stimuli in a prespecified order and by requesting a standardized response from the subject. Psychophysical methods specify the sequence of presentation of stimuli, and include the method of limits, the method of constant stimuli, as well as the adjustment and tracking methods. The response paradigms specify the standardized response the subject has to make to stimuli presented according to a specific format. The three major response paradigms are the yes-no, forced-choice, and rating paradigms. Sensory processes can be studied in humans as well as in animals by several techniques, such as classical conditioning, operant behavior, and reflex modulation. A critical review of the application of psychophysical methods and response paradigms is developed. Several examples illustrate the potential confusion brought about by unorthodox use of psychophysical terminology and methodology. Finally, a few suggestions are given for the evaluation of sensory processes in humans.

Psychophysical testing in human populations exposed to neurotoxicants.

Sensory organs can be studied from different points of view: biochemical, morphological, electrophysiological, clinical, and psychophysical. In this paper, the psychophysical and electrophysiological approaches are discussed and compared. Even though close parallels have been drawn between these two methodologies, discrepancies clearly indicate that they do not measure the same phenomenon. Vibration sensitivity is used to illustrate the role sensory assessment plays in neurotoxicology. Several psychophysical techniques are described to measure this sensory modality. Emphasis is placed on the need to quantitatively define stimulus parameters. The validity of vibration sensitivity as an indicator of sensory peripheral nerve dysfunction is established through a review of diseases, physical agents, pharmaceuticals, and other neurotoxicants known to alter the morphology and/or the physiology of the peripheral nervous system or sense organs. A series of factors or conditions affecting vibration detection thresholds, such as age, laterality, gender, temperature and others, are examined. Finally, a review of the psychophysical methods and procedures illustrates the advantages of the forced-choice paradigm and the tracking technique over other procedures and methods of stimulus presentation. The importance of psychophysical assessment of sensory functions in neurotoxicology is emphasized.

Acrylamide effects on the macaque visual system. I. Psychophysics and electrophysiology.

Oral acrylamide produces axonal swelling and later degeneration and gliosis in the distal optic tract, especially within the lateral geniculate nucleus, of macaque monkeys. Measures of visual thresholds and cortical-evoked potentials were used to study the time course of visual changes during exposure to acrylamide in macaque monkeys. Contrast sensitivity, visual acuity, and flicker fusion frequency were reduced during exposure, and only flicker fusion recovered rapidly and completely after exposure. Pattern-reversal-evoked responses exhibited increased latency and reduced amplitude during dosing but substantially recovered after exposure. Visual acuity and contrast sensitivity for high spatial frequencies were decreased throughout the 140 days of testing after dosing. These results suggest an acute general depression of visual capacities as the initial effect of acrylamide exposure, whereas later effects were confined to high spatial frequencies.