Neurotoxicologic examination of rats exposed to 1,1,2,2-tetrachloroethylene (perchloroethylene) vapor for 13 weeks.

Large evoked potential and EEG changes occurred in a pilot study in Fischer 344 rats during exposure to 800 ppm of 1,1,2,2-tetrachloroethylene [perchloroethylene (Perc)], a cleaning solvent with anesthetic properties. In the main study, rats were evaluated for persistent nervous system effects the week following exposure to 0, 50, 200, or 800 ppm Perc for 6 h/day, 5 days/week, for 13 weeks. The only effect related to treatment was in the flash evoked potential (FEP-V), recorded from the visual cortex. The longer latency potentials (N3) of the FEP-V had a greater amplitude in the 800 ppm Perc group. The FEP-Vs were of normal shape and latency. Although mild neurotoxicity could not be ruled out completely, amplitude changes in N3 can occur for a variety of psychophysiological reasons other than neurotoxicity. Consequently, as a stand-alone finding, the toxicologic significance of the larger FEP in the 800 ppm exposure group was unknown. Other data did not support a diagnosis of neurotoxicity. No treatment-related alterations were noted in expanded clinical observations, in the FEP recorded from the cerebellum (as opposed to visual cortex FEP-V), or in auditory, somatosensory, or caudal nerve evoked potentials. No treatment-related lesions were noted during histopathologic examination of eyes, optic nerves, optic tract, or multiple sections of brain, spinal cord, peripheral nerves, or limb muscles. The no-observed-effect-level (NOEL) was 200 ppm, based on increased amplitude of the longer latency potentials of the FEP at 800 ppm.

Dichloroacetylene: effects on the rat trigeminal nerve somatosensory evoked potential.

Humans overexposed to trichloroethylene (TCE), under specific conditions, were reported to develop trigeminal nerve dysfunction. A degradation byproduct dichloroacetylene (DCA), however, has been suggested as the probable neurotoxicant rather than TCE. Studies in mice, rats, and rabbits support the hypothesis of DCA-induced trigeminal neurotoxicity. This study, therefore, was conducted to characterize DCA-induced trigeminal nerve dysfunction in rats using the electrodiagnostic procedure trigeminal nerve-stimulated somatosensory evoked potential (TSEP). A group of six rats was exposed once to DCA (approximately 300 ppm) or room air for 2.25 h and a separate group of six rats was not exposed and served as controls. Trigeminal nerve somatosensory evoked potentials (TSEPs) were collected before exposure and 2, 4, and 7 days postexposure. Because DCA was manufactured from TCE with acetylene added as a stabilizer, another group of rats was exposed to TCE and acetylene without generation of DCA. TSEPs from DCA-exposed rats were smaller and slower compared to their baseline recordings and to the concurrent negative controls. TSEPs from the controls and the TCE/acetylene-exposed rats were unchanged. Neuropathology did not reveal treatment-related lesions. It was concluded that the rat is mildly to markedly susceptible to DCA-induced trigeminal nerve dysfunction as assessed by TSEP, but that the kidney was the likely target organ based on gross observations and the DCA literature.

Neurotoxicologic examination of rats exposed to 1,1,1-trichloroethane vapor for 13 weeks.

Large evoked potential and EEG changes occurred in a pilot study in Fisher 344 rats during exposure to 2000 ppm of 1,1,1-trichloroethane (1,1,1-T; a cleaning solvent with anesthetic properties). In the main study, rats were evaluated for persistent nervous system effects the week following exposure to 0, 200, 630, or 2000 ppm 1,1,1-T for 6 h/day, 5 days/week, for 13 weeks. Rats were clinically examined regularly and were given a functional observational battery monthly (FOB, including forelimb and hindlimb grip performance testing). After 13 weeks of exposure, the rats were evaluated by FOB and by visual, auditory, somatosensory, and caudal nerve-evoked potentials. After functional testing, a subgroup of rats had histopathologic examination of brain, spinal cord, peripheral nerves, and limb muscles. There were no post-exposure treatment-related findings in any parameter (FOB observations plus 39 dependent variables) except for a slightly smaller forelimb grip performance in the 2000-ppm exposure group. There was no recognized toxicologic significance for the difference in forelimb grip performance; a lack of findings in any other clinical, evoked potential or morphologic parameter did not support a diagnosis of neurotoxicity.

Neurotoxicity screening methods are sensitive to experimental history.

Toxicity studies commonly include unavoidable environmental differences (experimental history) among test groups, such as chemical taste, odor and irritation. The influence of environmental variables on USEPA guideline neurotoxicity tests was evaluated using an environmental enrichment model. 6-week-old male Fischer 344 rats were housed for 13 weeks in pairs with access to an exercise wheel, trained to run on a rotating rod and handled frequently. Control animals were housed singly, lacked the exercise wheel and rotating rod training, and had only routine interaction with caretakers. At the end of 13 weeks, flash evoked potentials (FEPs), somatosensory evoked potentials (SEPs), auditory brainstem responses (ABRs), grip performance, motor activity (MA), elements of the functional observational battery (activity and reactivity to handling/restraint) and brain histopathology with glial fibrillary acidic protein immunohistochemistry (GFAP IHC) were evaluated. Animals from the enriched group demonstrated changes (P < 0.05) in FEPs, SEPs and grip performance. Enriched animals were more active and reactive to their surroundings, and were highly reactive to physical restraint. Control (unenriched) animals showed little to no exploratory behavior and were more tolerant of restraint. Differences in experimental history can be detected using elements of standard guideline tests and may confound interpretation of such data if not taken into consideration.

Abnormal auditory brainstem responses and cochlear pathology in rats induced by an exaggerated styrene exposure regimen.

Groups of 12 male 42-day-old rats were exposed to 0 or 800 ppm styrene vapors for 14 hr/day, 5 days/week for 3 weeks. Tone-pip auditory brainstem responses (ABRs) at 4, 8, 16, and 30 kHz were obtained after the last exposure. ABRs were minimally affected at 4 kHz and moderately to severely affected at 8, 16, and 30 kHz as indicated by waveforms which had a decreased amplitude and increased latency as compared to the controls. Missing outer hair cell(s) were evident in the basal and lower middle turns of the organ of Corti. Outer hair cell loss was least in the first row and greatest in the second and third rows. Occasional inner hair cells were also missing in regions of severe outer hair cell loss. The distribution of hair cell loss within the cochlea was consistent with the pattern of ABR alterations. These data document mid-frequency auditory dysfunction in styrene-exposed young adult rats with significant damage to the organ of Corti following an exaggerated styrene exposure regimen.

Neurotoxicologic evaluation of rats after 13 weeks of inhalation exposure to dichloromethane or carbon monoxide.

Male and female Fischer 344 rats were exposed to dichloromethane (methylene chloride, DCM) or carbon monoxide (CO) for 6 hr/day, 5 days/week, for 13 weeks. Since oxidative metabolism of DCM to CO and CO2 is a saturable process, DCM exposure concentrations were selected clearly below saturation (50 ppm), just below saturation (200 ppm), and well above saturation (2000 ppm). At saturation of metabolism, metabolic CO causes about 10% carboxyhemoglobinemia (COHb). Therefore, as a control for CO effects, a separate group of rats was exposed to 135 ppm CO to induce approximately 10% COHb. Postexposure functional tests included an observational battery, hindlimb grip strength, and a battery of evoked potentials (flash, auditory brainstem, somatosensory, caudal nerve). After functional tests were completed, rats from all groups were perfused with fixative and a comprehensive set of nervous tissues from the high DCM exposure group and from controls were examined by light microscopy. Although some miscellaneous functional and morphologic variations were recorded, none were related to treatment. Thus, subchronic exposures as high as 2000 ppm DCM or 135 ppm CO had no deleterious effects on any of the measures of this study.

Evoked potential changes from 13 weeks of simulated toluene abuse in rats.

Fischer 344 rats were exposed to 8000 ppm toluene vapor in an 'abuse' paradigm for 13 weeks to develop an animal model for 'solvent neurotoxicity.' Exposures to toluene were multiple and short (15 to 35 min), adjusted according to tolerance. Although body weight was reduced 23% from controls, the toluene-exposed rats appeared healthy. Evoked potentials taken postexposure were, however, mildly to severely affected. Flash-evoked potentials were slow and topographically disorganized; 10 kHz tone-pip auditory brainstem responses (ABRs) had severe loss of power and loss of detail. Click and 30 kHz ABRs, somatosensory-evoked potentials, and caudal nerve action potentials were less affected. No neuropathologic changes were detected by light microscopy (perfusion fixation, special stains). Thus, postexposure multimodal functional effects were readily detected after subchronic, severe episodic exposures to toluene.

Spontaneous lesions in subchronic neurotoxicity testing of rats.

Male and female Fischer 344 rats, 30 weeks of age, were examined for neuropathologic changes after a 13-week inhalation neurotoxicologic study. Tissues were preserved by whole-body perfusion with 1.5% glutaraldehyde/4% formaldehyde solution. An extensive set of neural tissues was embedded in paraffin, sectioned, and stained with hematoxylin and eosin, luxol fast blue/periodic acid-Schiff/hematoxylin, Sevier-Munger silver, and cresyl echt violet. Lesions in the central and peripheral nervous system were comparable between sexes and between control and treated animals. Bilateral swollen axons were present in the medial aspect of the nucleus gracilis adjacent to the area postrema. Occasional swollen axons also were observed in the dorsal and ventral funiculi of the spinal cord. Degeneration of individual nerve fibers was present in the trapezoid body, vestibular nerve root, trigeminal nerve, cerebellar peduncles, and the funiculi of the spinal cord. Individual nerve fiber degeneration also was present in the spinal nerve roots, sciatic and tibial nerves. Nerve fiber degeneration was characterized by myelin disruption and degeneration, vacuoles and axonal fragmentation. Similar spontaneous neuropathology may be encountered in rats from other subchronic neurotoxicologic studies and must be differentiated from treatment-related toxicity.

Screening for neurotoxicity: complementarity of functional and morphologic techniques.

Our philosophy is that screening tests should be applicable across species and emphasize complementarity to neuropathology. Within this context, electrophysiological tests comparable to those in human clinical neurology are powerful screening tools. For example, while histopathologic evaluation of the cochlea for ototoxicity is difficult, evoked potential audiometry is fast and easy. In this instance, one might routinely screen for deficits in auditory function, and reserve morphologic techniques for a characterization role rather than one of discovery. Lesions of neurons, axons and myelin are, however, readily assessed by light microscopy. A suitable combination of functional and morphologic screening tests, therefore, enhances the ability to discover neurotoxicity, and these data often are ideal for generation of refined hypotheses for subsequent characterization studies.

Neurological consequences of congenital hypothyroidism in Fischer 344 rats.

Congenital hypothyroidism was induced in rat pups by treating pregnant and lactating dams with an antithyroid drug, methimazole. Methimazole (0.00, 0.01, 0.03 or 0.10 mg/ml) was added to the drinking water of female Fischer 344 rats from gestational day 17 through lactational day 10. The same animals as pups and adults were evaluated with a developmental neurotoxicological test battery. Pups were evaluated for physical measures of maturation, thermoregulation, flash evoked potential (FEP), motor activity, and morphology of brain, thyroid and kidneys. Parameters evaluated in the same animals as adults were body weight, functional observational battery, grip strength, body temperature, and neurological tests (FEP, auditory brainstem response to 4 and 16 kHz tone pips (ABR4, ABR16) and clicks (ABRc), somatosensory evokes potentials recorded from the somatosensory cortex (SEP-S) and the cerebellum (SEP-C), and caudal nerve action potential to single and paired stimuli (CNAP). Treatment-related findings in pups included slightly decreased body weight, slightly increased kidney weights, altered thyroid morphology, delayed incisor eruption, decreased thermoregulation, and FEP changes. Although a pup no effect level was not determined, effects at 0.01 mg/ml were minimal. Adult ABR4 and ABR16 waveforms were slower than controls and had altered shapes; ABRc, SEP-S, and SEP-C waveforms exhibited reduced power, increased latency and altered shape. Effects were detected in adults at all doses and thus, the neurological characteristics of rat congenital hypothyroidism were clearly detected with this developmental neurotoxicological test battery. The effects on body weight, kidney weight and thyroid morphology, however, suggest a general developmental effect and nervous system function did not appear to be preferentially affected.

Similarities of toluene and o-cresol neuroexcitation in rats.

Exposure to high concentrations of toluene vapors, or to intravenous o-cresol (a toluene metabolite) at about 0.9 mg/min, caused excitation of the somatosensory evoked potential (SEP) and EEG of Fischer 344 rats. SEP excitation was characterized by a large increase in a positive waveform at about 20-50 msec. Prolonged exposure to either compound caused numerous oscillations to appear from 20 msec to the end of the recording (150 msec). Both substances induced an increase in EEG beta activity and caused a large increase in activity at 5 Hz. Toluene exposed rats were lightly anesthetized, while o-cresol rats were conscious but hyperreactive. If exposure was continued, both sets of rats had involuntary muscle movements and tremors. Benzoic acid and hippuric acid, also metabolites of toluene, were similarly tested. Neither caused neuroexcitation (about 2.4 mg/min IV, 144 mg total dose). It was concluded, therefore, that metabolically derived cresols are plausible candidates for the neuroexcitatory properties of toluene.

Sensory evoked potentials in neurotoxicology.

This paper provides a summary of routine evoked potential tests used with rats, with elaboration on the cochlear microphonic portion of the auditory brainstem response, the effects of chemicals on high frequency (above 40 Hz) components of the somatosensory evoked potential, on cerebellar recording of sensory evoked potentials, and on central conduction time. An alternative to peak-valley amplitude and latency measurements is discussed, wherein a computer analyzes evoked potentials for differences from control in waveform shape, latency, and power. Since multiple use of statistics is common, resulting in an inflated false positive rate, an alpha criterion of less than 0.05 is recommended. Instead of dividing alpha by the number of statistical tests (Bonferroni), a less severe correction of dividing alpha by the square root of the number of tests is proposed.

Subchronic neurotoxicity in rats of the structural fumigant, sulfuryl fluoride.

Inhalation exposure of male and female Fischer 344 rats to sulfuryl fluoride [Vikane (Dow Chemical Company) gas fumigant] at 300 ppm for 6 hr/day, 5 days week, for 13 weeks caused diminished weight gain, dental fluorosis, a slight decrease in grooming, decreased flicker fusion threshold, slowing of flash, auditory and somatosensory evoked potentials, mild nasal and pulmonary inflammation, mild kidney effects, and mild vacuolation in the brain. Auditory brainstem responses (ABRs) and brain histology were evaluated two months postexposure in 2 male and 2 female rats. Both the ABRs and brain histology were within normal limits at this time, indicating that these treatment effects were, to at least a great extent, reversible. Exposure to 100 ppm resulted in dental fluorosis and very minor slowing of some evoked responses; all other measures, including brain histology, were normal. No treatment effects were noted at 30 ppm.

Neurobehavioral effects of dietary restriction in rats.

The effects of reduced body weight gain on nervous system function of young male Fischer 344 rats were examined. The rats were fed 15% ('mild') or 50% ('severe') less than the controls. Mild and severe dietary restriction resulted in 9% and 38% lower body weight compared to the controls. Mild dietary restriction caused slight changes in flash evoked potentials, auditory brainstem responses, caudal nerve action potentials, and body temperature. Severe dietary restriction increased the magnitude of the effects noted in the mild group, as well as causing a significant decrease in grip strength. Somatosensory evoked responses were not affected by either mild or severe restriction. Diet restricted rats were more excitable while restrained for testing. Thus, dietary restriction has significant effects on numerous behavioral and neurophysiological parameters that should be considered in the interpretation of neurotoxicological data when body weight differences are present.

Incapacitation and treatment of rats exposed to a lethal dose of sulfuryl fluoride.

Rats exposed to 4000 ppm sulfuryl fluoride (VIKANE gas fumigant, SO2F2) were incapacitated within 45 min and died within several hours after exposure. Exposure to higher concentrations resulted in a shorter time to incapacitation and death occurred within minutes. Treatment with calcium gluconate before exposure to 4000 ppm SO2F2 for 45 min resulted in 80% survival. However, calcium gluconate did not alleviate SO2F2-induced convulsions. Administration of phenobarbital before or after exposure to 4000 ppm SO2F2 for 45 min effectively reduced the frequency and severity of convulsions and resulted in survival of all animals. Exposure of rats to 10,000 ppm SO2F2 for 15 min followed by treatment with phenobarbital reduced the frequency of convulsions and delayed death, but did not prevent death. Diazepam was less effective than phenobarbital while diphenylhydantoin had no beneficial effect and, in fact, made the convulsions more severe and longer in duration. The results of this study indicate that phenobarbital was effective in ameliorating the acute toxic effects of an overexposure to SO2F2 in rats.

Neurotoxicologic examination of rats dermally exposed to 2,4-D amine for three weeks.

There was no evidence of peripheral neuropathy or other neurotoxicity in rats dermally treated with a 12% aqueous solution of the amine salt of 2,4-dichlorophenoxyacetic acid (2,4-D amine). Male and female Fischer 344 rats were treated on the skin of all four limbs with 2,4-D amine for 2 hr/day, 5 days/week, for 3 weeks. Measurements were: body weights, hindlimb grip strength, accelerating rod performance, single and paired pulse electrophysiology of the caudal and sciatic nerves, hindfoot H-reflexes, light microscopy of brain, spinal cord, sciatic nerve, tibial nerve, digital nerve, and electron microscopy of the tibial nerve. The experiment continued for up to one month postexposure. Treatment caused a weight loss in both male and female rats and caused minor skin changes during treatment in both sexes. No other treatment-related effects were found.

Lack of neuropathologic consequences of repeated dermal exposure to 2,4-dichlorophenoxyacetic acid in rats.

A 24% aqueous solution of the dimethylamine salt of 2,4-dichlorophenoxyacetic acid (2,4-D amine) was applied to the legs of male Fischer 344 rats 2 hr/day, 5 days/week, for 2 weeks. Because this concentration caused severe skin lesions, a second group of rats was treated similarly with a 12% solution of 2,4-D amine for 3 weeks. The 12% solution caused only mild skin changes. The plasma 2,4-D content, at the end of exposure, was nearly five times greater in the rats exposed to the 24% solution than to the 12% solution (323 vs 66.5 micrograms/ml). The severe skin changes probably facilitated absorption in the rats treated with the 24% solution. Rats treated with either concentration weighed less than controls. Although histologically normal, kidneys of treated rats weighed more than controls. The increased kidney weights were attributed to physiological adaptation due to active excretion of absorbed 2,4-D. Light microscopic examination of tissues, other than skin, revealed no differences between treated and control animals. There were no nervous system pathologic changes although the rats were exposed to sufficient amounts of 2,4-D amine to cause severe skin lesions, decreased body weights, and increased kidney weights.

H-reflex waveform and latency variability in rats.

The right tibial nerve of each of 11 anesthetized male Fischer 344 rats was electrically stimulated at the ankle, and H-reflexes were recorded simultaneously from four locations in the hind foot. Needle electrodes were used for stimulating and recording. The mean (+/- SD) latency to the dominant peak of the H reflex was 10.21 +/- 0.75 msec. Individual rats had a 10% difference in latency from the "fastest" to the "slowest" locations in the foot, although any of the four locations could produce the fastest (shortest latency) response. The H reflexes varied from simple, single peak waveforms to complex multiple peak waveforms. The dominant peak polarity was unpredictably either positive or negative. These variations in complexity and latency occurred among rats and among the four locations within an individual rat. The dominant peak was identified in complex waveforms by adjusting the stimulating intensity and by recording from more than one location in the foot. The smallest coefficient of variation (5.57%) resulted from selecting data from the fastest electrode locations.

Visual evoked response testing method for neonatal rats.

Visual evoked responses (VER's) were recorded from pentobarbital-sedated neonatal Fischer 344 rats at 10, 12, 14 and 22 days of age. Subcutaneously placed needle electrodes were used to record VER maturation within individual animals. The mean latency to the first negative peak was 190.8 msec at 10 days of age, 117.4 msec at 12 days of age, 84.2 msec at 14 days of age and 39.4 msec by 22 days of age. Larger pups had shorter latencies, and males had shorter latencies than females. When latencies were adjusted for body weight differences (females were lighter), there were no sex differences. There were significant latency differences between litters. Repeatability was demonstrated by the persistence of pup-to-pup order (fastest to slowest latency), from 12 to 14 days of age.

Subchronic toxicity of acrylamide administered to rats in the drinking water followed by up to 144 days of recovery.

Groups of male and female Fischer 344 rats were administered acrylamide in their drinking water at 0, 0.05, 0.2, 1, 5, or 20 mg/kg/day for up to 93 days. Following the administration of acrylamide in the drinking water, male rats from each dose level were held for up to 144 days of recovery. The 20 mg/kg/day groups had definite treatment-related effects after 92 (males) and 93 (females) days. They were dragging the rear limbs, body weights were decreased, serum cholinesterase activity was decreased in top dose females, and packed cell volume, red blood cell, and hemoglobin values were slightly decreased in males and females. In the 20 mg/kg/day groups, the primary target tissue was the peripheral nerve with lesions consisting of severe degeneration characterized by demyelinization and axonal loss. Slight spinal cord degeneration was observed. Other effects included atrophy of skeletal muscle, testicular atrophy, and distended urinary bladders; these were probably secondary to the nerve degeneration. After 144 days of recovery, the lesions had partially or completely reversed. Parameters affected at the 5 mg/kg/day dose level after 92 (males) and 93 (females) days consisted of peripheral nerve degeneration which were of a lesser degree of severity than those seen in the 20 mg/kg/day groups and appeared to have completely reversed after 111 days of recovery. In rats given 1 mg/kg/day, a minimal treatment-related effect was observed in males after 92 days, and this was limited to very slight nerve degeneration using electron microscopy (females were not examined by electron microscopy). This observed effect appeared to have reversed after 25 days of recovery. No treatment-related effects were seen in any of the parameters monitored in the rats given 0.05 or 0.2 mg/kg/day of acrylamide.