Ralph R. Albee

A performance standard for clinical and functional observational battery examinations of rats

Clinical examinations are a key component of all toxicology studies and are a major component of the functional observational battery (FOB). The FOB is a core feature of the USEPA neurotoxicity screening guideline of 1991. The need for well-trained technicians is recognized, but technician competency is virtually always judged subjectively by their supervisors. Although subjective evaluation of performance cannot be replaced, what constitutes satisfactory performance is nebulous and therefore difficult to communicate to trainees and difficult to document. Consequently, a laboratory performance standard, with a clear pass-fail criterion, was developed to address this deficiency. The performance standard was an idealized composite of FOB data from experienced laboratory personnel, each person tested on a separate set of four groups of rats. The rats were examined in random order, and treatments were either (a) saline, (b) chlorpromazine, (c) atropine followed by physostigmine, or (d) amphetamine. Thus, the performance standard was the pattern of scores generated by the four treatments. After training, each technician was individually tested on four groups of rats similarly treated (random order, blind examination). The pattern of scores generated by the technician was compared to the performance standard by calculating a Pearsons cross-correlation coefficient. An r > 0.8 was considered passing. The use of the standard meets several laboratory goals: (a) good science (improved training leads to better studies), (b) documentation of observer competency required by the USEPA neurotoxicity guideline, (c) improved documentation of training for Good Laboratory Practices, and (d) objective documentation of performance for purposes of personnel management.

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.

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.

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.

An inhalation toxicity study of chlorine in Fischer 344 rats following 30 days of exposure

Abstract A subacute study was completed in groups of 10 male and 10 female Fischer 344 rats exposed to air (controls), 1, 3, or 9 ppm chlorine for 6 hr/day, 5 days/week, for 6 weeks. Concentration related decreases in body weight gain were seen at all exposure concentrations in females and at 3 and 9 ppm in males. Additionally, three females at 9 ppm died before the end of Day 30 of exposure. Urinalysis, hematology, and clinical chemistry evaluations were completed on the surviving animals. The urine specific gravity was elevated at all exposure concentrations in the females and at 3 and 9 ppm in the males. The hematocrit and white blood cell count were increased in the females exposed to 9 ppm. Elevations in alkaline phosphatase activity, blood urea nitrogen, γ-glutamyl transpeptidase, and serum glutamic-pyruvic transaminase occurred at 9 ppm; alkaline phosphatase was elevated at 3 ppm in rats of both sexes. Widespread evidence of inflammation was seen throughout the respiratory tract with hyperplasia and hypertrophy of epithelial cells of the respiratory bronchioles, alveolar ducts, and alveoli of male and female rats exposed to 9 ppm. Changes in male rats at 3 or 1 ppm consisted of focal inflammation of the nasal turbinates and a slight to moderate inflammatory reaction around the respiratory bronchioles and alveolar ducts. Increased eosinophilic cytoplasmic homogeneity and decreased granularity of the epithelial cells of the proximal convoluted tubules were seen in the kidneys of male rats exposed to 9 ppm. The livers of rats of both sexes at 9 or 3 ppm had an increased hepato-cellular cytoplasmic vacuolation. These data indicated that repeated exposure of Fischer 344 rats to chlorine resulted in pulmonary effects at all levels of chlorine used, and hepatic and renal effects at 9 and 3 ppm.

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.

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/Thematoxylin, Seviwr-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.

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.

Neurological Approach to Neurotoxicological Evaluation in Laboratory Animals

The neurotoxicological potential of a compound is best evaluated with information from a wide variety of tests. Our philosophy is that tests should be applicable across species and emphasize complementarity to neuropathology. Electrophysiologic tests comparable to those in human clinical neurology fulfill these requirements. Animals are not anesthetized but are physically restrained during the tests. Routine tests include flash evoked potentials, auditory brainstem responses to clicks and tone pips, somatosensory evoked responses, and caudal nerve action potentials. Tests can be added (e.g., H-reflex, EMG, or EEG) or deleted as circumstances warrant. A careful clinical examination of all animals (functional observational battery) and, for rats, a test of grip strength are utilized as well. Neuropathology typically includes perfusion fixation and special stains of histological sections from the central and peripheral nervous systems. Approximately half the animals are retained as a recovery group for subsequent tests if warranted by the results of the neurotoxicological evaluation. The neurological approach, in conjunction with standard toxicological studies, provides data with the necessary breadth and depth for a comprehensive evaluation of neurotoxicity.

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.