Pamela J. Spencer

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.

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 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.

Lack of Trigeminal Nerve Toxicity in Rats Exposed to Trichloroethylene Vapor for 13 Weeks

Male and female Fischer-344 rats were exposed to 1,1,2-trichloroethylene (TCE) at 250, 800, or 2500 ppm for 6 h/day, 5 days/week, for 13 weeks. Weekly body weights and daily clinical observations were recorded and a functional observational battery (FOB) was performed monthly. Postexposure neurotoxicological evaluations included an electrodiagnostic evaluation of auditory function, the trigeminal nerve, and a comprehensive neuropathological examination. After 8 weeks of exposure, female, but not male, rats exposed to 2500 ppm were slightly more reactive to handling than the controls but not after 13 weeks of exposure. After 13 weeks, female rats exposed to 2500 ppm TCE were slightly more active during the 1-min observation period than the controls. There were no treatment-related differences in grip performance, landing foot splay, or on the trigeminal nerve–evoked potential at any dose. At 2500 ppm TCE, mild frequency-specific hearing deficits were observed, including elevated tone-pip auditory brainstem response thresholds. Focal loss of hair cells in the upper basal turn of the cochlea was observed in 2500 ppm–exposed rats. Except for the cochleas of 2500 ppm–exposed rats, no treatment-related lesions were noted during the neuro-histopathologic examination. The no-observable-adverse-effect level for this study was 800 ppm based on ototoxicity at 2500 ppm.

Propylene glycol monomethyl ether (PGME): inhalation toxicity and carcinogenicity in Fischer 344 rats and B6C3F1 mice.

A series of inhalation studies with propylene glycol monomethyl ether (PGME) vapor were undertaken to characterize its subchronic toxicity in mice and chronic toxicity/oncogenicity in rats and mice. Groups of male and female Fischer 344 rats and B6C3F1 mice were exposed to 0,300, 1,000, or 3,000 ppm vapor from 1 week to 2 years. Primary treatment-related effects included: initial sedation of animals exposed to 3,000 ppm and its subsequent resolution correlating with induction of hepatic mixed function oxidase activity and S-phase DNA synthesis; elevated mortality in high-exposure male rats and mice (chronic study); elevated deposition of alpha2U-globulin (α 2U-G) and associated nephropathy and S-phase DNA synthesis in male rat kidneys; accelerated atrophy of the adrenal gland X-zone infemale mice (subchronic study only); and increased occurrence and/or severity of eosinophilic foci of altered hepatocytes in male rats. No toxicologically relevant statistically significant increases in neoplasia occurred in either species. A numerical increase in the incidence of kidney adenomas occurred in intermediate-exposure male rats; however, the association with α 2U-G nephropathy, a male rat specific effect, indicated a lack of relevance for human risk assessment.

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.

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.

The Tg.AC Transgenic Mouse as a Screening Tool for Anticarcinogens: Broccoli Juice Protected Against 12-O-Tetradecanoylphorbol-13-Acetate (TPA) But Not Benzo[a]Pyrene (B[a]P)-Induced Skin Tumors

The Tg.AC mouse model was used to assess the utility of a short-term screening assay to evaluate compounds with suspected carcinogenic/anticarcinogenic activity. Crude broccoli juice (BROC) was evaluated for antitumorigenic effects against 12-Otetradecanoylphorbol-13-acetate (TPA) and benzo[a]pyrene (B[a]P)-induced tumors. Groups of female mice were dosed three times a week (200 μL/mouse) with one of the following: acetone vehicle control, TPA (2.5 μg/mouse), B[a]P (250 μg/mouse), BROC/TPA, or BROC/B[a]P. BROC (200 μL, 1:1 acetone) was dermally administered 1 h prior to the administration of either TPA or B[a]P to evaluate anticarcinogenic activity. Papilloma numbers were recorded weekly for each mouse. Following 13 weeks of treatment, samples from the dermal test site from all mice were examined histologically. B[a]P-induced tumors were evaluated for transgene expression by RT-PCR and immunohistochemically for cyclin D1 and p53 proteins. TPA and B[a]P induced tumors in all surviving mice. BROC showed effective antitumorigenic activity against TPA but not B[a]P. Tumor development was distinct between TPA (small, benign papillomas) and B[a]P (large, ulcerated, squamous cell carcinomas). The transgene v-Ha-ras, cyclin D1, and p53 proteins were highly expressed in B[a]P tumors where progression to malignancy was rapid (< 13 weeks). The effects induced by B[a]P appeared to cooperate with transgene expression to enhance conversion to malignancy and could serve as a phenotypic indicator for genotoxic versus nongenotoxic carcinogens. The model distinguished differences in tumor response for carcinogenic and anticarcinogenic agents. The Tg.AC mouse model offers a potentially useful screen for identifying new anticarcinogenic agents and directing future mechanistic evaluations.

Evaluation of a novel assay of potential toxicity/neurotoxicity of carpet emissions (VOCs) in mice

A private testing laboratory utilizing the whole-body plethysmograph/head-only exposure apparatus outlined in the respiratory irritation assay ASTM E981-84, along with a novel exposure regimen, has reported neurotoxic effects and mortality in mice exposed to relatively low levels of volatile organic compounds (VOCs) emitted from a number of consumer products. This methodology was evaluated by exposing groups of mice, including unrestrained and sham-treated animals, to VOCs generated from a sample of carpet reported to be neurotoxic using the modified assay. General toxicological (haematological measurements, organ weights, gross pathology, histopathology) and specific neurotoxicity (functional observations, body temperature, histopathology of nervous tissues) parameters were evaluated. No effects related to exposure to carpet VOCs were observed in the mice. However, despite careful handling, a number of effects were observed which were attributed to the repeated restraint of mice in the ASTM E981 apparatus. These included a number of minor physical injuries, decreased body weights, altered thymus weights, compression damage to the liver and haemorrhage of the pituitary gland. It was concluded that the modification of the original ASTM E981 methodology may result in physical injuries and stress which may significantly affect any evaluation of toxicity and neurotoxicity in treated animals and result in inaccurate conclusions.

Subchronic toxicity and genotoxicity of diiodomethyl-p-tolylsulfone (DIMPTS) in laboratory animals

These studies were conducted to determine subchronic toxicity and genotoxicity of the biocide diiodomethyl-p-tolysulfone (DIMPTS) in rats and dogs. Male and female Sprague-Dawley rats and Beagle dogs were administered DIMPTS for 90-days via the diet at 0, 5, 20, and 80 mg/kg/day to rats and via capsules at 0, 2, 10, and 60 mg/kg/day to dogs. In rats, the only treatment-related finding was squamous metaplasia of the salivary gland duct in the 80 mg/kg/day group. In dogs, female body weights in the high-dose group were significantly lower than controls. Altered clinical pathology parameters were considered secondary to inflammatory changes observed in some of the dogs. Treatment-related alterations were found in the thyroid glands, salivary glands, GI-tract in the mid- and/or high-dose groups. DIMPTS was negative in the four in vitro and one in vivo genotoxicity assays. The toxicological effects noted in the two mammalian species are consistent with the principal toxic effects of iodine, and are proposed to arise from release of iodide from the DIMPTS molecule with toxic sequelae.