Long-lasting central nervous system (CNS) neurotoxicity of 4-tert-butyltoluene (TBT) has been investigated using electrophysiology, behaviour, and neurochemistry in Long Evans rats exposed by inhalation to 0, 20, or 40 p.p.m. TBT 6 hr/day, 7 days/week for 4 weeks. Flash evoked potentials and somatosensory evoked potentials were not affected by TBT. In Auditory Brain Stem Response there was no shift in hearing threshold, but the amplitude of the first wave was increased in both exposed groups at high stimulus levels. Three to four months after the end of exposure, behavioural studies in Morris water maze and eight-arm maze failed to demonstrate any TBT induced effects. Exposure was followed by a 5 months exposure-free period prior to gross regional and subcellular (synaptosomal) neurochemical investigations of the brain. TBT reduced the NA concentration in whole brain minus cerebellum. Synaptosomal choline acetyltransferase activity increased and acetylcholinesterase activity was unchanged suggesting increased synaptosomal ability for acetylcholine synthesis. The relative and total yield of synaptosomal protein was reduced suggesting reduced density and total number of synapses in situ, respectively. We hypothesise that a reduced yield of synaptosomal protein reflects a more general effect of organic solvent exposure on the software of the brain. The synaptosomal concentration per mg synaptosomal protein and the total amount of 5-hydroxytryptamine were not affected whereas the total amount of synaptosomal noradrenaline decreased. The concentration and the total amount of synaptosomal dopamine decreased. The noradrenergic and dopaminergic parts of CNS may be more vulnerable to TBT than the serotonergic, and these long-lasting effects may cause or reflect TBT-compromised CNS function.

Rats were exposed to 1200 ppm or 0 ppm toluene (CAS 108-88-3) for 6 h per day from day 7 of pregnancy until day 18 postnatally. Developmental and neurobehavioral effects in the offspring were investigated using a test battery including assessment of functions similar to those in the proposed OECD TG for Developmental Neurotoxicity Study, i.e., physical development, reflex ontogeny, motor function, motor activity, sensory function, and learning and memory. The exposure did not cause maternal toxicity or decreased viability of the offspring. Lower birth weight, delayed ontogeny of reflexes, and increased motor activity in the open field was registered in the exposed offspring. Impaired cognitive function was revealed in the exposed female offspring at the age of 3.5 months, i.e., they used more time to locate the hidden platform in the Morris water maze after platform relocation. The difference was not related to poorer swimming capabilities, because swim speeds were similar to control values. The results show that exposure to 1200 ppm toluene during brain development caused long-lasting developmental neurotoxicity in rats.

To examine whether it is possible to enhance the level of 22:6(n-3) in the central nervous system, newborn rats were fed dietary supplements containing oils with either specific or random triacylglycerol structure, but similar concentrations of polyunsaturated fatty acids. In the specific structured oil, 22:6(n-3) was located in the sn-2 position, whereas it was equally distributed among the three positions in the triacylglycerol molecule in the randomized oil. A reference group was fed rat milk before weaning and nonpurified diet after weaning. After 12 wk, the levels of 22:6(n-3) in brain and liver phospholipids were higher in the groups fed the experimental diets than in the reference group. The specific structured oil resulted in the highest level of 22:6(n-3) in the brain, whereas the level of 22:6(n-3) was highest in the liver of the group fed randomized oil, indicating differences in metabolism of fatty acids resulting from their position in the dietary triacylglycerol molecule. The higher levels of 22:6(n-3) were accompanied by significantly lower levels of the long-chain (n-6) polyunsaturated fatty acids compared with the reference group. The fatty acid profiles, including the level of 22:6(n-3), in the retina phospholipids were not affected by the three different diets apart from a lower level of 20:4(n-6) in rats fed the experimental diets, indicating a strong tendency to maintain a high level of 22:6(n-3) in the retina. The changes in the fatty acid profiles did not result in differences in learning ability, but caused changes in visual function, evidenced by higher latency of the b-wave and lower oscillatory potential, and in auditory brainstem response, evidenced by generally greater amplitude of wave Ia in the group fed specific structured oil.

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b. wt.; 5000 mg PGA/I: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. in the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pens and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pens, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/-0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA. (C) 1998 Inter Press, inc.

The persistence of neurobehavioral effects in female rats (Mol:WIST) exposed to 500 ppm technical xylene (dimethylbenzene, CAS-no 1330-20-7) for 6 hours per day on days 7-20 of prenatal development was studied. The dose level was selected so as not to induce maternal toxicity or decreased viability of offspring. Investigations of learning and memory abilities were performed using a Morris water maze. This task requires rats to spatially navigate, using distal extramaze cues to locate a small platform under the surface of the water in a large pool. At the age of 16 weeks, the exposed offspring showed impairments when the platform was relocated in the pool. Impaired performances after platform relocation were also observed in exposed offspring at 28 and 55 weeks of age, although the difference was not statistically significant at 55 weeks. These data could indicate that the effect was partly reversible, although over a long time period. However, another explanation could be that the animals became more practised at solving the problem (finding the platform) as continued testing occurred and therefore were able to compensate for the neurotoxic effect of the prenatal xylene exposure. Further studies are planned to investigate whether neurobehavioral effects resulting from prenatal xylene exposure can interact with neurophysiological aging processes.

Long-lasting effects of inhalation exposure to p-cymene (p-isopropyl-toluene; CAS No. 99-87-6) on regional and subcellular brain neurochemistry were studied. Male Long-Evans rats were exposed to 0, 50, or 250 p.p.m. p-cymene 6 hr/day, 5 days/week for four weeks followed by an exposure-free period of 8 weeks. Synaptosomes were isolated from whole brain minus cerebellum and used as an ex situ model for in situ conditions at the level of the presynaptic nerve terminal. There was no persistent effect on wet weight (regional) or regional noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) concentrations owing to exposure. Yield of synaptosomal protein was statistically significantly reduced in an exposure concentration-related manner (Control: 16.6 +/- 3.1; 50 p.p.m.: 9.2 +/- 2.1; 250 p.p.m.: 8.6 +/- 1.7 mg protein/g tissue, mean +/- I.S.D.). Synaptosomal NA and DA concentrations and acethycholinesterase, butyrylcholinesterase, and lactate dehydrogenase activities were statistically significantly increased when expressed relative to synaptosomal protein. It is hypothesized that a reduced density and number of synapses in situ are functionally compensated for by increased NA and DA release from noradrenergic and dopaminergic presynaptic nerve terminals. The applicability of the synaptosome as an ex situ neurochemical research model for the presynaptic CNS nerve terminal in situ for the study of solvent neurotoxicity in rats was further supported.

A strategy for delineating risk factors from use of neurotoxic chemicals was applied to the Danish working environment. An analysis using this strategy disclosed the need for internationally adopted criteria for neurotoxicity, and consequently a working group was established by the Nordic Council of Ministers with the task to propose criteria for neurotoxicity. Functional effects on the nervous system, such as reduction in memory and learning ability, decrease in attention, and alteration of behavior due to toxic chemicals in the environment is now being acknowledged as an important public health problem. This change in concern from obvious effects of high dose exposure to the more subtle effects of environmental exposure to neurotoxicants was incorporated in the criteria. The new criteria for evaluating neurotoxicity data has subsequently been used on 79 common industrial chemicals. The results indicate that numerous persons are exposed in the working as well as in the general environment to several chemicals, for which almost no data on the effect on subtle neurophysiological functions are available. Development of an approach to risk assessment dealing with this problem is a major challenge in the nineties. Different approaches to risk assessment are discussed, the quality of the databases available for hazard assessment are evaluated, and the needs for further research are identified.

Exposure for 6 h per day, 5 days per week, during a period of 6 months to the organic solvent dearomatized white spirit (0, 400, and 800 ppm) was studied in rats that were 3 months old when the repeated exposure was initiated. After an exposure-free period of 2–6 months duration, neurophysiological, neurobehavioral, and macroscopic pathologic examinations were performed. The study revealed exposure-related changes in sensory evoked potentials and a decrease in motor activity during dark (no light) periods but no white spirit-induced changes in learning and memory functions. The measurements of the flash evoked potential (FEP), somatosensory evoked potential (SEP), and auditory brain stem response (ABR) all demonstrated dose-dependent increases of the amplitudes of the early latency peaks of the sensory evoked potentials (EPs). Furthermore, an increase of the dose showed that the measurements of FEP and SEP revealed changes in the later-latency peaks, which reflect the more associative aspects of sensory processing. The results demonstrated that 6 months of exposure to dearomatized white spirit induced long-lasting and possible irreversible effects in the nervous system of the rat.

Pregnant rats (Mol:WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease viability of offspring. In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal and motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.

The effects of exposure to 800 or 4000 p.p.m. of n-heptane, CAS No. [142-82-5]) 6 hr per day during a period of 28 days, on the function of the auditory system were examined by measurements of auditory brain stem response (ABR) in Long Evans rats. The ABR was measured simultaneously with both needle electrodes and implanted electrodes. The wave forms recorded with the two types of electrodes were similar, but the amplitudes were largest on the recordings with implanted electrodes. The overall ratio between the amplitudes obtained with implanted electrodes and with needle electrodes was 1.4 for peak Ia and 2.5 for peak IV of the ABR. The exposure to n-heptane (4000 p.p.m.) reduced the amplitudes of components Ia and IV of the ABR. The reduction was most consistent for component IV and most pronounced at higher frequencies and intensities. The reduction in ABR corresponds to an increase in the auditory threshold of approximately 10 dB at all frequencies. Neither the latencies nor the interpeak latencies of components Ia and IV were changed. No significant changes in ABR were observed in the group exposed to 800 p.p.m. The mechanism behind the ototoxicity of organic solvents is discussed.

The effects of repeated exposure to 20 p.p.m. 4-tert-butyltoluene (CAS No. [98-51-1]) 6 hr/day for 14 days on the function of the intact nervous system were examined by measurements of flash evoked potentials in Wistar rats. The exposure to 4-tert-butyltoluene induced changes in the amplitudes of the flash evoked potentials. The changes were significantly different from controls on day 2, 19 and 26 after cessation of the exposure, but not on day 5 and 12. No significant difference in body weight gain between groups was found during the experiment. These results indicate that repeated exposure to 20 p.p.m. 4-tert-butyltoluene causes persistent changes in the function of the central nervous system measured as changes in the flash evoked potential. A reevaluation of the present TLV value of 10 p.p.m. for 4-tert-butyltoluene is suggested.

This text is the result of the authors' involvement in a working group on criteria for the identification and classification of neurotoxic chemicals. (The work of the group does not necessarily represent the official stand of the affiliated institutes.) A definition of neurotoxicity and criteria for evaluating studies dealing with neurotoxicology are presented. The evaluation is a stepwise process that ends with assigning the chemicals to groups depending on the available evidence for neurotoxicity (ie, neurotoxic, probably neurotoxic, possibly neurotoxic, probably not neurotoxic, or not classifiable). Finally, the description of the potency of neurotoxic chemicals is briefly discussed. The model has been tested by evaluating selected research papers on the following 10 chemicals: manganese, aluminum, tetrahydrofuran, cyclohexanone, dichlorvos, trichloroethylene, formaldehyde, tri-ortho-cresyl phosphate, n-hexane, and vinyl chloride. There was sufficient evidence for classifying five of the ten chemicals (aluminum, manganese, n-hexane, trichloroethylene, tri-ortho-cresyl phosphate) as definitely neurotoxic to humans, and three were considered to be possibly neurotoxic to humans (dichlorvos, tetrahydrofuran, vinyl chloride). Cyclohexanone and formaldehyde were not classifiable according to the model.

The use of evoked potentials to measure neurotoxicity was evaluated using 4-tert-butyltoluene (TBT) as a test compound. Male Wistar rats were habituated to the recordings of auditory- and flash-evoked potentials until the combined waveform of the evoked potentials reached a steady state. The rats were then divided into three groups and exposed to 0,50 and 150 ppm TBT for 6 h, and the auditory- and flash-evoked potentials were measured for up to 288 h after exposure. Event-related potentials specifically associated with the temporal pairing of auditory and visual stimuli were not apparent in the recordings. The peak-to-peak values of selected components, integrated amplitude and power spectra of the waveforms in exposed rats were significantly different from control values for at least 288 h in the group exposed to 150 ppm TBT and for 120 h in the group exposed to 50 ppm TBT. It is concluded that evoked potentials may be used for detection and characterization of minor neurofunctional changes due to low-dose exposure to chemicals.

A method for delineating the risks due to exposure to neurotoxic chemicals based upon the linkage of four national computer-based registers is described. The four registers are: the Danish Product Register database, PROBAS; Register of air pollution measurements in Danish workplaces, ATABAS; Register of notified occupational diseases; and Register of work force. Based on the information from the four registers, risk profiles for neurotoxic chemicals in 69 industrial groups were generated. The risk profiles describe the number of neurotoxic chemicals, their potency, the number of exposure measurements exceeding the occupational exposure limit (OEL), and the total and relative number of diseases caused by the chemicals in each group. Based on this linkage, twelve industries are noted to be potentially at high risk due to exposure to neurotoxic chemicals; therein, 18 chemicals are identified as "risk chemicals," primarily used in 8 main groups of products. The goal of the evaluation is to develop a tool for priority setting of preventive measures. The evaluation may also serve as a tool for assessing improvements and selecting areas for further epidemiological studies.

At two scientific conferences in 1985, one in Copenhagen sponsored by the Nordic Council of Ministers and the World Health Organization (WHO), the other in Raleigh, NC, it was concluded that chronic toxic encephalopathy may develop following long-term occupational exposure to organic solvents (1,2). The terms organic affective syndrome, mild and severe chronic toxic encephalopathy were suggested for this condition describing increasing severity. In May 1990, a conference on organic solvents and the nervous system was held in Copenhagen sponsored by the Commission of the European Communities and the Danish Ministry of the Environment (3). Scientists and representatives from the governments, industries, and labour organisations from the EEC and US participated.