Behavioural effects and kinetics in brain in response to inhalation of constant or fluctuating toluene concentrations in the rat

The toxicity of exposure by inhalation to organic solvents may not only be related to the total external dose, but also to the pattern of exposure. In this study the impact of the exposure scenario on the behavioural effects of the model solvent toluene in rats was investigated. Rats were exposed for 7.5 h to either a constant concentration or fluctuating concentrations at total external dose levels of 20,000 ppm h and 10,000 ppm h. Different effects on measures of visual discrimination performance were observed in rats exposed to a constant or fluctuating concentrations, and when rats were tested immediately or sometime after the end of exposure to fluctuating concentrations. Motor activity was also differently affected by different exposure scenarios. Physiologically based toxicokinetic (PBTK) modelling was used to predict the toxicokinetics of toluene induced by these different exposure scenarios. The model was calibrated by measuring toluene concentrations in blood and brain during and after exposure. The results show that the acute effects of toluene on behaviour do not depend only on the concentration and duration of exposure, but primarily on the pattern of exposure.     

Neurobehavioural evaluation and kinetics of inhalation of constant or fluctuating toluene concentrations in human volunteers

The health risks of inhalation exposure to volatile organic solvents may not only depend on the total external dose, but also on the pattern of exposure. It has been suggested that exposure to regularly occurring peak concentrations may have a stronger impact on the brain than constant exposure at the same average level. Recent animal experimental studies conducted in our laboratory using relatively high concentrations of toluene have shown different effects on discrimination performance and motor activity during and after exposure, depending on the exposure scenario. Relevance of these findings for man was evaluated in a volunteer study in which 11 healthy men (age 20–49 years) were exposed by inhalation for 4 h to either a constant concentration of 40 ppm toluene or to three 30-min exposure peaks at 110 ppm during this 4 h period. Selected tests from the Neurobehavioural Evaluation System (NES) were performed repeatedly during and after exposure. Blood concentrations of toluene as well as urinary o-cresol excretion were measured at relevant time points.The results show that toluene concentration in blood increased during constant exposure and fluctuated during occupationally relevant peak exposures. Presumably, brain concentrations showed similar qualitative patterns. No clear changes were observed on neurobehavioural measures of motor performance, attention, perceptual coding and memory, or on measures of mood and affect. The exposure conditions do not seem to induce significant acute changes in central nervous system function similar to those observed at much higher concentrations in animals, although a statistical correlation was found between one motor performance test (Finger Tapping Test with alternating hands) and blood toluene concentrations. Urinary o-cresol excretion appeared to be significantly higher during the first 2 h after exposure.     

Impact of heat on biological concentrations of toluene and acetone resulting from exposure by inhalation: A pilot study

Climatic conditions raise new concerns about the potential impact of heat on the absorption and kinetics of certain chemicals. The impact of 3 temperatures (21, 25 and 30 °C WBGT) on the toxicokinetics of toluene and acetone was therefore evaluated in five human subjects during controlled exposures in an inhalation chamber. Biological samples were collected and analyzed by GC–MS/MS. Increases between 4 and 85 % were observed for solvents concentrations in blood (30 vs 21 °C) while decreases in urine samples for acetone and o-cresol were measured at the end of the exposure period (4 h). Mean blood concentrations at 4 h are well correlated with temperature. Results suggest an increased absorption and/or a decreased elimination of volatile chemicals in the presence of heat. Higher increases of blood chemical concentrations were observed in heavier individuals. Further studies should include physiologically based toxicokinetic models to help in better understanding the mechanisms involved and their respective contribution.     

Selective inhibition of gamma-aminobutyric acid type A receptors in human IMR-32 cells by low concentrations of toluene

Effects of the neurotoxic organic solvent toluene on human neuronal nicotinic acetylcholine (nACh) and γ-aminobutyric acid type A (GABA_A) neurotransmitter receptors were investigated in whole-cell voltage-clamped IMR-32 neuroblastoma cells. Ion currents evoked by near maximum effective concentrations of 1 mM acetylcholine (ACh) and 1 mM γ-aminobutyric acid (GABA) are inhibited by toluene in a concentration-dependent way. Concentration–effect curves of toluene yield IC₅₀ values of 276±26 and 39±6 μM and slope factors of 1.4±0.2 and 0.8±0.1 for inhibition of the ACh- and GABA-induced ion currents, respectively. The results demonstrate the selective inhibition of human GABA_A receptors by toluene at concentrations comparable with brain concentrations associated with occupational exposure.     

Regional brain distribution of toluene in rats and in a human autopsy

Toluene concentrations in 9 brain regions of acutely exposed rats and that in 11 brain regions of a human case who inhaled toluene prior to death are described. After exposure to toluene by inhalation (2000 or 10000 ppm) for 0.5 h or by oral dosing (400 mg/kg), rats were killed by decapitation 0.5 and 4 h after onset of inhalation and 2 and 10 h after oral ingestion. After each experimental condition the highest range of brain region/blood toluene concentration ratio (BBCR) was in the brain stem regions (2.85–3.22) such as the pons and medulla oblongata, the middle range (1.77–2.12) in the midbrain, thalamus, caudate-putamen, hypothalamus and cerebellum, and the lowest range (1.22–1.64) in the hippocampus and cerebral cortex. These distribution patterns were quite constant. Toluene concentration in various brain regions were unevenly distributed and directly related blood levels. In a human case who had inhaled toluene vapor, the distribution among brain regions was relatively similar to that in rats, the highest concentration ratios being in the corpus callosum (BBCR: 2.66) and the lowest in the hippocampus (BBCR: 1.47)     

Visual evoked potentials in individuals exposed to long-term low concentrations of toluene

 An investigation of visual evoked potentials was carried out in two groups of subjects; 49 workers employed in a printing-press where toluene has been used exclusively as an organic solvent for the last 30 years, and 59 workers not occupationally exposed to any known neurotoxic substances. The average length of work service in the printing-press was 21.4 years. The level of exposure was assessed by determination of the concentration of toluene in peripheral blood, the concentration of hippuric acid and ortho-cresol in urine in subgroups of subjects chosen at random from both groups. N75, P100 and N145 waves of the pattern reversal visual evoked potentials were analyzed. In the group of exposed subjects, significantly greater amplitudes were found in all waves, with significantly longer latency of the P100 wave. Received: 12 September 1994 / Accepted: 15 November 1994     

Detection and measurement of S-benzyl-N-acetylcysteine in urine of toluene sniffers using capillary gas chromatography

We examined the urinary excretion of S-benzyl-N-acetylcysteine (SBAC) of toluene sniffers using capillary gas chromatography. SBAC was extracted from 10 ml urine with chloroform and backextracted into 1 M sodium bicarbonate solution. After acidification, the aqueous solution was reextracted with ethyl acetate, and then derivatized to its methyl ester (ME). The peak appearing in the gas chromatogram was identified as SBAC-ME by mass spectrometry. The calibration curve was constructed by plotting the peak height ratio of SBAC-ME and internal standard (S-phenethyl-N-acetylcysteine)-ME against analyte concentration using 10 ml toluene unexposed urine. It showed good linearity over the range of 0.05–3.0 mg/l (r = 0.99). We have applied this technique to urine samples from toluene sniffers. SBAC was detected in all urinary samples of sniffers (n = 30, 0.11–47.13 mg/l), but not at all in the urine of toluene unexposed subjects (n = 60). These results prove that SBAC is also formed from toluene by human metabolism, and detection of SBAC is considered a useful marker for inhalation of toluene.Some of these results were presented at the 30th International Meeting of the International Association of Forensic Toxicologists (Fukuoka) in 1992.     

Toluene concentrations in various tissues of rats after inhalation and oral administration

The uptake, distribution, and elimination of ³H-toluene in various tissues of rats were studied after inhalation or after gastric intubation.The maximum radioactivity was measured 2–3 h after gastric intubation in tissues, except in white adipose tissue, where the peak radioactivity was reached at 5 h.After inhalation exposure, the uptake to various tissues was very rapid. The maximum radioactivity in most tissues was reached in 15–30 min. The accumulation was slowest in white adipose tissue, where it took 1–2 h.The radioactivity in tissues decreased after inhalation exposure more rapidly than after gastric intubation. Brown adipose tissue and white adipose tissue were different from other tissues in their ability to retain toluene. Twenty-four hours after exposures, only 1% or less of the initial radioactivity was found in tissues other than white adipose tissue, in which the corresponding value was 3.5–5%.The results show a very rapid absorption and distribution of toluene after inhalation and a retention of radioactivity in white adipose tissue. After oral ingestion the distribution showed a similar but much slower pattern.

---

Zusammenfassung Nach Inhalation und nach oraler Gabe wurden Aufnahme, Verteilung und Ausscheidung von ³H-Toluol in verschiedenen Geweben der Ratte untersucht.Bei oraler Gabe (mittels Schlundsonde) wurde das Maximum an Radioaktivität im weißen Fettgewebe nach 5 Std erreicht, während es in allen anderen untersuchten Organen bereits nach 2–3 Std auftrat. Nach Inhalation wurde ³H-Toluol wesentlich schneller aufgenommen. Die höchste Radioaktivität wurde hierbei im weißen Fett nach 1–2 Std und in den meisten anderen Geweben nach 15–30 min gemessen. Ebenso war die Abnahme der Radioaktivität nach Inhalation schneller als nach oraler Gabe, wobei sich auch in diesem Fall das braune und das weiße Fettgewebe von anderen Organen unterschieden. Vierundzwanzig Stunden nach Applikation enthielt das Fett noch 3,5–5% der initialen Radioaktivität, während in allen anderen Geweben nur noch 1% oder weniger gemessen wurden.Die Ergebnisse dieser Studie zeigen, daß Toluol nach Inhalation sehr viel rascher absorbiert und wieder ausgeschieden wird, als nach oraler Aufnahme, während das Verteilungsmuster nach beiden Applikationsarten sehr ähnlich ist.

     

Distribution of c-Fos immunoreactivity in the rat brain following abuse-like toluene vapor inhalation

Inhalation of vapors from toluene-containing products results in euphoria accompanied by a variety of cognitive impairments and motor dysfunctions. The profound behavioral changes observed during and following toluene inhalation suggest changes in the activity of cells in potentially many brain regions; however, a comprehensive assessment of the neuroanatomical structures activated by toluene vapor has not been completed. Thus in the present study we systematically mapped in over 140 brain structures the distribution of c-Fos immunoreactivity (c-Fos IR), a proxy for neural activation, following exposure to an abuse-like concentration (~ 5000 ppm) of toluene vapor for 0, 5, 10 or 30 min. Quantitative analyses revealed increases in c-Fos IR in about one-third of the brain structures examined, with most of these structures significantly activated only after prolonged toluene exposure. The majority of brain structures activated by toluene were found in the forebrain and midbrain, with particularly pronounced activation in nuclei implicated in the processing of rewarding, emotional, and olfactory stimuli, and those controlling motor output. These structures included the ventral tegmental area, nucleus accumbens, select regions of the amygdala and hypothalamus, cingulate cortex, olfactory nuclei, piriform cortex, secondary motor cortex and caudate-putamen. In contrast, all subregions of the hippocampus and most thalamic nuclei were not significantly activated by toluene vapor. In the brainstem, effects of toluene vapor were restricted to select nuclei in the pons. The pattern of c-Fos IR evoked by inhalation of toluene vapor appears distinct from other psychoactive substances, consistent with the unique and complex behavioral outcomes associated with acute toluene inhalation.     

Maternal and fetal blood and organ toluene levels in rats following acute and repeated binge inhalation exposure

Inhalation of organic solvents is a persistent form of drug abuse with particular concern being the abuse of inhalants by women of child-bearing age. While studies have begun assessing postnatal outcomes of offspring exposed prenatally to inhalants, relatively little is known about the distribution of toluene in blood and body tissues of pregnant, inhalant-abusing women, or in the fetuses. The present study assessed the tissue toluene levels attained following brief toluene exposures using a pre-clinical rat model of maternal inhalant abuse. Timed-pregnant Sprague–Dawley rats were exposed to toluene at 8000 or 12,000 parts per million (ppm) for 15, 30 or 45 min/exposure. Exposures occurred twice each day from gestational day 8 (GD8) through GD20. Immediately following the second exposure on GD8, GD14 and GD20 blood was taken from the saphenous vein of the dams. Following saphenous vein blood collection on GD20, dams were sacrificed and trunk blood was collected along with maternal tissue specimens from cerebellum, heart, lung, kidney and liver. The placenta, amniotic fluid and fetal brain were also collected. Results demonstrated that maternal saphenous blood toluene levels increased as the inhaled concentration of toluene and duration of exposure increased. The maternal cerebellum, heart, kidney and liver appeared to be saturated after 30 min on GD20 such that toluene levels in those organs were equivalent across all ambient concentrations of inhaled toluene. Toluene levels also increased in fetal brain as the inhaled concentration of toluene increased and in placenta and amniotic fluid as the duration of exposure increased. Toluene levels in all tissues at GD20, except maternal lung and amniotic fluid, were higher than in maternal saphenous blood suggesting that toluene concentrated in those organs. Measurement of toluene levels in blood and other tissues following repeated toluene exposure demonstrated that toluene readily reaches a variety of potential sites of action throughout the maternal–placental–fetal unit.     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat II. Developmental toxicity

The developmental toxicity of toluene was evaluated via whole body inhalation exposure, in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) from gestation day (GD) 6–15 inclusive, 6 h/day, at concentrations of 0, 250, 750, 1500 and 3000 ppm (0, 938, 2812, 5625 and 11250 mg/m³). Doses were selected from a preliminary study performed over a range of concentrations from 0 to 5000 ppm, in which maternal and fetal toxicity were observed at 2000 ppm and above. This study has been cited in various regulatory documents and is presented here to allow greater accessibility to results and conclusions.

Toluene induced clinical signs in pregnant dams (ataxia, hyper-responsivity, increased water intake, decreased food consumption) at 3000 ppm, ataxia and hyper-responsivity at 1500 ppm, and reduced maternal body weight gain at 1500 during the exposure period only and at 3000 ppm from initiation of exposure to GD20. At Caesarean section on GD20, no adverse effects on implantation, number and viability of fetuses, or fetal sex distribution were observed. Litter weight and mean fetal weight was reduced at 3000 ppm and mean fetal weight was reduced at 1500 ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. Mean fetal weight was also reduced at 250 ppm but not at 750 ppm. Extensive statistical analysis of fetal body weight data support the conclusion that there is no toxicologically significant dose-related effect on fetal body weight at or below 750 ppm. Low incidences (≤2.5%) of various malformations occurred in the 250, 1500, and 3000 ppm groups; there was no increase in the incidence of specific or total malformations with increased exposure and thus these were not attributed to toluene.

In this Toluene study, the maternal toxicity NOAEL was 750 ppm with a defined maternal and developmental toxicity LOAEL of 1500 ppm.     

Sex differences in urinary levels of several biological indicators of exposure: a human volunteer study

The aim of the study was to quantify the variability on biological indicators of exposure between men and women for three well known solvents: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile.Controlled human exposures were carried out in a 12 m³ exposure chamber for each solvent separately, during 6 h and at half of the threshold limit value. The human volunteers groups were composed of ten young men and fifteen young women, including ten women using hormonal contraceptive.An analysis of variance mainly showed an effect on the urinary levels of several biomarkers of exposure among women due to the use of hormonal contraceptive, with an increase of more than 50% in metabolites concentrations and a decrease of up to 50% in unchanged substances concentrations, suggesting an increase in their metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele.Our study suggests that not only physiological differences between men and women but also differences due to sex hormones levels can have an impact on urinary concentrations of several biomarkers of exposure. The observed variability due to sex among biological exposure indices can lead to misinterpretation of biomonitoring results. This aspect should have its place in the approaches for setting limits of occupational exposure.     

Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y cells

The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.     

Risk of human exposure to paralytic toxins of algal origin

The most significant neurotoxins produced by harmful algal blooms (HABs) are paralytic shellfish toxins (PSTs) found in shellfish and freshwater. Human exposure to neurotoxins through the food consumption represents a severe hazard to human health and the exposure through contaminated water represents an added risk often difficult to recognize. Furthermore, there is an insufficient knowledge of toxicokinetics of these complex toxins produced by HABs. If human acute exposure occurs, the diagnosis of intoxication is typically based upon symptomatology and analysis of shellfish tissue by mouse bioassay, HPLC-FLD analysis and mouse neuroblastoma assay. However, the health risks due to chronic exposure should also be considered and its prevention could be reached with a better understanding of sub-lethal doses of these toxins. In this context, information required for development of a diagnostic protocol should include knowledge about toxicokinetics and toxicodynamics of these neurotoxins. We emphasise the importance of research on biomarkers to prevent, predict and diagnose acute and chronic human exposure to PST.     

Washout kinetics of inhaled hydrogen cyanide in breath

Hydrogen cyanide (HCN) intoxication causes or contributes significantly to many of the fatalities among fire victims. To enable fast treatment of HCN poisoning, a more rapid diagnostic method than currently available is required. One possibility would be measurement in exhaled air. However, as HCN is highly water soluble, it may be absorbed during inhalation and reabsorbed during exhalation. If this, so-called, washin-washout effect is substantial it may interfere with the diagnosis, as a major part of breath HCN may originate from the respiratory tract, due to recent exposure, and not from systemic exposure. The aim of this study was to estimate the importance of the washin-washout effect of HCN. The time-course of cyanide in exhaled air was measured with an electrochemical detector in 10 volunteers during and after a 1 min x 10 ppm exposure to HCN. The experiment revealed an average half-life of 16s (range 10-24s) in breath. Extrapolating the results to higher exposures suggests that the contribution from washin-washout from the airways will be negligible even at fatal exposures. The results support the use of breath HCN as a potential indicator of systemic intoxication.     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat. I. Reproductive toxicity

The reproductive toxicity of toluene was evaluated in a 2-generation test in which male and female Sprague–Dawley rats, parental (F0) and first generation (F1), were exposed to toluene via whole body inhalation, 6 h/day, 7 days/week for 80 days premating and 15 days of mating at concentrations of 0, 100, 500 and 2000 ppm (0, 375, 1875 and 7500 mg/m³). Toluene was administered at 2000 ppm to both sexes, or to females or males only to be mated with untreated partners. Pregnant females at all dose levels were exposed from gestation day (GD) 1–20 and lactation day (LD) 5–21. At LD5, females were removed from their litters for daily exposure and returned when 6 h of exposure was completed. F1 pups selected to produce the F2 generation were treated for 80 days beginning immediately after weaning (LD21) and initially mated at a minimum of 100 days of age. F2 pups were not exposed to toluene by inhalation.

Toluene exposure did not induce adverse effects on fertility, reproductive performance, or maternal/pup behaviors during the lactation period in males and females of the parental or first generation, but did inhibit growth in F1 and F2 offspring in the 2000 ppm (both sexes treated) and 2000 ppm (females only treated) groups. Caesarean section of selected 2000 ppm (both sexes treated) dams at GD20 showed reduced fetal body weight and skeletal variations. Exposure to toluene caused decreased pup weights throughout lactation in F1 and F2 2000 ppm (both sexes treated), and 2000 ppm (females only treated) groups. Exposure at 2000 ppm to male parents only did not induce similar weight inhibition in offspring. The toluene offspring NOAEL is 500 ppm in groups in which maternal animals were exposed, and 2000 ppm for male only treated groups.     

Inhalation and epidermal exposure of volunteers to ethylene glycol: kinetics of absorption, urinary excretion, and metabolism to glycolate and oxalate

Ethylene glycol (EG) is a widely used liquid. Limited data are published regarding inhaled EG and no data regarding transdermal EG uptake in humans. In order to gain information on the quantitative fate of EG, four male volunteers inhaled between 1340 and 1610 micromol vaporous 13C-labeled EG (13C2-EG) for 4h. Separately, three of these subjects were epidermally exposed for up to 6h to liquid 13C2-EG (skin area 66 cm2). Plasma concentrations and urinary amounts of 13C2-EG were determined by gas chromatography with mass selective detection. Additionally, plasma was assayed for 13C-labeled glycolic acid 13C2-GA) and urine for 13C2-GA and 13C-labeled oxalic acid (13C2-OA). Both EG metabolites were nephrotoxic in animals and humans and embryotoxic in rodents. 13C-labels enabled to differentiate from also determined endogenous EG, glycolic acid (GA), and oxalic acid (OA). Of 13C2-EG inhaled, 5.5+/-3.0%, 0.77+/-0.15%, and 0.10+/-0.12% were detected in urine as 13C2-EG, 13C2-GA, and 13C2-OA, respectively. The skin permeability constant of liquid EG was 2.7 x 10(-5)+/-0.5 x 10(-5)cm/h. Of the dose taken up transdermally, 8.1+/-3.2% and up to 0.4% were excreted in urine as 13C2-EG and 13C2-GA, respectively. It is calculated that equally long-lasting exposure to 10 ppm vaporous EG or wetting of both hands by liquid EG leads to about the same body burden by EG and metabolites. The amounts of GA and OA excreted daily in urine as a result of exposure (8h/day) to 10 ppm EG are about 15% and 2%, respectively, of those excreted from naturally occurring endogenous GA and OA.     

Further evaluation of the kinetics of white spirit in human volunteers

This paper gives supplementary informations on the kinetics of aliphatic white spirit in humans. Eight volunteers were exposed to 600 mg/m3 (100 p.p.m.) white spirit during 3 hrs. Calculated pulmonary uptake (dose) was 392 +/- 38 mg, residence time 47.5 hrs, volume of distribution 749 1, and total body clearance 263 ml/min. Then 7 volunteers were exposed to 600 mg/m3 white spirit 6 hrs daily in 5 consecutive days. Calculated pulmonary uptake was 3464 +/- 329 mg. A mathematic model is presented which uses the blood concentrations to calculate the tissue concentrations during repeated doses administered over an extended period. By means of this model and the measured concentrations in blood and fat, the partition coefficient fat: blood for white spirit was calculated to 47. Estimated redistribution phase of white spirit in adipose tissue was approximately 20 hrs for the first 5 exposures, and half-life in adipose tissue after redistribution 46-48 hrs. During exposure to 600 mg/m3 white spirit 6 hrs daily, 5 days a week, calculated maximum steady state concentrations were 55 mg/kg fat and 5 mg/kg brain, minimum steady state concentrations 35 mg/kg fat and 0.6 mg/kg brain. These values for half-life and steady-state concentrations are considered more correct than our previously reported values, which did not take into account the existence of a redistribution phase.     

Effects of subchronic exposure to toluene on working and reference memory in rats

Rats that had inhaled 600 ppm of toluene vapor 24 h a day for 50 days after weaning at 3 weeks of age were trained in a radial-arm maze with a 4-out-of-8 baiting procedure, and their performance based upon reference and/or working memory was compared with that of air-exposed control animals during the early stage of acquisition. Pharmacological challenge testing was also conducted after completing a total of 48 training sessions; the effects of scopolamine and methylscopolamine on the maze performance were measured after acute IP administration to determine the long-lasting effects of toluene exposure. During the acquisition stage, toluene-exposed rats made a significantly smaller number of reference memory errors (entries into “never-baited” arms) and total arm entries than the control rats. No significant effects of exposure were observed for working memory errors (reentries into “already-entered” arms). During the pharmacological challenge testing, only scopolamine increased both types of errors significantly. No significant differences due to toluene exposure were revealed.