Excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene in an exposure chamber and in workshops, with specific reference to repeated exposures

Ogata, M., Takatsuka, Y., and Tomokuni, K. (1971).Brit. J. industr. Med.,28, 382-385. Excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene in an exposure chamber and in workshops, with specific reference to repeated exposures. Four male volunteers were exposed to 200 p.p.m. of toluene for five one-hour periods separated by one-hour intervals. The excretion curve of hippuric acid showed multi-peaks, and almost concided with a theoretical curve previously described. The fraction of the toluene absorbed which was accounted for as hippuric acid was only slightly lower than after a single exposure.

In a paint spraying shop exposure was measured both from the concentrations of toluene in the air by a Kitagawa detector and from the exceretion of urinary hippuric acid. The results were in general agreement, with a correlation coefficient of 0·67.

Urinary hippuric acid and methylhippuric acid were determined on urines from two workers in a shipbuilding yard who used paint thinned with toluene and xylene. The concentrations of the acids varied from day to day depending on the kind and the duration of work. From the concentrations found the mean concentrations to which the workers were exposed were calculated as a fraction of the maximum allowable concentration (M.A.C.). One worker was, on this evidence, exposed to more than the combined M.A.C. on three days out of six.

     

Quantitative determination in urine of hippuric acid and m- or p-methylhippuric acid, metabolites of toluene and m- or p-xylene

Ogata, M., Tomokuni, K., and Takatsuka, Y. (1969).Brit. J. industr. Med.,26, 330-334. Quantitative determination in urine of hippuric acid and m- or p-methylhippuric acid, metabolites of toluene and m- or p-xylene. Improved and more specific methods for the quantitative determination of hippuric and m- and p-methylhippuric acids in urine are described. The acids were extracted from urine with ether/ethanol, which was dried with silica gel, or with ethyl acetate. After removing the solvent by evaporation coloured azlactones were formed by reaction with p-dimethylaminobenzaldehyde in acetic anhydride (DAB, only usable after ether/ethanol extraction) or benzenesulphonyl chloride in pyridine, and the absorbances were measured. The sensitivities were about 4 μg./ml. of urine using DAB, and 20 μg./ml. of urine using benzenesulphonyl chloride reagent. Separation of hippuric and methylhippuric acids was achieved by paper and thin-layer chromatography before estimation. A spot test is also described.     

Urinary excretion of hippuric acid and m-methylhippuric acid after administration of toluene and m-xylene mixture to rats

Summary Male Wistar rats were injected intraperitoneally (i.p.) with toluene alone, m-xylene alone, and in combination with both of them. Urinary excretion of hippuric acid or m-methylhippuric acid of rats injected with solvents of various concentrations was investigated from the end of the injections up to 48 h later, at appropriate intervals. The amounts of increased excretion of HA and m-MHA in 0–24h urine samples of the rats given simultaneous injections of toluene and m-xylene were not lower than those of the rats given single injections of toluene alone or m-xylene alone, in the same amount. Data indicated that simultaneous administration of toluene and m-xylene did not significantly interfere with the metabolism of toluene or m-xylene.Read before the 50th Annual Meeting of Japan Industrial Health Association, Kurume, April 5, 1977The work was supported in part by grant No. 357188 of the Ministry of Education Agency     

Three fatal cases of thinner-sniffing,and experimental exposure to toluene in human and animals

Summary Three fatal cases of organic solvent abuse revealed high levels of toluene in blood and alveolar air and a high level of hippuric acid, metabolite of toluene, in urine. The lethal concentration of toluene was estimated to be 2,000 ppm.Furthermore, 10 male and female volunteer students were exposed to 107 ±12 ppm toluene for 4 hours. Hippuric acid in urine increased with the exposure time and reached maximum 2 hours after initiation of toluene exposure and remained at the same level thereafter. Following cessation of exposure to toluene, hippuric acid in urine showed a rapid decrease and recovered almost to the normal level 4 hours after cessation of exposure.Urinary excretion of hippuric acid in 7 rabbits exposed to 350 ppm for 100 minutes or to 4,500 ppm toluene for 10 minutes, reached its maximum 1.5–2 hours after initiation of exposure and decreased rapidly after cessation of exposure to toluene to recover to the normal level 4 hours later.Read before the 43rd Annual Meeting of Japanese Association of Industrial Health at Tokushima on April 2, 1970, and the 18th Annual Meeting of North Kanto Medical Association at Maebashi on November 14, 1971.     

Urinary methylhippuric acid isomer levels after occupational exposure to a xylene mixture

Summary The quantitative relationship between exposure to xylene vapor and urinary excretion of methylhippuric acid (MHA) isomers were studied in the second half of a working week. The participants in the study were 121 male workers engaged in dip-coating of metal parts who were predominantly exposed to three xylene isomers. The intensity of exposure measured by diffusive sampling during an 8-h shift was such that the geometric mean vapor concentration was 3.8 ppm for xylenes (0.8 ppm for o-xylene, 2.1 ppm for m-xylene, and 0.9 ppm for p-xylene), 0.8 ppm for toluene, and 0.9 ppm for ethylbenzene. Urine samples were collected at the end of the shift and analyzed for metabolities by HPLC. The statistical analysis showed that there is a linear relationship between the intensity of exposure to xylenes and the concentration of MHA in urine, that the regression line passes very close to the origin, and that the increment in observed (i.e., noncorrected) MHA concentrations as a function of increasing xylene concentration was 17.8 mg × 1^–1 ppm^–1. Further examination on the basis on individual xylene isomers showed that the slopes of the regression lines for o- and m-isomers were similar (i.e., 17.1 and 16.6 mg l^–1 ppm^–1, respectively), whereas that for p-xylene was larger (21.3 mg l^–1 ppm^–1).     

Effect of various exposure scenarios on the biological monitoring of organic solvents in alveolar air

Summary The present study was undertaken to investigate the influence of different exposure scenarios on the elimination of toluene and m-xylene in alveolar air and other biological fluids in human volunteers. The study was also aimed at establishing the effectiveness of physiologically based toxicokinetic models in predicting the value of biological monitoring data after exposure to toluene and m-xylene. Two adult male and two adult female white volunteers were exposed by inhalation, in a dynamic, controlled-environment exposure chamber, to various concentrations of toluene (21–66 ppm) or mxylene (25–50 ppm) in order to establish the influence of exposure concentration, duration of exposure, variation of concentration within day, and work load on respective biological exposure indices. The concentrations of unchanged solvents in end-exhaled air and in blood as well as the urinary excretion of hippuric acid and m-methylhippuric acid were determined. The results show that doubling the exposure concentration for both solvents led to a proportional increase in the concentrations of unchanged solvents in alveolar air and blood at the end of a 7-h exposure period. Cumulative urinary excretion of the respective metabolites exhibited a nearly proportional increase. Adjustment of exposure concentration to account for a prolongation of the duration of exposure resulted in essentially identical cumulative urinary excretion of the metabolites. Induced within-day variations in the exposure concentration led to corresponding but not proportional changes in alveolar concentration for both solvents, depending on whether or not sampling preceded or followed peak exposure to solvent. At the end of repeated 10-min periods of physical exercise at 50 W, alveolar air concentrations of both solvents were increased by 40%. Experimental data collected during the present study were adequately simulated by physiologically based toxicokinetic modeling. These results suggest that alveolar air solvent concentration is a reliable index of exposure to both toluene and m-xylene under various experimental exposure scenarios. For clinical situations likely to be encountered in the workplace, physiologically based toxicokinetic modeling appears to be a useful tool both for developing strategies of biological monitoring of exposure to volatile organic solvents and for predicting alveolar air concentrations under a given set of exposure conditions.     

A new derivatization procedure for the analysis of hippuric acid and m-methyl-hippuric acid by gas chromatography

Summary The industrial solvents, toluene and xylene, have physicochemical properties that can be hazardous to the workers exposed. Since hippuric acid and m-methyl-hippuric acid represent the products of toluene and xylene biotransformation in urine, they are used as biological markers in studies on occupational exposure to these solvents. Several methods have been used to determine hippuric acid and m-methyl-hippuric acid —either based on gas chromatography or on high-performance liquid chromatography. In this study we propose the derivatization of hippuric acid and methyl-hippuric acid using methanol in acid medium (HCl), a low-cost reagent with a low level of toxicity. The method has been routinely used in our laboratory for 1 year and has proven to be a reliable procedure for the biological control of occupational exposure to toluene and/or xylene.     

Significance of urinary hippuric acid determination as an index of toluene exposure

Ikeda, Masayuki, and Ohtsuji, Hatsue (1969).Brit. J. industr. Med.,26, 244-246. Significance of urinary hippuric acid determination as an index of toluene exposure. Urine samples from 118 male workers in photogravure printing factories were analysed for hippuric acid. The urinary levels of hippuric acid were proportional to the environmental concentrations of toluene, although within wide variations. The urinary concentration of hippuric acid corresponding to 200 p.p.m. of toluene was 3·5 g./litre (specific gravity 1·016) or 4·3 g./g. creatinine.     

Toxicokinetics of toluene and urinary excretion of hippuric acid after human exposure to 2H8-toluene.

Nine male volunteers were exposed to 2H8-toluene (200 mg/m3 for two hours during a workload of 50 W) via inspiratory air with the aid of a breathing valve and mouthpiece. Labelled toluene was used to differentiate between hippuric acid originating from exposure to toluene and hippuric acid normally excreted in urine. The total uptake of toluene was 2.2 (standard deviation (SD) 0.2) mmol, or 50% of the amount inhaled. Four hours after the end of exposure 1.4 (SD 0.3) mmol or 65% of the total uptake had been excreted in urine as 2H-hippuric acid and 20 hours after the end of exposure the cumulative excretion of 2H-hippuric acid was 1.8 (SD 0.3) mmol, or 78% of the total uptake. By contrast the cumulative excretion of labelled plus unlabelled hippuric acid exceeded the total uptake of toluene already after four hours. The excretion rate of 2H-hippuric acid was highest, about 5 mumol/min, during exposure and the SD between the subjects was low. The background concentrations of unlabelled hippuric acid in urine were high, however, and there were large differences between subjects. These findings confirm earlier indications that for low exposure, urinary hippuric acid concentration cannot be used for biological monitoring of exposure to toluene.     

Comparative evaluation of biomarkers of occupational exposure to toluene

Objectives This study was initiated to make comparative evaluation of five proposed urinary markers of occupational exposure to toluene, i.e., benzyl alcohol, benzylmercapturic acid, o-cresol, hippuric acid and un-metabolized toluene. Methods In practice, six plants in Japan were surveyed, and 122 Japanese workers (mostly printers; all men) together with 12 occupationally nonexposed control subjects (to be called controls; all men) agreed to participate in the study. Surveys were conducted in the second half of working weeks. Time-weighted average exposure (about 8 h) to toluene and other solvents were monitored by diffusive sampling. End-of-shift urine samples were collected and analyzed for the five markers by the methods previously described; simultaneous determination of o-cresol was possible by the method originally developed for benzyl alcohol analysis. Results The toluene concentration in the six plants was such that the grand geometric mean (GM) for the 122 cases was 10.4 ppm with the maximum of 121 ppm. Other solvents coexposed included ethyl acetate (26 ppm as GM), methyl ethyl ketone (26 ppm), butyl acetate (1 ppm) and xylenes (1 ppm). By simple regression analysis, hippuric acid correlated most closely with toluene in air (r = 0.85 for non-corrected observed values) followed by un-metabolized toluene (r = 0.83) and o-cresol (r = 0.81). In a plant where toluene in air was low (i.e., 2 ppm as GM), however, un-metabolized toluene and benzylmercapturic acid in urine showed better correlation with air-borne toluene (r = 0.79 and 0.61, respectively) than hippuric acid (r = 0.12) or o-cresol (r = 0.17). Benzyl alcohol tended to increase only when toluene exposure was intense. Correction for creatinine concentration or specific gravity of urine did not improve the correlation in any case. Multiple regression analysis showed that solvents other than toluene did not affect the levels of o-cresol, hippuric acid or un-metabolized toluene. Levels of benzylmercapturic acid and un-metabolized toluene were below the limits of detection [limit of detections (LODs); 0.2 and 2 μg/l, respectively] in the urine from the control subjects. Conclusions In over-all evaluation, hippuric acid, followed by un-metabolized toluene and o-cresol, is the marker of choice for occupational toluene exposure. When toluene exposure level is low (e.g., 2 ppm), un-metabolized toluene and benzylmercapturic acid in urine may be better indicators. Detection of un-metabolized toluene or benzylmercapturic acid in urine at the levels in excess of the LODs may be taken as a positive evidence of toluene exposure, because their levels in urine from the controls are below the LODs. The value of benzyl alcohol as an exposure marker should be limited.     

Are women more sensitive than men to 2-propanol and m-xylene vapours?

Aims: To evaluate possible differences between men and women in acute health effects after controlled short term chamber exposure to vapours of two common organic solvents.

Methods: Fifty six healthy volunteers (28 per sex) were exposed to 150 ppm 2-propanol, 50 ppm m-xylene, and clean air for two hours at rest. The subjects rated symptoms on a visual analogue scale before, during, and after the exposure. Blinking frequency was measured continuously during exposure. Pulmonary function, nasal swelling, inflammatory markers (lysozyme, eosinophilic cationic potein, myeloperoxidase, albumin) in nasal lavage and colour vision (Lanthony D-15 desaturated panel) were measured before and at 0 and 3 hours after the exposure.

Results: There were no significant sex differences in response to solvent exposure with respect to blinking frequency, lung diffusing capacity, nasal area and volume, inflammatory markers in nasal lavage, and colour vision. Increased symptoms were rated by both sexes for nearly all 10 questions during exposure to 2-propanol or m-xylene, most increases being significant at one time point at least. The rating of "discomfort in the throat or airways" increased more in women during exposure to 2-propanol or m-xylene. During exposure to 2-propanol the rating of "fatigue" was more increased in men after one hour, but more increased in women after two hours of exposure. With regard to pulmonary function, women had small but significant decreases in FVC, FEV₁/FVC, and FEF₇₅ three hours after exposure to m-xylene, but only the decrease in FVC was significantly different from that in men.

Conclusion: Our results suggest that women are slightly more sensitive than men to the acute irritative effects of 2-propanol and m-xylene vapours.

     

Hippuric acid, phenol, and trichloroacetic acid levels in the urine of Japanese subjects with no known exposure to organic solvents

Ikeda, M., and Ohtsuji, H. (1969).Brit. J. industr. Med.,26, 162-164. Hippuric acid, phenol, and trichloroacetic acid levels in the urine of Japanese subjects with no known exposure to organic solvents. Urine samples from 36 male and 30 female university students and 31 male factory workers with no known exposure to industrial organic solvents were analysed for hippuric acid, phenol, and trichloroacetic acid, which are the major metabolites of toluene, benzene, and trichloroethylene respectively. The normal levels were less than 1·4 g./l. for hippuric acid, less than 80 mg./l. for phenol, and less than 1 mg./l. for trichloroacetic acid. No evidence was obtained to suggest that correction for urine concentration with either specific gravity or creatinine concentration minimizes individual variation of metabolite levels.     

Hippuric acid and ortho-cresol as biological indicators of occupational exposure to toluene

Industrial exposure to toluene was studied in a group of 18 subjects working in a printing plant, exposed only to this solvent. Environmental monitoring was carried out using personal samplers for the whole work-shift. Urine samples were collected for the determination of hippuric acid and ortho(o)-cresol before toluene exposure, at the end of the work-shift, and 5, 9, and 17 h after the end of the work-shift. The values of two metabolites in all the urinary samples were corrected for g creatinine and specific gravity (1.024). Toluene time weighted average (TWA) concentrations ranged from 51 to 221 mg/m³ (7-h samples; two samplings lasting 3.5 h each). Urinary hippuric acid and o-cresol values at the end of the work-shift were significantly higher than the prework-shift values. Both hippuricuria and o-cresoluria end-of-work-shift values, corrected for creatinine and specific gravity, were significantly related to the mean daily environmental concentration of toluene, the correlation being weaker for o-cresol. Correlation coefficients were 0.88 and 0.84 for hippuric acid and 0.63 and 0.62 for o-cresol after correction for creatinine and specific gravity, respectively. No significant relationship was observed between environmental exposure and the values of the two urinary metabolites 5, 9, and 17 h after the end of the work-shift. Extrapolated values from the linear regression analysis at 375 mg/m³ were in good agreement with the biological exposure index (BEI) suggested by ACGIH for hippuric acid. We conclude that determination of hippuric acid in urine samples collected at the end of the work-shift can be used for routine biological monitoring of exposure to toluene, even at low levels. O-cresol seems to be a less reliable indicator of toluene exposure.     

Urinary hippuric acid concentration after occupational exposure to toluene.

The results of industrial investigations have shown a correlation between the rate of hippuric acid excretion in a single urine sample collected after daily occupational exposure and the amount of toluene absorbed. The rate of hippuric acid excretion and the average concentration of toluene vapour during exposure time were also related. The quantitative range of the test has been limited to amounts exceeding 425 mg of toluene and concentrations exceeding 69 ppm of toluene in the air because of the physiological presence of hippuric acid in urine. The rate of hippuric acid excretion in urine depends on diuresis and is constant for urinary fractions with diuresis of 30 ml/h. The physiological excretion rate was 20 mg/h with a standard deviation +/- 4.3 mg/h, maximal physiological level 33 mg/h.     

Occupational chronic exposure to organic solvents XVI. Ambient and biological monitoring of workers exposed to toluene

Object Ambient air and biological monitoring of an occupational toluene exposure was carried out on a group of 33 workers. Method The biological monitoring of the workers was based on determination of the concentration of toluene in blood and on quantification of the urinary metabolites o-cresol and hippuric acid. All blood and urine samples were collected post-shift. Results The average toluene concentration in the workplace air was 65?ppm, ranging from 13 to 151?ppm. An average concentration of toluene in blood of 911?μg/l was found, corresponding to an average urinary concentration of 2.9?mg/l (2.3?mg/g creatinine) o-cresol and 2.4?g/l (1.9?g/g creatinine) hippuric acid. Both urinary metabolites can be correlated with the concentration of toluene in ambient air and blood, respectively. Conclusions The results of our study indicate that the determination of the urinary o-cresol excretion represents a diagnostically specific and sensitive parameter for the estimation of an individual toluene uptake. In contrast, monitoring of the concentration of hippuric acid in urine cannot be recommended for assessment of individual exposure. To set up a biological tolerance value (BAT) for o-cresol, a urinary concentration of 3?mg/l o-cresol should be in accordance with the current MAK value of 50?ppm toluene.     

Chronic occupational exposure to toluene

Summary Chronic occupational exposure to toluene was studied in a factory preparing tarpaulins. Seventy-eight workers were studied; 46 were exposed to various concentrations of toluene in air (20–200 ppm), 32 were unexposed workers in the same factory. In many cases the exposure had lasted for 10–20 years. The urinary hippuric acid excretion at the end of work shift showed good correlations to toluene concentrations in air, and it seems to be a good measure of exposure. The hippuric acid in urine samples collected overnight showed that elimination of toluene still occurs several hours after exposure. Most of the biological parameters measured showed no correlation to toluene exposure. The blood leukocyte count did show slight positive correlations to toluene exposure, but even this parameter stayed inside the range of normal values. The occurrence of chronic diseases, drug using habits, and drinking and smoking habits did not show any correlations to toluene exposure.This study has been supported by the grant of Y. Jahnsson Foundation in Finland     

A rapid colorimetric method for the determination of phenylglyoxylic and mandelic acids: Its application to the urinalysis of workers exposed to styrene vapour

Ohtsuji, H., and Ikeda, M.(1970).Brit. J. industr. Med.,27, 150-154. A rapid colorimetric method for the determination of phenylglyoxylic and mandelic acids. Its application to the urinalysis of workers exposed to styrene vapour. A rapid colorimetric method has been developed for the determination of the styrene metabolites, phenylglyoxylic and mandelic acids, in urine. Ether extracts of acidified urine containing the two acids were evaporated to dryness in a test tube, and a mixture of sulphuric acid and formalin (100:1, v/v) was added for colour development. Factors necessary to convert optical extinction to gravimetric units were determined. When urinesamples from workers exposed to up to 30 ppm of styrene were analysed for phenylglyoxylic and mandelic acids together with hippuric acid, it was found that the phenylglyoxylic acid level provided the most sensitive index of styrene exposure and that the optical extinction at 350 nm was practically proportional to phenylglyoxylic acid concentration. No significant increase in hippuric acid levels was observed.     

Limited validity of o-cresol and benzylmercapturic acid in urine as biomarkers of occupational exposure to toluene at low levels

This study was initiated to evaluate o-cresol and benzylmercapturic acid in urine in comparison with other biomarkers, as tools to estimate the intensity of occupational exposure to toluene at low levels. In total, 108 solvent exposed workers (engaged in tape production) and 17 non-exposed controls (all men) participated in the study. The surveys were conducted in the second half of working weeks. Diffusive sampling was conducted to measure 8-h time-weighted average intensity of occupational exposure to toluene. Blood and urine samples were collected at the end of a working shift. Blood samples were subjected to analysis for toluene (Tol-B), and urine samples were analyzed for benzyl alcohol (BeOH-U), benzylmercapturic acid (BMA-U), o-cresol (o-CR-U), hippuric acid (HA-U) and toluene (Tol-U) by the methods previously described. The toluene concentrations in workplaces were low in general, with a geometric mean (GM) and the maximum concentration of 1.9 ppm and 8.8 ppm, respectively. The statistical analyses of the six biomarkers for correlation with air-borne toluene showed that both Tol-B and Tol-U gave a high correlation coefficient of 0.58 to 0.61 (p<0.01), whereas the coefficients for BeOH-U and BMA-U together with HA-U were all low (up to 0.22, depending on the correction for urine density) and statistically insignificant (p>0.10) in most cases. o-CR-U had an intermediary coefficient of 0.20 (p<0.05). Comparison with previous publications disclosed that BeOH-U, BMA-U and HA-U correlate with toluene in air when the exposure is intense (e.g., 50 ppm or above), but no longer proportional to air-borne toluene when the exposure is low, e.g., 2 ppm. Such appeared to be also the case for o-CR-U. In over-all evaluation, the validity of o-CR-U in monitoring occupational exposure to toluene at low levels (e.g., 2 ppm) appear to be limited, and BMA-U is not an appropriate biomarker. BeOH-U and HA-U are also inappropriate for this purpose. Only Tol-B and Tol-U may be employed to estimate toluene exposure at low levels.     

Effects of ethanol and phenobarbital treatments on the pharmacokinetics of toluene in rats.

Rats were exposed to toluene at a wide range of concentrations from 50 to 4000 ppm for six hours, and the effects of ethanol and phenobarbital (PB) treatments on the pharmacokinetics of toluene metabolism were investigated. Ethanol treatment influenced toluene metabolism mainly at low exposure concentrations. Thus ethanol accelerated the clearance of toluene from blood only when the blood concentration of toluene was not high (less than 360 microM), and ethanol increased hippuric acid (HA) excretion in urine more significantly at low (less than 250 ppm) than at high atmospheric toluene concentrations. Ethanol also expressed a similar effect on p-cresol excretion as on HA, but had little effect on o-cresol. Phenobarbital treatment promoted the urinary excretion of all of the metabolites of toluene, especially after exposure to high toluene concentration. As well as HA, benzoylglucuronide (BG) and free benzoic acid were found in urine. These are the products of the side chain metabolism of toluene. Amounts of BG could be detected when the urinary excretion of free benzoic acid exceeded 5 mumol/kg/6 h, indicating that a great deal of benzoic acid is required for the formation of BG. The Michaelis constant (Km) and the maximum rate of metabolic excretion in urine during six hours exposure (Vmax) of isozymes involved in the excretion of toluene metabolites were calculated, and correlated with the subtypes of cytochrome P-450. The significance of the result was suggested in the biological monitoring of exposure to toluene.     

Toluene itself as the best urinary marker of toluene exposure

Head-space gas chromatography (GC) and high-performance liquid chromatography (HPLC) (with fluorescence detectors) methods were developed for toluene (TOL-U) and o-cresol (CR-U) in urine, respectively. In order to identify the most sensitive urinary indicator of occupational exposure to toluene vapor (TOL-A) among TOL-U, CR-U, and hippuric acid in urine (HA-U), the two methods together with an HPLC (with untraviolet detectors) method for determination of HA-U were applied in the analysis of end-of-shift urine samples from 115 solvent-exposed workers (exposed to toluene at 4 ppm as geometric mean). Regression analysis showed that TOL-U correlated with TOL-A with a significantly higher correlation coefficient than did HA-U or CR-U. With regard to the TOL-A concentrations at which the exposed subjects could be separated from the nonexposed by the analyte, TOL-U achieved separation at < 10 ppm TOL-A, whereas both HA-U and CR-U did so only when TOL-A was 30 ppm or even higher. The ratio of the analyte concentrations at 50 ppm TOL-A to those at 0 ppm TOL-A was also highest for TOL-U. Overall, the results suggest that TOL-U is a better marker of exposure to toluene vapor than HA-U or CR-U.