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     

Toluene in blood as a marker of choice for low-level exposure to toluene

The validity of two new biological exposure markers of toluene in blood (TOL-B) and toluene in urine (TOL-U) was examined in comparison with that of the traditional marker of hippuric acid in urine (HA-U) in 294 male workers exposed to toluene in workroom air (TOL-A), mostly at low levels. The exposure was such that the geometric mean for toluene was 2.3 ppm with a maximum of 132 ppm; the workers were also exposed to other solvents such as hexane, ethyl acetate, styrene, and methanol, but at lower levels. The chance of cutaneous absorption was remote. Higher correlation with TOL-A and better sensitivity in separating the exposed workers from the nonexposed subjects were taken as selection criteria. When workers exposed to TOL-A at lower concentrations (< 50 ppm, < 10 ppm, < 2 ppm, etc.) were selected and correlation with TOL-A was examined, TOL-B showed the largest correlation coefficient which was significant even at TOL-A of < 1 ppm, whereas correlation of HA-U was no longer significant when TOL-A was < 10 ppm. TOL-U was between the two extremes. The sensitivities of TOL-B and TOL-U were comparable; HA-U showed the lowest sensitivity. Thus, it was concluded that TOL-B is the indicator of choice for detecting toluene exposure at low levels.     

Urinary levels of proteins and metabolites in workers exposed to toluene

Summary We measured urinary excretion of albumin and retinol-binding proteins to investigate the occurrence of early renal dysfunction in 45 paint workers exposed principally to toluene, and in the same number of unexposed control subjects matched individually for sex and age. Two biological indicators of personal toluene absorption, namely urine hippuric acid and o-cresol, were also measured in the exposed subjects. A significantly higher level and increased prevalence of elevated retinol-binding protein in the urine of exposed workers was found, whereas no significant difference in urinary albumin concentration was seen between the two groups. Urinary concentrations of retinol-binding protein was correlated (r = 0.399, P < 0.006) with that of o-cresol, but not with hippuric acid or employment duration. The results suggest a dose-dependent early tubular effect due to toluene exposure that might be useful for monitoring individuals exposed to toluene at work.     

Evaluation of biomarkers of occupational exposure to toluene at low levels

Objectives The purpose of the present study was to compare validity of various biomarkers of occupational exposure to toluene (Tol) at low levels. The focus was placed on the comparison of un-metabolized toluene in urine (Tol-U) and peripheral blood (Tol-B) with hippuric acid in urine (HA-U). Methods Surveys were conducted in 16 workplaces on the second half of working weeks, with participation of male solvent workers. Urine and peripheral blood samples were collected at the end of the shifts. After exclusion of cases with dense or diluted urine samples, 473 valid sets of samples were obtained for statistical evaluation. Time-weighted average exposure (for about 8-h) were monitored by diffusive sampling for toluene and other four solvents. Blood samples were subjected to the analyses for Tol-B, whereas urine samples were analyzed for HA-U and Tol-U. Results The solvent exposures were low, i.e., a grand geometric mean (GM) Tol concentration was 1.6 ppm, and the GM for the SUM in the additiveness equation was 0.12. The correlation analyses of the biomarkers in urine and blood with Tol exposure showed that Tol-U and Tol-B were more closely [correlation coefficients (r) being 0.67 and 0.60, respectively] related than HA-U (r = 0.27). Results of receiver operator characteristic analyses were in agreement with the correlation analysis results. Conclusions Taking the non-invasive nature of sampling together, Tol in the end-of-shift spot urine sample appears to be the marker of choice for biological monitoring of occupational exposure to Tol at low levels such as <2 ppm as a geometric mean.     

Experimental human exposure to toluene

Summary The relationship between the individual toluene uptake and the urinary hippuric acid excretion was studied under experimental conditions. Six healthy male subjects were exposed to various concentrations in inspired air (50, 100, 125, 150, and 200 ppm) at rest or under different levels of physical effort.The hippuric acid excretion near the end of the exposure appeared under all circumstances directly proportional to the time-weighted uptake rate of toluene. The correlation between respiratory uptake rate and the rate of metabolite excretion near the end of the exposure period proved not to be systematically influenced by personal factors such as body weight, amount of body fat, urine flow rate and urinary pH. The relatively pronounced differences in background excretion of hippuric acid and, perhaps, distribution phenomena of toluene between different tissues under heavy workload conditions, can partly explain the greater variability in metabolite excretions as compared to the individual uptake rates.The correlation between the individual uptake rate of toluene and the hippuric acid excretion proved substantially better when using the end exposure excretion rate as exposure parameter as compared with the end exposure hippuric acid concentration, even after correcting the latter for urine density.Reasonable biological limit values complying to an acceptable time-weighted toluene dose were found to be 3000–3500 mg/l and 2.0–2.5 mg/min, resp. for average hippuric acid concentrations and excretion rates in spot samples during the second half of a complete work shift.     

Urinary levels of proteins and metabolites in workers exposed to toluene

We measured urinary excretion of albumin and retinol-binding proteins to investigate the occurrence of early renal dysfunction in 45 paint workers exposed principally to toluene, and in the same number of unexposed control subjects matched individually for sex and age. Two biological indicators of personal toluene absorption, namely urine hippuric acid and o-cresol, were also measured in the exposed subjects. A significantly higher level and increased prevalence of elevated retinol-binding protein in the urine of exposed workers was found, whereas no significant difference in urinary albumin concentration was seen between the two groups. Urinary concentrations of retinol-binding protein was correlated (r = 0.399, P less than 0.006) with that of o-cresol, but not with hippuric acid or employment duration. The results suggest a dose-dependent early tubular effect due to toluene exposure that might be useful for monitoring individuals exposed to toluene at work.     

An enzyme-linked immunosorbent assay for hippuric acid: its potential application for biological monitoring of toluene exposure

An enzyme-linked immunosorbent assay (ELISA) for hippuric acid (HA) was developed using polyclonal anti-HA antibodies. Anti-HA antibodies were obtained by immunizing rabbits with N-benzoyl-cysteine (B-Cys) or N--benzoyl-lysine (B-Lys). An antibody with highest reactivity to HA was obtained from anti-B-Lys antiserum by affinity chromatography with B-Cys-Sepharose. The ELISA system was composed of solid-phase B-Cys, anti-HA antibody, and horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody. The detection limit of the ELISA for HA was around 1 g/ml. The urinary HA concentration determined by the ELISA system correlated well with that obtained by high-performance liquid chromatography (HPLC). The ELISA system was considered to be useful in the biological monitoring of toluene exposure, and to be more advantageous than time-consuming HPLC, especially when measuring a large number of samples.     

Experimental human exposure to toluene

Summary Six healthy male subjects exposed to various concentrations of toluene in inspired air (50, 100, and 150 ppm) under controlled conditions of rest or physical exercise, showed markedly little differences in the rate of respiratory solvent uptake. On the whole the intra-individual variations proved as important as the between subjects variations. For a given level of physical exercise the lung clearance appeared most affected by fluctuations in respiratory minute volume. In our experimental group the uptake rate was not significantly influenced by the amount of body fat.Toluene concentrations in expired air (C_E) during the first 4 h after an exposure cannot be considered a reliable measure for the individual toluene uptake. This parameter appears to reflect the influence of a number of host factors which do not affect — or at least not in the same way — the toluene uptake by itself. As a consequence the observed variability of toluene in the expired air was always much greater than for the related lung clearances. The single most important factors explaining differences in respiratory solvent excretion, were the respiratory minute volume in the post-exposure period and, after exposures at rest, the amount of body fat.The mean excretion amounted to about 4 % of the total uptake within 24 H after the end of the exposure.     

Alveolar air and blood toluene concentration in rotogravure workers

Summary Eleven workers exposed to toluene inhalation in a rotogravure plant were examined for the determination of the concentration of toluene in alveolar air, the excretion of urinary hippuric acid, and the concentration of toluene in the blood both before and after the 7-h work shift: the research has shown that these three paramters were correlated.The partition coefficient of toluene for blood and air was between 7 and 15.     

The validity of urinary metabolites as indicators of low exposures to toluene

Summary Exposure to toluene was studied in a group of 14 subjects working in a printing industry, who were exposed to this solvent only. Environmental monitoring was carried out using personal samplers for the whole workshift over three consecutive days. Toluene TWA concentrations ranged from 37 to 229 mg/m³. At the end of the workshift on each day of investigation, urine samples were collected for the determination of hippuric acid and ortho-cresol. Hippuric acid was also determined for urine before the workshift and on the Saturday and Monday mornings after the end of exposure; hippuric acid was also determined in 16 controls over the same five-day period. At the end of the workshift, hippuricuria levels in exposed workers always turned out to be statistically different from pre-workshift levels and those of the controls.The end-of-workshift hippuricuria levels of exposed workers were significantly correlated with the mean daily environmental concentration (TWA): in the three days of comparative study, we found r = 0.63 (P < 0.05) on Day 1, r = 0.90 (P < 0.001) on Day 2, and r = 0.87 (P < 0.001) on Day 3. Orthocresol turned out to be correlated with daily exposure less significantly than hippuric acid: r = 0.49 (n.s.) on Day 1; r = 0.78 (P < 0.001) on Day 2, and r = 0.65 (P < 0.05) on Day 3. Using all available data (41 observations), a very significant correlation (P < 0.001) was found between the TWA and both metabolites (r = 0.80 for hippuric acid; r = 0.68 for o-cresol). The values of the two metabolites in the end-of-workshift urine samples (41 observations) also turned out to be well correlated (r = 0.70; P < 0.001). The authors conclude that hippuric acid is a valid test for evaluating even low exposures to toluene.This work was supported by research grant no. 83.02580.56 from the Italian National Research Council (CNR), applied project Preventive and Rehabilitative Medicine     

Quantitative determination of urinary hippuric acid and m- or p-Methylhippuric Acid as Indices of toluene and m- or p-Xylene exposure by high performance liquid chromatography

Summary A high performance liquid chromatographic method for the determination of hippuric and m-(p-)methylhippuric acids in urine, metabolites of toluene and m-(p-)xylene, is described. A stainless-steel column packed with octadecyl silanized silica gel was used and the mobile phase was a mixed solution of methanol/water/acetic acid (20/80/0.2). The method is simple and specific. Urine can be analyzed directly, without solvent extraction or pretreatment. The method has a lower detection limit of 0.2 g on column. All the analysis and quantitative determination can be performed within about 30 min. for samples containing m-(p-)methylhippuric acid.     

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.     

Urinary excretion of O-cresol and hippuric acid after toluene exposure in rotogravure printing

Summary In 62 male rotogravure printers, the time-weighted average (TWA) toluene exposure during one workweek ranged from 8 to 496 mg/m³ (median 96). Post-shift urinary excretion of hippuric acid showed a poor correlation with the air toluene concentration. Level of o-cresol excretion ranged from 0.08 to 2.37 mmol/mol creatinine and was associated with the exposure (r _s = 0.57, P<0.0001), although the variation was considerable. However, this metabolite was significantly influenced by smoking habits, both in the workers (0.34 vs 0.10 mmol/mol creatinine after adjustment to zero exposure for the smokers and non-smokers, respectively; P = 0.03) and in 21 unexposed controls (0.18 vs 0.06 mmol/mot creatinine; P = 0.002). The excretion of these metabolites was followed during vacation, when the workers were unexposed. The shared one-compartment half-time was 44h (± SE 30, 82). After 2–4 weeks of vacation, the concentration of o-cresol was significantly higher for the smokers than the non-smokers (0.14 vs 0.06 mmol/mol creatinine; P = 0.02).No smoking-associated difference was found for the urinary hippuric acid concentration. However, there was an association between alcohol consumption and hippuric acid excretion (P = 0.03); no such difference was shown for o-cresol. These results demonstrate that hippuric acid excretion is unsuitable for biological monitoring of toluene exposure when the exposure level is below 200 mg/m³. Also, in spite of the favourable excretion kinetics, the impact of smoking and the large interindividual variation warrant the same conclusion for o-cresol as a means of monitoring low level exposure in an individual worker.     

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.     

High-performance liquid chromatography for the quantitative determination of the urinary metabolites of toluene,xylene, and styrene

Summary A new high-pressure liquid chromatography (HPLC) method for simultaneous quantitative determination of the urinary metabolites of toluene, m-xylene, and styrene (hippuric acid, m-methylhippuric acid, phenylglyoxylic acid, mandelic acid) is described. The extraction procedure was performed on acidified urines, after addition of 4-hydroxybenzoic acid as internal standard, using a butylchloride/isopropanol mixture and drying 0.5 ml of the organic layer under nitrogen flow. The residue obtained was dissolved in 0.1 ml water/acetonitrile and 5 l were injected into an HPLC apparatus equipped with a 0.26 × 25 cm HC ODS SIL X column.Absorbance measures were performed at 225 nm throughout the investigation. All metabolites were clearly separated in a short time (12 min) and the amounts of other urinary compounds affecting the analysis were so small that the measurement of low concentrations of the urinary metabolites could be easily performed. Linear calibration curves were obtained from 0.1 to 3 mg/ml and a correlation coefficient greater than 0.99 was found between concentrations of the standards and areas of the peaks. Statistical analysis confirms that this method, which has a high reproducibility, is simple, reliable, and useful for the biologic monitoring of industrial exposure to aromatic compounds.     

Quantitative analysis of urinary glycine conjugates by high performance liquid chromatography: excretion of hippuric acid and methylhippuric acids in the urine of subjects exposed to vapours of toluene and xylenes

Summary A new method for the direct determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (MHAs) in the urine, metabolites of toluene and o-, m- and p-xylenes by high performance liquid chromatography (HPLC) is described. A stainless-steel column packed with silica gel having dinitrophenyl residue and a mixed solution of methanol/water/acetic acid (80/20/0.2) containing tetra-n-butylammonium bromide (0.2% w/v) as mobile phase was used. Concentrations of HA and MHAs were estimated from their peak height at a wave length of 225 nm. Urine can be analyzed directly without solvent extraction or pretreatment to obtain complete separation of HA and o-, m- and p-MHAs. Urine samples from male workers exposed to toluene or xylenes were analyzed for HA or MHAs. The urinary levels of HA and MHAs increased by exposure to toluene and xylenes in proportion to the environmental concentrations of the solvents, although there is a considerable variation in metabolite concentrations. The slope of regression line between toluene and HA and that between m-xylene and m-MHA were similar. The urinary concentrations of HA and MHAs corresponding to 100 ppm (TLV) of toluene was 2.35 g/g creatinine and that of m-MHA corresponding to 100 ppm (TLV) of m-xylene was 2.05 g/g creatinine. The warning levels of the urinary metabolite concentrations of a group of workers and that of an individual worker corresponding to TLV of organic solvent concentration is discussed.     

Cytogenetic effects of low-level exposure to toluene,xylene, and their mixture on human blood lymphocytes

Summary The present study was undertaken to investigate, both in vitro and in vivo, the genotoxic potential of short-term low-level exposure to toluene, xylene, and their mixture, for which information is limited at the present time. Five adult healthy white men were exposed for 7 consecutive hours per day over 3 consecutive days to 50 ppm toluene and 40 ppm xylene either alone or in combination in a controlled exposure chamber. Such an exposure was repeated three times at intervals of 2 weeks. Blood samples were taken before and after the termination of such exposure. Three different cytogenetic end-points were evaluated using peripheral blood lymphocytes: number of sister chromatid exchanges (SCEs), cell cycle delay, and cell mortality. No significant effects on SCEs, cell cycle delay, and cell mortality were observed following such exposure to toluene or xylene or their mixture. Similarly, exposure of human blood lymphocytes in vitro to either toluene (0–2.5 mM) or xylene (0–2 mM) or their mixture for 72 h did not result in any significant cytogenetic effects at lower concentrations, while at higher concentrations, only cell mortality was found to be significantly affected. Thus our present study indicates that simultaneous exposure to low levels (within the admissible limits) of toluene, xylene, or their mixture for a short period does not pose any potential mutagenic threat to humans.     

Comparison between blood and urinary toluene as biomarkers of exposure to toluene

Objectives: To compare blood toluene (TOL-B) and urinary toluene (TOL-U) as biomarkers of occupational exposure to toluene, and to set a suitable procedure for collection and handling of specimens. Method: An assay based on headspace solid-phase microextraction (SPME) was used both for the determination of toluene urine/air partition coefficient (λ_urine/air) and for the biological monitoring of exposure to toluene in 31 workers (group A) and in 116 non-occupationally exposed subjects (group B). Environmental toluene (TOL-A) was sampled during the work shift (group A) or during the 24 h before specimen collection (group B). Blood and urine specimens were collected at the end of the shift (group A) or in the morning (group B) and toluene was measured. Results: Toluene λ_urine/air was 3.3 ± 0.9. Based on the specimen/air partition coefficient, it was calculated that the vial in which the sample is collected had to be filled up to 85% of its volume with urine and 50% with blood in order to limit the loss of toluene in the air above the specimen to less than 5%. Environmental and biological monitoring of workers showed that the median personal exposure to toluene (TOL-A) during the work-shift was 80 mg/m³, the corresponding TOL-B was 82 μg/l and TOL-U was 13 μg/l. Personal exposure to toluene in environmentally exposed subjects was 0.05 mg/m³, TOL-B was 0.36 μg/l and TOL-U was 0.20 μg/l. A significant correlation (P < 0.05) was observed between TOL-B or TOL-U and TOL-A (Pearson's r=0.782 and 0.754) in workers, but not in controls. A significant correlation was found between TOL-U and TOL-B both in workers and in controls (r=0.845 and 0.681). Conclusion: The comparative evaluation of TOL-B and TOL-U showed that they can be considered to be equivalent biomarkers as regards their capacity to distinguish workers and controls and to correlate with exposure. However, considering that TOL-U does not require an invasive specimen collection, it appears to be a more convenient tool for the biological monitoring of exposure to toluene. Received: 20 October 1999 / Accepted: 4 March 2000     

Effect of ethanol,cimetidine and propranolol on toluene metabolism in man

Summary In a climatic exposure chamber four healthy volunteers were exposed to 100ppm toluene, 100ppm toluene + ethanol, 100ppm toluene + cimetidine, and 100ppm toluene + propranolol for 7h each at random over four consecutive days. A control experiment and 3.5 h of exposure to 200 ppm toluene were also performed. Ethanol inhibited toluene metabolism by 0.5 as expressed by the urinary excretion of two of the metabolites of toluene, namely o-cresol and hippuric acid. In agreement with this, the mean alveolar concentration of toluene was greater by 1.7 during ethanol exposure; 45 min after discontinuation of exposure the increase was by 3.3. Neither cimetidine nor propranolol changed toluene metabolism significantly. The results indicate that ethanol may prolong the time interval in which toluene is retained in the human body in persons simultaneously exposed to ethanol and toluene. When using o-cresol or hippuric acid in biological monitoring of persons occupationally exposed to toluene, the consumption of ethanol should be considered.Supported by grants from the Working Environment Fund, Denmark     

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