Leukemia risk assessment of exposure to low-levels of benzene based on the linearized multistage model.

Objectives: To assess leukemia risk in occupational populations exposed to low levels of benzene. Methods: Leukemia incidence data from the Chinese Benzene Cohort Study were fitted using the Linearized multistage (LMS) model. Individual benzene exposure levels, urinary S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t, t-MA) were measured among 98 benzene-exposed workers from factories in China. Subjects were categorized into four groups by rounding the quartiles of cumulative benzene concentrations (< 3, 3-5, 5-12, ≥12 mg/m3·year, respectively). The risk of benzene-induced leukemia was assessed using the LMS model, and the results were validated using the EPA model and the Singapore semi-quantitative risk assessment model. Results: The leukemia risks showed a positive correlation with increasing cumulative concentration in the four exposure groups (excess leukemia risks were 4.34, 4.37, 4.44 and 5.52 × 10-4, respectively; Ptrend < 0.0001) indicated by the LMS model. We also found that the estimated leukemia risk using urinary t, t-MA in the LMS model was more similar to those estimated by airborne benzene compared to S-PMA. The leukemia risk estimated by the LMS model was consistent with both the Singapore semi-quantitative risk assessment model at all concentrations and the EPA model at high concentrations (5-12, ≥12 mg/m3·year), while exceeding the EPA model at low concentrations (< 3 and 3-5 mg/m3·year). However, in all four benzene-exposed groups, the leukemia risks estimated by these three models exceeded the lowest acceptable limit for carcinogenic risk set by the EPA at 1 × 10-6. Conclusion: This study demonstrates the utility of the LMS model derived from the Chinese benzene cohort in assessing leukemia risk associated with low-level benzene exposure, and suggests that leukemia risk may occur at cumulative concentrations below 3 mg/m3·year.

Simultaneous determination of benzoic acid and sorbic acid in non-alcoholic beverages by a validated HS-GC-MS method with reduced waste.

A headspace gas chromatography-mass spectrometry (HS-GC-MS) method is presented for the simultaneous determination of benzoic acid (BA) and sorbic acid (SoA) in different types of non-alcoholic beverages. Sensitive and reliable results were achieved together with minimising consumption of reagents and samples. Salicylic acid (SalA) was used as internal standard (IS). It was necessary to derivatise BA, SoA and SalA to their methyl esters for HS-GC-MS measurement and extensive optimisation studies for in-vial derivatisation were carried out on the temperature, incubation time, injection time of the loopless HS, as well as on the concentration of sulphuric acid used as a catalyst. Validation studies carried out under optimum conditions after mixing 50 µL of sample and IS solutions with 200 µL of 4.5 M sulphuric acid in 22 mL HS vials revealed that the developed method was both very precise (relative standard deviation < 5%) and accurate (average recovery%: 101.0% for BA and 100.4% for SoA). The validated method was applied to a wide range of beverage types and the results compared with the relevant regulation and product label declarations.

In-syringe ionic liquid-dispersive liquid-liquid microextraction coupled with HPLC for the determination of trans,trans-muconic acid in human urine sample.

trans,trans-Muconic acid has been widely used as a biomarker in biological monitoring of benzene-exposed workers during routine occupational health services. In the present study, a novel microextraction technique, in-syringe ionic liquid-dispersive liquid-liquid microextraction, was implemented for preconcentration of trans,trans-muconic acid followed by analytical determination by high-performance liquid chromatography with ultraviolet detection. Moreover, the important variables affecting the performance of applied microextraction technique including needle diameter, volume of the spiked sample, volume of the ionic liquid, salt addition, rotation speed of centrifugation, centrifuge time, and ultrasonic time were optimized by experimental design. A good linear relationship was observed at the range of 0.032-10 µg/mL between the peak area and the concentration levels (R2  = 0.9997). The limit of detection and extraction recovery for trans,trans-muconic acid were 0.011 µg/mL and >96.2%, respectively. This method provided easy and rapid analysis of low amounts of trans,trans-muconic acid in human urine with simple equipment.

Determination of phenol biodegradation pathways in three psychrotolerant yeasts, Candida subhashii A011, Candida oregonensis B021 and Schizoblastosporion starkeyi-henricii L012, isolated from Rucianka peatland.

In this study, three psychrotolerant phenol-degrading yeast strains Candida subhashii (strain A011), Candida oregonenis (strain B021) and Schizoblastosporion starkeyi-henricii (strain L012) isolated from Rucianka peatland were examined to determine which alternative metabolic pathway for phenol biodegradation is used by these microorganisms. All yeast strains were cultivated in minimal salt medium supplemented with phenol at 500, 750 and 1000 mg l-1 concentration with two ways of conducting phenol biodegradation experiments: with and without the starving step of yeast cells. For studied yeast strains, no catechol 2,3-dioxygenase activities were detected by enzymatic assay and no products of catechol meta-cleavage in yeast cultures supernatants (GC-MS analysis), were detected. The detection of catechol 1,2-dioxygenase activity and the presence of cis,cis-muconic acid in the analyzed samples revealed that all studied psychrotolerant yeast strains were able to metabolize phenol via the ortho-cleavage pathway. Therefore, they may be tested in terms of their use to develop biotechnology for the production of cis,cis-muconic acid, a substrate used in the production of plastics (PET) and other valuable goods.

Biological Monitoring of Exposure to Benzene in Port Workers.

Port workers are exposed to a wide range of occupational hazards that can cause injuries and occupational diseases. Among these, exposure to benzene is one of the most important but least studied. The highest occupational exposures for port workers occur during the filling and loading of gasoline, and cleaning of tanks and receptacles. The aim of the study was to evaluate occupational exposure to low levels of benzene by measuring trans,trans-muconic acid (t,t-MA) in urine samples from workers operating at fuelling stations in a tourist port of Southern Italy. The overall sample was composed of 43 port workers of a tourist port in Southern Italy. In 2018, each participant provided two (morning and evening) urine samples for the determination of urinary t,t-MA. Urinary excretion of t,t-MA was always higher at the end of the work shift than at the beginning with significant difference (p = 0.002). In smokers, median t,t-MA urinary excretion is higher than non-smokers both at the beginning (90.5 µg/g creatinine vs. 61.45 µg/g creatinine) and at the end of the work shift (128.2 µg/g creatinine vs. 89.5 µg/g creatinine). Urinary excretion of t,t-MA is higher at the end of the work shift than at the beginning in both smokers and non-smokers, but the difference is significantly higher in non-smokers (p = 0.003) than in smokers (p = 0.05). In conclusion, our results showed that the role of inhaled benzene at fuelling stations in a tourist port can be relevant. On the basis of these results and the known adverse effects of benzene on human health, we encourage the use of personal protective equipment in the fuelling area of ports in order to minimize exposure to benzene to workers.

Simultaneous determination of benzoic acid, sorbic acid, and propionic acid in fermented food by headspace solid-phase microextraction followed by GC-FID.

A simple and environmentally friendly method was developed for simultaneous determination of benzoic acid, sorbic acid, and propionic acid in fermented food samples. The analytes were extracted and pre-concentrated by headspace solid-phase microextraction (HS-SPME) and analysed by GC-FID. Central composite design (CCD) was conducted for the optimization of HS-SPME conditions. Under optimal conditions, a good linear range was obtained in the range of 5-150 mg L-1. The limit of detection (LOD) values were 1.1-1.7 mg L-1. The developed method was successfully applied to determine the concentration of three organic acid preservatives in various fermented food samples, including thai shrimp paste, pickled vegetables, soy sauce and fish sauce, with high accuracy (recoveries were between 83% and 109%) and good precision (%RSD was less than 6.0% and 4.5% for intra-day and inter-day, respectively).

Combination of large volume sample stacking with polarity switching and cyclodextrin electrokinetic chromatography (LVSS-PS-CDEKC) for the determination of selected preservatives in pharmaceuticals.

In this study, a large volume sample stacking (LVSS) with polarity switching (PS) and cyclodextrin electrokinetic chromatography (CDEKC) method has been developed for the simultaneous separation and determination of 8 preservatives: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), isobutylparaben (IBP), sorbic acid (SA), benzoic acid (BA), p-hydroxybenzoic acid (PHBA) in pharmaceuticals. The effects of some typical parameters such as sample volume, applied voltage, composition and pH of the running buffer and organic modifier concentration were examined and optimized. Moreover, the impact of type and concentration of cyclodextrin as electrolyte modifiers was also investigated. The detection limits of analytes for the elaborated LVSS-PS-CDEKC method were found to be in 0.8-5 ng mL-1 range, which were around 500 times lower than normal CDEKC without preconcentration technique. All analytes were completely resolved in less than 11 min in an uncoated fused-silica capillary of 75 µm internal diameter (I.D) x 50 cm length. The electrophoretic separation was performed in a 2 mM α-cyclodextrin and 25 mM tetraborate system (pH = 9.3) with an applied voltage of 25 kV. The established method was validated and confirmed to be applicable for the determination of the preservatives in a quality control of pharmaceuticals.

Factors Affecting Urinary tt-Muconic Acid Detection among Benzene Exposed Workers at Gasoline Stations.

Trans, trans-muconic acid (tt-MA) is a metabolite that is widely used as a biomarker to identify low exposure to benzene, a human carcinogen. This study aimed to investigate occupational factors related to the urinary tt-MA detection of benzene exposed workers in gasoline stations. Spot urine samples were collected and analyzed for tt-MA using a high performance liquid chromatography. Additional data were collected via subject interviews using a structured questionnaire. The personal benzene concentration was measured and analyzed by gas chromatography with a flame ionization detector. Results showed that, among the 170 workers, tt-MA was detected in 24.7% of workers and the concentration ranged from 23.0 to 1127.8 µg/g creatinine. Over 25% of those detections possessing tt-MA exceeding the recommended 500 µg/g creatinine was safe. A multiple logistic regression analysis identified that factors significantly associated with the detectable tt-MA were having no other part-time jobs (ORadj = 4.2), personal benzene concentrations of 0.05 ppm or higher (ORadj = 10.3), close to fuel nozzle during refuelling (ORadj = 93.7), and no job training (ORadj = 2.74). Safety training is recommended for those tt-MA detected workers or under a reference benzene concentration of 0.05 ppm or higher. The proposed reference of occupational action level to benzene exposure is 0.05 ppm and compliance could be assessed tt-MA for biomonitoring of those benzene exposed workers.

Benzene Exposure and Biomarkers in Alveolar Air and Urine Among Deck Crews on Tankers Transporting Gasoline.

INTRODUCTION: Increased rates of leukaemia have been found among tanker crews. Occupational exposures to the leukomogen benzene during loading, unloading, and tank cleaning are possible causes. Studies on older types of tankers carrying gasoline with most handling being done manually have revealed important exposures to benzene. Our study explores benzene exposures on tankers with both automatic and manual systems. Correlations between benzene exposure and benzene in alveolar air (AlvBe), benzene in urine (UBe), and trans,trans-muconic acid (ttMA) in urine were investigated. METHODS: Forty-three male seafarers (22 deck crewmembers and 21 not on deck) on five Swedish different product and chemical tankers transporting 95- or 98-octane gasoline were investigated between 1995 and 1998. The tankers used closed systems for the loading and unloading of gasoline but stripping and tank cleaning were done manually. Benzene in respiratory air was measured using personal passive dosimeters during a 4-h work shift. Samples for biomarker analyses were collected pre- and post-shift. Smoking did occur and crewmembers did not use any respiratory protection during work. RESULTS: The average 4-h benzene exposure level for exposed was 0.45 mg m-3 and for non-exposed 0.02 mg m-3. Benzene exposure varied with type of work (range 0.02-143 mg m-3). AlvBe, UBe, and ttMA were significantly higher in post-shift samples among exposed and correlated with exposure level (r = 0.89, 0.74, and 0.57, respectively). Smoking did not change the level of significance among exposed. DISCUSSION: Benzene in alveolar air, unmetabolized benzene, and ttMA in urine are potential biomarkers for occupational benzene exposure. Biomarkers were detectable in non-exposed, suggesting benzene exposure even for other work categories on board tankers. Work on tankers carrying gasoline with more or less closed handling of the cargo may still lead to significant benzene exposure for deck crewmembers, and even exceed the Swedish Occupational Exposure Limit (OEL; 8-h time-weighted average [TWA]) of 1.5 mg m-3.

Fast and simple method of simultaneous preservative determination in different processed foods by QuEChERS and HPLC-UV: Method development, survey and estimate of daily intake.

A fast and simple method, which employs QuEChERS and HPLC-UV, was developed to determine preservatives in processed foods from different classes. The method showed correlation coefficients above 0.99, LOQs between 0.13 and 0.33 mg kg-1 and recoveries between 91 and 107%, with RSD ≤ 5.3%. Levels of preservatives were up to 2040 mg kg-1 for benzoates, up to 3185 mg kg-1 for sorbates and up to 452 mg kg-1 for methylparaben. Only four out of 82 samples under analysis were above the maximum level allowed by the legislation. Additionally, daily intakes of preservatives were estimated. Regarding benzoates, relatively high intakes were estimated (25% of the acceptable daily intake - ADI) in comparison with sorbates (5% of ADI) and parabens (<1% of ADI), when mean consumption is considered. This method is a good alternative to determining preservatives in different food samples.

Boron doped carbon dots as a multifunctional fluorescent probe for sorbate and vitamin B12.

Boron doped carbon dots (B-CD) were synthesized by a one-step hydrothermal method using phenylboronic acid as the starting material. They have an average size of about 3.3 nm, with excitation/emission wavelength of 247/323 nm and a quantum yield of 12%. The B-CD is shown to be viable fluorescent probe for sorbate (PS) and vitamin B12 (VB12). The fluorescence (FL) of the B-CD is quenched in the presence of PS or VB12 mainly coming from inner filter effect (IFE), but Förster resonance energy transfer (FRET) from the B-CD (as a donor) to PS/VB12 (as an acceptor) cannot be excluded. The probe enables PS to be detected by fluorometry with a linear response in the 0.20-24 µM concentration range and a 6.1 nM detection limit (at 3σ/slope). For VB12, the data are 0.20-30 µM and 8.0 nM. Graphical abstract Boron doped carbon dots (B-CD) as a probe was prepared by phenylboronic acid as single starting material via one-step hydrothermal method, which has remarkable selectivity and high sensitivity for monitoring PS/VB12. The fluorescence quenching of B-CD by PS/VB12 mainly comes from inner filter effect.

Association of benzene exposure with insulin resistance, SOD, and MDA as markers of oxidative stress in children and adolescents.

Benzene is a ubiquitous environmental pollutant with various health effects. It is reported that benzene exposure might be associated with insulin resistance in elderly adults. The aim of this study is to investigate the association between urinary benzene metabolite, trans, trans-muconic acid (t,t-ma) and markers of oxidative stress and insulin resistance in children and adolescents. This cross-sectional study was conducted in 2017 among 86 children and adolescents, aged 6-18 years, living in Isfahan, Iran. t,t-ma was measured as urinary benzene metabolite and homeostasis model assessment (HOMA-IR) was determined as an index of insulin resistance. Moreover, malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed as oxidative stress markers. We found significant association between insulin resistance, fasting blood glucose, and fasting blood insulin with t,t-ma (p values = 0.002, 0.03, and 0.001, respectively). Results of this study indicate that benzene metabolite in higher concentrations in comparison with lower concentrations is associated with increased risk of insulin resistance. Moreover, after adjustment for age, sex, and household passive smoking, statistically significant increase were documented in SOD and MDA (4.49- and 3.54-fold, respectively) in intermediate levels of t,t-ma vs. low levels of t,t-ma (p values = 0.01 and 0.034, respectively). To the best of our knowledge, this is the first study in its kind in the pediatric age group. It showed that benzene exposures, even in environmental levels, might be associated with insulin resistance and oxidative stress in children and adolescents. Further longitudinal studies are necessary to assess the clinical impacts of this finding.

Assessment of sodium benzoate and potassium sorbate preservatives in some products in Kashan, Iran with estimation of human health risk.

The purpose was to assess of sodium benzoate (SB) and potassium sorbate (PS) preservatives in 103 samples of cake, toast bread, tomato paste, mayonnaise sauce, carbonated soft drink and Olovieh salad in Kashan, by spectrophotometry and high performance liquid chromatography (HPLC) methods. The chronic daily intake (CDI), target hazard quotient (THQ) and hazard index (HI) of SB and PS for Iranian population were calculated. The results showed that SB and PS were not detected in the tomato paste samples. SB and PS concentrations for all samples were less than regulatory limits except for PS in one cake sample (3.57%). CDI and THQ of PS for mayonnaise sauce, Olovieh salad and cake products, except toast bread, were less than the acceptable daily intakes (ADIs) and one, respectively. While HI value of PS for the selected products was more than one, indicating that the non-carcinogenic risk represent a threat to consumers. THQ and HI values of SB for mayonnaise sauce and carbonated soft drink products were more than one through consumption of these products, indicating considerable non-carcinogenic risk. Therefore, the results highlighted the importance of a more attentive monitoring of these preservatives by the public and food health authorities in Iran.

Comparison of personal air benzene and urine t,t-muconic acid as a benzene exposure surrogate during turnaround maintenance in petrochemical plants.

Previous studies have shown that biomarkers of chemicals with long half-lives may be better surrogates of exposure for epidemiological analyses, leading to less attenuation of the exposure-disease association, than personal air samples. However, chemicals with short half-lives have shown inconsistent results. In the present study, we compared pairs of personal air benzene and its short-half-life urinary metabolite trans,trans-muconic acid (t,t-MA), and predicted attenuation bias of theoretical exposure-disease association. Total 669 pairs of personal air benzene and urine t,t-MA samples were taken from 474 male workers during turnaround maintenance operations held in seven petrochemical plants. Maintenance jobs were classified into 13 groups. Variance components were calculated for personal air benzene and urine t,t-MA separately to estimate the attenuation of the theoretical exposure-disease association. Personal air benzene and urine t,t-MA showed similar attenuation of the theoretical exposure-disease association. Analyses for repeated measurements showed similar results, while in analyses for values above the limits of detection (LODs), urine t,t-MA showed less attenuation of the theoretical exposure-disease association than personal air benzene. Our findings suggest that there may be no significant difference in attenuation bias when personal air benzene or urine t,t-MA is used as a surrogate for benzene exposure.

Epigenetic and Transcriptional Modifications in Repetitive Elements in Petrol Station Workers Exposed to Benzene and MTBE.

Benzene, a known human carcinogen, and methyl tert-butyl ether (MTBE), not classifiable as to its carcinogenicity, are fuel-related pollutants. This study investigated the effect of these chemicals on epigenetic and transcriptional alterations in DNA repetitive elements. In 89 petrol station workers and 90 non-occupationally exposed subjects the transcriptional activity of retrotransposons (LINE-1, Alu), the methylation on repeated-element DNA, and of H3K9 histone, were investigated in peripheral blood lymphocytes. Median work shift exposure to benzene and MTBE was 59 and 408 µg/m³ in petrol station workers, and 4 and 3.5 µg/m³, in controls. Urinary benzene (BEN-U), S-phenylmercapturic acid, and MTBE were significantly higher in workers than in controls, while trans,trans-muconic acid (tt-MA) was comparable between the two groups. Increased BEN-U was associated with increased Alu-Y and Alu-J expression; moreover, increased tt-MA was associated with increased Alu-Y and Alu-J and LINE-1 (L1)-5'UTR expression. Among repetitive element methylation, only L1-Pa5 was hypomethylated in petrol station workers compared to controls. While L1-Ta and Alu-YD6 methylation was not associated with benzene exposure, a negative association with urinary MTBE was observed. The methylation status of histone H3K9 was not associated with either benzene or MTBE exposure. Overall, these findings only partially support previous observations linking benzene exposure with global DNA hypomethylation.

An Orthogonal and pH-Tunable Sensor-Selector for Muconic Acid Biosynthesis in Yeast.

Microbes offer enormous potential for production of industrially relevant chemicals and therapeutics, yet the rapid identification of high-producing microbes from large genetic libraries is a major bottleneck in modern cell factory development. Here, we develop and apply a synthetic selection system in Saccharomyces cerevisiae that couples the concentration of muconic acid, a plastic precursor, to cell fitness by using the prokaryotic transcriptional regulator BenM driving an antibiotic resistance gene. We show that the sensor-selector does not affect production nor fitness, and find that tuning pH of the cultivation medium limits the rise of nonproducing cheaters. We apply the sensor-selector to selectively enrich for best-producing variants out of a large library of muconic acid production strains, and identify an isolate that produces more than 2 g/L muconic acid in a bioreactor. We expect that this sensor-selector can aid the development of other synthetic selection systems based on allosteric transcription factors.

Sorbic, benzoic and propionic acids in fishery products: a survey of the South Korean market.

This study was conducted to provide basic data as part of a project to distinguish naturally occurring organic acids from added preservatives. Accordingly, we investigated naturally occurring levels of sorbic, benzoic and propionic acids in fish and their processed commodities. The levels of sorbic, benzoic and propionic acids in 265 fish and their processed commodities were determined by high-performance liquid chromatography-photodiode detection array (HPLC-PDA) of sorbic and benzoic acids and gas chromatography-mass spectrometry (GC/MS) of propionic acid. For propionic acid, GC-MS was used because of its high sensitivity and selectivity in complicated matrix samples. Propionic acid was detected in 36.6% of fish samples and 50.4% of processed fish commodities. In contrast, benzoic acid was detected in 5.6% of fish samples, and sorbic acid was not detected in any sample. According to the Korean Food and Drug Administration (KFDA), fishery products and salted fish may only contain sorbic acid in amounts up to 2.0 g kg-1 and 1.0 g kg-1, respectively. The results of the monitoring in this study can be considered violations of KFDA regulations (total 124; benzoic acid 8, propionic acid 116). However, it is difficult to distinguish naturally generated organic acids and artificially added preservatives in fishery products. Therefore, further studies are needed to extend the database for distinction of naturally generated organic acids and added preservatives.

Direct quantitation of the preservatives benzoic and sorbic acid in processed foods using derivative spectrophotometry combined with micro dialysis.

The preservatives benzoic acid and sorbic acid are generally quantified with separation techniques, such as HPLC or GC. Here we describe a new method for determining these compounds in processed food samples based on a narrowness of the UV-visible spectral band width with derivative processing. It permits more selective identification and determination of target analytes in matrices. After a sample is purified by micro dialysis, UV spectra of sample solutions were measured and fourth order derivatives of the spectrum were calculated. The amplitude between the maximum and minimum values in a high-order derivative spectrum was used for the determination of benzoic acid and sorbic acid. Benzoic acid and sorbic acid levels in several commercially available processed foods were measured by HPLC and the proposed spectrometry method. The levels obtained by the two methods were highly correlated (r2>0.97) for both preservatives.

Correlation between benzene and testosterone in workers exposed to urban pollution.

AIM: Many studies have examined the effects of benzene on testosterone. The purpose of this study was to evaluate the possible correlation between the blood levels of benzene and the levels of testosterone. MATERIALS AND METHODS: The study involved a group of 148 subjects. For every worker have been made out a blood sample for the evaluation of benzene and testosterone levels and an urine analysis for the evaluation of the levels of trans, trans-muconic acid and S-phenylmercapturic acid. We estimated the Pearson correlation coefficient between the variables in the sample and the urinary metabolites, age, length of service, gender, BMI. For the analysis of the major confounding factors it was performed a multiple linear regression. RESULTS: The Pearson correlation coefficiet showed: 1. a significant inverse correlation between the S-phenyl mercapturic acid and free testosterone; 2. a significant direct correlation between trans-trans muconic acid and BMI. After dividing the sample according to the median of blood benzene (161.0 ng / L), Pearson correlation coefficient showed a significant inverse correlation between the S-phenyl mercapturic acid and free testosterone in the group with values below this median. CONCLUSIONS: Our results, to be considered preliminary, suggest that occupational exposure to low levels of benzene, present in urban pollution, affect the blood levels of testosterone. These results need to be confirmed in future studies, with the eventual possibility of including more specific fertility tests.

Evidence for non-linear metabolism at low benzene exposures? A reanalysis of data.

The presence of a high-affinity metabolic pathway for low level benzene exposures of less than one part per million (ppm) has been proposed although a pathway has not been identified. The variation of metabolite molar fractions with increasing air benzene concentrations was suggested as evidence of significantly more efficient benzene metabolism at concentrations <0.1 ppm The evidence for this pathway is predicated on a rich data set from a study of Chinese shoe workers exposed to a wide range of benzene concentrations (not just "low level"). In this work we undertake a further independent re-analysis of this data with a focus on the evidence for an increase in the rate of metabolism of benzene exposures of less than 1 ppm. The analysis dataset consisted of measurements of benzene and toluene from personal air samplers, and measurements of unmetabolised benzene and toluene and five metabolites (phenol hydroquinone, catechol, trans, trans-muconic acid and s-phenylmercapturic acid) from post-shift urine samples for 213 workers with an occupational exposure to benzene (and toluene) and 139 controls. Measurements from control subjects were used to estimate metabolite concentrations resulting from non-occupational sources, including environmental sources of benzene. Data from occupationally exposed subjects were used to estimate metabolite concentrations as a function of benzene exposure. Correction for background (environmental exposure) sources of metabolites was achieved through a comparison of geometric means in occupationally exposed and control populations. The molar fractions of the five metabolites as a function of benzene exposure were computed. A supra-linear relationship between metabolite concentrations and benzene exposure was observed over the range 0.1-10 ppm benzene, however over the range benzene exposures of between 0.1 and 1 ppm only a modest departure from linearity was observed. The molar fractions estimated in this work were near constant over the range 0.1-10 ppm. No evidence of high affinity metabolism at these low level exposures was observed. Our reanalysis brings in to question the appropriateness of the dataset for commenting on low dose exposures and the use of a purely statistical approach to the analysis.