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Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles.

Data source

Reference Type:
An integrated approach to biomonitoring exposure to styrene and styrene-(7,8)-oxide using a repeated measurements sampling design
Fustinoni S., et al.
Bibliographic source:
Biiomarkers 13 (6): 560-578

Materials and methods

Study type:
biological exposure monitoring
Endpoint addressed:
not applicable
Principles of method if other than guideline:
The aim of this work was to investigate urinary analytes and haemoglobin and albumin adducts as biomarkers of exposure to airborne styrene (Sty) and styrene-(7,8)-oxide (StyOX) and to evaluate the influence of smoking habit and genetic polymorphism of metabolic enzymes GSTM1 and GSTT1 on these biomarkers.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:


Type of population:
Details on study design:
Study population:
The study entailed air and biological monitoring of two groups of workers exposed to styrene, namely, 13 varnish workers and eight reinforced plastics workers, and of a group of 22 automobile mechanics, who served as control workers. Styrene is the major solvent and monomer for the resin system used in the production of reinforced plastics and is also used as ingredient for the production of these particular varnishes.
All subjects were informed of the aims and protocol of the study and provided written consent to be included as human subjects. Detailed information about work activities, demographic and lifestyle factors, and medical histories was obtained using a questionnaire administered by a physician specialized in occupational health.

Air and biological sampling:
Personal exposures to Sty and StyOX were assessed over the entire work shift (about 8 h), using passive samplers. Among exposed subjects, air sampling was repeated three or four times during 6 consecutive weeks. Personal exposures were measured once among a subset of seven control subjects; all measurements from controls were below the limits of detection. For exposed subjects, spot urine samples were collected both before the work shift (BS about 08:30 h) and at the end of the work shift (ES about 16:30 h) on the same days that air sampling was performed. Six to eight urine samples, consisting of sets of three or four (each) BS and ES specimens, were collected from each exposed subject. A single blood sample (about 10 ml) was collected from the cubital vein prior to the last investigated work shift. For control subjects, single BS samples of urine and blood were obtained. Urine samples were partitioned in the infirmary of the worksites into two plastic vials (about 7 ml urine each) for the determination of MA, PGA, PHG, S,R-M2, R,RMI, R,R-M2, S,R-ML, VP-G, and VP-S. For StyU, a further 7 ml aliquot was transferred to a pre-cleaned glass vial, promptly closed with a butyl rubber/PIFE septum, and sealed with an aluminium septum cap. All samples were coded, chilled at 4°C, and delivered to the laboratories within 4 h. Here blood samples were centrifuged to separate plasma from red blood cells for the determination of Alb and Hb adducts, respectively. DNA was prepared from white blood cells. After lysis and proteinase digestion DNA was isolated by a solvent free commercially available kit (Puregene, Gentra Systems, Minneapolis, MN, USA). Samples were stored at -20 °C prior to analyses that were performed without knowledge of samples' origins.

Analysis of air and biological samples:
The air concentrations of Sty and StyOX were determined by eluting the passive monitors with ethyl acetate and subsequent analysis by either gas chromatography with flame ionization detection or gas chromatography-mass spectrometry (GC-MS).

Unmetabolized styrene in urine (StyU):
Levels of StyU were determined by headspace solid-phase microextraction (SPME) followed by GC-MS analysis as described by (Fustinoni et al. 1999), with modifications.

Styrene metabolites in urine:
Urinary metabolites were assayed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), as previously described (Manini et al. 2002).

Protein adducts:
Cysteinyl adducts of StyOX with Alb and Hb were assayed as described previously (Yeowell-O'Connell et al. 1996, Fustinoni et al. 1998).

The wide gene deletion polymorphisms of glutathione transferases GSTM1 and GSTT1 were determined by polymerase chain reaction (PCR) according
to methods described previously (Arand et al. 1996).

Results and discussion

Study population:
The workers tended to be male, aged 30-40 years, with about a fourth to a third being cigarette smokers. According to GSTM1 genotype, 11 controls, six varnish workers and five fibreglass workers had the null genotype. According to GSTT1 genotyping, three controls, three varnish workers, and three fibreglass workers bad the null genotype. These genotype frequencies are in agreement with allele frequencies previously reported for other European populations. No differences were detected in the frequencies of genotypes among the three study groups (X2 test).

Sty, StyOX and biomarkers:
Personal exposure to Sty and StyOX decreased in the order: fibreglass reinforced plastic workers > varnish workers >> control workers. Among reinforced plastic workers the ratio of airborne Sty to StyOX was 1000:7.3, which was about twice that observed in varnish workers (1000:3.6). Comparing subjects who used and did not use peroxide catalysts, the ratio Sty:StyOX decreased from 1000:10.9 to 1000:2.7, suggesting that the use of peroxides contributed to StyOX exposure. Comparing BS urine samples among the three groups of workers, controls always had lower levels of StyU and Sty metabolites than the exposed workers. Median BS levels of metabolites between varnish workers and reinforced plastics workers were comparable except for MA and PGA, which were much greater in the reinforced plastics workers. Median levels of urinary analytes in ES samples tended to be greater in reinforced plastic workers than in varnish workers; these differences were significant (p <0.05) for all analytes except Sty-U, all but one of the mercapturic acids (levels of R,R-M2 were not different), and VP-S. Considering only exposed subjects, levels of the urinary analytes were about 2.5 times higher in ES samples than in BS samples. In ES samples among varnish workers and reinforced plastics workers, respectively, MA accounted for 48.1 % and 63.4% of total uninary analytes, followed by PGA (48.4% and 33.4%), VP (2.1% and 1.6%), PHG (1.2% and 1.4%), M1+M2 (0.25% and 0.11%) and StyU, (0.005% and 0.003%). In BS samples the relative proportions of MA and PGA were inverted, with the highest excretion for PGA (56.8% and 57.8%), followed by MIA (36.6% and 37.2%), PHG (2.2% and 3.3%), VP (4.0% and 1.6%), Ml +M2 (0.34% and 0.12%) and StyU (0.009% and 0.002%). The different proportions of MIA, PGA and PHG between ES and BS samples reflect the fact that MA is the precursor of PGA, which is the precursor of PHG. Among urinary mercapturic acids S,R-M2 and RR-M1 represented about 84-95% of the total mercapturic acids, with S,R-M2 in ES samples predominating in both reinforced plastic workers and in varnish workers (49% and 59%, respectively). Considering only styrene exposed workers, and assuming a urinary creatinine concentration of 1.0 g /l, the daily urine volume was 1.44 1 (0.001 l/min urinary excretion rate x 1440 min per day). Assuming further that half of the daily urine volume contained biomarkers at median concentrations observed in BS samples while the other half contained biomarkers at median concentrations observed in ES samples, then the median total excretion of urinary biomarkers was 1.53 mmol per day for reinforced plastics workers (i.e. 0.72 l x (0.58 + 1.55) mmol g/l creatinine x 1 g creatinine/l) and 0.45 mmol per day for varnish workers. These values are similar to estimated median values of Sty uptake, assuming low to moderate rates of exercise, namely 1.01-2.02 mmol daily for reinforced plastics workers (18.2 mg/m3 x 0.9 or 1.8 m3/h x 8 h per day x 0.8 (fraction of styrene absorbed)/104 mg styrene mmol/l) and 0.19-0.38 mmol per day for varnish workers. Regarding protein adducts, median levels of 1PE-Alb and 2PE-Alb were significantly higher in exposed workers than in control workers (p <0.05), but did not differ significantly between reinforced plastics workers and varnish workers. Moreover, Hb adducts did not differ significantly across the investigated groups.

Influence of job, cigarette smoking, and OST genotypes:
All urinary biomarkers significantly increased with job category in the order: control workers <
Pearson correlations:
Highly significant positive correlations were found between all the urinary biomarkers and Sty, with values of r ranging from 0.789, for Ml +M2 vs Sty, to 0.974, for MA vs Sty; slightly lower correlation coefficients were observed between the urinary biomarkers and StyOX. Correlations between Sty or StyOX and protein adducts were not significant (p > 0.05). The urinary biomarkers were all highly correlated with each other, with values of r as high as 0. 958 (between MA +PGA and VP-S +VP-G). The only significant or borderline significant correlations observed between urinary biomarkers and protein adducts were the negative correlations between the mercapturic acids and 2PE-Alb. Levels of 2PE-Alb were also significantly correlated with LPE-Alb. Somewhat smaller correlations were observed using BS instead of ES values.

Effects of exposure to Sty and StyOX
For each urinary and blood analyte (dependent variable), values of model R 2 (adjusted) and regression coefficients are indicated for the predictor variables, Sty and StyOX. All urinary analytes were significantly associated with Sty exposure, after adjusting for co-exposure to StyOX (p <0.0 1), while levels of only StyU and PHIG were significantly associated with StyOX exposure, after adjusting for co-exposure to Sty (p <0.05). However, opposite directions of the associations between StyOX exposure and levels of StyU (-) and PHG (+) were observed. Since these multiple linear regression models were performed on the logged air and biomarker levels, effect coefficients close to one indicate that the biomarker level should be proportional to the particular exposure variable in natural seale, while effect coefficients less than one indicate downward concavity in the relationship between the analyte and the exposure variable in natural scale (Rappaport et al. 2002). As styrene was an important predictor variable for all urinary analytes, it is, therefore, interesting that the regression coefficients for Sty in models of and the mercapturates were not significantly different from one, suggesting linear production of these analytes with increasing levels of Sty exposure. Yet, the corresponding models of the other urinary metabolites, including the products of phenylethylene glycol (i.e. MIA and PGA) and of 4-vinylphenol (i.e. VP-G and VP-S), all had values that were significantly less than one (p <0.05), suggesting saturable metabolism along the corresponding pathways. Regarding the protein adducts of StyOX, no significant effects of exposure to Sty or StyOX were observed for levels of Alb and Hb adducts in the exposed workers.

Applicant's summary and conclusion