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Genotoxic risk in rubber manufacturing industry: A systematic review Claudia Bolognesi a,∗ , Angelo Moretto b,c a
Environmental Carcinogenesis Unit, IRCCS AUO San Martino IST-Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10, 16132 Genoa, Italy Department of Biomedical and Clinical Sciences, University of Milano, Luigi Sacco Hospital, via GB Grassi 74, 20157 Milano, Italy c International Centre for Pesticides and Health Risks Prevention (ICPS), Luigi Sacco Hospital, via GB Grassi 74, 20157 Milano, Italy b
h i g h l i g h t s • A systematic review on genotoxic risk on rubber manufacturing industry was carried out. • Data on genotoxic activity of exposure mixture evidence large differences among industries and processes. • Data on human biomonitoring studies suggests that a genotoxic hazard may still be present in certain rubber manufacturing industries.
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Rubber manufacturing industry Tire manufacturing Occupational exposure Genotoxicity Mutagenicity DNA adducts
a b s t r a c t A large body of evidence from epidemiological studies among workers employed in the rubber manufacturing industry has indicated a significant excess cancer risk in a variety of sites. The International Agency for Research on Cancer has recently classified the “Occupational exposures in the rubber-manufacturing industry” as carcinogenic to humans (Group 1). A genotoxic mechanism for the increased cancer risk was suggested on the basis of the evidence from the scientific literature. Exposure assessment studies have shown that workers in the rubber manufacturing industry may be exposed to different airborne carcinogenic and/or genotoxic chemicals, such as certain aromatic amines, polycyclic aromatic hydrocarbons, N-nitrosamines, although the available information does not allow to establish a causal association of cancer or genotoxic risk with particular substances/classes of chemicals or specific jobs. The aim of this paper is to critically evaluate, by conducting a systematic review, the available biomonitoring studies using genotoxicity biomarkers in rubber manufacturing industry. This systematic review suggests that a genotoxic hazard may still be present in certain rubber manufacturing industries. A quantitative risk assessment needs further studies addressing the different, processes and chemicals in the rubber manufacturing industries. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A large body of evidence from epidemiological studies among workers employed in the rubber manufacturing industry has indicated a significant excess cancer risk in a variety of target tissues. The International Agency for Research on Cancer (IARC) has recently published a Monograph reporting the results of a working group on “Occupational exposures in the rubber-manufacturing industry” (IARC, 2012) that updates previous monographs (IARC, 1982, 1987). The evaluation in this Monograph was restricted to rubber-manufacturing industry, and excluding the production of synthetic polymers in chemical plants. Under the commonly used term “rubber manufacturing industry”, different production processes are included. The IARC working group classified the
∗ Corresponding author. Tel.: +39 105558304; fax: +39 105558344. E-mail addresses: [email protected], [email protected] (C. Bolognesi).
occupational exposures in the rubber manufacturing industry as “carcinogenic to humans” (Group 1). This conclusion was based on sufficient epidemiological evidence for leukemia, lymphoma, and cancers of the urinary bladder, lung and stomach, whereas the evidence was considered limited for cancers of the prostate, esophagus, and larynx. It should also be noted that tumor incidence in cohorts of workers, when observed, are related to past exposures, and that current exposures are significantly different due to technological changes and adoption of more rigorous risk management measures (de Vocht et al., 2008). While animal experimental data relevant to the rubber manufacturing industry were not available, the working group concluded that the evidence for genetic and cytogenetic effects among workers was strong enough to support genotoxicity as one mechanism for the observed increases in risk for several cancer types. The outcome of recent cytogenetic studies were considered of concern by the IARC working group, although it conceded that other mechanisms may also have likely played a role because the exposure is too complex and variable mixtures of compounds are involved.
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The manufacturing process of rubber goods is complex (Lewis, 1999). In general, the main operations involved include handling raw materials, milling, extruding and calendering, assembling/building, ‘curing’ or vulcanizing, inspection (finishing), storage and dispatch. In addition to the different operations, the assessment of workers’ exposure and of consequent health effects is complicated by the fact that, while the composition of the mixture used to produce the rubber good is well known, what happens and what compounds are produced during milling/extruding/calendering, and especially vulcanization, is at least partly unknown. Exposure assessment studies have shown that workers in the rubber manufacturing industry have been or are potentially exposed to different airborne carcinogenic and/or genotoxic chemicals, such as aromatic amines, polycyclic aromatic hydrocarbons, N-nitrosamines. However, the absence of detailed information prevents in some cases the identification of specific compound(s) or jobs that can be related to either cancer or genotoxic risk. The reviews on environmental exposure in rubber manufacturing industry, carried in the late 1970s and early 1980s, focused on inhalable particulate exposure to rubber dust, fumes and solvents (Parkes et al., 1975; van Ert et al., 1980; Williams et al., 1980). Later on, more specific surveys were conducted for exposure to specific classes of chemicals, such as nitrosamines and several polycyclic aromatic hydrocarbons (Spiegelhalder and Preussmann, 1982; Oury et al., 1997; Fracasso et al., 1999). In a number of exposure assessment studies, the mutagenic activity of ambient rubber dust, fumes and surface contamination wipes for different industrial processes in the rubber manufacturing industries was evaluated in in vitro systems (Fracasso et al., 1999; Monarca et al., 2001; Vermeulen et al., 2001). Genotoxicity biomarkers of exposure, and of effect in different surrogate tissues were applied in several recent studies of exposure in the rubber manufacturing industry. The aim of this paper is to critically evaluate the available studies on biomonitoring of exposures using biomarkers of genotoxicity in the rubber manufacturing industry. The analysis involved consideration of the good being manufactured, the biomarker(s) used, and the availability of environmental monitoring or biological monitoring of exposure, in the attempt to identify specific chemical compounds or manufacturing processes associated with the observed genotoxic effects and to define if a genotoxic risk could be still attributed to the modern rubber manufacturing industry.
eligibility. The literature search was carried out on March 2013. Its results and the stepwise exclusion process are described in Fig. 1. Twenty nine studies were included in the analysis, 3 papers were assessed as correlated studies, reporting information on airborne genotoxic chemicals or mixture of chemicals in rubber manufacturing processes (de Vocht et al., 2008; Cemeli et al., 2009; Talaska et al., 2012).
3. Results 3.1. Exposure to genotoxic mixtures Table 1 reports the studies on the genotoxic activity of airborne particulates and fumes collected by ambient and personal samplers for different processes in rubber manufacturing industries. All studies applied Ames test using common strain TA 98 and TA 100 and specific strains of Salmonella typhimurium (YG1021, YG1024, YG1041) in presence or absence of metabolic activation fraction, to evaluate the possible mutations induced by the presence of nitroarenes and aromatic amines. Two studies, carried out in Italy (Fracasso et al., 1999; Monarca et al., 2001) in factories producing different types of rubber articles, describe direct and indirect mutagenicity of both ambient and personal air samples. In one study (Monarca et al., 2001) gene mutation by Ames test and DNA-damaging activity by comet assay in human leucocytes were evaluated in parallel: the results show that the dichloromethane extracts of particulate matter, particularly those obtained from the finest fractions, have genotoxic effects with both assays. Another study was carried out in rubber tire industry, involving two companies in Sweden and in The Netherlands using comparable production processes. The mutagenic profile of rubber dust and fumes showed substantial differences between the companies probably associated with different industrial processes and control measures (Vermeulen et al., 2000a,b). A further study, carried out in The Netherlands in nine rubber manufacturing companies, reports large differences in median mutagenicity of total suspended particulate matter (TSPM) and of wipe samples from contact surfaces (Vermeulen et al., 2001). These studies do not allow the identification of any classes of chemicals or industrial processes specifically associated with the mutagenic activity of environmental samples. The main determinant for the exposure to mutagenic compounds seems to be the “company”, involving the use of different chemicals and industrial practices.
2. Materials and methods This systematic review follows the methodology described in the PRISMA statement (Moher et al., 2009).
3.2. Biomonitoring studies
2.1. Eligibility criteria Eligible for the inclusion in the present review were all genotoxicity studies on environmental samples and biomonitoring studies among workers in the rubber manufacturing industry involving the use of validated genotoxicity assays. Only studies in English where the full text was available were considered. 2.2. Search strategy Studies on genotoxic biomonitoring among workers in rubber manufacturing industry were identified by using the MedLine/PubMed database (National Library of Medicine, National Institutes of Health, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/pubmed/). The terms genotoxic, DNA damage, chromosomal damage, chromosomal aberration, micronucleus (i), DNA strand break, Ames, mutation, HPRT, Comet were used as medical subject heading (MeSH) associated to rubber or tire industry using AND operator. 2.3. Study selection All retrieved studies were reviewed and assessed for inclusion in the present analysis. A number of publications were discarded on the basis of the criteria of
Table 2 summarizes the biomonitoring studies carried out in groups of workers from rubber manufacturing industries. Twentyfive studies published between 1980 and 2011 carried out in different countries are described: 9 of them are related to the tire manufacturing industry involving the application of different biomarkers of exposure and effect. Small sample sizes with low statistical power are common for the large majority of the studies. Different biomarkers are considered, assessing transient and permanent genotoxic responses: (a) urinary mutagenicity, as marker of exposure; (b) DNA adducts in different surrogate tissues, peripheral lymphocytes and urothelial exfoliated cells, as a measure of the dose reaching the target; (c) DNA single strand breaks determined by Comet assay in peripheral lymphocytes, as markers of early genotoxic effect; (d) chromosomal alterations, SCE and micronuclei frequency as marker of chromosomal damage; (e) frequency of hprt mutants in peripheral lymphocytes, as marker of expressed genetic damage.
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Individual samplers
Exposure/substance/conc
Type of test
Results (FR)a Statistical significance
Country/Ref.
Two fixed positions: (a) mixing area (Bambury mixer), (b) calendering area and (c) reference area not subject to chemical exposure. Total dust collected over a period of 3 h. Ambient and personal samplers taken on the same day.
Personal samplers from 5 workers engaged in different manufacturing duties (mixing, weighing, calendering, compounding and extruding) Total dust collected over a period of 2 h.
Fixed positions: above average values for pyrene and dibenzo(a,h)-anthracene. Personal filters: low or undetectable levels of PAHs.
Mutagenicity of air particulate extracts in: Salmonella t TA98, Salmonella t TA100, TA98NR(deficient in reductase), TAYG1021 (nitroreductase overproducing) in presence and absence of S9 mix.
Significant mutagenic effect in TA98, TA98NR, YG1021. No effect in TA100. Fixed positions without S9 mix: (FR 5–15); with S9 mix significant results only in calendering area (FR 3–7). Personal samplers without S9 mix(FR 10–20) with S9 mix (FR 3–13)
Italy/Fracasso et al. (1999)
Rubber industry: 4 factories selected according to nitrosamine exposure: (A) ethylene-propylene rubber; (B) and (C) acrylonitrile-butadiene rubber; (D) styrene-butadiene rubber. Different processes
Fixed positions for particulates and PAH. Samplings for 8 h on two non consecutive days
Personal samplers: 6–7 workers/factory. Samplings: 6 h for nitrosamines and PAH
N-nitroso-dimethylamine: 0.10–0.98 mg/m3 ; N-nitroso-morpholine: 0.77–2.28 mg/m3 . Highest conc of total PAH: 11.35–66.1 ng/m3 )
Mutagenicity of the dichlorometane extracts of particulate matter. Salmonella t. strain TA98 with and without S9 mix DNA damage by Comet assay using peripheral blood leukocytes with and without S9 mix In situ study: micronucleus test in Tradescantia
Particulate < 0.5 Mutagenicity; without S9 mix:23.3–63.8 rev/mg vs 17.8 rev/mg (FR 1.3–3.6) with S9 mix24.1–198.2 rev/mg vs 18.3 rev/mg (FR: 1.3–10.8). Comet assay tail moment: without S9 mix: 0.35–5.22 vs 0.34 (FR 0–15); with S9 mix: 0.57–2.71 vs 0.34 (FR: 1.7–7.9). Particulate 0.5–10 m Mutagenicity; without S9 mix: 0–41.2 vs 17.8 rev/mg (FR 0–2.31) with S9 mix:5 vs 18.3 rev/mg (FR 0–2.4). Comet assay tail moment: without S9 mix: 0.38–1.32 vs 0.34 (FR 1.1–3.9) With S9 mix 0.39–21.15 vs 0.34 (FR 1.14–62). Micronucleus test in Tradenscantia MN/100 tetrads 1.7–13 vs 5.2
Italy/Monarca et al. (2001)
Rubber industries. Company A (Netherlands) Company B (Sweden) Mixing and curing departments
Total suspended particulate matter (TSPM) Total Soluble Matter (TSM) Cyclohexane Soluble Matter (CSM) Company A: 2 locations within the mixing and curing department. Samples collected during 3 consecutive days Company B: 5 locations in mixing dept, 3 locations in curing dept Repeated 8 h samples within 1 week.
ND
Company A Mixing: TSPM 0.20–0.30mMg/m3 ; TSM 101–170 g/m3 , CSM 45–108 g/m3 Curing: TSPM 0.29–0.30mMg/m3 ; TSM 226–268 g/m3 , CSM 164–177 g/m3 Company B Mixing: TSPM 0.03–0.34 mg/m3 , TSM 9–44 g/m3 , CSM: 1–16 g/m3 ; Curing TSPM 0.04–0.23 mg/m3 ; TSM 25–81 g/m3 , CSM: 2–16 g/m3
Mutagenicity of airborne particulate samples. Salmonella t: YG 1021 nitroreductase overproducing, YG 1024 O-acetyltransferase overproducing YG1041, nitroreductase and O-acetyltransferase overproducing.
Company A Filter extracts Salmonella t YG1021: No mutagenic activity Salmonella t YG1024. combined filter extracts and fumes: mixing 721–989 rev/m3 ; curing 684–1292 rev/m3 . Salmonella t. YG1041: mixing 308–735 rev/m3 , curing 224–346 rev/m3 . Company B Filter extracts Salmonella t YG1021: No mutagenic activity Salmonella t. YG1041 mixing 3–48 rev/m3 , curing 6–139 rev/m3 .
Netherland Sweden/Vermeulen et al. (2000)
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Stationary samplers
Rubber industry, different types (no tire) weighing, mixing, calendering, compounding, extruding
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Table 1 Assessment of exposure to genotoxic mixture(s) in different rubber manufacturing industries and processes.
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Frequency ratio (FR) = mean value in tested samples/mean control value.
3.2.1. Urinary mutagenicity Six papers on urinary mutagenicity, using Ames test applied in Salmonella TA 98, TA 100 ed Escherichia coli WP2 are available in rubber manufacturing workers: 4 of them report significant increase associated with the exposure in the workplace. The most recent studies reporting positive results were carried out in general rubber goods production industries, while no recent study is available on tire manufacturing plants. In the former, significant associations with the exposure to mutagenic inhalable particulates, and contaminated surfaces were observed; highest urinary mutagenicity was detected in mixing and curing workers (Peters et al., 2008). Large interindividual variation was observed in the different groups due to the influence of lifestyle factors, such as environmental and mainstream tobacco smoke (Vermeulen et al., 2000a,b; Vermeulen et al., 2003). A statistical significant increase of revertants in S. typhimurium TA98 and E. coli WP2 uvrA after treatment with urine samples from workers in a tire manufacturing plant was observed with highest values in smokers (frequency ratio 5.9 vs 2.4) (Falck et al., 1980). Another mutagenicity study in a tire plant in Italy, including 73 workers and 23 controls, reported negative results in S. typhimurium TA 1535, TA 98, TA 100. A parallel analysis of raw materials used in workplace showed only a weak mutagenicity of a minor fraction (3/22) of the technical products tested (Crebelli et al., 1984, 1985). No difference in urine mutagenicity was claimed in a further study carried out in 19 rubber manufacturing workers from 4 rubber different factories: no data are reported (Moretti et al., 1996). Three more recent studies he in workers in rubber manufacturing industries in The Netherlands, showed increased urinary mutagenicity associated with inhalable dust exposure during the work-week compared with the week-end (Vermeulen et al., 2000a,b; Vermeulen et al., 2003; Peters et al., 2008).
a
Netherland/Vermeulen et al. (2001) TSPM samples 76/83 (95%) showed mutagenic activity: 19–1263 rev/m3 . highest effects in curing process: 43/104 (41.3%) Wipe samples mutagenic activity: 17–665 rev/cm2 Mutagenicity of TSPM extracts and surface contamination with Salmonella TA YG1041 TSPM 0.03–2.75 mg/m3 ND Rubber (various, not tire) industries. The study was conducted in 1997 9 companies: Different processes: mixing, pre-treating, molding, curing, finishing, shipping, engineering service, laboratory
8 h total suspended particulate matter (TSPM) in air 83 sampling sites 145 repeated samples 104 wipe samples
Country/Ref. Results (FR)a Statistical significance Type of test Exposure/substance/conc Individual samplers Stationary samplers Type of industry/processes
Table 1 (Continued)
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3.2.2. DNA adducts DNA adducts, were determined in urothelial cells and in peripheral blood lymphocytes in a number of studies carried out in rubber workers (Talaska et al., 2002). A first study did not reveal any increase of DNA adducts in peripheral lymphocytes of workers from the vulcanizing department of a rubber manufacturing company in Hungary when compared with controls associated with a low urinary 1-hydroxypyrene level (Schoket et al., 1999). Two further studies, conducted in different years in the same rubber manufacturing factories in The Netherlands, report DNA adducts in urothelial and blood cells (Vermeulen et al., 2002; Peters et al., 2008). A different DNA adduct pattern was observed in the two surrogate tissues with four specific DNA adducts reliably detected in both cell types. Total urothelial cell DNA adducts were related to urinary hydroxypyrene and mutagenicity with highest values found in workers engaged in compounding, mixing and curing departments. No correlation between bladder and blood cell DNA adduct levels was found, suggesting the involvement of different chemicals and/or different metabolic pathways in bladder and blood cells. 3.2.3. DNA damage DNA damage, measured by Comet assay in peripheral lymphocytes, was found in workers employed in rubber manufacturing factories in different countries. The use of different experimental protocols and of different DNA damage parameters prevent any comparison among the studies. A statistical significant increase of DNA damage, as single strand breaks and oxidatively damaged DNA bases, was observed in a small group of workers employed in a rubber tire factory in Czech Republic in two different samplings at interval of 1 year. Significant correlation between values of DNA damage and micronuclei
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Identification
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Search strategy (19/10/2012): Terms: (genotoxic, DNA damage, chromosomal damage, chromosomal aberration, micronucleus(i), DNA strand break, Ames, mutation, HPRT, Comet) AND rubber or tyre industry. Studies retrieved (n = 87)
Included
Eligibilty
Screening
Studies selected
5
Studies excluded (n = 46) • Experimental studies on single compounds • Reviews
(n=41)
Abstracts excluded:9 3 Reviews reporting the same data 6 Not in english, not relevant
Studies screened (n = 41)
Full-text articles assessed for eligibility (n= 31)
Correlated studies( n = 3) • Experimental in vitro study on combination of chemical compounds relevant in rubber industry (Cemeli et al Mut Res 664; 69-76, 2009) • Data on inhalable dust in rubber industry in Europe (de Vocht et al Occup Env Med, 65, 384-391, 2008)
Articles on genotoxicity biomonitoring studies included in the review (n= 28 (17 reported in IARC monograph)
• Exposure to PAH in rubber workers (Talaska et al Toxicology Lett, 213, 45-48, 2012)
Fig. 1. Study selection flow chart.
frequency was reported for both the samplings (Somorovská et al., 1999). A study, with some methodological shortcomings, carried out in a small group of workers in rubber manufacturing industries in Italy, shows a non statistically significant increase of the DNA migration length measured by Comet assay (Moretti et al., 1996). A decrease of DNA damage was described in workers from a Portuguese tire factory showing higher levels of urinary thioethers compared with a group of controls (Laffon et al., 2006). This result could be attributed to the crosslinking effect induced by some compounds in the exposure mixtures of rubber production (Cemeli et al., 2009). Finally a large study carried out in China, involving 371 workers in a factory processing natural rubber to manufacture tires, report a weak, but statistical significant increase of DNA strand breaks, expressed as tail moment, in rubber workers compared with managerial workers. The highest values were observed in mixers exposed in poor hygienic conditions since the toluene concentrations exceeded the Chinese maximum allowable concentration by four folds (Zhu et al., 2000). 3.2.4. Chromosomal damage The large majority of biomonitoring studies on genotoxic risk in rubber manufacturing industry refers data on chromosomal damage, including chromosomal aberrations, micronuclei frequency and SCE: 6/10 studies report positive results. All the studies except one (Musak et al., 2008) are of small size and seven of them were carried out before 2000. The extent of increase is higher for the frequency of chromosomal aberrations (frequency ratio range 1.5–5.0) compared to micronuclei frequency (F.R. 1.66–3.0) and SCE (F.R.1.27–1.61). Two studies carried out in Poland in small groups of vulcanizers from a tire factory report borderline increase of chromosomal aberrations and SCE associated with a decrease of the proliferation index (Sasiadek, 1992, 1993). A further study in a tire factory in the Czech Republic, using as a raw material styrene-butadiene rubber shows significant increase of chromosomal aberrations and
micronuclei frequency associated with DNA single strand breaks in peripheral lymphocytes (Somorovská et al., 1999). The only statistically powerful study was carried out in 177 tire plant workers in the Slovak republic and shows a borderline increase of chromosomal aberrations with high interindividual variability associated with genetic polymorphism in genes encoding biotransformation DNA repair enzymes (Musak et al., 2008). A recent study reports a significant reduction in telomere length, as a marker of chromosomal instability, in a group of 157 workers in a rubber manufacturing industry in Sweden correlated with increased exposure to N-nitrosamines in air, based on individual measurements or on work task (Li et al., 2011). The large interindividual variability observed in exposed subjects as well as in controls, potentially associated with the large number of confounding factors described for this biomarker, such as age, smoke, diet, genetic susceptibility, prevents a clear interpretation of the study. 3.2.5. Gene mutations A number of studies were carried out between 1992 and 1999 on workers from synthetic rubber manufacturing plants using as raw materials polymerized polybutadiene rubber and styrenebutadiene copolymers in Texas to evaluate the frequency of mutant lymphocytes using HPRT mutant lymphocyte assay (Table 2). Statistically significant increase in mutant frequency were observed in workers exposed to butadiene at concentration higher that 150 ppb (Abdel-Rahman et al., 2001). The most recent study (Wickliffe et al., 2009) where the large majority of exposures were below of method detection limit did not show any increase of the frequency of mutant lymphocytes. 4. Discussion The aim of our study was to critically review the available scientific literature in order to identify potential genotoxic chemicals or manufacturing processes in the rubber manufacturing industry
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Type of test
Results (FR)a Statistical significance
Country/Ref.
Tire manufacturing Different processes
20/16
NA
Urinary mutagenicity: Salmonella t TA98; E. coli WP2 uvrA with S9
Mutagenic activity Salmonella t TA98: Smokers 717/298 (FR: 2.4) Non-smokers 149/25 (FR: 5.9) E. coli: Smokers 727/13.2 (FR: 55) Non-smokers 501/34.7 (FR: 14.7)
Finland/Falk et al. (1980)
Rubber industry 2 Rubber plants Factory A: heavy and light tires weighing and mixing, tire building vulcanizing Factory B: various technical rubber products weighing, mixing, cleaning, vulcanizing,
Factory A: 28/17 Factory B: 27/18
NA Factory A: 1.5–11.7 y Factory B: 1–14.5 y
CA and SCE in peripheral lymphocytes
Factory A Smokers: CA3.1/4.8 (NS) SCE 13.1/11.5 (NS) Non smokers: CA 2.6/2.4 (NS) SCE 11.2/10.4 (NS) Factory B Smokers: CA 5.2/5.7 (NS) SCE 13/12.6 (NS) Non smokers: CA 3.8/3.7 (NS) SCE: 12.2/10.7 (NS)
Finland/Sorsa et al. (1983)
Tire manufacturing vulcanizing
34/15
total airborne particulate (area samplers) 0.5–3.4 mg/m3 8.2 y
CA and SCE in peripheral lymphocytes
CA: 1.9 ± 1.4/2.1 ± 1.5 (NS) SCE 5.2 ± 1.3/5.2 ± 0.7 (NS)
Italy/Degrassi et al. (1984)
Tire manufacturing mixing, weighing, compounding, extruding, calendering, tire building, vulcanizing
72/23
total airborne particulate (area samplers) 0.7–2.73 mg/m3 7–12 y
urinary mutagenicity: Salmonella t TA98, TA100, TA1535 with S9
No effect of exposure Mutagenic samples (smokers/non smokers): 20/56 v 3/34 p < 0.001
Italy/Crebelli et al. (1984)
Tire manufacturing Styrene-butadiene rubber Vulcanizing
21/14
Styrene, butadiene, PAH, nitrosamine, carbon oxide and sulfur dioxide 2–35 y
CA and SCE in peripheral lymphocytes
CA 2.2 ± 1.06/0.9 ± 1 (FR:2.4) p < 0.01 SCE 16.1 ± 3.5/10 ± 1.5 (FR:1.61) p < 0.001
Poland/Sasiadek (1992)
Tire manufacturing Styrene-butadiene rubber Vulcanizing
26/25
Styrene, butadiene, PAH, nitrosamine, benzene, toluene 0.5–30 y
SCE in peripheral blood
SCE 13.3 ± 2.9/9.8 ± 1.8 (FR: 1.34) NS
Poland/Sasiadek (1993)
Rubber industry 4 factories: (A) ethylene-propylene rubber; (B) and (C) acrylonitrile-butadiene rubber; (D) styrene-butadiene rubber. Different processes
19/20 Selected according to nitrosamine exposure
N-nitroso-dimethylamine, N-nitroso-morpholine, PAH. > 10 y
Comet assay MN test and SCE in peripheral blood lymphocytes
Comet assay: MD (migration distance) 43.08 ± 12.49/38.92 ± 8.06 NS Micronucleus test MN/1000 BN cells: 22.84 ± 15.82/13.74 ± 4.42 (FR: 1.66) NS SCE SCE/metaphase 6.06 ± 1.15/5.51 ± 0.84 (FR: 1.19) NS
Italy Moretti et al. (1996)
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N. exposed subjects/controls
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Table 2 Biomonitoring studies in groups of workers from rubber manufacturing industries: genotoxic effects.
Exposure substances/concentration/duration
Type of test
Results (FR)a Statistical significance
Country/Ref.
Tire manufacturing Styrene-butadiene rubber Different processes
27/22 industry controls and 22 lab controls
Styrene, toluene, butadiene, PAHs, alkanes and alkenes. B(a)P = 3.65 ng/m3
Comet assay CA and MN test in peripheral lymphocytes
Comet assay % DNA in tail: 1◦ sampling 1996: 33%; 2◦ sampling 1997: 45% vs 13%(industry controls) and 22% (lab controls) (FR: 2.5–3.4) p < 0.00001. Micronucleus test MN/2000 cells 6.5 vs 2.1 (FR: 3) Chrom aber % 1 vs 0.2 (FR: 5) p < 0.00001
Czech Rep./Somorovska et al. (1999)
Rubber industry vulcanizing
61/76
PAH, aromatic amines, N-nitroso compounds, organic solvents
1-OH-pyrene in urine. DNA adducts (32 P postlabelling) in peripheral blood lymphocytes. GPA assay
mol 1-OH-PY/mol creatinine: 0.22 ± 0.18/0.32 ± 0.48 NS DNA adducts/108 nucleotides: 5.6 ± 2.6/6.4 ± 3.3 NS GPA variant cell frequency: 8.7 ± 5.4/8.0 ± 6.9 NS
Hungary/Schoket et al. (1999)
Rubber industry different processes
29/87
Aromatic solvents, dust, tar and lubrificating oils 3–20 y
CA, premature centromere division (PCD) and aneuploidy in peripheral lymphocytes.
CA: 3.38 ± 0.26/1.60 ± 0.62 (2.1) p < 0.01 PCD < 3 Chr: 11.45 ± 1.43/1.57 ± 0.44 (7.29) p < 0.01 PCD > 3Chr: 6 ± 1.18/0.32 ± 0.10 (18.7) p < 0.01 Aneuploidy: 5.64 ± 0.44/6.20 ± 0.43 NS
Hungary/Major et al. (1999)
Tire manufacturing finishing, maintainance calendering, vulcanizing mixing
281/90
Dust, toluene, xilene, gasoline, H2 S, S02
Comet assay in peripheral lymphocytes
Comet assay Tail moment (m): All workers: 1.77 (1.64–1.90)/1.52 (1.36–1.71) NS Mixers: 2.54 (1.95–3.31)/1.52 (1.67) p < 0.002
China/Zhu et al. (2000)
Tire manufacturing Different processes
32/32
No data 1–34 y
Urinary thioethers Comet assay, SCE and MN in peripheral lymphocytes
Urinary thioethers mM 0.41 ± 0.05/0.24 ± 0.02 (FR1.7) p < 0.01 Comet assay: Tail moment (m) 44.72 ± 0.66/48.25 ± 0.71 NS SCE: 4.35 ± 0.20/4.38 ± 0.17NS MN: 2.34/1.84 NS
Portugal/Laffon et al. (2006)
Tire manufacturing butadiene rubber mixing, extruding, calendering, tire building, tube repair
177/172
Personal samplers Mixing dept BD 2.4–2.8 mg/m3 Styrene (8.1–13.2 mg/m3 ) and sulphur dioxide (0.6–1 mg/m3 ); Pressing dept: BD (0–5.8 mg/m3 ) and sulphur dioxide (0.6–1 mg/m3 ) 19.4 ± 8.5 y
CA in peripheral lymphocytes
CA/100 metaphases: 2.5 ± 1.8 (exposed smokers) vs 1.7 ± 1.2 (non smoker controls) (1.47)
Slovak rep/Musak et al. (2008)
Rubber industries 9 companies Different processes: compounding and mixing, pretreating, molding, curing, finishing
105 subjects employed full time in rubber manufacturing industries 38 smokers/67 non smokers
ND
Urinary mutagenicity with S. typhimurium strain YG1041 with S9 on Sunday and week days. Adjustement for cotinine
Urinary mutagenicity non smokers/smokers Sunday: 4416/4664 rev/g creat Weekday 5913/6470 rev/g creat
The Netherlands/Vermeulen et al. (2002)
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N. exposed subjects/controls
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Table 2 (Continued)
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Type of test
Results (FR)a Statistical significance
Country/Ref.
9 companies Different processes: compounding and mixing, pretreating, molding, curing, finishing
The study was conducted in 1997. 52 subjects 3 exposure categoriesb : 18 Low TSPM Low SMEL; 18 High TSPM Low SMEL; 16 HH High TSPM high SMEL
Total Suspended Particulate Matter (TSPM): high ≥ 210 rev/m3 ; low ≤ 210 rev/m3 . Surface Mutagenic Exposure Level (SMEL): high ≥ 25 rev/cm3 ; low ≤ 25 rev/cm2
DNA adducts (32P postlabelling) in exfoliated bladder cells
11 possible DNA adducts identified. 46 (88%) samples had positive results for 3 main adducts; adduct1 in 41 samples, adduct 2 in 13 samples; adduct 3 in 29 samples. The dept mixing and curing showed the highest level of DNA adducts and % of positive samples.
The Netherlands/Vermeulen et al. (2002)
Rubber industries Same companies as in Vermeulen et al. (2002) Different processes
The study was conducted in 1997. 116 rubber manufacturing workers.
Hydroxypirene in urine (116 subjects). Urinary mutagenicity (104 subjects) DNA adducts by P32 postlabelling in: urothelial cells from 24 h urine (52 subjects) and in peripheral mononuclear cells (PBMC)(48 subjects).
Results in non-smokers: Hydroxypyrene 0.17–0.20 (weekdays)/0.12 (Sunday) mol/mol creatinine(curing 0.31 mol in weekdays) Urinary mutagenicity: rev/g creatinine weekday/Sunday: compounding and mixing: 10511/6522 (curing 11527/10447 p < 0.05). 11 possible DNA adducts were identified in urothelial cells: 38 (73%) samples had positive results for 4 main adducts: adduct 1 in 30 (58%); adduct 2 in 12 (23%), adduct 3 in 23 (44%); adduct 11 in 2 (4%). 4 adducts were identified in PBMC: adduct A in 5 (10%); adduct B in 38 (79%) adduct C in 3 (6%); adduct D in 23 (48%).
The Netherlands/Peters et al. (2008)
Rubber industry Styrene-butadiene polymer plant
Pilot study carried out in 1991. 8 subjects high expo; 5 subjects low expo, 6 controls
Exposure to BD by personal samplers: 150 (mean 2244.2 ± 749.1); low expo < 150 (mean18.4 ± 5.5)
HPRT variants
hprt Vf per million cells: high expo: 9.25 ± 1.56;/low expo:4.82 ± 0.73 high vs low p = 0.02
Texas USA/Abdel-Rahman et al. (2001)
Rubber industry Styrene-butadiene polymer plant
The study was carried out in 1999. 30 subjects
Exposure to BD (ppb): from non detectable to 1683.5 (mean 93.5) 1–39 y
HPRT variants
no relationship between BD exposure and HPRT mutants. A single subject with the highest expo estimate has high HPRTvf
Texas USA/Wickliffe et al. (2009)
Rubber industries Different processes
157 subjects
Air measurement for nitrosamines: 0.07–35.5 mg/m3 Urinary markers: 1-HP: 0.0020–0.85 mmol/mol creatinine TTCA: 1.7–690 mmol/mol creatinine toluidine: orto 0.025–108 ng/ml; meta 0.025–108 ng/ml; para 0.025–4.7 ng/ml
telomere lenght analysis (PCR)
TL: 0.16–1.3 nitrosamine levels are highly correlated with the telomere lenght
Li et al. (2011)
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Type of industry/Process
a
Frequency ratio (FR) = mean value in tested samples/mean control value. The four a priori exposure groups were defined by: (1) dermal exposure, based on the median levels of mutagenic activity detected on likely skin contact surfaces (high (>25 rev/cm2 ), low (210 rev/m3 ), low (
ARTICLE IN PRESS Toxicology Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
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Genotoxic risk in rubber manufacturing industry: A systematic review Claudia Bolognesi a,∗ , Angelo Moretto b,c a
Environmental Carcinogenesis Unit, IRCCS AUO San Martino IST-Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10, 16132 Genoa, Italy Department of Biomedical and Clinical Sciences, University of Milano, Luigi Sacco Hospital, via GB Grassi 74, 20157 Milano, Italy c International Centre for Pesticides and Health Risks Prevention (ICPS), Luigi Sacco Hospital, via GB Grassi 74, 20157 Milano, Italy b
h i g h l i g h t s • A systematic review on genotoxic risk on rubber manufacturing industry was carried out. • Data on genotoxic activity of exposure mixture evidence large differences among industries and processes. • Data on human biomonitoring studies suggests that a genotoxic hazard may still be present in certain rubber manufacturing industries.
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Rubber manufacturing industry Tire manufacturing Occupational exposure Genotoxicity Mutagenicity DNA adducts
a b s t r a c t A large body of evidence from epidemiological studies among workers employed in the rubber manufacturing industry has indicated a significant excess cancer risk in a variety of sites. The International Agency for Research on Cancer has recently classified the “Occupational exposures in the rubber-manufacturing industry” as carcinogenic to humans (Group 1). A genotoxic mechanism for the increased cancer risk was suggested on the basis of the evidence from the scientific literature. Exposure assessment studies have shown that workers in the rubber manufacturing industry may be exposed to different airborne carcinogenic and/or genotoxic chemicals, such as certain aromatic amines, polycyclic aromatic hydrocarbons, N-nitrosamines, although the available information does not allow to establish a causal association of cancer or genotoxic risk with particular substances/classes of chemicals or specific jobs. The aim of this paper is to critically evaluate, by conducting a systematic review, the available biomonitoring studies using genotoxicity biomarkers in rubber manufacturing industry. This systematic review suggests that a genotoxic hazard may still be present in certain rubber manufacturing industries. A quantitative risk assessment needs further studies addressing the different, processes and chemicals in the rubber manufacturing industries. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A large body of evidence from epidemiological studies among workers employed in the rubber manufacturing industry has indicated a significant excess cancer risk in a variety of target tissues. The International Agency for Research on Cancer (IARC) has recently published a Monograph reporting the results of a working group on “Occupational exposures in the rubber-manufacturing industry” (IARC, 2012) that updates previous monographs (IARC, 1982, 1987). The evaluation in this Monograph was restricted to rubber-manufacturing industry, and excluding the production of synthetic polymers in chemical plants. Under the commonly used term “rubber manufacturing industry”, different production processes are included. The IARC working group classified the
∗ Corresponding author. Tel.: +39 105558304; fax: +39 105558344. E-mail addresses: [email protected], [email protected] (C. Bolognesi).
occupational exposures in the rubber manufacturing industry as “carcinogenic to humans” (Group 1). This conclusion was based on sufficient epidemiological evidence for leukemia, lymphoma, and cancers of the urinary bladder, lung and stomach, whereas the evidence was considered limited for cancers of the prostate, esophagus, and larynx. It should also be noted that tumor incidence in cohorts of workers, when observed, are related to past exposures, and that current exposures are significantly different due to technological changes and adoption of more rigorous risk management measures (de Vocht et al., 2008). While animal experimental data relevant to the rubber manufacturing industry were not available, the working group concluded that the evidence for genetic and cytogenetic effects among workers was strong enough to support genotoxicity as one mechanism for the observed increases in risk for several cancer types. The outcome of recent cytogenetic studies were considered of concern by the IARC working group, although it conceded that other mechanisms may also have likely played a role because the exposure is too complex and variable mixtures of compounds are involved.
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The manufacturing process of rubber goods is complex (Lewis, 1999). In general, the main operations involved include handling raw materials, milling, extruding and calendering, assembling/building, ‘curing’ or vulcanizing, inspection (finishing), storage and dispatch. In addition to the different operations, the assessment of workers’ exposure and of consequent health effects is complicated by the fact that, while the composition of the mixture used to produce the rubber good is well known, what happens and what compounds are produced during milling/extruding/calendering, and especially vulcanization, is at least partly unknown. Exposure assessment studies have shown that workers in the rubber manufacturing industry have been or are potentially exposed to different airborne carcinogenic and/or genotoxic chemicals, such as aromatic amines, polycyclic aromatic hydrocarbons, N-nitrosamines. However, the absence of detailed information prevents in some cases the identification of specific compound(s) or jobs that can be related to either cancer or genotoxic risk. The reviews on environmental exposure in rubber manufacturing industry, carried in the late 1970s and early 1980s, focused on inhalable particulate exposure to rubber dust, fumes and solvents (Parkes et al., 1975; van Ert et al., 1980; Williams et al., 1980). Later on, more specific surveys were conducted for exposure to specific classes of chemicals, such as nitrosamines and several polycyclic aromatic hydrocarbons (Spiegelhalder and Preussmann, 1982; Oury et al., 1997; Fracasso et al., 1999). In a number of exposure assessment studies, the mutagenic activity of ambient rubber dust, fumes and surface contamination wipes for different industrial processes in the rubber manufacturing industries was evaluated in in vitro systems (Fracasso et al., 1999; Monarca et al., 2001; Vermeulen et al., 2001). Genotoxicity biomarkers of exposure, and of effect in different surrogate tissues were applied in several recent studies of exposure in the rubber manufacturing industry. The aim of this paper is to critically evaluate the available studies on biomonitoring of exposures using biomarkers of genotoxicity in the rubber manufacturing industry. The analysis involved consideration of the good being manufactured, the biomarker(s) used, and the availability of environmental monitoring or biological monitoring of exposure, in the attempt to identify specific chemical compounds or manufacturing processes associated with the observed genotoxic effects and to define if a genotoxic risk could be still attributed to the modern rubber manufacturing industry.
eligibility. The literature search was carried out on March 2013. Its results and the stepwise exclusion process are described in Fig. 1. Twenty nine studies were included in the analysis, 3 papers were assessed as correlated studies, reporting information on airborne genotoxic chemicals or mixture of chemicals in rubber manufacturing processes (de Vocht et al., 2008; Cemeli et al., 2009; Talaska et al., 2012).
3. Results 3.1. Exposure to genotoxic mixtures Table 1 reports the studies on the genotoxic activity of airborne particulates and fumes collected by ambient and personal samplers for different processes in rubber manufacturing industries. All studies applied Ames test using common strain TA 98 and TA 100 and specific strains of Salmonella typhimurium (YG1021, YG1024, YG1041) in presence or absence of metabolic activation fraction, to evaluate the possible mutations induced by the presence of nitroarenes and aromatic amines. Two studies, carried out in Italy (Fracasso et al., 1999; Monarca et al., 2001) in factories producing different types of rubber articles, describe direct and indirect mutagenicity of both ambient and personal air samples. In one study (Monarca et al., 2001) gene mutation by Ames test and DNA-damaging activity by comet assay in human leucocytes were evaluated in parallel: the results show that the dichloromethane extracts of particulate matter, particularly those obtained from the finest fractions, have genotoxic effects with both assays. Another study was carried out in rubber tire industry, involving two companies in Sweden and in The Netherlands using comparable production processes. The mutagenic profile of rubber dust and fumes showed substantial differences between the companies probably associated with different industrial processes and control measures (Vermeulen et al., 2000a,b). A further study, carried out in The Netherlands in nine rubber manufacturing companies, reports large differences in median mutagenicity of total suspended particulate matter (TSPM) and of wipe samples from contact surfaces (Vermeulen et al., 2001). These studies do not allow the identification of any classes of chemicals or industrial processes specifically associated with the mutagenic activity of environmental samples. The main determinant for the exposure to mutagenic compounds seems to be the “company”, involving the use of different chemicals and industrial practices.
2. Materials and methods This systematic review follows the methodology described in the PRISMA statement (Moher et al., 2009).
3.2. Biomonitoring studies
2.1. Eligibility criteria Eligible for the inclusion in the present review were all genotoxicity studies on environmental samples and biomonitoring studies among workers in the rubber manufacturing industry involving the use of validated genotoxicity assays. Only studies in English where the full text was available were considered. 2.2. Search strategy Studies on genotoxic biomonitoring among workers in rubber manufacturing industry were identified by using the MedLine/PubMed database (National Library of Medicine, National Institutes of Health, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/pubmed/). The terms genotoxic, DNA damage, chromosomal damage, chromosomal aberration, micronucleus (i), DNA strand break, Ames, mutation, HPRT, Comet were used as medical subject heading (MeSH) associated to rubber or tire industry using AND operator. 2.3. Study selection All retrieved studies were reviewed and assessed for inclusion in the present analysis. A number of publications were discarded on the basis of the criteria of
Table 2 summarizes the biomonitoring studies carried out in groups of workers from rubber manufacturing industries. Twentyfive studies published between 1980 and 2011 carried out in different countries are described: 9 of them are related to the tire manufacturing industry involving the application of different biomarkers of exposure and effect. Small sample sizes with low statistical power are common for the large majority of the studies. Different biomarkers are considered, assessing transient and permanent genotoxic responses: (a) urinary mutagenicity, as marker of exposure; (b) DNA adducts in different surrogate tissues, peripheral lymphocytes and urothelial exfoliated cells, as a measure of the dose reaching the target; (c) DNA single strand breaks determined by Comet assay in peripheral lymphocytes, as markers of early genotoxic effect; (d) chromosomal alterations, SCE and micronuclei frequency as marker of chromosomal damage; (e) frequency of hprt mutants in peripheral lymphocytes, as marker of expressed genetic damage.
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Individual samplers
Exposure/substance/conc
Type of test
Results (FR)a Statistical significance
Country/Ref.
Two fixed positions: (a) mixing area (Bambury mixer), (b) calendering area and (c) reference area not subject to chemical exposure. Total dust collected over a period of 3 h. Ambient and personal samplers taken on the same day.
Personal samplers from 5 workers engaged in different manufacturing duties (mixing, weighing, calendering, compounding and extruding) Total dust collected over a period of 2 h.
Fixed positions: above average values for pyrene and dibenzo(a,h)-anthracene. Personal filters: low or undetectable levels of PAHs.
Mutagenicity of air particulate extracts in: Salmonella t TA98, Salmonella t TA100, TA98NR(deficient in reductase), TAYG1021 (nitroreductase overproducing) in presence and absence of S9 mix.
Significant mutagenic effect in TA98, TA98NR, YG1021. No effect in TA100. Fixed positions without S9 mix: (FR 5–15); with S9 mix significant results only in calendering area (FR 3–7). Personal samplers without S9 mix(FR 10–20) with S9 mix (FR 3–13)
Italy/Fracasso et al. (1999)
Rubber industry: 4 factories selected according to nitrosamine exposure: (A) ethylene-propylene rubber; (B) and (C) acrylonitrile-butadiene rubber; (D) styrene-butadiene rubber. Different processes
Fixed positions for particulates and PAH. Samplings for 8 h on two non consecutive days
Personal samplers: 6–7 workers/factory. Samplings: 6 h for nitrosamines and PAH
N-nitroso-dimethylamine: 0.10–0.98 mg/m3 ; N-nitroso-morpholine: 0.77–2.28 mg/m3 . Highest conc of total PAH: 11.35–66.1 ng/m3 )
Mutagenicity of the dichlorometane extracts of particulate matter. Salmonella t. strain TA98 with and without S9 mix DNA damage by Comet assay using peripheral blood leukocytes with and without S9 mix In situ study: micronucleus test in Tradescantia
Particulate < 0.5 Mutagenicity; without S9 mix:23.3–63.8 rev/mg vs 17.8 rev/mg (FR 1.3–3.6) with S9 mix24.1–198.2 rev/mg vs 18.3 rev/mg (FR: 1.3–10.8). Comet assay tail moment: without S9 mix: 0.35–5.22 vs 0.34 (FR 0–15); with S9 mix: 0.57–2.71 vs 0.34 (FR: 1.7–7.9). Particulate 0.5–10 m Mutagenicity; without S9 mix: 0–41.2 vs 17.8 rev/mg (FR 0–2.31) with S9 mix:5 vs 18.3 rev/mg (FR 0–2.4). Comet assay tail moment: without S9 mix: 0.38–1.32 vs 0.34 (FR 1.1–3.9) With S9 mix 0.39–21.15 vs 0.34 (FR 1.14–62). Micronucleus test in Tradenscantia MN/100 tetrads 1.7–13 vs 5.2
Italy/Monarca et al. (2001)
Rubber industries. Company A (Netherlands) Company B (Sweden) Mixing and curing departments
Total suspended particulate matter (TSPM) Total Soluble Matter (TSM) Cyclohexane Soluble Matter (CSM) Company A: 2 locations within the mixing and curing department. Samples collected during 3 consecutive days Company B: 5 locations in mixing dept, 3 locations in curing dept Repeated 8 h samples within 1 week.
ND
Company A Mixing: TSPM 0.20–0.30mMg/m3 ; TSM 101–170 g/m3 , CSM 45–108 g/m3 Curing: TSPM 0.29–0.30mMg/m3 ; TSM 226–268 g/m3 , CSM 164–177 g/m3 Company B Mixing: TSPM 0.03–0.34 mg/m3 , TSM 9–44 g/m3 , CSM: 1–16 g/m3 ; Curing TSPM 0.04–0.23 mg/m3 ; TSM 25–81 g/m3 , CSM: 2–16 g/m3
Mutagenicity of airborne particulate samples. Salmonella t: YG 1021 nitroreductase overproducing, YG 1024 O-acetyltransferase overproducing YG1041, nitroreductase and O-acetyltransferase overproducing.
Company A Filter extracts Salmonella t YG1021: No mutagenic activity Salmonella t YG1024. combined filter extracts and fumes: mixing 721–989 rev/m3 ; curing 684–1292 rev/m3 . Salmonella t. YG1041: mixing 308–735 rev/m3 , curing 224–346 rev/m3 . Company B Filter extracts Salmonella t YG1021: No mutagenic activity Salmonella t. YG1041 mixing 3–48 rev/m3 , curing 6–139 rev/m3 .
Netherland Sweden/Vermeulen et al. (2000)
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Stationary samplers
Rubber industry, different types (no tire) weighing, mixing, calendering, compounding, extruding
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Table 1 Assessment of exposure to genotoxic mixture(s) in different rubber manufacturing industries and processes.
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Frequency ratio (FR) = mean value in tested samples/mean control value.
3.2.1. Urinary mutagenicity Six papers on urinary mutagenicity, using Ames test applied in Salmonella TA 98, TA 100 ed Escherichia coli WP2 are available in rubber manufacturing workers: 4 of them report significant increase associated with the exposure in the workplace. The most recent studies reporting positive results were carried out in general rubber goods production industries, while no recent study is available on tire manufacturing plants. In the former, significant associations with the exposure to mutagenic inhalable particulates, and contaminated surfaces were observed; highest urinary mutagenicity was detected in mixing and curing workers (Peters et al., 2008). Large interindividual variation was observed in the different groups due to the influence of lifestyle factors, such as environmental and mainstream tobacco smoke (Vermeulen et al., 2000a,b; Vermeulen et al., 2003). A statistical significant increase of revertants in S. typhimurium TA98 and E. coli WP2 uvrA after treatment with urine samples from workers in a tire manufacturing plant was observed with highest values in smokers (frequency ratio 5.9 vs 2.4) (Falck et al., 1980). Another mutagenicity study in a tire plant in Italy, including 73 workers and 23 controls, reported negative results in S. typhimurium TA 1535, TA 98, TA 100. A parallel analysis of raw materials used in workplace showed only a weak mutagenicity of a minor fraction (3/22) of the technical products tested (Crebelli et al., 1984, 1985). No difference in urine mutagenicity was claimed in a further study carried out in 19 rubber manufacturing workers from 4 rubber different factories: no data are reported (Moretti et al., 1996). Three more recent studies he in workers in rubber manufacturing industries in The Netherlands, showed increased urinary mutagenicity associated with inhalable dust exposure during the work-week compared with the week-end (Vermeulen et al., 2000a,b; Vermeulen et al., 2003; Peters et al., 2008).
a
Netherland/Vermeulen et al. (2001) TSPM samples 76/83 (95%) showed mutagenic activity: 19–1263 rev/m3 . highest effects in curing process: 43/104 (41.3%) Wipe samples mutagenic activity: 17–665 rev/cm2 Mutagenicity of TSPM extracts and surface contamination with Salmonella TA YG1041 TSPM 0.03–2.75 mg/m3 ND Rubber (various, not tire) industries. The study was conducted in 1997 9 companies: Different processes: mixing, pre-treating, molding, curing, finishing, shipping, engineering service, laboratory
8 h total suspended particulate matter (TSPM) in air 83 sampling sites 145 repeated samples 104 wipe samples
Country/Ref. Results (FR)a Statistical significance Type of test Exposure/substance/conc Individual samplers Stationary samplers Type of industry/processes
Table 1 (Continued)
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3.2.2. DNA adducts DNA adducts, were determined in urothelial cells and in peripheral blood lymphocytes in a number of studies carried out in rubber workers (Talaska et al., 2002). A first study did not reveal any increase of DNA adducts in peripheral lymphocytes of workers from the vulcanizing department of a rubber manufacturing company in Hungary when compared with controls associated with a low urinary 1-hydroxypyrene level (Schoket et al., 1999). Two further studies, conducted in different years in the same rubber manufacturing factories in The Netherlands, report DNA adducts in urothelial and blood cells (Vermeulen et al., 2002; Peters et al., 2008). A different DNA adduct pattern was observed in the two surrogate tissues with four specific DNA adducts reliably detected in both cell types. Total urothelial cell DNA adducts were related to urinary hydroxypyrene and mutagenicity with highest values found in workers engaged in compounding, mixing and curing departments. No correlation between bladder and blood cell DNA adduct levels was found, suggesting the involvement of different chemicals and/or different metabolic pathways in bladder and blood cells. 3.2.3. DNA damage DNA damage, measured by Comet assay in peripheral lymphocytes, was found in workers employed in rubber manufacturing factories in different countries. The use of different experimental protocols and of different DNA damage parameters prevent any comparison among the studies. A statistical significant increase of DNA damage, as single strand breaks and oxidatively damaged DNA bases, was observed in a small group of workers employed in a rubber tire factory in Czech Republic in two different samplings at interval of 1 year. Significant correlation between values of DNA damage and micronuclei
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
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Identification
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Search strategy (19/10/2012): Terms: (genotoxic, DNA damage, chromosomal damage, chromosomal aberration, micronucleus(i), DNA strand break, Ames, mutation, HPRT, Comet) AND rubber or tyre industry. Studies retrieved (n = 87)
Included
Eligibilty
Screening
Studies selected
5
Studies excluded (n = 46) • Experimental studies on single compounds • Reviews
(n=41)
Abstracts excluded:9 3 Reviews reporting the same data 6 Not in english, not relevant
Studies screened (n = 41)
Full-text articles assessed for eligibility (n= 31)
Correlated studies( n = 3) • Experimental in vitro study on combination of chemical compounds relevant in rubber industry (Cemeli et al Mut Res 664; 69-76, 2009) • Data on inhalable dust in rubber industry in Europe (de Vocht et al Occup Env Med, 65, 384-391, 2008)
Articles on genotoxicity biomonitoring studies included in the review (n= 28 (17 reported in IARC monograph)
• Exposure to PAH in rubber workers (Talaska et al Toxicology Lett, 213, 45-48, 2012)
Fig. 1. Study selection flow chart.
frequency was reported for both the samplings (Somorovská et al., 1999). A study, with some methodological shortcomings, carried out in a small group of workers in rubber manufacturing industries in Italy, shows a non statistically significant increase of the DNA migration length measured by Comet assay (Moretti et al., 1996). A decrease of DNA damage was described in workers from a Portuguese tire factory showing higher levels of urinary thioethers compared with a group of controls (Laffon et al., 2006). This result could be attributed to the crosslinking effect induced by some compounds in the exposure mixtures of rubber production (Cemeli et al., 2009). Finally a large study carried out in China, involving 371 workers in a factory processing natural rubber to manufacture tires, report a weak, but statistical significant increase of DNA strand breaks, expressed as tail moment, in rubber workers compared with managerial workers. The highest values were observed in mixers exposed in poor hygienic conditions since the toluene concentrations exceeded the Chinese maximum allowable concentration by four folds (Zhu et al., 2000). 3.2.4. Chromosomal damage The large majority of biomonitoring studies on genotoxic risk in rubber manufacturing industry refers data on chromosomal damage, including chromosomal aberrations, micronuclei frequency and SCE: 6/10 studies report positive results. All the studies except one (Musak et al., 2008) are of small size and seven of them were carried out before 2000. The extent of increase is higher for the frequency of chromosomal aberrations (frequency ratio range 1.5–5.0) compared to micronuclei frequency (F.R. 1.66–3.0) and SCE (F.R.1.27–1.61). Two studies carried out in Poland in small groups of vulcanizers from a tire factory report borderline increase of chromosomal aberrations and SCE associated with a decrease of the proliferation index (Sasiadek, 1992, 1993). A further study in a tire factory in the Czech Republic, using as a raw material styrene-butadiene rubber shows significant increase of chromosomal aberrations and
micronuclei frequency associated with DNA single strand breaks in peripheral lymphocytes (Somorovská et al., 1999). The only statistically powerful study was carried out in 177 tire plant workers in the Slovak republic and shows a borderline increase of chromosomal aberrations with high interindividual variability associated with genetic polymorphism in genes encoding biotransformation DNA repair enzymes (Musak et al., 2008). A recent study reports a significant reduction in telomere length, as a marker of chromosomal instability, in a group of 157 workers in a rubber manufacturing industry in Sweden correlated with increased exposure to N-nitrosamines in air, based on individual measurements or on work task (Li et al., 2011). The large interindividual variability observed in exposed subjects as well as in controls, potentially associated with the large number of confounding factors described for this biomarker, such as age, smoke, diet, genetic susceptibility, prevents a clear interpretation of the study. 3.2.5. Gene mutations A number of studies were carried out between 1992 and 1999 on workers from synthetic rubber manufacturing plants using as raw materials polymerized polybutadiene rubber and styrenebutadiene copolymers in Texas to evaluate the frequency of mutant lymphocytes using HPRT mutant lymphocyte assay (Table 2). Statistically significant increase in mutant frequency were observed in workers exposed to butadiene at concentration higher that 150 ppb (Abdel-Rahman et al., 2001). The most recent study (Wickliffe et al., 2009) where the large majority of exposures were below of method detection limit did not show any increase of the frequency of mutant lymphocytes. 4. Discussion The aim of our study was to critically review the available scientific literature in order to identify potential genotoxic chemicals or manufacturing processes in the rubber manufacturing industry
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Type of test
Results (FR)a Statistical significance
Country/Ref.
Tire manufacturing Different processes
20/16
NA
Urinary mutagenicity: Salmonella t TA98; E. coli WP2 uvrA with S9
Mutagenic activity Salmonella t TA98: Smokers 717/298 (FR: 2.4) Non-smokers 149/25 (FR: 5.9) E. coli: Smokers 727/13.2 (FR: 55) Non-smokers 501/34.7 (FR: 14.7)
Finland/Falk et al. (1980)
Rubber industry 2 Rubber plants Factory A: heavy and light tires weighing and mixing, tire building vulcanizing Factory B: various technical rubber products weighing, mixing, cleaning, vulcanizing,
Factory A: 28/17 Factory B: 27/18
NA Factory A: 1.5–11.7 y Factory B: 1–14.5 y
CA and SCE in peripheral lymphocytes
Factory A Smokers: CA3.1/4.8 (NS) SCE 13.1/11.5 (NS) Non smokers: CA 2.6/2.4 (NS) SCE 11.2/10.4 (NS) Factory B Smokers: CA 5.2/5.7 (NS) SCE 13/12.6 (NS) Non smokers: CA 3.8/3.7 (NS) SCE: 12.2/10.7 (NS)
Finland/Sorsa et al. (1983)
Tire manufacturing vulcanizing
34/15
total airborne particulate (area samplers) 0.5–3.4 mg/m3 8.2 y
CA and SCE in peripheral lymphocytes
CA: 1.9 ± 1.4/2.1 ± 1.5 (NS) SCE 5.2 ± 1.3/5.2 ± 0.7 (NS)
Italy/Degrassi et al. (1984)
Tire manufacturing mixing, weighing, compounding, extruding, calendering, tire building, vulcanizing
72/23
total airborne particulate (area samplers) 0.7–2.73 mg/m3 7–12 y
urinary mutagenicity: Salmonella t TA98, TA100, TA1535 with S9
No effect of exposure Mutagenic samples (smokers/non smokers): 20/56 v 3/34 p < 0.001
Italy/Crebelli et al. (1984)
Tire manufacturing Styrene-butadiene rubber Vulcanizing
21/14
Styrene, butadiene, PAH, nitrosamine, carbon oxide and sulfur dioxide 2–35 y
CA and SCE in peripheral lymphocytes
CA 2.2 ± 1.06/0.9 ± 1 (FR:2.4) p < 0.01 SCE 16.1 ± 3.5/10 ± 1.5 (FR:1.61) p < 0.001
Poland/Sasiadek (1992)
Tire manufacturing Styrene-butadiene rubber Vulcanizing
26/25
Styrene, butadiene, PAH, nitrosamine, benzene, toluene 0.5–30 y
SCE in peripheral blood
SCE 13.3 ± 2.9/9.8 ± 1.8 (FR: 1.34) NS
Poland/Sasiadek (1993)
Rubber industry 4 factories: (A) ethylene-propylene rubber; (B) and (C) acrylonitrile-butadiene rubber; (D) styrene-butadiene rubber. Different processes
19/20 Selected according to nitrosamine exposure
N-nitroso-dimethylamine, N-nitroso-morpholine, PAH. > 10 y
Comet assay MN test and SCE in peripheral blood lymphocytes
Comet assay: MD (migration distance) 43.08 ± 12.49/38.92 ± 8.06 NS Micronucleus test MN/1000 BN cells: 22.84 ± 15.82/13.74 ± 4.42 (FR: 1.66) NS SCE SCE/metaphase 6.06 ± 1.15/5.51 ± 0.84 (FR: 1.19) NS
Italy Moretti et al. (1996)
G Model
Exposure substances/concentration/duration
C. Bolognesi, A. Moretto / Toxicology Letters xxx (2013) xxx–xxx
N. exposed subjects/controls
ARTICLE IN PRESS
Type of industry/Process
TOXLET-8542; No. of Pages 11
6
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Table 2 Biomonitoring studies in groups of workers from rubber manufacturing industries: genotoxic effects.
Exposure substances/concentration/duration
Type of test
Results (FR)a Statistical significance
Country/Ref.
Tire manufacturing Styrene-butadiene rubber Different processes
27/22 industry controls and 22 lab controls
Styrene, toluene, butadiene, PAHs, alkanes and alkenes. B(a)P = 3.65 ng/m3
Comet assay CA and MN test in peripheral lymphocytes
Comet assay % DNA in tail: 1◦ sampling 1996: 33%; 2◦ sampling 1997: 45% vs 13%(industry controls) and 22% (lab controls) (FR: 2.5–3.4) p < 0.00001. Micronucleus test MN/2000 cells 6.5 vs 2.1 (FR: 3) Chrom aber % 1 vs 0.2 (FR: 5) p < 0.00001
Czech Rep./Somorovska et al. (1999)
Rubber industry vulcanizing
61/76
PAH, aromatic amines, N-nitroso compounds, organic solvents
1-OH-pyrene in urine. DNA adducts (32 P postlabelling) in peripheral blood lymphocytes. GPA assay
mol 1-OH-PY/mol creatinine: 0.22 ± 0.18/0.32 ± 0.48 NS DNA adducts/108 nucleotides: 5.6 ± 2.6/6.4 ± 3.3 NS GPA variant cell frequency: 8.7 ± 5.4/8.0 ± 6.9 NS
Hungary/Schoket et al. (1999)
Rubber industry different processes
29/87
Aromatic solvents, dust, tar and lubrificating oils 3–20 y
CA, premature centromere division (PCD) and aneuploidy in peripheral lymphocytes.
CA: 3.38 ± 0.26/1.60 ± 0.62 (2.1) p < 0.01 PCD < 3 Chr: 11.45 ± 1.43/1.57 ± 0.44 (7.29) p < 0.01 PCD > 3Chr: 6 ± 1.18/0.32 ± 0.10 (18.7) p < 0.01 Aneuploidy: 5.64 ± 0.44/6.20 ± 0.43 NS
Hungary/Major et al. (1999)
Tire manufacturing finishing, maintainance calendering, vulcanizing mixing
281/90
Dust, toluene, xilene, gasoline, H2 S, S02
Comet assay in peripheral lymphocytes
Comet assay Tail moment (m): All workers: 1.77 (1.64–1.90)/1.52 (1.36–1.71) NS Mixers: 2.54 (1.95–3.31)/1.52 (1.67) p < 0.002
China/Zhu et al. (2000)
Tire manufacturing Different processes
32/32
No data 1–34 y
Urinary thioethers Comet assay, SCE and MN in peripheral lymphocytes
Urinary thioethers mM 0.41 ± 0.05/0.24 ± 0.02 (FR1.7) p < 0.01 Comet assay: Tail moment (m) 44.72 ± 0.66/48.25 ± 0.71 NS SCE: 4.35 ± 0.20/4.38 ± 0.17NS MN: 2.34/1.84 NS
Portugal/Laffon et al. (2006)
Tire manufacturing butadiene rubber mixing, extruding, calendering, tire building, tube repair
177/172
Personal samplers Mixing dept BD 2.4–2.8 mg/m3 Styrene (8.1–13.2 mg/m3 ) and sulphur dioxide (0.6–1 mg/m3 ); Pressing dept: BD (0–5.8 mg/m3 ) and sulphur dioxide (0.6–1 mg/m3 ) 19.4 ± 8.5 y
CA in peripheral lymphocytes
CA/100 metaphases: 2.5 ± 1.8 (exposed smokers) vs 1.7 ± 1.2 (non smoker controls) (1.47)
Slovak rep/Musak et al. (2008)
Rubber industries 9 companies Different processes: compounding and mixing, pretreating, molding, curing, finishing
105 subjects employed full time in rubber manufacturing industries 38 smokers/67 non smokers
ND
Urinary mutagenicity with S. typhimurium strain YG1041 with S9 on Sunday and week days. Adjustement for cotinine
Urinary mutagenicity non smokers/smokers Sunday: 4416/4664 rev/g creat Weekday 5913/6470 rev/g creat
The Netherlands/Vermeulen et al. (2002)
C. Bolognesi, A. Moretto / Toxicology Letters xxx (2013) xxx–xxx
N. exposed subjects/controls
ARTICLE IN PRESS
Type of industry/Process
G Model
TOXLET-8542; No. of Pages 11
Please cite this article in press as: Bolognesi, C., Moretto, A., Genotoxic risk in rubber manufacturing industry: A systematic review. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.11.013
Table 2 (Continued)
7
Type of test
Results (FR)a Statistical significance
Country/Ref.
9 companies Different processes: compounding and mixing, pretreating, molding, curing, finishing
The study was conducted in 1997. 52 subjects 3 exposure categoriesb : 18 Low TSPM Low SMEL; 18 High TSPM Low SMEL; 16 HH High TSPM high SMEL
Total Suspended Particulate Matter (TSPM): high ≥ 210 rev/m3 ; low ≤ 210 rev/m3 . Surface Mutagenic Exposure Level (SMEL): high ≥ 25 rev/cm3 ; low ≤ 25 rev/cm2
DNA adducts (32P postlabelling) in exfoliated bladder cells
11 possible DNA adducts identified. 46 (88%) samples had positive results for 3 main adducts; adduct1 in 41 samples, adduct 2 in 13 samples; adduct 3 in 29 samples. The dept mixing and curing showed the highest level of DNA adducts and % of positive samples.
The Netherlands/Vermeulen et al. (2002)
Rubber industries Same companies as in Vermeulen et al. (2002) Different processes
The study was conducted in 1997. 116 rubber manufacturing workers.
Hydroxypirene in urine (116 subjects). Urinary mutagenicity (104 subjects) DNA adducts by P32 postlabelling in: urothelial cells from 24 h urine (52 subjects) and in peripheral mononuclear cells (PBMC)(48 subjects).
Results in non-smokers: Hydroxypyrene 0.17–0.20 (weekdays)/0.12 (Sunday) mol/mol creatinine(curing 0.31 mol in weekdays) Urinary mutagenicity: rev/g creatinine weekday/Sunday: compounding and mixing: 10511/6522 (curing 11527/10447 p < 0.05). 11 possible DNA adducts were identified in urothelial cells: 38 (73%) samples had positive results for 4 main adducts: adduct 1 in 30 (58%); adduct 2 in 12 (23%), adduct 3 in 23 (44%); adduct 11 in 2 (4%). 4 adducts were identified in PBMC: adduct A in 5 (10%); adduct B in 38 (79%) adduct C in 3 (6%); adduct D in 23 (48%).
The Netherlands/Peters et al. (2008)
Rubber industry Styrene-butadiene polymer plant
Pilot study carried out in 1991. 8 subjects high expo; 5 subjects low expo, 6 controls
Exposure to BD by personal samplers: 150 (mean 2244.2 ± 749.1); low expo < 150 (mean18.4 ± 5.5)
HPRT variants
hprt Vf per million cells: high expo: 9.25 ± 1.56;/low expo:4.82 ± 0.73 high vs low p = 0.02
Texas USA/Abdel-Rahman et al. (2001)
Rubber industry Styrene-butadiene polymer plant
The study was carried out in 1999. 30 subjects
Exposure to BD (ppb): from non detectable to 1683.5 (mean 93.5) 1–39 y
HPRT variants
no relationship between BD exposure and HPRT mutants. A single subject with the highest expo estimate has high HPRTvf
Texas USA/Wickliffe et al. (2009)
Rubber industries Different processes
157 subjects
Air measurement for nitrosamines: 0.07–35.5 mg/m3 Urinary markers: 1-HP: 0.0020–0.85 mmol/mol creatinine TTCA: 1.7–690 mmol/mol creatinine toluidine: orto 0.025–108 ng/ml; meta 0.025–108 ng/ml; para 0.025–4.7 ng/ml
telomere lenght analysis (PCR)
TL: 0.16–1.3 nitrosamine levels are highly correlated with the telomere lenght
Li et al. (2011)
C. Bolognesi, A. Moretto / Toxicology Letters xxx (2013) xxx–xxx
Type of industry/Process
a
Frequency ratio (FR) = mean value in tested samples/mean control value. The four a priori exposure groups were defined by: (1) dermal exposure, based on the median levels of mutagenic activity detected on likely skin contact surfaces (high (>25 rev/cm2 ), low (210 rev/m3 ), low (
