Assessing cancer hazards of bitumen emissions – a case study for complex petroleum substances

Abstract

When assessing cancer hazard and risk associated with a complex petroleum substance, like bitumen emissions, there are often conflicting results related to human, animal and mechanistic studies. Validation of the complex composition to assure that it matches real-world exposures and control of confounders are pivotal factors in study design to allow the necessary read-across during assessments. Several key studies on bitumen emissions in two-year dermal cancer assays reported variable outcomes ranging from high cancer incidence to no cancer incidence. Here, we synthesize findings from published studies to explain the differences and discuss critical factors in cancer hazard evaluation for complex petroleum substances. Using these critical factors, we reviewed relevant human genetic toxicity, mammalian toxicity and mechanistic studies with bitumen to understand the divergence in results. We assess the most reliable and scientifically supported information on the potential carcinogenic hazards of bitumen emissions and comment on quality and completeness of data. Human hazard data are typically considered highest priority because they eliminate the need for interspecies extrapolation and reduce the range of high -to low-dose extrapolation during the risk assessment process. Finally, two well-conducted comprehensive animal studies are discussed that have well-defined test material, exposure concentration and composition representative of worker exposure, evidence of systemic uptake, no confounding exposures and provide consistency across all elements within both studies. Studies that allow effective read-across from human, animal and mechanistic components, control for confounders and are well-validated analytically against workplace exposures, provide the strongest evidence base for evaluating cancer hazard.

Keywords:

• Asphalt
• fumes
• human
• mammalian
• dermal
• inhalation
• mechanistic
• mutagenicity
• risk assessment
• genetic toxicity
• read-across

Introduction

Evaluating the potential health effects caused by simultaneous exposure to many constituents in a complex petroleum substance is of considerable importance, but fraught with challenges, for example, ensuring that experimental exposures are representative of actual exposure in the workplace. We examine experimental studies with bitumen and bitumen emissions as examples of this challenge.

Several organizations have reviewed the health aspects of bitumen and bitumen emissions including International Agency for Research On Cancer (IARC), National Institute for Occupational Safety and Health (NIOSH), German Committee for the Determination of Occupational Exposure Limits (MAK Commission), American Conference of Governmental Industrial Hygienists, Inc. (ACGIH), Internal Program on Chemical Safety (IPCS) and Institut National de la Recherche Scientifique or National Institute of Scientific Research (INRS). It is especially challenging to assess the carcinogenic potential of these materials due to the complexity of their chemical composition and the possible presence of small amounts (parts per million) of potentially carcinogenic/genotoxic constituents. Some organizations, notably IARC, have attempted to incorporate mechanistic and genotoxicity data into their hazard evaluation. However, the lack of a standardized approach to evaluating mechanistic data, and its significance for cancer hazard evaluation, has resulted in some criticism and recommendations for a more systematic and transparent approach (Goodman and Lynch 2017).

Results of mammalian studies vary broadly from benign (Fuhst et al. 2007; Clark et al. 2011) to carcinogenic (Sivak et al. 1997). With studies showing such vastly different outcomes, it is often difficult to reach sound scientific conclusions on the health effects of a complex substance without an in-depth understanding of the material, how it behaves under different conditions and what factors can influence exposure.

Here, we look at how carcinogenicity and genotoxicity data on complex substances could be evaluated and reviewed to better explain the outcomes in the face of apparently conflicting data. We began with published large-scale hazard reviews, considered the individual papers cited within those reviews and expanded our assessment to include new additions to the literature.

When assessing hazard, it is important to look for research that includes human, animal and mechanistic studies conducted in well-validated, linked investigations. This allows read-across from one approach to another to better understand the mechanisms of health outcomes. It is important in assessing such studies that proper analytical validation of test material composition against workplace exposure samples is conducted. This ensures that hazard evaluations studies reflect true exposure potential. Studies which lack validation need to be reviewed separately to determine how the composition and biological activity of complex substances can change with end use, such as temperature or environmental conditions.

Our review is focused on experimental data and therefore excludes review of the extensive dataset of epidemiological studies; primary outcomes have been published elsewhere (Boffetta et al. 2003; Rhomberg et al. 2015). Boffetta et al. concluded that it was not possible to confirm a causal link between exposure to bitumen emissions and cancer of the lung, head, and neck. Furthermore, no association was identified between exposure to bitumen emissions and any other neoplasm. Rhomberg et al. (2015) have used various methods illustrating that cancer risks to roofers from dermal and inhalation exposure to built-up roofing asphalt (BURA) are within acceptable risk thresholds, concluding that the risk is extremely low.

Complex petroleum substances

Bitumen is a complex substance, derived from crude oil, that contains tens of thousands of different individual constituents, many of which are likely to present different physicochemical and biological properties (Asphalt Institute, Eurobitume 2015). When bitumen is heated to liquefy the product, it can release low parts per million (<10 ppm) levels of volatile and semivolatile compounds into the atmosphere in the form of aerosols, gases and particulates, to which workers can be exposed. These compounds are initially trapped in the bitumen because of the incomplete distillation of crude oils. Where bitumen is cut-back or fluxed with lower boiling hydrocarbons, additional volatile and semivolatile compounds may be released upon heating.

The product, bitumen, is used in many ways due to its engineering properties for building roads, waterproofing roofs and in hydraulic applications such as pond liners. Bitumen is a non-distillable residuum obtained from the distillation of suitable crude oils (Asphalt Institute, Eurobitume 2015). The distillation process normally involves atmospheric distillation followed by either vacuum distillation or steam distillation. Additional processing, such as air oxidation, solvent stripping or blending of petroleum residua of different stiffness characteristics, may be needed to form a material whose physical properties meet the technical requirements for commercial applications.

A significant amount of research has been conducted to chemically characterize bitumen emissions under normal application conditions (NIOSH 2000; Kriech et al. 2002). Emissions comprise predominantly straight chain hydrocarbons with lesser amounts of cycloalkanes (approximately 70% aliphatic/30% aromatic). The aromatic portion includes alkyl benzenes, polar and semipolar compounds. Much of the polycyclic aromatic compound (PAC) composition is in the form of alkylated species (primarily alkyl-naphthalenes, alkyl-fluorenes and alkyl-anthracenes) and includes sulfur heterocyclic materials (primarily alkyl benzothiophene and alkyl-dibenzothiophenes). The most prominent of the parent, unalkylated PACs detected in workplace exposures for paving and roofing workers include acenaphthene, anthracene, fluoranthene, fluorene, naphthalene, phenanthrene and pyrene (Kriech et al. 2007; Cavallari et al. 2012a). Normal paving worker exposures contain only trace levels of parent unalkylated PACs, primarily of the 2- and 3-ring variety. Some 4-ring PACs have been reported in fume condensates generated to mimic paving worker exposures (pyrene and triphenylene (Clark et al. 2011) and eight 4–6 ring PACs have been reported in fume condensates generated to mimic roofing worker exposures (Clark et al. 2011), all at trace levels (<0.08–8 ppm) except for pyrene, a noncarcinogenic 4-ring PAC, at ≤33 ppm.

Temperature plays a significant role in determining the amount and composition of bitumen emissions. As the temperature of the bitumen increases, so does the concentration of emissions released as a logarithmic function (Asphalt Institute, Eurobitume 2015). Also, with increased temperature, the proportion of higher molecular weight compounds increases, which can lead to the increased presence of 4–6 ring PACs. Although minor data variations occur, the emissions generated from air rectified (partially oxidized) and straight-run vacuum distilled bitumen, appear similar in composition and physical properties (Asphalt Institute, Eurobitume 2015) when heated to the same temperature. The chemical composition of bitumen emissions is dependent on a wide variety of other variables such as crude oil source, processing, handling, oxidation (Trumbore et al. 2015), exposure levels and distance from the emission source.

In 1981, Thayer et al. conducted a two-year mouse dermal cancer assay on condensates of built up roofing asphalt (BURA) emissions, derived from oxidized bitumen. Tumors were observed with fume condensates generated at 232 °C (450 °F) and 316 °C (601 °F) in a laboratory. The higher the temperature of preparation of the bitumen emission condensates, the greater tumorigenic activity observed. Kriech et al. (2007) showed that these emissions were not the same as worker exposures and that the high temperature and continuous agitation contributed to significantly higher levels of 4–6 ring PACs than seen in worker exposures. A follow-up mouse study was conducted in 1989 by Sivak et al. on five fractions of this same fume condensate – with increasing polarity A, B, C, D and E. The carcinogenic activity was limited to Fractions B and C, which represented only 10.2% of the total fume condensate. These studies provided valuable insight into the complex composition and helped identify potential causative components of the potential carcinogenicity of bitumen emissions.

For road paving, conventional hot-mix asphalt is a mixture of bitumen and mineral aggregate (stone) materials that is typically produced and applied at temperatures in the range of 140–160 °C. In Europe, the term is synonymous with asphalt, whereas the petroleum portion (the binder) is referred to as bitumen. Warm-mix asphalts have also been developed to reduce the energy required to build roads and to reduce worker exposures. Warm-mix asphalts are applied at temperatures typically 10–40 °C lower than conventional rolled or dense-graded asphalt. Use of diesel oil as a releasing/cleaning agent is widespread in the hot mix asphalt paving industry and can contribute significantly to worker hydrocarbon exposures, including PACs, which may also be present in bitumen emissions (Cavallari et al. 2012a, 2012b; Osborn et al. 2013).

Mastic asphalt is an asphalt mixture in which the volume of filler (normally finely crushed rock) and bitumen binder exceeds the volume of remaining voids producing a stiffer material requiring higher mixture temperatures of >200 °C. Mastic asphalt is only used in Europe (mainly in Germany and France) and contains aggregates <2 mm in size. The mastic industry represents only 1.1% of the worldwide asphalt market (Asphalt Institute, Eurobitume 2015; IMAA 2013). Mastic asphalt pavement systems include a leveling course called pulver asphalt placed over a concrete base. Once the pulver layer is laid, the crew pours a semi-fluid, high temperature (250 °C) mastic layer. This is troweled down and then pre-coated stone chips are spread before rolling. Coal tar contamination from the subbase (pulver layer) has been shown to be a significant confounder in evaluations of the hazard from mastic asphalt use (Raulf-Heimsoth et al. 2008; Blackburn et al. 1999).

Roofing products containing bitumen include bitumen shingles (applied at ambient temperatures), rolled roofing (often torch applied), Built-Up Roofing Asphalt (BURA), which is hot mopped at temperatures of 200–250 °C (392–482 °F) and has kettles that hold bitumen that can be heated up to 288 °C (550 °F), polymer-modified membranes, saturated felt underlayment and roofing adhesives. These are the highest temperature roofing application uses for bitumen. Hot-applied bitumen is a small and shrinking part of the roofing market. Exposures for the other bitumen roofing products are far lower (torch jobs) to negligible (shingles). Studies of hot applied bitumen applications are of little meaning for most of the roofing industry. Manual removal of old roofing materials (“roofing tear-off”) containing coal tar is one of the primary confounders involved in assessing workplace roofing worker exposures and is associated with a sixfold increase in relation to asphalt emission exposure alone (McClean et al. 2007a).

Critical factors in cancer hazard evaluation for complex petroleum substances

Petroleum-derived substances, such as bitumen and bitumen emissions, can contain many thousands of individual hydrocarbon species, many of which are likely to present different physico-chemical, toxicological and environmental properties. The properties and exposure profile of the complex petroleum substance will depend upon the nature and amount of the chemical species present and the operational/environmental conditions. Release of individual constituents from the complex substance and hence potential for occupational and environmental exposure will be determined by the overall properties of the material and conditions of use.

This complexity of composition and properties presents a significant challenge when designing toxicological studies or evaluating existing data, to determine the carcinogenic hazards of complex petroleum substances. Review of the experimental methodology used in the studies is therefore critically important to ensure that experimental results, to the greatest extent possible, are scientifically robust and that conclusions reached are based on actual hazard properties, rather than hazard predictions based solely on the presence of individual hazardous constituents. Consideration of the following factors provides a structured approach to the design of toxicological studies and the evaluation of both individual studies and the overall data set that contributes to the final hazard conclusion.

Data quality

An analytical approach for assessing the quality of data from all experimental and occupational/environmental health effect studies is required to ensure that conclusions reached are solely based on sound and reliable scientific evidence. The approach should include assessment of data “Relevance”, “Reliability” and “Adequacy” for each study.

Under Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), the regulatory program for registration of chemicals in Europe a systematic approach (Klimisch et al. 1997) is used to evaluate the quality of toxicological and ecotoxicological data for hazard and risk assessment. Klimisch is a widely used and accepted method for assessing the quality and adequacy of individual studies. It is recognized that human data do not readily fit the Klimisch scheme, so a modified approach has been proposed (Money et al. 2013). Both approaches result in individual studies being assigned to one of four categories (Table 1). Only studies that meet the criteria for Category 1 (Reliable without restrictions) or 2 (Reliable with restrictions) should be considered adequate for the purposes of reliable hazard and risk assessment (Klimisch et al. 1997).

Table 1. The four categories of review (adapted from Money et al. 2013).

Category 1 – Reliable without restriction Toxicology/ecotoxicology data  This includes studies or data from the literature or reports that were carried out or generated according to generally valid and/or internationally accepted testing guidelines (preferably performed according to GLP) or in which the test parameters documented are based on a specific (national) testing guideline (preferably performed according to GLP) or in which all parameters described are closely related/comparable to a guideline method Human Data (chronic, nonspecific outcomes) Applies to human data (HD) where the study design was appropriate to the research question under consideration and was executed with high attention to the details of study protocols and procedures. Specifically: (1) subjects were selected to represent the appropriate exposure distributions of persons or person time at risk, with emphasis on measuring and reporting response parameters, and adequate recruitment and follow-up procedures in place to maximize participation and reduce loss to follow up with cohort studies; (2) exposure assessment was made independent of outcome, with as little measurement error as possible (relative to between subject variation), using well-established methods appropriate to the study design; quantitative, validated, individual-level data will be essential for exposure–response assessment; (3) outcome data were collected independent of exposure status and ascertained with equal probability for both cases and noncases (or controls in a case–control study), using standardized data collection procedures (e.g. registries); (4) the possibility for serious biases have been reduced by design, controlled through statistical adjustment and/or quantified through sensitivity analyzes; (5) the sample size/exposure range was sufficient to study the question under investigation so that effect estimates are not constrained by high imprecision, and (6) these data were analyzed comprehensively, using appropriate statistical techniques to address the research questions and model assumptions, if applicable, were met. In addition, the methodology and results were comprehensively and transparently reported according to relevant guidelines e.g. the STROBE guidelines for observational data (von Elm et al. 2007) Human data (acute or specific outcomes) Similar principles to those applying for chronic or nonspecific outcomes also apply to this type of study, although the focus of the review should lie more with how well exposure has been characterized and the disease/effect outcome is defined, (e.g. no restrictions: precise exposures measured on an individual basis, outcome verified via objective criteria, etc.) and less emphasis on whether there are minor confounders and/or biases. However, in the case of very rare outcomes, reports of cases among clearly exposed population may be interpretable without restrictions although such reports would be categorized as “‘not reliable” in the case of chronic, nonspecific outcomes

Category 2 – Reliable with restriction Toxicology/ecotoxicology data  This includes studies or data from the literature, reports (mostly not performed according to GLP), in which the test parameters documented do not totally comply with the specific testing guideline but are sufficient to accept these data or in which investigations are described which cannot be subsumed under a testing guideline, but which are nevertheless well documented and scientifically acceptable. Similar principles to those applying for chronic or nonspecific outcomes also apply to this type of study, although the focus of the review should lie more with how well exposure has been characterized and the disease/effect outcome is defined, (e.g. no restrictions: precise exposures measured on an individual basis, outcome verified via objective criteria, etc.) and less emphasis on whether there are minor confounders and/or biases. However, in the case of very rare outcomes, reports of cases among clearly exposed population may be interpretable without restrictions although such reports would be categorized as “not reliable” in the case of chronic, nonspecific outcomes Human Data (chronic, nonspecific outcomes) Applies to HD which possess most of the elements of a “Type 1” quality study, but where their overall quality has been compromised due to minor, but obvious, methodological limitations. Examples of such limitations include well-designed and well-conducted studies, but where there are limited measurement data available to validate estimated individual-level exposure data, some residual confounding is possible or there is some imprecision because of a small sample size or low exposure range. Alternatively, the study design may not be optimal, forexample, a cross-sectional design that does not allow inferences to be made about the time order of events. Another example would be a study in which subject selection procedures and/or postentry loss to follow up introduces the possibility of selection bias. Human data (acute or specific outcomes) Likely to apply to HD which possess most of the elements of a “Type 1” study, but where some restrictions on interpretation are appropriate, for example, individual-level exposure data are lacking; effects derive from self-reported outcomes, etc. As in the case of type-1 studies, some studies with serious methodological limitations may provide reliable information for an acute or specific outcome. It is also possible for the same study with specific and nonspecific outcomes to have a different Klimisch scores for these outcomes.

Category 3 – not reliable Toxicology/ecotoxicology data  This includes studies or data from the literature/reports in which there are interferences between the measuring system and the test substance or in which organisms/test systems were used which are not relevant in relation to the exposure (e.g. nonphysiologic pathways of application) or which were carried out or generated according to a method which is not acceptable, the documentation of which is not sufficient for an assessment and which is not convincing for an expert judgment Human data These studies fail to meet one or more of the most basic standards necessary to interpret epidemiologic research, such as appropriate study design for the research question of interest or adequate selection of study subjects. Nevertheless, many such studies will be well-conducted investigations and be useful for the purpose intended, for example, hypothesis generation, but their design renders them uninformative for hazard identification or risk assessment. Examples of such studies include ecological studies and linkage studies using census job titles as a surrogate for exposure. Studies classified as not reliable will often have serious methodological flaws that make the results uninterpretable regarding a causal association, particularly with respect to chronic, nonspecific outcomes

Category 4 – not assignable This includes studies or data from the literature, which do not give sufficient experimental details and which are only listed in short abstracts or secondary literature (books, reviews, etc.)

Test material composition

The complex composition of the “bulk” petroleum substance will differ significantly from the emissions to which workers and others are likely to be exposed upon heating bitumen (Asphalt Institute, Eurobitume 2015). The individual hydrocarbon constituents present in the substance each have specific and different physical–chemical properties, which determine its behavior and availability for exposure under a given set of conditions. It is important therefore to understand the nature and the amounts of individual constituents released under realistic, operational and environmental conditions. Analytical characterization of the test material used in experimental studies is critical to ensure that it is representative of the material workers and others may be exposed to under normal working conditions.

Experimental exposure conditions

An understanding of how complex petroleum substances are used/handled, and how operational conditions/environmental conditions can affect release of, and exposure to constituents, is a key consideration. This will help determine the most appropriate route of exposure and the nature of the test material. It is also important to “validate” the composition of the test material used against collected occupational “field” samples, to ensure the test material reflects human exposure under realistic conditions.

Evidence of exposure and uptake

When evaluating data from experimental animal studies or human occupational/environmental health studies, it is important to ascertain, either directly or indirectly, whether exposure has resulted in systemic uptake and exposure of the target cells. This can be confirmed with toxicokinetic data, if available, or indirectly by methods such as the presence of urinary metabolites or the identification of DNA adducts such as in white blood cells of workers or animals, and in skin, nasal and lung tissue samples in animal studies.

Data consistency

For some petroleum substances, an extensive set of health effects data are available, particularly for genotoxicity and carcinogenicity endpoints. Different studies can provide conflicting results, so careful evaluation is needed to determine whether factors such as use of unrepresentative test materials, different operational/environmental conditions, confounding exposures or experimental design bias can help explain conflicting or contradictory results.

Confounding exposures

In laboratory studies, it is possible to control the composition of exposure and how much the organism is exposed to. Unfortunately, in health effect studies of complex petroleum substances in exposed populations, possible confounding exposures to occupational or environmental hydrocarbons from other sources occur frequently and have the potential to influence study outcomes. Strategies to eliminate or control for possible confounding exposures are critically important in both study design and data analysis.

With these critical factors guiding the path forward, we discuss human toxicology studies, genetic toxicity, validated animal cancer studies, mechanisms and other human studies relevant to bitumen emissions.

Bitumen human genetic studies

Human studies investigating the genotoxic potential of exposure to bitumen emissions have been reviewed and assigned modified Klimish scores. Criteria to identify studies suitable for hazard assessment included characterization of bitumen/asphalt samples, exposure levels and duration of exposure, adequate group size of exposed subjects and unexposed control populations, background data on subjects (e.g. work experience, health, smoking, alcohol consumption), workplace monitoring data and urinary metabolites and confounding variables (e.g. diesel oil, diesel exhaust, coal tar pitch). In general, all studies included were representative of workplace environment. Sample size ranged from 20 to 60 subjects with concurrent controls, except for the extensive and much larger German Human Bitumen Study (2001–2008), which included more than 300 subjects. Details about the bitumen samples used were often limited to general descriptive terms without compositional characterization, but the studies typically included air sampling or personal exposure monitoring of total or individual PACs and urinary 1-OH pyrene content, or other biomarkers (e.g. hydroxylated phenanthrenes) used to quantify and characterize worker exposure. Studies are briefly summarized in text tables, with assigned modified Klimisch scores and discussed in greater detail in Supplemental materials.

DNA adducts and DNA strand breaks

Studies involving roofers exposed to bitumen vapor and aerosols were generally positive for DNA adducts/strand breaks in peripheral white blood cells (WBC) (Herbert et al. 1990a, 1990b; Fuchs et al. 1996; Toraason et al. 2001), whereas DNA adducts observed in paving workers and asphalt painters demonstrated merely suggestive evidence of possible genotoxic effects (Fuchs et al. 1996) (Table 2). Sample sizes were small, but exposures were monitored and worker backgrounds provided. Toraason et al. (2001) demonstrated that when roofers were exposed to coal tar pitch dust as well as bitumen emissions in roof tear-off operations, breathing zone measurements of total particulates, benzene-soluble and total PAC content were higher and DNA damage was statistically significantly greater (p < .05). Exposure to bitumen alone was associated with a small but significant increase in DNA stand breaks. Thus, studies evaluating DNA damage among roofing workers must correct for the potential presence of coal tar dust (confounding exposure) in evaluating positive results.

Table 2. Summary of human mechanistic studies – DNA adducts and strand break studies.

References	Test material	Results	Klimisch score and comments
Herbert et al. (1990a,b)	Tear-off of coal tar pitch roof, application of asphalt roof Roofers (12), Control (12)	Positive DNA adducts in roofers postshift correlated with skin levels of total PAH and B(a)P but not personal air samples	Klimisch 4. Coal tar confounds relevance to bitumen. All subjects exposed to both coal tar pitch and asphalt.
Fuchs et al. (1996)	Roofing fumes (350 °C); paving fumes (150 °C); bitumen paint. Roofers (7), pavers (18), painters (9), Control (34)	Positive strand breaks for roofers, negative overall for pavers and painters. Low levels of DNA adducts in pavers and 2/9 painters	Klimisch 2. Well-performed study albeit small sample size for roofers (7) and painters (9). Pavers and painters DNA effects comparable to controls.
Toraason et al. (2001)	Steep asphalt. Some Coal tar pitch exposure Roofers (26), Control (15)	Positive for strand breaks. No oxidative damage. Coal tar + asphalt exposed workers had higher PAH exposure and slightly higher strand breaks	Klimisch 2. Demonstrated coal tar tear-off exposure a factor in increasing DNA damage in roofers. Well performed study, good monitoring
Marczynski et al. (2006)	Mastic asphalt for paving indoor garages. Pavers (66), Control (45)	Positive increased strand breaks before and after shift compared to control. OxodGuo adducts did not correlate with strand breaks, or exposure	Klimisch 2. Cross shift study with large sample size. Part of German Human Bitumen Study from 2001–2008.
Marczynski et al. (2010)	Mastic asphalt for paving indoor garages. Pavers (42), no control cited	Negative for increased strand breaks in induced sputum [IS] leukocytes post-shift. DNA damage overall high but did not change during shift. Correlation with IL-8 measure of inflammation	Klimisch 2. Follow-up to large German study
Marczynski et al. (2011)	Mastic asphalt for paving indoor garages. Pavers (320), Control (118)	Positive: Higher 8-oxodGuo adducts and DNA strand break in pre- and postshift blood samples compared to concurrent controls but within range of nonexposed healthy individuals.	Klimisch 1. Large cross-sectional, cross-shift study. Authors considered increases in genotoxic effect not be attributed to bitumen exposure due to lack of association with exposure levels
Raulf-Heimsoth et al. (2011a)	Mastic asphalt for paving indoor garages. Pavers (320), Control (118)	Indicators of lower airway irritation and inflammatory effects in induced sputum samples seen preshift in exposed workers did not increase postshift.	Klimisch 2. Large cross-sectional, cross-shift study No significant effect on upper airway by NALF. Lung function factors in normal range with slight decrease in FEV1 & FEC during shift
Raulf-Heimsoth et al. (2011b)	Rolled asphalt followed by mastic asphalt in tunnel pavers (6)	DNA strand breaks higher pre-shift, decreased during shift. Increased 8-oxodGuo independent of asphalt application.	Klimisch 2. Small longitudinal study compared results with larger asphalt exposed population to identify influence of tunnel setting.
Cavallo et al. 2006	Concrete asphalt fumes, 8 h shifts Pavers (19), Controls (22)	Positive for increased DNA strand breaks, some oxidative damage in WBC. Negative for SCE	Klimisch 2. Well-performed study, background data. Did not address specific asphalt product
McClean et al. (2007a,b)	Paving asphalt some high levels recycled asphalt product Pavers (49), Controls (36) over 4 seasons.	Equivocal: Increases in adduct levels during work week. Levels similar in spring, summer and fall, higher in winter.	Klimisch 3. Complex results, multiple confounders and the presence of diesel oil used in cleaning tools
Kendzia et al. (2012)	Road construction workers nonbitumen exposed (118)	Positive: Nonsmokers pre-DNA stand breaks preshift; more DNA adducts and apoptosis postshift. Conc. of IL-8 in lower airways enhanced in smokers.	Klimisch 2. Cross-sectional cross-shift data set for comparison asphalt exposed workers in German Human Bitumen study

Bitumen emission-induced DNA adducts have been characterized as various benzo[a]pyrene diol epoxide adducts (Wey et al. 1992). Herbert et al. (1990a, 1990b) identified one major adduct and several fainter ones from peripheral white blood cells of roofing workers. When mixed with BPDE-1-DNA, this principal unnamed DNA adduct did not comigrate with the major N²-guanine-adduct of B[a]P diol epoxide [BPDE-I guanine N² adduct], suggesting that B[a]P in bitumen emissions is not the principal source of adducts.

Most of the studies have addressed exposure of paving workers. Many conventional paving worker studies show the incidence of DNA strand breaks in bitumen-exposed workers was higher than unexposed controls and sometimes increased with work week exposure. McClean et al. (2007a, 2007b) designed a longitudinal study evaluating DNA adduct formation in hot mix asphalt paving workers (49) and nonpavers (36) over 12 months during the spring, summer and autumn working seasons and nonworking season (winter). They reported that DNA adducts increased during the work week, consistent with absorbed dose measured by urinary 1-hydroxypyrene (1-OHP) and the magnitude of response varied with paving tasks. However, the adduct levels were no higher among paving workers than among nonpaving workers, suggesting that either the presumed unexposed controls were inadvertently exposed to asphalt dust or the apparent correlation between bitumen emissions exposure and DNA adducts is not a definitive measure for health hazard identification. Adding to this question was the observation that for both groups DNA adduct levels were higher in non-working winter months indicating the potential importance of seasonal variations and other environmental factors in contributing to DNA damage. The complexity of these results and multiple confounding factors brings into question the reliability of this study for predicting genotoxic effects.

Concern for high exposure, at higher application temperatures of bitumen in the small population of mastic workers in Germany resulted in the performance of the German Human Bitumen Study evaluating mastic asphalt workers (320 workers, 118 unexposed controls) employed in paving indoor garage spaces from 24 to 180 months. This extensive study produced six DNA adduct and strand break studies (Marcyznski et al. 2006, 2010, 2011; Raulf-Heimsoth et al. 2011a, 2011b; Kendzia et al. 2012), one cytogenetic study (Welge et al. 2011) and two biomarker studies (Pesch et al. 2011; Lotz et al. 2016). DNA strand breaks were higher in bitumen-exposed workers before and after work-shift compared to unexposed controls but strand break levels did not increase significantly during the work week. The higher levels of strand breaks detected preshift suggest DNA cross-links from the previous week’s exposure were repaired over the weekend leaving more strand breaks pre-shift (Marcyznski et al. 2006, 2010). Interestingly, some unexposed controls showed a similar pattern of strand breaks albeit at lower levels. In a subsequent publication evaluating all subjects in the study, Marcyznski et al. (2011) reported that elevated levels of DNA strand breaks and 8-oxo-dG adducts in bitumen exposed mastic asphalt workers compared to reference construction workers, were considered within the range of non-exposed healthy individuals, leading the authors to conclude that there was “no positive association between DNA damage and the magnitude of bitumen emissions or between urinary metabolites and DNA damage in the blood”.

Conventional paving asphalt is handled at lower temperatures [180 °C] than mastic asphalt [250 °C]. To evaluate differences in response to exposure to vapors and aerosols, Raulf-Heimsoth et al. (2011a) examined a small subgroup of 6 workers employed at a tunnel construction site over 2 weeks, one week working with conventional paving asphalt, the second week applying mastic asphalt. Subjects were sampled mid-week. Although mastic asphalt application led to higher exposure to PAC and bitumen – containing vapor and aerosol than conventional paving asphalt workers, no differences were seen in urinary metabolite excretion, lung function impairment or genotoxic markers between examination days.

In a subset of 42 mastic asphalt workers (no controls) from the German Human Bitumen Study, induced sputum (IS) was collected pre- and postshift and evaluated for DNA strand breaks in sputum leukocytes compared with DNA strand breaks in blood lymphocytes; interleukin-8 (IL-8) levels, a marker of irritation, were also measured (Marcyznski et al. 2010). DNA damage in IS leukocytes was high overall but did not increase during workshift. The values correlated with total cell and neutrophil count pre- and postshift but did not correlate with DNA strand breaks in blood lymphocytes, which were higher before work shift than after work shift. DNA stand breaks in IS cells did correlate with IL-8 levels. Raulf-Heimsoth et al. (2011b) followed this study by evaluating the irritating effects of bitumen-containing vapor and aerosol exposure for all 320 mastic asphalt workers and 118 reference controls using spirometry, nasal lavage fluid (NALF) and induced sputum. IS concentrations of interleukin 8, total protein and matrix metalloproteinase-9, all biomarkers of inflammatory effects in lower airways were statistically significantly higher (p < .05) in exposed workers before work-shift and did not increase during work-shift. No significant effects were seen between groups for upper airways by NALF and lung function factors were within normal range for both pre- and post-shift groups. Results suggest a chronic irritation effect in lower airways, which does not seem to be exacerbated by additional workplace exposure.

In 2012, Kendzia et al. expanded the German Human Bitumen study by evaluating pre- and post-shift levels of inflammatory biomarkers and DNA-damage in non-bitumen exposed constructions workers (59 smokers, 59 nonsmokers). Values for DNA strand breaks and 8-oxodGuo adduct from peripheral white blood cells were like those reported for controls in the Marczynski et al. (2011) publication evaluating DNA damage in bitumen exposed workers. The pattern of response for nonsmokers indicated more DNA strand breaks preshift than postshift while more DNA adducts and greater percentage of apoptotic cells were seen postshift. Cross-shift differences were also seen in concentrations of preinflammatory IL-8 in lower airways with higher levels in the morning before work-shift. Concentration of IL-8 in smokers was enhanced both pre- and postshift compared to nonsmokers. This study indicated the potential influence of circadian rhythm on biomarkers. When considered with Marczynski et al. (2011), a similar shift-work effect from general occupational exposure was observed, marginally enhanced by bitumen exposure but within the acceptable range of healthy individuals.

Pesch et al. (2011) and Lotz et al. (2016) reported that PAC metabolites in urine of mastic asphalt workers could not be considered specific biomarkers of bitumen exposure in the workplace due to the weak correlation with airborne bitumen exposure levels. Smoking however showed strong impact on PAC metabolite concentrations. Pesch et al. (2011) found only a weak association between airborne concentrations of bitumen during a working shift and post-shift concentrations of urinary 1-OHP and hydroxy-phenanthrene metabolites and no association with hydroxy-naphthalene metabolites in 317 mastic asphalt workers compared to 117 unexposed reference controls. Lotz et al. (2016) showed similar results investigating diol epoxide pathways of phenanthrene (PHE) and phenolic pathways of PHE and pyrene in urine of 91 mastic asphalt workers compared to 42 unexposed reference controls, before and after work-shifts. Urinary diol epoxides of PHE and pyrene increased during and after shift and were highest in smokers. Metabolites of diol epoxides of PHE were excreted in higher concentrations than phenolic metabolites. Overall, correlations of these PAC metabolites with bitumen exposure were weak or negligible and likely result from low concentrations of PACs (0.05%) in airborne bitumen emissions.

Table 2 shows that most studies meet criteria for use for hazard assessment with only McClean et al. (2007a, 2007b) considered unacceptable due to the complexity of results, multiple confounders and Herbert et al. (1990a, 1990b), unassignable due to exposure to coal tar pitch exposure of all subjects. Although most studies show primarily positive results for induction of DNA damage, the extensive German Human Worker study and even the McClean et al. (2007a) study with its limitations suggest that these results fall within the range of values seen in populations of normal, healthy adults and may not be predictive of potential health effects from exposure to bitumen.

Cytogenetic studies

Cytogenetic studies evaluated the occurrence, or increases in sister chromatid exchange (SCE), micronuclei formation (MN) or chromosome aberrations (CA) primarily in paving workers (Table 3).

Table 3. Summary of human mechanistic studies – cytogenetic studies.

References	Test material/subjects	Results	Klimisch score and comments
Burgaz et al. (1998) [Turkey]	Paving asphalt steam-refined Pavers (21), plant (7) Control (28)	Positive: SCE; % micronucleated [MN] cells higher in asphalt workers than controls	Klimisch 3. Use of diesel oil to clean tools in Turkey confounds results.
Murray and Edwards, (2005)	Paving asphalt. Road pavers (12), Control (18)	Positive: Significant micronucleated cell frequency in urethral cells and lymphocytes	Klimisch 3. Study designed to compare events in urethane workers (12) with asphalt workers; design weak, no exposure data, potential use of diesel oil.
Järvholm et al. (1999)	Paving asphalt from one source. Road pavers (28); Controls (30)	Negative: No significant increase in SCE or micronucleated cells in lymphocytes.	Klimisch 2. Adequate study. Asphalt single sourced in Sweden. No coal tar present.
Karaman and Pirim, (2009)	Concrete asphalt. Pavers (26), Controls (24)	Positive: SCE and % MN correlated with increasing years of exposure	Klimisch 2. SCE and MN higher than controls pre- and post- shift but comparable to each other
Major, Jakab and Tompa, (2001)	Paving Asphalt tar-free [Hungary] Pavers (8), drivers (14), Concurrent (6) & historical (101 + 87) controls	Positive for chromosome aberrations (CA) and SCE in 1996 with decreasing results to control levels in 1999	Klimisch 1. Study demonstrated from 1996–1999 improved working conditions, better ventilation replacement of crude oil for cleaning equipment
Tompa et al. (2007)	Paving asphalt tar-free follow up to Major et al. (2001) above	CA levels at control levels in 1999 began to increase in 2005–2005 with use of crude oil again for cleaning and inadequate ventilation in finisher cabs	Klimisch 1. Mutagenic activity may be related more to organic solvents and diesel exhaust than asphalt fume exposure. Immunomodulation as shifts in lymphocyte subpop’n and activation of T & B cells
Sellapa et al. 2011 [South India]	Road paving asphalt Pavers (36), Controls (37)	Positive: MN and DNA damage in WBC. Smokers and heavy drinkers had higher MN and DNA damage. Increased with years of employment.	Klimisch 3: Diesel oil likely present based on typical work practices, confounder of effects, no urinary exposure measurements.
Welge et al. (2011)	Hot application mastic asphalt Mastic pavers (225), Controls (69)	Negative: MN frequency comparable exposed and control preshift and slightly elevated for both postshift; regression modeling suggested slight weak group difference postshift.	Klimisch 2. Large cross-sectional, cross-shift study from German Human Bitumen Study
Celik et al. (2013) [Mersin Turkey]	Paving asphalt Road pavers (40), Controls (40)	Positive: Increase in MN in buccal mucosal cells, % karyorrhectic cells higher and repair index for cytogenetic damage lower. BMCyt assay	Klimisch 3: absence of specific detail on asphalt composition, air monitoring for PAC exposure or urinary measurements [e.g. 1-OHP]

Although studies of the large population from the German Human Bitumen study showed DNA strand breaks and DNA adduct formation with exposure to bitumen vapor and aerosol, a micronucleus study indicative of actual cytogenetic damage produced comparable results for mastic asphalt workers and reference controls pre- and postshift (Welge et al. 2011). Regression modeling did show a weak and nonstatistically significant group difference in the postshift mode but age was the strongest predictor of increased micronuclei formation in both control and mastic asphalt workers. Similarly, Cavallo et al. (2006) evaluating 18 pavers exposed to bitumen emissions compared to 22 unexposed controls reported statistically significant increased DNA strand breaks and increased oxidative damage (p < .05), but no increase in sister chromatid exchanges. Järvholm et al. (1999) in comparing 28 road paving workers to 30 unexposed controls, all nonsmokers, reported no statistically significant increase in SCE or MN (p > .05). Urinary 1-OHP levels were statistically significantly higher in bitumen workers than in unexposed controls (p < .01) but levels did not increase during shift. The bitumen used came from a single manufacturer, who provides all the bitumen in Sweden.

Statistically significantly positive SCE and MN results (p < .05; p < .001, respectively) were reported by Burgaz et al. (1998) in peripheral blood samples from paving workers exposed for a workplace duration of 9.8 ± 3 years. Smoking habits did not have a statistically significant effect among exposed and control subjects (p >.05). Murray and Edwards (2005) also found statistically significantly increased SCE and MN (p < .01) in peripheral blood lymphocytes and exfoliated urethral cells in a comparative study of paving and urethane workers with controls (all subjects sampled once); smoking did not affect results within exposure groups (p > .05). Karaman and Pirim (2009) determined that Turkish road paving workers exposed to bitumen at 170 °C had similar magnitude of increased levels of SCE and MN (p < .001) compared to controls before and after shift. A positive correlation existed between duration of exposure (avg. 11.16 ± 8.86 years) and the ratio of SCE and MN frequencies (p < .05). Urinary I-OHP levels were comparable to controls before shift but were increased in the exposed group (p <.001) after shift. These changes, however, did not correlate with cytogenetic markers (p > .5). Confounding factors such as coexposure to diesel oil or smoking history were not reported. These workers wore personal protective equipment, which may explain why the levels of cytogenetic damage remained the same before and after shift. The stable levels of SCE and MN before and after shift may suggest fixed cytogenetic damage in the bitumen paving workers from other sources, which were not reported and thus renders this study of questionable significance. Major et al. (2001) demonstrated the value of proper ventilation, personal protective equipment and safer tool and equipment cleaning solvents in a study of Hungarian paving workers exposed to tar-free bitumen from 1996–1999. Minimizing exposure to diesel exhaust in closed cabs for finishers, providing personal protective equipment to paving workers and replacing diesel oil used for cleaning tools and equipment with safer detergents reduced the incidence of SCE and chromosome aberrations back to control levels by 1999. However, these levels were found to increase again as safety precautions were relaxed and diesel oil was again used as a cleaning agent in 2004–2005. The investigators further reported a shift in lymphocyte subpopulations and activation of T and B cells, indicative of immunomodulation, linked to bitumen exposure (Tompa et al. 2007). Exposure to bitumen emissions in road paving operations was reported to increase the frequency of micronuclei (statistically significant at p < .001) in samples of buccal mucosa cells of pavers (40, one half smokers) compared to controls (40, one half smokers). No statistically significant difference in micronuclei incidence (p > .05) between smokers and nonsmokers (Celik et al. 2013) was observed. Similar increases in micronuclei frequency and other nuclear abnormalities were observed in exfoliated buccal mucosa cells of foundry workers (100 exposed, 100 controls) in India. Here smoking was associated with increased micronuclei frequencies (Singaravelu and Sellappa 2015).

Oxidative damage

Whether bitumen exposure causes genetic damage by producing oxygen-free radicals remains unclear. Urinary 8-hydroxy-2′-deoxyguanosine [8–0H-dG] is a by-product of DNA repair and oxidative damage. Halter et al. (2007) reported no evidence of 8-OH-dG adducts in an extensive rat inhalation study, concluding that oxidative damage was not a component of DNA adduct or strand break induction. The Toraason et al. (2001) study of roofing workers did not show an increased leukocyte DNA 8-OHdG/dG ratio and urinary 8-OHdG levels were comparable to those of controls. In the German Human Bitumen Study, increased levels of 8-OHdG nonspecific adducts in peripheral blood WBC did not correlate with strand breaks, bitumen exposure or urinary metabolites and were within the range of levels in non-exposed workers (Marcyznski et al. 2006, 2011; Raulf-Heimsoth et al. 2011a). In other studies, Cavallo et al. (2006) reported DNA strand breaks and increased oxidative damage measured by the formamido pyrimidine glycosylase (FPG)-modified comet assay in 7/19 (37%) of bitumen paving workers, some of whom were smokers. The authors acknowledged that in the absence of cytogenetic damage these oxidative effects could relate to the level of physical activity or heat exposure. Serdar et al. (2016) examined levels of phosphorylated histone H2AX [yH2AX] in peripheral blood lymphocytes along with urinary 8-OHdG in 40 Colorado Springs roofing workers (20/week) (no unexposed controls) from one company. Information on roofing bitumen composition, temperature or possible exposure to coal tar was not provided. Subjects were sampled before and after work-shift on Monday and Thursday. Personal air monitoring vests were worn during the shifts. Markers of DNA damage were approximately 1.7-fold higher after work shift than before. Preshift levels were similar for both days indicating no likely carry-over from earlier damage from Monday to Thursday. However, exposure measurements did not show an association with biomarkers or measures of DNA damage. The authors noted that urinary 8-OHdG is highly affected by urine dilution and roofing workers are subject to bitumen skin burns (as reported by Serdar et al. 2012) and excessive heat and can dehydrate during a single workday. In addition to low levels of bitumen emissions, the authors cited that subjects were exposed to a widespread range of PACs, diesel exhausts, UV, heat and other potential confounding contaminants in the environment.

A study with Turkish subjects who worked with hot bitumen (34 nonsmoking exposed, 35 nonsmoking unexposed) reported that the disulfide/thiol ratio as a measure of oxidative stress was elevated in serum of blood from bitumen exposed workers and correlated with levels of 1-OHP in the urine (Yilmaz et al. 2016). However, diesel oil is commonly used in Turkey as a cleaning agent for tools and equipment by bitumen workers and may confound results of this study (Ayberk Özcan, personal communication, July 2, 2013).

In a review of mechanistic studies relevant to the potential carcinogenicity of bitumen, Schreiner, (2011) pointed out that in animal and human studies, DNA adducts have been identified in subjects in which more direct evidence of genotoxicity (gene mutations in transgenic mice, micronuclei or sister chromatid exchanges) have not been observed. In some worker studies, DNA adduct levels were lower at the beginning of the week and increased during the work week suggesting that adducts were either unstable or were efficiently repaired over the weekend when occupational exposure did not occur. Seasonal variations in background DNA adduct levels of the population can impact predictive values. For example, McClean et al. (2007a) and Wiencke (2002), reported higher DNA adduct levels in winter (when there was minimal bitumen exposure occupationally) than during the summer working season. Poirier (1997) reported that background levels of DNA adducts are essentially universal and appear identical to adducts formed by exposure to exogenous agents which, when present at low levels, are unlikely to exceed DNA repair capacity.

Table 3 indicates most of the studies meet criteria for reliability while several studies were discarded due to regular practice of using diesel oil to clean equipment and limited monitoring data. Those studies provide interesting results but must be interpreted with caution for health effects assessments.

Experimental genetic toxicity (in vitro and in vivo mammalian)

In general, genetic toxicity studies performed in vitro and in vivo employed uniform test procedures. The characterization of the bitumen samples generally identified source and heating temperature for emission generation but lacked validation to actual worker exposure emissions. The summation of plus and minus (positive/negative) designations over a series of studies on a test material (e.g. bitumen) does not always adequately characterize what is occurring in the assays and the relevance to health hazard evaluation. For example, bitumen fume condensate-induced adducts in calf thymus DNA have been reported by De Méo et al. 1996; Genevois et al. 1998; Akkineni et al. 2001, predictive of genotoxicity and potential mutagenicity. However, when Akkineni‘s group compared the adduct results for roofing (227 °C) and paving fume condensate (156, 163 or 146 °C) metabolically activated by induced rat liver or uninduced human liver, with DNA adducts induced by a noncarcinogenic severely hydrotreated petroleum base oil, adducts from the roofing sample were comparable to the base oil-induced adducts and paving fume condensate produced fewer adducts, suggesting a weak potential for genotoxicity from these bitumen fume condensates (Schreiner 2011).

In vitro study results can vary as a function of cell lines used. Genies et al. 2013 highlighted these differences in responses of human cell lines to B[a]P diol epoxide [BPDE]-induced DNA adducts. Dose-dependent adducts were induced in human hepatocyte cell line [HEPG2] but not in bladder [T24] cell line. In the lung cell line [A549] adduct formation was most efficient at the lowest concentration. Results were explained by differences in induction and activity of phase-1 metabolizing enzymes, as well as by proteins eliminating the B[a]P epoxide in the lung cell line at higher doses. Oxidative damage (strand breaks, oxidized purines) was produced only in minute amounts in all three cell lines. B[a]P-induced stable adducts but appeared to cause little oxidative damage. A similar biphasic dose response has also been reported for immunological responses resulting from B[a]P exposure (Burchiel and Luster 2001; Booker and White 2005) as discussed later in this paper under Mechanistic Studies.

Qian et al. (1996, 1998, 1999) identified genetic effects from exposure to type I or type III roofing bitumen emissions generated at 316 °C which had been employed by Sivak et al. 1997 to induce tumors in mouse skin painting studies. Exposure caused increased micronuclei formation in Chinese hamster V79 cells (Qian et al. 1996, 1999) and dose-dependent increases in DNA adducts in lung cells of male rats exposed by intratracheal instillation (Qian et al. 1998). No adducts were seen in blood lymphocytes. These bitumen emissions were not representative of worker exposure (Kriech et al. 2007). Fume generation temperature was at the extreme end of the fume-generation spectrum for bitumen and furthermore intratracheal instillation is an extreme exposure route by which to evaluate pulmonary DNA adduct–forming potential of bitumen emissions. Zhao et al. 2004 detected DNA single-strand breaks and DNA adducts in alveolar macrophages and in lungs (Comet assay) of female Sprague–Dawley rats exposed by whole-body inhalation to road paving Class 1 bitumen emissions generated at 120–170 °C 6 h/day for 1–5 days but did not find increased micronuclei in bone marrow erythrocytes, suggesting an organ-specific toxicity. As part of the same study, Zhao et al. 2004 found in the Ames test that incubation with S9 prepared from the lungs of bitumen-exposed rats (6 h/day, 5-day exposure) increased the mutagenic activity of 2-aminoanthracene [2-AA] but not that of benzo[a]pyrene. The authors show that exposure to bitumen emissions in altering pulmonary P450 enzymes (induced levels of CYP1A1 and reduced levels of CYP2B1) can have a profound effect on handling of different toxic chemicals in the lung as demonstrated by enhanced activity of some mutagens/carcinogens (e.g. 2-AA) in vitro but not others such as B[a]P, the carcinogen most frequently discussed in the context of bitumen emission exposure. Wang et al. 2003 also identified DNA adducts in the lungs of mice exposed to paving bitumen emissions representative of Midwest formulations generated at 180 °C using the NIOSH/Heritage fume-generating system. However similar DNA adduct results from Halter et al. 2007, in the absence of increased micronuclei in bone marrow erythrocytes and with no increase in 8-oxo-dG adducts from exposure to semiblown paving bitumen emissions, produced no respiratory tumors in rats after 2 years of exposure as discussed more thoroughly later. Studies performed with transgenic rodents exposed by nose-only inhalation to emissions from road paving bitumen [Venezuelan 50/70 pen generated at 170 °C] for 6 h/day, 5 days produced no adducts in the lungs of male Lac1 mice and no change in the mutant frequency for lac 1 gene or CII, a neural reporter gene (Micillino et al. 2002). In a follow-up study in transgenic rats with CII in the genome exposed by nose-only inhalation, 6 h/day for 5 days, only one adduct was observed at 3 and 30 days after treatment in the lung, with no increase in mutation frequency of CII (Bottin et al. 2006). Although a slight but nonstatistically significant modification of the mutation spectrum, an increase in G:C to T:A and A:T to C:G transversions was observed in the treated rats these transversion data failed to demonstrate a pulmonary mutagenic potential for bitumen emissions generated at road paving temperature despite the presence of a DNA adduct.

Dhondge et al. 2012 conducted a cell transformation study in a human osteosarcoma cell line using a laboratory bitumen extracted in dichloromethane and resuspended in dimethyl sulfoxide. The resulting transformed cells were then injected into nude, severe combined immunodeficiency mice to assess the biological activity in vivo. Proteomic analysis had revealed the existence of 19 differentially expressed proteins typically involved in cancer progression, angiogenesis, and cell adhesion. Transformed cells showed characteristics of anchorage independence and chromosomal anomalies, but when injected into immunodeficient mice did not form tumors, a result similar to the absence of tumors in the 2-year inhalation study in rats (Fuhst et al. 2007).

Validated animal cancer studies

Table 4 describes four validated animal cancer studies and includes consideration of test material characterization, test species, exposure route, duration and frequency, treatment levels, results and study strengths.

Table 4. Validated bitumen emission animal cancer studies – all with a Modified Klimisch Score of 1.

Clark et al. 2011
Test material	Paving and type-III built up roofing bitumen [BURA] fume condensates and a sample of NIOSH laboratory generated fume condensate
Route	Dermal
Representativeness	Detailed measures were taken to ensure representativeness of these test materials to actual worker exposures. Extensive chemical analysis was conducted to match test material to worker exposures.
Confounders	Confounders were eliminated to ensure only the bitumen emissions were being evaluated in the study.
Test Species	C3H/HeNcRL mice. 80 male mice/treatment group
Exposure Details	Dermal application of liquid fume condensate in mineral oil, to clipped dorsal mouse skin. Total weekly dose of 50 mg/animal. Study duration was 104 weeks with interim sacrifices after 26, 52 and 78 weeks to evaluate dermal irritation.
Exposure Duration	104 weeks
Treatment levels	Paving fume condensate: daily applications of 7.14 mg in mineral oil BURA fume condensate: twice/week applications of 25 mg in mineral oil Lab generated fume condensate: twice/week applications of 25 mg in mineral oil
Results	Control (mineral oil): one animal with a basal cell carcinoma Paving fume condensate: one animal with a benign, squamous cell papilloma BURA fume condensate: 11 affected animals, showing 8 squamous cell carcinomas, 1 keratoacanthoma and 4 squamous cell papillomas Lab generated fume condensate: 37 affected animals, showing 35 squamous cell carcinomas, 2 keratoacanthoma and 3 squamous cell papillomas
Study strengths	The paving and BURA fume condensates were “matched” analytically with industrial hygiene workplace samples collected at paving and roofing work sites using the same bitumen. The fume condensates were characterized for individual and total polycyclic aromatic hydrocarbons, UV fluorescence activity, simulated distillation range and mutagenic activity (modified Ames). Dose and application frequency were based on results of pilot studies, to control/minimize the potential confounding effect of dermal irritation on tumor incidence. The inclusion of a laboratory-generated fume condensate provides a reference point to a previous positive study on a material fume condensate with markedly different composition.
Freeman et al. 2011
Test material	Type-III built-up roofing bitumen [BURA] fume condensate
Route	Dermal
Representativeness	Detailed measures were taken to ensure representativeness of these test materials to actual worker exposures. Extensive chemical analysis was conducted to match test material to worker exposures.
Confounders	Confounders were eliminated to ensure only the bitumen emissions were being evaluated in the study.
Test Species	Crl:CD1 mice. 30 male mice/group
Exposure Details	Dermal initiation/promotion study. Initiator: DMBA Promotor: TPA Dermal application of liquid fume condensate in mineral oil, to clipped dorsal mouse skin. Total weekly dose of 50 mg/animal
Exposure Duration	Initiation: 2 weeks Promotion: 26 weeks
Treatment levels	Twice/week applications of 25 mg in mineral oil
Results	Control (mineral oil): No tumors. BURA fume condensate Initiated: Five animals affected showing 5 squamous cell papillomas BURA fume condensate Promoted: Two animals affected showing one benign keratoacanthoma, two squamous cell papillomas
Study strengths	The BURA fume condensate was “matched” analytically with industrial hygiene workplace samples collected at roofing work sites using the same bitumen. The fume condensate was characterized for individual and total polycyclic aromatic hydrocarbons, UV fluorescence activity, simulated distillation range and mutagenic activity (modified Ames) Dose and application frequency was based on results of pilot studies, to control/minimize the potential confounding effect of dermal irritation on tumor incidence. The sample tested was weakly positive in a conventional 2-year mouse skin painting study
Fuhst et al. 2007
Test material	Bitumen fume condensate. Mixed aerosol and vapor
Route	Inhalation
Representativeness	Detailed measures were taken to ensure representativeness of these test materials to actual worker exposures. Extensive chemical analysis was conducted to match test material to worker exposures.
Confounders	Confounders were eliminated to ensure only the bitumen emissions were being evaluated in the study.
Test species	Wistar rats Control and high-dose group – 86 males and 86 females. Low and mid dose groups – 50 males and 50 females
Exposure details	OECD Guideline 451. Nose only exposure to mixed aerosol and vapor, generated from a liquid emission condensate, using an evaporation condensation generator
Exposure duration	6h/day, 5 days/week, 24 months
Treatment levels	0, 6.8, 34.4, 172.5 (mg/m^3 total hydrocarbon)
Results	No significant increase in tumor incidence. Treatment related, minimal to slight, non-neoplastic irritation of the nasal epithelium
Study strengths	Exposure atmospheres validated against workplace measurements. Animal exposures representative of human exposures in the workplace. Interim sacrifices (7 days, 3 month and 12 months) Bronchiolar lavage, cell proliferation investigations PAH profiling of exposure atmosphere Concurrent investigations (Halter et al. 2007) – urinary PAH metabolites – micronucleus formation (blood and bone marrow) – DNA adduct formation – gene expression (lung, nasal epithelium, WBC)
Halter et al. 2007
Test material	Bitumen fume condensate. Mixed aerosol and vapor
Route	Inhalation
Representativeness	Detailed measures were taken to ensure representativeness of these test materials to actual worker exposures. Extensive chemical analysis was conducted to match test material to worker exposures.
Confounders	Confounders were eliminated to ensure only the bitumen emissions were being evaluated in the study.
Test species	Wistar rats Control, high-, mid- and low-dose groups – 24 males and 24 females
Exposure details	OECD Guideline 451. Nose only exposure to mixed aerosol and vapor, generated from a liquid emission condensate, using an evaporation condensation generator
Exposure duration	6h/day for either 5 days, 1 month or 12 months
Treatment levels	0, 6.8, 34.4, 172.5 (mg/m^3 total hydrocarbon)
Results	Urinary metabolites confirm uptake and metabolism of PAHs following inhalation exposure. No increase in erythrocyte or bone marrow micronucleus formation Repression of bone marrow RBC count – high dose after 12 months Increased DNA adduct formation in lung, nasal and alveolar epithelium. No increase in WBC adducts Altered expression of genes involved in inflammation, asthma, and PAH metabolism in lung, and nasal epithelium. No consistent dose/time/sex dependent pattern of regulation
Study strengths	PAH metabolites confirm exposure, uptake and metabolism of PAHs following inhalation exposure. No evidence of genotoxicity in blood cells or bone marrow, despite evidence of target tissue exposure. Clear DNA adduct formation in respiratory tissues but no evidence of a carcinogenic effect in concurrent study (Fuhst et al, 2007) Clear alterations in gene expression in respiratory tissue but no evidence of significant inflammatory change or progressive disease in concurrent study (Fuhst et al, 2007)

Case study 1 – evaluating carcinogenic potential of bitumen emissions following inhalation exposure

In a two-year chronic toxicity/carcinogenicity study (Fuhst et al. 2007) with bitumen fume condensate validated against workplace exposure data, groups of rats were exposed, by nose only inhalation, to an atmosphere derived from a mixture of air-rectified and straight-run paving bitumen. One of two principal routes of occupational exposure in the workplace is inhalation of emissions generated from heated bitumen, for example during paving or roofing applications. To ensure that the results of the animal study were directly relevant for the hazard assessment of human exposure to bitumen emissions, a significant amount of prework was required prior to the main toxicology study.

The bitumen selected for study represented the most commonly used paving bitumen grade in Germany. To evaluate workplace exposure, Rühl et al. 2006 evaluated industrial hygiene data and determined average total hydrocarbon (THC) exposures of 2.8, 3.8 and 1.3 mg/m³ for the paver operator, screedman and roller operator, respectively. Further work to characterize and quantify workplace exposure to the bitumen emissions chosen for study was undertaken to compare workplace samples with samples of condensed fume collected from a heated storage tank (Preiss et al. 2006). Consistent with the typical application temperature in the workplace, condensate was collected from the headspace of a storage tank at 175 °C. Workplace and condensate sample results were comparable based on predetermined acceptance criteria that included boiling point distribution, UV fluorescence (Osborn et al. 2001) and PAC profile.

Pohlmann et al. 2006a showed the feasibility of collecting samples of condensed emissions from the headspace of a heated bitumen storage tank that matched closely emissions to which paving workers were exposed. Further validation work (Pohlmann et al. 2006b) demonstrated it was possible to use a tank bitumen fume condensate to regenerate an exposure atmosphere representative of paving worker exposure for the toxicity study.

The experimental design of the chronic toxicity/carcinogenicity study was in accordance with OECD TG 451 (2009). Animals were exposed 6 h/day, 5 days/week for 2 years to mixed aerosol and vapor at concentrations of 6.8, 34.4 or 172.5 mg/m³, measured as THC. Control animals were exposed to filtered air only. In addition to the guideline parameters/investigations, groups of animals were included in the study design to allow interim sacrifices to assess irritation of the respiratory tract, following 7 days, 90 days and 12 months exposure. Animals from the interim sacrifice groups were subject to the following investigations:

• Bronchiolar lavage [BAL] – to assess respiratory tract inflammatory changes

• Immunohistochemistry – to assess cell proliferation in the respiratory tract

• Histopathology – to assess damage to the nasal/paranasal cavities, larynx and lung

Comprehensive results of the two-year, chronic toxicity/carcinogenicity study have been reported elsewhere (Fuhst et al. 2007). In summary, nose-only inhalation exposure of rats to bitumen emissions, did not show any evidence of treatment-related systemic toxicity nor an increase in total or organ-specific tumor incidence. Treatment-related findings were restricted to minimal/slight local irritant effects on lung and nasal cavity tissues, as evidenced by BAL, cell proliferation and histopathological findings.

In a parallel series of investigations, as part of the same chronic toxicity/carcinogenicity study, the following parameters were examined in groups of rats exposed to bitumen emissions (Halter et al. 2007):

• Micronuclei formation in peripheral red blood cells and bone marrow samples, following 5 days, 1 month and 12 months of exposure.

• Urinary metabolites of naphthalene and phenanthrene – the predominant PACs in the exposure atmosphere, and pyrene – a minor constituent, following 5 days, 1 month and 12 months of exposure.

• DNA adduct formation (³²P-postlabeling) in lung, nasal and alveolar epithelium, and WBC, following 5 days, 1 month and 12 months of exposure.

• Oxidative DNA damage (8-oxo-dG adduct formation) in lung, nasal and alveolar epithelium and WBCs, following 5 days, 1 month and 12 months of exposure.

• Altered gene expression in lung, nasal and alveolar epithelium and WBC following 5 days, 1 month and 12 months of exposure.

Increased levels of urinary metabolites of both naphthalene and phenanthrene were observed following exposure at the mid- and high-emission concentrations, particularly after 12 months. Levels of pyrene metabolites were, however, close to the limit of detection and therefore could not be reliably determined. These results confirm systemic uptake and metabolism of PACs following inhalation exposure to bitumen emissions.

Potential genotoxic effects (micronuclei formation) were assessed in erythrocytes and bone marrow. No evidence of an increase in micronuclei formation was detected. Exposure of bone marrow cells was confirmed by slight repression of bone marrow RBC count in four of six animals at the highest concentration after 12 months.

There was no evidence of oxidative DNA damage in lung, nasal or alveolar epithelium or WBCs at any exposure concentration. An exposure concentration- and time-dependent increase in DNA adducts was observed in nasal, lung and alveolar tissue but not in WBCs. Three DNA adducts were detected in nasal and lung tissue and four in alveolar epithelial cells, with adduct levels being highest in nasal epithelium. Gene expression studies showed altered regulation of genes involved in metabolic activation of polycyclic aromatic hydrocarbons and cellular inflammatory processes. These findings were consistent with the histopathology results and the presence of PAC metabolites in the urine.

In conclusion, this series of experimental studies provides a comprehensive view of the carcinogenic potential from exposure to bitumen emissions and their relevance to worker health. Exposure of rats by inhalation for two years, to an atmosphere qualitatively representative of workplace exposure, did not result in an increase in either systemic damage or tumor incidence. Although parallel studies (urinary metabolites and altered gene expression) confirmed systemic exposure, they failed to show any evidence of genotoxicity. Moreover, despite evidence of treatment- related DNA adduct formation, no evidence of a tumorigenic effect was observed following exposure for 2 years, possibly because of DNA repair.

Case study 2 – evaluating the carcinogenic potential of condensed bitumen emissions following dermal exposure

A second example of a validated read-across study investigated the carcinogenic potential of bitumen fume condensates in a series of mouse dermal skin-painting studies (Clark et al. 2011). The paving fume condensate was straight run and the type-III BURA was oxidized. The mouse skin-painting study is a sensitive indicator of dermal carcinogenic potential, particularly for complex petroleum substances, where carcinogenicity may be mediated by PACs (Chasey and McKee 1993; Kriech et al. 1999a).

Roofing and paving grade bitumens are heated to facilitate application. Skin exposure of workers to condensed emissions can occur. The composition of the exposure atmosphere, however, represents only the volatile fraction released on heating, not the base bitumen. To ensure that the experimental cancer data reflect the true carcinogenic hazard potential of this material, considerable prework was again undertaken to validate the test material against occupational hygiene data.

The objective of the study was to investigate the carcinogenic potential of US Performance Grade (64–22) paving grade bitumen and type III, built-up roofing [BUR] bitumen fume condensates and compare this with results of laboratory-derived roofing fume condensate; samples of the latter have previously proved carcinogenic in skin painting studies (Niemeier et al. 1988; Sivak et al. 1997), although their composition was not representative of normal worker exposure (Reinke et al. 2000; Kriech et al. 2007). To select the paving and roofing grade bitumen samples for the study, four commercially available products of each type, representing four different crude oils from four distinct regions of the USA were collected and analyzed (Kriech et al. 2007). For the laboratory-derived condensate samples, generation of emissions was performed on the four type-III BURA products according to the method described by Sivak et al. 1989.

Samples of liquid fume condensates from each of the bitumens were collected from the headspace of heated storage tanks using a method like that used for the chronic toxicity/carcinogenicity study (Preiss et al. 2006; Pohlmann et al. 2006a).

To understand the nature of worker exposure, personal air samples were collected, under normal operating conditions, on workers at paving and roofing work sites handling each of the four-commercial paving (average 150 °C) and roofing products (average 230 °C in the mop buckets – average 273 °C in the kettle). Samples were analyzed to provide both quantitative and qualitative data for comparative purposes. Parameters measured included boiling point distribution (simulated distillation using ASTM D2887), UV fluorescence, individual PAC measurements, selected ion GC/MS fingerprinting, total particulate [TP] matter and benzene-soluble fraction [BSF].

To validate that the tank fume condensates mimicked worker exposure, predetermined acceptance criteria were established by a scientific advisory committee. Workplace exposures were compared to tank fume condensates from the same bitumen source based on simulated distillation, UV fluorescence, PAC profile and selected ion GC/MS fingerprinting. Tank emissions were also tested for mutagenicity index (Blackburn et al. 1984, 1996). The tank roofing and paving samples selected for skin painting closely matched the composition of worker exposure samples.

Laboratory-derived roofing fume condensate was collected by drawing air through a flask containing heated and stirred oxidized bitumen at 232 °C, replicating conditions used by Sivak et al. 1989.

For the skin-painting studies (Clark et al. 2011), a single sample of each paving, roofing and laboratory-derived fume condensate was selected from the materials collected during the validation phase (Kriech et al. 2007), based on meeting key predetermined parameters. To preclude confounding dermal irritation responses, comprehensive pilot studies were undertaken to identify appropriate diluent vehicles and dosing frequency, to minimize skin irritation during the two-year skin-painting study. As these studies showed a clear difference in irritation potential between paving and roofing condensates, the main studies used fume condensates diluted in refined mineral oil but with different dosing frequencies (Table 5).

Table 5. Dermal dose and application frequency – Clark et al. (2011).

Test material	Application frequency	Volume (μl)/ application	Condensate (mg)/ application	Total weekly dose (mg)
Paving condensate	Daily	37.5	7.14	50
Roofing condensate	2x/week	37.5	25	50
Laboratory condensate	2x/week	37.5	25	50

Test materials were applied topically to groups of male mice for 104 weeks. During the treatment phase, dermal irritation was assessed weekly by visual inspection and histologically in sub-groups of mice sacrificed after 26, 52 and 78 weeks.

In the paving fume condensate study, no significant adverse effects were observed between treated animals and vehicle controls. Levels of dermal irritation were comparable throughout and no treatment-related tumorigenic response was evident.

Both field-matched and laboratory generated roofing fume condensates treatment resulted in some mortality during the first six months of treatment; four animals (out of 80) in the field- matched and 10 animals (out of 80) in the laboratory-generated groups. Thereafter mortality incidence in the field-matched group was comparable with vehicle controls. For the laboratory generated condensate, group mortality increased significantly after 60 weeks. Compared to vehicle controls, dermal irritation in the field-roofing fume condensate group was increased to moderate after 53 weeks and was maintained through to study termination. In the laboratory-generated condensate group, marked irritation was apparent beyond week 46 and remained for the duration of the study. Microscopic examination of skin from field-matched and laboratory-generated roofing condensate treated animals confirmed the difference in dermal irritation severity.

Skin masses were observed in both field-matched and laboratory-generated roofing fume condensate groups from around week 50, with the number of masses and the rate of increase being significantly greater in the laboratory-generated condensate exposed animals. Histological examination confirmed tumorigenic effects in both groups (Table 6) with squamous cell carcinomas being the most prevalent tumor type.

Table 6. Dermal tumor incidence and type – Clark et al. (2011).

Treatment group Number examined/at risk	Refined mineral oil 80	Field-matched roofing condensate 80/62	Laboratory-generated roofing condensate 80/64
Carcinoma, squamous cell	0	8 (13%)	35 (55%)
Keratoacanthoma	0	1	2
Papilloma, squamous cell	0	4	3
Papillomas and carcinomas	0	11 (18%)	37 (58%)
Time to first tumor (latency)	n/a	62 weeks	50 weeks
Mean latency	n/a	90 weeks	76 weeks
Latency range	n/a	62–104 weeks	50–104 weeks

n/a = not available.

The results of skin-painting studies in mice show clearly that paving bitumen fume condensate, produced under conditions representative of normal working practice, is not carcinogenic to mouse skin and is corroborated by analytical data used to characterize the material, which show relatively low levels of 4–6 ring PACs (34 mg/kg), low fluorescence activity (30 EU/g) and a mutagenicity index in the modified Ames test of 0.69 (Kriech et al. 2007). Conversely, field-matched roofing fume condensate produced an increase in the incidence of dermal tumors (18%), with a relatively long mean latent period [90 weeks], suggesting a weak carcinogenic potential; this result was not surprising as the sample showed higher levels of 4–6 ring PACs (74 mg/kg), higher fluorescence (157 EU/g) and a mutagenicity index of 1.2. The most active material was the laboratory generated roofing fume condensate, which produced tumors in a significant number of animals, with a shorter mean latency period [76 weeks]. This material showed significantly higher levels of 4–6 ring PACs [232 mg/kg], high fluorescence activity (336 EU/g) a mutagenicity index of 3.3 and the tumorigenic response was consistent with that seen previously (Niemeier et al. 1988). Mutagenicity Index, derived using the modified Ames test (Blackburn 1984, 1996), is predictive of carcinogenic potential in mouse skin; complex petroleum substances with values >1.0 are considered to be carcinogenic, while those <1.0 are unlikely to be active.

To investigate the weak tumorigenic response observed by Clark et al. 2011 with field-matched roofing fume condensate, a dermal initiation–promotion study was undertaken (Freeman et al. 2011). Groups of male mice were exposed by skin application, to the same fume condensate tested by Clarke et al. twice per week for either two weeks, during the initiation phase, or 26 weeks during the promotion phase. A total dose of 50 mg fume condensate/week, diluted in mineral oil was applied to clipped dorsal skin. Positive control materials were 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator, or 12-O-tetradecanoyl-PHORBOL-13-acetate (TPA), as a promoter. During the study, mice were observed for mortality, signs of toxicity, dermal irritation and the development of dermal masses. Samples of treated skin, dermal masses and any gross lesions were examined histologically.

Data on tumor incidence and type are shown in Table 7. Animals initiated with roofing fume condensate, followed by promotion with TPA, showed mild- to moderate-dermal irritation and a statistically significant increase in the incidence of benign squamous cell papillomas. In the group initiated with DMBA and promoted with roofing fume condensate, two animals developed skin tumors. Histology confirmed one benign keratoacanthoma and two benign squamous cell papillomas, a response significantly less than that seen with DMBA initiation and TPA promotion.

Table 7. Tumor-response – mouse skin tumor initiation–promotion study with type-III BUR bitumen fume condensate (AFC) (Reproduced from Freeman et al. 2011).

				Histologically identified tumors
Group #	Test group	Grossly observed tumors	Time to first tumor (weeks)	Malignant squamous cell carcinoma	Benign keratoacanthoma	Benign squamous cell papilloma
1	MO/MO	0	NA	0	0	0
2	DMBA/TPA	29	7	3	3	27
3	DMBA/MO	0	NA	0	0	0
4	DMBA/AFC	2	16	0	1	2
5	MO/TPA	1	23	0	0	1
6	AFC/TPA	8*	12	0	0	5
7	AFC/MO	0	NA	0	0	0

AFC : type-III BUR fume condensate; DMBA: 7,12-Dimethylbenzanthracene; MO: mineral oil; NA: not applicable; TPA: 12–0-tetradecanoyl-13-acetate.

* neoplasms were not observed microscopically in three animals, although hyperplasia and inflammation were seen suggesting exophytic portions of papillomas were lost.

Results from this dermal initiation promotion study suggest that field-matched roofing fume condensate acts as a tumor initiator in mouse skin. There was no apparent relationship between dermal irritation and tumor development, but a contributory role for fume condensate acting as a skin promotor cannot be excluded based on these results.

In conclusion, a series of studies using the same test materials, investigating the potential of bitumen fume condensates to cause skin cancer in mice, show that field-matched paving bitumen fume condensate is not carcinogenic to mouse skin. In contrast, type-III built up roofing [BUR] bitumen fume condensate proved weakly carcinogenic when applied repeatedly to mouse skin for two years. The tumorigenic activity was confirmed in a follow-up initiation/promotion study, where the roofing fume condensate was shown to act as a tumor initiator. The low tumor incidence and long latency period suggest, however, that the type III [BUR] should be considered of low carcinogenic potency, compared to results reported for individual PACs and PAC-rich petroleum streams.

In a skin-painting study with laboratory-generated roofing fume condensate, a significant number of animals developed skin tumors, replicating the tumorigenic activity seen previously (Niemeier et al. 1988). The difference in response between “field” and “laboratory” fume condensates confirms the importance of temperature and fume generation methodology, in determining the chemical composition and biological activity of bitumen fume condensates. To avoid erroneous hazard conclusions, it is critically important that the composition of materials used in experimental studies matches as closely as possible the composition of the material to which workers are exposed under representative, that is, realistic conditions.

Mechanistic studies

Exposures to bitumen emissions have been investigated to determine changes in enzyme systems, proteins, altered gene expression and other mechanisms involved in metabolism of xenobiotics, cell cycle regulation, apoptosis and immune responses; changes which arguably link bitumen emissions exposure as a weak initiator or tumor promoter to potential carcinogenesis in humans. Studies with bitumen emission test materials that have been characterized are discussed below.

The cytochrome P450 mono-oxygenase system is required for the bioactivation of polycyclic aromatic hydrocarbons. Genevois et al. 1996, 1998 explored this activity in adduct formation with calf thymus DNA and skin painting of rats with bitumen fume condensate collected at 160 or 200 °C from Venezuelan 45/60 pen. CYP1A isoforms and aryl hydrocarbon receptor (AHR) both played important roles in PAC activation, although CYP1A2 played a lesser role than CYP1A1. Ma et al. 2002 tested male Sprague–Dawley rats by intratracheal instillation with bitumen fume condensate collected from the top of a paving bitumen tank (PC64–22 similar in composition to condensates collected from road paving operations (Kriech et al. 1999b)) and reported that exposure to bitumen fume condensate did not affect total cytochrome P450 content, cytochrome c reductase or CYP2B1 levels or enzyme activity in the lung. However, CYP1A1 levels and activity were significantly increased. Micronuclei formation in bone marrow polychromatic erythrocytes was increased only at the highest dose tested (8.88 mg/kg). A subsequent study by Ma et al. 2003a confirmed the increased CYP1A1 levels and activity in female Sprague–Dawley rats exposed to emissions generated at paving temperatures (120–150 °C) with concomitant downregulation of CYP2B1 and increased quinine reductase activity but without exhibiting acute inflammation or lung injury.

AHR is a ligand-activated cytosolic transcription factor central to upregulation of phase-I and phase-II genes encoding xenobiotic-metabolizing enzymes. PAC exposure produces AHR-ligands that regulate CYP1-metabolizing enzymes and can also impact DNA replication, cell cycle and cell proliferation and immune responses. Unliganded AHR is widely expressed in mouse tissues and is high in human lung, thymus, kidney and liver. PAC-induced full functional ligands trigger signal transduction pathways that may “cross-talk” with protein kinases involved in gene expression, phosphorylation and chromosomal regulation (Puga et al. 2009). Nebert et al. 2000 suggested that AHR-mediated oxidative stress generated by the induction of P450 enzymes may be a critical event in the apoptosis pathway. Most of the studies specifically investigating PAC-induced AHR ligand activities have been performed with individual PAC – dioxin [TCDD] being the principle material – although the immunomodulatory role of AHR has been demonstrated with exposure to 7,12-dimethylbenzanthracene [DMBA] and B[a]P. For example, Yamaguchi et al. 1997 demonstrated apoptotic activity of an AHR ligand of DMBA in a murine Pre-B-cell developmental model, which highlighted the interactive role of AHR ligands and bone marrow stromal cells in regulating lymphopoiesis. Apoptosis could be blocked by α-naphthoflavone, an AHR and cytochrome P450 inhibitor blocker. Burchiel and Luster, 2001 suggested that B[a]P and other PACs exert their effects by binding to AHR, upregulating the AHR-controlled metabolic enzymes [P450 1A1 and 1B1] inducing immunotoxic PAC metabolites altering signaling pathways in human T and B cells. Both Burchiel and Luster, 2001 and Booker and White, 2005 state that B[a]P is immunosuppressive at high doses but can enhance immune response at low doses. Upregulation of AHR in rat lung is seen with exposure to bitumen fume condensate despite low levels of PACs in the aerosol and vapor, but expression of these changes has not been reported to result in lung tumors in rats (Fuhst et al. 2007) or increased incidence of lung tumors in studies of humans (Olsson et al. 2010).

The extensive microarray gene expression profile of rat lung tissue (Gate et al. 2006) following nose-only inhalation to rats to road paving bitumen emissions (Venezuelan 50/70 pen at 170 °C, 100 mg/m³, 6 h/day for 5 days) demonstrated altered expression of 363 of 20,500 probes with the largest overexpression of CYP1A1 and CYP1B1 genes, genes involved with cellular response to oxidative stress and upregulation of inducible genes with AHR binding sites in their promoter. No genes involved in DNA damage and repair except for XRCC5 were upregulated in exposed lungs. This absence of upregulation of DNA damage and repair genes may be associated with the constitutive expression of the genes or possibly the extent of DNA damage induced by bitumen emissions may be insufficient to trigger gene expression. Pulmonary inflammation was observed with broncho-alveolar lavage associated with increases in pro-inflammatory cytokines and chemokines and overexpression of the inflammation-related genes in microarray. The authors explained this transitory inflammatory response as the result of sacrifice immediately after termination of exposure, allowing the observation of particles being cleared by alveolar macrophages. Halter et al. 2007 also reported up regulation of CYP1A1 and CYP2G1 genes or actual isoforms (cytochrome P450 monooxygenase 1B1 and 2G1) in nasal and lung tissue of rats exposed for 1 year to partially oxidized bitumen fume condensate as well as up and downregulation of genes with known functions in inflammation/immune responses. However, no lung tumors were observed at the end of two years of exposure.

Because bitumen emissions may contain low concentrations (ppm or less) of known carcinogenic PACs, it is theorized that contribution to potential carcinogenesis is at least partially related to nongenotoxic tumor promotion. The impact of fractions of roofing bitumen fume condensate (class II) generated at 316 °C on intercellular communication via gap junction was explored in Chinese hamster V79 cells (Toraason et al. 1991) or cultured human epidermal keratinocytes (Wey et al. 1992). All five well-characterized fractions, comparable to the NCI/EPA/NIOSH sample set, produced statistically significant (p < .05) concentration-dependent inhibition of gap junction intercellular communication to varying degrees. Only two of these fractions caused skin cancer in mice (Sivak et al. 1997).

Dermal exposure to bitumen emissions may stimulate gene and protein expression in the skin. Ma et al. 2003b examined bitumen fume condensate generated at 150 °C from hot performance grade bitumen [PG64–22] to stimulate activity of AP-1, the activator protein regulating the expression of a diverse array of genes, including those involved in cell growth, proliferation and transformation and frequently associated with the promotion of skin carcinogenesis. The test condensate was generated at the NIOSH inhalation facility by standardized methods and validated to worker exposure. Increased activity was seen in both the mouse JB6P + epidermal cell line and in tissue from transgenic mice expressing the AP-1 luciferase reporter gene suggesting a role in formation of mouse skin tumors. Exposure of bitumen emissions to JB6P + cell line promoted basal and epidermal cell growth indicative of anchorage independent cell transformation and activation of PI3K/Akt and other kinases but blockage of P13K/Akt activation eliminated bitumen emissions stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. Similar results were seen in tail skin of treated transgenic mice and epidermal keratinocytes from newborn AP-1 transgenic mice exposed in vitro.

Human studies

Ellingsen et al. 2010 examined pneumoproteins and inflammatory biomarkers in bitumen paving workers exposed to standard paving bitumen emissions from Norway’s largest construction company (Table 8). They measured lung function and a wide range of reactive proteins, interleukin-6, fibrinogen, intercellular and vascular cellular adhesion molecules in 72 pavers, 12 plant operators and 19 engineers over an entire paving season April–October. Smoking, BMI and levels of physical activity were considered confounding factors (details in Supplemental materials). Nevertheless, increased systemic inflammation and endothelial activation were not detected across the paving season, suggesting that exposure to paving bitumen emissions under modern working conditions may not contribute significantly to respiratory issues.

Table 8. Summary of human mechanistic studies – other human studies – urinary Ames test and urinary metabolites; other measures of inflammatory and cellular effects I think this table should be placed and numbered nearer the text it illustrates after the cancer tables, to be located appropriately in the final publication.

References	Test material/subjects	Results	Klimisch score and comments
Pasquini et al. 1989	Road paving asphalt Pavers (16), controls (27) urinary study	Positive: TA98 +S-9, neg TA100. urinary thioether unaffected by bitumen exposure alone	Klimisch 3. Small sample size, exposure to diesel exhaust; possible diesel oil for cleaning confounders
Yilmaz et al. 2016	Hot mix asphalt, Workers pavers and mixers (34), Controls (35)	Positive: Disulfide/thiol ratio higher in pavers (may reflect oxidative stress). Total and native thiols comparable to controls	Klimisch 3. Lack of exposure data, potential diesel oil used in cleaning as confounder.
Pesch et al. 2011	Mastic asphalt Pavers (317), Controls (117)	Measured 1-OHP, hydroxylated metabolites of phenanthrene, naphthalene. During shift, PAH metabolite levels increased in all exposed and non-exposed workers.	Klimisch 2. German Human Bitumen Study, extensive data set, possible diesel oil confounder not identified
Lotz et al. 2016	Mastic Asphalt Pavers (91), Control (42)	Urinary PAH metabolites [1-OHP, PHE, PYR] higher after shift. Association with bitumen weak or negligible due to small PAH content	Klimisch 2. German Human Bitumen Study, extensive data set, possible diesel oil confounder not identified
Serdar et al. 2016	Hot Asphalt Roofers (40), no unexposed controls	Positive. Increased yH2AX in blood and 8-OHdG in urine after work shift. Urinary 8-OHdG highly affected by urine dilution.	Klimisch 3. Low level of exposure, no unexposed controls, exposure to other PAC and contaminants. Dehydration of roofers may alter 8-OHdG results.
Ellingsen et al. 2010 [Norway]	Paving asphalt pavers (72); Plant operators (32), minimal exposed Engineers (19)	Negative: Increased systemic inflammation and endothelial activation not detected in asphalt workers across season	Klimisch 2. Large sample size. Asphalt single sourced at Norway’s largest construction Co. good exposure monitoring, unknown use of diesel oil for cleaning.
Fenga et al. 2000	Paving bitumen fumes Pavers (16), controls (10)	Positive: heat shock protein 27 upregulated. Reactive products in all skin layers. Protect against stress factor in inflammatory disease	Klimisch 4. Small sample size. Limited characterization of bitumen. See Loreto et al. 2007 & Rapisarda et al. 2009 below for other endpoints
Loreto et al. 2007	Paving bitumen fumes Pavers (16), controls (10)	Dermal biopsy: Epidermal thinning, flattened dermal papillae, overexpression of bax, under expression of bcl-2	Klimisch 4. Limited characterization of bitumen. Results suggest bitumen fume induces activation of apoptosis as cellular defense.
Rapisarda et al. 2009	Paving bitumen fumes Pavers (16), controls (10)	Overexpression of TRAIL (apoptosis ligand), DRS death receptor, caspse-9 and enhanced TUNEL (terminal deoxynucleotydl transferase); mediated dUTP nick-end labeling positively	Klimisch 4. Limited characterization of bitumen. Results suggest bitumen fume induces activation of apoptosis as cellular defense.
Neghab et al. 2016	Paving asphalt Pavers (80), Controls (130) single source sample Turkey	Statistically significant association between exposure to asphalt fumes and changes in blood count parameters, liver and kidney function tests at non-pathological, subclinical levels.	Klimisch 3. Adequate subject size, personal monitoring (particulates, benzene soluble fractions), Confounders not addressed. Urinary 1-OHP not monitored. Uncertain of relevance for health effects as values within normal ranges.

Bitumen exposure to the human skin can activate protein sufficient to induce apoptosis as a defense mechanism against skin disease and potential skin cancer. Loreto and colleagues used histochemical techniques to examine skin biopsies from a small group (16) of nonsmoking road paving workers with an average occupational exposure to bitumen emissions of 13.3 ± 5.7 years, matched with 10 unexposed controls. Chemical characterization of the paving bitumen was not reported. Results of the biopsies demonstrated statistically significant (p < .05) changes such as upregulation of heat-shock protein 27 (HSP 27) a cellular mechanism that protects against stress factors in inflammatory disease (Fenga et al. 2000), overexpression of cytokeratin protein, and BAX (a pro-apoptotic peptide) and under expression of BEL-2 (an anti-apoptotic peptide), all components of an intrinsic pathway of apoptosis (Loreto et al. 2007). From the same specimens, Rapisarda et al. 2009 identified statistically significant overexpression of components of an extrinsic pathway of apoptosis, the tumor necrosis factor related apoptosis inducing ligand [TRAIL] and its death receptor [DR5], caspase-3, and enhancement of terminal deoxynuceleotidyl transferase-mediated dUTP nick ending labeling [TUNEL]. All workers wore protective clothing, gloves and safety shoes, but bitumen emissions were still absorbed sufficiently to induce statistically significant changes in the protein and ligand levels. In addition to bitumen, subjects were exposed to polymers, solvents, oils, sand and gravel, crushed rocks, mineral wad, ultraviolet light and heat. These exposures appear to stimulate the skin to induce apoptotic mechanisms as protective responses to insult; such programed cell death could also eliminate fixation of genetic damage leading to mutation. Exposure to such a variety of materials makes it difficult to directly link changes in these mechanisms to bitumen alone.

Neghab et al. 2016 reported that occupational exposure of Iranian paving workers (80 exposed, compared to 130 nonexposed subjects) to levels of total particulate (0.9 mg/m³) and benzene-soluble fractions (0.22 mg/m³) of bitumen emissions below the ACGIH 2012 threshold limit values is associated with nonpathological, subclinical hematologic, liver and kidney changes that may be early indicators of potential organ dysfunction. Limiting factors may be that blood samples were taken prework shift but not postwork shift and no analysis of urinary 1-OH pyrene, an internal biomarker of PAC-absorbed dose, commonly used to confirm exposure in bitumen workers was reported. All elevated values fell within the generally acceptable ranges reported for human studies, which may also suggest that these results are a general response to exposure to any xenobiotic.

Most of these studies listed in Table 8 have designations of unreliable (Klimisch 3) or not assignable (Klimisch 4) primarily due to limited characterization of samples, lack of exposure data and details on confounders, but the data provided are useful for metabolic research.

With much of the work addressing PAC mechanisms performed with individual well-characterized compounds, it is tempting to use additivity as the method for determining hazard from these complex substances. Recently, toxicogenomics has been used to address mode of action and points of departure for human health risk assessments. Labib et al. (2016 manuscript submitted; Labib, 2016 thesis) exposed MutaTM Mouse by oral gavage for 28 days to coal tar extract, or to a PAC mixture of 4 or 8 PACs found in coal tar. Three days after termination of exposure, microarrays were prepared to identify genes differentially expressed in lung tissue; cancer related pathways were identified and specific dose-response modeling was conducted to calculate gene/pathway benchmark doses [BMD]. Pathway BMD derived from coal tar were comparable to BMD from published coal tar-induced mouse lung incidence data but concentration addition modeling overestimated responses. Although this study addressed coal tar components, use of toxicogenomics with appropriate modeling could be useful in evaluating complex PAC substances and bitumen emissions concentrate specifically.

Discussion and conclusions

Evaluating the health hazards of complex petroleum substances presents a significant challenge. The compositional complexity and the physicochemical properties of the substance, coupled with differing release/emission profiles for constituents under varying use or environmental conditions add to the complexity.

Researchers have for decades investigated the carcinogenic/genotoxic potential of bitumen and bitumen emissions, primarily due to concerns about the presence of and thus potential exposure to trace levels of carcinogenic/genotoxic PACs in the bulk bitumen. As a result, a wealth of peer-reviewed published data is available.

We have applied an analytical approach to the review of key studies (animal, experimental/human genotoxicity and mechanistic) to evaluate the potential cancer hazard of bitumen and its emissions. This approach, focusing on use of high quality data that are both biologically significant and relevant to human health, seeks to address, at least in part, some of the concerns expressed by other authors (Goodman and Lynch 2017), on the inappropriate use of mechanistic data in overall cancer hazard evaluation.

Cancer induction from dermal exposure to bitumen and its emissions has been studied in animals for many years, but many of these studies are acknowledged to be of poor quality (IARC 1985, 2013). In 2013, IARC concluded that there was inadequate evidence in experimental animals for the carcinogenicity of straight-run bitumen and fume condensates. For oxidized bitumen, they concluded there was limited evidence, while for oxidized bitumen condensates the evidence of carcinogenicity was considered sufficient. In their monograph, IARC also highlighted the importance of application temperature in determining the amount and composition of emissions during use and dilution in solvents, both of which significantly influence bioavailability and potential health outcomes.

The results of well conducted and validated dermal studies by Clark et al. 2011 and Freeman et al. 2011, are consistent with IARC’s conclusions – straight run paving bitumen condensate was not carcinogenic to mouse skin whereas oxidized roofing bitumen condensate was weakly carcinogenic and shown to be an initiator of dermal tumorigenesis. Moreover, these studies demonstrated that carcinogenic effects are correlated with composition, temperature at which emissions were generated, dermal irritation, duration and frequency of exposure.

Experimental (in vitro and in vivo) and human studies indicate that dermal exposure can induce genetic damage in peripheral blood cells and alter protein and gene expression in the skin, in genes involved in cell death, cell growth, proliferation and transformation. Despite these “indicators”, a significant incidence of skin cancers was not identified in animal studies with straight run paving bitumen condensate and only weak activity was seen following exposure to roofing fume condensate. Furthermore, an increased incidence of skin tumors was not observed in the IARC Multicenter epidemiology case-controlled cohort study (Olsson et al. 2010).

The effects of inhalation exposure to bitumen emissions has been investigated in a well conducted and validated rat study (Fuhst et al. 2007). The applicability of this study for evaluating carcinogenic/genotoxic potential was enhanced by the inclusion of additional endpoints, including DNA adducts, urinary PAC metabolites, micronuclei formation and altered gene expression (Halter et al. 2007). Despite evidence of PAC uptake and metabolism, altered gene expression, and DNA adducts (respiratory tract), no evidence of micronuclei formation, oxidative DNA damage or an increase in overall or organ-specific neoplasms was observed. Under the conditions of study, investigators concluded that repeated inhalation exposure to bitumen emissions was not carcinogenic to rats.

In their final evaluation (IARC 2013) IARC considered “mechanistic and other relevant data” from studies in exposed paving workers to provide “strong” evidence for a genotoxic mechanism, despite there being inadequate evidence of carcinogenicity in experimental animals and in exposed human populations. The mechanistic data were used to elevate bitumen used in paving from Group 3: “not classifiable as to its carcinogenicity to humans’ to Group 2B: ‘possibly carcinogenic to humans”.

Our review of published in vitro and in vivo genotoxicity studies with bitumen has, however, shown variable, and often conflicting results. The IARC subgroup review of mechanistic data focused on identifying “positive” findings, but critical evaluation of individual studies suggests that the mixed picture of positive and negative results likely reflects differences in test material chemical composition (due to higher fume generation temperature or agitation or both), thus limiting the usefulness of most studies for predicting genotoxicity hazard under normal occupational field conditions.

Several genotoxicity markers, including DNA adducts/strand breaks, oxidative DNA damage, micronuclei formation, chromosomal aberrations and sister chromatid exchanges, have been investigated in studies of worker populations exposed to bitumen emissions. Many of these studies were of limited utility because of experimental design deficiencies such as small study population size, no apparent control for confounding coexposures such as diesel exhaust, diesel fuel, organic solvents, smoking etc., thus limiting the reliability of conclusions reached about the relationship between adverse health effects and exposure to bitumen emissions. As part of our analysis, modified Klimisch ‘quality’ ratings have been adapted and assigned to each of the reviewed human mechanistic studies (Supplemental materials), resulting in only a limited number being considered adequate for assessing the genotoxicity potential of bitumen or bitumen emissions.

As part of a well-conducted, extensive study of German workers exposed to bitumen emissions during mastic asphalt application, Marcyznski et al. 2011 reported elevated levels of DNA strand breaks and 8-oxo-dG adducts when compared to reference (unexposed) construction workers, but these elevated levels were within the range of nonexposed healthy individuals. Furthermore, the authors concluded “no positive association between DNA damage and the magnitude of bitumen emission exposure or between urinary metabolites and DNA damage in the blood” was observed. In a follow-up micronucleus study (Welge et al. 2011), comparable results were reported for mastic workers and reference controls pre- and postshift, indicating a lack of cytogenetic damage.

In summary, several critical factors for evaluating data quality have been identified and systematically applied to the review of data available on the carcinogenic and genotoxic hazards of the complex petroleum substances, bitumen and bitumen emissions. Data from well-conducted and validated animal and human studies have been evaluated to assess the hazards to workers posed by exposure to bitumen and bitumen emissions. Overall, the data available for bitumen used in paving operations indicate that exposure to bitumen emissions does not present a carcinogenic or genotoxic hazard under normal operating conditions. On the other hand, application of oxidized bitumen, at higher temperatures in roofing, results in increased bitumen emissions coupled with a slightly different chemical composition including proportionally more 4–6 ring PACs. The experimental data from the available studies indicate that oxidized bitumen emissions are potentially weakly carcinogenic.

While recognizing the importance of in vitro and in vivo genotoxicity data in the identification of potential hazards and possible mechanisms of action, individual studies must be evaluated critically to identify relevant and scientifically robust data. Our analysis also suggests that the primary use of such data should be in support of, rather than driving hazard conclusions. This is particularly important, where good quality animal or human evidence suggests low or negligible cancer hazard.

Furthermore, Rhomberg et al. 2015, using quantitative risk assessment methodology, concluded that the cancer risk from occupational exposure of roofing workers to built-up roofing asphalt [BURA] emissions is within a range “typically deemed acceptable within regulatory frameworks” (Rhomberg et al. 2015).

Declaration of interest

The employment affiliation of the authors is as shown on the cover page. This paper was prepared with some financial support from the Asphalt Institute (AI), Lexington Kentucky. AI is an international trade association promoting the quality use of asphalt through their members, training and research (http://www.asphaltinstitute.org/). Heritage Research Group authors received no funding for their contributions. The two consulting authors were compensated by AI for their time devoted to this publication. Although an early draft was reviewed by AI’s HSE committee members, the authors have the sole responsibility for the writing and contents of this paper.

Anthony J. Kriech is VP and Director of Research at Heritage Research Group (HRG) in Indianapolis, IN and a long-time member of the HSE committee for AI. He has published numerous studies on bitumen emissions many of them sponsored by the Asphalt Institute and has presented on numerous occasions around the world on this topic. These included a series of studies sponsored by the National Asphalt Pavement Association (NAPA). HRG, a private, for profit business, is part of The Heritage Group which includes Asphalt Materials, Inc and a diverse set of national and international companies involved in highway construction and materials, environmental services, petroleum refining and chemical manufacturing. Tony has represented the Asphalt industry in many capacities, including as an expert witness for Prop 65 in 2017, and as an industry observer in October 2011 at the IARC Working Group meeting for Monograph 103 evaluating the carcinogenic risks of bitumen and bitumen emissions.

Ceinwen A. Schreiner, Ph.D., ATS is retired from C&C, Consulting in Toxicology, Inc., a consulting group with expertise in petroleum and petrochemical toxicology and environmental safety working with businesses, trade organizations and regulatory agencies to address health effects from exposure to a range of petroleum and products for 17 years. She retired from Mobil Corporation in 2000 with 20 years of service. Dr. Schreiner co-authored the Closure Assessment Document on Asphalts (2010) for the EPA High Production Volume testing program and the subsequent publications. Dr. Schreiner provided a review of reproductive and mechanistic studies for the Asphalt Institute in preparation for their observer role at the IARC Working group meetings on October 2011 (monograph 103) evaluating carcinogenic potential of bitumen and bitumen emissions.

Linda V. Osborn has been a researcher with HRG for 22 years and an analytical chemist with Heritage Environmental Services for 11 years prior. Currently Senior Project Chemist, Linda has designed, implemented, and published numerous bitumen emissions related studies often working in conjunction with government and academia. These included studies sponsored by AI and NAPA. Specialties include analytical chemistry and industrial hygiene related to bitumen emissions. Linda was a contributor to a joint publication between AI and Eurobitume, The Bitumen Industry – A global Perspective: Production, chemistry, use, specification and occupational exposure 3rd edition.

Anthony J. Riley is a retired Senior Toxicologist, having worked in the oil and petrochemical industry for 37 years. A substantial period was spent working for BP, the UK based Oil Company, including work on evaluating health hazards and risks associated with the production and use of bitumen. This included representing the European industry in meetings with regulatory authorities and other scientific bodies, including as an Observer at the IARC Working Group meeting in October 2011 (Monograph 103) evaluating the carcinogenic risks of bitumen and bitumen emissions.

Supplemental material

Supplemental data for this article can be accessed here.

Acknowledgements

The authors gratefully acknowledge the comments of six reviewers selected by the Editor who were anonymous to the authors. The comments helped improve the clarity of the paper. We thank Jacqueline Bartek for her assistance in literature searching, procurement, and distribution.

Notes

Supplemental data for this article can be accessed here.