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

Figures & data

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.)

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

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]

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)

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

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.

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.

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.

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