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Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

ther oxygenate additives in gasoline reduce toxicity of exhausts

.A. Westphala,∗, J. Krahlb, T. Brüninga, E. Hallierc, J. Büngera

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance - Institute of the Ruhr-University Bochum (IPA),ürkle-de-la-Camp-Platz 1, 44789 Bochum, GermanyUniversity of Applied Sciences, Friedrich-Streib-Strasse 2, 96450 Coburg, GermanyInstitute of Occupational and Social Medicine, Georg-August-University Göttingen, Waldweg 37, 37073 Göttingen, Germany

r t i c l e i n f o

rticle history:eceived 6 November 2009eceived in revised form4 December 2009ccepted 15 December 2009vailable online xxx

eywords:asoline engine emissionsther oxygenatesutagenicity

ytotoxicity

a b s t r a c t

Fuel additives can improve combustion and knock resistance of gasoline engines. Common additivesin commercial fuels are “short-chain, oxygen containing hydrocarbons” such as methyl tert-butyl ether(MTBE) and ethyl tert-butyl ether (ETBE). Since these additives change the combustion characteristics,this may as well influence toxic effects of the resulting emissions. Therefore we compared toxicity andBTEX emissions of gasoline engine exhaust regarding addition of MTBE or ETBE.

Non-reformulated gasoline served as basic fuel. This fuel was supplemented with 10%, 20%, 25% and 30%ETBE or 15% MTBE. The fuels were combusted in a gasoline engine at idling, part load and rated power. Con-densates and particulate matter (PM) were collected and PM samples extracted with dichloromethane.Cytotoxic effects were investigated in murine fibroblasts (L929) using the neutral red uptake assay andmutagenicity using the bacterial reverse mutation assay. BTEX emissions were analyzed by gas chro-matography.Results: PM-extracts showed mutagenicity with and without metabolic activation. Mutagenicity was

reduced by the addition of MTBE and ETBE, 10% ETBE being most effective. The condensates produced nosignificant mutagenic response. The cytotoxicity of the condensates from ETBE- and MTBE-reformulatedfuels was reduced as well. The BTEX content in the exhaust was lowered by the addition of MTBE andETBE. This effect was significantly related to the ETBE content at rated power and part load.Conclusions: Addition of MTBE and ETBE to fuels can improve combustion and leads to decreased toxicityand BTEX content of the exhaust. Reduction of mutagenicity in the PM-extracts is most probably caused

ycycl

by a lower content of pol

. Introduction

A considerable part of environmental air pollution and a broad

Please cite this article in press as: Westphal, G.A., et al., Ether oxygenatedoi:10.1016/j.tox.2009.12.016

pectrum of health hazards arise from motor vehicle emissionsCaprino and Togna, 1998). This prompted strong legislative effortso reduce vehicle exhaust. Significantly reduced traffic emissionsere achieved by new engine technologies, exhaust aftertreat-

Abbreviations: ACGIH, American Conference of Governmental Industrial Hygien-sts; BTEX, benzene, ethyl-benzene, toluene, and xylene; CO, carbon monoxide;MSO, dimethyl sulfoxide; EPA, Environmental Protection Agency; ETBE, 2-thoxy-2-methylpropane (tert-butyl ethyl ether); GC, gas chromatography; HC,otal hydrocarbons; IARC, International Agency for Research on Cancer; MTBE,-methoxy-2-methylpropane (tert-butyl methyl ether); NOX , nitrogen oxides par-icular; NRU, neutral red uptake; NTP, National Toxicology Program; FID, flameonization detector; PAH, polycyclic aromatic hydrocarbons; PM, particulate matter;pm, parts per million (equivalent to ml/m3).∗ Corresponding author. Tel.: +49 234 3024577; fax: +49 234 3024505.

E-mail addresses: westphal@ipa-dguv.de (G.A. Westphal), krahl@hs-coburg.deJ. Krahl), bruening@ipa-dguv.de (T. Brüning), ehallie@gwdg.de (E. Hallier),uenger@ipa-dguv.de (J. Bünger).

300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2009.12.016

ic aromatic hydrocarbons.© 2009 Elsevier Ireland Ltd. All rights reserved.

ment, and newly developed, reformulated fuels. However, onlya part of the exhaust compounds are legally regulated, such asnitrogen oxides (NOX), carbon monoxide (CO), total hydrocarbons(HC), and particulate matter (PM). In particular, carcinogenic com-pounds such as benzene and polycyclic aromatic hydrocarbons(PAH) remain unregulated. A screening of possible hazards fromexhaust can be performed by short-term tests. Since a broad spec-trum of health hazards are concerned, various methods have to beapplied to cover the range of toxic effects. Toxicity of exhaust from“normal and high emitter gasoline and diesel vehicles” for exam-ple showed strong differences concerning mutagenic effects in vitroand toxic effects in the lungs of instilled rats, yet there was no cor-relation between mutagenicity in the bacterial reverse mutationassay and lung toxicity (Seagrave et al., 2002, 2003). It was shownthat the compounds which were associated with pulmonary toxic-

additives in gasoline reduce toxicity of exhausts. Toxicology (2010),

ity were different from those compounds which induced mutageniceffects (such as nitro-PAH, oxy-PAH and quinones) (McDonaldet al., 2004). This shows that cytotoxicity and mutagenicity aresuitable endpoints for a comparative investigation of differentexhausts.

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Table 1BTEX content of basic fuel and fuel containing MTBE.

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2.5. PM extraction

Extraction of the soluble organic fraction of the filters was done with 150 mldichloromethane (Merck, Darmstadt, Germany) using a soxhlet apparatus (Brand,Wertheim, Germany) for 12 h in the dark (cycle time 20 min at 65 ◦C). The extractswere concentrated by rotary evaporation (Heidolph, Kehlheim, Germany) to a

Table 2Operating parameters of the 3-point tests.

Benzene [%] Toluene [%]

Basic fuel 0.9 10Optimax® <0.5 17.4

Fuel additives are a promising approach to reduce gaso-ine engine emissions. Ether oxygenates, such as MTBE (methylert-butyl ether, 2-methoxy-2-methylpropane, CAS 1634-04-4,C 216-653-1) or ETBE (ethyl tert-butyl ether, 2-ethoxy-2-ethylpropane CAS 637-92-3, EC 211-309-7) increase the oxygen

ontent of the fuel, enhance the efficiency of combustion, andeduce emissions of CO, HC and benzene (Health Effects Institute,996). This so-called reformulation of fuels may also influencehe non-regulated exhaust compounds and lead to altered toxicffects. MTBE is manufactured from methanol and isobutylene.he production capacities in Europe amount to approximately 4illion tons MTBE and 1 million tons ETBE per annum (Koenen

nd Püttmann, 2005). Since huge amounts of fuels are combusted,hanges in the toxicity of exhausts from reformulated or alternativeuels and additives may generate new hazards to humans and to thenvironment. Therefore the development of new fuels and addi-ives must be accompanied by research to identify possible healthazards (Swanson et al., 2007).

Increased ambient MTBE exposure was measured at gasolinetations and among workers who were employed in the man-facture and transport of MTBE (Health Effects Institute, 1996).iomonitoring showed increased levels of MTBE in tank truckrivers in Finland (Vainiotalo et al., 1998). High levels of MTBEere discovered in the water wells and in the groundwater of many

egions in North America (Brown, 1997).Several reports on toxic MTBE effects are available. Concentra-

ions at and above 30 �g/L MTBE have a strong unpleasant odor andxposed individuals reported headache, nausea, and sensory irri-ation (Health Effects Institute, 1996). Nevertheless, no increase ofeurobehavioral or neurophysiologic effects could be substantiated

n MTBE exposed subjects (Fiedler et al., 2000).MTBE was in the focus of public interest following reports on

arcinogenic effects in experimental animals (Mehlman, 2002; Deeyster et al., 2003; Belpoggi et al., 1995; Phillips et al., 2008).owever possible carcinogenic effects of MTBE are still a matter ofebate: according to the National Toxicology Program (NTP) there

s “some evidence for carcinogenicity” of MTBE. NTP has not recom-ended MTBE for listing in the “Report on Carcinogens Nominatedgents” (NTP, 2005). The American Conference of Governmental

ndustrial Hygienists (ACGIH) (2008) classified MTBE as “confirmednimal carcinogen with unknown relevance to humans”. Accord-ng to the International Agency for Research on Cancer (IARC, 1999)here is “limited evidence of carcinogenicity” of MTBE in experi-

ental animals.These known or suspected health hazards caused in part

replacement of MTBE by ETBE. It was speculated that theower volatility and water solubility of ETBE compared to otherxygenates would help minimize possible environmental pollu-ion. Studies on mutagenicity of ETBE yielded negative resultsMcGregor, 2007). Studies on the metabolism of MTBE and ETBEuggest that no reactive or potentially toxic metabolites are formeduring biotransformation of these ethers (Dekant et al., 2001;cGregor, 2006, 2007).Despite numerous reports on the toxicology of ETBE and MTBE

Please cite this article in press as: Westphal, G.A., et al., Ether oxygenatedoi:10.1016/j.tox.2009.12.016

hemselves, we found no studies that dealt with the alteration ofngine exhaust caused by fuel reformulation with MTBE or ETBE.his induced us to investigate the mutagenic and cytotoxic effectsf PM and condensates which are produced during combustion ofuels containing ETBE and MTBE. Since separate chemical analysis

Ethyl-benzene [%] Xylene [%] � BTEX [%]

2.7 11.9 44.30.3 2.8 Max. 42

of all exhaust compounds is impracticable, we assessed the cyto-toxic and genotoxic effects of the whole emissions using in vitroassays as a screening tool. MTBE was proposed to reduce BTEX emis-sions (benzene, ethyl-benzene, toluene, and xylene) of gasolineengines (Health Effects Institute, 1996). Therefore we additionallyanalyzed the BTEX content of the exhaust with particular respectto the carcinogen benzene which is not mutagenic in any in vitrogenotoxicity assay (Westphal et al., 2009).

2. Materials and methods

2.1. Fuels

“Shell, ROZ 95®” served as basic fuel. This basic fuel was reformulated with 10%,20%, 25% and 30% ETBE. Additionally, a fuel was investigated containing 15% MTBE(Optimax® , ROZ 99, Shell AG, Hamburg, Germany). BTEX content of the basic fueland the fuel reformulated with 15% MTBE are compiled in Table 1. Biological effectswere measured for basic fuel, 10%, and 20% ETBE only.

2.2. Combustion

We used a spark ignition engine (GM, Opel Astra 1.6i, type C16NZ, four cylinders,66 kW) which was equipped with a 3-way catalytic converter. The load was suppliedby coupling the engine to a dynamometer. The investigations were performed atthree different load modes (idling power, part load and rated power). The biologicalassays were only performed with exhaust samples yielded at rated- and idling power(Table 2).

2.3. Sampling of condensates and PM

Exhausts were sampled directly at the tail pipe. The exhaust was cooled to lowerthan 20 ◦C and semivolatile compounds were sampled as condensates. PM was sam-pled onto glass fibre filters coated with PTFE (Teflon) (T60 A20, Pallflex ProductsCorp., Putnam, CT, USA). Filters were conditioned at 20 ◦C and 50% relative humid-ity and weighed before and after sampling to determine total PM. Used filters andcondensates were stored at 4–8 ◦C. Each of the six different fuels was tested in trip-licate at both load modes, resulting in 54 test samples each (54 particle filters and54 condensates).

2.4. Measurement of BTEX emissions

For the analysis of benzene [CAS 71-43-2], ethyl-benzene [CAS 100-41-4],toluene [CAS 108-88-3], and xylene [CAS 1330-20-7] samples were taken directly atthe tail pipe after reduction of the temperature to lower than 150 ◦C. Measurementwas done online using a gas chromatograph (GC) (Varian Star 3600 CX, Darmstadt,Germany) and flame ionization detection (FID). GC analysis was done using a CP-Sil5CB column (Chrompack, Engstingen, Germany). Hydrogen 5.0 was used as carriergas at a flow rate of 5 ml/min. Septum flow rate was 2 ml/min. The sample wasinjected into a 0.25 ml sample loop with a split ratio of 5. Injector temperature wasset to 150 ◦C and detector temperature to 250 ◦C. The following temperature pro-gram was applied: the initial temperature of 50 ◦C was held for 1 min. The initialrate of temperature increase was 5 ◦C/min up to 125 ◦C, followed by 25 ◦C/min up to200 ◦C.

additives in gasoline reduce toxicity of exhausts. Toxicology (2010),

Revolutions perminute [rpm]

Torsional [Nm] Power [kW]

Rated power 3600 79.6 31.6Part load 2500 44.2 116Idling power 2050 1.3 2.8

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Fig. 1. GC/FID analysis of benzene emissions at rated power, part load, and idlingpower. Basic fuel (0% ETBE) was reformulated with 10%, 20%, 25% and 30% ETBE. Inaddition benzene emissions resulting from the combustion of commercially avail-able fuel which contained 15% MTBE are shown. Each data point represents the

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olume of 10 ml, divided into two 5 ml aliquots, and dried under a stream of nitro-en.

.6. Cell and bacterial cultures

Media and chemicals for cell cultures were obtained from ICN BiomedicalsMeckenheim, Germany) and Fetal Calf Serum from Biochrom (Berlin, Germany).utrient media and most chemicals for the bacterial reversion test were purchased

rom Difco Laboratories (Detroit, USA) and Sigma/Aldrich (Deisenhofen, Germany).eutral red dye (toluylene red) [CAS 553-24-2], methyl methanesulfonate [CAS 66-7-3], 2-aminofluorene [CAS 153-78-6], �-naphthoflavone [CAS 6051-87-2], andhenobarbital [CAS 50-06-6] were obtained from Sigma/Aldrich. Dimethyl sulfox-

de (DMSO), spectrometric grade, [CAS 67-68-5] was provided by Merck (Darmstadt,ermany).

The bacterial reverse mutation assay (Ames test) was performed according tohe revised standard protocol (Maron and Ames, 1983) with the tester strains TA98nd TA100. These strains have been shown to be particularly sensitive for mutagensf organic extracts of diesel and gasoline engine particles (Clark and Vigil, 1980;laxton, 1983). Tests were performed with and without metabolic activation by aicrosomal mixed-function oxidase system (S9 mix).

PM-extracts were re-dissolved in 4 ml DMSO immediately before use. The fol-owing dilutions were applied: 1.0, 0.5, 0.25, and 0.125, corresponding to 100, 50, 25,nd 12.5 l of exhaust. Every concentration was investigated in duplicate both withnd without 4% S9 mix. Reverse mutations (revertants) were recorded after 48 hncubation in the dark at 37 ◦C. Counting was performed using an electronically sup-orted colony counting system (Cardinal, Perceptive Instruments, Haverhill, Greatritain).

Acceptance criteria for a positive test result were solvent control and positiveontrols within the range of published data and an at least twofold dose-dependentncrease of spontaneous mutations (Maron and Ames, 1983; Mortelmans andeiger, 2000). Methyl methanesulfonate (MMS: 10 �g/ml in aqua dest.) and 2-minofluorene (2-AF: 100 �g/ml in DMSO) served as positive controls.

The neutral red uptake (NRU) assay was performed according to Borenfreundnd Puerner (1984). In brief, L929 mouse fibroblasts were used, revealing at least5% viability according to trypan blue staining. 10,000 cells/0.2 ml medium per wellere seeded in 96-well plates and incubated for 24 h to achieve approximately 70%

onfluence. After 24 h the medium was replaced by the extracts which were freshlyiluted in 0.2 ml serum-free medium containing 2% DMSO. The corresponding stockolutions were 100% toxic and served as positive controls and for the determinationf non-specific staining. Following incubation for another 24 h the cells were washednd lysed by the use of 0.5% formaldehyde and 1% CaCl2. The dye was extracted (1%lacial acetic acid, 50% ethanol), and NR uptake was analyzed in a microplate readert 540 nm (Spectra SLT, Tecan, Crailsheim, Germany). Each concentration was testedn 8 wells/plate on two 96-well plates.

.7. Statistics

The differences between the results from reformulated fuels and the basic fuelere calculated using two-sided Student’s t-test. The standard deviation (SD) and

he regression coefficient (R) were calculated using standard software (MS Excelersion 1997–2003).

. Results

.1. BTEX emissions

Benzene emissions were significantly reduced by the additionf ETBE at rated power and part load. A linear trend concerning theelationship between the ETBE content in the fuels and the benzeneontent in the corresponding exhausts was apparent. A reductiony 35% in the exhaust compared to the basic fuel was achieved byhe addition of 30% ETBE at rated power and a reduction by 15%t part load. At idling power no clear trend was observed concern-ng BTEX and in particular concerning benzene emissions. HoweverTEX emissions at this load are relatively low anyway (Fig. 1).

Toluene, ethyl-benzene and xylene emissions were reduced byhe addition of ETBE at rated power up to 40%, 30% and 91%, respec-ively. This effect depended linearly on the ETBE content. A similar

Please cite this article in press as: Westphal, G.A., et al., Ether oxygenatedoi:10.1016/j.tox.2009.12.016

ut less clear effect was seen at part load: toluene, ethyl-benzenend xylene were reduced by up to 35%. This was clearly related tohe ETBE content in the case of toluene and xylene. We observedo clear trend concerning BTEX emissions at idling power (data nothown).

mean of three independent samplings and measurements. R = regression coefficient,SD = relative standard deviation, P = level of significance.

3.2. Bacterial reverse mutation assay

PM-extracts induced mutagenic effects in TA98 and TA100dose dependently (Figs. 2–5). At rated power mutagenic effectswere considerably stronger without metabolic activation (Fig. 3)compared to the experiments which were performed with theaddition of S9 mix (Fig. 2). A reduced mutagenicity of the PM-extracts was achieved with both oxygenates. Addition of 10%ETBE was apparently most effective. The number of reverse muta-tions which were induced in TA98 by the non-reformulated basicfuel was nearly up to sixfold higher compared to the fuel con-

additives in gasoline reduce toxicity of exhausts. Toxicology (2010),

taining 10% ETBE (Fig. 3). Condensates showed no mutageniceffects according to the acceptance criteria of the assay (data notshown).

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Fig. 2. Mutagenicity of PM-extracts which were sampled at rated power. The bacterial reversion assay was carried out using TA98 and TA100 with metabolic activation.Reverse mutations induced by PM-extracts which were obtained following 398 combustion at rated- and idling power of fuels which contained 0% (basic fuel), 10% and 20%ETBE and 399 15% MTBE. Two independent experiments each were performed.

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ig. 3. Mutagenicity of PM-extracts which were sampled at rated power. The bacteeverse mutations induced by PM-extracts which were obtained following 398 comTBE and 399 15% MTBE. Two independent experiments each were performed.

.3. Neutral red uptake assay

The cytotoxicity of the condensates at idling and rated poweraried according to the ETBE contents of the corresponding fuels.0% ETBE reduced toxicity most effectively. However toxicity var-

ed in a relatively narrow range between 35–80 ml at idling and0–90 ml at rated power. We were not able to generate PM-extractshich were concentrated enough for the NRU assay, since L929

Please cite this article in press as: Westphal, G.A., et al., Ether oxygenatedoi:10.1016/j.tox.2009.12.016

ells do not tolerate more than 2% DMSO or other solvents. Pre-iminary data from assays with low concentrations suggest thatytotoxicity of the particles is about one order of magnitude lowerhan the ED50 of the condensates (data not shown), but this effect

ay be influenced by the poor solubility of the extracts.

ig. 4. Mutagenicity of PM-extracts which were sampled at idling power. The bacterialeverse mutations induced by PM-extracts which were obtained following 398 combustiTBE and 399 15% MTBE. Two independent experiments each were performed.

version assay was carried out using TA98 and TA100 without metabolic activation.on at rated- and idling power of fuels which contained 0% (basic fuel), 10% and 20%

4. Discussion

Despite the widespread prevalence of gasoline engine emissionsin the environment, and numerous studies – which were mostlyconducted prior to 1980 – relatively little is known as to theirtoxicity. Recent studies point towards an involvement in devel-opment of allergic diseases, cardiovascular effects and oxidativestress (McDonald et al., 2007). The benzene and PAH content of the

additives in gasoline reduce toxicity of exhausts. Toxicology (2010),

exhaust is suspected to cause genotoxic and possibly carcinogenichealth effects. Improvements in the combustion process may helpto reduce health hazards from the exhaust.

According to our data a significant reduction of BTEX emis-sions and of mutagenic effects of PM-extracts from gasoline engine

reversion assay was carried out using TA98 and TA100 with metabolic activation.on at rated- and idling power of fuels which contained 0% (basic fuel), 10% and 20%

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ig. 5. Mutagenicity of PM-extracts which were sampled at idling power. The bacteeverse mutations induced by PM-extracts which were obtained following 398 comTBE and 399 15% MTBE. Two independent experiments each were performed.

xhaust can be achieved by the addition of ETBE or MTBE. Themount of BTEX emissions was related to the concentration ofTBE in the corresponding fuels at rated power and at partoad.

A reduction of BTEX emissions is highly desirable. Environmen-al benzene exposure is a particular concern (Mehlman, 2004). Thiseed has recently been stressed by two independent Europeantudies that showed an association of traffic related benzene expo-ure with childhood leukemia (Crosignani et al., 2004; Steffen etl., 2004). Acute hematotoxicity in adults was reported at atmo-pheric concentrations below 1 ppm benzene (Lan et al., 2004). Lesslear are the consequences of environmental exposure towardslkylbenzenes. Neurotoxic, fetotoxic and teratogenic effects areiscussed (Caprino and Togna, 1998). According to our data, addi-ion of ETBE and MTBE seems to achieve a reduction of BTEXmissions and in particular a reduction of the benzene con-ent.

The reduction of mutagenic effects of the particle extracts wasost pronounced at 10% ETBE content. No mutagenicity occurred

n the corresponding condensates. Previous studies show thatutagenic effects of engine emissions are mainly caused by PM-

ssociated PAH, including nitro- and oxy-PAH. Traffic is knowno be a major source of PAH emissions (Larsen and Baker, 2003).AH mainly occur as co-pollutants with PM during combustionrocesses in engines (Spencer et al., 2006) and are consequentlyreferentially found particle bound (Westerholm et al., 2001; Bentet al., 2009). Accordingly our own and previous data show thatutagenic effects of gasoline engine exhaust are rather associatedith PM than with condensates.

The strongest mutagenicity occurred at rated power withoutetabolic activation. Similar results for gasoline engine exhaustere reported previously. Yang et al. (2008) for example reported

M2.5 associated mutagenic effects of motor cycle exhausts withnd without addition of S9 mix. Direct mutagenic effects of the par-icle extracts which were seen without addition of S9-mix pointowards the occurrence of oxy- and nitro-PAH in the exhaustMcDonald et al., 2004; Liu et al., 2005; Bamford et al., 2003; Arlt,005; Havey et al., 2006; Heeb et al., 2008). However, the PM-ssociated mutagenic effects of gasoline engine emissions in ourresent study are low in comparison to those which are obtainedrom diesel engine emissions although the mutagenicity of diesel

otor emissions has been strongly reduced during the last decadeBünger et al., 1998, 2006, 2007). Therefore the reduction of toxi-ity by the addition of ETBE and MTBE in our experiments is most

Please cite this article in press as: Westphal, G.A., et al., Ether oxygenatedoi:10.1016/j.tox.2009.12.016

robably caused by a reduction of the amount of PM-bound PAHnd nitro-PAH. Any initiative to reduce these emissions is worth theffort because of the strong mutagenic and carcinogenic propertiesf these compounds.

version assay was carried out using TA98 and TA100 without metabolic activation.on at rated- and idling power of fuels which contained 0% (basic fuel), 10% and 20%

5. Conclusions

Additives can improve the combustion process. In case of ETBEand MTBE this leads to a reduction of PM-associated mutageniceffects and BTEX in the exhausts. The reduction of mutagenicitydepends on the additive and its proportion in the fuel. Direct muta-genic effects are most probably caused by PM-bound nitro-PAH.Risk assessment of PM should therefore include particle bound PAHand nitro-PAH. The bacterial reverse mutation assay is shown to bean appropriate, quick and cost effective method to evaluate thetoxicity of the exhaust.

Conflict of interest statement

All authors declare that they disclose any actual or potentialconflict of interest including any financial, personal or other rela-tionships with other people or organizations within three (3) yearsof beginning the work submitted that could inappropriately influ-ence (bias) their work. This includes employment, consultancies,stock ownership, honoraria, paid expert testimony, patent applica-tions/registrations, and grants or other funding.

Acknowledgements

The authors gratefully acknowledge the grant by the Bun-desministerium für Bildung und Forschung (BMBF, Federal Ministryof Education and Research), Friedrichstraße 130 B, 10117 Berlin,Germany. Support Code 1705999.

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