Characterization of Automotive Emissions by Bacterial Mutagenesis Bioassay: a Review


EPA 600/J-83-096
                                                                      PB84-117407
       Characterization of Automotive Emissions by
       Bacterial  Mutagenesis Bioassay:  A Review
        (U.S.) Health Effects Research  Lab.
       Research  Triangle Park, NC
       1983
    U.S. Department of Commerce
    ISstkmsl Technkal infermatior

-------
TECHNICAL REPORT DATA
(Please read instructions on the reverse before coinplc'
I. REPORT NO.
EPA-600/J-83-096
2.
JOURNAL ARTICLE
4. TITLE AND SUBTITLE
Characterization of Automotive Emissions by Bacterial
Mutagenesis Bioassay: A Review (Journal Version)
5. REPORT DATE
1983
6. PERFORMING ORGANIZATION CODE
-11 7407
7. AUTHOR(S)
Larry D. Claxton
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Genetic Toxicology Division
Health Effects Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711
10. PROGRAM ELEMENT NO.
A9XA1C
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12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research & Development
Health Effects Research Laboratory
US Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
Published In: Environmental Mutagenesis 5: 609-631, 1983
16. ABSTRACT

Due to the growing numbers of diesel passenger autobiles in the United States, there has
been an expanded effort to understand the health effects of airborne pollutants arising
from increased automotive emissions. Bacterial mutagenicity testing has played an im-
portant role in the characterization of genotoxic effects and components arising from
these combustion products. This review examines published material concerning the
bacterial mutagenicity of automotive emissions. In addition, the paper explores factors
that modify the mutagenicity of mobile-source emissions, the use of bacterial tests for
the comparison of various mobile source emissions, and the use of bacterial tests to ex-
amine the phenomena of mammalian uptake and metabolism.
17.
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EPA-600/J-83-096
JOURNAL ARTICLE

Environmental Mutagim'sts 5:609-631 (1983)
Characterization of Automotive Emissions

by Bacterial EViutagenesss Bioassay: A

Review

Larry D. Ciaxton

Genetic Toxicology Division, U.S. Environmental Protection Agency. Research Triangle
Park. North Carolina

Due Jo the growing numbers of dicscl passenger automobiles in the United Slates.
there has been an expanded effort to understand the health effects of airborne
pollutants arising from inereased automotive emissions. Bacterial mutagenieity
testing has played an important role in the chanicieri/atioh of genotoxic effects
and components arising from these combustion products. This review examines
published material concerning the bacterial imiiagenicity of automotive emissions.
In addition. iJw paper explores factors that nuidify the mutagenicity of mobiJe-
source emissions, the use of bacterial tests lor the comparison of various mobile
source cmi>s»ons. and the use of bacterial ICM> to examine the phenomena of
mammalian uptake and metabolism.

Key words: dicscl. gasoline, Ames test. Salmoiiflh. fuel, combustion

INTRODUCTION

The United Stales has approximately 130 million passenger cars and light-duty
trucks, nearly one light-duty vehicle registered for each adult. The sales and servicing
of automobiles and trucks account for about 25 S of the US retail market [Gray and
von Hippo!. 19811. In 1980 this fleet of vehicles consumed approximately 2 billion
barrels of oil. Since engineering tests have shown a 25% or greater improvement in
fuel economy in light-duty vehicles equipped vviih dicscl engines versus those equipped
with gasoline engines, diesel vehicle sales are expected to increase from 4$ (1980)
to 159r. (1985) of the new car market. This "dieseli/ation" has sparked new interest
in the health effects of mobile-source emissions. Although earlier work JKotin ct al.
195-1, 1955) had demonstrated that diesel and gasoline emissions have potential
carcinogenic activity, it was .not until 1978 that industry and government expanded
their efforts toward understanding whether or riot mobile-source emissions could

Received July I. IVS2; revised and accepted January 13, IVS3.

Address reprint requests to Larry 1). CLixlon. Genetic Toxicology Division. U.S. Environmental
Protection Agency. Research Triangle Park. NC 27711.
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610 Claxton

show any potential genotoxic health effects. This issue received priority after Hui-
singh et al 11978| and a cautionary notice for laboratory workers issued by the US
Environmental Protection Agency (EPA) (Gage. I977J reported that organic fractions
from the exhaust particles of diescl vehicles were mutagenic in the Ames Salmonella
typhimurium plate incorporation assay (Ames et al, I975J. The purpose of this review
is to recognize and document the role that bacterial mutation tests have played in
characterizing mobile source emissions for genotoxic activity. A summary of this
review was given at the EPA 1981 Diesel Emissions Symposium held October 5-7,
1981, in Raleigh. NC (Claxton, I98lbj.
Although the original report by Huisingh et al [I978J was quite extensive-
examining, for example, chemical and physical properties of fractions from exhaust
organic*, multiple vehicles, anil multiple fuels—many questions remained unan-
swered. Researchers in government and industry-, in the service of the public interest.
have used bacterial mutagenicity tests to answer several critical questions concerning
sample generation, collection, extraction, fractior.ation, bioassay. statistical signifi-
cance, and relevance. For example, Wei et al 11980) postulated that "... controver-
sies on the biological hazards of diesel emissions will remain unresolved until more
is known about the chemical identities of the direct-acting mutagens." Fractiorwiion
directed by bacterial bioassax results enables investigators to follow the distribution
of genotoxic activity among different chemical classes before compound identification
is complete. In addition, microbia! tests allow comparative measurements of geno-
toxic activity from roadside-exhaust, smog-chamber, and dilution-tunnel samples.
The purpose of this paper, therefore, is to rccopni/.e anil document the role that
bacterial mutation tests have played in characterizing mobi1..-source emissions for
genotoxic activity. The bacterial mutagcniciiy of mobile-source emissions is consid-
ered according to the following schema: (I) generalized observations; (2) generation
of emissions: (3> collection of omissions samples: (4) cx'raction of paniculate sam-
ples*. (5) fractionation and identification of individual chemical components: (6)
applicability and relevance of bacterial bioassays; and (7) data transformation and
statistical analysis of research data,

GENERALIZED OBSERVATIONS

Huisingh et al |I978| and most other investigators have used the Salmonella
typhimurium plate incorporation assay f Amos et al. I975J a.> the primary test protocol.
Although some investigators employ all five tester strains recommended by Ames for
general screening, many investigators work exclusively with strains TA98 and TAIOO.
primarily for two reasons: First, the sample amounts available have been relatively
limited: and second. TA9X and TAIOO have been the strains mo
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Bioassay of Automotive Emissions 611

or (2) TA98 detects frameshift promutagens thai arc S9-dcactivated. which TA1538
is not capable of delecting. Researchers may thus be underestimating the importance
of indirect-acting frameshift mutagcns within mobile emissions.
Using the knowledge of mulagcns available at that time. Wei et al 11980)
surmised that nhro-substitutcd polycyclic aromatic hydrocarbons (PAH) were the
probable mutacens associated with diesel exhaust emissions. Nitro derivatives of
some PAHs have been identified in ambient air samples [Jiigcr, 1978; Wang et al,
1980; Talcott and Harger. 1981; Pitts et al. I982bj. Rosenkranz el al |1980| and
Mcrmelstcin ei al J1981] characterized niiro reductase-deficient bacterial-strains that
allowed for the initial recognition of nitfoarenes in mobile emissions. Nitroarcncs
were identified in diesel exhaust through the use of these strains by Claxton 11981a).
Claxton and Kohan 119811. and Lofroth 11981 j.
The 8-a/aguanine mutation system has also shown positive results with various
mobile source emissions. It can be used to give a more quantitative approach and
identify a wider spectrum of compounds (Claxton and Kohan. 1981: Liber ct al. 1980;
Barfknccht et al. I98lb|. However, this system has not been generally employed.
The widely used liver homogcnate systems generally reduce the mutagenic
response of die*el organic* (excepting TAI538). However, exogenous activation in
gasoline exhaust organk*s produces an enhanced mutagenic response, a fact that has
received litlle emphasis in the literature. Thus, diesel and gasoline vehicles are
demonstrated to emit different mutageni»: compounds.

FACTORS THAT MODIFY THE GENERATION OF MUTAGENS WITHIN
MOBILE SOURCE EMISSIONS

In the generation of emissions from a combustion system, there are five funda-
mental components to consider: the fuel, the fuel's oxidunt. the fuel's diluents, the
type and degree of combustion, and the atmospheric and environmental conditions.

Fuels
Fuels could influence the mutagenicity of exhaust organics by cither the direct
contribution of miitagcns or by supplying (he precursors for mutagcns created during
the combination process. The diesel fuel used by Httisingh et. al 11978) was negative
when tested directly in the Salmonella bioassay. Lebowit/ et al 11979| also reported
that diesel fuel was negative. The diesel fuel JP-4 and two types of gasoline were
reported negative by Wang et al 11978a.b| when tested with TA98. Various crude oils
and some of their distillates, however, were observed as positive in the Ames test
jBrusick and Malheson. 1978a.hj. Positive results, for example, were reported for
some natural, syncrudc. and shale oil crudes (and some of their distillates) by Calkins
et al II98UJ and Calkins and Krahn |1979|. In each case, however, the naph'.ha
distillate was negative. I:pier et al | I978b| and Ciucrin et a! [ 1980) demonstrated that
coal-derived petroleum substitutes could provide a tenfold increase in bacterial muta-
genicity over a similar natural pr
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612 Claxton

using strain TAIOO. These varying reports may indicate vast differences between
sources of dicse! fuel and hioassay techniques employed. A further possible variable
is the presence of minor components not detected unless fractionated components of
the fuel are used for testing. In addition. Henderson el al (1982) noted that exposure
to nitrogen dioxide (NO>) dramatically increased the response of both fractions. This
finding is supported by the work of Pitts 11979|. who exposed an indirect-acting
mutagen, ben/o(a)pyrene, to NO* and generated a direct-ac.ing derivative. Such a
correlation was not unexpected, since sonic nitrous acid also could have been present
during ihesc exposure conditions. The precursor effect of the fuel appears to have
been demonstrated by Huisingh el al (1978|. Upon testing the effects of seven different
fuels in two different vehicles, they found a wide range of mutagenic activity in the
emission organics. The results of McClellan | I980b| are similar, also suggesting that
fuels high in aromatic content produce a more notable mutagenic response. Although
results support the hypothesis that fuels mainly supply precursor material for the
mutagens in exhaust emissions, more research would be needed to rule out any major
concentrating effect.

The Fuel's Oxidant and Diluent
The oxidant for both spark-ignited (gasoline) engines and compression-ignited
(dicsel) engines is. of course, oxygen. Nitrogen, which composes approximately
78tf of the atmosphere, is the most common diluent. Water vapor, other inert gases.
and some inorganic ash are the other diluents present. At high temperature, some
inert nitrogen enters into the combustion reaction, and nitrogen oxides are produced.
In addition, excess oxygen, lubricating oils, and/or fuel behave as diluents. (For an
introduction to combustion and emission chemistry, sec the text by lidwards 119771.)
As will be .shown in a later section, nitrated and oxygenated components of incom-
plete combustion contribute to the muiagenicity of emission products. Crankcase oils
have ai>o been investigated for possible mutagenic activity. Wang et al | I978a).
Hermann et al (I9XOJ. and Lofroth [I98|| each reported that unused crankcase oils
are nonmutagenic, but that used crankcase oils from gasoline engines give a positive
response. In addition, Lofroth (1981j stated (hat (I) metabolic activation increased
any muiagenic response seen; (2) the response increased with vehicle mileage: and
(3> this positive response was not seen xvtih used oil recovered from a dicscl engine.

Type and Degree of Combustion
Within mobile sources, the type of combustion depends upon the type of power
source that is used. In the Untied States, the most common power train for light-duty
vehicles is the typical spark-ignited gasoline engine. Diesels, which are recipn»cat-
ing-compression ignition engines, are most often the power source for heavy-duty
trucks, buses, locomotives, and vessels. Other engines that have shown some utility
or are undergoing further research are: gas turbine and Wankel inlerna'-combtislion.
Rankins and Stirling cycle external-combustion, and electric.
The effect of type and degree of combustion on mutagenicily is examined by
comparing the results from different power sources and/or vehicles. Although a few
authors did not describe the engine or vehicle used in their research, most gave at
least a limited description. Huisingh et al |I°7X| employed two heavy-duiy engines
and three light-duty engines. Although direct comparison of differing sources was
not the primary purpose of thai research, it provided a mobile source comparison
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Bioassay of Automotive Emissions 613

based on bacterial mutagenicity. Particle exhaust organics from heavy-duty engines
were tested in TA98. TAI535. TAIOO. TA1537, and TA1538, both with and without
exogenous activation. Both engines showed very similar qualitative results, with the
four positive strains having decreasing activity in the order TAIOO > TA98 >
TA1538 > TAI537. Without activation, TAI535 was negative with samples from
both engines: however, with activation, one engine (Caterpillar 3208, four-stroke V-
8) produced a marginally positive response. Given the sample amounts available.
exhaust organics from the three light-duty engines were tested using only strain
TA1538. In this study, in which fuel comparison was a primary component, results
for even a single vehicle (using different fuels) could vary greater than 100 times.
Qualitatively, the.results from other studies [Claxton, 1980, 198la; Claxton and
Kohan. 1981; Lofroih. 1981; Dukovitch et al, 1981; Dietzman et at. 1981J are in
agreement with the report of Huisingh ct al (1978].
The results of Claxton and Kohan [1981] demonstrating the effects of three
different sample parameters are given in Table I. The three comparisons were made
between (1) different runs with the same diesel engine; (2) gasoline vehicles of the
same make, model, and configuration: and (3) different makes of diesel vehicle. The
coefficients of variation for the revcrtants per mile for these three cases were 0.11,
•0.49, and 0.59, respectively. Assuming normal distribution and that the coefficient
of variation was. in this case, a good estimation of the true standard deviation, one
can estimate confidence limits in all three cases. For the above three cases, a value
could fall within 997< confidence limit values and vary by 33%. 1477&, and 177%,
respectively. If multiple testing facilities, fuels, and bioassay laboratories were used.
the variation between results would be expected to increase. Because the Ames assay
is a semiquuntitativc test tor screening substances over a dynamic range of — I0h in a
dose-response slope and because other parameters (such as percent of the particles
cxtracinblc) show broad variation, the variation encountered here for a complex
TABLE I. Comparison of Summary Data Demonstrating the Effect of Differing Sampling
Parameters

Sli»pe'
rev/
r plate/
,
-------
614 Claxton

testing situation should not be considered excessive. Together, these studies indicate
the degree to which quantitative comparisons can be made within a single study and
show that cautious qualitative comparisons can be made using results from multiple
studies.

Ambient Environmental Conditions
Ambient conditions are known to affect the condensation of organic compounds
onto particles, influence the interaction of organic compounds, alter the organic
species emitted by a source, and provide the conditions for various other interactions
(Pitts et al, 1982aj. Dilution-tunnel experiments examining the effect of crankcase oil
temperatures upon test results were reported by Braddock (198JJ. After the vehicles
were maintained overnight at various ambient temperatures (ranging from 23°F to
82°F). the vehicles were tested at ambient temperatures. For the soluble organic
fraction, a mild correlation belveen mutagenic activity and conditioning temperature
was noied: however, this correlation did not exist for comparisons on a rcvcnant per
mile basis. Only a few investigators have explored ambient effects. Claxton and
Barnes 119811 used the Calspan smog chamber to examine a variety of ambient
factors. They found that the.presence of ozone in the chamber tended to reduce the
mutagenic response to the organic material collected. Those results also showed that
ambient like irradiation without other mitigating factors such as ozone did not alter
the mutagenic response.
Ohnishi et al | I980J examined road side particles collected in a highway lunnel.
They found a 60- to 88-revertanis/m* response for panicles collected during daytime
hours and tested with TA100 in the presence of an activating system. In the same
study, panicles collected at night with a high density of dicsel traffic exhibited 121 U>
238 revertanls/nv\ Alfheim and Mollcr JI98J) found that the contribution of traffic
lo the mutagenicity of air samplers is significant by comparing saniplcs from a
roadside site, on a roof, and at a park. In an Allegheny tunnel siudyrconducted by
Pierson ct al (1982). the diesel aerosol organics were similar in activity lo organic*
recovered in dilution tunnel studies. Furthermore, it was shown that ihc mutagenicity
of diesel engine exhaust is several times that of gasoline engine exhaust whin
expressed as revcrtunts per mile. Studies such as these demonstrate that the produc-
tion, chemical alteration, distribution, and concentration of mutagenic mobile source
particles are dependent upon traffic patterns, amounts of reactive gases and vapors.
level of ozone present, meteorological conditions, and the presence or absence of
other ambient air particles.

Effect of Sample Collection Upon the Mutagenicity of Mobile Source
Emissions
The influence of particle collection methcxls upon (he chemical composition and
biological activity of diesel-particle extracts was investigated by Chan et al |I98|),
They noted that filter sampling allowed potential chemical conversion «»f organic
compounds by the nitrogen oxides in the exhaust gases, whereas electrostatic precip-
itation (ESP) collection methods provided for ozone generation and interaction at the
time of collection. In their experimental results, they found 11% extractablc organics
for the ESP sample and 6.2% for the filler collected sample. The chemical profiles
for the two collection methods were similar except that the ESP sample contained
greater amounts of an acid salt fraction. Although the overall biological activity of
the ESP and filter samples was comparable, subtle but consistent differences sug-
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Bioassay of Automotive Emissions 615

gested that different direct-acting mutagcns could be found in the two sample types.
As seen from the studies of Chan et al j 1981], the most serious obstacle in collection
methodology is the generation of artifacts, ie, the generation of substances that do not
exist in the natural situation or the elimination of substances that would normally
exist. A number of investigators |Claxton. 1980; Claxton and Kohan, 1981; Lofroth,
1981; Gibson ct al. 1980: Pcderson and Siak. 1980, I98la.b| have demonstrated that
mutagenie nitroarenes are contained in organic extracts of filter-collected particles.
However, since diesel and gasoline also emit varying levels of nitrogen oxides that
pass across the filters and collected particles, these nitroarenes may be artifacts. They
may be produced under three possible circumstances: (I) the combustion process: (2)
the exhaust process, as organics interact and condense upon the particles; or (3) the.
collection process as an artifact. The passage of nitrogen oxides across a PAH
compound upon a filter can generate a nitroarene (nitrogen dioxide (NO^J-PAHj that
is direct-act ing in the Ames bacterial assay 1 Pitts, 1979J.
Henderson et al (1981) generated direct-acting mutagens for strain TAIOO by
exposing l-g samples of fuel aromatics and fuel aliphatics to excess NO; at 25°C.
The aromatic NO; fraction was the most active and nilro-PAH compounds were
identified in this fraction. In some preliminary experiments, Bradow |I980| and
'Claxton | I9SOJ reported passing artificial gas streams containing high levels of NO;'
across filters with diesel particles and observing increased mutagenie activity of the
extracted organics. Gibson et al 11980| recxposed filter-collected diesel particles to
the gas-phase portion of similar diesel emissions and found increased levels of I-
nitropyrene. nitrobcnzo(a)pyrene. and mulagenic activity. Although the issue of the
extent and relex'ance of artifacts has not been fully resolved, hacteriul testing has
paved the way in identifying and providing methods for examining the problems.
Since sample collection must occur during some type of test cycle (running test
modes of the vehicle or engine), the test cycle may affect the generation, transfor-
mation, condensation, and collection of emitted particles and organics. Only a few
researchers have published any direct comparison of test cycles. When reporting
data as rcvertants per microgram of organic material, Gabcle et al |1981J found no
great differences between six different test cycles. Gibbs ct al 11980) examined five
different cycles with six different automobiles. When expressing the data as rever-
tants per gram of particulates, they found "widely divergent" results: however, when
expressing the data as revertants per mite, "cycle-to-cycle" trends were more
pronounced and reproducible. For cycles ranked by revertants per mile, activity
decreased in the order Federal Testing Procedure (FTP) > Congested Freeway
Driving Schedule (CFDS) > Highway Fuel Economy Test (HFET) > 50-Milc-an-
Hour Cruise Procedure (50C), and a general reduction in revcrtants per mile was
found as the mileage of the vehicle increased. Upon close examination of the data of
Gibbs et al 11980|. it was noted that very low-mileage cars (< 4.000 miles)
demonstrated a greatly enhanced mutagenie response for all cycles except idle.
McClellan j I98()bj examined four test cycles using a single automobile and reported
that ihc cycles with lower speeds and more stops and starts resulted in higher
mutagenie activity.

INTEGRATION OF PHYSIOCHEMICAL INFORMATION AND PROCEDURES
WITH BACTERIAL BIOASSAY PROCEDURES .
A review of the literature prior to 1979 provided a list of 184 chemicals
identified as being in diesel exhaust jClaxton, 1982). Of these 184 compounds. 44
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616 Claxton

were listed in published mutagenicity reports and 21 were acceptable positives in one
or more mutagenicity assays. Seven of the 184 were reported as carcinogens. Since
1979. more research activity has been devoted to bioassay-dirccted fractionaiion than
to the pairing of chemical and biological literature reports. For showing the mutagenic
re.sponse of different chemical fractions from the organic emissions of a diesel engine,
an organic extract from emission particles of two heavy-duty engines was initially
used (Huisingh et al, 1978|. The two most active fractions, the transitional and the
oxygenate, were eluted from a silica gel column after dichloromethane (DCM)
extraction from the exhaust particles. Choudhury and Doudney |1981) fractionated
organic emission into three primary fractions—acid, basic, and neutral—and subse-
quently fractionated the neutral fraction into seven subtractions. All three major
fractions and five of the seven subfractions showed some type of mutagenic activity.
The puruffinic subtraction was negative.
Upon examining emissions from both a diesel and a gasoline vehicle, LSfroth
119811 noted that the aromatic and an oxygenate fraction were the most mutagenic.
McClcllan's work | I980aj, using a Fiat under varying conditions, showed that upon
Sephadex fractionation three of five fractions were mutagenic to bacteria. The classes
of compounds reported as contributing to the mutagenicity of these fractions were
alkyl-subsiiiute
-------
Bioussay of Automotive Kmissions 617

associated with the argon/oxygen sample.was observed when the experiment was
conducted with a cracked piston ring allowing entry of nitrogen-containing air lo the
<:".• imber.) These results suggest that the formation of nitroarcncs is not dependent
u^/on fuel-hound or lubricant-hound nitrogen and that some of the PAH compounds
are products of the combustion process.
At EPA's 1981 Diesel Emissions Symposium, Raleigh, NC, several investiga-
tors provided lists of compounds recently identified in diescl exhaust. Since a listing
of these results may not be readily available, a compilation of the compounds is
given in Table II. Table il also provides a summary of the bacterial mutagenicity
associated with the compounds tested and reported. It is interesting to note that in
bioassay-directcd fractionation, vcr-y- few investigators used the indicator strains
TA1535 and TAI538; therefore, some mutagens that cause base pair substitution and
that need activation to be framesbift mutagens could be overlooked. In any event,
bioassay-direeled fractionation has aided in the identification of several mutagens
that previously have not been recognized in mobile source emissions.

USE AND EFFECT OF VARIOUS BIOLOGICAL AND ASSAY PROCEDURES

Since initial testing involved organic chemicals extracted from particles with
strong organic solvents, researchers questioned whether chemicals bound to carbon-
aceous particles would be released into physiological fluids in vivo. McGrath et al
(1978J. using the Ames bioassay, tested whole particles suspended in dimethytsulf-
oxide (DMSO) and obtained results ranging from negative to moderately positive.
However, DMSO is a moderately effective solvent. Siak et al |I981) reported
extracting particles with four simulated biological fluids: fetal calf serum. 0.5%
bovine serum albumin, lung surfactant, and saline. The assay of each biological fluid
in the Ames test was negative except for a positive response with the fetal calf serum.
The fetal calf scrum extract provided only about 6% of the response found with
extraction by DCM. Brooks et al 11980) found similar results with.xlog scrum, lung
lavage fluid, saline, dipalmitoyl lecithin, and albumin. However, they state that "the
mininuil mutagcnic activity . . . may be due to a lack of removal of mutagens from
the particles or an inactivation of removed mutagens by binding or some other
process." Clark and Vigil (1980J tested a DCM dicsel extract under the following
conditions: Aroclor 1254-induced -rat liver 5»9, an uninduccd S9. an S9 without
nicolinamide adenine dinucleotidc (NAD), bovine scrum albumin, and fetal calf
scrum. They found a decreased mutagcnic response in each case. That result suggests
that protein binding of mutagenic components .was at least partially responsible for
the lack of activity seen with incubated particles. By following the mutagenic activity
of the DCM extracts in serum, lung cytosol, protease-treated serum, protcasc-treated
lung cytosol, and extracted particles. King et al |1981J demonstrated the release of
mutagens from diescl particles and postulated that the lack of mutagenic response is
due to cither protein binding or metabolism. Siak and Strom 119811 exposed rats to
diescl particles, recovered the lung macrophagcs, and extracted the macrophages
with DCM. They showed that although the particles continued to contain mutagens,
"seven days after exposure. DCM extracts of alveolar macrophages had no detectable
mutagcnic activity, even though more dicsel particles were recovered." These effects
may be due to either protein binding or metabolism. Wang and Wei 119811 and Wang
ct al 119811 gave evidence that the antimutagcnie effect of S9 is not enzymatic by
-------
TABLE II. Salmonella Mutagenicity Results Tor Compounds Identified in Diesel Exhaust Emissiuns and Presented at the EPA's
1981 Diesel Emissions Symposium. Raleigh. NC
No.
1.

6.
7.
Compound
Acenaphthalcne

Accnaphihulenc, nitro

Accnaphthalene.
nitronicthyl
Anthracene

Anthracene, methyl
2-methyl
9-methy)

Anthracene, dimethyl
Anthracene, irimcthyl
CAS No.
34493-60-2

—

102-12-7

—
613-12-7
779-02-2

29063-00-1
27358-28-7
Reference for dicscl
identification3
Yergcyet all 19811
Riley ci al| 1982|
Riley MalJ I982|
XuetaHI9X2a|
Rilcy et al 1 1982)

"Prater and Schuet/.le 1 19821
Ycrgcyct al| 1981]

Praicr and Schuctzlc |1982|
—
—
—
—
—
Prater and Schuct/.Ic [ 1982 1
Prater and Scheuulc 11982J
Bioassay reference15
Kadcnct all 19791

—

Anderson and Stvles 1 19781
Eplerctal|l978a. 1979)
Florin ei alj 1980)
Gibson ct al 1 1978|
Lavoic ct al 1 1979|
Probst and Hill I1980J
Salamonc et al |I979|
—
Gibson ct al| 1978|
Kadcn ci al \ 1979}
Epler ct al 1 1978a|
Gibson ct all 1978)
Kadenet all 19791
HubK:rdeiaIlI9XI|
—
Bioassay
result1"
+ (8-Az)

—

Neg
Ncg
Neg
Neg
Neg
Ncg
Neg
_
Ncg
•f (8-A/>
?
Ncg
+ (8-Az)
+
—
8. Anthracene, iciramcthyl
9. Anthracene, niiro

9-ni»ro
2-nitro
10 Anthracene, nitronicthyl
Prater and Schuctzle [ 19821
Rilcy ct al | I982J
Xu ct al 11982al
602-60-8
Rileycta!ll982|
Hoeta!|19SI|
Matsushita 11980]
Pederson and Siak (1980.
198 Ui]
Tokiwaet al| 19811
ClaxtonandKohan|198IJ
n
-------
11. Anthracene, nitrodimcthyl —
12. Anthracene. . —
carboxyaldchydc
13. Anthracene, —
carboxyaldchydc,
nitromcihyl
14. • Anthracene, bcnz(a) 56-55-3
15. Anthracene, dionc. benz —
16.- 9, 10-anthraquinone 84-65-1
17. Anthraquinonc. nitro —
l-nitro 82-34-8
18. Amhronc 90-44-8
19. Amhronc, nitro —
20. Anthrone, methyl —
21. Amhronc, dimethyl —
22. Anthrone, trimcthyl —
23. Bcnzo(a)pyrcnc 50-32-8
Rilcyet al[ 1982]
Prater and Schuetzlc |J982)

Rilcyet alf 1982]
Prater and Schuctzlc [19821
Prater and Schuctzlc [1982]
Ericksoncial|1982J
Xuctal(I982a]

Prater and Schuctzlc [19821
Erickson et al 11982]
Rilcyet al{ 19821
Prater and Schuct/le 11982]
Prater and Schuclzlc |1982|
Prater and Schuctzlc ] 1982]
Prater and Schuelzlc 11982]
dc Flora 11981] +
Glattetal]1981] +
Probst et al| 19811 +

Anderson and Styles [ 1978) Neg
Brown et al 11977] Neg
Gibson el al [ 1978} , Neg
Kaden ci al [ 1979] Nq:
Salamonc ct al 11979| Neg

Matsushita 11980] +
Anderson and Styles |I978] Neg
Brown ct al 11977] Neg
Gibson ctal[ 1978] Neg
Kadcn ct al ] 1979] Neg
Eplcr ct al 11978a] +
Florin et al [ 1980) +
Pcdcrson and Siak 1198la| +
Lavoiccial[1979) +
Pitts et al| 1978} +
Pins 11979] +
Saiamone ct al 11979] +
••»
re
m
§
Continued
-------
TAIH.K II. Salmonella Mutiigvnirity Results for Compounds Idcnlifii-d in Diesel Kvhaust Emissions and Presented at the KI'A's
1981 Diesel Kriissions Symposium. Raleigh. XC (Continued)
No.
24.
25.

37.

38.

39.
Biphenyl. niiromethyl

2-mcihyl-4-niin>
3-methyl-4-nim>
Biphcnyl, diniiromcthyl
Biphonylenc
Chryseno
Riley ct ;il 119H2|
Xuctal(l982a|
Chmcne, nilromeihyl
Cinnoline. bcn/n(c|
Uilx:n/o(h.d)ilii(»plu.'nc
Diben/oihinphene, methyl
Ribentt « hiophenc,
dimethyl
Dihcn/oihiophcne.
ictramcthvl
Diben/oihiophene.
carhoxy aldehyde
Fluoranihcnc
33350-73-1
69314-47-2

259-79-0
218-01-9
230-17-1
U2 <•? 0
30995-64-3
70021-47-5
206-44-0
Ycrgeyaal|l98l|
Yerpoyetal(i98l|

Praier and Schuc(/.!c |I9S2|
Ycrgcyctal|l98l|
Ycrt;cyclalfl9K||
Prater and Schuet/.le 1 1982]
Prater and Schuctelc 1 1982)
and Sehuet/lc ( 1VS2J

Praier and Sc'-
Riley etall.vS1'
Yerj-cyelaljmiJ
El-Bayoumyet all 1981]
F.I-Buynct all I978|
Uvoie ct al 11979|.
McCoy and Rasenkran/
| I9HO|
Salanw meets!!
Ncg

Neg

•f
ict al| I979| NcjjiH-Ac.)
N'akainura and Kashimulo . Ncg
J1979)
Barfknechtetalil9Hla|
Kpler et al 11979)
Florin ct al | I9KO|
Gzifehoute 11980}
Kaden cl al 11979|
Lav
-------
TABI.K II. Salmonella Mutagenicity Results Tor Compound* Identified in
I98t I)U-M?| Emissions SyrnpoMurri. Raleigh. XC
!>i<%d Kxhauvt Kmissions and Presented at the KPA's

No.
40.

41.
42.

43.

44.

45.

46.

Compound
Fluoranihcnc. methyl
. 1 -methyl
2 -methyl
3-meihyl
7-mcth) 1
8-methyl
Fluoranihcnc. nitromcihyl
Fluoramhenc.
qviirumc. nilro
Fluoramhenc.
hcn/ojghh
Fluorcne

Fluorrnc. ni'ro
2-niiro

Fluorcne. dinitro
2.5-dinitro
2.7-dmiiro

Reference lor dioel
CAS No. idcnlirieaiion"
— Prater and Schuctzle 1 1982)
25889-60-5 -
33543-31-6
1706-01-0 -
23339-05-1 -
20485-57-8 -
- Rilcyct al| 19X2)
- Ycrgcyct al| 198I|

203-12-3 Verge)- ei al 1 1981}

86-73-7 YergcyctaI|I98l|

- Xuctal|l982a!
609-57-8 —

- Xueial|1982al
15 110-74-4 -
5405-53-8 -

Lavnicct al| 1979)

Ncg
Ncg
Neg
Ncg
Ncp
Ncg
—
4
4
4
4
4
—
4
4 •
4
4
4
4
4
G
-------
47.
48.
49.

50.
51.
52.
53.
54.
55.
56.
57.
58.

59.

60.

61.

62.

63.
Fluurenc. nitrnmcfhyl
Fluorcne. quinonc
Fluorenone

Fhiorenone. hcn/o
Fluitrenone. nitron)

3-mini
2.7-dimtrn

Fluorenonc. nilromethyl
Furan. ilibcn/<»
Furan. 7-methy I bvn/o
InJcrvc. nitfn
lrhk"ne-I-onc. dihydri*
Naphthalene
N'jphihjlcnc.
dinitnnncfhyl
nitrorncihyl
l-n'tn.-2-nKihyl
l-mcthyl-2-niini
3-mcth\l-2-ni»rn
Naphthalene.
niinxJihyiJr«i!iy
Naphthalene.
nilrotrimethyl
Naphthalene
dicarhoxylic acid, nilro
Naphthuquinonc. nilro
4W>-25-9

76723-yct:il|19K2i
Rilcy « al| 19821
Xuctal|l9K2:t|
- cl al
cycia
VctgcyciatllVSII
\etfc\ i-«al|19S!J
Yeryc) iMal|l9K|}
HcndvrMinctal|l9K2|

Hen»krM»n ct a) 119H2]
Xuctal|l9K2a]
Rilcy el all 19821

.Rik->etal|f982|

Rilcy rial JI9821

IV81 DifM'l Kmissions Sjmpmiuin. Kiilvijjh. M.' ((.'ontinurd)

No.
W.

65.
66.

67.

AH.
69.

70.
71.
72.

Reference for die^el
I'ompound CAS N«». kk'ntifk'jiHm'*
PrKTunthn-no . K< 01- X Place,' jml Sthucc/lc |M2|
Yerfcy cl al| WI |

Phcnamhrene. nitro — Henderson ei al |19K2|
Phenanthrene, methyl — Henderson cl al | IMC |
Pulcr and Schud/lc | I9H2|
l-nu.-th\l X32-69 9 -

? -methyl 25XI-S4-2 -

Hioassav refereik-c1'
|{jram.vhCc(at|IVXIhi
I-pleret al |!97Xa. 19791
Murin cl al | IVX()|
Prohsi and Hill | IVX()|
—
—

Cihv«nctal|l97X|
UvoiectaI|l9X|h|
Gihx.net al| I978|
I^ivoicctal j!9S|h|
l^aM'ic et al | !9Xlh|
l^t\ iiie et al | I9X 1 h|
l^iect'all'Sl
—

—
_

. —
_
Amk-rvm and St>le> |197K|
Florin cl al |'|9KO]
Ho ct al| 1980}
Uvoiccl&l|l979|
Salamone et al 1 1979|
Bwuissay
resuti'

'?
Ncg
Neg
—
—

Neg
+
Ncp
Nep
Ncg
Ncg
Kef
—

_
—

—
—
•4-
+
+
•f
Ncg
Q
ET
-------
7.V
74.
Perylene. nitro
3-nitro
Pyrcnc
20589-63-3
'.9 1)0-0
Rilcyetal 1 19X2|
Prater and Schuet/le 119X2|
Vergeyet jl||9SI|
Hoc«al|l9SI|
Piti>ct«il|»978)
Pitts 11979|
r.plcrc(iil||97Xa. 1979)
Morin et al | I9W»|

75.

76.

77.

78.
79.
80.
81.

82,

Pyrene. cyclopenia
(c.d>
Pyrene. cyclopenseno
(c.d)
• Pyrenc. methyl
1- methyl
Pyrenc. niiromclhyl
I*yiL-ncquinoncs. nitro
Thioxamhones
Triphcnyicnes

Xanihone.s

27208-37-3 Ycrpey ct al 1 19X11

- Barn,ncthlclalil>»<>r*!

—

—
Kadcn cl al 1 1979|
—
_
—
Eplere«at|l979|
Gibson cl al 1 1978|
~

Neg
Neg
H-
NCj!
Neg
+

—

—
+ (8-A/)
_
—
—
•4-
Nep
„

"All references for dic'-el identificalion are from Smith 11982|.
''References.resulting frotn a Environmental Mutagcn Inforniatum Center (EMIC) search in September 1982. References arc fnund within
the paper's reference list.
''Bittis.vay results as reported by the authors and sumntari/cd a\ follows: +. posiiivc: Nep. negative: ?. quesiionablc or ± result: (8-A/>.
8-a/uquaninc forward mutation system used.
a
|
*+
i
c
•f.
-------
626 Ctaxton

examining S9. heat-deactivated S9. S9 minus cofactors, and albumin effects. Some-
what in ami rust. Pederson and Siak | I98lbj used a nitroreductase-defieient bacterial
strain to show that some mutagens in diesel particle extracts are activated by S9 and
that 1-niiropyrcnc is also activated by NAD phosphate-dependent S9 cnxymes.
These, studies demonstrate that substances are released from diesel exhaust
particles into certain physiological fluids and cells. Physiological fluids and S9
apparently decrease the niutagenic activity of diesel extracts and particles primarily
because of protein binding; however, some mutagenic components (eg. l-nitropyrene)
are activated by the microsomal fraction of S9, while other components are activated
by the eytosol fraction.
In addition to the above concerns of scientists regarding biological parameters.
investigators have questioned whether the Salmonella bioassay correlates well enough
with other bioassays to use as a routine screen. This knowledge would be useful in
the development of new combustion and control technologies'. Lewtas showed in her
review J1981J that when no exogenous activation system was used, the Salmonella
bioassay data had a greater than 90$ degree of correlation vviih the following assay
data: mouse Jynphoma. sister chromatid exchange in Chinese hamster ovary cells.
viral enhancement, and skin tumorigenesis. When exogenous activation was used.
however, the correlation with viral enhancement and .skin umiorigcniciiy data was
79% and 72 /?. (respectively). It will he interesting to see whether these high correla-
tions are maintained, as the body of data and information grows.

SUMMARY

In summary, the work presented demonstrates that rapid, in vitro indicators of
genotoxicity continue to play a valuable role in our understanding of the toxicity of
mobile source emissions. Bacterial assays have had tremendous importance in the
charaeterixatton of mobile source emissions. Specifically they have had four major
uses: (I) comparative screening. (2) analy/.ing factors that alter the genotoxicams
found in emission products, (3) directing the chemical fraclionaiion of emission
organics for the identification of specific genotoxicants. and (4) analy/.ing the inter-
action of complex emission products with vurious mammalian systems.

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