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

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                                    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
                             11. CONTRACT/GRANT NO.
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/
,
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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
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                                        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

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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.

2.

3.

4.






5.





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.

26.
 27.
          Compound
CAS No.
                                          Reference for die*:!
                                            identification'
         Be.n/oU'>79|
                                                 Pitt.sctalII97K|
                                                 Tokiwactalll98l|
                                                 Wei cl al 11978J
Anderson and St> les 1 1978|
Bron^ctti etal J198I|
                                                                                    Kawachi ct al |
                                                                                    Probst and Hill 1 1980|
                                                                                    ProhMd al| 1981)
                                                Anderson and Sl)lcs 1 1978)
                                                EI-Bayoutny el all 19811
                                                Matsushita  [ I980J
                                                McMalxm ci al I I979J
                                                ProhMandHill|l980|
                                                Tokiwa el al | I9S1 1
                                                lil BdyinJmycialll9Hl)
                                                Matsushita  | I9K(>)
                                                McMahonct al| 1979)
                                                Anderson and Si) les | I97H|
                                                                                   Matsushita | I9S(I|
                                                                                   McMahon etal 1 1979|
                                                                                   Probst and Hill 1I980|
                                                                                   Prohstetal|I9XI|
                           Bioassay
                            result"-'
                                                                                                      Ncg
                                                                                                      Ncj:
                                                                                                      Neg
                                                                                                      Ncg
                                                                                                      Ncg
                                                                                                      Ncp
                                                                                                                Ncg
                                                                                                                Ncg
                                                                                                                Ncg
                                                                                                                Neg
                                                                                                                Ncg
                                                                                                                -f
                                                                                                                +
                                                                                                                                 F
                                                                                                                                 x
                                                                                                                                 I

-------
28.
30.
31.
32.
33.
34.
35.
36.

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 -






Hioussav reference1*
—
Lavoic ci al 1 1979|
Lavoicci al| 1979]
La voice! al| 1979|
Lavoic ci al | I979J
Lavoic et all 19791
_
—

Lavnicct al| 1979)

F-plerctal[l978a|
Gihvin ct al 1 1978|
Kawaehi ci al J 1980]
Lavoic ct al| 198 U|
Probst el al| 198 1|
Probst and Hill 1 19»)|
—
Anderson and Styles (I978J
McCoy ct al| 1981 1
Pedersi>nandSiak|198la|
. Probst and Hill 1 19801
Tokiwact al| I9XI|
—
Matsushita | I9HO|
U-vin ci al | I979J
Matsushita 1 1980)
McCoy et al| 1981 )
PcdtTMin and Siak 1 19X1 a]
Prohst and Hill ] I9XO|
Tokiwactal |I9XI |
Bioassay
result'
—
4
4
4
4
4
—
—

+

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

                 Rileyctal|t9K2|
                                Florin ct al[ I980|
                                                 Pcdcrvm and Siak 1198la|
                                                 U» in ct al| I979|
                                                       and Hill
                                Arxlcrvm and St\!o 11978|
                                Fli>rin ct al | I9X<)|
                                Ho et all 198 J |
                                Kadcn ct al 11979|
                                Kplcr ci al | I979J
                                I£l-Bayinimyctal|l98!|
                                Matsushita 11980)
                                El-Rayoumy ct al | I98IJ
                                F.I-Bayoumyctal|198IJ
Ncg
                                                                                                                 Ncg
                                                                                                                 Ncg
                                                                                                                 Nvg
                                                                                                                 Nog
                                                                                                                 Ncp
                                                                             •f-
                                                                             •*•
                                                                                        I
                            £
                                                                                                                   Continued

-------
T.-VB1.K II. Salmonella Mutajirnicit) KrMilts for CompiMiiMK Idrntifird in DM-SC! Kthausl KmU>k»n\ and Prr\cnJcd al  the KP/Vs

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 -

3-iiK'thyl M.^-71-3 -
4-nKth)l X32-W-4 -
9-mcthyJ KSO-20 -5 -
Phciumhrcnc-5-<»nc. — Vcrpey cl al |I9S1|
cyek>pcnta
Pheiumhrrrt:. qu>n>nc — Verjvy « al JI'JXlJ
Phcnanthinric — Erk-k.son et al |1''K2|
Prater and «K-huc(/lcll9S2|
Pncnanihretw. nu:lh>l - Prater and Sv-huit/lc | I9X2|
Phenanthronc. nitro — Rilcy cl at |IVK2|
pcryk-nc I98:55-0 Prater and SehaeUlc II9K2|





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*!

- Prater and Schuei/le 1 19821
\crsoctaim.HII
RileyclaltI19X2|
Rileye! Ill 19821
— Pmter and Schuct/lc 1 1982)
- Riley .rial 11^821
Ycrpeyclaf I1981J
— Ericdson etal |I9X3 1
Prater ami Schuei/le |1982|
dihson et ul |I978|
Ho dull Wl|
Kuwachi ei al | I'>XO|
Lavoiect al| I979|
Probst ad>iiil|l9KO|
Gild ;ind HLsenstadt 1 1980|

—

—
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.


 REFERENCES

 Allhciiii S. Muller N tl'JXI): Mulugcnicity of airborne partieuUte mailer  in relation u» traffic and
      meteorological conditions.  In Waters MI).  Sandhu SS. HmMtigh Jl.. Claxton  I.I).  Nesnow S
      (eds): "Short-Term  Bioussays in the Analysis of Complex l:m jnmmcnial  Mi.xturcs II." New-
      York: Plenum Presv pp X5-W.
 Ames UN.  McCanii 1. Yaituvikt II < 1°75>: Methods fur dffvc'irftc carcinogens and nmtaircns wiih the
      .SalmonelJa<'immmaIian-micn»ome muiujicmciiy lesi. Mutat Rc.t 3I:437-.V»4.
 Anderson I).  Siyles JA (I*)7X>:  An evaluation >.t. Br J Cancer .\7:924-9?t).
 Rurfknecht TR.  Andon BM. Bishop W\V. Tliilly \VG (l')Kla): R;idislmi(ocht andmutation in bacteria and human
      cells. In  C'ooke  M. IX'imis AJ  (ed.s): "Chcrtiical Analy>j>  and Biological l-'ale: f'oh iiuvlear
      Aromatic HydrocarUms." Columbus. Ohio:  Baiellc Pros, pp 231-242.
•Braddock JN (1981): iimisNions of diesel puniclo and paniculate mutacens at low ambient temperatures.
      Paper presented at. l-PA Sympo>ium on (he Application of Short-Term Bioasvays in the AnaKsts
      of Complex (Invimnmenial Mixtures. Chapel Hill.  North Carolina. January 25-27. 1981.

-------
                                                 Ilioasvay of Automotive Kmissions      627

Bradow Rt. I W«t)>: Diesel particle emissions. Bull NY AcaJ Mod 56:7: Comparative Mudy of hiphenyi and diphenyl ether
       in sea urchin. S. t* phimurium ami S. cere»'isiae. Mutat Res H5:233.
Bnioks AL. Hold KK. Roxer RE. Clark CR. Sanche* A. McClcILn RO (IVKW: Biological availability
       of mutujicnic chemicals associated with dioct exhaust particles. In IVpctko WJ-.  IXinncr KM.
       Clarke NA (cdsi: "Health liflects of Diesel l:nj:it««r  Emissions:  Proceedings of an International
       Symposium." l-PA-MIOiy-M)-057a.  Cincinnati. QhKf. U.S.  linvironmental Protection Agency.
       pp.U5-35».
Broun JP, Dietrich PS. Brown  RJ lllJ77»: Framcs.hift mutajienicity of certain  naturally  ivcurring
       phenolic coinrnHinds in the Salmonella*microMimc test. Bioehcm Soc Trans 5:14X9-1W2.
Bruxick O. Ma(hes«m 1>W  (l97Sj»: Mutajrcn and »nco$cn  -4udy «»n JP-8. AMRL-TR-78-20. Wriohi-
       Pattcrson Air I-'orce Base. Ohio: Acrmpacc Medical Research l^aNwuiurj-.
l)ru>ici D, Maiheson OW  H»)7Xb»: Mulapcn aiu) oncogen  Mudy on JP-K. AMRl.-TR-78-24. \Vright-
       PjHerxon Air Force Base. Ohio; Aerospace Medical Research Lar*>raior>'-
Calkins WH. Krahn l)l; < IV7S>i: Synlheitc cruJe  carciwo^eriicify screening lests: Progress rvpufl.
       rK)i:»-CCXJ-475.S-2.  WashHtyton. IK': U.S. IVpf ttf Ilncrjiy.
Calkins W. tJeyc C. King  C (IVifl.li: S>uthetkr crude oii> carcin<>j:enicity  scrcvntng lest. E.I. Huftwit
       Rerx>n  t"ni:7-.Vl-7.
Chan Tl., Lee PS. SiA JS (IWI»:  Diesel-paniculate collection for biological testing.  Comparison of
       electrttslatic precipitation and UltfalKm. l:nviron Tccttnol  15(1 (:S*> 93.
Cltoudhun DR, l)oudne> CO ir'KI): Muu^cnic  :n,-ti\u>  of dicsc! emission  particulutv extracts and
       isolation of mutjccnic  lrav.'lioiis. l:,n\uon I [it 5(4-5l:3W-?l*2.-
Clark CR. Vigil CI- i )s>S: Influence of rat lunj.' and liver homosicnale.s on the nmtapenicity of diescl
       cxh.iuxi [xtflkuljic c.tiracts. Toxicul Appl J'hannjc^J  5r»:II()-II5.
Cbvt»n t.l> ): IXtvVtion and  cSlai: Mutagenic and carcinogenic potency <«t diescl ;IIK!  related en\ ironinental emissions:
       S;ifnv»nellj luojssas. In Pcpelko WI-. (tanner RM. Clatke NA  (eds»: "Health (• fleets of Diesel
       Hnpine liuiisxions: Proceedings of art International S\mpt«siiiin."t:PA-Mkh'V>-KO-05ih. CiiKJn-
       nati. Ohio: U.S. l:m ironnicnUl Protection Agcrwy. pp SOI-KtW.
Claxton LD (I'Wlhi: The uiihty of hacierUI i«uiaj!enc>is icstmj;  in the chat.icten/atton ofmohilc vtorce
       emissions. In Leilas JL (edi:  "To\ic«»k»j;ical litfevis of Emissions From Diesel P.nymcs." Ne»
       York: l-lscvier. pp<>'»-K2.
Cl.iOun I.I.) (I4><2t: Thi- integration ot'hiojssjy ami phvMochcmical information for CfM»plev mixtures.
       Paper  presented  at the KPA Suiiposiom on (he Application of Short-Term  Bioassays in the
       Analysis of Complex  l:n*ironmental Mixtures.  Chapel  Hill. North  Carolina. January 25-27.
       i««o'.
Cl.ixtoi' I.I).  B.HIVS HM (I'Wli: The ruuia|:cnieiiy 4>t" dxrsel-exhaust  particle extracts collected under
       ••i,«og chmnhcr conditions usinj: tde Sjlnumclla l>phmuirium test  s\stcm. Mutat Res SS:255-272.
CUxton I.I).  Kohan M i ll>SI t: Bacterial ini;tj^ciK>is ur»t ihe esaluation oi mohile  source emissions. In
       Waters MD. Sandhu SS. Huisinth Jl.. Claxton IJ>.  Nest^i*  S  ledsi:  "Sn«s in
       the Analvsis of Complex Km ironmental Mixture^  II." New York: Plenum Press,  pp 2W-.M7.
de |-'J«ira S HMX}): A "spiral tcsi" applied to Kactenal nnnjj:emlsi.s assa\s. Mutal Res S2:2H-227.
Diet/man Hli. PiirjK-s» MA. Brado«  Kl. (J'WJc l:nii>-».vjs  from gasoline and dtesel delivery trucks by
       chassis transient cycle. ASM!.: PuW \. Mulat Res 81:143-153.
F.pler JL. Larimer I;\V.  RaoTK. Nix CI:. HoT(|y/Sjr t:wri:y-rel»ieil p«>!lulant.s it> the environment:
       Use of >liort-lcrin tests (or Hui(aj.'cnicity in the is«»5.itK>n and idcnlirtcation ofhioha/ards. Environ
       Healtti Perspcct27:ll-2
-------
62H      CliixUm

liplcr JL. Young JA.  Hardigree  AA.  !<.'..  »K. Guerin MR. Rubin  JB.  Ho CM. Clark Bk . Newton DL. Savior MC. Tomer KB. Pelli//ari El>. X.weidinj-er KB. Tepda S U9H2»:
       Fractional ion and identification of organic components in diesel exhaust paniculate. In Smith JR
       (cd>:  "Diesel Emissions Symposium Pri»ccedings." EPA-MW-X2-OM. Research Triangle Park.
       Nonh Carolina: U.S. Environmental Protection Agency, pp 509-512.
Florin 1.  Ruthcrg  L. Curvali M,  En/ell CR II9SO): Screening of tobacco \moke constituents for
       niwagcniciiv using the Antes test. Tlogy 15:219-232.
Gabclc PA.  Black |:M. King FG,  Xvveidinger RB.  Brillain KA (t'WIl: Exhaust emission patterns from
       two light-duty dicM-l uutotnobiles.  SAl: Technical  Rerxirl SKXJS], Wyrrendule. Pennsylvania:
       Society «if Autonuiive liiijiincvrN.
Gage SJ (I977c 1'rec.mtioiuiry notice on laboratory handling of exhaust pnxlucis from diescl engines.
       Washington. ItC: Office «f Kesv.irehanJ IX^clopttteni. U.S. Ka\ ironmental Projection Agency.
Gjteh(uise I) ( WHO): Mutagenicity of 1.2 ring-fused acenapthenes against  S. lyphiinuriuin 1A1537 and
       TA153X: Siruciure-aciivity  relationships. Mutat Res  78M2I-I.VS.
Gihl>> Rh'. Mule Jl>. B>er SM ||'^7.S.
       Warren. Michigan: General Motors. Inc.
(ilatt  II.  Vogel K.  IWntley P. Sims P.  Oe.sch F Ul>Kh: Large dilfcrcnco in tiK*tah«>lic *.'tivaiion and
       ina>:livaii:  Mcl.ihohc activation  t>f  c>cli'|Kmatcdip>renc  to  3.4-epi«\>  cyclo-
       pentateiltpvreiv h> rat li*et microsumes. C'anccr Res Jtl:.VWO-3'>44.
Cira> C. von Hip|vl l: < I'WJ >; The fuel cc."iottty oflight vehicles. Svi  Ant 24-4:4N-59.
Guerin MR. Ho CH.  Rao JK. Clark  BR. l-pler JL  tl"S«)>: Polvcvclk aromatic priiv^;r>  amines as
      determinant ctk.-inn.jl iinitagens in (vtroleuin suhstiiuics. 1-jtviron Res 23:42-53.
Henderson TR. l,i A P. Rover RE. Clark I'K tl'JXIi: Increased cvi-noxieiiy and mutageniciiy of diesel
       fuel after reaction wnh NO:. F.nviron Mutagen 3:211-220.
UeiHicivin TK. Sun JO. Rover  RII. Clark CR. Hjrvey TNt. Hum OF. Fulford/E. Lovett AM. f>avtdMin
       WR < |«JX2i: t»CMS and MS MS studies ofdireci-actinj: mutagens in diesel emissions. In Smith
       JR  (edi: "Diesel Emissions Svrnposium Proceedings."  I-.PA-M1O 9-K2-U14. Research Triangle
       Park. Nonli Carolina: U.S. Hnvirontrienral Protection Agcncj. pp 523-527.
Hermann  M. Chaude <.').  Weil) S. Hedouelle  H. llofnting N! th'Stl): Adaptation t»f the Salmonella'
       matiinulian-microsome  lest 10 the determination of the mut.igcmc piofX'rties of mineral oils.
       Mtiui Res 77:327-339,
Ho C-H. Clark BR. Guerin MR.  B.:rkenhus.BD.  Rao TK. l-pler JL  il*»8l»:  Analytical and biological
      analyses of lest material from the synthetic fuel technologies. Mutat Res 85:335-345.
Htihhard  SA. Bridges JW. Green NtHL (1'^XIi: Freshly isnlaied hepaiix'ytes for nietaK>lic activation in
      a bacterial mutation assay. Mutat Re^ X5:2M.
Huisingh JL. BraJow R. Jongers  R. ClaUon  Ll>.  Xweidinger R. Tcjjxia S. Burngarner J.  DutlicM F.
       Waters  M. Simmon VF. Hare C. RodrigiKY. C. Smm L i l')7Si: Application of biiwssay  lo ihc
      cliarav-ten/alion of diesel panicle emissions.  In Waters MO. Ncsnow S. Hui.singh JL.  Sandhu SS.
      C'laxlon LI)  (e»Js);  "Applicaiion of Short-Term Bioassays in the Fruciioruition aixl Arulysi', of
      Complex  Environmental Mixtures II."  I-PA-WX) 9-7X 027. Research Triangle Paikl  North Car-
      olina: U.S. Environmental Protection Agency, pp 1-32.
J jger J (I'J7X(: IX-leclion  ami charactei i/ation of mtrodcrivativcs of S«>IIK jx»tycytlie aromatic hvdnvar-
      Nms  by lluorescenee quenching  alter  thin-layer  chromasography.  Application to air pollution
      analysis, rhromatography 152:575 57X.
Kadcri DA.  lines RA. Thillv  WG t|«>79>: Mtitageniciiy  ofso.it ami asvviaied p»lye>clic aromatic
      hydrocarhons to Salmonella typhimuriuni. Cancer Res 3^:4152-4159.

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                                                Bioassay of Automotive Emissions       629

 Kuwachi T, Yahugi T. Kada T.  Ta/ima V. Ishidate M, Sasaki M. Sugiyama T (1980): Cooperative
       program on short-term assays tor earcinogenicity in Japan. IARC Set Publ  27:323-330.
 King LC, Kohan MJ. Austin AC. Claxton LD. Huisingh JL (1981): Evaluation of Ihc release of mutagcns
       from diesel particle* in the presence of physiological fluids. Enviton Mutagcn 3:109-121.
 Kolin P. Falk  HL. Thomas M (1954): Aromatic hydrocarbons, II. Presence in the paniculate phase of
     - gasoline-engine exhausts and the carcinogcnicity of exhaust extracts.  AMA-Arch Ind Hyg Qecup
       Med 9:164-177.                                  •
 Kotin P. Falk  HL, Thomas M (1955): Aromatic hydrocarbons. III. Presence in the paniculate phase of
       dicscl-enginc cxhauMs and the carcinogcnicity of exhaust extracts. AMA ArcH Ind Health 11:113-
       120,
 Lavoie E.  Bedenko V. Hi rota N. Hecht SS. Hoffman D (1979): A comparison of the mutagcnicity.
       tumor-initiating activity and complete carcinogen icily of polynuclear aromatic hydrocarbons. In
       Jones PW, l.eber P (ed>): "Polynuclear Aromatic Hydrocarbons." Michigan: Ann Arbor Science
       Publishers. Inc.. pp 705-721.  '
 Lavoie E,  Tulley L, Bcdcnko  V. Hoffman D (I981a): Mutagcniciiy of  methylated fluorenes  and
       hctttofluorencs. Muui Res 91:167-176.
 Lavoie E. Tulley -Freilcr L. Bcdcnko V. Holt man D (I981b): Mutagenicity. tumor-initiating activity, and
       metaboliNm of methyl phcnamhrcncs. Cancer Res 41.'3441-3447.
 Lchuu-iiz H. Brusick D.  Matheson 0. Jagannath DR. RecJ M. G: "Toxicological Effects of
       Emi..sions  from Diesel F.ngmes." New York: Elsevier. pp 243-264.
 Liher HL.  AnJon BM. Hues KA. Thilly SVG (1980): Diesel MHII: Mui;»fii>n mca.M»renwnts in haclerial
       and  human cells.  In Pepelko WE, Danner RM.  Clarke  NA (cds|:  "Health Effects of Diesel
       Engine  Emissions: Proceedings of an International Symposium." EPA-6(X)/9-KO-U57a, Cincin-
       r»aii. Ohio: U.S. Environmental Protection Agency, pp 404-412.
 Lofroth G   (I98(b>: "Diesel Exhaust Emissions." Toxicology  program status  report—January
       1980. DOI:/EV-76-C-(M-l013. SS'ashinj-ton. DC: U.S. Dept of Energy.
 McClelland RO ted) (19ROh): "Diesel Exhaust  Emissions." Toxicology program status report—July
       19X0. DOI:/DE-AC(M-7fiVO-1013. Washington. DC: U.S IX'pl ol Energy.
 McCoy EC. Ro\enkran/.  HS < I9W)|: Activation of pofycyclic ..mmatic hydnvarhons  to mutagens by
      single oxygen: An  enhancing effect of atmospheric pollutants? Cancer Leu 9:35-42.
 McCoy EC. Rosenkran/.  EJ. Roscnkranz HS. Merrnelstcin R (19811: Nitrated ftuorenc derivatives are
      potent framcshift rnutagens. Mut;it Res 90:11-20.
 McXJralh Jt, .Schreck'RM.  Siak. JS (1978): Nfutagenic screening of diesel  paniculate  material.  Paper
      presented at the 71st Annual Meeting of the Air Pollution Control Association. Reprint No.  78-
      33-6. Houston. Texas. June 25-30. 1978.
 McMahon RE. Cline JC.  Thompson C7. (1979): Assay of 855 test chemicals in ten tester strains using a
      new  nwKlification of the Antes lest  for bacterial mulagcns.  Cancer Res 39:682-693.
 Mcrrr.clstcin R. Rosenkran/ HS. McCoy EC (1981): The microhial mutagenicily of nitroarencs. In Tice
      RR.  Costa  1)1.. Schaich KM (ed.s): "Genoloxic Effects of Airb!>rne Agents." New York: Plenum
      Press, pp 369-396.
 N'akamuru A. Kashimoto T (1979): Toxicological assessment of heavy o«l und sulfur-containing compo-
      nents by various conventional biological tests. 2. Studies on heavy oil  components in food.
      Shokuhin Eiscigaku Zasshi 0 Food Hyg Soc Jpn) 20:161 -165.
Ohnishi Y.  Kachi  K. Sato K. Tahara S. Takeyoshi H. T«>fciwa H (1980): Detection of mutagcnic activity
      in automobile exhaust. Mutat Res 77:229-240.
 PcdcrMin JC. Siak JS (19HO): Characterisation of direct-acting mutagens in diesel exhaust particulatcs
      by thin layer  chrorrutography. Research  Puhl  No  GMK-3265.  Wjirren.  Michigan: General
      Motors. Inc.

-------
 630      Ciaxion

 Pcdcrson 1C, Siak JS (19813): Role of nitroaromatic compound* in the direct-acting mulagcnicity of
       diescl panicle extracts. J Appl Toxico) 1:54-60,
 Pcdcrson JC. Siak JS (I98lb): The activation of mutagcns in dicsel panicles with rat liver S9 enzymes.
       J Appl Toxicol  1:61-66.
 Pierson WR, Gorsc RA. SAailai AC. Brachaczek WW. Japar SM. Lee SC.  7.weidingcr RB..CIaxton
       LD (I9K2): Mutagenicity «f extracts of paniculate matter from vehicles on the road. In Smith JR
       (ed): "Diesel Emissions Symposium Proceedings." EPA-<>00/9-82-Oi4. Research Triangle Part.
       North Carolina: U.S. Environmental Protection Agency, pp 453-456.
• Pins )N Jr 11979): Photochemical and biological implications of t)ie atmospheric reactions of amines and
       bctuo(a)pyrcnc. Phitos Trans R Stic ton (a|290:551-556.
 Pitts JN Jr. van Cauwcnberghc KA. Gmsjcan D. Schmtd JP. Fit/. DR. Bclscr WL. Knudson G, Hynds
       P (1978):  Atmospheric reactions of  polycyclic hydrocarbons:  Facile formation of mutagenic
       nitro derivatives. Science 202:515-519.
 Pitts JN Jr. Harger W. Lokensgard DM. Fit/. DR. Scor/iell GM. Mejia V (I982a): Diurnal variations in
       •he niutagcnicity of airborne paniculate organic mutter in California's south coast air basin. Mutat
       Res  104:35-41.
 Pitts JR Jr. Lokensgard DM. Harger W. Fisher TS. Mcjia V, Schulcr JJ. Scomell GM, Katzenstcin YA
      . (I982b): Muiagcns  in diescl exhaust  paniculate, Identification and direct  activities of 6-nitro-
       ben«Xa)pyrene. 9-nitroanthracene'. l-niiropyrene. and 5H-phenan:hro|4.5-bcdlpyran-5-one. Mu-
       lat Res 103:241-249.
 Prater TJ. Sehuct/.lc D U982): Capillarj- ailumn GC/.MS characieri/ation of diescl exhaust paniculate
       extracts. In Smith JR  :  Chemically-induced DNA repair synthesis in primary' rat ncpaiocylcs:
       Correlation with bacterial mutagenicity. Ann NY Acad Sci 349:405-406.
 Probst  GS.  McMahon RE. Hill LE. Thompson C7..  Epp JK. Seal SB (1981).  Chemically-induced
       unscheduled DNA synthesis in primary rat hcpatocyic cultures: A comparison  with bacterial
       inutjigenicity usirip 218co»)poundv Environ Muugcn 3:11-32.
 Rao TK. Ruhin  IB. Guerin  MR, Eplcr JL < 1976): Environmental mujagenc>is of energy-related effluents.
       Genetic;. K3(5'):«>.
 Rappupon  SM. Wang YY.  Wei EJ, Sawyer R, Walkings BE. Rappapon  If (1980);  Isolation  and
       identification of a direct-acting  inutagcn in diesel cthausi  panicul''ites.  Environ Sci Icchnol
       14(12): 1505-1509.
 Riley T, Prater T, Schuet/lc  D. Harsx-y TM, Hum  DF (1982):  The analysis of nitrated  poly'nuclear
       aromatic hydrocarbons in dtesel exhaust paniculaic.v by MS/MS technology. In Smith JK led):
       "Diesel Emissions Symposium Proceedings." EPA-60/9-82-014. Research Triangle Park, North
       Carolina: U.S. Environmental Protection Agency, pp 115-119.
 Rnscnkran/. HS, McCoy EC. Sanders DK, Butler M, Kina/.ides OK. Merniestein R (1980):  Nitropyr-
       enes: i.solaiion. idemiiication and reduction of mutagenic impurities in carbon black and  tonc.t.
       Science 209:1039-KM 3.
 Salamone  MF.  Hcddle JA,  Kat/.  M (1979): The mutagcnic activity  of thirty  polyeyclic aroniatic
       hydrcK-arbons and oxides in urban airborne particulars. Environ Inl 2:37-43.
 Schuetzlc D. Riley TL. Prater TJ (19X2): Analysis of nitrated polycyclic aromatic hydrocarbons in Diesel
       particulates. Anal  Chcin 54:265-271.
 Siak JS. Chan TL. l-cc PS i 1979): Diesel particular extracts in bacterial lest systems. Research Pub)
       No. GMR-3171. Warren. Michigan: General Motors. Inc.
 Siak JS. Chan JL. Lee PS (1981): Diesel-paniculate cxlracts in bacterial icM systems. Environ Int 5(4-
       5):243-24«.     .                   .                                 .
 Siak JS. Strom KA (1981): Biological fate of inhaled diescl panicles.  Paper presented  at the Annual
       Meeting  of the Society of Toxicology.  San Diego. California. Feb 1981.
 Smith JR (cd»-(!9«2»: Dtesel Emissions Symposium  Proceedings. EPA-600/9-82-014.  Research Tri-
       angle Park, North Carolina:  U.S. Environmental Protection Agency.
 Talcott RE, Harger W  (1981): Chemical characterization of direct-acting  airborne mutugcns: The
       Functional Group. Mutal Res 91.-433-436.        .                    •
 Tokiwa  H, Nakagawa R.  Ohmshi Y  (I9KI):  Mutagcric  assay of aromatic nitro  compounds with
      .Salmonella lyphimurium. Mulat Res 91:321-325.
 WangCY, l.ce MS. King CM, Warner PCM 1980): Evidence for nilroaronialics as direct-acting muiagcas
       of airborne paniculate*. C'hemospherc 9:83-87.

-------
                                               Bioassay of Automotive Emissions

 Wang YY. Wei ET (1981): Ability of liver homogeriutcs and proteins to reduce the mulapcnic effect of.
       diesel exhaust .particulars, in Waters  MD, Sandhu SS. Kuisingh JL. Claxton LD, Nesnow S
       ): "Short-Tcni> Bioassay in the Analysis of Complex Mixtures II." New York: Plenum Press,
       pp 359-368
 Wang YY, Rappaport SM. Sawyer RF, Talcott RD, Wei LJ (I978a): Direct-acting mulagens in automo-
       bile exhaust. Cancer Lett 5:39-47.
 Wang YY. Sawyer RF. Wei ET (I978b): Mulagens in automobile exhaust. In Waters MD, Nesnow S.
     .  Hubtngh JL.. Sandhu  SS, Claxton LD. (eds):  "Application  of  Short-Term  Bioassays in the
       Fractionaiion and Analysis of Complex Environmental Mixtures." EPA-600/9- 78-027, Research
       Triangle Park. North Carolina: U.S. Environmental Protection Agency, pp 587.
. Wang YY. Takott RE. Seid DA. Wei EJ (1981): Antiniutagenic properties of liver homogenate  protein
       and gluiathione «m diesel exhaust purticulies. Cancer Lett 11:265-275.
 Wei ET. Wang YY, Rappaport SM (1980): Diesel emissions and the Ames test:  A commentary. J Air
       Pollui Control Assoc 30(3):267-271.                                             r
 Wei ET. Wang YY, Taleott RE. Sawyer RF.  Ruppaport SM( 1978): Mutagea< in automobile exhaust.
       Fed Proe Fed Am Soc E\p Biol 37:247.
 Xu XH. N'achtinan JP. Jin 7.L, Wei ET. Burlingarnc AL (I982a): Isolation and identification of mutagenic
       nilroarcnes "in Uirsel exhaust particulates.  Jn Smith JR (ed):  "Diesel Emissions Symposium
       Proceedings." EPA-WXt/9-82-014, Research Triangle Park, Norlh Carolina: U.S. Environmental
       Protection Agency, pp 556-558. •               •                     '
 Xj XB. Nachtman JP. Jin ZL. Wei ET, Ruppaport  SM (I982h): Isolation arid identHcation of mutagenic
       nitro-PAH in dicsel-exhuust paniculatcs..Anul Chim Acia 136:163-174.
 Yergey JA.  Risby TH,  l^rstz SS (1981): The  chemical characterization of diesel paniculate matter, tn
       Smith JR  (ed):  "Diesel  Emissions Symposium  Proceedings."  EPA-600/9-82-OI4.  Research
       Triangle Park.-North Carolina: U.S. Environment! Prolection Agency, pp 111-114.
 Yergey JA. Risby TH.  Lest/. SS (1982):  Chemical characterization of organic ad.sorbates  on diesel
       paniculate matter. Anal Chem 54(3):354-357.

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