Retrospective View of the Value of
      Short-Term Genetic Bioaasays in
      Predicting the Chronic Effecta of Diesel Soot
       (U.S.)  Health Effects Research Lab.
      Research Triangle Park, NC
      Aug  86
                                                                     PB86-240934
* ;'"r 1 fTCf--' *"-*^»--* -••Y^iiftj-^fcy
                    s^,ii.ffiSK7'?^^

                           K^

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                                   TECHNICAL REPORT DATA
                            If lease read /mtrucnons on iHe reverse before completing)
1. REPORT NO.
   EPA/600/D-86/189
                              z.
             3. RECIPIENT'S ACCESSION NO.
               PB86-2409347AS
4. ' ITLE AND SUBTITLE
A RETROSPECTIVE REVIEW OF THE VALUE OF SHORT-TERM
GENETIC BIOASSAYS  IN PREDICTING THE CHRONIC EFFECTS
OF DIESEL SOOT
             5. REPORT DATE
                 August  1986
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Joellen Lewtas and (Catherine Williams
                                                           8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
USEPA, HERL/GTD/GBB, RTP,  NC 27711
             10. PROGRAM ELEMENT NO.

                  ANNA1E
                                                            11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory - RTP, NC
Office of Research and Development
U.S. Environmental Protection  Agency
Research Triangle Park, NC 27711
                                                            13. TYPE OF REPORT AND PERIOD COVERED
             14. SPONSORING AGENCV CODE


                  EPA/600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
    • In retrospect, it is now safe  to  conclude that short-term mutagenicity assays
were not only useful but instrumental  in:  (1) indicating that diesel soot was potentially
carcinogenic and should be evaluated in chronic animal cancer bioassays, (2) identifying
V02~PAHs as potential carcinogens in this  very complex mixture, (3) providing initial
evidence that the mutagens were btoavailable, and (4) estimating the relative importance
of various sources and fuels  and other factors which can influence human exposure to
carcinogens.  This is not to  say that  short-terra bioassays used alone can accomplish all
af this.  However, used in combination with chemical/analytical methods and toxicological
tools, short-term genetic bioassays have become a critical component of many environmental
lealth studies.  Although substantial  advances in our knowledge of the toxicology of
diesel emissions have been made since  1978 when the initial observation that the organics
extracted from diesel soot were mutagenic, a number of important questions remain not
only for diesel emissions but for other combustion sources as well.  Are the chemicals
*hich induce positive results in the short-term bioassays the same agents which cause
tumors in chronic animal bioassays? Which phase of the diesel emissions (gaseous or
particulate) is carcinogenic  in the animal inhalation studies?  With advances in our
understanding of the molecular mechanisms  involved In producing chronic effects such as
cancer, it is possible that new genetic tools and short-term bioassays will continue to
contribute to our ability to  answer these  and other questions as they arise.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTOR?
                                              b.lDENTlFIEPS/OPEN ENDED TERMS
                           c.  COSATi Field/Group
18. DISTRIBUTION STATEMENT

 RELEASE TO PUBLIC
19. SECURITY CLASS (Tliii Report/
JNCLSSSIFIED
                                                                          21. NO. OF PAGES
                                               20. SECURITY CLASS iThit pagtl'
                                                UNCLASSIFIED
                                                                          22. PRIC
EPA Fern 2220-1 (R*«. 4-77)    PREVIOUS EDITION i< OBSOLETE

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                                                         PB86-2<*093<*
                                                        EPA/600/D-86/189
                                                        August  1986
A RETROSPECTIVE VIEW OF THE VALUE OF SHORT-TERM GENETIC  BIOASSAYS IN
            PREDICTING THE CHRONIC EFFECTS OF DIESEL SOOT
                                 by
                Joellen Lewtas and Katherine Williams
                 Health Effects Research Laboratory
                U.S. Environmental Protection Agency
                  Research Triangle Park, NC 27711
                 HEALTH EFFECTS RESEARCH LABORATORY
                 OFFICE OF RESEARCH AND DEVELOPMENT
                U.S. ENVIRONMENTAL PROTECTION AGENCY
                  RESEARCH TRIANGLE PARK.' NC 27711

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                      NOTICE

This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication.  Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
                       11

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 A  RETROSPECTIVE VIEW OF  THE VALUE OF SHORT-TERM  OENETIC
 BIOASSAVS  IN  PKEDICTING THE CHRONIC EFFECTS OF DIESEL SOOT
 JOELLEN  LEV.TAS  AND KATHERINE WILLIAMS
 Genetic Bioas5ay Branch. Genetic Toxicology  Division. Health  Effects Research
 Laboratory,  U.S. Environmental  Protection'Agencv. MD 68, Research Triangle
 Park. NC 27711  (U.S.A)                          -
  The organic matter extractable from diesel soot particles was first reported to be
 mutagcnic in bacteria in  1978 (1-3).  This finding was rapidly confirmed by many
 groups (-1-6).  These organics were also  found to cause gene mutations. DNA
 damage, and chromosomal effects  in several  mammalian cell systems (7-11).
 Bioassay directed fractionation and characterization studies using bacterial
 mutagenicity  assays  indicated that compounds more  polar than polynuclear
 aromatic hydrocarbons (PAHs) »ere responsible for  most  of  the  nutagenicity  in
 these organics (1, 4-6. 12). By 1982 a  number  of potent nitrated PAHs (NO-,-
 PAHs) had been identified in diesel soot (13-16).
••
  Short-term genetic  bioassays were used in studies designed  to  determine the
 bioavailability and metabolism of diesel soot  mutagens (4, 17. IS).  Concern that
 bacterial mutagenesis assays may "overestimate'* (he mutagenic activity of diesel
 soot due  to the presence  of NOi-PAHs.  led to studies  on  the  mammalian
 metabolism  and DNA-binding of NO-,-PAHs alone and associated with diesel soot.
 These studies showed that the bacterial mutagens and  NO,-PAHs were rapidly
 released from diesel  particles.  Metabolism  studies in  both  whole animals and
 mammalian target cells (e.g..  tracheal ceils)  have  demonstrated that  NOi-PAHs
 are metabolized by both  oxidathe and reductive pathways to  produce  metabolites
 that  bind covjlently to DNA (19-21). In diesel  soot extracts,  the concentration of
 certain NO->-PAHs is highly correlated with the mutagenicity  and tumor-initiating
 activity of the extracts (22).   Soots from many other combustion  sources (e.g.,
 wood  stoves  and   gasoline  automobiles)   contain substantially less NO-,-
 PAHs than diesel  emissions (15).
  Short-term genetic bioassays have been  used  in a series of studies designed to
--"determine  (he comparative potency and  characteristics of  various  diesel soot
 extracts compared  (o other  combustion sources  (7, 23. 24).   Bacterial and
 mammalian cell assays have been compared to mouse skin tumor initiation assays.
 These studies suggest  that the relative potency of diesel  soot extracts  in bacterial
 or mammalian cell assays can  be used to piedict the relative  carcinogenic potency
 in rodent  assays and in  humans  under  certain  conditions and assumptions.
                                 19
 Comparative studies  of (he  mutagenic emission  rates of various automobiles and
 fuels provide a simplified method for directly comparing alternative sources and
 technologies.

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 Studies to evaluate the mutagenicity of whole diesel emissions containing both
the soot particles and gases hare been conducted in plants, insects, and mammals
(8, 25).  Bacterial assays were used to evaluate the gaseous organics that could be
collected  by adsorption or condensation. These studies demonstrate that the
gaseous components also contain mutagens. In vivo mutagenesis studies of rodents
after relatively short inhalation exposures, however, show much less nu'tagenicity
and demonstrated  no heritable effects (25).  This suggests that  either the
oiutagenic components of diesel emissions did not reach the gonads under these
exposure conditions,  that  these assays are insensitive  to the   mutagens present in
diesel emissions, or the effects were helow the level of detection.
 Incomplete combustion of many types of fuels result in the production of soot.
The International Agency  for Research  on Cancer's (IARC) Monograph on Soot
(26) concludes  that there  is sufficient evidence that soot is carcinogenic to
humans. Very few studies of humans exposed to diesel soot, however, have clearly
shown evidence of increased cancer risk.  Many soot extracts  have also been shown
to be carcinogenic in experimental animals; however, few rodent inhalation studies
of combustion emissions have demonstrated carcinogenicity.  New studies,  reported
in this volume,  showing that diesel emissions are carcinogenic to rodents after
chronic inhalation are consistent with the IARC conclusion that soot is a human
and animal carcinogen.  These rew results are also consistent with  the positive
short-term genetic bioassay results reported over five  years earlier.  In this paper
we take a retrospective  vie* of the short-term genetic bioassay data developed
over the  past  eight years  and their value in predicting chronic carcinogenic
effects.

WHAT WERE  THE EARLY BIOASSAY CLUES REGARDING THE RISK OF
DIESEL SOOT?
  We found  the  e.vtractable organics (tar) from diesel particle emissions to
consistently cause positive responses in short-term mutagenesis and carcinogenesis
bioassays (8, 11) as summarized in  Table I.  Other investigators have reported
similar samples to be mutagenic  in human cells  (9, 27).  We have  recently
evaluated  the genetic activity (potency) data from diesel and gasoline automotive
emission.'; in a  number of bioassays  using  the genetic activity  profile method of
Waters (28, 29).  These profiles provide a graphic representation of bioassay data
that facilitates both visual and computer-assisted comparative assessment.  The
concept of a genetic activity profile for a chemical originated from  the need to
represent  in a  single  two-dimensional configuration  the  qualitative  and
quantitative data from a large number of genetic  bioassay  systems (currently more
than 200).  The x-axis values  of the profiles shown here (Fig. 1) represent the
bioassays  in an  endpoint/pliylogenetic sequence, and the  y-axis values represent

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TABLE I
SUMMARY OF THE RESULTS UF SHORT-TESM BIOASSAYS USED IN THE EVALUATION OF DIESEL
PARTICLE EXTRACTS
                                                                      Results for
                                                                        3iesel
                                                             Genetox   Particle
Assays                                                         Code    Organics

Mutagenesis Bioassays

    Gene Mutation Assays
        Bacterial
            Salmonella typhimurium                              SA9         +
            Escherichia coli WP2                                EC2         *
            Escherichia coli K12                                ECK         +
        Mammalian cell
            Mouse lymphoma, L5178Y/TK*/- locus                  G5T         +
            Chinese hamster ovary, CHO, HGP3T locus             GCO         «•
            Chinese hamster lung, V79, HGPRT locus              G9H        (-)
            Mouse embryo fibroblasts, Balb/c 3T3, Ouar          GIA        (*)

    DMA Damage Assays
        Yeast
            Saccnaromyces cerevisiae
                03 mitotic recombination assay         .         SC3        (+)
                03 preincubation mitotic recombination assay    SCP
                04 mitotic gene conversion astiy                SCG
                07 induced mitotic crossing over                SCH        (+)
                07 reverse mutation                             SCR
                07 gene conversion assay                        SCG
        Mammalian Cell
            Unscheduled ONA repair in liver cells               UPR         *
            Sister chromatid exchanges in CHO cells             SIC         *
            ONA strand breaks in SHE cells               .       OIA

    Chromosomal Aberrations
        Mammalian Cells
            CHO cells                                           CIC         *
            Human lymphocytes                                   CHL        (+)
Carcinogenesis Bioassays

    Oncogenic Transformation Assays
        Chemical Transformation
            Mouse embryo fibroblasts, Balb/c 3T3
        -   Mouse embryo fibroblasts, C3H10T1/2
            Syrian hamster embryo, SHE, focus assay
        Viral Enhancement of Transformation
            SA7 virus enhancement in SHE cells
    Skin Tumor Initiation
            SENCAR mice
            C57 Black mice
            C3H/HeJ mice
T8M
TCM
TFS

TEV
SKT
(*)
() indicates the response was either:  weak, not reproducible, or was observed  in
   only one sample

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-------
the potency or linear slope of the dose response.
 These results all suggest (hat diescl soot contains mutagens and carcinogens and
that inhalation of diesel soot is very likely to pose an excess cancer risk to whole
animals and humans.  The negative results reported in  the first several inhalation
carcinogenesis studies (30-33) led to questions  about  the use of short-term
bloassays and inhalation studies such as: 1) Were the carcinogens associated  with
the particles not  bioavailable? 2) Did  the  short-term  mutagenesis  bioassays
"overestimate"  the potential carcinogenicity of the diesel emissions?  3) Were
there mitigating  factors present in the whole emissions that had anti-carcinogenic
properties?  or 4) Were the animal inhalation models or  study designs employed
simply inappropriate?
 Several of these questions were addressed by using genetic bioassay methods as
described below.

ARE THE MUTAGENS BIOAVAILABLE?
 Initial studies reported that the mutagens  associated with diesel particles  were
not "bioavailable" when incubated with  lung fluids or other physiological fluids
(34, 35).   Subsequent studies demonstrated that the  mutagens  and specific
components (e.g.  1-nitropyrcne) were removed from  particles'after incubation  with
lung fluids and lung cells (36, 17); however, the proteinacious fluids were directly
antimulagenic by a protein  binding mechanism in the Ames assay  (36).  Other
investigators have demonstrated that the antimutagenic activity of 59 with diesel
extracts was non-enzymatic (37).
 A number of studies have been reported that directly  assess the mutagenicity and
bioavailability of mutagens  from whole particles.  Cultured cells attached to a
surface will readily engulf small particles; and in two independent studies, whole
diesel  particles induced mutations in CHO cells (38) and human fibroblasts  (27).
The most convincing studies  on the bioavailability  of  mutagens  from diesel
particles  have been  conducted in  vivo either  by inhalation  or iniratracheal
instillation.  The inhalation studies (39—11) are described in more detail in this
volume; however, they generally support the  Unavailability of mulagens observed
lo  the short-term bioassay studies.  We have found  that    C-nitropyrene  vapor
coated  onto diesel particles and intratracheally instilled  into rats was readily
released from the particles both  in the lung and CI  tract (18).

WHAT ARE THE MUTAGENS  IN  DIESEL SOOT?
 The discovery that  orgaalcs from  diesel soot were mutagenic  (1) resulted  from
studies  to fractionate and chemically characterize the mutagenic constituents.  We
Initially used  a fractlonation scheme developed by Swain et al (42)  for cigarette
smoke condensate.  Ths moderately polar and highly polar fractions contained

-------
most of the bacterial mutagenic activity as shown in Table II.  The moderately
polar fraction, which was the most mutagenic in Salmonella, induced mutation and
oncogenic  transformation  in  Balb/c-3T3  cells  but  did not  induce mitotic
recombination la Saccltaromyces cerrerisiae D3  (Table III). The fraction which
contains the most polar and highly oxygenated species, was also mutagenic In
mammalian cells in  the absence of S9  and  Induced mitotic recombination and
oncogenic transformation.  Conventional gas  chromatography/mass  spectroscopy
identified many  ftuorenones and methylated ftuorecones as major constituents of
these mutagenic fractions.   None  of  these or other identified  constituents
accounted  for the direct-acting frameshift mutagenic activity observed. Studies
with  nitroreductase-deflcient  strains of Salmonella  lyphimtirium  showed  a
substantial reduction in the mutagenicity suggesting that nitrated corn-pounds
contributed to this direct-acting mulagenicity (43). Nitrated polycyclic aromatic
hydrocarbons (NOj-PAHs) are  potent direct-acting frameshift mutagens initially
detected in xerographic toners  (44).  A series of NOj-PAHs were later  identified
and quantilated in diesel extracts In order to estimate their contribution  to the
mutagenic  activity 31* diesel paniculate  emissions (15, 45). These studies showed
that NO2-PAHs, di-NO,-PAHs, and hydroxy-NOj-PAHs together account for
much of the mutagenicity observed in Salmonella lyphimurium.   Particulate
emissions from  catalyst-equipped gasoline-engine vehicles using unleaded fuel
contain  significantly  less of these NC>2-PAHs (15).  The  mutagenic activity of both
leaded- and unleaded-gasoline emissions is substantially  Increased  with the
addition of an exogenous metabolic activation  (MA) system, suggesting that the
unsubstituted PAHs  may play  a more important role than do NO,-PAHs  in the
mutagenicity and carcinogenicity of gasoline emissions (8, 11).
  Although NOt-PAHs were  identified  in extracts of diesel particles and urban
air paniculate matter,  the  quantification of these compounds at low  levels has
posed problems  for analytical chemists because the  conventional analytical
techniques for quantifying PAHs are relatively Insensitive to  nitro-substituted
PAHs.   The dinitropyrene homers are  so highly  mutagenic in the Ames (TA98 -59)
assay that  trace concentrations of these compounds, if present, could account  for
a  major   proportion  of  the   observed  mutagenic activity.    Therefore,   a
capillary   column GC/MS  analytical  technique   using  on  column  injection
and  negative  chemical ionization   (NCI)  detection  was  developed to  detect
these and  other nitro-PAHs at very low concentrations (15).  This technique »at
applied  to  extracts of soot  particles  from  diesel  and gasoline vehicles and
urban air particles.  Over twenty different NO->-PAHs  were  identified  in the
diesel  engine extracts.  1-Nitropyrene  was the  NO^-PAH detected in greatest
quantity in the  diesel  extracts  (107-1590  ppm relative  to the  weight  of  ilu-
extract),  followed by  the nitrophenanthrcne/anthracene isomers. The   onl>

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TABLE II

DISTRIBUTION OF THE MASS AND BACTER1AL/MUTAGENIC ACTIVITY  OF  FRACTIONATED  DIESEL
PARTICLE OP.3ANICS

                                         SpecificDistribution  of
                                    Mutagenic Activity         Mutagenic  Activity
     Fraction           Mass             (n
                                      -S3
Organic acids
Organic bases
Ether insolubles
Paraffins
Aromatics
Moderately Polar
Highly Polar
Unfractionatad (DCM)
»
TABLE III
14.9
0.3
3.9
36.7
6.9
5.0
26.9
—


193
43.8
53.9
Neg.
49.5
7520
629
2557


248
132
80.9
"eg.
30.1
2620
798
1625


4.9
0.02
0.36
0.0
0.60
64.9
29.2



9.5
0.10
0.80
0.0
0.54
33.5
55.4



COMPARISON OF THE 8IOASSAY ACTIVITY OF THE TOTAL ORGANICS AND TWO FRACTIONS IN
FIVE SHORT-TERM 8IOASSAYS
Bioassay
Salmonella typhimurium
(revertants/ug)
Mitotic Recombination in
Total
Extract3
-59
2.6
0.3
+S9
1.6
0.1
Moaerately Polar
Fraction
-S9
7.5 •
Neg.
+39
2.6
Neg.
Hignly Polar
Fraction
-S3
0.6
0.2
0.8
0.5
Saccharomyces cerevisiae 03
  (mitotic recombination/
         xlO'6)
Gene Mutation in L5178Y              NT                 NT          5.7      0.7
Mouse Lymphoma Cells
  (mutation frequency/
   ug/ml x 10-6)

Gene Mutation in Balb/c         0.6     0.05      1.2      1.6      1.6      1.4
3T3 Cells
  (mutation frequency/
         x 10-6)
Oncogenic Transformation        0.3     0.06      1.4      0.6      0.8      1.0
in Balb/c 3T3 Cells
  (transformation frequency/
   ug/ml xlO"5)                     *
aTotal extract and fractions are those shown in Table II.

                             .    7

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dinitro-PAHs  for which analytical  standards  were available, the dinitropyrene
isomers, were delected in one diesel extract sample at sub-ppm concen:r?:ions (0.4-
0.6 ppm).

NO,-PAHs. ARE THEY  THE CULPRITS?
   Quantification of the concentration of the NO->-PAHs in diesel soot and
determination  of  their contribution to the  direct-acting trutagcnicity in Salmonella
lyphimiiritim TA 98 (Table IV) shows that  although  1-nitropyrene was present at
the highest concentration  (107-1590 ppm) in the  diesel particle extracts,  it
accounted  for  only  3-13% of   the   mutagenicity.    3-Nitrofluoranlhene
present at 1 ppm to 7 ppm  in the diesel samples accounted for 0.8% to 1.4% of
the mutagenicity.   By   using  the mutagenicity  values  determined in separate
experiments  for  2-nitrofluoreue and vnose  reported  in  the  literature  for  1-
nitronaphthalene.  these  compounds were estimated to account for less  than
0.01%  of  the  mutagenic  activity.    Although  the dinitropyrene isomers (1,3:
1,6; and 1.8) were detected  in only one diesel sample (Auto 2) at 0.4-0.6 ppm (sum
of 1.6 ppm), their mutagenic  activity  (496,000;  629,000;  and  870,000  rev/mg,
respectively)  was high enough  to   account  for  26%  of  the mutagenicity of
this  sample.   The total  "direct-acting" mutagenic  activity  In  Salmonella
typhimurium TA98 that  can be accounted for by  the  23  nitro-PAHs  quantified
In Diesel  2 is 40%.  This estimation  is  supported by the loss of 50%  of  the
mutagenic activity  of  this extract when it was  assayed  in   TA98NID  a
classical  nitrorcductase-deficienl Salmonella lypliimiirinni tester strain obtained
front H. Rosenkranz (49).
  The fact that dinitropyrenes at concentrations below the ppm level can account
for nearly one-third of the mutagenic activity (50) suggests that the presence of
other highly potent dinitro-PAHs may account  for  even more of the mutagenic
activity  in the moderately polar neutral  fraction.  A recent application of S.
typhimurium tester strains developed to exhibit resistance to dinitropyrenes (e.g.,
TA98/l,8DNPg)  lias led to  even larger  estimations  of the contributions  of
dinitropyrenes to the  mutagenicity of diesel particle extracts.   The lack  of
quantitative data on the concentrations  of the dinitropyrene isomers in such
samples has previously  made it impossible  to confirm whether the concentrations
of the dinitropyrenes  are indeed  sufficient to account for the  contribution
predicted  by  the resistant tester strains. Pederson (51) has  reported  that in
several light-duly  diesel particle  extracts,  50%  to  90% of  the TA98 (-S9)
mutageniclly Is lost when the  extracts are  tested  in TA98/1.8DNPg.  Because these
strains may show a resistance to other dinitro-PAHs,  it is possible that highly
potent dinitro-substituted isomers of other parent  PAHs present in  the particle
extracts  may also contribute to the  mutagenicity of  the moderately polar neutral

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   TABLE IV

   CONTRIBUTION OF NO^-PAHS TO THE  MUTAGENIC ACTIVITY OF PARTICLE EXTRACTS  IN
   SALMONELLA TYPHIHURIUM TA93 (-S9)

Extract
Sample

Diesel
Auto 1
Auto 2
Auto 3
Gasoline
Auto 4
Mutagenic
Activity
(rev/wg)
TA98 -S9

13.
3.9
3.5
1.6
1-nitropyrene nitrofluoranthene dinitrooyrene isomers
(ppm) {•.)« (ppm) (*.} (ppm) ('.)

1590 11. 7.0 1.4
589 13. 1.2 0.8 1.6 26.
107 2.7 0.9 0.8
2.5 0.1
   aPercent  contribution  to the total  mutagenicity  in  Salmonella  based  on  the
    concentration and mutagenicity  of  the  individual MUg-PAH.
ifr1 '
   TABLE V

   NITSOPYRENt AND NITROFLUORANTHENE CONCENTRATIONS  IN  DIESEL  PARTICLE  EXTRACTS
   AND CORRELATION ANALYSIS WITH HUTAGENIC AND TUMORIGENIC ACTIVITY
Particle Extract

Diesel Auto 1
Diesel Auto 2
Diesel Auto 3
Gasoline Auto 4
Correlation Coef.
r2 with 1-r;?
r2 rfith 3-KF
1-Npa
ppm
1590
589
107
2.5


3-NF&
ppm
7.0
2.9
1.2
0.9


Ames
TA98 (-S9)
rev/pgt
13.0
3.9
3.5
1.6

0.91
>0.99
Mouse
Lynphoma (-59)
MF/yg/mld
4.2
0.98
1.2
0.38

0.9P,
0.99
Tunor
Initiation
pap/mouse/-^
590
210
310
170

U.82
0.95
   al-Nitropyrene
   &3-NitrofIuoranthene
   cRevertants per ug
   ^Mutation frequency (mutants p^r 106 survivors) per yg per ml
   ePapillomas per mouse per ug in SENCAR mice

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fraction.
  Good correlations (r~>0.90) were observed when the slope of the dose-response
for the mutagenic activity of this same seies of automotive particle extracts in S.
typhimurium strain TA98(-S9) was plotted versus the mutagenic  activity  in the
mammalian cell assays and the skin tumor initiating activity.  The correlation of
mutagenic and skin  tumor initiating  activity  with the concentration of selected
nitro-PAHs  was examined for these diesel and gasoline samples.  Table V" shows
the high correlations observed between the concentrations of 1-nitropyrene  and 3-
nitrofluoranthene and the mutagenic activity in Salmonella typhimurium (-59),
L517SY mouse lymphoma cells (-S9), and skin tumor initiating activity in SENCAR
mice.  The  r   correlation coefficient in the presence  of S9 (not shown) was
somewhat lower. The mutagenicity and tumor initiation  activity (-S9) also
correlated well (r >C.9) with the concentrations of the nilro-252 isomer,  the nitro-
228 isomers, nitromethylpyrenes, and nitrofluorenes: while no correlation was
observed with several of the other less mutagenic, lower molecular weight nitro-
PAHs (nitro-phenanthrenes and nitronaphthalenes).  The quantified mono-NO-,-
PAHs account for less than 20% of  the direct-acting bacterial mutagenicity of
these samples.  The high correlations  observed between .the concentration of these
compounds and the mutagenic activity of the total extract, therefore, suggest that
the unidentified mutagens responsible for  the remainder of the mutagenic activity
and possibly the mutagenic and carcinogenic activity in other bioassays is directly
related to the relative concentrations of these mono-NOt-PAHs. Because the
remaining unidentified mutagens appear to be located primarily in the chemical
fractions that are more  polar than the fraction that contains the unsubstituted
and   mono-NO2-PAHs,  it  is   possible  that  other  di-NO->-PAHs   (e.g..
dinitrofluoranthenes) or  other oxygenated NO-»-PAH*s (e.g., hydroxy-nilro-PAHs)
species are responsible for the unidentified mutagenic activity.

ARE   THE  ORCANICS   FROM  DIESEL  SOOT SIMILAR  TO  OTHER
COMBUSTION ORGANICS?
  Characterization of  the mutagenicity of  emissions from other combustion  sources
shows some  general similarities (46).  In both wood and diesel combustion. 82-99%
of the mutagenicity was in the neutral fraction. Very  little mass or mutagenic
activity was observed  in the organic bases.  Differences between these sources were
observed in the distribution of mass and mutagenicity in the neutral subfractions.
  Bioassay-directed fractionation and chemical characterization studies also hate
been used to characterize and compare the complex organic emissions from  roofing
tar pots, coke ovens, and cigarette smoke. To obtain a gross characterization of
the chemical classes present in the samples, the chemical class distribution was
determined  by solvent partitioning the orga-nics into acidic, basic, neutral, and
                                  10

-------
cyclohexane insoluble fraciions.  The  ceutral fractions were further separated
into  the  ftonpolar neutrals,  aromatic*   (nitre-methane soluble) and  polar
neutrals. Characterization  of  the  distribution of  bacterial mulaqcnic activity
(Ames Salmonella lyphimiirium  bioassay) in each of  these  fraciions (47.  -IS)
showed significant differences  between the diesel.   coke  o»en  main,  roofing
tar.  and cigarette  smoke condensate samples as shown in  Fig. 2.  In the  diesel
samples, over  90%  of the mutagenic actiiity  was located in the aromatic and
polar-neutral  fractions,  and  a  significant portion of  this activity can  be
accounted  for by NO-»-PAHs.  The cigarette  smoke condensate.   coke  o»en
main,  and roofing tar samples  did  not contain  detectable amounts of  NO-,-
PAHs  (48).  Most of  the mutagenicity of  coke  oven main sample was found in
the  basic fraction (37%) and polar neutral fraction (39%).   The  cigarette smoke
condensate  sample  also had significant activity  in  the basic  fraction (66%).
but chemical analysis indicated  that  the components  differed significantly from
those of the coke  oven main sample.  The roofing tar sample contained aromatic
(14%) and  polar (75%) mutagenic con-stituents that were  not NO^-P.AH's. The
PAH  subfraction  of  each of these samples accounted for   only  a small portion
of the mutagenicity [e.g.  diesel (0.2%), cigarette smoke  condensate (0.1%).
roofing tar  (5%). and coke oven main (S%)|.
  Although  the specific mutagens in  these different sources  are not identical, they
all cause frameshift  mutations  and  appear  to  be compounds that could  be
classified as polycyclic  organic matter.  Chemical characterization  suggests that in
addition to nitrated  NC^-PAHs found  in  the slightly and moderately  polar
neutrals, hydroxylated and carboxylated polycyclic organics are found in the
Organic acid fraction, aromatic  amines and nitrogen  heterocycles are  found  in the
organic bases, and  highly oxygenated quinones. diones. and  nitro-ox\ genated
compounds  aro found in the polar neutral fractions.

CAN GENETIC  BIOASSAYS  BE  USED  TO  ESTIMATE RISK FROM NEW-
ENGINES.  FUELS OR CONTROL  TECHNOLOGIES?
  A comparative potency method has been developed  for cancer risk assessment of
diesel particle emissions faasec1 on a constant  relative potency  hypothesis and using
data  from a  battery  of  short-term  mutagenesis  bioassays  and  animal
tumorigtnicity studies on a series  of diesel vehicles emissions (23.24).  These same
bioassays were used to evaluate three complex  emissions for which  human lung
cancer risk estimations were available (emissions from coke ovens,  roofing tar
pots, and cigarette smoke).
  The comparative potency  method for cancer risk assessment  is based on the
hypothesis that there  Is a constant  relative  potency between two different
carcinogens (Cl and C2) across different bioassay systems (Bl and B2).  This

                           11

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  partitioning (48).  Fractions are as follows:  organic acids (Acid), organic
  bases (Base), nonpolar neutral-alphatics (NPN), polynuclear aromatic hydrocarbons
  (PNA1), moderately polar fractions separated from the PNA fraction  (PNA2,  PNA3,
  PNA4), polar neutrals (PH) and highly polar cyclohtxane insoluahles (CI).

-------
constant relative potency  assumption  has been tested most extensively for diesel
emissions  where the  largest series  of emissions has  been tested  in seteral
mutagenesis and carcinogenesis bioassays. Tne test of this model is whether there
is a constant relationship (k) between the relative potencies in the two bioassays
beinrc compared such that:
     Relative Potency (C1/C2) in  Bioassavf 1) = constant (k)
      Relatue Potency (C1/C2) in liioassay(2)
Based upon the data avialable on diesel  emissions, it appears that this  assumption
holds when comparing the relative potency  in the Ames Salmonella typhimurium
(TA  98) assay, the  mouse  lymphoma assay,the sister chromatid exchange assay in
CHO cells, and mouse skin tumor initiation in SENCAR mice. A  number of short-
term bioassays that did not result in quantitative dose-response data or in which
the responses were very weak could not be evaluated.
 In order to adequately test this hypothesis for  prediction of human cancer risk,
a more extensive comparative data base is needed for human lung cancer.  This
hypothesis was tested  for only  the three complex  organic emissions from a coke
o»
-------
TABLE VI

COMPARATIVE MUTAGENIC AND TUMORIGENIC EMISSION RATES
                   Organic     Potency of Organicsb
                  Emission  Ames   House   SCE   Skin
Vehicles3           Rate    TA98   Lymph.  CHO   Tumor
                            (+S9)  (+59)   (+S9) Init.

                  (mg/kn)
                                           Activity
                                        Emission Ratesc
                                   Ames   Mouse  SCE   Skin
                                   TA98   Lymph. CHO   Tumor
                                   (+S9)   (+S9)  (+S9)  Init.

                                   (xlo5)  (xlO4)  (xlO3)
Diesel
Car
Car
(Mercedes)
(VW Rabbit)
Truck (Ford/Cat)
Bus
(GM)
20
52
312
362
.2
.2
.0
.0
12
6
1
0
.0
.1
.7
.1
1
0
0
0
.5 0.16 0.37
.72 0.03 0.24
.23 	
.35 	
2.4
3.2
5.3
.4
3
3
8
13
.0
.8
.5
.0
3.2 7.5
1.6 12.5
...
—
Gasoline
Non-catalyst
  (Ford Van)

Catalyst
  (Mustang 11)
5.61   32.0   5.7     0.47    0.20     1.8    3.2     2.6     1.1
3.67    8.6   1.1
0.16    0.3    0.4
0.5
aThe methods for collecting these emissions and performing the mutagenesis Dio-
 assays have been reported-elsewnere (7,3,46).

bThe mutagenicity of the organics in the Ames bioassay is expressed in revert-
 ants/ug (rev/ug) in the mouse lymphoma bioassay as mutants/10^ survivor/yg/ml
 (mut.freq./yg/ml).  SCE as SCE/cell/ug/ml and skin tumor initiation as papil-
 lomas/mouse at 1 mg.

cThe rcutagenic and turaorigenic emission rates were determined from multiplying
 the mutagenicity of the organics times the organic emission rate to give
 activity emission rates per km driven.
                                  14

-------
 gasoline automotive emissions to unit risk  estimates  for  various combustion
 emissions or other complex mixtures through the data  base established  linking
 human, animal, and short-term genetic bioassay data.  A simplified comparative
 approach to evaluating alternative energy sources is  to employ  parallel bioassay
 studies of the alternative (a) and conventional (c) source emissions and determine
 a relative  risk  by direct comparison as follows: increased risk (a/'c) = relative
 bioassay potency   (a/c).   Because there is no one   conventional   standard
 petroleum-derived  fuel or one standard combustion  source,  such studies  need Co
 consider the range  of  mutagenic and  carcinogenic potency  between  different
 conventional sources and  fuels.  The establishment  of  such a range could  then
 serve as a  guide for evaluating alternative fuels or sources.
   In order to evaluate a battery of short-term  genetic  bioassays for their utility in
 testing and  assessment of unregulated automotive emissions, a matrix of bioassays
 and particle extract samples from various engines and fuels was constructed  (54).
 Three heavy-duty vehicles, each fueled  by three to five grades of diesel  fuel,  were
 operated on the '83 transient driving  cycle.  The light-duty automobiles  were
 fueled  by gasoline (leaded or unleaded) or the No.  2  diesel fuel. The short-term
*
 bioassays included  those shown in Fig. 1 and included both mutagenesis (gene
 mutation, DNA damage, and chromosomal effects in procaryotcs and eucaryoles),
 oncogenic transformation, and mouse skin  tumor initiation.  The mutagenesis
 bioassays were of three types:  gene mutation in both bacterial and mammalian
 cells, DNA damage assays in yeast and  mammalian cells, and chromosomal effects
 in mammalian  cells.  The carcinogenesis assays included  oncogenic transformation
 in two  lines  of mouse embryo fibroblasts and the mouse skin tumor initiation assay
 in SENCAR mice.  One sample from a diesel Mercedes car was tested in all 16
 assays (Fig  1).  The  data has been compared as mutagenic emission rates.
   This data base of mutagenic emission  rates has been evaluated  by an analysis of
 variance (ANOVA) method  to determine  if any of  the  emissions  produced
 significantly different (p-0.05  level) responses (54).  Because the ANOVA did not
 detect any significant difference with or without S9  activation, all of the results
 were combined for this analysis. Only the data from the  following  three bioassays
 where adequate for such ANOVA analysis:  Ames Salmonella typhimuriiim assay,
 mouse lymphoma gene mutation assay, and sister chromaioid exchange assay in
 CHO cells.   Among the three tests, the Ames test exhibited greatest differences
 between the samples as shown in  Fig. 3. Samples 5  (heavy-duty Caterpillar
 engine) and  IS (light-duty Nissan engine),  both run on minimum grade diesel  fuel,
 hoc" significantly higher  mutagenic emission rates than the other vehicles and
 fuels. The  two mammalian cell bioassays showed higher standard deviations, and
 none of the emissions were found to'be significantly different, possibly due to the
 lack of replicate bioassays.
                                    15

-------
        ANALYSIS OF VARIANCE FOR ARTiES TEST ASSAYS

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 The increased or decreased human cancer risk  from combustion emissions may be
of greater concern than the absolute risk from  these emissions when we consider
the effect of alternative energy technologies and fuels. The data of greatest value
In  determining this increased or decreased risk  are comparative bioassay data  on
the conventional and alternative technology being evaluated. Such studies should
be useful  in providing  direction for engineers and chemists to design alternative
energy  sources  and fuel:; that result  in  less mutagenic and potentially less
carcinogenic emissions.
 The comparative potency  method for cancer  risk assessment described in this
paper cannot be employed without uncertainty or without invoking the constant
relative potency assumption inherent in the method.  It  is important to recognize
that the assumptions and uncertainties are different from those employed in using
either low-dose extrapolation techniques with human data or scaling factors with
animal data.  This makes it  possible, therefore,  to perform  quantitative cancer risk
assessments  using more than one approach  with  different  methodological
assumptions.  In the future, more information may be  gained by comparing the
quantitative assessments for one  source using several  independent cancer risk
assessment methodologies.

ARE DIESEL PARTICLE EMISSIONS  A SIGNIFICANT SOURCE OF HUMAN
EXPOSUE TO MUTAGENS  AND CARCINOGENS?
 The relative contribution of different sources  to ambient particle concentrations
has been determined using either dispersion or receptor modeling.  These studies
show that combustion emissions  account for  most of  the respirable (<2.5 um)
particles  in the air (55). We have recently used these  methods to estimate the
contribution of  various combustion sources to  the airborne mutagenicity due to
these particles (56).   Since a  much larger data base is  available  on the Ames
Salmonella assay, this data base  was used for the emission factors.  Table VII
summarizes the findings from these studies.  Using simply the  information in
EPA's publication of the annual U.S. fuel use by category, we estimate that 44%
of the mutagenicity emitted into  the air was  derived from diesel vehicles, 22°/o
from gasoline vehicles,  and 32% from residential heating. Dispersion modeling was
used to estimate-ambient  concentrations in a theoretical city and in actual cities
where more detailed source inventories and meterological factors can be considered
in a more complex dispersion model. In the examples shown in Table VII, 33-51%
of the mutagenicity of these locations was estimated to  arise from diesel vehicles.
Receptor  modeling uses data from ambient concentrations of tracer signature
chemicals (e.g., lead for automobiles) to determine the  contribution  from various
sources.  The data shown  for Denver, Colorado was  derived from a study to
                                  17

-------
  apportion the particles and organic; using receptor  modeling (57).  By applying
  data on  the mutagenicity  of  each of  these  source emissions,  assuming no
  atmospheric  transformation of the mutagenicity, only 5% of the mutagenicity was
  estimated to be derived from  diesel trucks and 56% from gasoline  vehicles.
  Recently we  have actually  used mutagenicity  as a parameter for apportionment in
  a receptor modeling study  in Albuquerque, Nc.w Mexico. Although this study was.
  not  designed tc separate diesel from gasoline vehicle emissions, 50%  of the
  mutagenicity of  this air shed in wintertime was due to automotive emissions.  In
  future studies we plan to improve our ability to  directly measure the contribution
  of various automotive and residential heating sources to the airborne mutagenicity
  and  tumorigenicity.
   The impact of automotive emissions on the total human exposure is generally less
  than that estimated by considering only  outdoor ambient air.  Most individuals
  spend over 80% of  (heir time indoors at home or work.  In a recent study of ten
  homes, we found environmental tobacco smoke to be the principal source of indoor
  particle-assocoated  mutagenicity  (59).
TABLE VII
CONTRIBUTION  OF MOBILE SOURCES AND  RESIDENTIAL HEATING TO  THE  AIRBORNE MUTAGEN-
ICITY ASSOCIATED WITH RESPIRA8LE  PARTICLES
Source
Mobile Sources
Diesel
Gasoline
Emissions from
U.S. Annual
Fuel Use

44
22
Dispersion
Modeling
Theoretical Site A Si
% %

49
22

51
23

Ce B
%

38
17
Receptor
Model ina
Denver Albuquerque
% %

56
5



     (TOTAL)         (66)             (71)        (74)     (55)     (61)         50
Residential
Heating
32
24 25 43 39 50
W
                                   18

-------
CONCLUSIONS ON THE VALUE OF SHORT-TERM GENETIC BIOASSAVS
  In retrospect, it is now  safe to conclude that short-term mutagenicity assays
were not only useful but instrumental in:
I)  Indicating  that  diesel soot was  potentially  carcinogenic  and should  be
evaluated on chronic animal cancer liioassays.
2) Identifying NO,-PAHs  as potentiil carcinogens in this very complex mixture.
3) Providing initial evidence that the mutagens were bioavailable.
4)  Estimating the relative importance of various sources and  fuels and other
factors which can influence human exposure to carcinogens.
  This  is not to say that short-term bioassays  used alone  can accomplish all of
this.   However, used in  combination  with  chemical/analytical methods and
toxicological tools, short-term  genetic bioassays have become a critical component
of many environmental health studi-:.
  Although  substantial advances in our knowledge of the toxicology of diesel
emissions have been made since 1978 when the ini.'ial observation that the  organics
extracted from diesel soot were mutagenic, a number of important questions
remain not only for diesel  emissions but for other combustion sources *is well.  Are
the chemicals  which induce positive results in the short-term bioassays the same
agents  which cause tumors in chronic animal bioassays? Which phase  of the diesel
emissions (gaseous or paniculate) is  carcinogenic in the animal inhalation studies?
  With  ad.vances  in our understanding of the molecular mechanisms involved in
producing chronic effects  such as cancer, it is possible (hat new genetic tools and
short-term bioassays will continue to contribute to our ability to answer these and
other questions as they arise.

 ACKNOWLEDGMENTS
 'The authors acknowledge the assistance if Dr. Larry Claxion and Ms. Jackie
Finley  in the preparation of the manuscript   The research  described in this paper
has been reviewed by  (he Health Effects  Research Laboratory, U.S. Environmental
Protection Agency and approved for publication.  Approval does not signify that
the contents necessarily reflect the views and policies of the  Agency.

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