EPA/AA/CTAB/PA/81-19
       Summary of EPA and Other Programs on the Potential
                Carcinogenlclty of Diesel Exhaust


                               by


                         Penny M. Carey


                          August 1981
                             NOTICE
Technical  reports  do  not  necessarily  represent  final  EPA
decisions  or   positions.    They   are   intended  to   present
technical  analyses  of  issues  using data  which are  currently
available.  The  purpose in the release  of such reports  is  to
facilitate the exchange of technical information and  to inform
the public of  technical developments  which may form  the  basis
for a final EPA decision, position or regulatory action.
    Control Technology Assessment and Characterization Branch
              Emission Control Technology Division
          Office of Mobile Source Air Pollution Control
              U.S. Environmental Protection Agency
                       2565 Plymouth Road
                   Ann Arbor, Michigan  48105

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                              Table of Contents

                                                                    Page

I.    Summary and Conclusions	       4

II.   Introduction  	       8

III.  Summary of Major Diesel Health Effects Publications ....      10

      A. EPA	      10
      B. National Academy of Sciences (NAS) 	      10
      C. Health Effects Institute (HEI) 	      11

IV.   Description of EPA's Diesel Emissions Research Program  . .      12

V.    Mutagenicity	      14

      A. Iti vitro studies	      14
      B. In vivo studies	      19

VI.   Carcinogenic! ty	      22

      A. In vitro studies	      22
      B. In vivo studies	      24
      C. Bioavailability	      30

VII.  Non-genetic Effects 	      31

VIII. Characterization  	      33

      A. Gas-phase	      34
      B. Particulate	      37

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IX.   Epidemiology	            39






X.    Risk Assessment	  .  .            42






References	            47






Tables	            54

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I.    Summary and Conclusions

Much research  has  been performed to  evaluate the health  effects  associated
with exposure  to Diesel emissions.   The  research performed falls  into  five
general   areas:   mutagenicity,    carcinogenicity,    non-genetic    effects,
characterization  and epidemiology.   EPA is  conducting a massive  research
program that includes  studies  in each area.  Since epidemiological  data for
Diesel  emissions are  limited,  a  major  portion  of   EPA's  research  effort
involves  determining the  relative mutagenic and carcinogenic  potency  of
Diesel  emissions compared to  potencies  of comparative  emissions  for which
epidemiological  data are  available.   EPA will  use  the  results  of these
studies with epidemiological data for the comparative sources to  assess the
human health risk associated with exposure to Diesel emissions.

This report summarizes  most  of the studies EPA plans  to use to formulate  a
Diesel risk assessment  including  significant  studies  by researchers  outside
EPA.  The National Academy of  Sciences (NAS) has  completed an evaluation of
the  existing   health effects  data  base  on  Diesel   exhaust  products;   the
general conclusions reached in this study are also included.

Based on the studies summarized, the following generalizations can  be made:

1.    Mutagenic  compounds, both direct and indirect acting  in the Ames and
      other  bioassay   tests,   are  associated   with  Diesel  particulate
      emissions.  Most of these particulate emissions  represent  particulates
      which are  small enough  to  be  inhaled  and  deposited  deep within the
      human lung.

2.    Diesel exhaust particulate  extracts and  whole particulates  contain
      some  known  carcinogenic   materials   such  as  benzo(a)pyrene.    The
      extracts have been shown to be  mutagenic and carcinogenic in a number
      of  j.n   vitro  and  in_  vivo  bioassay  tests.    Whether   whole (i.e.
      unextracted)  engine  exhaust  particulates  are   carcinogenic  to   any
      significant extent  is not  known yet;  however,  work  is  currently  in

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      progress  on  two  studies:  the  intratracheal  instillation  of  Diesel
      particulate   and   extract  with  Syrian  Golden   hamsters  and   the
      intraperitoneal  injection  of  Diesel  particulate  and  extract  with
      Strain A  mice.   The results  of  these studies could  provide  important
      information   about   the   bioavailability  of   the  organics  on   the
      particulate,  in addition  to  determining  the  carcinogenicity of  whole
      Diesel exhaust particulates.

3.    To date, whole Diesel exhaust  (particulate and gas phase) has  not been
      found  to   be  carcinogenic  when   inhaled   by   laboratory   animals.
      Inhalation  experiments  with  dilute  Diesel  exhaust  possibly  do  not
      permit a sufficient dose  of the  active portion to enter the lung.   In
      addition,   inhalation  experiments   designed   to   detect   in_   vivo
      mutagenicity  have  produced   generally  negative  results  with   the
      exception of one study designed to detect sister chromatid exchange.

4.    The  mutagenic and  carcinogenic  potencies  of the Diesel  particulate
      samples are  generally but not always less than  the  potencies of  the
      comparative  samples  (coke oven  emissions, roofing tar emissions,  and
      cigarette smoke condensate) based on  a  variety of  tests including skin
      tumorigenesis initiation;  however, they all fall within the same  order
      of magnitude (per unit weight  of material tested).   The four  Diesel
      samples tested  (Caterpillar  3304, Datsun  Nissan 220C, Oldsmobile  350
      and  Volkswagen  Rabbit)  exhibited  a  wide range  of  potencies  (i.e.
      sometimes an order of  magnitude difference).   The  potencies  of  the
      Diesel and  comparative  samples  appear  to be  two  to  three orders  of
      magnitude less  than that  of  pure benzo(a)pyrene,  a carcinogenic  and
      mutagenic polynuclear aromatic hydrocarbon (PAH).

5.    While  extensive Ames and  other  bioassay  testing  for mutagenicity  is
      being  performed on the organics extracted from  the particulate,  the
      relative  mutagenicity  of  the  gas  phase organics  remains   unknown
      primarily because  an analytical method for  collection had  not  been
      developed.   EPA is  currently  working to develop  artifact-free  methods
      to  collect   gas  phase  hydrocarbons  in exhaust   for  future  bioassay
      testing.  Some work has been done by  EPA-OMSAPC  in Ann  Arbor,  with the
      bulk of the work being performed at EPA-ORD-RTP.

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6.    The chemical  composition of  the  organics adsorbed  on Diesel  exhaust
      particulate is  complex.  Polynuclear aromatic  hydrocarbons (PAH)  and
      numerous PAH  derivatives have  been identified thus  far.  It  appears
      that  nitro-PAHs  may   account   for  a  significant  portion   of   the
      direct-acting mutagenicity, as measured by the Ames test.

7.    Few conclusions  can be made  regarding  non-carcinogenic  pulmonary  and
      systemic effects  of Diesel particulates.  In  one  study,  mice  exposed
      to  diluted   Diesel  exhaust  exhibited  enhanced   susceptibility   to
      infection  when   subsequently  exposed  to a  bacterial  pathogen.   The
      significance  of  this  finding  is not  yet clear  and requires  further
      research.

8.    Epidemiological  data for  Diesel emissions  are limited.   The  London
      Transit Worker  study,  an epidemiology study  of Diesel bus workers  in
      London, has  been cited  as  a strong  indication that Diesel emissions
      result  in  no  excess cancer  risk.  There are many inconsistencies  in
      this  study including  the lack of  considering the  "healthy   worker"
      effect, as well  as  some doubt as to  whether  the study population  was
      exposed  to a  greater  amount  of  Diesel emissions  than  the   general
      population to which it  was  compared.   EPA's  statistical analysis  of
      this study indicates it would  still  be possible  to  have lung  cancer
      deaths numbering in  the  thousands each year  in the U.S. due to  Diesel
      engine emissions and not be  inconsistent with the results obtained  in
      the London Transit Worker study.

9.    A  risk  assessment  performed  by Lovelace  Research  Institute  for  the
      Department of Energy estimates  30 lung  cancer  deaths  per year may  be
      attributable  to   exposure   to  light-duty   Diesel   exhaust.    Their
      calculations were based  on  the  year 1995 and beyond,  assuming 20%  of
      the light-duty vehicle  fleet  will  be Diesel-powered and controlled  to
      0.16  gm/mile  particulate.   An  earlier  preliminary  risk assessment
      performed  by  EPA's  Carcinogen  Assessment Group   (GAG)  estimated   346
      lung  cancer   deaths attributable   to  exposure  to  light-duty   Diesel

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      exhaust and  668  lung cancer deaths due  to heavy-duty Diesel  exhaust.
      EPA's calculations were  based  on 1990,  assuming 10% of  the  light-duty
      vehicle   fleet   will   be   Diesel-powered   (a  best   estimate)   and
      uncontrolled   (1.08   gm/mile   particulate).    Both   the   EPA   and
      DOE-Lovelace assessments  hypothesized that  Diesel  emissions  were  as
      potent as coke oven  emissions.  Allowing for the different  population
      exposure  estimates,  the  two   assessments  agree reasonably  well.   It
      should be mentioned  that  both  the EPA and Lovelace  Research Institute
      risk assessments  are  tentative.

      EPA intends to release a  revised risk assessment in the future,  based
      on the results of  the relative potency  study.   The data available  to
      date indicate that coke  oven emissions  are,  in fact, more potent  than
      Diesel particulate extract.  Based on the potencies obtained  from  the
      skin tumorigenesis assay  only, a reevaluation  of  the preliminary  EPA
      risk assessment  was  made  by   GAG.   It  was  estimated that  19  cancer
      deaths per  year  in  the  U.S.   may  be  attributable  to  Diesel  exhaust,
      assuming  15%   of   the   automotive   fleet   is   Diesel-powered   and
      uncontrolled (1.0 gm/mile  particulate).   It  should  be  noted  that  the
      revised risk  assessment  that  EPA  plans  to  release in  the  future  is
      expected  to  incorporate  the results  of a variety  of mutagenesis  and
      carcinogenesis assays for estimation  of  relative potencies.

10.   The most  apparent  research gaps in  the Diesel health  program are  in
      the following areas:

      0  determination of  the  iii vitro  and i^ vivo  bioavailability of  the
         organics  adsorbed  on inhaled or ingested Diesel  exhaust  particulate,

      0  determination  of the mutagenic and carcinogenic activity  of the  gas
         phase components;  identification of the major components  or classes
         of compounds in the gas phase of Diesel exhaust,

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      0  effect  of  inhaled  or  ingested  Diesel  exhaust   on  susceptible
         subpopulations (e.g. pulmonary and cardiovascularly impaired),

      0  synergistic and  potentiative effects of  Diesel exhaust with  other
         environmental pollutants,

      0  short-  and  long-term   deposition  and  clearance   of   inhaled   or
         ingested Diesel exhaust particulates,

      0  further   characterization   of   the   particulate  soluble   organic
         fractions responsible  for  the mutagenic and  carcinogenic  activity,
         and

      0  a  more  definitive  estimate of  the  potential  carcinogenic  risk
         associated with Diesel particulate emissions.

II.   Introduction

The  projected  increase   in  the  production  of  light-duty  Diesel-equipped
vehicles has  raised  concern over possible adverse health effects associated
with  this  increase.  Attention has  been primarily focused  on  particulate
emissions  from Diesel-equipped vehicles.   The particles  in Diesel  exhaust
differ  both  in quantity  and composition from  particles in  gasoline  engine
exhaust.  Currently, Diesel-equipped  vehicles emit from  30  to 100 times more
particulate mass  (grams  per  mile)  than  gasoline-powered,  catalyst-equipped
vehicles.  These  Diesel  exhaust particulates  are small enough  to be  inhaled
and  deposited deep within  the  lungs.  Gasoline  particulate  emissions  from
catalyst-equipped vehicles  using  unleaded fuel are primarily  sulfates,  while
Diesel  exhaust particulates  are composed  of  carbonaceous  soot  with high
molecular weight organic compounds adsorbed on  the surface.   These  "particle
bound organics", or PBO,  can account for 10-50% of the  particulate weight.

In 1977, EPA  tested organic extracts  of Diesel  exhaust particulate  and  found
that  the extracts  contained materials  that  were  mutagenic to  strains  of

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Salmonella typhimurium in  the  Ames bioassay.  Since the Ames assay  has been
shown  to be  indicative  in  detecting  substances  that  are carcinogenic  (or
non-carcinogenic) in  whole animal studies,  EPA  felt that the  positive test
result  warranted  the issuance  of an informal  Precautionary  Notice  in 1977
(1)*.   This  notice  mentioned EPA's  preliminary  findings and suggested that
persons  working  with Diesel  emissions  in  a  laboratory  setting  exercise
caution to avoid exposure to the emissions.

EPA  has since launched  a  massive research program to  evaluate the  health
effects associated with  exposure  to  Diesel emissions.   Since  epidemiological
data for Diesel emissions are very limited,  a major  portion of  this  research
effort  involves determining  the relative mutagenic and  carcinogenic potency
of Diesel emissions  (specifically,  the  particle-bound organics)  compared  to
potencies  of  particle-bound   organics   from  other  emissions  for   which
epidemiological data  are available.  The  comparative  sources  selected were
coke oven  emissions,  roofing  tar emissions  and  cigarette smoke  condensate
(CSC).   Benzo(a)pyrene,  a known  carcinogen, was  used  as  a  standard.   The
mobile  source  samples  evaluated  included  a  heavy-duty  Diesel  engine
(Caterpillar  3304),  three light-duty  Diesel passenger  cars  (Datsun  Nissan
220C,  Oldsmobile  350  and  Volkswagen  turbocharged  Rabbit)  and  a  gasoline
catalyst-equipped car  (Ford  Mustang II).   The results  from  this study  and
others  will  be  used  to  assess the  human health  risk associated  with
increased use of the Diesel engine.

An extensive  amount of  research  has been performed by EPA  and others.   A
comprehensive  summary of  the  existing  health  effects  data  base on  Diesel
exhaust  products  entitled,  "Health  Effects of Exposure  to Diesel  Exhaust"
(prepublication) was  completed  by the National Academy of Sciences  (NAS)  in
1980 (2).
^Numbers in parentheses designate references listed at end of paper.

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The purpose  of  this report  is  to summarize  the significant health  effects
studies of  Diesel exhaust with an emphasis on  1)  research EPA will use  to
formulate  a  risk  assessment,   2)  research  results  available  after  the
printing  of  the NAS  report,  and  3)  significant studies  by  researchers
outside EPA.   Important  ongoing  research  will  also  be discussed.   Topics
covered in  this  report   include  mutagenicity,   carcinogenicity,  non-genetic
effects, characterization, epidemiology and risk assessment.

III.  Summary of Major Diesel Health Effects Publications

A.    EPA

EPA has published much information  on the health  effects  of Diesel  engine
emissions.  A description of some parts of  the program is  contained in  a
pamphlet  entitled,  "The  Diesel  Emissions  Research  Program" (3).   An  EPA
review of the health  effects data entitled,  "Health Effects  Associated with
Diesel Exhaust Emissions" was published in November 1978 (4).   EPA's  Health
Effects  Research   Laboratory  (HERL)  sponsored   the   first  International
Symposium on  the Health  Effects of  Diesel  Emissions  in December  1979.   The
proceedings  of  this international  symposium are contained  in  a  two  volume
publication (5,  6) and are referenced throughout this  report.

B.    National Academy of Sciences (NAS)

NAS recently published a report  entitled,  "Health Effects  of  Exposure  to
Diesel Exhaust"  (2).   This report was  prepared  by  the Health Effects  Panel
of  the  Diesel Impacts Study Committee under  contract to  the Environmental
Protection  Agency,   the   Department  of   Energy   and  the  Department   of
Transportation.   The report  is a  comprehensive summary  of the health  effects
of  exposure  to  Diesel exhaust  in four areas -  mutagenesis,  carcinogenesis,
pulmonary and systemic effects  and epidemiology.   The  principal conclusions
drawn by the Panel are summarized below.

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  0   The available epidemlologic information does not reveal an excess  risk
      of human  cancer  of  the  lung or  any other  site  in  the populations
      studied.  This  information  is based  entirely  on occupational  studies
      that have numerous deficiencies in the research design.

  0   There is  no  convincing evidence that  inhaled  whole Diesel exhaust  is
      mutagenic or  carcinogenic  in laboratory  animals.   However,  in  animal
      cell  and  whole  animal skin  application  tests,  organic  extracts  of
      Diesel exhaust particulates have been found to  contain substances  that
      have  mutagenic   and  carcinogenic  potencies  similar  to  extracts  of
      gasoline  engine  exhaust,  roofing tar, and coke oven effluent.  It  is
      possible  that Diesel  exhaust is carcinogenic  or  mutagenic in  animals
      or humans exposed by inhalation  but at a  level too low to be  detected
      in studies conducted to date.

  0   From available epidemiologic, clinical, and laboratory animal  studies,
      no firm conclusions can be drawn about possible pulmonary and  systemic
      effects  of  Diesel  exhaust  exposure.    However,   evidence  based  on
      laboratory animal studies suggests that inhaled Diesel exhaust  affects
      the  lung  clearance  mechanisms,  produces  nonspecific histopathologic
      changes in the  lung  that  may or may  not  be  reversible,  and adversely
      affects the pulmonary defense mechanisms.

The panel emphasized  that much of  the  information  and data evaluated  in the
report were incomplete  or  unpublished;  therefore,  the conclusions  should  be
regarded as tentative.

C.    Health Effects Institute (HEI)

In late  1980, HEI  was formed to  conduct  health research on  mobile  source
emissions.  HEI is  jointly  funded by  industry  and  EPA but is  set  up as  an
independent  entity,   i.e.  HEI Panel members  will  not  be  affiliated  with
either industry or EPA.  HEI  represents a  unique  opportunity for  EPA  and
industry  to  function together in a  cooperative  mode  to  complete  needed
research.

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At this  time,  HEI is formulating plans for  the  type of research  they  would
Implement.   EPA and industry  will  be suggesting  the type  of  work that  is
important from  their perspectives.   While HEI has not  initiated  any studies
to date, it was included here because it  is  expected  to  be  a major source  of
Diesel health effects data in the future.

IV.   Description of EPA's Diesel Emissions Research Program

For  the  past several years, EPA has conducted a  large research  program  to
assess the  potential carcinogenicity of  Diesel exhaust.  A  major  segment  of
EPA's health  effects research on Diesel  exhaust involves the  comparison  of
the  mutagenic  and carcinogenic  activity  of  various Diesel  samples  and coke
oven, roofing  tar and cigarette smoke samples.   If a consistent  pattern  of
data  can be obtained  on relative potencies among  these  samples,  then  the
epidemiological  data  from  coke oven,   roofing  tar,  and  cigarette  smoke
probably  can be  used  to  estimate  human effects  from exposure  to  Diesel
exhaust.   This  approach  is used  because EPA has  found no usable  Diesel
epidemiologic data on which to base a risk assessment.

The  mobile  source  samples  tested  include  three  light-duty  Diesel-powered
vehicles  (Oldsmobile 350,  VW  turbocharged  Rabbit,  and Nissan  220C),  one
heavy-duty Diesel-powered engine (Caterpillar  3304)  and  one  gasoline-powered
catalyst-equipped vehicle  (Ford Mustang  II).  Coke oven emissions,  roofing
tar  emissions,  cigarette  smoke  condensate  and  benzo(a)pyrene (B(a)P)  were
selected  as  the  comparative   samples.   The  organics  extracted  from  the
particulates  emitted from  these sources were  used to determine  relative
potencies.

The  light-duty  vehicles were  operated  on a  chassis dynamometer, using  the
highway  fuel  economy test  cycle (HWFET).   The  HWFET cycle  has  an  average
speed of  48 miles per  hour over a  length  of 10.24 miles.  The  heavy-duty
Caterpillar  engine was  mounted on  an engine dynamometer  and operated  at
steady-state  using  mode  2   of the  13-mode  heavy-duty   test   procedure.
Operation at  this lightly  loaded mode (2% load) apparently resulted in  low

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activity  for  the Caterpillar  sample  in the  mutagenesis and  carcinogenesis
bioassays.  These results  are  consistent with those from other  samples  from
heavy-duty Diesels  at lightly loaded modes.   Additional information on  the
collection of the mobile source samples can be found in reference 6.

The   gasoline-powered  vehicle   was   operated  at   a   richer-than-normal
stoichiometry in  order to generate enough  sample;  however, the  hydrocarbon
and extractable organic emission rates  are  not considered high when  compared
to in-use catalyst-equipped vehicles.   The  Nissan Diesel-powered  vehicle  had
an injector  design  defect resulting  in considerable  "after  injection"  of
Diesel fuel.  Newer Nissan vehicles have  redesigned injectors  to  eliminate
this  problem. When  the injector  was corrected,  the biological  activity  as
measured by the Ames  bioassay  was about 2  revertants/ug extract  versus  10-15
revertants/ug  extract  with  the  earlier   injector  system.   These   factors
should be taken into consideration when evaluating the results.

The coke oven samples were collected on  top of  a coke  oven  at  Republic  Steel
in Gadsden,  Alabama.   The samples were collected  in a location  that  was
upwind of  the coke  ovens  a large  fraction (as much  as 98%)  of the  time;
however, large sample masses  were obtained for the  2% of  the time  that  the
wind  was  blowing in  the right direction.   It  is also thought that some  of
the sample was from road  traffic; there was  a paved road  about  750 to  1000
feet  from the sampler.  Thus,  an unknown portion of  the sample may have  been
from  the urban environment rather than the coke oven.

There  was  not enough  ambient  coke oven sample for  every  type  of  In  vivo
carcinogenesis testing  required  so additional  samples  were obtained in  the
coke  oven main.   Workers  have not been  and are not exposed to  the  material
in the main.   The main contains  the volatilized  organics  emitted  from  the
coke  oven as  coal  is  coked.   Biological testing  of  the coke oven  main
reveals that the coke oven main sample  is  slightly more active than  the  coke
oven  ambient sample.

The   cigarette   smoke  condensate   was  generated  by  Oak  Ridge   National
Laboratory.   Kentucky  Reference  2R1   cigarettes  were  used  (non-filter,
approximately 30  mg tar per cigarette).

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The roofing  tar emissions were obtained  by collecting a particulate  sample
above a hot  pot of roofing tar.  Pitch-base  tar  was used which is the  type
of tar in use at the time the epidemiological data were generated.

The  dichloromethane  extracted organics  were  subjected  to  a  battery  of
mutagenesis  and, carcinogenesis bioassays.   The  results  of  these  bioassays
are  reported  in  this  paper.  This  work  involves  studies  on  particulate
extract rather  than total  vehicle  exhaust containing  both  particulate  and
gas phase  emissions.   EPA has also  done  some animal  inhalation  experiments
with whole  exhaust (discussed later) but  these inhalation experiments  were
generally negative.

EPA is developing a method  to collect the gas phase  hydrocarbons present in
Diesel  exhaust  for  mutagenicity testing  so  that  an  assessment  of  the
relative mutagenicity  of  the  particulates versus the gas phase  hydrocarbons
in Diesel exhaust can be made.  EPA has also  done some  work to  identify some
of  the  compounds  present  in  the extractable  organics.   A summary  of  the
progress in these areas can be found in section VIII.

V.    Mutagenicity

A.    In vitro studies

Chemical mutagens  are  toxic  substances  that cause  changes in the  primary
structure of the DNA.   A high correlation exists between an agent's  ability
to cause mutations  in  bacteria and  cancer in animals.  Two bacterial  assays
designed to  detect  gene mutations are the Salmonella  typhimurium (Ames)  and
Saccharomyces cerevisiae assays.  They are discussed below.

One  of  the  most   frequently used   bacterial  assays  is  the  Ames   assay,
developed by Ames  et  al.  (7).  The Ames test involves  specially  constructed
strains  of  the bacterium  Salmonella  typhimurium.   Each  strain  contains
unique  types   of   DNA   damage   (base   pair  substitutions,   frame   shift
mutations).   The  tester  strains  all  require  an exogeneous supply  of  the
amino acid  histidine  for  growth.  Different  doses  of  the  material  to  be

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                                    -15-
tested are  combined  directly on a Petri dish  along with a  bacterial  tester
strain.  A  trace of  histidine,  which is  not  enough  to  permit colonies  to
form but which  will  allow sufficient growth for expression of mutations  is
added.  Homogenates of rat liver (S-9 mix) can also  be added directly  to  the
Petri  plates   to  detect  carcinogens   that   require   metabolic   activation
(metabolic  conversion  to  an  active mutagenic form).   The bacteria  will grow
only if the material is mutagenic, since a mutagen will cause one  or more  of
the  tester  strains  to revert  so  that  they  no  longer  require  exogeneous
histidine for  growth.   The  number  of  revertant  bacteria  are measured  by
counting  the  revertant   colonies  on  the  plate  after  two  or  three days
incubation.    The potency  of compounds  can  be  compared  by determining  the
number of revertants per microgram of sample generated in  the  linear portion
of the dose-response curve.

EPA  has  measured  the mutagenic  activity of  particle  bound  organics from
Diesel  and  related  environmental  emissions   using  the  Ames  assay (8).
Dichloromethane was used  to  extract  the  organics from  the  particulate.  Four
strains (TA98,  TA100,  TA1535 and TA98-NRD) were used  in the study.   Strain
TA98  is  used to  detect  frameshift  mutagens and was  chosen because  of  its
high overall  sensitivity  to  mutagens.  Strains TA100 and  TA1535 are designed
to  detect   mutations  due  to base-pair  substitutions  and  tend  to respond
selectively  to  mutagens   such  as  alkylating  agents.  Strain  TA98-NRD   is
nitroreductase  deficient, i.e. not sensitive to nitro compounds.

The  results can be found  in Tables  I and 2 (following the text).  Table  1
gives  the   specific  activities  for  each sample.  The specific activity  is
defined as  the  expected  response at 100 ug of  organic material and is used
as  a  convenient  method  of  comparison.    The  activity  of  each  sample  was
compared to the activity of the Nissan  sample by arbitrarily  assigning  the
Nissan sample a relative value of  100.   These  comparisons  are referred to  as
"relative potency" and are found in Table 2.   Results for  strains TA98  and
TA100  are  included in  the  tables.  All samples  were negative with  strain
TA1535.  Only two samples, the  Diesel Nissan and  cigarette  smoke  condensate
samples, provided sufficient material  for testing  with the  nitroreductase
strain, TA98-NRD.

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                                    -16-
As   seen   In  Table   1,   cigarette   smoke   condensate,   roofing   tar   and
benzo(a)pyrene   required   metabolic   activation   to   achieve  a   positive
response.  These  samples  are said to contain indirect  acting  mutagens.   The
other samples,  including  the Diesel samples contain direct  acting  mutagens;
they  do  not  require  activating  agents.   The  majority  of  the  activity
associated  with  the   Diesel samples  appears  to be   direct  acting.   This
indicates  that  the  mutagenic  activity does  not  reside primarily in  the
polynuclear  aromatic  hydrocarbon  (PAH)  fraction because  the PAH's require
addition  of  activating agents to  produce  responses.   The negative response
in  strain TA1535 suggests that most  of the activity  is  due  to  polynuclear
frameshift mutagens rather than alkylating agents.  When the Diesel  Nissan
and  cigarette  smoke  condensate samples were  tested  with the  nitroreductase
strain, TA98-NRD  the responses differed.  The responses of TA98  and TA98-NRD
were  quite different  with  the Diesel Nissan  sample  but similar   with  the
cigarette  smoke  condensate  sample.   This  suggests that  the  particle  bound
organics  from  the Nissan  Diesel  contain  nitroarene  compounds.  In general,
results  from  the Ames  bioassay  indicate that  mutagenic  compounds,  both
direct   and    indirect   acting   adhere   to  carbonaceous   Diesel   exhaust
particulates.

The  Saccharomyces cerevisiae (yeast)  D3 assay  was used  to  evaluate  the  iji
vitro mutagenic  effects of  extracted  particle-bound organics  of Diesel  and
related environmental emissions.   The work was  performed  for EPA by Stanford
Research  Institute  (SRI)  International (9).  The  samples were  from similar
engines used in  the  Salmonella typhimurium assay  with the exception  of  the
heavy-duty Caterpillar sample.

Homozygous mutants of  the yeast S_. cerevisiae  D3  can  be  generated  from  the
heterozygotes  by  mitotic   recombination.  The homozygous  mutants are  easily
distinguished  because  they produce a  red pigment.  The frequency of mitotic
recombinations may be  increased  by  incubating the organisms with  various
carcinogenic or   recombinogenic  agents.   The  recombinogenic  activity  of  a
test  sample  is determined by recording the number of  red-pigmented colonies
appearing  on the test plates.  All testing was  performed  with and without
metabolic activation.

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                                    -17-
The results for the seven emission samples Indicate a  slight  Increase in the
number  of  recombinants at  one  or two  concentrations; however,  the  results
were  neither reproducible  nor  dose-related.   It  was  concluded that  this
assay was not sufficiently sensitive for this evaluation.

The mammalian cell  in_ vitro assays used by  EPA to compare the  potencies  of
Diesel  and  related  environmental emissions  are the following:  L5178Y  mouse
lymphoma assay,  Balb/c 3T3  fibroblast  assay and  the  Chinese hamster  ovary
(CHO) cell assay.  They are discussed below.

The L5178Y mouse  lymphoma  assay was  performed  for EPA by  SRI  International
(9).  EPA supplied  the samples for testing.  The L5178Y  mouse lymphoma assay
measures the effects  of  chemicals on the  forward  mutation frequency  of the
cells at  the thymidine  kinase  (TK)   locus.  Unlike the heterozygous  cells,
mutated homozygous  cells can not utilize  exogenous thymidine.   The  mutated
homozygous  cells  can  not  utilize  thymidine  analogs   as  well,  such  as
trifluorothymidine  (TFT),   but  are  able  to   survive  and  grow  in  their
presence; the heterozygous cells, on  the  other hand,  can not survive  in the
presence of  thymidine analogs.   Hence,  the mutagenic  activity of a  chemical
in  this assay  is  determined by the number of  colonies found growing  in the
presence of the thymidine analog, TFT.

All of  the  emission  sample  extracts  gave positive mutagenic responses  with
and without  metabolic activation.  When B(a)P  was tested, mutagenicity was
detected  only   in  the  presence  of   activation.   Good  dose-dependent  dose
response  curves  were  found.    The   comparative  potency  rankings  for  the
samples using  the  L5178Y  mouse  lymphoma assay  can  be  found  in Table  3.
Table 3 also includes comparative potency  rankings  for other  mutagenesis and
carcinogenesis  assays and will  be referred to frequently throughout  this
document.

As  seen in  Table 3,  the  comparative  potencies have been  evaluated for the
mutagenesis assays  using the data obtained with metabolic  activation.   This
is  because  better  dose  response curves  have   been  obtained when metabolic
activation  was  used.   Both  the Ames  and  lymphoma   assays  show  the  same
relative potency within the Diesel samples with NissanX)lds>Rabbit>Cat.

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                                    -18-
The  BALB/c   3T3   assay,   like  the  mouse  lymphoma   assay,   detects   gene
mutations.   This  assay  was performed  by Microbiological  Associates  under
contract  to  EPA using  the Diesel  and  comparative samples  provided by  EPA
(10).  The comparative potency rankings for this assay  can be  found in  Table
3.   The  BALB/c 3T3  assay  responded  in  a  dose-dependent fashion when  the
B(a)P was used as  the positive control.   However,  when  the complex emissions
extracts  were  assayed   none  yielded  good  dose-response  curves.   It  was
determined that  this particular  assay does  not  seem  to work for  complex
mixtures and should not be used for comparative potency.

The sister chromatid exchange  (SCE) assay was  performed by SRI International
under contract to  EPA  (9).  The SCE assay uses Chinese hamster ovary  cells
to  detect  DNA damage.   The induction of DNA  lesions  by chemical  mutagens
leads  to  the formation  of  sister  chromatid exchanges.   In  this study,
particulate  extracts from Diesel  and  related environmental  samples   were
tested to determine whether  they increase SCE  frequencies.   The samples were
tested both with and without metabolic activation.

DNA damage was induced by most of  the emission samples  both  with and without
metabolic activation.  Table 3 presents  the  potency rankings  when metabolic
activation was used.  The  cigarette smoke condensate and two Diesel samples,
the Caterpillar and  Oldsmobile,  gave  a negative response in  the  SCE assay,
unlike the Ames and mouse lymphoma assays.   According to  the  researchers,
the negative  response may be related to  the  relatively short  exposure time
used  for SCE  testing  with  activation.   The  SCE  assay did  not  seem  very
sensitive  or responsive,  in  comparison  with  the mouse lymphoma and  Ames
assays.

From  the above  data,  it  can  be   concluded  that,  in   general,  the Diesel
samples gave positive responses in the in vitro mutagenesis  assays.   Further
work is  needed  to  determine which  portion of  the  Diesel exhaust  causes  the
mutational events to occur.  Another major area of concern is how the  above
mutagenesis  data  can be  used,  if  at  all,  to  determine human  carcinogenic
risk to current and future levels of Diesel  exhaust emissions.

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                                    -19-
B.    In vivo studies

A number of  in vivo studies have  been  conducted to determine  the  mutagenic
effects  of  Diesel  exhaust.   Biological  endpoints  include  gene  mutation,
sister  chromatid  exchange  and clastogenicity.   A  selection  of  iia  vivo
studies are discussed below.

Two in  vivo  studies designed  to  detect gene  mutations were conducted  with
male  fruit  flies  of  the  species  Drosophila  melanogaster.  In one  study,
approximately  200  flies were  exposed  eight  hours to  whole Diesel  exhaust
(i.e.  gases  and particulate)  diluted  five-fold with  filtered ambient  air
(11).  The flies were  mated  and successive generations  were  Investigated for
the occurrence  of  recessive  lethal events.  The  Diesel engine  used  in  this
study was  a  6-cylinder Nissan engine.  Particulate levels in the  Drosophila
                        3
chamber  were  2.2  mg/m .   Results  indicate  that,   under  the  conditions
tested,  the  Diesel exhaust  did not increase  the  mutation frequency of  the
exposed flies when compared to the control flies.

In another study,  Drosophila  were fed 1  mg/ml  in a  sugar  solution of  the
most  polar  neutral  (oxygenated)   fraction  of  Diesel  particulate  extract
generated  from a Caterpillar 3208  heavy-duty engine (12).  This was done for
three  days.    Difficulties were  encountered  in administering  the  organic
fraction and the results were negative.

Male  Chinese hamsters  were  exposed  by inhalation to  whole diluted  Diesel
exhaust daily (8 hrs) for  6  months  to determine  if  the  exposure would result
in sister  chromatid exchange (SCE)  in the  bone marrow cells  of  these animals
(13).   The  Diesel  exhaust  was generated  from  a  Nissan 6-cylinder  Diesel
engine.  Six animals were exposed  to Diesel  exhaust,  six animals  to  clean
air and  four animals to benzo(a)pyrene  (B(a)P)  as a positive  control.   The
Diesel  exhaust  was diluted  to achieve a  particulate  mass  concentration  as
near  6  mg/m  as  possible.    The   B(a)P  positive control  group  showed  an
increase in  the number  of  SCE/cell compared to the clean air control group.
Diesel  exhaust, on  the   other  hand,  did not  cause  an  increase  in  the
frequency of SCE in Chinese hamster bone marrow cells.

-------
                                    -20-
Another  in  vivo  assay  with  sister  chromatid  exchange  as  a  biological
endpoint  uses  Syrian  Hamster  lung cells  (14).   Like  the  previous  assay
discussed, a Nissan 6-cylinder Diesel engine was used  to generate the Diesel
particulate.  One  set  of animals was  intratracheally  Instilled  with Diesel
particulate,  administered in a  range  from 0  to 20 mg  per  hamster over  a
24-hour  exposure  period.   Lung  tissues  from  these  animals  were  later
analyzed   for  chromatid   exchange.    SCE  increases   were   induced   with
intratracheal instillation  of Diesel exhaust particulate,  producing  a linear
SCE dose response.

A separate group of animals were chronically exposed to Diesel  exhaust  for 8
hrs/day,  7  days/week  for  a period  of  about 3  months.   The  particulate
concentration  in  the  exhaust  emission  chambers   during the  exposure  was
                    3
approximately 6 mg/m .   The results indicated  that  a  3-month exposure  to  6
mg/m    of    Diesel   exhaust  particulates   was  insufficient   to   produce
measurable  mutagenic  changes in lung cells.   Results  for  the exposed  and
control groups were similar.

A  subsequent study involved  sister chromatid exchange  analysis of  Syrian
hamster lung cells from a total  of  33 treated  and control animals (15).  The
                                                                        3
treated animals were  exposed via  inhalation  for  3 months to  12 mg/m   of
Diesel  exhaust  particulate  (DEP)  in  contrast  to  6  mg/m   DEP   in  the
previous  study.   Preliminary results  indicate  a positive  response for  the
                            3
animals  exposed  to 12  mg/m  DEP.  The  SCE le
was about double that for the control hamsters.
                            3
animals exposed  to 12 mg/m  DEP.  The  SCE level for  the treated  hamsters
An  attempt  was made  recently to measure  the effect  of Diesel  particulate
extract upon SCE  (16).   Instillation  of  extract  in  dimethyl  sulfoxide  (DMSO)
resulted  in a  very  short  residence  time in  the  lung  due  to  its extreme
solubility, and resulted in only a marginal  increase  in SCE.  By  attaching
the extract to an inert  carbon carrier particle, however, they were able  to
once again achieve a  dose  response  curve.  This  test could be regarded as  a
useful  ill  vivo   test   for  determining  comparative  potencies  of   Diesel
particulate  extract   and  extracts  from  other comparative  sources.   Future

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                                    -21-
work  could  thus  involve  using  other  samples,  Including more  Diesel  and
possibly  coke  oven, cigarette  smoke,  and roofing  tar samples*  A  complete
analysis of the SCE work should be available later this year.

The  Chinese  hamster  bone marrow bioassay  to  detect  SCE  was  discussed
earlier.   This bioassay  was  a  portion  of  a  larger study  using  Chinese
hamster bone marrow (IS).   Various endpoints were  examined.   Clastogenicity
endpoint  bioassays  included  the  chromosome aberrations  bioassay  and  the
micronucleus bioassay.  Male Chinese hamsters were  exposed to  diluted Diesel
exhaust daily  (8 hrs/day,  7 days/wk)  for  6 months.  There was  some  increase
in  the  frequency   of  micronuclei  in  the animals exposed  to  the  Diesel
exhaust.   No  increase  in  chromosomal  abnormalities  was  observed  in  bone
marrow cells of the hamsters exposed to Diesel exhaust.

In  another study  to assess the  potential risk  from  heritable  effects  in
human populations, mice were exposed by inhalation  to  diluted  Diesel exhaust
(particulate and gas phase)  and a  number  of  genetic  endpoints were studied
(17).  The  genetic endpoints  included point  mutations in males,  chromosome
damage  in males  and  chromosome  damage in  females.   In  addition,  various
parameters  were used  to  assess  reproductive  performance  in  females  and
histological analyses of germ-cell  survival were  done  in males.   The results
of this study have recently been released.

A six-cylinder  Nissan  engine was used to  generate  the exhaust;  the exhaust
was  diluted  with  air  at  the  ratio  of  1:18.   The  Diesel  particulate
                                               2
concentration  in  the chambers  averaged  6 mg/m   during the exposure  period
of 8 hours per  day  and  7  days  per week.   Exposure  times in different groups
varied from  5  to  10 weeks.  The  authors  calculate  that,  during  the 10-week
exposure  period, the total intake  of  exhaust per mouse was  about  85  times
what a person  in  an average U.S.  environment (urban-rural)  would intake  in
30 years.

The  results  of  all genetic  assays in  both  sexes were  negative.   Slight
effects  on  the reproductive  performance  of  females of  one  strain  were

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                                    -22-
observed,  consisting  of a  decrease  in  the  number  of  ovulations and  an
increase  in the interval between mating opportunity  and  copulation.  There
was  no detectable effect of  Diesel exposure on the  number and distribution
of cell types  in  the  testis.

The  results  indicate  that transmitted genetic  effects are not a major hazard
from exposure  to  Diesel  exhaust;  however,  the  authors  stress  that  the
findings  should not be used  to draw any  conclusions  about possible risks to
•the  exposed  individual himself.

In  contrast to the ill  vitro  results, results for the in  vivo mutagenesis
assays  were generally negative with  the  exception of one  study designed to
detect  sister.chromatid  exchange.

VI.    Carcinogenici ty

A  number  of  iia  vitro  and  ill  vivo  tests  for  carcinogenic!ty  have  been
performed;   selected   studies   will be   described  in  this  section.   Also
included   in  this  section  are   the  results   of  studies  examining  the
bioavailability of  the organics bound  to  Diesel particulate.

A.     In  vitro studies

Michigan  State University researchers have performed an JLn vitro study on
the  effects of Diesel particulate  on  human normal and xeroderma pigmentosum
cells  (18).   Their investigation  found  that normal human  fibroblasts  and
excision  repair deficient xeroderma pigmentosum  fibroblasts are sensitive to
the  cytotoxic  (toxic  to cells) action of a direct-acting agent(s) of Diesel
particulate.  The  xeroderma  fibroblasts  were significantly more  sensitive
than normal fibroblasts to both  the  organic solvent  extracts  of the Diesel
particulate  and  the  whole  particulate  itself.    The organic  extracts  from
Diesel exhaust particulate  and the non-extracted organics on the particulate
interfere with the  human  cell  DNA.   The  xeroderma pigmentosum  cells  are

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                                    -23-
subsequently unable to repair  the  DNA damage caused by  the  Diesel organics.
In  the  case  of  whole  particulate,  the  cells absorb  the  particulate  as
demonstrated  by  electron  microscopy  and  the  blackish appearance  of  the
Diesel particulate-treated cells.   The  surface  of the cells  is  almost  clear
of  particulate.   Since  the   differentially  cytotoxic  material  in  Diesel
particulate is  only  slightly  soluble  in tissue  culture  medium  containing
serum, the  data  suggest that  the  Diesel particulates are  taken  up by  the
cells  and  then the  differentially cytotoxic material elutes from  the  cell
surface and attacks the cell's DNA.

As  part   of  EPA's  comparative  potency  study,  particulate  extracts  from
Diesel,  gasoline  and comparative sources  were  tested  for  morphological
transformation  in BALB/c  3T3  cells  (10).  The  extracts were also  tested
simultaneously  for  mutagenic  activity  in  BALB/c   3T3  cells;   this   was
discussed earlier in the mutagenesis section.   This assay  did not yield  a
dose  dependent  increase  in   either  mutation  frequency  or  transformation
frequency.  As  mentioned  previously,  this particular  assay, as  performed,
does  not  work for complex mixtures and should not  be used  for comparative
potency.

Another  in_  vitro  carcinogenic!ty  test,  performed   as   part   of   EPA's
comparative  potency  study,   is  designed to  detect  enhancement  of  viral
transformation  in Syrian hamster embryo  cells  (19).   This assay  produced  a
positive dose-response with  all samples;  however, the  particulate extracts
prepared from Caterpillar  emissions failed to induce a  significant increase
in  the transformation  frequency.   The  comparative  potencies  of  the  test
samples for this assay  can  be found in  Table 3.   The  Diesel  and gasoline
particulate extracts,  with  the  exception of  the Caterpillar  appear to  be
within the  same range, with the Nissan  the highest.   The comparative sources
were  clearly  more active, particularly the roofing  tar.   A  substance  that
repeatedly  scores positive in one or more  transformation  assays  is  highly
suspect of being  carcinogenic in vivo (2).

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                                   -24-
B.    In vivo studies

Many  of   the   jLia  vivo  tests  for  carcinogenicity   are   currently  being
performed.  This  section summarizes the available  results  from a  selection
of  ill vivo  studies, with  an  emphasis  on  those  studies  EPA will  use  to
perform a risk assessment.

The  objective  of  a study  performed  at the  EPA-Cincinnati  facility is  to
determine the relative  carcinogenicity  of  Diesel exhaust participates  using
the pulmonary adenoma assay on  Strain A mice.  In  the  initial  study, Strain
A  mice  were  exposed  to  Diesel  particulate by  intraperitoneal  injection
(20).  The  animals were injected  3  times  weekly for 8 weeks to the Diesel
particulate.  At  the  time  of  this study,  Strain A  mice  were  also  being
exposed   to  diluted   Diesel   exhaust  by   inhalation.   The   particulate
concentration  in  the  inhalation  exposure  chambers   was   approximately  6
    3
mg/m .   In  an   attempt  to  correlate  the  inhalation  and  intraperitoneal
injection   studies,   the   highest   particulate   dose   level    for   the
intraperitoneally  injected  mice was chosen to  correspond  to the  inhalation
dose  assuming  50%  retention.    Thus,  the  highest  dose group  received  235
ug/injection, 705  ug/week.  The animals were sacrificed after  approximately
9 months.  Results  showed no significant difference between  the incidence of
tumors in the  injected and control  mice.   (The  inhalation study will  be
discussed in more detail later in this section.)

The  intraperitoneal  injection   study  with  Strain A   mice  has  since  been
expanded.   More  animals and  test materials  have  been employed.   The  test
materials  include:   control  injected  (control  chemicals  are  urethane  and
benzo(a)pyrene),  cigarette  smoke  condensate,   roofing  tar,  coke  oven,
Oldsmobile Diesel particulate extract, Nissan Diesel particulate extract  and
Nissan particulate.   Except for  the  control chemicals,  the test  materials
are injected three times per week for eight weeks (1 mg of  test material  per
injection).  Fifty-five  (55) mice  per test material are being injected.   The
animals are to  be sacrificed  at 9 months  of  age to determine the  number  of
pulmonary adenomas.  Currently,  sacrifice on all but one group  of animals is
complete.  Results should be available later this year.

-------
                                   -25-

An intratracheal  instillation  lifetime  study with Syrian golden hamsters  is
currently  underway  to  compare  the  potential  carcinogenicity  of  Diesel
particulates,  Diesel  particulate extract,  coke oven  main extract,  roofing
tar extract,  cigarette smoke condensate, and  benzo(a)pyrene.   The  study  is
being performed  by the Illinois  Institute  of  Technology Research  Institute
(IITRI)  and   sponsored  by  EPA.   Hamsters   were  used   for   intratracheal
instillation  because  they are  susceptible  to  lung  tumors, yet  have a  low
spontaneous   rate  of  lung  tumors.   The   advantages   of   intratracheal
instillation as a route of exposure are  that it allows high doses  of samples
when compared  to inhalation studies and is a natural route of  exposure (21).

The lifetime  study includes  positive, solvent, and untreated controls.   The
treatment  schedule is once  per week for  15  weeks,  beginning  at  12 to  13
weeks of age.  Each material is being tested at three  dose  levels,  1.25,  2.5
and  5  mg/week.   These  doses   were  chosen  after  performing  a  preliminary
dose-range  study  with  Diesel  particulates (22).   The  Diesel  particulate
extract, coke  oven main  extract,  cigarette smoke condensate and roofing  tar
extract  are  being  administered with  a ferric  oxide carrier.   The  ferric
oxide carrier offers the advantage of keeping  the sample  in contact with  the
lung tissue longer  than an emulsion would because the particles take longer
to be  cleared out  of  the lung.   Also,  if  the  same amounts of extract  in
emulsion  and   extract  plus  carrier  are injected,  the  sample  plus  carrier
represents  a  lower dose  of  extract  due  to the slow  release  of the  extract
from the carrier  (21).   The Diesel particulate and solvent are  administered
with and without  the ferric  oxide carrier.   The study is being  conducted  in
two replicates of half the animals in each test or  control group.

As of May  30, 1981, the  first  replicate is in the  forty-ninth week  of  the
study (23).  The hamsters in the first replicate are 14 months of  age. When
the hamsters were 12 months of age, a scheduled sacrifice of 245 animals  was
conducted.  All treatment groups and  control groups were  represented.  Organ
weight  determinations failed   to  indicate  a   toxic  effect  that  could   be
attributed  directly to  the test  articles used  in  this  study.   IITRI   is
working on the histopathological evaluation.

-------
                                   -26-
The  second replicate  is  in  the  thirtieth week.   The hamsters  are now  10
months of  age.  All  dose  levels  have continued to gain weight;  however,  the
Diesel particle group  (5  mg),  the Diesel extract plus ferric  oxide  group  (5
mg and  1.25 mg) and  the  benzo(a)pyrene plus ferric  oxide  group (2 mg)  are
below the  colony controls with respect  to the percent increase  in mean body
weight.

During the period  July 20-30, 1981, 255 randomly selected,  twelve-month  old
hamsters  from the  second replicate  were  sacrificed for  histopathological
evaluation.  Selective organ weight determinations will also be  made at this
time.   The remainder  will  be allowed  to  live  out  their  normal  lifespan.
Final results for this study are not expected until  1983.

Both  the  intratracheal instillation  study  with Syrian  golden hamsters  and
the  intraperitoneal  injection study with Strain A  mice include  the testing
of Diesel  particulate  and Diesel particulate extract.  The results  of  these
studies could provide  important information about the bioavailability of  the
organics  on the particulate,  in  addition  to  providing comparative potency
data.

An important ill vivo assay in progress that will be used to help formulate a
risk assessment is the mouse  skin carcinogenesis assay.  This work  is  being
sponsored  by  EPA  and conducted  at  Oak  Ridge  National  Laboratory.   The
objective  of  this  study  is  to evaluate  the ability of  Diesel  particulate
extracts  to act as   tumor  initiators,  tumor promoters,  cocarcinogens and
complete carcinogens and  to  compare the potency of  these  extracts  to  other
emission extracts and pure carcinogens.

Tumor initiation  is  the  first  step  of  the carcinogenic  process.   A  tumor
initiator  converts a normal cell  to a pre-malignant  cell.   A  tumor  promoter
is  one  which,  when  applied  repeatedly  after  a  single  dose  of  a   tumor
initiator,  will result   in  tumors.   Tumor  promoters can  be  either  weak
carcinogens or  noncarcinogens.   Pre-malignant (initiated)  cells can become
malignant,  even after  1  year  from the time the  initiator  is applied to  the
time  the  promoter  is  applied. To  determine if an agent  is a  cocarcinogen,

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                                    -27-
the  agent  Is  given  concurrently  with  a  tumor  initiator.    A  complete
carcinogen  is  both  an initiator  and  a  promoter.   A  good qualitative  and
quantitative correlation  exists  between complete  carcinogenesis and  tumor
initiation in mouse skin (24).

Two strains  of mice are being  used, one sensitive  strain (SENCAR) and  one
resistant  strain  (C57 black).    The  mobile  source  samples  consisted  of
particulate   extracts   from   three   light-duty   Diesel-fueled   vehicles
(Datsun-Nissan   220-C,  Volkswagen  Rabbit   and   Oldsmobile   350),    one
gasoline-fueled  vehicle  (Mustang  II)  and  one  heavy-duty  Diesel engine
(Caterpillar 3304).   The  comparative sources  employed were cigarette  smoke
condensate, coke oven  ambient samples, roofing tar  emissions and  residential
home furnace samples.  The pure carcinogen  tested was  benzo('a)pyrene.   Forty
male and forty female mice per dose  were  used  for each strain.  The extracts
were applied to the mouse skin at five dose levels per agent.

Recent results with  the C-57  black mice were negative  (i.e. no carcinogenic
response) with  the exception of  one dose of  the roofing tar  samples  (25).
Work with the more sensitive Sencar  mouse does show a  positive  response.   At
the present  time,  the tumor initiation papilloma data  are available(24,26).
A  statistical  analysis was performed with  these data.   Slopes of  the  dose
response curves were determined for  each  complex mixture  sample (in terms of
papillomas/mouse/mg dose).  A square root analysis  was then performed.   The
data  did  not   follow a  Poisson distribution  (which  assumes  independent
events) because it was discovered that  the  chances  are greater that a  mouse
with an existing tumor will get another versus a mouse with no  tumor getting
an  initial  tumor.   Hence,  the Probit  model was  used to  rank  the complex
mixtures.

The samples  have  been scored 26  weeks  after treatment according  to potency
(papillomas/mouse/mg)  and  then  ranked.   The results  are  included in  Table
3.   Both  the  cigarette  smoke  condensate   and  the  heavy-duty  Caterpillar
sample  gave a  negative  response.   The  cigarette  smoke  condensate was  not
concentrated to  the  same  extent  as the other  samples.    The  detectability
limit of this  particular assay  is above  the doses  and concentrations tested
for the cigarette smoke condensate (24).

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                                   -28-
The ranking  indicates that  the  potency of  pure benzo(a)pyrene was  greater
than the coke oven  sample, which was,  in turn,  greater than the roofing  tar
and Nissan samples.  The Diesel samples exhibited a wide range  of potencies.

Samples  from  other  sources are  currently  being  tested.   These  sources
include  a  Mercedes  Diesel,  1970  Ford  van  (non-catalyst),  roofing  tar
condensate, coke  oven main,  and cigarette  smoke condensate.  The  cigarette
smoke  condensate  sample is  being  retested at  10  times  the  original  dose.
The coke  oven main  sample  has been  discussed  in  section  IV.  The  initial
roofing tar sample  consisted of  an extract of roofing  tar  emissions  trapped
in  the collecting  device.   This  sample  was subsequently  replaced with  an
extract of roofing tar emissions which condensed on the funnel just prior to
the  collecting  device.   This  new  sample  has  been  called  roofing  tar
condensate.

Improved statistical  models  have since been  developed to analyze  the  data.
Tumor  initiation  data  for  the coke  oven main  and  roofing   tar  condensate
samples  should  be  available later  this  year.   Carcinoma  data for  the
original samples, which will supplement  the papilloma initiation data, will
be  presented  at  a  Diesel emissions  symposium  in  October  1981.   Pathology
results will also be presented.

General  Motors   is   sponsoring  a  skin  carcinogenesis  study  on  Diesel
particulates and  Diesel  particulate  extract.  The  study  is being  conducted
by the Bushy Run  Research Center,  Carnegie-Mellon University.  Male C3H mice
are being used in three types of studies,  initiation,  promotion  and complete
carcinogenesis.   Studies  have been  in progress  for   close  to  2  years  and
results should be available in about  a year.

Other iji vivo carcinogenesis  studies have  been performed  using inhalation as
the route of exposure.  In inhalation studies, the  test animal is exposed to
the whole exhaust (particulate and gas phase) rather  than just one  component
of the exhaust.  A selection of studies are described  in detail below.

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                                   -29-
A  number  of  inhalation studies have  been conducted  at the  EPA-Cincinnati
facility.   Strain  A mice were  exposed to either  diluted Diesel exhaust  or
clean control air  8 hours  per  day,  7  days per week for up to 30 weeks.   The
particulate  concentration in  the  Diesel exhaust  inhalation  chambers  was
                    3
approximately 6  mg/m  .   There was  no  increase in  incidence of lung  tumors
                                    .3
in  the  animals  exposed  to  6  mg/m    Diesel particulate  compared  to  the
control animals (20).
In a  subsequent study, Strain A  mice were divided  into the following  four
exposure  groups:  control  (no  treatment),  control  plus  urethane  (5  mg),
Diesel exhaust  (particulate concentration of  12 mg/m )  and Diesel  exhaust
plus urethane (5  mg).   The urethane was administered by  the intraperitoneal
route.   The  animals  were  exposed  30  weeks.   Results  indicate  that  the
urethane  treatment  had more effect  than the Diesel exhaust  treatment.   The
number of tumors  per mouse  for the  Diesel exhaust  group were similar  to the
control group.  The  groups  given urethane had  more  tumors than the  control
group.  The group given urethane alone had the  greatest  number  of  tumors per
mouse.
Additional Strain  A mice were exposed  to either clean air,  clean air  plus
                                                                     3
urethane,  Diesel  exhaust  (particulate  concentration  of  12  mg/m  ),   or
Diesel exhaust  plus urethane.  This study  differed  from the previous  study
in  that  the mice were  exposed  during  darkness.   This  was  expected  to
increase  the  exposure  since the animals  were  awake  and  active   during
exposure.  The  animals  have  been sacrificed  and gross  adenomas  have  been
counted.   The  final  analysis  has  not  yet   been  performed;  however,  the
preliminary  results are  similar  to  those  of  the  previous  study.    There
appear to be  significantly fewer  tumors in  the Diesel exhaust group and  the
Diesel  exhaust  plus  urethane group  compared to  the group  given urethane
alone.  The reasons for this are  not clear yet.

Sencar mice are being  tested  to determine   the effect of  lifetime  inhalation
                                                                 3
of diluted Diesel  exhaust (particulate concentration of  12 mg/m ) on  tumor
induction.  The mice were exposed  15 months to the exhaust.  One  test  group
was also given an  initiator (urethane) and another was also given  a promoter

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                                    -30-
(butylated hydroxytoluene).   The animals have  been sacrificed.  Tumors  and
histology examinations are currently being done.  Gross  lung  examination  did
not show any obvious difference  between  the  exposed and  control animals with
respect to lung surface tumors.

General Motors Research  Laboratories  is  sponsoring a  large-scale  inhalation
study  with  Strain  A/Jackson mice,  Fischer  344  rats  and  Syrian  Golden
hamsters.   The work  is  being  conducted  by  the  Southwest  Foundation  for
Research and  Education and  the  Southwest Research Institute,  San  Antonio,
Texas.   In  a  preliminary  short-term study,  the three  species are  exposed
                                             3
either  to  diluted Diesel  exhaust  (1500 ug/m   particulates)  or to  filtered
air for 20 hours per day, 7 days per week for 3 months.  The  animals will be
monitored -for 6  months  following  exposure to  determine  recovery  rates.
Subsequently,  a  large-scale, 15 month  study has  been initiated with mice,
rats and hamsters.   The  animals  will be exposed  to filtered air or diluted
                                                     3
Diesel  exhaust  containing  250,  750  or  1500  ug/m  of  particulate. This
study is in progress.   Histopathologic  examination of the rats exposed  for
up to one year of exposure revealed no lung tumors.

When the NAS  Health  Effects Panel reviewed  this  study,  they  suggested that
the  investigators  consider  increasing  the  concentration  of  Diesel  exhaust
particulates because studies have  shown  that Strain A mice will tolerate up
             3
to 6400  ug/m  particulates when exposed 20  hours  per day,  5 days  per week
(20).   Syrian  golden hamsters have been shown  to  accept  approximately 7000
to 8000 ug/m  when exposed 7-8 hours per day, 5  days per  week (27).
C.    Bioavailability

An important  issue is the  bioavailability  of the  organics  bound to  Diesel
particulate.  As  mentioned  previously,  both  the intratracheal  instillation
study and  the intraperitoneal  injection  study include  in their design  the
testing  of  Diesel  particulate  and  the  Diesel  particulate  extract.   These
in vivo  studies   should  provide   some   insight  into   the   question   of
bioavailability.

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                                    -31-
A  number  of   In  vitro  studies  related  to   bioavailability   have  been
performed.  McCormick et  al.  (18) showed that  the organic extract  from the
Diesel exhaust particulate and  the non-extracted  organics  on  the  particulate
interfere with the human cell DNA when xeroderma pigmentosum cells were used.

Siak, et al. (28) used the Ames mutagenicity assay to  examine the effects of
various extraction  solvents and biological  fluids on the  mutagenie  activity
of Diesel  particulate extracts.  Of  the various  solvent  extracts  examined,
the dichloromethane extract exhibited  the highest  activity in the Ames test,
although  methane1  yielded   the  largest   extractable  mass.    (EPA  uses
dichloromethane to  extract the  organics from the  particulate.)   The results
of this  study  indicate that  the  mutagenic  activity in Diesel particles  is
not  readily  removable  by simulated  biological  fluids.   Fetal  calf  serum
(FCS)  was  the  only  simulated  biological  fluid  which  eluted  mutagenic
activity from the particles;  however, FCS only extracted 3.6  to 12.6% of the
activity found in the dichloromethane extracts.

The objective of a  study  by King  et  al. (29) was to evaluate  the release  of
mutagens bound to Diesel  particles in  the presence of  organic solvents,  lung
fluids  and  human  serum.   The  mutagenic   activity  of  the  organics  was
evaluated using the Ames  assay.   Organic  solvents were found to  be  the  most
efficient at removing mutagens from  Diesel particles,  with  dichloromethane
extracted organics  having the  greatest mutagenic activity  of   the  solvent
systems  examined.   The  mutagenic  activity  of  Diesel  particle  organics
pre-extracted with  dichloromethane  is greatly reduced  upon the  addition  of
serum and lung cytosol to organics.  Subsequent  incubation of serum  and lung
cytosol  bound  Diesel  organics  with  protease   (an   enzyme  that   digests
proteins) increases the  mutagenic activity.   This research suggests  that
substantial  mutagenic  activity  is  released  from Diesel  particles  upon
incubation with serum and lung cytosol.

VII.  Non-genetic effects

Non-genetic  effects  include  pulmonary  and  systemic   effects.    Inhalation
studies are  generally used to  determine the pulmonary and  systemic  health
effects of Diesel exhaust.

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                                    -32-
One  study was  designed  to  determine  the effect  of  inhalation of  Diesel
exhaust  on  sperm-shape  abnormalities  in mice  (30).   Strain A mice  were
exposed   to   either   clean  air  or  diluted  Diesel   exhaust  (particulate
                         3
concentration  of  6  mg/m  )  for  31  or  39  weeks.    The  results  show  that
inhalation  exposure  to  diluted Diesel  exhaust  did  not  increase  spermhead
abnormalities in the mice.
Twenty-five male  Fischer  344 laboratory rats were exposed  to  diluted Diesel
                                                 3
exhaust  (particulate  concentration of  1500  ug/m ) for  20  hours/day, 5  1/2
days/week  for  267  days  to  determine  the effect of  chronic  inhalation  of
Diesel exhaust on pulmonary  function (31).   Twenty-five  control  animals  were
exposed  to clean,  filtered air.   There was  no  significant  difference  in
pulmonary function between the control and experimental animals.
A series of experiments was conducted to determine  if mice  exposed  to dilute
Diesel  exhaust  exhibit enhanced  susceptibility to  infection  (32).   Female
albino  mice were  first  exposed  for  various  durations  (involving  acute,
subacute  and  chronic  exposure periods)  to diluted  Diesel  exhaust with  a
                                       3
particulate  concentration  of  6  mg/m .   The  animals  were  then  briefly
exposed to  a  bacterial pathogen  (Streptococcus).   Typically,  post-infection
mortality was significantly greater in  groups exposed to  Diesel exhaust  than
in their corresponding control groups exposed to purified air only.

Limited data  on acute  tests  of  N02  and acrolein vapor  alone suggest  that
the infectivity-enhancing effect  of Diesel exhaust could  be  accounted for  in
large  part  by  these  components.   Comparison  of  these   data with  past
experiments involving Diesel-powered and gasoline  catalyst-equipped vehicles
indicate  a  somewhat  greater  excess mortality  from bacterial infection  in
mice exposed to  Diesel  exhaust than in those  exposed to catalytic gasoline
exhaust.  Exposures  to  Diesel exhaust,  N02,   or  acrolein  did not  enhance
the mortality response to a viral pathogen (A/PR8-34).

Chronic inhalation  studies are  currently being  conducted  with  cats  (33).
The  cats  have  been  exposed  to  diluted  Diesel  exhaust  emissions  for
approximately  27 months.   Toxicological  effects  to  be  examined  include

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                                   -33-
pulmonary function, pathology,  blood  enzyme levels and  sperm abnormalities.
No important changes  in pulmonary function were  detected after one year  of
exposure; however, since that time significant pulmonary function  decrements
have occurred.  Half the cats were sacrificed following  completion of  the  27
month exposure.  The other half will  be  sacrificed after six months recovery
in  clean air.   The  complete  pathological evaluation  is  scheduled  to  be
completed December 1981.

The deposition and  clearance of inhaled Diesel particles  in the respiratory
tract was  studied by Chan et  al. (34).  Twenty-four  male Fischer 344  rats
were  exposed  in a  "nose-only"  inhalation  chamber   for  40-45  minutes  to
diluted Diesel exhaust  generated  from Diesel engines  burning fuel  containing
either     Ba  or    C  radioactive  tracers.    Immediately  after   exposure,
the deposition efficiency of  inhaled  Diesel  particles  in the respiratory
                                                        131
tract  was  determined  to  be   15+6%   by  counting  of     Ba  and   17+2%  by
             14                                     14
counting of    C  in the lung tissue  samples.   The   C  tracer was used for
the  long-term  clearance  study.   Two distinct  phases  of  clearance  were
evident  in  the   experimental  data  collected up to  105  days.    Clearance
half-times  of  1  day  and  62 days were  found for mucociliary and  alveolar
clearance,  respectively.   Approximately  6% of  the   initial deposition was
found in the  mediastinal lymph nodes  after 28  days,  demonstrating that the
lymphatic system  was  also  involved in  the  removal  of Diesel  particles  from
the pulmonary airways.  After the monitoring  period  of 105  days,   27%  of the
initial dose still remained in the lung.
VIII. Characterization

Diesel  exhaust  consists of gaseous  compounds  and particulate matter.   Much
research has  been performed in  an attempt to characterize  these  emissions.
Of particular interest  is  the  identification of  the  mutagenic organics  bound
to  the  exhaust particulate.   This section gives  a brief  overview of  this
work, summarizing some of the significant results obtained to date.

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                                   -34-
A.    Gas-phase

The  principal combustion  gases  (hydrocarbons,  nitrogen  oxides  and  carbon
monoxide) emitted  from  Diesel-powered vehicles are similar to  those  emitted
from gasoline-powered vehicles.  Table 4 gives  the emission rates of  these
regulated   compounds   along   with   some    unregulated    compounds   from
Diesel-powered  light-duty vehicles  and  heavy-duty  engines  studied  in  EPA
programs.  Gasoline counterparts  are  also included  for  comparison.  The data
in  this  table  were  taken  from  a  study performed  by  Southwest  Research
Institute under contract to EPA (35).

The Diesel-powered light-duty vehicles appear  to  emit more hydrocarbons than
their  gasoline  counterparts  but, in turn,  emit less  carbon monoxide  and
oxides   of    nitrogen.    Particulate  emissions   are   much   higher   from
Diesel-powered  vehicles  (This  will  be  discussed  in  more  detail  in  the
following  section).   The   unregulated  emissions  of  sulfates   and  total
aldehydes  were  higher  from the light-duty  Diesel-powered  vehicles.   As
expected, the Diesel-powered vehicles  are more  fuel  efficient  than  their
gasoline counterparts.

The  heavy-duty   engines   can  be   examined   in  the  same   fashion.    The
Diesel-powered  Caterpillar  3208  engine  and  the gasoline-powered  Chevrolet
366   engine   are   used  in  many   identical   truck   applications.    The
Diesel-powered  engine  emits   more   oxides  of   nitrogen,   particulate  and
sulfates  than the  gasoline-powered  engine.   The  gasoline-powered  engine,  in
turn,  emits  more  hydrocarbons, carbon monoxide  and total aldehydes.   The
brake   specific  fuel   consumption   (BSFC)  was   lower   for  the   heavy-duty
Diesel-powered engines.

EPA's Office  of Research and Development  (ORD) has  attempted  to identify the
individual  hydrocarbon  compounds    in   the   gas  phase   of  Diesel-   and
gasoline-powered vehicle  exhaust.  This work  has been  described  by ORD  in
several  publications  and was  recently summarized  in a  report prepared  by
EPA's Office  of Mobile Source Air Pollution Control (OMSAPC) (36).

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                                    -35-
In  this  report, gas  phase hydrocarbon  emissions  from light-duty  gasoline-
and  Diesel-powered vehicles  were examined  and compared.   The  hydrocarbon
composition of  gasoline  exhaust  consists to  a large extent of  components  of
carbon numbers 1 through about 10.  In  contrast, the gas  phase  organics from
Diesel-powered  vehicles range  from  C,  to  about  C,Q,  the  majority  being
below  C25«   The  C,-C,Q   hydrocarbons  result  almost  entirely  from  the
combustion  process.    It   is  postulated  that  the   C,n-C9,-   hydrocarbons
result,  to   a   large  extent,   from   uncombusted  fuel,  and   the  C,,--C,0
hydrocarbons from lubricants  (37).

It is possible  to  identify  the individual hydrocarbons  with carbon  numbers 1
through 10 with a gas  chromatograph;  therefore,  the gas phase hydrocarbons
emitted from  gasoline-powered vehicles can be  readily  identified.   The  gas
chromatograph used to measure the hydrocarbon compounds with carbon  numbers
greater than  10 does not  have  adequate resolution to  permit  identification
of  each  individual compound  in  this  range.   It  is,   however, possible  to
determine the  molecular weight  distribution  of the compounds  of  interest.
Since  it  is  not  currently possible  to identify  individual   components  of
Diesel-powered  vehicle  exhaust  above C,0, Diesel  hydrocarbon  analyses  must
be  done  in  terms  of carbon  number.   Results  Indicate  that  the  gas  phase
emissions  from  Diesel-powered  vehicles  contain  small quantities  of  high
molecular weight organics.  These organics have not, as  yet, been identified.

While extensive Ames  and  other  bioassay testing for  mutagenicity  is  being
performed  on the  organics  extracted  from   the  particulate,  the   relative
mutagenicity  of the  gas  phase  organics remains  uncertain.   Methods are
currently  being developed  to collect  artifact-free samples   of  gas  phase
hydrocarbons in motor vehicle exhaust  for bioassay testing.    These  methods
are discussed below.

EPA-ORD has  done  some   preliminary  testing  with  both a condensate  and  a
filter  cartridge  method.   The  condensate  method  involves   filtering  the
exhaust participates  and then condensing the  components  in the  gas  stream.
The  condensate  appears  to have low  Ames  test  activity.   If  the  filter
upstream of  the condenser  is removed,  the condensate  contains  some  Diesel
particulate and has somewhat higher Ames test  activity.

-------
                                   -36-
The  filter  cartridge method  involves  passing a  gas stream  sample after  a
conventional particulate filter  through  a cartridge or bed of  treated XAD-2
resin.  After  the  gas stream is  passed  through  the XAD-2, the hydrocarbons
absorbed  onto  the XAD-2 are  removed  from the resin  by methylene  chloride
extraction.  The  lower  molecular weight  hydrocarbons  (e.g.  below C-10)  are
sufficiently volatile that they are  probably lost during  the  extraction.
However, hydrocarbons above C-10 are retained and  can then be  subjected  to
the  Ames  test.   Since  the conventional particulate  filter  will  generally
retain  hydrocarbon compounds above  C-15, the XAD-2 traps could provide  a
good  method  to   collect  hydrocarbons   in   the  C-10   to  C-15  range.    A
preliminary result of Ames testing on  the gas phase hydrocarbon collected  by
this  method for  a VW  Diesel Rabbit  shows  that  the  activity may  be  low
compared to that of the particulate.

EPA-OMSAPC  has  also been  developing a  technique to collect  Diesel  gaseous
hydrocarbons.  The procedure  involves collecting particulate on 20"  by  20"
filters using  dilute  Diesel exhaust from a  vehicle run  on  the EPA  Highway
Fuel Economy Test  (HFET).   A portion of  the  loaded filters  were  then baked
in an oxygen-free  oven to  drive  off  extractable  organics.  Two  of  the baked
filters were then installed behind  a  double  primary filter and an FTP was
run.  The particulate is  caught  by  the  double primary filter,  enabling  the
gas  phase hydrocarbons to pass  through to  the  pre-baked back-up  filters.
The procedure was  repeated for the HFET  cycle.  As  a control, background air
was  passed  through the  filters with no  car  installed.  Filter  weights were
measured  during  each  step of  the process.   In addition,  filters from each
step of the process were extracted and submitted  for Ames testing.

Preliminary Ames  results  with strain TA98  indicate that, without  metabolic
activation,  the mutagenic activity  of  the   extractables  from  the  loaded
(unbaked)  filters  is roughly  equal  to  the extractables  from  the  baked
back-up  filters   (used  to  collect   the gas phase   hydrocarbons).   When
metabolic activation was used, the mutagenic activity of the loaded filter
extractables  was  much  greater   that  that  of   the  baked  back-up  filter
extractables.  The same trends were  apparent  with strain TA100.  With strain
TA1538, the mutagenic activity  of  the  loaded filter  extractables was much

-------
                                   -37-
greater  than that of  the baked  back-up  filter extractables,  regardless  of
whether  or  not metabolic activation  was  used.  It  must be  emphasized  that
these  results  are very preliminary;  however,  they appear  to indicate  the
presence of direct-acting mutagens in the gas phase of Diesel exhaust.

B.    Particulate
The  chemical  composition  of  particulate  matter  from  Diesel  exhaust  is
complex.   Diesel  particulate consists  of a carbonaceous  core with  organic
compounds  adsorbed on  the  surface.   Particulate  emission rates  for  some
Diesel-powered vehicles can be found in Table 5.  The  sources  in  Table  5 are
those   being  tested   as  part   of   EPA's  health   effects   program.    A
gasoline-powered  vehicle  is  also  included  for comparison.  It can  be  seen
that  the  particulate emission  rates  of  the  Diesel-powered vehicles  differ
from one another  but, in  all cases, exceed the particulate emission  rate of
the gasoline-powered vehicle  by more than an order  of  magnitude.   Typically,
more  than  85%  of the  Diesel particulate  emitted  is under  1 micron  (10
meter)  in  size; as a result, the  particulate is  small enough   to be  inhaled
and deposited deep within the lungs.

Because  Diesel particulate  is easily  respirable,  considerable  effort  has
been spent in  an  attempt to  identify the organic compounds adsorbed on the
particulate  surface, in   particular  those  responsible  for  the  mutagenic
activity observed in the Ames test.   The organic  compounds   are  extracted
from  the  particulate  using  a solvent   such as  dichloromethane.   Table  5
includes  the percentage  of  particulate.  composed  of  extractable  (soluble)
organics  for   each   sample.    Like  the  particulate  emission  rate,   the
percentage of extractable matter varies  from vehicle to vehicle.

The  soluble  organic fraction (SOF) of  particulate  from  Diesel  exhaust  has
been  separated  by high  performance liquid chromatography  into  three  major
fractions: acid,  base and neutral.  The neutral fraction, in  turn, has  been
further separated  into  nonpolar, moderately polar and  highly polar  fractions
designated as   the  polynuclear aromatic  hydrocarbon  (PAH),  transition  and
oxygenate fractions, respectively.  The Ames test has  been used to  determine

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                                   -38-
the mutagenicity  of  these  fractions.  It was  found that the  transition  and
oxygenate  fractions  account  for most  of  the  Ames  test  activity.   The
transition fraction  alone  accounts for more  than  65%  of the direct  acting
mutagenicity for  the total extract.  The mutagenicity of the total  extract
was found to be equivalent to the summation of its fractions.

The nonpolar (PAH) fraction  has  been well characterized and consists  of  PAH
and aliphatic  hydrocarbons.   Benzo(a)pyrene (B(a)P) is  a PAH that has  been
identified.  Table 5  reports the  results  of  B(a)P  analyses performed  on
those samples.

The transition and polar fractions are more difficult  to  characterize.   The
polar fraction consists primarily of carboxylic acid PAHs.

Approximately  60% by  weight  of  the  material  in  the  transition  fraction
consists   of   oxygenated  PAH  derivatives   (including  hydroxy,   ketone,
carboxaldehyde, quinone,  dihydroxy,  acid anhydride and nitro derivatives).
The  carboxaldehyde  PAH  derivatives  were  among   the   most  abundant   PAH
derivatives found  in the transition  fractions.   A total of about  eighty  PAH
derivatives have  been identified  in this fraction,  including a  nitro-PAH,
1-nitropyrene.   The  1-nitropyrene was found  to account  for  roughly 45%  of
the direct-acting mutagenicity for  the  transition fraction and  30% of  the
direct-acting mutagenicity for the total  extract.  Two other  nitro-PAH were
tentatively identified in  the transition  fraction but their mutagenicity  is
not known.  The investigators conclude that the nitro-PAH may account for a
significant  portion of  the   direct-acting  mutagenicity  for  the  transition
fraction (38).

General Motors conducted a study  to  determine whether nitro-PAH  are  formed
in the  combustion process or by  chemical  reactions during exhaust  sampling
by filtration (39).  They found  that  the  levels of nitro-PAH were higher  in
particulate samples  collected over  longer  sampling  times or reexposed  to
additional Diesel exhaust gases.   The samples  reexposed  to  exhaust gases
also showed higher direct-acting  mutagenic  activity in the Ames  test  (using
strain  TA98).   The  authors  conclude that  much of the  nitro-PAH could  be
formed as artifacts of filter sampling.

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                                   -39-
EPA  has   conducted   experiments   recently  in  which  additional  NO,,   was
introduced into  the  dilution tunnel  to  determine if high  levels  of N02  in
the  dilution  tunnel  would  result  in a reaction of N02  with  some  of  the
hydrocarbons present in the gas stream or  on  the  particulate  filter.  If  so,
the  reaction  products  (artifacts),  including various  nitro-PAH  compounds
would cause an  artifically high  Ames test response.  Results indicate  that
artifact  (e.g.   excess  1-nitropyrene)  is  formed when  the  NO   levels  are
above 5  ppm  in  the  dilute exhaust.  The  N0» levels in the dilute  exhaust
are normally no higher than 3 ppm (40).

EPA has also conducted experiments with a  single  cylinder Diesel engine with
artificial combustion  air containing no nitrogen and  a nitrogen free  fuel.
Subsequently,  no NOx  would  be formed in the  exhaust gas.   By comparing  the
results  of these  tests   to  tests with  conventional air  (79%  N2,  21%  0,)
and  regular  Diesel   fuel  (which  contains   traces  of  nitrogen),   one  can
determine  if  there  were artifact  formation  due to N0_.    The  results  of
these tests have not yet been published.
IX.   Epidemiology

The following  section  summarizes  selected epidemiology studies performed  to
date.

Of  the several  epidemiological  studies  evaluating  the  effects  of  Diesel
exhaust the London  Transit  Worker study  has  received much attention  in  the
last few  years.   Lung cancer  incidence  among male  employees  of  the  London
Transport Authority, aged 45  to 64,  was  reported  during  1950  to 1974  (41).
(The results for 1950 to 1954 were originally reported by Raffle in  1957  and
updated by  Waller  in  1979  to  cover  the period from 1950 to  1974.)   In  a
presumed  highly  exposed  group,  i.e.,  the  "engineers"  servicing  buses  in
garages who were  exposed  to Diesel exhaust in an  enclosed  area,  a  total  of
177 cases  of   lung  cancer  were observed  in 86,054 man-years  at  risk  where
197.1  were  expected based  upon greater London  death rates in the  1950  to
1974 time frame.

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                                   -40-
Both  studies,  the  original and  the  follow-up,  suffer  many weaknesses  in
design.  When the NAS  Diesel Health Effects Panel reviewed  this  study,  they
cited the following weaknesses:

      0  no measure  of individual worker exposure,  merely a gross  estimate
         of pollutant  concentrations in  the  garage on  a few separate  days
         over the 25 years,
                                                     V,
      0  no smoking habits/histories were known,

      0  not following employees who left the London Transport Authority for
         other jobs,

      0  not  considering the  impact on  lung  cancer  of  social  and  ethnic
         differences  between  the  workers and  the  general  population  (the
         comparison group), and

      0  not investigating the "healthy worker effect".

In addition,  the cause  of  death of  employees  who  retired  from  the  London
Transport  Authority was  not investigated.    If a  retired employee  learned
that  he had  lung  cancer  the day  after his  retirement,  he would  not  be
included in the data base.

This  epidemiology  study  has  been  cited by  many  individuals  as  a  strong
indication  that  Diesel  emissions result  in no  excess  cancer  risk.   Todd
Thorslund, of EPA's Carcinogen Assessment Group evaluated  the London  Transit
Worker  Study  (LTWS) (42).  His analysis  has been  reviewed  both  inside  and
outside  EPA by  various  experts.   Thorslund  analyzed  the study parameters
statistically to obtain  an  upper  bound potency estimate which could  then be
translated into an upper bound measure  for  the  total potential cancer effect
in the U.S. population.  He  concluded that  "...it would  still be  possible to
have lung cancer deaths numbering in the thousands each  year  in the U.S.  due
to Diesel engine emissions and not be  inconsistent with  the  results obtained
in the LTWS.".

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                                   -41-
Dr.  Jeff  Harris  of  the Analytical  Panel of  the NAS  Diesel Impacts  Study
Committee  also performed  an analysis  of the  LTWS  (43).   Like  Thorslund,
Harris obtained  a statistical upper bound potency estimate.  He  concludes,
with  95  percent  confidence,  that the  undetected incidence  of  lung  cancer
among  Diesel bus  garage workers  was  no  greater than  160  percent  of  the
incidence of lung  cancer among other unexposed  employees.   Harris  then asked
the  question:   with 95% confidence  that the risk will not  be  any  higher,
what is the possible lung cancer risk in  the general  population, based on my
treatment  of the  uncertainies  present in  the  London  Transport data  base?
Harris found that the  upper 95% confidence  limit represents a  0.05  percent
increase in  lung cancer incidence per  unit  of  exposure, where  one unit  of
exposure  is  equivalent to  inhaling  a  concentration  of  1 microgram  of
particulates per cubic meter for one year.

Based  on  the  analyses  by  Thorslund  and  Harris,   it  is  possible  that
significant excess cancer deaths could result in  the  general  population even
though the LTWS showed no excess cancer deaths in the  exposed group.

Two  major  epidemiology studies  are  currently   planned.   One is a study  of
heavy equipment operators in  the Operating Engineers  Union  (44).   This study
is sponsored by  the  Coordinating Research Council (CRC).   Eligible subjects
are  those  who have  worked  for  one  year or more between  January 1958  and
December 1978.   It  is  estimated that  there will be  from  25,000  to  40,000
subjects with  up  to 500,000 man-years  of  work  experience.   Mortality  and
cancer incidence will be recorded.

The  second  study,  sponsored by EPA under a  Research  Grant with the  Harvard
School  of  Public  Health,  will  examine  railroad workers  exposed  to  Diesel
exhaust (45).   Railroad Retirement Board  records will  be  used  to identify
about 80,000 subjects exposed to Diesel exhaust.   These  subjects have  worked
10 to 19 years as railroad workers  in 1964 and will be followed through 1978.

Both proposed  studies  have attempted to  eliminate the  shortcomings  present
in the London Transit Worker Study.

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                                   -42-

X.    Risk Assessment

This  section  summarizes  the  assessments  that  have  been performed  in  an
attempt to determine  the  risk associated with exposure  to  Diesel emissions.
EPA's revised risk assessment will also be discussed.

Dr.  Harris  has  estimated  the  potential  risk  of  lung cancer  from  Diesel
emissions (43).   His  results, based on  information from the London  Transit
Worker Study, were given  in the  previous section.  Estimates of  the  risk  of
human lung  cancer from exposure  to Diesel emissions were  also made  using
relative  carcinogenic potencies  for Diesel  emissions  and  two  comparative
source emissions,  coke oven and roofing  tar.   Data  from three  short-term
bioassays (using  the  soluble organic fraction)  were used  to  estimate  the
relative carcinogenic potencies.  The bioassays included tumor initiation  in
SENCAR mice  by  skin  painting, enhancement of viral  transformation  in Syrian
hamster embryo cells,  and mutagenesis  with and without  metabolic  activation
in L5178Y mouse lymphoma  cells.   (The bioassays  and sources were  tested  as
part of EPA's  Diesel emissions research  program.   More information on  this
work  can  be  found  in previous  sections.)  The  95%   confidence  limit  of
potential risk was found  to be a  0.03 percent proportional increase  in  lung
cancer incidence  per  unit  of  exposure.   This  is  comparable  to  the  0.05
percent increase in lung  cancer  incidence per unit of exposure derived  from
the EPA analysis of the LTWS.

Lovelace Inhalation Toxicology Research  Institute  prepared a report for  the
Department of  Energy  summarizing the  potential  environmental  effects and
human health risks for projected  increased use of  Diesel-powered  light-duty
vehicles in  the United States (46).  The  authors  estimated the lung  cancer
deaths  associated  with  exposure   to  light-duty   Diesel  exhaust.    Their
calculations were based  on 1995  and  beyond.   It  was  assumed  that all
Diesel-powered automobiles  would  be controlled to  0.16  gm/mile  (0.1  gm/km)
particulate.   The  projected increase in  Diesel-powered light-duty vehicles
used  was  20%  of  the  light-duty vehicle  fleet,  an  upper level  based  on
restrictions  on  oil  refinery   processes.   Estimates  were  made  of   the

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                                   -43-
concentration  of  Diesel  exhaust particulate  to  which various  numbers  of
people would  be exposed.  The routes  of  exposure estimated were  inhalation
and  ingestion.   Only  inhalation and  resulting  lung  cancer  deaths  were
quantified.

To  estimate  lung  cancers  from  light-duty  Diesel  exhaust,  coke  oven  and
smoking  epidemiological data were  used.   These  data  were  standardized  to
                                                        3
annual  lung  cancer  risk  per 100,000  people  per  ug/m   of  benzo(a)pyrene
                       3
(B(a)P),  and  per mg/m  of  ambient  air particulate  matter.   Assuming  that
Diesel exhaust  was  not  significantly  more potent  than the worst  estimates
obtained  from the coke oven and smoking data,  estimates of  lung  cancers
associated with breathing Diesel exhaust were made.  These  values are:

      using B(a)P

         10 excess lung cancers from 20% Diesel-powered
         automobiles controlled to 0.16 gm/mile particulate,
         in 189 million people exposed

      using particulate

         30 excess lung cancers from 20% Diesel-powered
         automobiles controlled to 0.16 gm/mile particulate,
         in 189 million people exposed.

This report was issued in late December 1980 and is  the first of a  series  of
annual reports  on this  topic.   Future reports will include evaluations  of
additional health risks  and  examination of the health risks for people  with
existing respiratory diseases.

An  initial  risk assessment  was  performed by  EPA's  Cancer Assessment Group
(GAG)  in June  1979  (47).   The  GAG assessment was based  on  the  following
major assumptions:

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                                   -44-
1)    that  Diesel exhaust  products measured  by  organic  extractables,  and
      coke  oven  emissions  measured by  organic extractables  have the  same
      carcinogenic potency on a unit mass basis,

2)    that  the  entire  U.S.  population,  estimated  to be  220  million  in
      number,  is  exposed   and   that   the   exposures  were   log-normally
      distributed, and

3)    that the shape of the dose-response curve for lung cancer due  to  high
      level  coke oven industrial  exposures  are extrapolatable  in a  linear
      fashion to low-level environmental exposures.

The  following estimates  of  excess cancer  deaths  per year  were  obtained
assuming  two  Diesel market  penetration scenarios   (10%  and  25%  of  the
light-duty  vehicles  and   68%  and  99%   of  the  heavy-duty  vehicles  being
Diesel-powered by 1990, which represent  the best estimate and  maximum growth
estimate, respectively):

                                 Excess Cancer Deaths Per Year

                             Best Estimate       Maximum Growth Estimate
      Light-duty Diesels          346                    625
      Heavy-duty Diesels          668                   1185
The  light-duty  Diesel particulate  emission  factor used  was 1.08  gm/mile;
this assumes no  particulate  standard and  allows for a  penalty  in particulate
emissions   to  compensate   for   further  control  of  NOx   emissions.    The
particulate  emission factors used for  the heavy-duty  vehicles  equipped  with
2  stroke and 4  stroke  cycle engines,  and buses  were 4.08,  2.44 and  4.87
gm/mile,  respectively.  The GAG  intended this document  to  provide a  crude
estimate  of  the  magnitude  of the potential Diesel  exhaust problem  and  would
rely on  the  results  from  ongoing health effects research before an improved
analysis  could be made.

Both the EPA-CAG and DOE-Lovelace assessments assumed that  Diesel  emissions
were at  least  as potent  but not  significantly  more potent  compared  to  coke
oven emissions.   Allowing  for  the  different  population exposure  estimates,
the two assessments agree  reasonably well.

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                                   -45-
EPA has been  conducting  a massive Diesel health effects  research  program to
determine  the  carcinogenic  potency  of  the  particle-bound  organics  from
Diesel  emissions as  well  as the  potency  of  particle-bound organics  from
other  environmental  emissions  (including  coke oven emissions)  for  which
human  epidemiological  data  are   available.   The   mobile   source   samples
selected for  this program were  collected from a heavy-duty Diesel engine,  a
series of  light-duty  Diesel passenger cars and a  gasoline  catalyst-equipped
automobile.    The   comparative   source   samples   include  cigarette   smoke
condensate, coke oven emissions, roofing  tar emissions  and  benzo(a)pyrene.
Further information on  the mobile source and comparative source samples  can
be  found  in  section  IV.   The  latest  comparative  potency  rankings  are
presented  in  Table  3.   This  information,  together  with   the  available
epidemiological data for  the  comparative sources,  will be used  to  assess  the
human health  risk associated with increased use of  the Diesel engine.   The
major assumption to be used  in formulating the revised risk assessment  is
that the relative carcinogenic  potencies of Diesel engine emissions  and  the
related  environmental emissions  are  preserved  across  human and non-human
biological  systems   so  that the   available  data  can  be used  for  human
exposures.

Todd Thorslund of EPA's  Carcinogen Assessment Group has used  some  of  the
potency  data  to formulate  highly  tentative  first  cut  estimates  of  the
population risk  parameters (48).   The SENCAR mouse  skin tumorigenesis  data
were used to estimate relative potencies of  the Diesel samples and coke  oven
emissions.   These  data  indicate   that  Diesel  particulate  extract   has  a
potency  roughly  10%  of   that  of   coke  oven  emissions;    the  1979   EPA
preliminary  risk  assessment  assumed  the  two  substances   to  have  equal
potency.  The  unit  risk estimate developed for  coke oven emissions, together
with the relative potency  data were used  to  estimate  a unit  risk for Diesel
emissions.  The  unit  risk  estimate for individuals  living   in  cities of  1
million or  more is estimated to be  2.5 x  10   .   Applying  this  unit  risk
estimate with  rough population  exposure  estimates  of  Diesel  particulate  and
particle-bound  organics  (supplied  by  S.  Blacker,  EPA,  OMSAPC)  Thorslund
estimates  about  19  respiratory  cancer deaths/year  in  the  U.S.  population
will  be attributable  to  Diesel  exhaust.   This  is  based  on  uncontrolled
Diesel  automobiles   (i.e.  1.0  gm/mile  particulate,   15%  organics   on  the

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                                   -46-
particulate  by  weight)  comprising  approximately  15%  of  the  automobile
fleet.    This   estimate   is  probably   far  below   the   ability   of   an
epidemiological study to detect.

It  should be noted  that  this latest  risk estimate of  Todd Thorslund's  is
highly tentative and only uses the results  of one test,  skin  tumorigenesis
initiation  as  measured by  papillomas,  to estimate relative  potencies.   A
point to  consider  is that  skin  tumorigenesis may  respond more strongly  to
the  types of mutagens  present  in coke  oven emissions  than  the types  of
mutagens  present in  Diesel  particulate extract.  The revised risk  assessment
is  expected to  incorporate  the  results  of  a  variety of  mutagenesis  and
carcinogenesis tests for estimation of relative potencies.

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                                   -47-
                                 References

1.    "Precautionary Notice on  Laboratory  Handling of Exhaust  Products  from
      Diesel Engines", 4 November 1977.

2.    "Health  Effects  of  Exposure to  Diesel Exhaust",  the  report  of  the
      Health Effects Panel of the Diesel  Impacts Study Committee,  National
      Research Council, National Academy of Sciences,  1981.

3.    "The  Diesel  Emissions  Research  Program",   EPA-625/9-79-004,  December,
      1979.

4.    "Health Effects Associated  with  Diesel Exhaust Emissions -  Literature
      Review and Evaluation",  EPA-600/1-78-063, November,  1978.

5.    "Health  Effects  of  Diesel  Engine   Emissions:   Proceedings  of   an
      International  Symposium  -  Volume  1",  EPA-600/9-80-057a,   November,
      1980.

6.    "Health  Effects  of  Diesel  Engine   Emissions:   Proceedings  of   an
      International  Symposium  -  Volume  2",  EPA-600/9-80-057b,   November,
      1980.

7.    B.N.  Ames,  J.  McCann,  and  E.  Yamasaki,  "Methods   for   Detecting
      Carcinogens  and   Mutagens   with  the  Salmonella/Mammalian-Microsome
      Mutagenicity Test", Mutation Research, 31:347-364,  1975.

8.    Larry D.  Claxton,  "Mutagenic and  Carcinogenic  Potency  of  Diesel  and
      Related  Environmental  Emissions:  Salmonella  Bioassay", In:   "Health
      Effects  of  Diesel  Engine Emissions:   Proceedings  of an International
      Symposium - Volume 2", 1980.

9.    Ann  D.   Mitchell,  et   al.,  "Mutagenic  and  Carcinogenic  Potency  of
      Extracts  of Diesel  and  Related  Environmental  Emissions:   In_ Vitro
      Mutagenesis  and  DNA Damage",  In:    "Health Effects  of  Diesel Engine

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                                   -48-
      Emissions:   Proceedings  of  an International  Symposium -  Volume  2",
      1980.

10.   R.D. Curren,  et  al.,  "Mutagenic and  Carcinogenic Potency of  Extracts
      from    Diesel   Related    Environmental    Emissions:     Simultaneous
      Morphological  Transformation and  Mutagenesis  in Balb/c  313  Cells",
      In:  "Health  Effects  of  Diesel  Engine Emissions:   Proceedings of  an
      International Symposium - Volume 2",  1980.

11.   Ronald  L.  Schuler  and  Richard  W.  Niemeier,  "A  Study  of  Diesel
      Emissions  on  Drosophila"',  In:    "Health   Effects  of  Diesel  Engine
      Emissions:   Proceedings  of  an International  Symposium -  Volume  2",
      1980.

12.   J.   Lewtas   Huisingh,   "Short-term   Carcinogenesis  and  Mutagenesis
      Bioassays of  Unregulated  Automotive  Emissions", Bulletin of  the  New
      York Academy of Medicine, In press, 1981.
                  \
13.   M.A. Pereira, et  al.,  "In Vivo  Detection  of  Mutagenic  Effects  of
      Diesel  Exhaust  by   Short-Term  Mammalian  Bioassays",  In:    "Health
      Effects of  Diesel  Engine  Emissions:   Proceedings of an  International
      Symposium - Volume 2", 1980.

14.   R.R. Guerrero, et  al., "Sister Chromatid  Exchange  Analysis  of  Syrian
      Hamster Lung Cells Treated  In Vivo with  Diesel Exhaust Particulates",
      In:  "Health  Effects  of  Diesel  Engine Emissions:   Proceedings of  an
      International Symposium - Volume 2",  1980.

15.   EPA, Health Effects Research Laboratory -  Cincinnati  Quarterly Report,
      July-September, 1980.

16.   William E.  Pepelko,  EPA,  Letter  to  Gerald Rausa on Sister  Chromatid
      Exchange, 10 March, 1981.

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                                   -49-
17.   L.B.  Russell,  et  al.,  "Evaluation  of  Mutagenic  Effects  of  Diesel
      Emissions I. Tests for  Heritable and Germ-Cell Effects in  the  Mouse",
      24 December, 1980.

18.   J.  Justin  McCormick,   et  al.,   "Studies  on  the  Effects  of  Diesel
      Particulate on Normal  and Xeroderma  Pigmentosum  Cells",  presented  at
      EPA's Second Symposium  on Application of  Short-Term  Bioassays in  the
      Analysis  of Complex  Environmental Mixtures,  Williamsburg,  Virginia,
      4-7 March, 1980.

19.   Bruce  C.  Casto,  et  al.,  "Mutagenic  and  Carcinogenic  Potency  of
      Extracts  of Diesel  and  Related  Environmental  Emissions:   In  Vitro
      Mutagenesis and  Oncogenic  Transformation",  In:   "Health  Effects  of
      Diesel Engine Emissions:   Proceedings  of an International  Symposium -
      Volume 2", 1980.

20.   John  G.   Orthoefer,  et  al.,  "Carcinogenicity of  Diesel  Exhaust  as
      Tested  in  Strain  'A'  Mice",  In:   "Health  Effects  of  Diesel  Engine
      Emissions:  Proceedings  of  an  International  Symposium -  Volume  2",
      1980.

21.   Robert W.  Dickinson,  EPA, "Trip to Illinois  Institute of  Technology
      (IIT) Research Institute in Chicago", OMSAPC memo,  11  December,  1980.

22.   Alan M.  Shefner,  et  al.,  "Carcinogenicity of Diesel  Exhaust  Particles
      by  Intratracheal  Instillation  -  Dose   Range  Study",   In:    "Health
      Effects  of  Diesel Engine  Emissions:   Proceedings  of an  International
      Symposium - Volume 2", 1980.

23.   Bobby  R.   Collins,   "Respiratory   Carcinogenicity   of  Diesel   Fuel
      Emissions", IITRI  Quarterly Report  - March  1,  1981  through May  30,
      1981 (dated June, 1981).

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                                   -50-
24.   Stephen  Nesnow,  et  al.,   "Tumorigenesis  of Diesel  Exhaust,  Gasoline
      Exhaust, and Related Emission Extracts on  Sencar  Mouse  Skin",  EPA-HERL
      Report, 1981.

25.   Stephen  Nesnow,  "Report  on  Skin  Tumorigenesis  Studies  of  Diesel
      Emissions  and  Related  Samples  on C57  Black  Mice",  EPA-HERL  Report,
      December, 1980.

26.   Stephen  Nesnow,  "Report on  Skin Tumorigenesis  Studies of  Volkswagen
      Turbo  Rabbit  and  Home  Heater   Samples",  EPA-HERL  Report,  December,
      1980.

27.   U. Heinrich, et al., "Long Term  Diesel Exhaust Inhalation  Studies  with
      Hamsters",   In:     "Health  Effects  of   Diesel   Engine   Emissions:
      Proceedings of an International  Symposium - Volume 2",  1980.

28.   J.S.  Siak, et  al., "Diesel  Particulate  Extracts in  Bacterial  Test
      Systems",   In:    "Health   Effects   of   Diesel   Engine   Emissions:
      Proceedings of an International  Symposium - Volume 1",  1980.

29.   Leon  C.  King,  et  al., "Evaluation  of  the  Release of Mutagens  from
      Diesel   Particles    in  the   Presence   of   Physiological   Fluids",
      Environmental Mutagenesis,  in press,  1981.

30.   M.A.  Pereira,  et  al., "The  Effect of  Diesel Exhaust on  Spermshape
      Abnormalities  in   Mice",  In:    "Health   Effects  of  Diesel   Engine
      Emissions:   Proceedings of  an   International  Symposium -  Volume  2",
      1980.

31.   Kenneth  B.  Gross,  "Pulmonary Function Testing of Animals  Chronically
      Exposed  to Diluted  Diesel Exhaust", In:   "Health Effects of  Diesel
      Engine Emissions:   Proceedings of an International Symposium -  Volume
      2", 1980.

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                                   -51-
32.   K.I. Campbell,  et  al., "Enhanced Susceptibility  to  Infection in  Mice
      After Exposure  to  Dilute  Exhaust from Light Duty Diesel  Engines",  In:
      "Health  Effects  of   Diesel  Engine  Emissions:   Proceedings   of   an
      International Symposium - Volume 2", 1980.

33.   William E.  Pepelko, et  al., "Pulmonary  Function Evaluation  of  Cats
      After One Year of Exposure to Diesel  Exhaust",  In:   "Health  Effects of
      Diesel Engine Emissions:  Proceedings of  an International Symposium  -
      Volume 2", 1980.

34.   T.L.  Chan,  et  al.,  "Deposition  and  Clearance  of  Inhaled  Diesel
      Particles  in the  Respiratory  Tract",  presented at  the  Society  of
      Toxicology Poster Session, 3  March,  1981.

35.   Karl J. Springer,  "Characterization of  Sulfates,  Odor, Smoke, POM  and
      Particulates  from   Light  and  Heavy   Duty  Engines   -  Part   IX",
      EPA-460/3-79-007, June, 1979.

36.   Penny  Carey and Janet Cohen,  "Comparison  of  Gas  Phase Hydrocarbon
      Emissions  From  Light-Duty Gasoline Vehicles and Light-Duty  Vehicles
      Equipped with Diesel Engines",  EPA-OMSAPC  Report, EPA/AA/CTAB/PA/80-5,
      September, 1980.

37.   Frank Black  and Larry High,  "Methodology for Determining Particulate
      and  Gaseous  Diesel  Hydrocarbon  Emissions",   Society  of   Automotive
      Engineers Paper 790422, 1979.

38.   Dennis Schuetzle, et al.,  "The  Identification of Polynuclear  Aromatic
      Hydrocarbon  (PAH)  Derivatives   in  Mutagenic   Fractions   of   Diesel
      Particulate  Extracts", Intern.  J.  Environ. Anal.   Chem.,   9:93-144,
      1981.

39.   T.L. Gibson,  et al.,  "Determination of  Nitro  Derivatives  of PNA  in
      Diesel   Automobile   Exhaust",    presented   at  the   CRC    Chemical

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                                   -52-
      Characterlzation  of  Diesel  Exhaust  Emissions   Workshop,   Dearborn,
      Michigan, 2-4 March, 1981.

40.   Discussion  by  Dr.  Ron  Bradow,  EPA   during   the   Workshop  on  the
      Evaluation of Research  in Support of the Carcinogenic  Risk  Assessment
      for Diesel Engine Exhaust, Research  Triangle Park, NC,  24-25 February,
      1981.

41.   R.E. Waller, "Trends in Lung  Cancer  in London in  Relation to Exposure
      to  Diesel  Fumes",  In:   "Health  Effects of Diesel  Engine  Emissions:
      Proceedings of an International Symposium - Volume 2", 1980.

42.   Todd  Thorslund,  "Answer  to   the  posed  question:    '"Are  the  results
      obtained in  the London  Transit Worker Study sufficient  to dismiss  any
      concern regarding the potential cancer  hazard  for the  U.S.  population
      in  the future,  due  to  Diesel engine  exhaust?1",   EPA-ORD memo,   29
      January, 1981.

43.   Jeffrey E.  Harris,  "Potential Risk  of Lung Cancer from Diesel  Engine
      Emissions", National Academy Press, Washington,  D.C.,  1981.

44.   "Health Effects  of  Exposure  to Diesel  Exhaust",  National   Academy  of
      Sciences, 1980, p. 155.

45.   Marc  B.  Schenker  and Frank E.  Speizer,  "A Retrospective Cohort  Study
      of  Diesel  Exhaust  Exposure   in Railroad  Workers:   Study  Design  and
      Methodologic   Issues",    In:    "Health   Effects  of   Diesel   Engine
      Emissions:    Proceedings of  an International  Symposium -  Volume  2",
      1980.

46.   R.G.  Cuddihy,  et  al.,   "Potential Health and  Environmental  Effects  of
      Diesel  Light  Duty Vehicles",  Lovelace  Inhalation Toxicology Research
      Institute,  October,  1980.

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                                   -53-
47.   Roy  E.  Albert,  "Carcinogen  Assessment  Group's  Initial  Review  on
      Potential Carcinogenic  Impact  of Diesel  Engine  Exhaust", EPA  Report,
      11 June, 1979.

48.   Todd W.  Thorslund,  "A  Suggested Approach for the  Calculation of  the
      Respiratory Cancer Risk Due to Diesel Engine Exhaust",  EPA-ORD  Report,
      February, 1981.

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                                   -54-
    TABLE  1.   AMES TEST SPECIFIC ACTIVITIES  AT  100  ug OF ORGANIC  MATERIAL

TA98
Sample
Caterpillar
Nissan
Oldsmobile
VW
Mustang

Cigarette
Coke Oven
Roofing Tar
+S9
59.3
1367.1
318.7
297.5
341.9

98.2
251.6
98.7
-S9
Diesel
65.9
1225.2
614.8
399.2
Gasoline
137.8
Comparative
Neg
164.1
Neg
TA100
+S9
115.2
881.7
169.9
426.0
228.0
Samples
265.6
420.0
-S9
167.8
1270.1
247.5
641.6
196.5

Neg
259.4
Neg
Control Compound
B(a)P
15202.3*
NT
26438.0*
NT

*Extrapolation
NT = Not tested
Neg = Negative.

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                                   -55-
               TABLE 2.  RELATIVE POTENCY OF ORGANIC MATERIAL
                          BASED ON AMES TEST RESULTS

Sample
Caterpillar
Nissan
Oldsmobile
VW
Mustang
Cigarette
Coke Oven
Roofing Tar
B(a)P
+S9
4.3
100.0
22.3
21.8
25.0
7.2
18.4
7.2
1112.1
TA98
-S9
Diesel
5.4
100.0
50.2
32.6.
Gasoline
TA100
+S9
13.0
100.0
19.3
48.3
11.3 25.9
Comparative Samples
Neg ?
13.4 30.2
Neg 47.6
Control Compound
NT
2997.5*
-S9
13.2
100.0
19.5
50.6
15.5
Neg
20.4
Neg
NT

*Extrapolation.
NT = Not tested
Neg = Negative

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                                        -56-
                                         Table  3

                              COMPARATIVE POTENCY RANKINGS

CARCINOGENESIS
MUTAGENESIS


DIESEL: CAT
NISSAN
OLDS
VW RAB
GASOLINE:
MUSTANG
COMPARATIVE
SOURCES
CIGARETTE
COKE
ROOF TAR
HOME HEATER
STANDARDS :
B(a)P
AMESa

4.3
100
23
22

25


7
18
7


1112
SCEa

0
100
0
50

1


0
44
291


1750
L-5178Ya

lc
100
64
50

36


21
339
850


189
BALBa

0
100
750
NTd

750


300
15
750


25000
VIRAL
ENHANCE
MENT
0
100
25
50

50


200
800
2016


52000

BALBa

0
100
0
NT

200


200
500
500


16700
TUMOR
INITATIONb

0
100
28
6

16


0
355
120
7

16500

aln the presence of an Aroclor-1254 induced rat hepatic S-9
       skin tumor initiation in male and female sencar mice after 26 weeks
 of treatment

cTesting incomplete at this time.

dNot tested.

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                                                      Table 4
in
                       Exhaust Emissions From Diesel- and Gasoline-Powered Light-Duty Vehicles
                                               and Heavy-Duty Engines
       Light-duty Vehicles^3)

       Oldsmobile Cutlass
        350 Diesel
        260 Gasoline

       Volkswagen Rabbit
        Diesel
        Gasoline
       Heavy-duty Engines

       Diesel(b>
        Mack ETAY(B)673A
        Caterpillar 3208/EGR  1.163

       Gasoline(c)

        Chevrolet 366         2.49
Emission Rate
HC CO
0.47
0.24
0.23
0.14
1.24
1.34
0.49
2.30
Emission Rate
HC CO
0.476
1.163
1.588
6.200
, g/km
NOx
0.70
0.85
0.54
0.63
Part.
0.573
0.006
0.182
0.004
, g/hp-hr
NOx Part .
6.613
3.747
0.612
2.208
Emission
Sulfates
9.962
1.373
3.662
0.041
Emission
Sulfates
33.467
16.725
Rate, mg/km
Total Aldehydes
82.5
14.0
39.6
37.8
Rate, mg/hp-hr
Total Aldehydes
64.29
161.34
Fuel Econ.
mpg
21.7
15.6
42.7
24.6
BSFC
Ibs/bhp-hr
0.399
0.472
55.00   3.39
0.207
1.033
1190.12
0.761
       (a) 1975 FTP cycle used for all light-duty vehicles
       (b) 13-mode FTP cycle used for the Diesel-powered heavy-duty engines
       (c) 23-mode FTP cycle used for the gasoline-powered heavy-duty engine

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                                      -58-

                                      Table 5

                            Particulate Emission Rates
Sample

Diesel:
  Caterpillar
  Nissan
  Olds
  VW Rabbit

Gasoline:
  Mustang
Comparative sources:
  Cigarette
  Coke
  Roofing tar
Particulate            Extractable        B(a)P           B(a)P
Emission Rate (g/km)   Matter (%)    (ng/mg extract)   (ng/mg part)
0.72*
0.205
0.32
0.11
27
8
17
18
2
1173
2
26
0.5
96.2
0.4
4.6
       0.003
43
                           5-10
                         >99
103
                 478
                 889
44.1
                31.5
               889
* g/hp-hr. The Caterpillar is a heavy-duty engine.

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