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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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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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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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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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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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.
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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.
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-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.
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-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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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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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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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.
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-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
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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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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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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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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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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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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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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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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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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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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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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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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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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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