United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-011 Sept 1984
&EPA Project Summary
Use of Short-Term Genotoxic
Bioassays in the Evaluation of
Unregulated Automobile
Exhausts
David J. Brusick, R.R. Young, and D.R. Jagannath
The levels of several products of fuel
combustion in ambient air (nitrogen
oxides, hydrocarbons and carbon mon-
oxide) are currently regulated under the
Clean Air Act. Amendments ([202(a)(4|]
of 1977) also specify that new vehicles
shall not be certified if they generate
unregulated emissions which present a
potential risk to human health. In
addition. Section 211 of the Clean Air
Act as amended in 1977 specifies that
tests should be conducted to determine
the mutagenic and carcinogenic effects
(among other health effects) of auto-
motive fuels and fuel additives and their
emissions.
The objectives of this document are
to review the data from selected short-
term in vitro and in vivo bioassays to (a)
determine if there is evidence sugges-
ting potential human health risk either
from uncombusted emissions or from
emissions of combusted motor vehicle
fuels or fuel additives, (b) identify the
operational variables involved in gener-
ating products of concern for human
health, (c) determine the probable
nature of the health effects of concern,
(d) estimate the ability of short-term
tests to establish human risk estimates
and (e) develop a short-term bioassay
program to monitor the potential health
hazard of fuel/fuel additives and
unregulated combustion emissions.
This report was submitted in fulfill-
ment of Contract No. 68-02-2681,
Technical Directive No. 008, by Litton
Bionetics, Inc., under the sponsorship
of the U.S. Environmental Protection
Agency (EPA).
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research pro-ject which is fully docu-
mented in a separate report of the same
title (see project report ordering inform-
ation at back).
Introduction
Automotive emission products from
complete or partial combustion of fuels
such as diesel fuel or gasoline are asso-
ciated with genotoxic activity. Most of the
genotoxic effects are found in the solid-
phase (paniculate) fraction as mutagenic
and cell-transforming organic compounds
condensed onto the carbonaceous core of
the exhaust particle.
Air pollutants include both gaseous
and solid phases. Gaseous-phase pollu-
tants include carbon monoxide, ozone,
nitrogen oxides, sulfur oxides, hydrocar-
bons, and other volatile organic compounds
(e g., formaldehyde, benzene). The solid
phase contains condensed organic poly-
cyclic matter (POM) including polycyclic
aromatic hydrocarbons (PAHs) and sub-
stituted PAHs, which are suspected of
contributing to a potential lung cancer
risk associated with long-term exposure
to urban air pollution.
The possibility of human health risk from
motor vehicle emissions has been a
matter of concern for many years, and
there has been substantial research into
the effects of emissions from both
gasoline spark ignition and diesel engines
(1,2). Even with this data base, the extent
to which motor vehicle emissions con-
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tribute to human health problems is far
from understood. The complex interac-
tions between fuel (and its additives) and
gases in the combustion chamber, envi-
ronmental exposure conditions, and bio-
logy of the target species must be investi-
gated in order to apply most conventional
hazard assessment methods.
The objective of this report was to
summarize the current status of research
on unregulated automobile emissions
using short-term tests for genotoxicity
and to develop an approach for the
application of these tests to the develop-
ment of regulatory strategy. Based on the
results of extensive studies with pure
chemicals, short-term genetic tests are
believed to be reliable quantitative indica-
tors of carcinogenic and mutagenic
potential. Thus, such tests should be
useful in addressing the concern for
carcinogenic effects.
Current Status of the
Application of Genetic Assays
to Studies of Motor Vehicle
Emissions
Many of the characteristics of short-
term tests measuring genotoxicity appear
ideally suited to an analysis of automobile
exhaust emissions; in fact, the current
level of information available on the bio-
logical properties and possible health
risks associated with particulate emis-
sions has been accumulated largely
using short-term test data. Certainly, the
conduct of multi-dose experiments using
relatively small quantities of particles
collected from exhaust emissions was
one of the true breakthroughs in the
evaluation of motor vehicle emissions.
Many attributes of short-term tests are
important to the application of these tests
and are summarized in the full report. The
importance of the ability to conduct
assays of very small samples cannot be
overemphasized, since spark-ignition
gasoline engines equipped with catalytic
converters produce very low levels of
particulate. Without short-term assays,
valid comparisons of these samples with
those of other emissions would not be
possible
Application of Short-Term
Bioassays to Risk Assessment
of Automobile Emissions
Much of the data predicting qualitative
hazard have been derived from short-
term, especially//? vitro, assays, because
they have been found to be readily appli-
cable to an evaluation of the solid-phase
emissions. Risk analysis must draw upon
all data available. Quantitative estimates
developed for automobile exhaust emis-
sions by the U.S. Environmental Protec-
tion Agency (EPA) and the National
Academy of Sciences (NAS) have relied
heavily upon comparative data obtained
from in vitro assays for genotoxicity and
short-term animal tests (1,3). Both ap-
proaches have been drawn upon the
available bodies of short-term data and
made comparative analyses of laboratory
and epidemiological data on diesel and
gasoline engines as well as chemically
related environmental exposurescoke
oven emissions, roofing tar emissions,
and cigarette smoke condensate (CSC).
Both the EPA and the NAS risk
estimates included analyses of short-
term test results in terms of a linear, non-
threshold, extrapolation model. The
potency response of all sample emissions
evaluated in the short-term bioassays
was compared using linear extrapolation
models. Relative potencies for gasoline
and diesel emissions were then compared
to those for coke oven, roofing tar, and
cigarette smoke condensate. Assuming
comparability between relative potency
for the diesel emissions and the model
emissions (coke oven or roofing tar), a
relative potency value for humans was
constructed using human lung cancer
data from coke oven workers and roofers.
The estimate for risk of lung cancer
from exposure to diesel exhaust emissions
was derived from the ratio of short-term
test activities between the two emissions
and extrapolating that ratio to the
incidence of human lung cancers for the
two exposures after adjusting for dose.
The calculated risks were quite similar
In summary, short-term tests for geno-
toxicity are amenable to use in human
risk estimates in situations where
conventional animal modeling or human
epidemiology results cannot be obtained.
An evaluation of two approaches con-
ducted by EPA and NAS shows similar
quantitative risk estimates for diesel
exhaust emissions. These two values are
also similar to a worst-case estimate for
lung cancer in humans derived from
negative epidemiological data. Risk
estimation for heritable genetic effects
cannot be derived from the available data.
Review of Bioassay
Performance on Emission
Samples
Review of the bioassay data is arranged
by the type of toxic endpoint measured
and is subdivided into mammalian in
vivo tests and in vitro mammalian/sub-
mammalian tests. Table 1 outlines the
stratification of endpoints included in this
evaluation.
An evaluation of data from the bioassay
data base was examined using the
procedure outlined in Figure 1. The end
product of this procedure was selection of
a battery of tests amenable to routine
evaluation of emissions.
Sample Collection and
Preparation
Analysis of the published literature
was presented and recommendations
were made regarding collection and
preparation of samples for genetic
testing.
The full report suggested that a com-
panion engineering document be pre-
pared to establish specific test conditions
for engine operation and emission collec-
tion.
Use of particle extracts in formulating
comparisons of various engines, fuels, or
fuel additives is considered appropriate,
since most biological activity of the
emissions appears to be associated with
the particle-bound organics and suitable
methods for gas phase or whole particle
testing are not ready for routine applica-
tion.
Selection of Bioassays to Form
a Minimum Evaluation Matrix
Objective analysis of the current data
base for automobile combustion emis-
sions and test performance criteria re-
sulted in the selection of three bioassay
types which appear to merit further con-
sideration as screens for automotive
emission certification. The Ames Sa/mo-
ne//a/microsome reverse mutation assay
appears to be useful. Cytogenetic end-
points, especially sister chromatid ex-
change (SCE), showed very good sensiti-
vity to the genotoxic components of auto-
motive emissions. An in vitro mammalian
cell gene mutation assay, especially the
Mouse Lymphoma assay, is the third test
considered applicable to emission screen-
ing, because it responded in a quantita-
tive fashion to organic solvent extracts
and in some cases to whole unextracted
particles. Some of the information used to
support the selection of these three tests
is shown in Table 2, which identifies the
range of susceptibilities of various bioas-
says to the types of samples evaluated.
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Table 1. Stratification of Bioassay Endpoints Reviewed
Genotoxic Effects
A. Specific Locus Mutation in Prokaryotic and Eukaryotic Organisms
B. Chromosome Alterations (Including Aberrations, Sister ChromatidExchange, andAneuploidy)
C. Damage of the Primary DNA Level in Mammalian Cells In vivo and In vitro /Including DNA
Adduct Formation, DNA Repair Phenomena, Mitotic Crossing Over)
Cell Transformation and/or Tumor Induction
A. In vitro Cell Transformation in Mouse Cell Lines and Hamster Primary Cells
B Short-Term Tumor Induction Assays in Mice
C. Long-Term Tumor Induction Assays in Rodent Species
D. Co-Carcinogenesis Studies in Rodents
Step
Collect, collate and review
available bioassay data.
Analyze each data element in a
response matrix organized by
end point. Extract qualitative
response, dose range, test conditions,
sample source and quantitative effects.
Summarize all qualitative
responses /Positive and Negative).
Compare sample type, sample
source and quantitative response
by assays.
Identify bioassays capabale of
detecting genotoxic/carcinogenic
effect of automotive emissions.
Critique all bioassays
producing positive responses.
Approach to Emission Testing
for Comparisons of Biological
Activity
The procedures described m the full
report attempt to satisfy the need for data
comparisons. To be useful, a description
of biological activity must include an
estimate of the specific activity of a
sample in each bioassay employed. For
the three tests recommended as the
battery, the linear slope of the dose-
response curve, expressed as revertants/
ug, SCE/cells/jug/ml, and revertant
mutant frequency//yg/ml, has been
selected. The specific activity should then
be adjusted to an estimate of particle
potency by determining the level of
organic extractability (percent extractable)
from the material collected. The final
calculation should consider the particle
potency vs. the particle emission rate
(PER). The following formula gives the
relationship among these factors in
developing a Sample Activity Rate (SAR):
The determination of samples that had
significant biological activity employed an
analysis of variance which compared the
experimental means (using a .05 level of
significance) for each sample in each
bioassay. Several methods may be used to
ask the question of how to identify the
outliers among the samples. For example,
it is possible to group samples using
various statistical methods and find those
tests which differ significantly from the
remaining tests.
Several conclusions can be made
based on analysis of a pilot set of
samples,
1. Comparisons between nonactivated
and 59-activated tests indicated
that the set of samples studied in S9
had no effect on the responses.
2. The biological activities calculated
for the emission samples did not
appear to follow similar patterns in
the three tests.
3. Among the three tests, the Ames
assay was the most discriminating for
this set of emission samples. It is
possible, however, that greater use
of replicate trials in the mammalian
cell assays would increase their
discriminating properties.
4. As a consequence of the analysis of
this pilot data, it would appear that
replication of sample collection,
analysis, and biological testing is
essential. Recommendations for
replication of these components of
the proposed scheme are shown in
Table 3. By performing independent
trials, the variability arising from
replicating the collection and extrac-
tion procedures as well as bioassay
techniques are important data com-
ponents. Claxtoh and Kohan showed
that there are small but consistent
variances resulting from sample
preparation differences (4).
Conclusions
1. Bioactive chemicals that exist in
vapor and solid-phase exhaust emis-
Review distribution of positive
bioassay responses across
sample types.
Select candidate bioassays
for use in emission screening.
Figure 1. Bioassay data evaluation matrix.
Genetic Activity
jjg organics
t
Slope
(Specific Activity)
fjg organics
particles _ Genetic Activity
100//g particles
% Extractables
kilometer (km)
t
Particle Emis-
sion Rate (PER)
km
t
Sample Activity
Rate (SAR)
Particle Potency
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Table 2, Distribution of Responses in Susceptible Bioassays Across Sample Categories
B/oassays
Organic Solvent
Extracts of
Particles
Biological Fluid
Extracts of
Particles
Unextracted
Particles
Vapor Phase
Alone
Whole
Exhaust
Gene Mutation
Microbial
Mammalian Cells in vitro
Plants
Insects
Mammals in vivo
Cytogenetic Endpomts
Mammalian Cells in vitro
Plants
Mammals in vivo
Primary DNA Effects
In vitro Mammalian
In vivo/in vitro Mammalian
Carcinogenesis Endpoints
Mammalian Cells in vitro
In vivo Mammals (complete)
In vivo Initiation
In vivo Co-carcinogenesis
V
V
V
^
V
^
x/
x/
(\l) - data consist of limited experimentation that may not apply to all bioassays of this class.
Table 3.
Step*
Composition and Logistics of an Emission Assessment
Trial A"
Trial 5"
Particle Collection
from Filters(s)
I
Extraction of Organics
1
Bioassay
1
Particle Collection
from Filters(s)
1
Extraction of Organics
1
Bioassay
1
3
4
1
Ames Test
Conducted with
Triplicate
Plates/Dose
{
Mouse Lymphoma
Assay Conducted
with Duplicate
Cultures
Data Analysis
1
SCE Assay
Conducted with
Duplicate
Cultures
1
Ames Test
Conducted with
Triplicate
Plates/Dose
1
Mouse Lymphoma
Assay Conducted
with Duplicate
Cultures
Data Analysis
1
SCf Assay
Conducted with
Duplicate
Cultures
"Step 1 - Independent trials for determination of Particle Emission Rate (PER).
Step 2 - Independent trials to measure percent extractables.
Step 3 - Replicate data within independent trials for each of the three bioassays.
Step 4 - Data analysis and comparison with other emission samples.
^Definition of independent trials A and B has not been made. Trials may be different runs on the same day or different runs on two days. The total
paniculate required per trial is approximately 0.5 g.
sions are generated by combustion
and are believed to be derived from
mono- or poly-substituted PAHs or
nitroaromatics that can be activated
to genotoxic agents by bacteria or
animal cells (such as liver cells or
macrophages).
Mutagens are bound to particles but
can be extracted, to various degrees,
with organic solvents and biological
fluids. Thus, one can expect some
level of bioavailability.
3. The levels and composition of chemi-
cals found in emissions may vary
significantly with engine types, fuel
types, operating conditions, control
devices, and environmental condi-
tions of collection.
4. An efficient screening program to
compare quantitative variations in
one of the above parameters (by
holding all other parameters con-
stant) can be accomplished using a
series of short-term in vitro tests;
however, the assessment cannot be
conducted on whole-emission col-
lections; rather, it requires a standard
sample collection and processing
protocol for the preparation of
organic compounds recovered from
the solid phase.
A uniform data interpretation scheme
has been defined in which the
relative biological activity of each
bioassay can be quantitatively com-
pared to an existing data base
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developed from similar engines,
fuels, or fuel additives. Samples that
show statistically significant devia-
tions from the data base can be inter-
pretated as a signal that an engine,
fuel, or fuel additive might warrant
further investigation for possible
health impact.
The proposed approach should be
adequate for making quantitative com-
parisons of the biological activity of
emissions generated by different engines
or fuels under standardized running and
collection protocols and for identifying
reponses which fall outside the normal
range of engines, fuels, or fuel additives.
The results will not be suitable for use in
quantifying the absolute health risk of
emissions because of the many uncer-
tainties associated with bioavailability
and dosimetry of emissions to hetero-
geneous human populations under
normal exposure conditions and the lack
of formal linkages between the responses
in short-term tests and human disorders.
D. Brusick, R. Young, andD. Jagannathare with Litton Bionetics, Inc., Kensington,
MD 20895.
Joellen Lewtas is the EPA Project Officer (see below).
The complete report, entitled "Use of Short-Term Genotoxic Bioassays in the
Evaluation of Unregulated Automobile Exhausts," (Order No. PB 84-226 976;
Cost: $14.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
References
1. National Research Council. Health
Effects of Exposure to Diesel Exhaust.
National Academy Press, Washing-
ton, DC, 1981.
2. U.S. EPA, Office of Research and
Development. Diesel Emissions Sym-
posium Proceedings. A Compendium
of Manuscripts from the Symposium
held in Raleigh, North Carolina,
1981.
3. Albert, R.E., J. Lewtas, S. Nesnow,
T.W. Thorslund, and E. Anderson. A
comparative potency method for
cancer risk assessment: application
to diesel particulate emission. Risk
Analysis, 3-101-117, 1983.
4. Claxton, L. and M. Kohan. Bacterial
mutagenesis and the evaluation of
mobile-source emissions. In: Short-
Term Bioassays in the Analysis of
Complex Environmental Mixtures II,
M.D. Waters, S.S. Sandhu, J. Lewtas
Huisingh, L. Claxton, S. Nesnow,
eds.. Plenum Publishing Corp., 1981,
pp 229-317.
S. GOVERNMENT PRINTING OFFICE: 1984/759-102/10687
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