P.1 

OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

70) ll-J-13 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:17 AM Sheet 70 of 93 
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7

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MUTAGENIC AND V*^_glNOGENIC POTENCY OF 

EXTRACTS OF DIESEL AND RELATED ENVIRONMENTAL EMISSIONS: 

STUDY DESIGN, SAMPLE GENERATION, 

COLLECTI ON, AND PREPARATION 

J. L. Huisingh1, R. L. Bradow1, R. H. Jungers1, 

B. D. Harris1, R. B. Zweidinger1, K. M. Cushing2, 

B. E. Gill3, R. E. Albert1,

14 

'Office of Research and Development 

U. S.. Environmental Protection Agency 

Research Triangle Park, North Carolina 

2Southern Research Institute 

Birmingham, Alabama 

30ak Ridge National Laboratory 

Oak Ridge, Tennessee 

l4New York University Institute of Environmental Medicine 

New York, New York 

ABSTRACT 

A major diesel emissions research program has been initiated 

by the U.S. Environmental Protection Agency to assess the 

human health risk associated with increased use of diesel 

automobiles. This program is intended to establish the 

mutagenic and carcinogenic potency of complex organics 

associated with diesel particles as well as comparative 

particle-bound organics from other environmental emissions 

for which human epidemiological data are available. 

The mobile source samples selected for this study were collected from a heavy-duty diesel engine, a series of lightduty diesel passenger cars, and a gasoline catalyst automobile. The comparative source samples incorporated into 

the study were cigarette smoke condensate, coke oven emissions, roofing tar emissions, and benzo(a)pyrene. The 

samples were tested using three mutagenic assays and four 

carcinogenic assays as prescribed by a test matrix. 

This report describes the study design, particle generation, 

and sample collection and preparation. A brief summary of 

the bioassays is also included. 

ACKNOWLEDGMENTS 

The authors wish to acknowledge J. Smith for coordinating 

and managing the interlaboratory effort to obtain the comparative samples; S. Tejada, F. King, H. Becker, W. Ray, 

and the Northrop Services staff for assistance in generation and collection of the diesel particles; J. Sturm 

for generation of the VW Rabbit diesel sample; T. Pugh for 

assistance in collection of coke oven samples; W. Griest 

and R. Jenkins for assistance with the cigarette smoke condensate; A. Fowler for assistance in collection of the roofing tar samples; F. Butler, C. Rogers, and F. Toth for 

assistance in the samples extractions; L. King for assistance in coding and shipping the samples; and the Northrop 

Services' Technical Services staff for assistance in editing and preparing this report. 

INTRODUCTION 

The complex mixture of incomplete combustion organics 

associated with diesel particle emissions has been shown 

to be mutagenic in short-term bioassays (1). These organics 

contain known carcinogenic polycyclic aromatic hydrocarbons, 

as well as highly mutagenic polar neutral (oxygenated polycyclic) compounds. A variety of combustion emission products have been recognized as human carcinogens, including 

chimney soot (2) and coke oven emissions (3). At present, 

there is no direct evidence that diesel emissions are human 

carcinogens or that human exposure to environmental concentrations of diesel emissions will increase the incidence 

of cancer in exposed populations. The expected increase 

in the use of diesel-powered automobiles and resulting increased ambient air concentrations of diesel emissions have 

stimulated a major diesel emissions research program being 

conducted by EPA's Office of Research and Development (4). 

The information generated from this research will be used 

to assess the human health risk associated with increased 

use of the diesel engine. 

Diesel exhaust is a mixture of particles (carbonaceous soot) 

and gases. The principal gases (hydrocarbons, nitrogen 

°*ides, sulfur oxides, and carbon monoxide) are similar to 
If 

those currently emitted by gasoline engines and other com-

bustion sources. The particles emitted from diesel engines 

differ significantly in both quantity and composition from 

gasoline particle emissions. Diesel cars emit 30-to-100 

times more particulate matter than gasoline catalyst cars. 

Gasoline particulate emissions are primarily sulfur com-

pounds, while diesel particles are composed of carbonaceous 

soot with a large amount (5 - 50%) of complex organics ad-

sorbed to the surface. This study was designed to determine 

the mutagenic and carcinogenic potency of these complex, 

organics compared to particle-bound organics from other 

environmental emissions. This report describes the study 

design, particle generation, and sample collection and pre-

paration for biological studies. The results and a summary 

of the relative mutagenic and carcinogenic potency of these 

organics are included elsewhere in this volume. 

STUDY DESIGN 

The objective of this study was to determine the relative 

mutagenic and carcinogenic potency of the extractable or-

ganics from diesel emissions and other emissions for which 

human epidemiological data are available. 

Epidemiological studies have shown that a dose-response 

relationship exists between exposure to coke oven emissions 

and human lung cancer rates (5, 6, 7, 8). Increased in-

cidence of cancer has also been shown in epidemiological 

studies of humans exposed to roofing tar emissions (9) and 

cigarette smoke (10). Environmental exposure to various 

hydrocarbon combustion products, measured by the benzo(a)-

pyrene (BaP) concentration, has also shown a highly con-

sistent carcinogenic potency in different epidemiological 

studies for coke oven, roofing tar, and gas works emissions 

(11). Coke oven emissions, roofing tar emissions, and 

benzo(a)pyrene were, therefore, selected as comparative 

sources for this study. Cigarette smoke condensate (CSC) 

was selected as an additional comparative sample because 

of the large number of animal studies that have been con-

ducted with this source (12). 

The mobile source samples selected for this study included 

a heavy-duty diesel engine (Caterpillar 3304), a series of 

light-duty diesel passenger cars (Datsun Nissan 220C, Olds-

mobile 350, and Volkswagen turbocharged Rabbit), and a gaso-

line catalyst car (Fort Mustang II). 

The design of the sample and bioassay test matrix is shown 

in Table 1. The mutagenesis bioassays, selected to detect 

gene mutations, chromosomal effects and DNA damage, were 

all short-term jrj^ vitro assays performed in microbial or 

mammalian cells. The carcinogenesis bioassays included both 

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in vitro mammalian cell assays and in vivo bioassays. The 

in vivo assays included skin carcinogenesis (Senecar and 

C57 black mice), pulmonary adenoma formation (strain A mice) 

and intratracheal instillation (hamsters). 

SAMPLE GENERATION AND COLLECTION 

The mobile and comparative source samples were provided for 

this study by the organizations shown in Table 2. Sample 

collection techniques are summarized in Table 3. 

TABLE SAMPLE SUPPLIERS 

1 

]• 

j 

J 

. 

Sample 

Diesel 

Gasoline 

Comparative 

sources 

source 

CAT 

NISSAN 

OLDS 

VW RAB 

MUSTANG 

CSC 

Coke 

Roof tar 

R. 

R. 

R. 

R. 

R. 

J. 

B. 

B. 

EPA 

Bradow/ESRL 

Bradow/ESRL 

Bradow/ESRL 

Bradow/ESRL 

Bradow/ESRL 

Smith/HERL 

Harris/IERL 

Harris/IERL 

Other 

J. Sturm/DOT 

-

W. Griest/ORNL 

K. Cushing/SRI 

ESRL = Environmental Sciences Research Laboratory, DOT = 

Department of Transportation, HERL = Health Effects 

Research Laboratory, ORNL = Oak Ridge National Laboratory, 

IERL = Industrial Environmental Research Laboratory, 

SRI = Southern Research Institute 

TABLE 3. SAMPLE COLLECTION TECHNIQUES 

Sample source Sampling apparatus Media 

Diesel 

Gasoline 

CAT Dilution tunnel 

NISSAN Dilution tunnel 

OLDS Dilution tunnel 

VW RAB Dilution tunnel 

MUSTANG Dilution tunnel 

Pallflex T filter 

Pallflex T filter 

Pallflex T filter 

Pallflex T filter 

Pallflex T filter 

Comparative CSC Refrigerated con- Acetone 

sources denser 

Coke Massive Air Volume ESP plates 

Roof tar Baghouse Teflon filter bags 

DIESEL AND GASOLINE 

The mobile source particulate emission samples used in th 

study were generated from the sources shown in Table 4. 

diesel engine and vehicles were all operated on the same 

0f No. 2 diesel fuel. The gasoline vehicle was operated < 

unleaded gasoline at a richer-than-normal stoichiometry ii 

order to produce sufficient sample volume for limited 

studies. The particle emission rates are shown in Table '. 

TABLE 4. MOBILE SOURCE SAMPLE DESCRIPTIONS 

Sample source Description Fuel Drivii 

cycli 

Diesel CAT 

NISSAN 

OLDS 

VW RAB 

Caterpillar 3304 Diesel No. 2 Mode 

Nissan Datsun 

220C 

Oldsmobile 350 

Volkswagen 

turbocharged 

Rabbit 

Diesel No. 2 HWFET 

Diesel No. 2 HWFET 

Diesel No. 2 HWFET 

Gasoline MUSTANG 1978 Mustang II- Unleaded 

302* gasoline 

HWFET 

*V-8, equipped with catalytic converter and exhaust gas 

recirculation (EGR) 

TABLE 5. PARTICLE EMISSION RATES FOR MOBILE SOURCES 

Sample source Emission rate 

g/hp/h g/mi 

Diesel CAT 

NISSAN 

OLDS 

VW RAB 

Gasoline MUSTANG 

0.72 

0.33 

0.52 

0.18 

0.0053 

To simulate actual driving patterns, the vehicles (except 

for the Caterpillar) were operated on a chassis dynamomet 

using the highway fuel economy test cycle (HWFET): avera 

speed of 48 miles per hour in 12.75 minutes over 10.24 

miles. The Caterpillar engine was mounted on an engine 

dynamometer and operated at Mode II using a steady-state 

operation of 2200 rpm and an 85-pound load. All of the 

mobile source particulate emission samples, except those 

793 
from the VW Rabbit, were generated in the chassis dynamometer and engine dynamometer facilities of EPA's Environmental Sciences Research Laboratory in Research Triangle 

Park, North Carolina. The VW Rabbit samples were provided 

through an interagency agreement with the Department of 

Transportation. 

Particulate samples were collected using dilution tunnel 

sampling technique (13). The emissions were diluted to 

obtain realistic samples of particle-bound organics. Because of the high temperatures (>200° C) in the tailpipe 

of an operating diesel engine, organic materials are generally in the gas phase; thus, soot filtered at these temperatures would contain very little extractable organic material 

In the ambient air, however, particle and gaseous exhaust 

is quickly cooled and diluted; as the overall temperature 

is reduced, carbon particles begin to adsorb organic 

material. The dilution tunnel technique simulates this 

process. 

The heavy-duty Caterpillar engine exhaust was split before 

dilution in the tunnel to ensure that exhaust volumes did 

not exceed the capacity of the dilution tunnel system. The 

total exhaust from the passenger cars was diluted approximately 10-fold with filtered air prior to the sampling. 

After dilution, the particulate matter was collected by 

filtration in large-scale fiber samplers, using Teflon-coated glass Pallflex T 68-20 filters at a flow rate of 100 cfm. 

CIGARETTE SMOKE CONDENSATE 

The CSC was generated and collected by the Tobacco Smoke 

Chemistry Group of the Oak Ridge National Laboratory (ORNL) 

Analytical Chemistry Division for the Chemical Repository 

Program. It should be noted that humans are actually exposed to smoke particulate matter, not condensate, and that 

there may be significant chemical differences between the 

two. The cigarettes chosen for making the CSC were the 

Kentucky Reference 2R1, a remake of the widely referenced 

1R1. The cigarettes are 85-mm (king size), non-filter, 

typical of U. S. cigarettes consumed from 1962 - 1966. 

The CSC generator loads, lights, smokes, and ejects 2,000 

cigarettes per hour. A maximum of 10 puffs per cigarette 

is taken at a rate of 1 puff (2 sec, 35 cc) per minute. As 

the cigarettes, which are in a large rotating turret, pass 

smoking ports, a vacuum draws a puff from each of 40 ciga-

. rettes in different stages of consumption. The smoke is 

mixed and drawn through traps, which are partially filled 

with acetone and immersed in a refrigerated, dry ice-

•Isopropanol bath. 

,or this study, approximately 50,000 cigarettes were conutned to produce approximately 1 kg of CSC. The CSC was 

Amoved by rinsing the trapping system with a minimal amount 

If acetone. 

COKE OVEN 

The coke oven emissions were collected by sampling on top of 

coke oven battery at Republic Steel in Gadsden, Alabama, 

about 60 miles northeast of Birmingham. Two Massive Air 

Volume Samplers (MAVS) were positioned side-by-side on top 

0f the north end of the coke oven battery. Due to local 

wind conditions, various types of aerosols were sampled. 

The prevailing wind direction was north and north-northwest 

-from the direction of Gasden; thus, as unknown, but significant, portion of the emissions sampled may have been from 

the urban environment. 

Initially, the electrostatic precipitator (ESP) plates coated with coke oven ambient emissions were rinsed in methylene 

chloride. Later, however, the impactor and ESP plates were 

dry-scraped. A relatively small amount of material (about 

200 g) was recovered in more than 2100 hours of sampling, 

indicating that the prevailing wind direction decreased the 

concentration of suspended dust at the site. 

Since the amount of ambient coke oven sample obtained was 

not adequate to complete all of the in vivo bioassays, an 

additional large sample of coke oven mains was obtained. 

The collection and bioassay of this sample will be reported 

later. 

ROOFING TAR 

The roofing tar emissions were generated and collected by 

the Process Measurement Branch of EPA's Industrial Environment Research Laboratory in Research Triangle Park. The 

North Carolina Department of Transportation provided the 

tar pot and the pitch-based tar for these studies. The tar 

pot was a conventional unit with an external burner. A 

6-foot stack extension was added to the test unit to prevent 

the burner from entering the sampling hood, which was mounted over the tar pot. A Teflon-coated aluminum pipe led 

from the hood to a small baghouse. Special nonreactive 

Teflon filter bags were fitted in the baghouse to collec t 

the particulate emissions. 

The sampling was conducted for 8 hours, during which the tar 

temperature was held between 360° - 380° F to maintain the 

tar in liquid form. Particulate matter was collected from 

the Teflon filter bags, and by scraping the collector duct 

and pot bottom. 
IM 

•Ml; 

SAMPLE PREPARATION 

The mobile source and comparative emission samples, except 

the CSC, were soxhlet-extracted with dichloromethane (DCM) 

to remove the organics. DCM was found to be the most effec, 

tive of a series of solvents in removing mutagenically 

active organics from diesel particles in preliminary studies 

using the Salmonella typhimurium mutagenesis bioassay. 

Soxhlet-extraction of the mobile sources particle-laden fii_ 

ters was accomplished by placing 12 - 16 filters, without 

thimbles, in a 2.3 liter side-chamber extractor for 48 hours 

The Teflon filter bags containing roofing tar emissions were 

cut into sections and soxhlet-extracted for 72 hours (until 

clear). The coke oven emissions scraped from the ESP plates 

were soxhlet-extracted in thimbles in a standard size extractor for 24 hours. 

The soxhlet-extracted organics were filtered using Teflon 

millipore filters (0.2 urn) to remove particles. Large 

volumes of solvent (mobile source samples) were concentrated 

to a smaller volume by rotary evaporation under reduced pressure. Aliquots were evaporated to dryness under nitrogen 

and stored frozen in the dark. The percent extractable and 

benzo(a)pyrene analysis results are given in Table 6. 

TABLE 6. RESULTS OF EXTRACTION AND BENZ0(A)PYRENE ANALYSIS 

Sample source 

Diesel 

Gasoline 

Comparative 

sources 

CAT 

NISSAN 

OLDS 

VW RAB 

MUSTANG 

CSC 

Coke 

Root tar 

Extractab 

matter 

percent 

26-27 

4-8 

12-17 

18 

39-43 

_ 

5-10 

>99 

le Benzo 

ng BaP 

mg extract 

2 

1 173 

2 

26 

103 

<1 

478 

889 

(a)pyrene 

ng BaP 

mg particulate 

0.5 

96.2 

0.4 

4.6 

44.1 

31.5 

889 

The CSC, after being drawn through the refrigerated condenser, was removed from the condenser by rinsing with a minimal 

amount of acetone. The acetone suspension was then placed 

on a rotary evaporator and, at reduced pressure and 30° C, 

the semi-volatiles were removed; evaporation was continued 

until a constant weight was achieved. The tar content of 

the CSC-acetone suspension was adjusted to require levels by 

the addition of appropriate amounts of acetone. The acetone was removed from the necessary samples by evaporation 

in a vacuum desiccator over Drierite. This treatment remo 

the acetone solvent, but most of the CSC water remained wi 

the sample. 

The samples prepared as described above, were furnished to 

the Health Effects Research Laboratory in Research Triangl 

park, North Carolina, (HERL-RTP) by the suppliers noted 

earlier. At HERL-RTP, the samples were coded, using a dis 

tinguishing number for each source and then assigning rand 

numbers to each sample, creating a blind experiment. The 

coded samples were then aliquoted according to the bioassa 

requirements. 

All shipments were via air express, on dry ice, according 

to the transportation procedures for potentially hazardous 

biologicals/chemicals specified by the Department of Trans 

portation. To ensure stability, the samples were stored i 

the dark at or below -30° C and bioassayed soon after receipt. 

Prior to conducting the bioassay(s), the investigator(s) 

added dimethyl sulfoxide (DMS0) to the samples to achieve 

the desired concentration (mg sample/ml). The same DMS0 

solvent was used as a control. For some specified assays 

(i.e., skin carcinogenesis and oncogenic transformation), 

acetone was used as the solvent and the control. 

BIOASSAYS 

The mutagenesis bioassays were selected to detect gene 

mutations, chromosomal effects, and DNA damage. The resu 

of the mutagenesis test matrix reported in this volume ar 

for the following bioassays: 

reverse mutation in Salmonella typhimurium 

forward mutation in L5178Y mouse lymphoma cells 

forward mutation in Balb/c 3T3 mouse embryo fib 

blasts 

forward mutation in Chinese hamster ovary cell? 

mitotic recombination in Saccharomyces cerevisi 

DNA breakage in Syrian hamster embryo cells 

sister chromatid exchange in Chinese hamster o\ 

eel Is. 
The carcinogenesis bioassays were selected to provide data 

as early as possible from Jji vitro and short-term iji vivo 

assays (e.g., skin tumor initiation). The longer term in 

vivo assays (e.g., complete skin carcinogenesis and intratracheal instillation) will allow scoring and pathological 

examination for carcinomas. The results of the carcinogenic 

testing, completed to date and reported in this volume, including the following bioassays: 

• oncogenic transformation in Balb/c 3T3 cells 

T 

viral enhancement of 

hamster embryo cells 

transformation in Syrian 

• skin tumor initiation in Senear mice. 

The bioassays were performed, if possible, in such a way 

that dose-response data would demonstrate a positive increase (slope) above spontaneous levels for at least three 

doses. This usually requires a preliminary toxicity test 

over a wide dose range followed by a test conducted at 

5-to-7 doses spaced over half-logs. Where such dose-response 

data were obtained, the initial linear slope of the response 

curve was determined and used to provide comparative potency 

rankings of the samples. In several assays (e.g., the Balb/ 

c 3T3 cells), dose-response data were not obtained and the 

lowest effective (positive) dose tested was determined. Difficulties in obtaining good dose-response data may be due, 

in part, to difficulties in delivering increasing doses of 

the sample to the cells due to solubility problems. 

The short-term bioassays generally included positive and negative (solvent) controls. The assays were repeated and, in 

some cases, run in more than one laboratory. Samples were 

decoded after submission of the final results. The papers 

which follow describe the results determined by each bioassay 

laboratory. The final paper of this session summarizes all 

of the results to date (14). 

REFERENCES 

1. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. 

Zweidinger, S. Tejada, J. Bumgarner, F. Duffield, 

M. Waters, V. F. Simmon, C. Hare, C. Rodriguez, and 

L. Snow. Application of Bioassays to the Characterization of Diesel Particle Emissions. In: Application of Short-Term Bioassays in the Fractionation and 

^ . Analysis of Complex Environmental Mixtures, M. Waters, 

S. Nesnow, J. Huisingh, S. Sandhu and L. Claxton, eds. 

Plenum Press, New York, 1978. pp. 383-418. 

9. 

10. 

11. 

12. 

13. 

Kipling, M. D. Soots, Tars and Oils As Causes of 

Occupational Cancer. In: Chemical Carcinogens, 

C. Searle, ed. ACS Monograph 173, American Chemical 

Society, Washington, D.C, 1976. pp. 315-323. 

U.S. Environmental Protection Agency. Preliminary 

Report on Population Risk to Ambient Coke Oven Exposure. 

Carcinogen Assessment Group, Washington, D.C, 1973. 

U.S. Environmental Protection Agency. The Diesel Emissions Research Program. EPA-625/9-74-004, Center for 

Environmental Research Information, Cincinnati, Ohio, 

1979. 

Lloyd, J. W., F. E. Lund in Jr., C K. Redmond. Long 

Term Mortality Study of Steelworkers. IV. Mortality 

by Work Area. J. Occup. Med., 12:151-157, 1970. 

Lloyd, J. W. Long Term Mortality Study of Steelworkers. 

V. Respiratory Cancer in Coke Plant Workers. J. Occup. 

Med., 13:53-68, 1971. 

Redmond, C. K., A. Ciocco, J. W. Lloyd, and H. W. Rush. 

Long Term Mortality Study of Steelworkers. VI. Mortality from Malignant Neoplasms among Coke Oven Workers. 

J. Occup. Med., 14:621-629, 1972. 

Mazundar, S., C. K. Redmond, W. Sollecito, N. Sussman. 

An Epidemiological Study of Exposure to Coal Tar Pitch 

Volatiles Among Coke Oven Workers. J. Air Pol. Control 

Assoc, 25:382-389, 1975. 

Hammond, E. C , I. J. Selikoff, P. L. Lawther, and H. 

Seidman. Inhalation of Benzo-a-pyrene and Cancer in 

Man. Ann, N.Y, Acad. Sci,, 271:161-124, 1976. 

Wynder, E. L. and D. Hoffman. Tobacco and Tobacco Smoke. 

Academic Press, New York, 1967. 

U.S. Environmental Protection Agency. Preliminary Report on POM Exposures. Carcinogen Assessment Group, 

Washington, D.C., 1978. 

Gori, G. B., ed. Toward Less Hazardous Cigarettes, 

Volumes 1-3. (NIH) 76-905, (NIH) 76-111), (NIH) 

75-1280, National Cancer Institue, Washington, D.C, 

1976. 

Bradow, R. 

Particles. 

L. Chemical Composition of Diesel Exhaust 

This volume, 1980. 

799 
Nesnow, S. and J. Huisingh. Mutagenic and Carcinoqeni 

Potency of Extracts of Diesel and Related Environmental 

Emissions: Summary and Discussion of Results Th U 

volume, 1980. ' 

:is! 

MUTAGENIC AND CARCINOGENIC POTENCY OF DIESEL AND Rl 

ENVIRONMENTAL EMISSIONS: SALMONELLA BIOASSAY 

Larry D. Claxton 

Genetic Toxicology Division 

Health Effects Research Laboratory 

U.S. Environmental Protection Agency 

Research Triangle Park, North Carolina 2771 

ABSTRACT 

Due to the expected increase in the percentage of die 

vehicles in the United States, the Environmental Prot 

Agency must evaluate the health effects associated wi 

exposure to diesel emissions. Respirable particles f 

variety of combustion sources have the potential of b 

carcinogenic and mutagenic. The objective of these s 

was to determine the relative biological activity of 

organic material adsorbed on these particles in J_Q

 vi 

mutagenesis bioassays. The organic extracts from tfii 

lowing series of emission sources were quantitative t 

assayed in the Salmonella assay for mutagenic activii 

a light-duty Oldsmobile diesel 350 engine; (2) a hea\. 

Caterpillar diesel engine; (3) a light-duty Nissan et 

(4) a Volkswagen Rabbit diesel engine; (5) cigarette 

(6) roofing tar; (7) coke oven; and (8) a gasoline c; 

Mustang. This paper provides a comparison of these < 

within the Salmonella bioassay and also demonstrates 

bacterial systems can be used as a quality assurance 

in 2_Q vivo testing.