P.1 

OAR Box 1827 

Prepped by Charmelle Mathews 

Document Number: 

135) ll-A-5 

Docket Number: 

A-90-07 

Printed 11/19/2008 12:40:48 PM Sheet 135 of 147 
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k-^o-o-j JT-A-5" 

O024S 

EPA 

OCTOBER 1983 

E fiBJLSJL 

ii; AUG-91990 

EPA AIR DOCKET 

USE OF SHORT-TERM GENOTOXIC BIOASSAYS 

IN THE EVALUATION OF UNREGULATED 

AUTOMOBILE EMISSIONS 

by 

David J. Brusick, R.R. Young and D.R. Jagannath 

Litton Bionetics, Inc. 

5516 Nicholson Lane 

Kensington, Maryland 20895 

EPA Contract No. 68-02-3682 

Technical Directive No. 008 

Task Officer 

Joel 1 en Lewtas 

Genetic Toxicology Division 

Health Effects Research Laboratory 

Environmental Protection Agency 

Research Triangle Park, NC 27711 

Project Officer 

Raymond Merrill 

Process Measurements Branch 

Industrial Environmental Research Laboratory 

Environmental Protection Agency 

Research Triangle Park, NC 27711 

Prepared for: 

U.S. Environmental Protection Agency 

Office of Research and Development 

Washington, D.C. 20460 
DISCLAIMER 

This report has been reviewed by the Health Effects Research Laboratory, 

U.S. Environmental Protection Agency, and approved for publication. 

Approval does not signify that the contents necessarily reflect the views 

and policies of the U.S. Environmental Protection Agency, nor does mention 

of trade names or commercial products constitute endorsement or recommendation for use. 

ii 
P.4 

FOREWORD 

The many benefits of our modern, developing, industrial society are 

accompanied by certain hazards. Careful assessment of the relative risk 

of existing and new man-made environmental hazards is necessary for the 

establishment of sound regulatory policy. These regulations serve to 

enhance the quality of our environment in order to promote the public 

health and welfare and the productive capacity of our Nation's population. 

The complexities of environmental problems originate in the deep 

interdependent relationships between the various physical and biological 

segments of man's natural and social world. Solutions to these environmental problems require an integrated program of research and development 

using input from a number of disciplines. The Health Effects Research 

Laboratory, Research Triangle Park, NC and Cincinnati, OH conducts a 

coordinated environmental health research program in toxicology, epidemiology and clinical studies using human volunteer subjects. Wide ranges 

of pollutants known or suspected to cause health problems are studied. 

The research focuses on air pollutants, water pollutants, toxic substances, 

hazardous wastes, pesticides, and non-ionizing radiation. The laboratory 

participates in the development and revision of air and water quality 

criteria and health assessment documents on pollutants for which regulatory actions are being considered. Direct support to the regulatory 

function of the Agency is provided in the form of expert testimony and 

preparation of affidavits as well as expert advice to the Administrator 

to assure the adequacy of environmental regulatory decisions involving 

the protection of the health and welfare of all U.S. inhabitants. 

F. Gordon Hueter, Ph.D. 

Director 

Health Effects Research Laboratory 

iii 
PREFACE 

The Environmental Protection Agency, under the Clean Air Act, is 

charged with the responsibility for regulating emissions from new motor 

vehicles and for the regulation of fuels and fuel-additives for motor 

vehicles and their emissions. The Clean Air Act specifically requires 

EPA to determine the potential public health effects of these emissions 

and specifies that these effects include carcinogenic and mutagenic 

effects. 

Short-term genetic bioassays have now been widely employed by 

research scientists in government, industry, and independent research 

institutions to evaluate the mutagenic and potentially carcinogenic 

effects of motor vehicle emissions. The purpose of this document is 

primarily two-fold: (1) to summarize the current state-of-the-art 

related to applying short-term genetic bioassays to the evaluation of 

motor vehicle emissions and the assessment of potential human health 

effects, and (2) to recommend guidelines for evaluating unregulated 

combustion emissions from motor vehicle emissions using short-term 

genetic bioassays. 

In preparing the final recommendations and guidelines for using 

short-term genetic bioassays, a scientific review and advisory workshop 

was convened. Scientists with expertise in this field from industry, 

government, and private research institutes were asked to scientifically 

review this document and specifically provide the recommendations which 

are reflected in the final section of this document. 

David J. Brusick 

Joel 1 en Lewtas 

iv 
P.6 

ABSTRACT 

Exposure to fuels and fuel additives may occur through direct contact 

with uncombusted fuel or fuel additives as well as products of fuel/fuel 

additive combustion. Presence of combustion products in ambient air is 

the major source of human exposure to motor vehicle fuels. 

The levels of several products of fuel combustion in ambient air 

(nitrogen oxides, hydrocarbons and carbon monoxide) 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 automotive fuels and fuel additives and 

their emissions. I 

The objectives of this document are to review the data from selected 

short-term jjn vitro and i_n vivo bioassays to (a) determine if there is 

evidence suggesting potential human health risk from either uncombusted 

emissions or from emissions of combusted motor vehicle fuels or fuel 

additives, (b) identify the operational variables involved in generating 

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 the human risk estimates, and (e) develop a short-term 

bioassay program to monitor the health effects of fuel/fuel additives 

and unregulated combustion emissions. 

Meeting these objectives will involve analysis of each of the following areas: 

1. Present state-of-the-art of short-term genetic test procedures and 

their applicability to the evaluation of fuels/fuel additives and 

combustion products. 

2. Feasibility of developing risk estimates for human health effects 

from short-term bioassay data. 

3. Review of short-term test data as they relate to motor vehicle 

fuels/fuel additives and unregulated combustion emissions. 

4. Preparation of recommendations for the assessment of biological 

activity 1n fuels/fuel additives and combustion emissions using 

short-term bioassays. 

5. Development of short-term screening strategy for application to 

ongoing assessment of fuels/fuel additives or combustion emissions 

from new sources. j 

This report was submitted in fulfillment of EPA Contract No. 68-02-3682, 

Technical Directive No. 008, by Litton Blonetics, Inc., under the sponsorship of the U.S. Environmental Protection Agency. 
P.7 

CONTENTS 

Page 

DISCLAIMER -

FOREWORD 

PREFACE 

ABSTRACT 

LIST OF FIGURES 

LIST OF TABLES 

ACKNOWLEDGMENTS 

LEGISLATIVE BACKGROUND 

INTRODUCTION 

PART I 

1. A SUMMARY OF THE CURRENT STATE-OF-THE-ART OF GENOTOXICITY TESTING APPLICABLE TO STUDIES OF MOTOR 

VEHICLE EMISSIONS 

General Application — -

Specific Limitations to Short-Term Test Application — 

Special Applications -

Summary 

References 

2. THE CURRENT STATE-OF-THE-ART IN APPLICATION OF SHORTTERM BIOASSAYS TO AN ASSESSMENT OF HUMAN CANCER 

AND MUTATION RISK 

Detection of Hazard 

Quantification of Hazard - — —— — 

Application of Conventional Risk Assessment Methods 

to Automobile Emissions 

Application of Short-Term Bioassays to Risk Assessment 

of Automobile Emissions : 

References 

n 

iii 

iv 

v 

ix 

x 

xiv 

1 

3 

12 

12 

15 

16 

17 

19 

21 

21 

26 

26 

27 

34 

vi 
CONTENTS (Continued) 

PART II 

Page 

P.8 

SAMPLING TECHNIQUES FOR COMBUSTION EMISSIONS 

Direct Sampling of Raw Exhausts — 

Proportional Sampling 

Dilution Tunnel Sampling — 

Gas Phase Trapping 

References 

EXTRACTION METHODS FROM EMISSION PARTICULATES 

Extraction 

Concentration 

Fractionation 

Biological Extraction 

References 

REVIEW OF BIOASSAYS APPLIED TO UNREGULATED COMBUSTION 

EMISSION SAMPLES 

Introduction 

Overview of Bioassay Response 

Gene Mutation Bioassays 

Cytogenetic Bioassays 

Primary DNA Effects 

Carcinogenesis Studies 

Analysis by Type of Sample Tested 

Evaluation of Positive Responses 

Criteria for Bioassay Suitability for Evaluating 

Motor Vehicle Emission Samples 

Selection of Bioassays to Form a Minimum Evaluation 

Matrix 

References 

EFFECTS OF ENVIRONMENTAL CONDITIONS ON BIOLOGICAL 

ACTIVITY OF EMISSION SAMPLES 

Effect of Sampling Conditions 

Ambient Conditions — -

Effect of Temperature 

Artifact Formation Due to Collection Methods 

Solvent Systems and Storage Method 

References 

36 

!36 

37 

38 

'40 

43 

45 

45 

46 

46 

46 

48 

50 

50 

51 

59 

59 

59 

59 

59 

60 

60 

67 

69 

73 

73 

73 

75 

75 

75 

78 

vn 
CONTENTS (Continued) 

P.9 

EFFECTS OF ENGINE TYPE, FUEL, AND FUEL ADDITIVES ON 

BIOLOGICAL ACTIVITY OF EMISSION SAMPLES 

Introduction 

Effect of Engine Type on Exhaust Mutagenicity 

Effect of Fuels on Exhaust Mutagenicity 

Mutagenicity of Crude Oils and Fuels 

Mutagenicity of Lubricating Oils 

References 

Page 

80 

80 

80 

85 

88 

88 

89 

PART III 

1. SELECTION OF BIOASSAYS FOR ROUTINE SCREENING OF 

AUTOMOBILE EMISSIONS 

Introduction 

Procedures — —--— * 

Data Interpretation 

Potential Problems 

2. TEST PROCEDURES 

Protocol Recommendations 

Recommendations for Collection and Processing 

of Samples 

Recommendations for Data Analysis 

Approach to Data Analysis Used in Comparing Genetic 

Activity of Emission Samples 

Data Interpretation 

Conclusions 

References 

92 

92 

92 

93 

93 

95 

95 

96 

96 

98 

107 

107 

110 

viii 
P.10 

FIGURES 

Number Title ; Page 

1 Automobile Emissions and Human Health 6 

2 Hypothetical Steps Involved in Oncogenic 

Transformation — 14 

3 Considerations Involved in Quantitative 

Risk Assessment 24 

4 Dilution Tunnel System for Collection of 

Heavy Duty Diesel Particulate - 39 

5 Dilution Tunnel System for Collection 

of Gas and Particulate Phase Emissions — 39 

6 Cryogradient Sampling Equipment 42 

7 Bioassay Data Evaluation Matrix 52 

8 Characterization of Diesel Particle 

Emissions 77 

9 Flow Chart for Sample Collection and 

Processing 97 

10 Analysis of Variance for Ames Test Assays -- 104 

11 Analysis of Variance for Mouse Lymphoma 

Assays 105 

12 Analysis of Variance for Sister Chromatid 

Exchanges = 106 

13 Composition and Logistics of an Emmision 

Assessment 108 

ix 
P.11 

TABLES 

Number Title Page 

1 Estimated Emission Products from 

Vehicles in 1981 4 

2 Particles in Ambient A1r 5 

3 Use of Short-Term Tests to Investigate 

the Specific Areas of Interests 

Defined in Figure 1 7 

4 A Summary of the Possible Roles for 

Selected Genetic Tests in Chemical 

Hazard Assessment — 13 

5 Characteristics of Short-Term Bioassays 

Which Facilitate Their Application to 

Health Effects Evaluation of Automobile Emissions 18 

6 Comparisons of the Approaches used in 

Predictive Screening for Carcinogens 

and Mutagens 22 

7 Major DNA Lesions Produced by Chemical 

Interaction and Their Genotoxic Consequences 23 

8 A Comparison of Risk Assessment for 

Somatic and Germ Cells Iri Vitro 25 

9 Data Sources Used to Prepare Lung Cancer 

Risk Assessments for Automobile Exhaust 

Emissions by NAS and EPA 28 

10 Human Lung Cancer Risk Estimates for 

Diesel and Gasoline-Catalyst Particulate Emissions Based on Short-Term 

Test Data 30 

11 Chemicals Evaluated as Carcinogenic for 

Humans and Their Responses in Rodent 

and Short-Term Predictive Assays 32 
P.12 

TABLES (Continued) 

Number Title Page 

12 Predictive Value of Short-Term Genotoxic Tests for Mammalian Germ 

Cell Mutagens 33 

13 Polynuclear Aromatic Hydrocarbon (PAH) 

Emissions 37 

14 Emissions Obtained from FTP, Cold Start 

and Hot Start 38 

15 Emissions Obtained from FTP 41 

16 Emissions From FTP, Cold Start 41 

17 Stratification of Bioassay Endpoints 

Reviewed — 51 

18 Matrix of Positive Responses for Gene 

Mutation 53 

19 Matrix of Negative Responses for Gene 

Mutation 53 

20 Matrix of Positive Responses for Cytogenetics Assays 54 

21 Matrix of Negative Responses for Cytogenetics Assays 54 

22 Matrix of Positive Responses for Primary 

DNA Damage 55 

23 Matrix of Negative Responses for Primary 

DNA Damage 55 

24 Matrix of Positive Responses for In 

Vitro Transformation Assays 56 

25 Matrix of Negative Responses for Ln 

Vitro Transformation Assays — - 56 

26 Matrix of Positive Responses for Jji 

Vivo Tumor Studies 57 

27 Matrix of Negative Responses for In 

Vivo Tumor Studies 57 

xi 
P.13 

TABLES (Continued) 

Number Title Page 

28 Long-Term Studies of the Health 

Effects of Diesel Exhaust, Completed 

or in Progress 58 

29 Distribution of Biological Endpoints 

Examined Across Automotive Engine 

Types — - 61 

30 Motor Vehicle Emissions: A Summary 

of Positive Gene Mutation Bioassay 

Results 62 

31 Motor Vehicle Emissions: A Summary 

of Positive Primary DNA Damage 

Bioassay Results — 64 

32 Motor Vehicle Emissions: A Summary of 

Positive Cytogenetic Bioassay 

Results 65 

33 Motor Vehicle Emissions: A Summary of 

Positive Cell Transformation Bioassay 

Results 66 

34 Distribution of Responses in Susceptible 

Bioassays Across Sample Categories -- 68 

35 The Effect of Dilution Ratio on Particulate 

Mass Concentration and Composition -- 74 

36 Effect of Filter Sample Storage on 

Diesel Exhaust Particulate 76 

37 Mutagenicity of Spark-Ignition and 

Diesel Engine Exhaust Particle 

Extracts 81 

38 Variability in Mutagenicity of 

Repeated Collections of Diesel 

Particulate Exhaust 82 

39 Mutagenicity of Extracts of Exhaust 

Particles Collected from Five 

Different Oldsmobiie Diesel 

Automobiles 82 

40 Mutagenicity of Extracts of Exhaust 

Particles from Five Different Ford 

LTD Gasoline Automobiles (Claxton 

and Kohan, 1981) 83 

x11 
TABLES (Continued) 

Number Title Page 

41 Mutagenicity of Extracts of Exhaust 

Collected from Six Different Diesel 

Vehicles — 84 

42 Mutagenicity of Extracts of Particles 

Collected from the Exhaust of an 

Oldsmobiie Diesel Car Operated on 

Different fuels 86 

43 Mutagenicity of Extracts of Particles 

Collected from the Exhaust of a 

Peugot Diesel Car Operated on 

Different Fuels 86 

44 Influence on a Jet "A" and isoparaffinic Fuels on Mutagenicity of 

Exhaust Particle Extracts of an 

Oldsmobiie Diesel Car 87 

45 Difficulties Encountered in the 

Interpretation of Mobile Source 

Genotoxicity Studies 94 

46 Tier Study - Description of Sources 

for Fourteen Emission Samples 

Compared for Mutagenic Activity 100 

47 Tier Study - Ames Test - TA98 Analysis 

of Revertants per Kilometer 101 

48 Tier Study - Mouse Lymphoma - Analysis 

of Mutation Frequency per Kilometer - 102 

49 Tier Study - Sister Chromatid Exchange 

Analysis of Exchanges per Kilometer - 103 

xiii 
P.15 

ACKNOWLEDGMENTS 

This report was prepared under the direction of Joel 1 en Lewtas, 

Genetic Toxicology Division, Health Effects Research Laboratory, 

U.S. Environmental Protection Agency, Research Triangle Park, North 

Carolina, who was the Task Officer. 

This report was prepared by Litton Bionetics, Inc., under EPA 

Contract No. 68-02-3682, Technical Directive No. 008. Review and suggestions by the following individuals are gratefully acknowledged: 

Charles R. Clark, Union Oil Company of California 

Larry D. Claxton, U.S. Environmental Protection Agency 

Victor Hasselblad, U.S. Environmental Protection Agency 

Joseph 0. Irr, E.I. du Pont de Nemours & Company, Inc. 

Alan J. Katz, Illinois State University 

Albert Li, Monsanto Company 

Phil Lorang, U.S. Environmental Protection Agency 

Roger 0. McClellan, Inhalation Toxicology Research Institute 

Hugh McKinnon, U.S. Environmental Protection Agency 

Kay Williams, U.S. Environmental Protection Agency 

Kristien Mortelmans, SRI International 

Michael A. Pereira, U.S. Environmental Protection Agency 

Richard Rykowski, U.S. Environmental Protection Agency 

Joseph Somers, U.S. Environmental Protection Agency 

William G. Thilly, Massachusetts Instiute of Technology 

xiv 
P.16 

LEGISLATIVE BACKGROUND FOR REGISTRATION OF 

FUELS AND FUEL ADDITIVES 

Section 211 of the Clean Air Act as amended in 1977, charges the Administrator of the Environmental Protection Agency (EPA) with responsibility 

for regulation of fuel and fuel additives. Registration is required by 

the Agency of fuels and fuel additives prior to sale or introduction into 

commerce and requires the manufacturer of a fuel or fuel additive to: 

Conduct tests to determine potential public health effects of 

such fuel or additives (including, but not limited to, carcinogenic, 

teratogenic, or mutagenic effects); and 

Furnish the description of any analytical techniques that can 

be used to detect and measure any additive in such fuel,the 

recommended range of concentration of such additive, and the 

recommended purpose in use of such additives, and such other 

information as is reasonable and necessary to determine the 

emissions resulting from the use of the fuel or additives 

contained in such fuel, the effect of such fuel or additives 

on the emission control performance of any vehicle or vehicle 

engine, or the extent to which emissions affect the public 

health or welfare, with respect to each fuel or fuel additive 

which is registered on the date of promulgation of such regulations and with respect to each fuel or fuel additive for which 

an application for registration is filed thereafter. , 

In addition, manufacturers would be required to inform the Agency of 

mechanisms of action of additives, reactions between fuels and their 

additives, effects of the additives on emissions, identification and 

measurement of emission products, and effects of the emission products 

on health, welfare, and emission control devices. 

i 

Additive manufacturers wouTd be required to furnish additional information 

about the chemical composition and structure of additives and methods of 

analysis, any impurities, and sales volumes. Fuel manufacturers would 

be required to furnish additional information on such properties as hydrocarbon composition, polynuclear organic materials, sulfur, trace elements, 

vapor pressure, distillation temperatures, and octane numbers, including 

methods of analysis for each of these, information on variation by geographic region, and production volume. 
P.17 

The registration of fuels and fuel additives is provided for in 46 CFR 

Part 79, A Revision of 40 CFR Part 79, (Federal Register, Volume 39, 

No. 46 (March 7, 1974). As revised, Part 79 designates certain fuels 

and additives as requiring registration: namely, motor vehicle gasoline 

and motor vehicle diesel fuels, and/or in motor vehicle crank case oil. 

These substances are further identified and designated in paragraphs 

79.31, 79.32, 79.33 of Part 79 (46 CFR 79). 
P.18 

INTRODUCTION 

The assessment of damage to the DNA molecule (genotoxic activity) by 

uncombusted fuels used in motor vehicles such as diesel fuel, unleaded 

gasoline, kerosene, ethanol, and methanol have not revealed evidence of 

significant genotoxicity (Lebowitz, 1978; ICPEMC, 1983). Emission products 

from the complete or partial combustion of fuels such as diesel fuel or 

gasoline, on the other hand, are associated with genotoxic activity, and 

a major portion of the genotoxicity is found in the solid phase (particulate) 

fraction as mutagenic and cell transforming organic compounds condensed 

onto the carbonaceous core of the exhaust particle (Vuk et al., 1976). 

Atmospheric parameters, such as humidity, temperature an<TuV~irradiation 

existing at the time of collection, greatly influence the level and 

biological activity of organics bound to the particle core. Exhaust 

particles are one of many types of particles found as pollutants in ambient 

air. The current particle load for medium to large cities 1s approximately 

100 ug/m- (Cuddihy et a_L , 1981); it is believed that these particles 

represent a major category of unregulated air pollution affecting human 

health. 

Air pollutants include both gaseous and solid phases. Gaseous phase 

pollutants (i.e., carbon monoxide, ozone, nitrogen oxides and sulfur oxides) 

are recognized human health hazards and fall under regulatory standards 

of the Clean Air Act. The solid phase contains numerous organic compounds, 

polycyclic aromatic hydrocarbons (PAHs), and is suspected of contributing 

to the chronic toxicity (particularly lung effects) associated with longterm exposure to air pollution (i.e., the "urban factor") (Higginson et 

a_L, 1977; Higgins, 1977; Pike et a_L , 1975). Although the organic 

compounds associated with solid phase are not regulated, recent epidemiological evidence indicates that between 11 and 21 percent of human lung 

cancer may be caused by air pollution (Karch and Schneiderman, 1981). 

Motor vehicle emissions are a major source of air pollution of both the 

gaseous and solid types (Table 1). The specific contribution of motor 

vehicle emissions to ambient pollution or to its associated health effects 

cannot be quantified, but based on the present understanding of the physical, chemical, and biological effects of the solid-phase emissions that 

are found in ambient air (Table 2), motor vehicle emissions might well 

contribute a disproportionate amount of the potential mutagenic and 

carcinogenic pollutants. 

The possibility of human health risks 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 (National Research Council, 1981; USEPA, 1981). Even 
P.19 

with this data base, the extent to which motor vehicle emissions contribute to human health problems is far from understood. Investigations 

have shown that complex interactions between fuel (and its additives) 

and gases 1n the combustion chamber, environmental exposure conditions 

and biology of the target species prevent most conventional hazard assessment methods to be applied for a reliable assessment. The multiplicity 

of complex interactions responsible for the difficulties in hazard assessment are shown in Figure 1. Each box in the figure represents a separate 

investigational effort that eventually must be integrated into the final 

human risk analysis. Some of the parameters identified in Figure 1 can 

be studied with current state-of-the-art techniques whereas others will 

require new test methods as well as novel approaches to data analysis. 

The suitability of short-term tests to evaluate the many facets of an 

emission's toxicity are listed in Figure 1 and summarized in Table 3. 

Some of the important issues which have been addressed during the past 

several years of research are: 

1. The chemical and physical characteristics of motor vehicle emissions 

and information on how atmospheric conditions alter those characteristics. 

2. The influences of controllable parameters of fuel source, fuel additives and engine-running conditions on the physico-chemical and 

biological activities of emission products. 

TABLE 1. ESTIMATED EMISSION PRODUCTS FROM VEHICLES IN 1981 

(USEPA, 1981) 

Thousands of Tons/yr 

Pollutant Gasoline Diesel 

Particles 5,650 774 

Sulfur oxides 242 461 

Nitrogen oxides 5,199 4,026 

Free hydrocarbons 6,863 679 

Carbon monoxide 73,839 1,629 
TABLE 2. PARTICLES IN AMBIENT AIR 

Sources Chemical/Physical Properties Reported Biological Effects 

Coal flyash from coalfired power plants A range of particle sizes including 

those of respirable size. The 

carbonaceous core contains bound 

inorganic and organic materials. 

The level of bound organics is 

low and generally less than 1.0% 

by weight. 

Mutagenic activity has been 

demonstrated in prokaryotic 

microbial assays using organic 

solvent extracts of large 

quantities of stack-sampled 

particles. Level of biological activity is highly dependent 

upon the temperature at which 

the particles were collected. 

Ul 

Commercial and hoae oilfired heating units 

Spark ignition gasoline 

combustion engines 

Oiesel engines 

A range of particle sizes 

including a high proportion 

of respirable size. The 

carbonaceous core contains 

bound inorganic and organic 

materials. The level of bound 

organics is high often exceeding 

10% by weight especially for oilfired heating units and diesel 

engines. 

Mutagenic and transforming 

activity has been demonstrated 

for organic solvent extracts 

jn vitro using target cells. 

Organic extracts have also 

been shown to induce tumors in 

rodents under specific routes 

of administration (i.e., skin 

painting, intratracheal instillation and subcutaneous injection). The levels of biological 

activity appear to vary according to coabustion efficiency, 

atmospheric conditions during 

cooling, fuel composition, and 

in the case of aobile sources, 

the running conditions of the — 

engine. 
Dilution and 

Atmospheric 

Conditions 

F-T Engine Type 

and 

Operating Conditions 

Emission 

Control 

Tailpipe 

Emissions 

1

' Ambient Emission 

Products 

' > 

Human Exposure 

o» 

Effective Dose 

To Target Organ 

Respiratory Physiology 

Pharmacokinetics 

Bioavailability 

Probability and 

Severity of 

Toxicity 

Adapted from Health Effects Institute (HEI) 1982 

215 First Street 

Cambridge, Mass. 

Individual 

Susceptibility 

Risk 

Quantitation 

Figure 1. Automobile emissions and human health. 

S3 
TABLE 3. USE OF SHORT-TERM TESTS TO INVESTIGATE THE SPECIFIC AREAS 

OF INTERESTS DEFINED IN FIGURE 1 

Critical Parameters 

Identified in Figure 1 Application of Short-Term Test Methods 

Fuel variations* 

Engine operating conditions* 

Emission control* 

Tail pipe emissions 

Atmospheric conditions and 

dilution effects 

Ambient emissions 

A single test engine can be run on different fuels for the minimum 

time sufficient to collect samples for one or two short-term tests. 

Multiple samples of the emissions can be compared for biological 

activity in a single assay simultaneously under the same test 

conditions. Using this approach the only variables which might 

give rise to differences in biological activity would be the 

fuels or fuel additives. If changes were observed, these could 

be tied to changes in the chemical composition of the collected 

emission. 

Short-term tests could be applied as described above but the 

fuel source would be held constant and the engine or engine 

operating conditions would be varied. 

Emission samples from uncontrolled and controlled engines could be 

collected and compared directly for different activities. 

Small samples can be collected directly off the tailpipe, cooled, 

extracted and analyzed. 

Short-term tests cannot directly provide answers to the dynamics 

of these processes but can be used to compare emission samples 

collected under various real or simulated atmospheric and/or 

dilution conditions. 

Ambient air samples of sufficient size for short-term tests can 

be collected however the proportion of the sample or the measured 

biological activity contributed by vehicle emissions must be 

estimated from source tracer compounds (e.g., lead) using 

apportionment methodologies. 
TABLE 3 (Continued) 

Critical Parameters 

Identified in Figure 1 Application of Short-Term Test Methods 

Secondary emission products 

oo 

Human Exposure 

Respiratory physiology and 

pharmacokinetics 

Effective dose 

Individual susceptibility 

Probability and, severity of 

risk 

Secondary emission products, formed after atmospheric reactions 

(eg. peroxyacetylnitrate) can be studied in smog chambers. Short-term 

tests have been applied to samples taken from smog chambers and 

methods are being developed to directly bioassay gaseous mixtures of 

such products. 

Some short-term jn vitro tests utilize human derived target cells. 

Short-term tests are also being developed for application to jn vivo 

human exposures. 

Short-term tests are not currently applicable. 

Short-term tests are not currently applicable, although studies of 

phagocytosis and metabolic processing of organic laden particles can 

be studied in a limited number of the short-term tests. The exposure, 

however, is still jn vitro. Methods by which relevant target cells can 

be collected from animals exposed jn vivo and then subjected to jn vitro 

analysis should be available In the near future. Such tests may provide 

an effective probe to answer this question. 

Short-term tests are not currently applicable. 

Short-term tests can be applied to either qualitative hazard determinations or quantitative risk estimates. 

*The amount of emission sample available from an engine running for a reasonable period would not be sufficient 

to make comparisons over a dose response curve using conventional animal tests for mutation or tumor induction. 

The amount of sample from an engine with a pollution control device would be less than an uncontrolled engine 

making comparisons in animals even less likely. 
P.24 

3. The probable types of human health effects associated with exposure 

to motor vehicle emissions. 

4. The specific chemical components of motor vehicle emissions which 

are most likely responsible for the health effect of concern. 

5. Assessment of the potential for human risk will be derived from 

both j_n vitro and j_n vivo studies. 

The objective of this report is to summarize the current status of fuel/ 

fuel additive research using short-term tests for genotoxicity and to 

develop an approach for the application of these tests to the development 

of regulatory strategy. Short-term genetic tests are believed to be 

indicators of carcinogenic and mutagenic hazard for humans on the basis 

of extensive studies with pure chemicals; thus, such tests should be 

used in addressing the concern for carcinogenic effects. The use of the 

term test is not meant to imply that these methods are standardized or 

uniformly reproducible. Short-term methods are continually undergoing 

validation, refinement and reinterpretation. 

The final report will be divided into seven sections. The first section 

will summarize the current state-of-the-art of short-term genotoxicity 

testing as it applies to studies of motor vehicle emissions. The methods 

used in studies of vehicle emissions will be described, and their strengths 

and limitations in the context of the more conventional long-term toxicology techniques will be reviewed. 

The second section of the report will be a review of the application of 

short-term bioassays in assessing human risk. Human risk analyses are 

usually based upon epidemiology studies or extensive animal modeling. 

However, automobile emissions do not lend themselves to the current 

animal model techniques used for making risk assessments, nor are available human epidemiology studies adequate to develop a reliable risk 

estimate. Consequently, the initial attempts at risk analysis for motor 

vehicle exhaust emissions have relied heavily on the results of shortterm tests. 

The third section of the report will consist of a review of the research 

efforts directed toward the evaluation of motor vehicle emissions using 

short-term tests. Short-term bioassays have been used extensively to 

evaluate the solid phase pollutants (particles) and their extracts by 

determining the biological properties of whole exhaust and collected 

particles. The flexibility and adaptability of vn vitro assays were also 

evident from data involving assessment of extracts or leachates from 

collected particles and their subfractions. These studies have resulted 

in the identification of chemicals or chemical classes associated with 

the biologically active samples. 

Section four of the report will attempt to delineate critical unresolved 

issues related to possible human health risk. This section will also 

include recommendations for possible additional research that would help 

fill existing data gaps. 
P.25 

Section five will contain recommendations for guidelines that could be 

applied to the evaluation of "unregulated" vehicle emissions on a continuing basis. 

Section six will outline the types of tests that could be included in a 

testing program for certification of motor vehicles on the basis of genotoxic predictive tests. 

The final section will be a summary of the entire document with a statement of principles concerning the current level of understanding of the 

genotoxicity of automobile emissions. 

10 
P.26 

REFERENCES 

1. Cuddihy, R.G., R.O. McClellan, W.C. Griffith, F.A. Seiler and B.R. 

Scott. Potential health risks from increased use of diesel light 

duty vehicles. In: Diesel Emission Symposium, U.S. EPA, Office of 

Research and Development, Raleigh, North Carolina, October, 1981. 

pp. 436-450. 

2. Higgins, I.T.T. Epidemiology of lung cancer in the United States. 

In: Air Pollution and Cancer in Man, U. Mohr, D. Schmahl and L. 

Tomatis, eds. International Agency for Research on Cancer, Lyon, 

1977. pp. 191-203. 

3. Higginson, J. and O.M. Jensen. Epidemiological review of lung cancer 

in man. In: Air Pollution and Cancer in Man, U. Mohr, D. Schmahl 

and L. Tomatis, eds. International Agency for Research of Cancer, 

Lyon, 1977. pp. 169-189. 

4. ICPEMC report of Committee 1. Ethanol was without genotoxic effects 

in a series of 15 short-term assays. Mutation Res., 114:117-177, 1983. 

5. Karch, N.H. and M.A. Schneiderman. Explaining the Urban Factor in 

Lung Cancer Mortality. A Report to the Natural Resources Defense 

Council. Washington, D.C, 1981. 

6. Lebowitz, H., D. Brusick, D. Matheson, D.R. Jagannath, M. Reed, 

S. Goode, and G. Roy. Commonly used fuels and solvents evaluated 

in a battery of short-term bioassays. Environ. Mutagen. 1:172-173, 

1979. 

7. National Research Council. Health Effects of Exposure to Diesel 

Exhaust. National Academy Press, Washington, D.C, 1981. 

8. Pike, M.C., R.J. Gordon, B.E. Henderson, H.R. Menck and J. Soo Hoo. 

A1r pollution. In: Persons at High Risk of Cancer. An Approach 

to Cancer Etiology and Control, J.F. Fraumeni, Jr., ed. Academic 

Press, New York, 1975. pp. 225-239. 

9. U.S. EPA, Office of Research and Development. Diesel Emissions 

Symposium Proceedings. A Compendium of Manuscripts from the Symposium held in Raleigh, North Carolina, 1981. 

10. U.S. EPA, Office of Research and Development. 1981 National Emissions 

Report. National Emissions Data System of the Aeromatic and Emissions 

Reporting System, 1981. 

11. Vuk, CT. , M.A. Jones and J.H. Johnson. The measurement of and 

analysis of the physical character of diesel particulate emissions. 

SAE Tech. Pap. 760131, 1976. 

11 
P.27 

PART I 

SECTION 1 

A SUMMARY OF THE CURRENT STATE-OF-THE-ART OF GENOTOXICITY 

TESTING APPLICABLE TO STUDIES OF MOTOR VEHICLE EMISSIONS 

GENERAL APPLICATION 

Short-term genotoxicity testing fills the rather specific role in hazard 

assessment as a cost-effective predictor of nonreversible toxicity, namely 

cancer and genetic disease (Ames, 1979; Hollstein et aj., 1979; Brusick, 

1978). Utility of these tests is dependent upon their acceptance as reliable models for actual jn vivo responses. Consequently, many of them 

have been subjected to correlation investigations to determine their 

quantitative predictive coefficients for cancer and/or heritable genetic 

disease in animals (Purchase, 1982). Details of these comparative 

studies as they relate to specific bioassays will be discussed in 

Sections 2 and 3. While the assays are generally considered reliable 

indicators of carcinogens, false positive and negative responses have 

been generated. 

Bioassays which qualify as short-term predictive tests include submammalian, jn vitro mammalian and in vivo techniques to identify which 

chemicals induce alterations in~~D"NA or chromosomes or produce cell 

transformation. The most widely used tests are listed in Table 4. They 

are identified by their applicability to either carcinogenicity and/or 

mutagenicity prediction. 

A general consensus has evolved which describes cancer as a multi-stage 

process involving a variety of events including initiation, repair, fixation, promotion and proliferation (Figure 2) (Weisburger and Williams, 

1981). Because genotoxic steps are implicated early in this process, 

their detection has assumed the status of presumptive carcinogen identification. This assumption is not without some controversy and a good 

objective review of the benefits and problems associated with application 

of the short-term technology to carcinogen screening is given in ICPEVC 

Committee 2 Report, 1982. 

The range of bioassays which fall into the short-term category is very 

broad and the number of tests is quite large. The EPA Gene-Tox program 

has reviewed the published literature for approximately 35 separate tests 

(Waters and Auletta, 1981) and prepared a series of papers which evaluate 

the performance of each test. Ultimately this program will attempt to 

define the applicability of specific short-term tests as carcinogen and 

mutagen predictors and will recommend specific test batteries. Not all 

12 
TABLE 4. A SUMMARY OF THE POSSIBLE ROLES FOR SELECTED 

GENETIC TESTS IN CHEMICAL 

HAZARD ASSESSMENT 

Primary Roles 

General Assay Type TcT IMT 

Microbial Assays 

Ames Reverse Mutation Test •+ + 

Reverse Mutation in E. coli 

WP2 and Related Strains + + 

Bacterial DNA Repair Tests + NA 

Yeast Mutation Tests + -MYeast Mitotic Recombination + NA 

In Vitro Mammalian Cell Assays 

Mouse Lymphoma Assay (TK) 

CHO or V79 Mutation Assays (HGPRT) 

Unscheduled DNA Synthesis (UOS) 

Chromosome Aberrations 

Sister Chromatid Exchange (SCE) 

Cell Transformation 

In Vivo Mammalian Assays 

SCE 

Dominant Lethal Assay 

Cytogenetic Analysis (aberrations) 

Micronucleus Assay 

Spermhead Abnormality Assay 

Heritable Translocation Assay in Mice 

Specific Locus Assay in Mice 

DNA Adduct Formation 

UDS Assays 

In Vivo Submammalian Assays 

Drosophila Assays 

Plant Cytogenetics 

+ 

+ 

++ 

+ 

++ 

++ 

+ 

NA 

+ 

+ 

NA 

NA 

NA 

+ 

+ 

+ 

NA 

•+ 

•MNA 

++ 

NA 

NA 

NA 

++ 

++ 

+ 

<•) 

+ 

++ 

<•) 

(•) 

++ 

(•) 

ICP = Identifies Mammalian Carcinogenic Potential 

IMP = identifies Mammalian Mutagenic Potential 

+ = Applicable 

++ = Greater Applicability for this Role 

NA = Not Applicable 

(+) = Possible Application Under Limited Conditions 

13 
P.29 

Step Comment 

Initiating Event 

Fixation 

I 

Promotion/Enhancement 

Expression of Cell 

Transformation 

(Evades Immune 

Surveillance 

System of Host) 

Tumor 

This is probably the initial event and Is a mutation or 

0NA lesion affecting a particular gene or set of 

genes. This step Is irreversible but requires subsequent steps for expression. 

The genetic lesion requires specific metabolic pro* 

cesses to occur and/or cell division to fix the lesion 

into the genome of the cell. 

It is assumed that specific physiological or 

biochemical phenomena are required to facilitate 

the expression of the Initiating event. Other 

chemicals, such as phorbol esters, hydrocarbons, 

phenobarbitel, smoke eondeneate, etc., have been 

implicated in the promotion and enhancement of 

cell transformation. To a certain extent these processes appear reversible. 

The cell acquires a new set of genetic biochemical 

and Immunological properties. The properties give 

rise to the tumor properties of the particular 

neoplasm. All progeny cells of the tumor acquire 

these properties fully expressed and do not go 

through the transformation process. 

When a sizable population of transformed cells is 

formed by rapid proliferation, the neoplastic disease 

Ie Identified clinically as a tumor mass. 

Figure 2. Hypothetical steps involved in oncogenic transformation. 

14 
of these tests, however, will be applicable to the evaluation of motor 

vehicle emissions. In order for a bioassay, even highly predictive 

assays, to be suitable for this type of investigation it should have the 

following characteristics: 

1. The method should be sufficiently flexible to test both particles 

(1-10 urn size) as well as aqueous- or organic-solvent-soluble 

complex mixtures that are extractable from the particle core. 

2. The technique should have intrinsic metabolic activation/detoxification capabilities or be compatible with a cell-free exogenously 

supplied metabolic activation system. 

3. The test should be capable of generating a reproducible dose-related 

response following exposure to genotoxic agents and produce data 

that can be evaluated by conventional statistical procedures. 

4. The method at least should be responsive to PAH carcinogens since 

this class of agents predominates in emissions. 

5. The test should be amenable to assessment of small samples 

(10-500 mg quantities). 

Only a fraction of the total assay of genetic bioassays satisfies all 

five criteria. The last two criteria are not only very important but 

are also responsible for ruling out many otherwise suitable test methods. 

SPECIFIC LIMITATIONS TO SHORT-TERM TEST APPLICATION 

Exhaust particles are generally less than 1 micron in diameter and easily 

transported to the deep lung in humans (Huisingh et al., 1978). Because 

of the presence of organic carcinogens on the particles, solid-phase 

pollutants appear to be the most likely lung carcinogen candidates. The 

finding that essentially all relevant genotoxic effects of gasoline or 

diesel exhaust emissions were associated with the particle phase has 

significantly affected the applicability of short-term tests to studies 

of exhaust emissions (McClellan et a_K , 1981). One limitation of vn 

vitro tests is inability to conduct reliable aerosol exposures of 

biphasic emissions. Some success with vapor-phase exposures in microbial 

tests have been reported but, in general, mammalian cell in vitro tests 

have not been adopted to this exposure route. Genotoxic cRemicals are 

produced during fuel combustion but, shortly after the emissions leave 

the tailpipe, a significant portion of these agents are condensed onto 

the carbonaceous particle core. To a certain extent this lessens the 

requirement for testing using only whole-exhaust emissions. 

In addition to their limitations with respect to certain routes of exposure, short-term tests have other characteristics which reduce their 

applicability to some important emissions research problem areas. For 

instance, one most critical issue involved in the risk analysis question 

is that of target-organ dosimetry by the biologically active component 

of the emission. Understanding this part of the risk process for air 

15 
P.31 

pollutants must take into account data on target organ exposure to particles, deposition sites of particles in the respiratory system, and clearance kinetics. Also of critical importance to the assessment of possible 

health risks are in vivo desorption rates for bound, toxic organic and 

inorganic material! and transport of biologically active substances to 

susceptible target cells. Short-term, especially uj vitro, tests cannot 

be applied easily to the resolution of these problems, with the possible 

exception of intracellular transport of specific organic chemicals. 

Several other available techniques show cellular absorption of particles, 

but these types of short-term studies on whole exhaust are severely 

limited. 

SPECIAL APPLICATIONS 

The inability to study relevant exposure conditions using in vitro tests 

does not completely exclude the use of short-term tests in target organ 

dosimetry. It appears possible to screen for genotoxic effects in respiratory tract target cells following j_n vivo inhalation or instillation 

exposures in hamsters (Guerrero and Rounds, 1981). However, at the 

present time, these methods are developmental and little data from motor 

vehicle emissions are available. Exposure to genotoxic fractions in 

these bioassays is detected via DNA binding, SCE, or stimulation of DNA 

repair processes. These tests and their results are reviewed in Section 3. 

If these techniques can be developed further and validated, they would 

enhance considerably the opportunity to study target-cell dosimetry under 

well defined aerosol-exposure conditions. 

Interest has also been raised about the possible enhancement of the carcinogenicity effects caused by motor vehicle emissions as a result of 

interactions between the PAHs bound to the solid phase and some of the 

irritating gases present in exhaust emissions. Reports from Ziem et al_. 

(1982), and Laskin et al. (1970), indicated that S02, N02 and 03 vapors 

enhance the carcinogenic effects of known carcinogens. If these interactions are important for expression of the carcinogenicity of exhaust 

PAHs, U\ vitro experiments would not give accurate estimates of the 

associated hazard. However, results from ongoing in vivo studies of 

such interactions might reveal their significance Tn risk assessment. 

Heinrich et al_. (1982), for example, has shown that tumor rates in 

Syrian hamsters exposed to diethylnitrosamine are increased following 

exposure to particle-free diesel exhaust. Short-term tests addressing 

this phenomenon have not been reported. 

Metabolism, especially of PAHs, plays a major role in toxicity extrapolation across phylogenetic barriers. The ultimate biological activity of 

a chemical in vivo is a function of the three processes of activation, 

deactivation occurring together and reactivation. The balance of these 

processes is a major factor in determining the carcinogenic potential of 

a chemical. Integration of these functions under the conditions of J_n 

vitro metabolism used in short-term bioassays might well vary considerably 

with the metabolic processes in vivo. Well defined metabolic studies 

with complex mixtures are dif7Tcult because of the need to selectively 

label specific compounds among the hundreds present. 

16 
P.32 

SUMMARY 

In summary, many characteristics of short-term genotoxicity tests 

appear ideally suited to the analysis of automobile exhaust emissions. 

In fact, much of the information available on the biological properties 

and possible health risks associated with the particulate emissions has 

been accumulated largely using short-term test data. Certainly the opportunity to conduct multi-dose experiments on the relatively small quantities 

of particles collected from exhaust emissions was one of the significant 

breakthroughs in the evaluation of motor vehicle emissions. Many of the 

attributes of short-term tests are important to the application of these 

tests and are summarized in Table 5. The importance of the ability to 

conduct assays using very small samples cannot be overemphasized since 

spark-ignition gasoline engines equipped with catalytic converters 

produce very low levels of particulate; thus, significant comparisons of 

these samples with other emissions would be impossible without short-term 

assays. 

17 
TABLE 5. CHARACTERISTICS OF SHORT-TERM BIOASSAYS WHICH FACILITATE THEIR 

APPLICATION TO HEALTH EFFECTS EVALUATION OF AUTOMOBILE EMISSIONS 

Characteristic Application to Emission Testing Comments 

Sample size required to 

conduct the test is small. 

oa 

Biodirected fractionation 

of complex mixtures 

Test methods are amenable to 

quantitation of test results 

which involves dose response 

This characteristic permits the evaluation 

of particulate samples collected on filters 

under simulated ambient conditions. Most 

animal model studies would require substantially more collected particle samples or 

could be conducted only on whole or 

scrubbed exhaust containing both gaseous 

and solid phases. 

Some of the U\ vitro or submammalian shortterm tests can be used to direct sample 

fractionation and chemical characterization. 

This is possible because of the high degree 

of test sensitivity and the ability to 

perform tests on very small samples of 

fractionated test sample. Extracts and/or 

leachates of particles collected from 

emissions have been fractionated to identify 

which fractions retain genotoxic activity 

and which do not. 

To develop the types of information which 

might be applicable for hazard assessment, 

data from comparative studies will be 

required. Accurate comparisons can only be 

generated from dose-response curves, 

especially from replicate studies. 

Limitations of animal studies to 

whole exhaust is technically difficult because of the levels of toxic 

vapors present at high exposure 

conditions. In order to enhance 

exposure to the presumed carcinogens, exposure to particulates 

would be necessary. 

It will be necessary eventually to 

identify specific chemicals (or 

chemical classes) of concern 

in the collected emissions. Once 

these agents are identified and 

linked to the specific health effects, 

permissible levels can be established 

and emissions from new fuels, fuel 

additives or engines can be evaluated 

rapidly. 

Comparative data (e.g., two types 

of fuels combusted in one type of 

engine) cannot be developed readily 

using animal models because of technical facilities limitations. Yet 

without direct comparisons, risk 

analysis cannot be formulated. 
P.34 

REFERENCES 

1. Ames, B.N. Identifying Environmental Chemicals Causing Mutations 

and Cancer. Science, 204:587-593, 1979. 

2. Brusick, D.J. The role of short-term testing in carcinogen detection. Chemosphere, 5:403-417, 1978. 

3. Guerrero, R.R. and D.E. Rounds. Genotoxicity of Syrian hamster 

lung cells treated i_n vivo with diesel exhaust particles. Environment . ternational, 5:445-454, 1981. 

4. Heinrich, U., et al. Investigation of toxic and carcinogenic effects 

of Diesel exhaustTn long-term inhalation exposure of rodents. In: 

Toxicological effects of emissions from diesel-engines, J. Lewtas, 

ed. Elsevier Biomedical, N.Y., 1982. pp. 225-242. 

5. Hoi lutein, M., J. McCann, F. Angelosanto and W. Nichols. Short-term 

tests for carcinogens and mutagens. Mutat. Res., 65:133-266, 1979. 

6. 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 character-, 

ization 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. 

7. ICPEMC Committee 2 Report. Mutagenesis as an Approach to Carcinogenesis. Elsevier Biomedical Press, Amsterdam, 1982. 

8. Laskin, S., et al. Studies in pulmonary carcinogenesis. In: 

Inhalation Carcinogenesis, AEC Symposium Series No. 18, M.G. Hanna, 

et al., eds. Oak Ridge, TN, USAEC Division of Technicol. Inf., 1970. 

pp. "321-351. 

9. McClellan, R.O., A.L. Brooks, R.G. Cuddihy, R.K. Jones, J.L. Manderly, 

and R.K. Wolff. Inhalation Toxicology of Diesel Exhaust Particles. 

In: Diesel Emissions Symposium, U.S. EPA, Office of Research and 

Development, Raleigh, North Carolina, 1981. 

10. Purchase, I.F.H. An appraisal of predictive tests for carcinogenicity. Mutat. Res., 99:53-71, 1982. 

11. Waters, M.D. and A. Auletta. The Gene-Tox Program: genetic activity 

evaluations. J. Chem. Inf. Comput. Sci., 21:35-38, 1981. 

19 
P.35 

12. Weisburger, J.H. and G.M. Williams. Carcinogen testing: Current 

Problems and New Approach. Science, 214:401-407, 1981. 

13. Z1em, U., et aj. Induction of lung adenomas in mice after a short 

latency period. Arbeitstagung der Deutschen Gesellschaft fiir Hygiene 

und Mikrobiologie e.V., am 23. und 24.09.1982 in Mainz, 1982. 

20 
P.36 

PART I 

SECTION 2 

THE CURRENT STATE-OF-THE-ART IN APPLICATION OF 

SHORT-TERM BIOASSAYS TO AN ASSESSMENT OF HUMAN CANCER 

AND MUTATION RISK 

Genotoxic testing can be used to estimate the qualitative risk potential 

of chemicals as mutagens and carcinogens. Approaches to evaluate the 

two phenonema cannot be identical because of the mechanistic and methodological reasons cited in Table 6. However, since numerous aspects of 

the application of short-term tests to assessing the human risk of cancer 

and mutation are similar, these dysfunctions generally will be combined 

for the purposes of this discussion. 

Mechanistically, chemicals which alter the structure or function of DNA 

are capable of inducing cancer or genetic dysfunction in humans in that 

lesions which give rise to mutations or to the initiating lesions of 

cancer result from specific chemical-DNA interactions described in Table 7. 

The essential questions, then, are those of exposure levels of the appropriate target cells (somatic or germinal), intrinsic potency of the 

genotoxic material, and bioavailability of the carcinogenic moiety to 

susceptible target cells of the appropriate species at sufficient concentrations to induce genetic damage (Figure 3). The genotoxic risk to 

somatic and germ cells posed by effects of the same chemical can differ 

greatly, somatic cells appear to be more susceptible for several reasons 

listed in Table 8. 

In order to approach risk assessment from an operationally feasible pointof-view, the process needs to be separated into two functions (a) detection 

and (b) quantification. 

DETECTION OF HAZARD 

Qualitative determination of hazard is based on observation of (a) increased 

cancer or genetic effects in human populations exposed to a particular 

substance, (b) experimental data from animal model systems in which the 

tumor incidence or mutation rates are increased as the result of exposure 

to genotoxic chemicals, or (c) positive results from short-term predictive 

tests which rely upon mechanistic linkages and correlation coefficients 

to signify that such chemicals, when identified, may be shown in subsequent 

studies as carcinogenic/mutagenic hazards. 

21 
TABLE 6. COMPARISONS OF THE APPROACHES USED IN PREDICTIVE SCREENING FOR 

CARCINOGENS AND MUTAGENS 

Parameter Carcinogenesis Prediction Mutagenesis Prediction 

Animal Test Data 

Reference Standard 

Human Epidemiology 

ro 

ro 

Mechanism of 

Induction 

Risk Extrapolation 

Methods 

Consists of a rather large body of data 

(300-500 experimental carcinogens) in 

rodents with a diverse group of chemical 

classes. 

Substantial body of data available. In 

approximately twenty cases, direct cause/ 

effect relationships have been established 

between chemical exposure and cancer. 

Presumably initiated by genotoxic damage 

produced by exposure to carcinogenic 

agent; however, tumor response can be 

significantly modified by secondary 

phenomena and are often the consequence 

of an exposure to a second, nongenotoxic 

chemical (promoters, cocarcinogens). 

Many mathematical models available. 

The methods vary from linear nonthreshold to nonlinear threshold 

models depending on the likely 

mechanism involved in tumor induction. 

Consists of a small body of data (50-100 

germ cell mutagens at best) in rodents 

with a relatively narrow spectrum of 

chemical classes (largely direct-acting 

alkylating agents). 

Few epidemiology studies. Evidence for 

cause/effect relationship between specific 

agent and human genetic effects is 

virtually nonexistent for chemical mutagens. 

Mechanism involves altered DNA base 

sequence or loss of chromosome integrity 

in target cell. There is no evidence for 

modulation of mutation in germ cells by 

nonmutagenic chemicals. 

Current approach seems to be limited 

only to a linear nonthreshold mathematical treatment of the data. This 

is a result of the single hit theory 

of mutation induction. 

co 
P.38 

TABLE 7. MAJOR DNA LESIONS PRODUCED BY CHEMICAL INTERACTION 

AND THEIR GENOTOXIC CONSEQUENCES 

Primary Lesion Dm script ion Consequence 

Alkylation Covalent adduct formed Involving 

the genotoxic sgent and a ONA 

bate or phosphodlester bridge. 

Intercalation Noneovalent stacking ol the 

genotoxic agent between 

adjacent base pairs In the 

DNA helix. 

Alteration of base-pairing 

loss of the base, stimulation of error-prone repair. 

Alteration of ONA transcription, replication or 

repair. 

Cross-Linkage 

Breakage 

Formation of two covalent 

bonds between bases within 

(intrsstrsnd) or between 

(interstrand) DNA strands. 

Scission of either a single 

or both strsnds of the 

DNA helix 

Dimer formations, alteration of replication. 

DNA rearrangements forming 

chromosoms aberrations 

after mitotic cell division. 

23 
Ni 

(Intrinsic Genetic 

Potential of Chemical) 

N2 

(Opportunity for Hits 

on Target Molecule at 

Normal Exposure Levels) 

R (Risk) 

ro 

• DETECTION OF HAZARD 

Nt can be detected using various assay systems on a qualitative basis; however, potency is difficult to 

measure except on a relative scale. Extrapolation of qualitative results from species to species also introduces uncertainty Into the equation. 

• DETERMINATION OF EXPOSURE 

N2 cannot usually be quantitatively measured at the low environmental levels normally encountered. 

Even qualitative evidence for target molecule hit is difficult at low levels because of the magnitude of the 

populations that would be required. Short-term tests may be able to approach the determination of N2 

because of their increased sensitivity and resolving power. 

• RISKS CALCULATION 

R can be roughly estimated at high dose levels using some type of Nt and N2 values. The meaning of R at 

high Nt and N2 levels is difficult to interpret and may not be directly extrapolatable to low levels. 

Figure 3. Considerations involved In quantitative risk assessment. 

CO 
P.40 

TABLE 8. A COMPARISON OF RISK ASSESSMENT FOR 

SOMATIC AND GERM CELLS IN VITRO 

Comparison Parameter Somatic Cells Qerm Cells 

Selective Pressure on 

New Mutants 

Barriers to Mutagenic 

Agents 

Sex Differences 

Ease of Detection 

of Induced Effects 

Size of Exposed 

Sample that can be 

Scored 

Type of Genetic 

Lesion Detected 

Slight, since cells 

encounter only 

mitotic division. 

Few barriers If agent 

Is distributed 

systemically. 

Minimal; some 

influence of hormones 

on metabolism In 

specific organs. 

Each dosed animal 

represents a population of exposed 

cells. 

Extremely large population, generally 1(P 

to 10' surviving cells 

scored. 

Chromosome aberratlona 

and single gene mutations both easily 

detected. 

High, since melotic cell 

cell division acts to screen 

out specific types of 

mutations and chromosome 

effects. 

Barriers similar to those 

for somatic cells plus 

blood-gonadal barriers. 

Significant because of the 

different proceases involved 

In gemetogenesls between 

male and female mammals. 

Analysis generally requires 

large animal populations 

since each offspring represents a single exposed 

gamete analyzed. 

Generally small sample 

sizes; less thsn 1()5 animals 

In all cases. 

Chromosome sberrstions 

typically scored. Point 

mutations require extensive 

studies to detect with 

reliability. 

25 
P.41 

QUANTIFICATION OF HAZARD 

This phase of the risk analysis process involves an estimation of the 

incidence of adverse effects in humans under anticipated exposure conditions encountered by the population at risk. The data providing the 

foundation for this part of the risk analysis are generally weak because 

analysis is based on unverifiable assumptions and extrapolations of high 

dose responses to estimates of low dose response and often requires extrapolation across vast phylogenic gaps. The high-to-low-dose extrapolations evoke considerable uncertainty and are often the subject of 

scientific controversy (Munro and Krewski, 1980). 

APPLICATION OF CONVENTIONAL RISK ASSESSMENT METHODS TO AUTOMOBILE EMISSIONS 

Epidemiological evidence in humans provides the strongest possible risk 

assessment information when studies show a positive cause-effect relationship. All chemicals currently recognized as human carcinogens have been 

identified as such by epidemiological studies (Bartsch et a_L , 1982). 

Both the hazard detection and quantification information come from the 

relevant target species. Unfortunately, most risk estimates have not 

been derived from human epidemiological data because of the limited nature 

of valid human retrospective studies. One human retrospective epidemiological study of motor vehicle emissions employed bus garage workers (men 

45 to 64 years old employed by the London Transport Authority during the 

years 1950 to 1974) who showed no carcinogenic effects from long-term exposure to diesel exhaust emissions (National Research Council, 1981). The 

cohort consisted of bus garage engineers, bus drivers, bus conductors, central 

work engineers, motormen, and guards. Calculations made to estimate the 

possible magnitude of an undetected effect showed that an undetected incidence of lung cancer in unexposed workers could not be excluded by the study. 

Using the London bus garage study, an estimate of lung cancer risk under 

ambient conditions was calculated to be a 0.05 percent proportional 

increase in lung cancer incidence per unit of exposure to diesel engine 

emissions (1 unit = 1 ug inhaled particles/m3 per year). However, this 

study was limited in many respects and cannot be considered a completely 

valid risk assessment for exposures to ambient motor vehicle emissions. 

Attempts to apply animal models to the risk assessment of motor vehicle 

emissions have likewise not been very successful for the following reasons: 

1. The classes of chemicals identified in diesel or gasoline automobile 

exhaust emissions (PAHs) are relatively inactive in the typical 

rodent model systems used (mouse and rat). Inhalation studies using 

whole diesel exhaust or pure aerosolized PAH such as benz(a)pyrene 

(BaP) did not produce cancer in mice or rats (Battelle-Northwest 

Laboratories, 1982; Heinrich et al., 1982; GM Research Center, 1982), 

although lung tumors have been induced in hamsters using extremely 

high levels of a pure carcinogen (Thyssen et aj., 1981). Solvent 

extracts of gasoline and diesel exhaust particulates have been shown 

to produce weak tumorigenicity when administered in skin painting 

and subcutaneous injection studies in mice, in intratracheal instillation in hamsters and in two stage promotion systems in mice (Kotin 

et al., 1954; Wynder et al., 1962; Mohr et al., 1976; Brune et a_L , 

15787 Misfeld, 1980, STaga et aj., 1980). 

26 
P.42 

2. The levels of particles containing bound carcinogens in ambient or 

diluted exhaust emissions are so low that calculations based on the 

benz(a)pyrene content of diesel exhaust indicates that no more than 

1 M9 of BaP will be deposited in the lungs of hamsters exposed for 

two years to diluted exhaust under experimental test conditions. 

This level 1s considerably lower than the 10 mg/m3 of pure BaP 

required to induce lung tumors in hamsters exposed by inhalation 

and almost precludes the observation of carcinogenesis activity of 

exhaust emissions in reasonably sized animal experiments. j 

3. Experimental inhalation studies, which represent the most relevant 

exposure route for humans, are complicated by the presence of both 

irritant (N02/S02) and lethal (CO) gases. To obtain high levels of 

emission particles in the inhalation chambers without a high level 

of mortality among the test animals, the exhaust emissions from 

either gasoline or diesel engines must be scrubbed to remove CO, 

N02 and S02 which is likely to result in the alteration of the 

composition of organic chemicals bound to the particles. 

Thus, the general consensus is that conventional lifetime inhalation 

studies of diluted automobile exhaust emissions will not provide the 

necessary data to develop an accurate estimate of human risk. Other 

animal models such as skin painting in SENCAR mice and intratracheal 

instillation in hamsters may provide data of limited utility, but these 

tests suffer from limitations in relevant route of exposure and test 

system refractibility. 

APPLICATION OF SHORT-TERM BIOASSAYS TO RISK ASSESSMENT OF AUTOMOBILE 

EMISSIONS 

Because short-term, and especially in vitro, assays have been found to 

be readily applicable to an evaluation of solid phase emissions, much of 

the data predicting qualitative hazard has been derived from this type 

of research. Risk analysis must draw upon all available data. Quantitative estimates developed for automobile exhaust emissions by the Environmental Protection 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 (Albert et IT., 1982; National 

Research Council, 1981). Both approaches were based upon available shortterm data and comparative analyses were made of laboratory and epidemiological data on diesel and gasoline engine exposure as well as other 

chemically related environmental exposures—coke-oven-emission, roofingtar-emission and cigarette-smoke-condensate (CSC). Types of data integrated Into the two risk analyses are summarized in Table 9. The NAS 

analysis excluded the Ames data primarily because the test results from 

bacteria studies are enhanced when compared to mammalian studies because 

of microbial nitroreductases. Mammalian cells are generally less 

responsive per unit dose because of reduced nitroreductase activity. 

The SCE data used in the EPA analysis were not available for inclusion 

in the NAS assessment. 

The short-term data base for diesel exhaust emissions extends far beyond 

the bioassays listed in Table 9, but the information reported from these 

assays met the type of criteria required for biostatistical analysis. 

27 
TABLE 9. DATA SOURCES USED TO PREPARE LUNG CANCER RISK 

ASSESSMENTS FOR AUTOMOBILE EXHAUST EMISSIONS BY NAS AND EPA 

NAS* EPA' 

Bioassays Used 

Reverse mutation in Salmonella typhimurium 

Forward mutation at the TK locus in L5178Y 

mouse lymphoma cells 

Sister chromatid exchange (SCE) in Chinese 

hamster ovary cells 

Enhancement of viral (SA7) transformation 

of Syrian hamster embryo cells 

SENCAR mouse skin tumor initiation 

V 

V 

V* 

4 

V 

v* 

V 

V 

Related Environmental Exposures Used 

Coke oven emissions 

Roofing tar emissions 

Cigarette smoke condensate (CSC) 

V 

Emission Sources Compared 

• Nissan diesel (LD)C 

Volkswagen diesel (LD) 

• Oldsmobiie diesel (LO) 

• Caterpillar diesel (HD)d 

Ford Mustang II gasoline 

V 

J 

v* 

bNAS = National Academy of Science (National Research Council, 1981) 

EPA = Environmental Protection Agency (Albert et al_., 1982) 

dLD = Light Duty Engine 

HO = Heavy Duty Engine 

28 
P.44 

The reasons that the short-term data from the tests listed in Table 9 

were amenable to use in risk estimation are: 

1. Results were reproducible within a given bioassay. 

2. Experiments provided dose response results spanning a broad range 

of concentrations. 

3. Experimental results could be formatted in a manner amenable to 

statistical analysis. 

4. Each bioassay was considered a valuable correlate with carcinogenicity 

or mutagenicity in mammals. 

5. Each bioassay was suitable for analysis of other environmental emissions with related composition (roofing tar emissions and coke oven 

emissions). 

Both the NAS and EPA risk estimates analyzed the 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 the linear extrapolation modes. 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 the human lung cancer data from coke-oven workers and 

roofers. 

The estimate for lung cancer risk from exposure to diesel-exhaust emissions was derived by determining 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 are given in Table 10 and are quite similar even 

though the two groups of short-term bioassays studied were not identical. 

It is also of interest to note that the estimated risks determined by 

NAS and EPA were similar to the estimated lung cancer risk for individuals 

exposed to ambient diesel-exhaust emissions in the London bus garage 

workers study. However, the expression of NAS and EPA risks are in absolute 

risk while the London bus worker study gave proportional risks. 

The EPA and NAS efforts may have established a precedent for the use of 

short-term test results in combination with epidemiological data for 

surrogate materials in estimating human risk, especially in cases of 

complex mixtures. An analysis of each risk estimate is provided in the 

primary reference document which outlines the limitations and assumptions 

of the two approaches. Questions of how to apply the risk estimates 

remain unanswered because the lung cancer estimates were made on the 

basis of organic solvent extracts of emissions particles. It is suspected 

that the bioavailabilities of organic carcinogens from diesel emissions, 

gasoline emissions, coke oven emissions, roofing-tar emissions and 

cigarette smoke condensates are significantly different. Some of the 

29 
P.45 

1 

TABLE 10. HUMAN LUNG CANCER RISK ESTIMATES FOR OIESEL AND 

GASOLINE-CATALYST PARTICULATE EMISSIONS BASED ON 

SHORT-TERM TEST DATA 

Unit-Risk Estimates 

(Lifetime Risk/ug/m3) 

Emission Source NASa , b EPAC 

Nissan diesel 0.2-0.6 x IO-4 0.35 x IO-4 

Oldsmobiie diesel 0.1-0.24 x IO-4 0.2 x IO-4 

Volkswagen diesel 0.02-2.78 x IO-4 0.23 x IO-4 

Caterpillar diesel 0.01-0.25 x IO-4 0.018 x IO-4 

Ford Mustang gasoline — 0.60 x IO-4 

?NAS = National Academy of Science (National Research Council, 1981) 

A range of estimates was given for the NAS study. Both estimates are 

for emission particulates. Similar estimates made by the EPA based 

on total organics extractable from particles are five to ten times 

greater. 

EPA = Environmental Protection Agency (Albert et a_L , 1982) 

active compounds in the mixtures are also different. Bioavailability, 

then, is a major undetermined variable in exposure to automobile emissions. 

Another important unanswered question is that of susceptible subpopulations 

(age, sex, disease state). ' Most, if not all, relevent human epidemiology 

data for the surrogate emissions were obtained from adult males under 

occupational exposure conditions whereas diesel emission risks will 

include people of both sexes and of all age ranges and health conditions. 

The following 11st identifies areas of concern or uncertainity in the 

application of short-term test data to human risk analysis. 

1. Are differences in identity and bioavailability of carcinogens from 

the test and surrogate emissions sufficient to affect the risk 

calculations? 

2. Was the selected battery of short-term tests the most reliable for 

estimation of cancer risk? 

30 
P.46 

3. Considering that cancer potency can be substantially modulated HI 

vivo by promoting agents and cocarcinogens and that most short-term 

tests are refractory to these agents, is it possible that the risk 

estimates would change if promotion or cocarcinogenic activities 

were factored into the calculations? 

4. How reliable is the relationship between genotoxicity and carcinogenicity? 

There are no available data to apply to the answers to questions 1-3, 

but extensive studies have been conducted addressing question 4. 

Overlapping mechanisms and correlation studies provide the rationale for 

the application of genotoxicity results to human risk analysis. Most 

reports evaluating the reliability of short-term test batteries as predictors 

of cancer in mammals indicate a high level of predictive power (85 to 

95 percent). While there may be serious concerns for the uncertainity 

associated with the quantitative extrapolation of short-term test results 

to humans, on a qualitative basis (detection of hazard) short-term tests 

are as reliable, if not more reliable, than conventional animal models 

in human carcinogen detection (Table 11). Results from several short-term 

tests can be used as reasonably good predictors of heritable genetic risk 

in mammals (Table 12). 

In summary, results from short-term tests for genotoxicity are amenable 

to use in making human risk estimates in situations where conventional 

animal modeling or human epidemiology results cannot be obtained. An 

evaluation of two approaches conducted by EPA and NAS show similar quantitative risk estimates for diesel exhaust emissions. These two evaluations 

are also within two orders of magnitude of a worst case estimate for lung 

cancer in humans derived from negative epidemiological data. Risk estimation for genetic effects cannot yet be accomplished using similar methods 

because (a) a different set of short-term bioassays are needed to predict 

germ cell damage (Table 12) and (b) there are no human risk estimates for 

genetic effects derived from epidemiology data on surrogate emissions. 

31 
P.47 

TABLE 11. CHEMICALS EVALUATED AS CARCINOGENIC FOR HUMANS AND 

THEIR RESPONSES IN RODENT AND SHORT-TERM PREDICTIVE ASSAYS 

Group 1 Chemicals3 

4-Aminobiphenyl 

Analgesic mixtures 

containing phenacitin 

Arsenic and arsenic compounds 

Asbestos 

Azathioprine 

Benzene 

Benzidine 

N,N-Bis (2-chloroethyl-2) 

naphthylamine (chlornaphazine) 

Bis(chloromethyl)ether and 

chloromethyl methyl ether 

Certain combined lymphoma 

chemotherapy (MOPP) 

Chlorambucil 

Chromium and chromium compounds 

Conjugated estrogens 

Cyclophosphamide 

Diethylstilbestrol 

Melphalan 

Methoxysalen plus UV light (PUVA) 

Mustard Gas 

Myleran 

2-Naphthylamine 

Soot, tars and oils 

Treosulphan 

Vinyl chloride 

Human 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Mouse 

Bioassay 

+ 

-

-

• 

(+) 

-

ND 

+ 

+ 

ND 

+ 

-

ND 

+ 

+ 

+ 

+ 

+ 

(+) 

+ 

+ 

ND 

+ 

Rat 

Bioassay 

+ 

+ 

-

+ 

(+) 

-

+ 

+ 

+ 

NO 

+ 

+ 

-

+ 

+ 

+ 

ND 

ND 

-

-

-

ND 

+ 

Short-Term. 

Bioassays 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Not included are manufacturing or mining processes which do not involve discrete 

chemical entities. 

Bioassays include the Ames test, a cell transformation test and tests for 

chromosome aberrations (Bartsch et al., 1982; Nakamauro and Sayato, 1981; 

Siou et al., 1981; Barrett, 1982; IA&C Monographs, Supplement 4, October, 

1982)"! 

The results of these comparisons show that a battery of 3 or 4 short-term 

tests for genotoxic activity can detect the range of human susceptibility 

to chemical carcinogens whereas studies in neither mice nor rats (nor both 

combined) can correlate with the human susceptibility range. 

32 
P.48 

TABLE 12. PREDICTIVE VALUE OF SHORT-TERM GENOTOXIC TESTS FOR 

MAMMALIAN GERM CELL MUTAGENS 

Test 

Agreement Between 

Qualitative Effects 

in Short-Term 

Tests and Germ 

Cell Mutagen Tests 

(Percent) 

Cytogenetics j_n vitro 

Spot Test in Mice 

SCE 

Rodent Micronucleus 

Cytogenetics j_n vivo 

Drosophila SLRL 

Spermhead Abnormality 

Mammalian cell i_n 

vitro mutation 

E. colj. WP2 

Ames Test 

Yeast Mitotic 

Recombination 

Bacterial ONA Repair 

Test 

88 

83 

83 

82 

79 

76 

73 

72 

65 

64 

61 

60 

(28/32)a 

(15/18) 

(20/24) 

(28/24) 

(27/34) 

(31/41) 

(19/26) 

(22/32) 

(15/23) 

(28/44) 

(22/36) 

(21/35) 

a(Number of correct responses/number of comparisons). 

The interpretations possible from this table are limited because of 

those problems identified in Table 6. But even with these limitations, 

one can clearly see that the bioassays used by EPA or NAS to make 

cancer risk estimates would not be the most accurate in predicting germ 

cell mutation risk. 

Source: ICPEMC Committee 1 Final Report, 1983. 

33 
P.49 

1 

REFERENCES 

1. 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 emissions. Risk Analysis, 3:101-117, 

1983. 

2. Barett, C. Demonstration that diethylstilbestrol and asbestos 

produces cell transformation and aneuploidy in Syrian hamster embryo 

cells in culture. Presented at the Conference on Cellular Systems 

for Toxicity Testing, New York Academy of Sciences, October 5-7, 

1982. 

3. Bartsch H., L. Tomatis and C. Malaveille. Mutagenicity and Carcinogenicity of Environmental Chemicals. Regulatory Toxicology and 

Pharmacology, 2:94-105, 1982. 

4. Battelle-Northwest Laboratories. Diesel Exhaust Inhalation Studies, 

Richland, Washington, (unpublished, 1982). 

5. Brune, H., M. Habs, and D. Schmihl. The tumor-producing effect of 

automobile exhaust condensate and fractions thereof. Part II. Animal 

Studies. J. Env. Path. Toxicol., 1:737-746, 1978. 

6. GM Research Center, Warren Michigan, Diesel Exhaust Inhalation 

Studies, (unpublished, 1982). 

7. Heinrich, U., et aj. Investigation of toxic and carcinogenic effects 

of Diesel exhaust in long-term inhalation exposure of rodents. In: 

Toxicological effects of emissions from diesel-engines, J. Lewtas,

 J 

ed. Elsevier Biomedical Press, N.Y., 1982. pp. 225-242. 

8. ICPEMC Committee 1 Final Report, Mutat. Res., 114:117-177, 1983. 

9. Kotin, P., et aj. Aromatic hydrocarbons. II. Presence in particulate phase of gasoline engine exhaust and carcinogenicity of exhaust 

extracts. A.M.A. Arch. Indust. Hyg., 9:164-177, 1954. . 

10. Misfeld, J. The tumor-producing effect of automobile exhaust condensate and of diesel exhaust condensate. In: Health Effects of 

Diesel Engine Emissions: Proceedings of an International Symposium, 

Vol. 2, Plpelko, W.E., et al., eds. EPA-600/9-80-057b, U.S. Environmental Protection Agency, 1580. 

34 
P.50 

11. Mohr, U., H. Reznik-Schuller, G. Reznik, G. Grimmer, and J. Misfeld. 

Investigations on the carcinogenic burden by air pollution in man. 

XIV. Effects of automobile exhaust condensate on the Syrian golden 

hamster lung. Zbl. Bakt. Hyg. I. Abt., Orig. B, 163:425-432, 1976. 

12. Munro, I.C. and D.R. Krewski. The role of risk assessment in regulatory decision making. In: Emerging Horizons in Toxicology, Thirteenth Annual Symposium of the Society of Toxicology of Canada 

Proceedings, 1980. pp. 150-184. 

13. Nakamuro, K. and Y. Sayato. Comparative studies of chromosomal 

aberration induced by trivalent and pentavalent arsenic. Mutat. 

Res., 88:73-80, 1981. 

14. National Research Council. Potential Risk of Lung Cancer from Diesel 

Engine Emissions. National Academy Press, Washington, D.C , 1981. 

15. Siou, G., L. Concan and M. el Haitem. Evaluation of the clastogenic 

action of benzene by oral administration with 2 cytogenetic techniques 

in mouse and Chinese hamster. Mutat. Res., 90:273-278, 1981. 

16. Slaga, T.J., et al. Mutagenic and Carcinogenic Potency of Extracts 

of Diesel and~RYTated Environmental Emissions: Two-Stage carcinogenesis in skin tumor sensitive mice (SENCAR). In: Health Effects 

of Diesel Engine Emissions: Proceedings of an International Symposium, Vol. 2, Pepelko, W.E., et al., eds. EPA-600/9-80-057b, U.S. 

Environmental Protection Agency, 1550. 

17. Thyssen, J., et a h Inhalation studies with benzo(a)pyrene in Syrian 

golden hamsters. J. Natl. Cancer Inst., 66:575-577, 1981. 

18. Wynder, E.L., et al. Study of air pollution carcinogenesis. III. 

Carcinogenic activTty of gasoline engine exhaust condensate. Cancer, 

15:103-108, 1962. 

35 
P.51 

PART II 

SECTION 1 

. SAMPLING TECHNIQUES FOR COMBUSTION EMISSIONS 

DIRECT SAMPLING OF RAW EXHAUSTS 

The cumbersome sampling equipment of the mid 1950's to early 1970's 

necessitated voluminous extraction and clean-up procedures. At that time, 

large sample amounts were required for the analytical procedures used 

and biological tests were performed with skin painting on whole animals. 

In 1954, the extracts of gasoline engine particulate emissions recovered 

by filtering at the actual exhaust temperature were shown to cause cancer 

in mice by Kotin et al. Later, in 1957, Mlttler and Nicholson enriched 

some of the gaseous P"AH by condensation of exhausts from both gasoline 

and diesel engines, however, no particulate emission was recovered. 

Stenburg et a K in 1961 used condensation before filtering and applied 

this technique to different suspected PAH emission sources including the 

automobile. The principle of this method was employed by Begeman and 

Colucci in 1962 to collect both gaseous PAH and particle-associated PAH 

by placing a large water condenser before the filter package. This trapping system was dimensioned to enrich the total exhaust volume from a 

364 cubic inch V-8 engine operated on a chassis dynamometer. Gross 

(1972) using similar sampling system investigated the impact of parameters 

such as engine deposits, PAH content of the fuel, and emission control 

devices on the emissions (Table 13). For these sampling systems, very 

little or no information is available concerning optimization arrangements 

or recovery data. Also, these earlier studies did not take into consideration the possibility of losses of volatile species from filter, 

artifact formation or degradation during sampling. 

In 1972, Grimmer et a_L chose a vertical glass condenser with a cooling 

area of 0.25 m2 for collection of the total exhaust volume. They 

reasoned that the washing procedure would be simple, using the glass 

device, and that catalytic effects on the condensed material should be 

lower. Evaluation of this method indicates that the entrance temperature 

on the filter should not exceed 35°C if quantitative trapping of threering PAHs was intended. Prolonged sampling involves losses of certain 

species, caused either by volatilization or degradation in this method. 

The Grimmer method has been used extensively in The Federal Republic of 

Germany by car manufacturers. An improved vertical glass condenser was 

described in 1981 by Kraft and Lies for trapping total exhaust volume 

for both gasoline and diesel vehicles. 

36 
P.52 

TABLE 13. POLYNUCLEAR AROMATIC HYDROCARBON (PAH) EMISSIONS4 

BaP 

BaA 

Vehicle Ac 

Low . High 

PAH Fuel0 PAH Fuele 

2.4 5.8 

4.4 9.0 

PAH Emission gq/km 

Vehicle Bc 

Low . High 

PAH Fuel0 PAH Fuele 

0.7 1.3 

1.5 2.8 

Vehic 

Low . 

PAH Fuel0 

0.4 

1.0 

leC c 

High 

PAH Fuel6 

1.0 

2.5 

: . i 

aData of Gross (1972); condensation before filter. 

Gross' data originate from different fuel types and deposit levels in 

the engine and were originally reported as ug/gallon fuel used. 

Values above represent mean values from several fuels, and the recalculations in ug/km were made by the authors on an estimated mileage of 

21 miles/gallon from a 7 mode cycle. 

CA = vehicle without emission control, 1966 

B = vehicle with engine modification, 1968 

C = vehicle with engine modification, 1978. 

dLow PAH fuel: BaP < 0.1 ppm 

eHigh PAH fuel: BaP = 3 ppm 

PROPORTIONAL SAMPLING 

A proportional sampling system was used by Chipman and Massey in 1960 

for sampling such gaseous pollutants as HC, CO and NOx. With development of more sensitive analytical methods and biological tests im vitro, 

this technique has become an interesting alternative to large sampling 

equipment. Most proportional sampling systems use a reference signal 

which reflects the amount of emitted exhaust. Stenberg et al. (1981), 

used a proportional system connected to a glass condenser followed by a 

glass fiber filter. The resulting filter temperature was approximately 

50°C; the total exhaust volume that was sampled was typically 10X. Using 

this sampling equipment, Stenberg (1979) investigated the emissions from 

34 vehicles, representing the three most popular makes in Sweden. Fifteen 

different PAH's were identified from both the particulate and gas phase. 

The emission of five selected PAH's is summarized in Table 14. 

37 
TABLE 

CpcdP 

BaA 

BaP 

BghiP 

Coronene 

14. EMISSION OBTAINED 

Vehicle 

Cold 

51 

25 

38 

44 

21 

A

b 

Hot 

10 

15 

7 

8 

2 

FROM FTP, COLD START AND HOT START3 

Emissions 

Vehi 

Cold 

29 

9 

7 

13 

16 

cle 

ug/km 

B

b 

Hot 

11 

8 

2 

6 

4 

Vehicle Cb 

Cold Hot 

2 0.5 

8 3 

4 2 

13 5 

6 3 

Data of Stenberg (1979); condensation before filter. 

A = mean value of 12 vehicles 

B = mean value of 13 vehicles 

C = mean value of 12 vehicles 

DILUTION TUNNEL SAMPLING 

The sampling methods described above utilize raw gases for the determination of PAH, i.e., the emission has not been mixed with ambient air where 

possible reactions with the atmosphere might occur. When particles and 

gaseous exhaust enter the ambient air, as from automobiles and trucks, 

the mixture is quickly cooled and diluted. During this process, the 

overall temperature is reduced to the point that carbon particles begin 

to absorb condensing organic material. Still-further dilution may reduce 

the gas-phase hydrocarbon concentrations to the point that further absorption ceases to occur. A technique has been developed in order to simulate 

reactions with ambient air, I.e., a dilution tunnel. Dilution tunnel 

conditions may not achieve normal environmental conditions, however, 

because the rate of dilution 1s considerably less in the tunnel than under 

ambient conditions. A number of investigators have used air dilution 

tunnel techniques to achieve this simulation and a wide variety of systems 

has been shown to be reasonably effective in at least some sampling applications. Originally, the construction of such equipment was devoted to 

the measurement of lead emissions from gasoline vehicles (Habbi, 1973 

and Newhall et al., 1981). However, the utility of the dilution tunnel 

has been extended* and It 1s now used in almost all measurements of automotive emissions. Schuetzle (1983) and Huisingh et al. (1978) have described 

schematically the research dilution tube system used" in their laboratories 

(Figures 4 and 5). The dilution tube system described by Schuetzle (1983) 

includes an exhaust inlet manifold and mixing baffle, a dilution tube, 

and various down-stream probes and equipment. The dilution tube receives 

the exhaust as input, and supplies a known and reproducible quantity of 

filtered and humidity- and temperature-controlled ambient air for rapidly 

38 
•tWMlIM 

tACKHUOUM 

MeukATo* 

OAI 

AMAlVTICAl 

ICWMMMT 

womi 

IKNAUfT 

Figure 4. Dilution tunnel system for collection of heavy 

duty diesel particulate (Husingh et aj_., 1978). 

MTAKC DUJCNT AM 

GASSAM»l£ 

^^o TotAcse 

EXHAUST ANACW * 

INJECTION COAJ^MCNT OOQNCTIC rCOW MEASURING 

SAMfVNG OftWCE 

Figure 5. Dilution tube used for the collection of gas andj 

particulate-phase emissions (Schutzle, 1983). 

39 
P.55 

diluting and cooling the exhaust. The mixed stream flows at a measured 

volume rate through the dilution tube where fixed proportions are withdrawn from the tube isoklnetically into the sampling devices. In the 

dilution tube system used for collection of heavy-duty diesel particulate 

(Huisingh et aj_., 1978), the exhaust is first variably split, then diluted 

10-15 foldTn the dilution tunnel. The individual mode dilution ratios 

are determined by the ratios of gas concentrations in the dilute and raw 

exhaust streams for C02 and NO. Large samples of particulate material 

are obtained from this apparatus by filtering the whole dilution tunnel 

contents at a flow rate of 12000 liters/minute. 

The dilution technique has been used for a number of years. However, 

only limited data are available on measurements of PAH and related components (see Tables 15 and 16). 

Newhall et a_L (1981) have used another technique for sampling PAH. 

Dilution of the exhaust is performed in a small tube approximately 0.1x5 m 

long rather than in a dilution tunnel. Approximately 10X of the exhaust/air 

mixture was taken through two condensers in series, followed by a scrubbing 

tower and reaching finally an absolute filter. They have shown that the 

recovery of radioactive B(a)P added to the exhaust was better than 90X 

by this method. They also emphasize that the degradation of the exhaust 

would be minimum by trapping at subambient temperatures. 

GAS PHASE TRAPPING 

Gas phase components not trapped by Teflon membrane filter (or other 

filters) at 125°F are trapped, either by absorbant method (Spindt, 1974; 

Lee et al., 1979) or by a cryo-technique (Stenberg et al_., 1981; Pedersen 

et aT7,~l980). Lee et a_L (1979) used XAD-2 traps on gasoline emissions 

and^ound that a large portion of three-ring and four-ring PAH were present 

in the gas phase. A cryogradient system for the enrichment of gaseous 

PAH's and derivatives of these, in combination with conventional particle 

trapping on glass fiber filters, has been described in Stenberg et aJL 

(1983). Samples were taken from both gasoline and diesel exhausts, which 

were diluted in a tunnel. The sampling equipment (Figure 6) consists of 

a thermostatted filter holder, an ice/water condenser, a C02/ethanol 

condenser, a liquid nitrogen condenser, a pump system with flow elements, 

temperature/pressure sensors, valves and dry-gas meter. By using this 

system, they have shown that compounds like phenanthrene and pyrene were 

present at more than 50X in the gas phase after dilution of gasoline 

exhausts. The cryo-technique used by Pedersen et al_. (1980), consisted of 

two washing flasks immersed in liquid nitrogen and they have shown that 

five-ring PAH and larger molecules were absorbed to 100X on particles 

from gasoline emissions. Anthracene, f1uoranthene, and pyrene were predominant in the gas phase. 

40 
P.56 

TABLE 15. EMISSION OBTAINED FROM FTP a.b 

Pyrene 

BaP 

6 N02-B(a)P 

1 N02-Pyrene 

Diesel 

A

c 

15 

0.5 

<0.1 

2 

B

d 

NO® 

ND® 

NO® 

Emissions, 

Gasol 

A

c 

8 

3 

0.9 

0.1 

ine 

B

d 

ND® 

NO® 

NO® 

uq/km 

Gasol 

A

c 

0.7 

0.1 

<0.1 

<0.1 

ine, Catalyst 

B

d 

NDe 

<oei 

NO® 

ND® 

*Data of Williams and Swarin (1981) and Gibson (1982). 

Only particulate emission from dilution tunnel. 

°.A = Gibson, one vehicle, hot start. 

B = Williams and Swarin. Diesel: mean value of two vehicles; 

gasoline: mean value of seven vehicles; gasoline catalyst: 

mean value of four vehicles, cold start. 

ND - not determined. 

TABLE 16. EMISSIONS FROM FTP, COLD START a.b 

Emissions, pg/km 

Gasoline Gasoline, Catalyst Gasoline M15 Diesel 

Phenanthrene 

Fluoranthene 

Pyrene 

BghiF0 

CPcdP 

BaA 

Chrysene 

Bbk£e 

BePT 

BaP _ 

InsP9 

BghiP 

Coronene 

1.7 

12.0 

20.0 

6.9 

4.5 

6.2 

6.9 

6.8 

5.5 

1.7 

1.3 

3.9 

1.7 

0.06 

0.07 

0.2 

0.07 

0.06 

0.06 

0.2 

0.4 

0.3 

0.1 

0.3 

0.7 

0.8 

2.3 

3.3 

6.3 

4.7 

3.2 

2.0 

2.5 

3.0 

3.2 

1.1 

1.7 

5.2 

4.4 

223 

210 

263 

48 

3.4 

34 

46 

37 

33 

11 

14 

22 

12 

*0ata of Egeback (1982). 

Only particulate sample from dilution tunnel. 

tests; diesel two tests. 

^Gasoline blended with 15X methanol. 

°BghiF - Benzo(ghi)fluoranthene. 

xBbkF = Benzo(b and k)fluoranthene. 

BeP value includes also benzo(cd)pyrenone. 

9Ind P = Indeno (l,2,3-cd)pyrene. 

Mean value from three 

41 
P.57 

(J) <§)© (g 

(A) flexible metal tubing. 250 mm, (B) filter holder, 

diameter 127 mm, stainless steel; (C) Teflon bellows; 

(D) round-bottomed flask, 1000 mL; (E) normal glass 

joint; (F) 1ce/water condenser; (G) cryo vessel, dry 

ice/ethanol; (H) condenser cartridge; (I) ball glass 

joint; (J) cryo vessel, liquid nitrogen; (K) condenser 

cartridge; (L) Teflon/glass joint, temperature sensor. 

Figure 6. Cryogradient sampling equipment, Stenberg et aK , 1983. 

42 
P.58 

REFERENCES 

1. Begeman, CR . and J.M. Colucci. Apparatus for determining the 

contribution of the automobile to the benzen-soluble organics in 

air. National Cancer Inst. Monograph, 9:17-57, 1962. 

2. Chipman, J.C., and M.T. Massey. Proportional sampling system for 

the collection of an integrated auto exhaust gas sample. J. Air 

Pollution Control Assoc, 10:60-61, 1960. 

3. Egeback, K.E., ed. Chemical and biological characterization of 

vehicle exhaust emissions, when using different engine/fuel combinations. National Swedish Environment Protection Board. Stockholm, 

December 1982. 

4. Gibson, T.L. Nitroderivatives of polynuclear aromatic hydrocarbons 

In airborne and source particulate. Atmos. Environ., 16:2037-2040, 

1982. 

5. Grimmer, G., A. Hlldebrant, and H. Bohnke. Sampling and analytics 

of polycyclic aromatic hydrocarbons in automobile exhaust gas: 1. 

Optimization of the collecting arrangements. Zbl. Bakt. Hyg., I. 

Abt. B158:22-34, 1973. 

6. Gross, G.P. Gasoline composition and vehicle gas polynuclear aromatic 

content. CRC-APRAC project CAPE-6-68, July 30, 1972. 

7. Habbi, K. Characterization of particulate matter in vehicle exhausts. 

Environ. Sci. Technol., 7:223-234, 1973. 

8. 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. Kotin, P., H.L. Falk, and M. Thomas. Aromatic hydrocarbon II. 

Presence in the particulate phase of gasoline engine exhausts. 

Arch. Ind. Hyg. Occup. Hyg., 9:164-177, 1954. 

10. Kraft, J., and K.H. Lies. Polycyclic aromatic hydrocarbons in the 

exhausts of gasoline and diesel vehicles. Society of Automotive 

Engineers, Warrendale, PA, 1981, Paper No. 810082. 

43 
P.59 

'1 

11. Lee, F.S.C., T.J. Prater, and F. Ferris. PAH emission from a stratified-charge vehicle with and without oxidation catalyst: sampling 

and analysis evaluation. In: Polynuclear Aromatic Hydrocarbons, 

P.W. Jones and P. Leber, eds. Ann Arbor Science Press, Ann Arbor, 

MI, 1979. pp. 83-110. 

12. Mittler, S. and S. Nicholson. Carcinogenicity of atmospheric pollutants. Ind. Med. Surg., 26:135-138, 1957. 

13. Newhall, H.K., R.E. Jentoft and P.R. Ballinger. The effect of 

unleaded fuel composition on polynuclear aromatic hydrocarbon emissions, Society of Automotive Engineers. Warrendale, PA, 1981, 

Paper No. 7308334. 

14. Pederson, P.S., J. Ingversen, T. Nielson, and E. Larsen. Effects 

of fuel, lubricant and engine operating parameters on the emission 

of polycyclic aromatic hydrocarbons. Environ. Sci. Technol., 

14:71-79, 1980. 

15. Schuetzle, D. Sampling of vehicle emissions for chemical analysis 

and biological testing. Environ. Health Perspect., 47:65-80, 1982. 

16. Spindt, R.S. First Annual Report on Polynuclear Aromatic Content 

of Heavy Duty Oiesel Engine Exhausts Gases. Gulf Research and 

Development Co., CAPE-24-72, July, 1974. 

17. Stenburg, R.L., D.J. von Lehmden and R.P. Hangebrauck. Sample collection techniques for combustion sources - benzopyrene determination. 

Am. Ind. Hyg. Assoc. J., 22:271-275, 1961. 

18. Stenberg, U. The emission of polynuclear aromatic hydrocarbons 

from gasoline powered vehicles. Report to the Swedish EPA, February 1, 

1979. 

19. Stenberg, U., T. Alsberg and B.M. Bertilsson. A comparison of the 

emission of polynuclear aromatic hydrocarbons from automobiles using 

gasoline or a methanol/gasoline blend. Society of Automotive Engineers, Warrendale, PA, 1981, Paper No. 810441. 

20. Stenberg, U., R. Westerholm, T. Alsberg, U. Rannug, and A. Sundvall. 

Enrichment of PAH and PAH derivatives from automobile exhausts by 

means of a cryogradient sampling system. Paper presented at 6th 

International Symposium on PAH, Columbus, Ohio, October 23-27, 1981. 

21. Stenberg, U., T. Alsberg and R. Westerholm. Applicability of a 

Cryogradient Technique for the Enrichment of PAH from Automobile 

Exhausts: Demonstration of Methodology and Evaluation Experiments. 

Environ. Health Perspect., 47:43-51, 1983. 

22. Williams, R.L. and S.J. Swarin. Benzo(a)pyrene emissions from gasoline and diesel automobiles. Society of Automotive Engineers. 

Warrendale, PA, 1981 Paper No. 790419. 

44 
P.60 

PART II 

SECTION 2 

EXTRACTION METHODS FROM EMISSION PARTICULATES 

Diesel particulate emissions are a very complex mixture of carbonaceous 

matter containing adsorbed and/or condensed organic components from the 

combustion of fuel and lubricants. The organic solvent extractable fraction of these particulates is extremely complex and has been reported to 

contain hundreds of individual components. A typical experimental approach 

used in the characterization of the soluble organic fraction of diesel 

particulates involves extraction of the organic matter from the collected 

particulate using an organic solvent, concentration of the extract for 

subsequent analysis, fractionation into less complex non-polar and polar 

subgroups and, final concentration to facilitate chemical and biological 

analysis. 

EXTRACTION 

Two widely employed methods for removal of organics associated with diesel 

particulates are Soxhlet extraction and ultrasonic extraction using a 

suitable organic solvent. Petersen et al. (1980) have indicated that 

either technique can be used satisfactorily to measure the total soluble 

organic fraction. However, the total quantity of material extracted by 

these two methods is influenced by the type of particulate being extracted, 

contact time with the solvent, temperature of the solvent during extraction, and polarity of the solvent. Extraction by sonication is usually 

more rapid (about 2 hours) than using the Soxhlet technique (3-24 hours) 

but sonication requires several additional analytical steps to remove 

suspended soot and filter particles. Soxhlet extraction gives slightly 

higher mass and B(a)P recovery than ultrasonication extraction (Schuetzle 

and Perez, 1981). Soxhlet extraction can be conducted unattended and 

many particulate samples can be extracted simultaneously in separate 

apparatuses. 

The type of organic solvent used for extraction has received much attention. Solvents like methanol and aromatic-alcohol mixtures have been 

found to extract the largest quantities of material from the particulate, 

presumably because of increased extraction of inorganic material (Siak 

et aj,., 1979; Brooks et a_L , 1979; Swarin and Williams, 1979). Binary 

solvent systems using an aromatic solvent and alcohol give the highest 

recovery of mass and B(a)P when compared to most other solvent systems 

(Schuetzle, 1983). Aromatic solvents have been shown to extract the 

largest quantity of PAH (Choudhury and Ooudney, 1979). The largest 

quantity of biologically active material from particulates is extracted 

45 
P.61 

by dichloromethane and is usually the solvent chosen for biological 

screening (Huisingh et a_L , 1978; Brooks et al_., 1979). 

CONCENTRATION 

Concentration is necessary within various steps of the overall procedure. 

The solvent must be removed from the extract, or an aliquot of the extract, 

to determine the residue mass for calculating the total organic mass. 

Typical methods of concentration include removal of the solvent by rotary 

film evaporation (Petersen et al_., 1980), nitrogen stream blow-down (Swarin 

and Williams, 1979; Petersen et ah , 1980) and Kuderna-Danish concentration. 

For removing large quantities of solvent (1000 ml to 10 ml) rotary film 

evaporation is the most widely used method. Nitrogen stream evaporation 

is used to concentrate the solvent to several hundred microliters or to 

dryness. Kuderna-Danish concentration has not received much attention 

for the concentration of particulate extracts primarily because of the 

overall time of concentration. Another method of concentrating small 

volumes (10 ml to 1 ml or less) is by vortex evaporation. Using this 

method, the solvent is transferred to a centrifuge tube, which is placed 

in the vortex unit. During the vortex mixing, the solvent is removed 

while heating (20-60°C) under vacuum. 

FRACTIONATION 

Before chemical or biological characterization, fractionation of the 

particulate extract may be performed. For fractionation of the particulate extracts on the basis of chemical functionality, a variety of partitioning methods and chromatographic techniques can be used (Huisingh et 

al., 1978; Swarin and Williams, 1979; Choudhury and Dondney, 1979; Lyons, 

1562). 

By sequential liquid-liquid partitioning, acids and bases can be removed 

from the neutrals using aqueous solutions at low and high pH. Neutral 

organic compounds are further fractionated by solvent partitioning and/or 

column chromatography. Column techniques using Sephadex LH-20, silica 

gel and alumina have been developed to separate the neutrals based on 

compound classes. For separation of acids, bases, neutral nitrogen-containing compounds, saturated hydrocarbons and aromatic hydrocarbons, 

Jewell et al. (1972) have used a combination of ion-exchange coordination 

and adsorption chromatography. Rapid fractionation of particulate extracts 

by the use of high performance liquid chromatography (HPLC) has been 

reported by Huisingh et al_. (1978). This method was modified by Schuetzle 

and co-workers (1981) to produce six specific fractions. 

BIOLOGICAL EXTRACTION 

Organic solvents such as dichloromethane readily remove the organic 

compounds from the diesel exhaust particles; the extracted material is 

mutagenic to bacterial and mammalian cells. However, organic solvents 

used for extraction are not typical of the solvents present within biological systems such as the respiratory tract. Biological fluids have been 

used as solvents to extract the organic compounds, however, the removal 

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REFERENCES 

1. Brooks, A., R. Wolff, R. Royer, C. Clark, A. Sanchez and R. McClellan. 

Biological Availability of Mutagenic Chemicals Associated with Diesel 

Exhaust Particles. In: Health Effects of Diesel Engine Emissions, 

Vol. 1. W.E. Pepelko, R.M. Danner and N.A. Clarke, eds. U.S. EPA, 

Cincinnati, Ohio, EPA-600/9-80-057. 1979. 

2. Choudhury, D., and C. Doudney. Isolation of Mutagenic Fractions of 

Diesel Exhaust Particulates as an Approach to Identification of the 

Major Constituents. In: Health Effects of Diesel Engine Emissions, 

W.E. Pepelko, R.M. Danner and N.A. Clarke, eds. U.S. EPA, Cincinnati, 

Ohio, EPA-600/9-80-057. 1979. 

3. Huisingh, J., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, S. 

Tejada, J. Bumgarner, F. Duffleld, M. Waters, V. Simmon, C. Hare, 

C. Rodriguez, and L. Snow. Application of bioassays to 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. 

4. Jewel, D.M., J.H. Weber, J.W. Bunger, H. Plancher, and D.R. Latham. 

Anal. Chem., 44:1391-1395, 1972. 

5. King, L.C., M.J. Kohan, A.C. Austin, L.D. Claxton, and J. Huisingh. 

Evaluation of the release of mutagens from diesel particles in the 

presence of physiological fluids. Environ. Mutagen., 3:109-121, 

1981. 

6. King, L.C, K. Loud, S.B. Tejada, M.J., Kohan, and J. Lewtas. Evaluation of the release of mutagens and 1-nitropyrene from diesel 

particles in the presence of lung macrophages in culture. Environ. 

Mutagen., 5:577-588, 1983. 

7. L1, A.P., R.E. Royer, A.L. Brooks and R.0. McClellan. Cytotoxicity, 

mutagenicity and co-mutagenicity of diesel exhaust particle extracts 

on Chinese hamster ovary cells 1n vitro. Proc. 1981 EPA Diesel 

Emissions Symposium, Research Triangle Park, NC, October 5-7, 1981. 

8. Lyons, M. Comparison of Aromatic Polycyclic Hydrocarbons from Gasoline Engine and Diesel Engine Exhaust, General Atmospheric Dust, 

and Cigarette Smoke Condensate. Paper presented at the Symposium 

on the Analysis of Carcinogenic Air Pollutants. National Cancer 

Institute Monograph No. 9, 193-199. 1962. 

48 
P.64 

9. Petersen, B.A., C.C. Chuang, G.W. Kinzer, T.L. Hayes, P.W. Meehan, 

and D.A. Trayser. Diesel Engine Emissions of Particulates and Associated Organic Matter, NTIS No. PB-80-221963, 1980. , 

10. Schuetzle, D. Sampling of Vehicle Emissions for Chemical Analysis 

and Biological Testing. Environ. Health Perspectives, 47:65-80, 

1983. 

11. Schuetzle, D., F.S.-C. Lee, J.J. Prater, and S. Tejada. International 

Journal of Environmental Analytical Chemistry, 9:93-144, 1981. 

12. Siak, J.S., T.L. Chan, and P.S. Lee. Diesel particulate extracts 

in bacterial test systems. In: Health Effects of Diesel Engine 

Emissions, vol. 1, W.E. Pepelko, R.M. Danner, and N.A. Clarke, eds. 

U.S. EPA, Cincinnati, Ohio, EPA-600/9-80-057a, 1980. pp. 245-262. 

13. Swarin, S.J., and R.L. Williams. LC Determination of BaP in Diesel 

Exhaust Particulate: Verification of the Collection and Analytical 

Methods. Presented at the Fourth International Symposium on PAH, 

Columbus, Ohio, GM Research Paper GMR-3127, ENV #69. 1979. 

49 
P.65 . 

PART II 

SECTION 3 

REVIEW OF BIOASSAYS APPLIED TO UNREGULATED 

COMBUSTION EMISSION SAMPLES 

INTRODUCTION 

Most of the data describing the activity of automobile emissions in a 

broad spectrum of bioassays have been presented and analyzed in four 

publications. 

1. The Report of the Health Effects Panel of the Diesel Imports Study 

Committee, National Research Council, Health Effects of Exposure to 

Diesel Exhaust, National Academy Press, Washington, D.C , 1981. 

2. Toxicological Effects of Emissions from Diesel Engines, J. Lewtas, 

editor, Elsevier Biometical Press, New York, 1982/ Proceedings of 

the EPA 1981 Diesel Emissions Symposium held in Raleigh, North Carolina, 

October 5-7, 1981. 

3. Environmental Health Perspectives, volume 47, January, 1983. Mutagenicity and carcinogenicity of air pollutants. The proceedings of 

the Symposium on Biological Tests in the Evaluation of Mutagenicity 

and Carcinogenicity of Air Pollutants with Special Reference to Motor 

Exhausts and Coal Combustion Products, held in Stockholm, Sweden, 

February 8-12, 1982. 

4. Environmental International, Volume 5, 1981. A series of articles 

summarizing several years of investigation. 

The objective of this section is to summarize both the extent of testing 

and the qualitative responses for automobile emission products in short-term 

tests for mutagens and carcinogens. A concomitant objective is to attempt 

to select bioassays which are most suitable for routine analysis of 

emissions from new mobile sources based on the results of the tests. 

Review of the bioassay data is organized by the type of toxic endpoint 

measured and is subdivided into mammalian in vivo tests and in vitro 

mammalian/submammalian tests. Table 17 outlines the stratification of 

endpoints included in this evaluation. 

Evaluation of data from the bioassay data base will be examined using 

the procedure outlined in Figure 7. The end product of this procedure 

will be a battery of tests amenable to routine evaluation of emissions. 

50 
P.66 

Data relevant to the activity of specific fractions of particulate extracts 

and to extracts derived from various fuels and engine running conditions 

will not be addressed in this section because those parameters are reviewed 

in detail in other sections of the report. 

Tables 18-28 lists the reported responses of emission samples from diesel 

and gasoline powered engines. Each matrix consists of either the positive 

or negative data elements for a genetic or carcinogen bioassay. The 

absence of an entry in a square indicates a gap in the data base. 

OVERVIEW OF BIOASSAY RESPONSE 

The summary of positive and negative results by endpoint given in Tables 18 

through 28 includes the qualitative responses for all studies reported 

for automotive exhaust emissions. 

In addition to the obvious data gaps, some apparent significant contradictions in qualitative responses exist in the total data set shown in 

the previous set of tables. These are as follows: 

TABLE 17. STRATIFICATION OF BIOASSAY ENDPOINTS REVIEWED 

Genotoxic Effects 

A. Specific Locus Mutation in Prokaryotic and Eukaryotic Organisms 

B. Chromosome Alterations (Including Aberrations, Sister Chromatid 

Exchange, and Aneuploidy) 

C. Damage at the Primary DNA Level in Mammalian Cells In Vivo and I_n 

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-Tern Tumor Induction Assays in Rodent Species 

D. Co-Carcinogenesis Studies in Rodents 

51 
P.67 

STEP 

Collection, Collation and Review 

of Available Bioassay Data 

ACTION 

Preparation of Citation 

Index 

Analysis of Each Oata Element 

in a Response Matrix Organized 

by Endpoint 

Extraction of Qualitative 

Response, Dose Range, Test 

Conditions, Sample Source 

and Quantitative Effects 

Summary of all Qualitative 

Reponses (Positive and Negative) 

Comparison of sample type, 

sample source and quantitates response by assay. 

Tables 18 through 28 provide 

rapid analysis of data by 

end-point. 

Identify Bioassays Capable 

of Detecting Genotoxic/ 

Carcinogenic Effects of 

Automotive Emissions 

Table 29 summarizes 

the responses from 

Tables 18 through 28. 

Critique of All Bioassays 

Producing Positive Responses 

Tables 30 through 33 

show detailed analysis 

and critique of positive 

studies by endpoint. 

Distribution of Positive 

Bioassay Responses Across 

Sample Types 

Table 34 identifies tests 

most amenable to a broad 

range of sample types. 

Selection of Candidate Bioassays 

for use in Emission Screening 

Tests which meet all 

previously described 

criteria of a good 

screening test and are 

responsive to the broadest 

range of sample types and 

sources will be considered 

candidate screening tests. 

Figure 7. Bioassay data evaluation matrix. 

52 
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CM • CktMM MMtirmr y Mi l 11 M 

U t t • S«« Maud rMiul w IIUM I 

V79 « ChlMM tanUr IUM. ctll I U 

0) 

oo 
TAUE 20 . NATRIK Of POSITIVE RESPONSES FOR 

CYI06ERI1ICS ASSAYS 

TAILS 21. NATRIK OF NEGATIVE RESPONSES FOR 

CYTOGENETICS ASSAYS 

cn 

WHOLE 

EXHAUST 

VAPOR PHASE 

ONLY 

UNEKTRACTEO 

PARTICULATES 

BIOLOGICALLY 

EXTRACTED 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

OIESI 

LIGHT DUTY 

Trenascentta 

Nicronucleus 

Assay 

SCE tn Haasttr 

Lung Calls 

SCE tn Nouse 

Rone Narrow 

Cells 

• SCE tn HtMttr 

Lung Cells 

• SCE tn Nouse 

B O M Narrow 

Cells 

• SCE tn Fetal 

Liver Calls 

• SCE in Haaster 

Lung Cells 

• Aberrations in 

Lyaphocytes 

EL 

HTAvYDUTY 

• SCE in CHO 

Cells 

• Aberrations 

tn vitro in 

BkfctTU 

GASOLINE 

LIGHT DUTY 

• SCE in CHO 

Cells 

• Aberrations 

in CHO Cells 

HEAVY DUTY 

• SCE in CHO 

Cells 

OIESEL GASOLINE 

WHOLE 

EXHAUST 

'APOR PHASE 

ONLY 

NiEXIRACTEO 

•ARTICULATES 

BIOLOGICALLY 

EXTRACTEO 

PARTICULATES 

3RGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

LIGHT DUTY 

• Nouse Nicronucleus Assay 

• HMster Nicronucleus Assay 

• SCE tn House 

Bone Harrow 

Cells 

• S U tn Fetal 

Liver Cells 

• Nouse Dominant 

Lethal Assay 

- Nouse Hertlable 

Translocation 

Assay 

• SCE in Dog 

Lyaphocytes 

• Nicronucleus In 

Dog Lyaphocytes 

• Nouse Nicronucleus Assay 

• Haasttr Nicronucleus Assty 

• SCE in fetal 

Liver Cells 

kk ' 

- Haastar Micronucleus Assay 

• Nouse Nicronucleus Assay 

WAVY DUTY LIGHT DUTY "HEAVY DUTY 

SCE - Sister chromatid exchange 
TAIU 22. MATRIX Of MSITIVE RESPONSES fOR PRIMARY 

OIESEL GASOLINE 

TAIU 23. MATRIX Of NEGATIVE RESPONSES FOR 

PRINARV DMA DAMAGE 

in 

WHOLE 

EXHAUST 

VAPOR PHASE 

ONLY 

UNEXTRACTEO 

PMTICUUTES 

•1010C1CALIY 

EXTRACTED 

PMTICMUTES 

ORGANIC 

SOLVENT 

EXTRACTED 

PARTICULATES 

LIGHT eUTV 

• MA lapatr In 

NuaeaKP Cells 

• MA Repair tn 

k*P HUM* fibroblast Call* 

• MS la EUC 

M M M Calls 

• MS la lat 

Priaary Ntpatacytts 

• MS ia trtchtal 

apttMllM af 

MMtar* 

• Vttst (I?) 

NlUtlc AaceaAi 

Mtt M 

mm DUTY LIGHT DUTY 

• MS la Rat 

PrtMry 

Hapattcytes 

• MA Irtakage 

tn SHE Calls 

WAVY DUTY 

WHOLE 

EXHAUST 

VAPOR PHASE 

ONLY 

UNEXTRACTEO 

PARTICULATES 

BIOLOGICALLY 

EXTRACTED 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

OIESEL 

LIGHT DUTY 

• DMA Repair tn 

M M M Xf> Calls 

• Ytast Mitotic 

AtCMbtMtlM 

la Strata Dl 

• MAIraakat* ia 

SHE Calls 

HEAVY DOTY 

• MA Ireakage ta 

SHE Calls 

• MS ID WI-JR 

M M Calls 

GASOLINE 

LIGHT DUTY ICAVYIUTV 

XP » Xtroatraa ptgatntosua 

MPS - Uaicbadultd MA Synthesis 

Sat • SyrlM aaastar aaaryo 

XP • Xe rata r t ptgatntosua 

Sat • SyrtM MMter aabryo 
TAILE 24. NATIIX Of POSITIVE RESPONSES FOR 

JN VITRO TRAaVOMATION ASSAYS 

TABLE 2S. NATIIX OF NEGATIVE RESPONSES F M 

IN VITRO TRAMSFORNAT1M ASSAYS 

WHOLE 

EXHAUSI 

VAPOR PHASE 

ONLY 

UNEXTRACTEO 

PARTICULATES 

BIOLOGICALLY 

EXTRACTEO 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

OIESEL 

' LIGHT DUTY 

• Enhanctatnt of 

Viral Transforaation tn 

SHE Calls 

• lalb/c 31 J* 

Cell IransfortMtton 

KAVYDUTY 

GASOLINE 

LIGHT DUTY 

• Enhanceacnl of 

Viral Iransforforaation in 

SHE Cells 

• Balb/c 3T3 

Cell Iransforaat ton* 

HEAVY DUTY 

WHOLE 

EXHAUST 

VAPOR PHASE 

M L V 

UNEXTRACTEO 

PARTICULATES 

BIOLOGICALLY 

EXTRACTEO 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

OIESEL 

LIGHT DUTY 

• Balb/c 3T3 

Cell Trans forMtion 

HEAVY DUTY 

• Enhanctatnt of 

Vtrtl TransMtion la SHE 

Calls 

GASOLINE 

LIGHT DUTY- " 

• Balb/c 3T3 

Call TrensforMlioo 

aTAVYDUTY 

• Balb/c 3T3 

Ctll TransforMtioa 

SHE « Syrian haasttr bryo 

'Equivocal response 

SHE « Syrian baasler tanryo 

Tl 
TABLE 24. MATRIX Of POSITIVE RESPONSES FOR 

JN VIVO TM M STUDIES 

TAIU 27. MATRIX OF NEGATIVE RESPONSES f M 

IN VIVO TUMOR STMIES 

cn 

* *j 

WHOLE 

EXHAUST 

VAPOR PHASE 

ONLY 

UNEXTRACTEO 

PARTICULATES 

BIOLOGICALLY 

EXTRACTEO 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PARTICULATES 

OIESEL 

LIGHT DUTY 

• Rasprialory 

Co-Card oogenesis Studies 

utth OEN tn 

haasters 

• Subcutaneous 

Injection site 

tuaors to aice 

• Skin tuaor 

Initiation In 

Senear alee 

HUVYDUTY 

GASOL 

LIGHT DUTY 

- Skin tutor 

initiation 

in Senear aice 

NE 

"HEAVY DUTY 

OEN * OtathylnitrosMine 

WHOLE 

EXHAUST 

VAPOR PHASE 

M L V 

UNEXTRACTEO 

PARTICULATES 

IIOLOGICAIIV 

EXTRACTEO 

PARTICULATES 

ORGANIC 

SOLVENT 

EXTRACTEO 

PMIICULATES 

DIESE 

LIGHT DUTY 

• Liver focus 

essay tn atce 

• Haastar respiratory tract 

tuaors 

• lung tuaors in 

Strain A aict 

- Lung tuaors tn 

Senear aict 

• Haastar respiratory tract 

tuaors 

• Skin tuaor 

initiation in 

C3H aict 

• Skin tuaor 

initiation tn 

Senear aict 

• SAIa tuaor 

initiation tn 

C3H aice 

• Caaplete Carcinogen la 

Saucer aict 

L 

HTJWVDUTY 

GASOLINE 

LIGHT DUTY " aTAVYOUTV 

s 
P.73 

TABLE 28. LONG-TERM STUOIES OF THE HEALTH EFFECTS 

OF OIESEL EXHAUST, COMPLETED OR IN PROGRESS 

Laboratory 

Environmental 

Protection Agency 

Fraunhofer 

Institute 

General Motors 

Lovelace Inhalation 

Toxicology Research 

Institute 

Battelle-Northwest 

Southwest Research 

Institute 

Species 

Chinese Hamster, 

Mice, Rats, Cats 

Syrian Hamsters 

Rats, Guinea Pigs 

Mice, Rats 

Rats 

Syrian Hamsters 

Syrian Hamsters, 

Rats, Mice 

Particle 

Concentration (ug/m3) 

6000-12000 

4200a 

250, 750, 1500 

350, 3500, 7000 

8300 

7300 

1:60, 1:120, h 

1:360 dilution0 

Response 

Negative 

Negative 

Negative 

Negative 

Negative 

Negative 

?Also, exposures to gaseous emissions only without particles. 

Particle concentrations not given. 

From McClellan et al., 1982. 

58 
P.74 

GENE MUTATION BIOASSAYS 

Organic solvent extracts of light duty diesel particles were mutagenic 

in CHO cells but not V-79 cells. Both cell lines are derived from Chinese 

hamsters and generally respond similarly. Attempts should be made to 

resolve this apparent discrepancy, especially since there was also a 

contradictory response in CHO cells with heavy duty diesel solvent extracts. 

CYTOGENETIC BIOASSAYS 

One series of studies in which SCE was measured in bone marrow cells 

indicated a much stronger response for whole exhaust particles injected 

intraperitoneally in DMSO than for organic solvent extracts of the particles injected intraperitoneally. Considering that significant differences in bioavailability of mutagens was anticipated between these two 

samples, the responses reported are surprising because they contradict 

the anticipated responses. The test procedures and data from these two 

studies should be reviewed in detail. 

PRIMARY DNA EFFECTS 

Most of the responses reported were consistent throughout all bioassays. 

The only apparently inconsistent effects were reports of mitotic recombination. Studies using yeast strain 03 were reported as positive for organic 

solvent extracts of diesel emission particles whereas results for extracts 

using yeast 07 were reported to be weakly positive. The source of diesel 

emissions was different in the studies and may account for this apparent 

discrepancy. A comparison of both sets of data should be made in an 

attempt to resolve the question. 

CARCINOGENESIS STUOIES 

There appeared to be considerable variability among the responses for 

direct transformation studies in Balb/c 3T3 cells. The ability of organic 

solvent extracts to produce transformation varied considerably, which 

possibly indicated an engine type specificity. The variable results might 

also indicate test system variability, especially since the positive 

responses reported were neither generally dose dependent nor strong. A 

systematic analysis of this body of data should be made using a properly 

selected statistical approach. 

Among the in vivo studies it was established that organic solvent extracts 

from light""3uty diesel emissions could initiate skin tumors in SENCAR 

mice but not in C3H mice. Both mice were susceptible to PAH control 

agents, but the SENCAR mice were selected because they were sensitive 

to early and repoducible apperance of papillomas after PAH initiation 

and TPA protomotion. 

ANALYSIS BY TYPE OF SAMPLE TESTED 

The data base for whole exhaust, vapor phase, or unextracted particles 

is extremely limited and drawing generalizations from the data base will 

59 
P.75 

be extremely restrictive, especially since most of the findings are 

negative. Table 29 describes the distribution of positive responses 

according to parameters of bioassay endpoint, sample type, and engine 

type. It is clear that most of the bioassay data consist of studies with 

light duty diesel particulates extracted with organic solvents. 

EVALUATION OF POSITIVE RESPONSES 

In order to use the current bioassay experience, for the purpose of 

selecting useful tests of automobile emissions in a certification 

program, the results from those bioassays able to detect biological 

activity must be examined in detail. Tables 30 through 33 provide an 

analysis of the results from bioassays using one of the emission types 

listed in Table 29. The results are organized by endpoint, relevant 

references, test conditions employed, and test response; specific comments 

are made when appropriate. 

CRITERIA FOR BIOASSAY SUITABILITY FOR EVALUATING MOTOR VEHICLE EMISSION 

SAMPLES 

In making an assessment of bioassay suitability, several parameters were 

considered. 

These are as follows: 

» 

1. Test Sample Compatibility - demonstrated adaptability to samples 

other than organic solvent extracts is considered a desirable 

feature. 

2. Relevance of endpoint - this parameter is not critical in a screening 

mode but would become more important if the results from bioassays 

are extrapolated to specific toxic endpoints. Tests directly assessing tumorigenicity, for example, would be preferable in estimating 

the potential carcinogenic hazard of a sample than a test for unscheduled DNA synthesis (UDS). 

3. Relevance of exposure route - this parameter assumes significance 

in assessments that require extrapolation to ambient exposures and 

risk estimates. 

4. Relevance of the target cell - mammalian cells might well be expected 

to provide greater reliability for extrapolation purposes than would 

bacteria, yeast, insect or plant cells. While these latter cell 

types offer distinct advantages in soon instances, some bioassays 

using non-mammalian cells may be inappropriate for evaluating 

combustion emissions. 

5. Reproducibility and quantitation - assays which do not provide reproducible responses under identical treatment conditions with one sample 

would not be useful in routine screening of new emission sources 

where variables such as engine and fuel types may produce subtle 

and important variations in response patterns. Quantitative data 

analysis and induction of dose-dependent responses are also important 

criteria. 

60 
P.76 

TABLE 29. DISTRIBUTION OF BIOLOGICAL ENDPOINTS 

EXAMINED ACROSS AUTOMOTIVE ENGINE TYPES 

A. 

B. 

C. 

D. 

Endpoint 

Gene Mutation 

Chromosome 

Primary DNA Damage 

Carcinogenicity Studies 

WE 

X 

X 

1. In vitro Transformation 

2. In vivo Initiation X 

3. In vivo Co-carcinogen 

4. Jji vivo Promotion 

X 

Diesel Engine 

Light Duty 

VP UP OP BP 

X 

X 

X

b 

X 

X 

X 

X X 

X 

X 

X 

X 

X 

Heavy Duty 

OP 

X 

X 

X 

X 

X 

Gasoline 

Light Duty 

OP 

X 

X 

X 

X 

X 

Engine 

Heavy Duty 

OP 

X 

X 

WE = whole exhaust 

VP = vapor phase only 

UP = unextracted particulate phase only 

BP = biologically extracted particulate phase 

OP = organic solvent extracted particulate phase 

uMost of the data employed came from engines using leaded gasoline. 

When tested in cell capable of engulfing the particles. 

61 
TABLE 30. NOTOR VEHICLE EMISSIONS: A SUMMARY OF POSITIVE GENE NUTATION BIOASSAY RESULTS 

Bioassay Reference Test Conditions Response its 

Specific locus Mutation in coltfora 

bacteria 

Claxton, 1982 

Liber et al. 1981 

Claxton, T981 

Lewtas, 1983 

Wang et al., 1981 

ro 

Specific locus mutation in cultured 

aaaaalian cells 

1. Nouse Lymphoma (TIC) LS178V Lewtas, 1983 

Mitchell et al, 1981 

U.S. EPAri98l 

Forward and reverse autation 

tests have been reported for 

Salmonella typhi murium, and 

for reverse autation In |. 

coli WP2 uvrA. Nonacttvition and activation test 

conditions using organic 

solvent and biological 

fluid extracts of eaission 

particles have been run. 

Both LO and HD diesel and 

gasoline engines run on a 

range of different fuels 

were used. Recent studies 

have been conducted with 

vapor phase coaponents. 

This bioassay is a forward 

autation assay using an 

autosomal gene (TK). The 

test is conducted under 

nonactivation and activation test conditions. 

Tests were reported for 

organic solvent extracts 

of LO and HO diesel and 

gasoline engine emissions. 

The Salaonella assays indicate 

the presence of potent direct 

acting bacterial autagens in 

organic solvent extract* of all 

types of eaission particles. 

The response range is broad 

and depends to great extent 

on variables such as engine, 

fuel and collection conditions. 

Recent studies have also demonstrated the presence of bacterial autagens in the vapor 

phase of automotive eaission. 

This assay responds to the 

autagens present in organic 

extracts of all engine types. 

Dose related Increases are 

typically generated froa both 

activation and nonactivation 

test conditions. 

While the addition of 

S9 tended to reduce 

the biologial activity 

of dlesal »aaplat in 

the Salaonella tests, 

it virtually aliainated activity in 

the E. coli bioassay. 

Gasoline samples show 

enhanced Mutagenicity 

in the presence of S9. 

Diesel saaples generally do not require 

S9 for maximum mutagenicity. Gasoline 

saaples generally 

showed enhanced mutagenicity in the 

presence of S9. 

Chinese Hamster Ovary (CHO) 

Cells 

Li et al, 1982 

Casio et ai, 1981 

CheschTer, et a h 1980 

This bioassay uses an X1 inked gene (HGPRT) to 

assess mutagenicity. 

Tests on LO diesel and 

gasoline engine amissions 

(organic solvent extracted) 

were conducted under nonactive test conditions. 

CHO protocol aod!fled by Li 

gave rise to positive and generally dose-dependent responses 

for both engine types. Mutagenic responses were observed 

over a 20-50 ug/al dose range 

of the extract. 

Earlier studies using 

the exposure protocol 

of HSIE, resulted in 

very weak to negative 

responses In the CHO 

test system with 

particle extracts. 

Whole particles were 

shown to be even 

more mutagenic after 

CHO cell engulfment. 
TAILE 30. NOTOR VEHICLE ENISSIONS: A SUMMARY Of MSITIVE GENE NUTATION IIOASSAY RESULTS 

(Continued) 

Bioassay Reference Teat Conditions Response Consents 

3. lyaphoblaat cell* Liber et al, 1981 

•arfaecnt et aj., 1982 

This bioassay employ* the 

HGPRT locus described 

Studies with and without 

metabolic activation ware 

conducted using organic 

solvent extracted 10 

diesel particles froa 

one vehicle. 

Tha result* ware positive, but 

only under activation test 

conditions. The active dose 

range was 10-20 ug/al of the 

extract. 

Thl* la the only 

report of a aaamallan 

cell assay exhibiting 

an S>-dependant response for diesel 

emissions extract. 

XT fibroblast calls HcCoralck et al, 1980 

o» 

S. lala/C 3T3 fibroblast Curren et al, 1981 

Tradescaatla Schalrer et al, 1961 

This bioassay employs tne 

MGMff locus described above; 

however, the fibroblast 

cell* eapleyed were explantc 

froa aa Individual with 

xeroderaa plflaantasua 

and are deficient la ONA 

repair. Only amaactivatiea 

test cendttleas ware eaployed. 

Tha temple* te*tad were unextracted and organic solvent 

extracted LO diesel particles. 

Ouabain re*l»taaca (aaalnant autation) usIng 

organic solvent extracts 

of diesel and gasoline 

engines, lasts Included 

S9 and uoerS* stadias. 

Tradescaatla plaata exposed 

te LO diesel whale exhaust 

emission*. Stamen hair 

cells are scored for specific 

locus autation. 

The tests for both particles 

and extracts were reported 

positive In the absence of 

metabolic activation. 

•esults were equivocal. No 

dose response results reported. 

las assay was reported as 

positive. 

This is a report of 

uneatracted particles 

Inducing nutation la 

cultured aaamallan 

cells. 

The report Indicated 

possible positives 

for one diesel sample 

sample and ena gasoline sample. 

LO * Light duty 

HO * Heavy duty 

oo 
TABLE 31. NOTOR VEHICLE ENISSIONS: A SUMMARY OF POSITIVE PRINARV 

DNA I1ANAGE BIOASSAY RESULTS 

Bioassay Reference lest Conditions Coaaeott 

explants 

la huaan IP fibroblast larfnecht et al. 1982 

Unscheduled DNA Synthesis (UOS) la 

aaanalian cells 

National Acadeay Press, 

1901 

U.S. EPA. 1983 

Cfl 

Nitettc Recoabination in 

Sacchareayces 

U.S. EPA. 1983 

Unscheduled ONA Synthesis (UOS) In 

haaster tracheal organ culture 

Schlff et al, 1981 

LO diesel particles 

(unextrected) and organic 

solvent extracts froa the 

particles were tested for 

direct ONA daaige using a 

comparative cytotoxicity 

test. 

Organic solvent extracts 

froa LO diesel emitstons 

were tested In several 

mammaliaa cell lines and 

primary cells far their 

ability to ttlaulete the 

eniyaatic ONA repair systea. 

Tests In WI-38 and EUE cell 

lines were conducted with 

and without S9 ain. The 

priaary cells used In one 

study were aetabollcally 

coapetent liver hepatocytes. 

Organic solvent extracts 

froa e LO ealssian sample 

were tested la several yeast 

strains. The 07 strain 

combines three endpoints 

(mutation, mitotic fane 

conversion and altetic recoabination). Tests were all 

conducted under nonactivation 

and activation test conditions 

The ability af aa organic 

solvent extract froa LO 

diesel amissions to ellet 

UOS In priaary tracheal 

cells located tn an organ 

culture was analyzed using 

an autoradiographic tcchnlThe results for both saaples 

were positive. No activation 

was required. 

The results for the LO diesel 

saaples were reported te be 

positive. 

The priaary reference 

for this study is 

McCormick et el.. 

1980. ia MealtK 

Effects of Oiesel 

Engine Eaission, 

EPA syapotlua. 

Sample was weakly positive 

under nonactivation test 

conditions tn the altetic 

recombination endpoint. 

A siatlar study 

employing sane endpoint In a different 

yeast strain (03) 

was reported negative. 

The saaple was positive at 1.0 

and 10 ug/al of the extract. 

DNA Breakage in Syrian Hamster 

Eabryo (SHE) Cells 

Caste et al. 1981 Organic solvent extracts 

of gasoline engine particulate tested in vitro 

in SHE cells. The eSF~ 

point was the sedimentation shift for ONA in 

alkaline sucrose density 

gradients. 

Positive response at 250 ug/al 

of gasoline particulate emission extracts. 

Results from light 

duty diesel extracts 

negative. 
TABLE 32. MOTOR VEHICLE ENISSIONS: A SUMMARY OF POSITIVE CYTOGENETIC 

BIOASSAY RESULTS 

Bioassay Reference Test Conditions Response Comments 

Nicronucleus Assay in Tradescantia Na, et al 1981 

Sister chromatid exchange (SCE) 

tn hamster lung cells 

Sister chromatid exchange (SCE) 

In mouse bone marrow cells 

Guerrero and Rounds, 

1901 

Guerrero and Rounds, 

(in press) 

Perotra, 1982 

Ol Sister chromatid exchange (SCE) 

in cultured aaanaltan cells 

Chromosome aberrations in 

cultured mammalian cells 

Mitchell et al. 1981 

Lewtas. 1» 3 

U.S. EPA, 1983 

Lewtas. 1983 

U.S. EPA, 1983 

Sister chromatid exchange (SCE) 

in fatal liver cells 

Pereira, 1982 

Plants exposed to whole 

diesel exhaust (LO) and 

stamen hair cells scored 

for micronuclei. 

SCE observed in explanted 

lung cells following exposure by inhalation and 

Instillation to diesel 

emissions. 

Unextracted diesel particulates administered by I.P. 

at 300 mg/kg BU (DMSO was 

the solvent). Organic 

extracts of diesel particulates administered at 

800 mg/kg BW. 

Tests conducted using 

organic solvents of diesel 

(LO and HD) and gasoline 

engines. Most tests were 

conducted both with and 

without S9 mix. 

Tests were conducted without S9 in CHO cells 

using organic extracts of 

diesel and gasoline engines. 

Human lyaphocytes were 

exposed to diesel organic 

extracts with and without. 

S9 aix. 

Pregnant Syrian hamsters 

were exposed to LO diesel 

whole exhaust, unextracted 

particles and solvent 

extracts of particles. 

Liver cells were removed 

from developing embryo 

and screened for SCE. 

Positive 

Positive at 12 mg/m* to whole 

diesel exhaust; particles were 

positive at S.O mg/animal, and 

organic extract of particles 

were positive at 0.6 ag/aniaal. 

The whole particles Increased 

SCEs from 5.6/cell in the 

control to 11.3 at the active 

dose. The organic extracts 

increased the control value of 

5.6 to 9.4/cell. 

Organic solvent extracts of 

collected diesel and gasoline 

particulates are positive 

producing dose related increases. 

Metabolic activation was not 

required for SCE induction. 

Chromosome aberrations were 

observed without S9 mix 

tn both cell types. 

Active dose ranges 

were from about 1-10 ug/al. 

The presence of S9 significantly reduced the clastogenic 

activity of diesel emission 

extracts. 

Organic solvent extracts were 

positive. The other two exposures did not produce effects. 

The exposures for the negative 

studies were 12 mg/m*4 x 12 days 

and 300 mg/kg IN x 1 day. 

respectively. No dose level(s) 

were given for the extract. 

Statistical treatment 

of data showed significant p values. 

No statistical 

analyses were used 

to confirm these 

effects which were 

not found positive. 

Positive effects 

were statistically 

significant. These 

data are supported 

by jn vivo studies. 

These positive 

responses were not 

confirmed by in 

vivo studies scoring 

for chromoso 

aberrations. 

No data were shown 

for this study and 

its usefulness in 

evaluating the 

genotoxicity of 

diesel emissions 

must be seriously 

questioned. 

BW = Body Weight 

oo 

o 
TABLE 33. NOTOR VEHICLE ENISSIONS: A SUMMARY OF POSITIVE CELL TRANSFORMATION 

BIOASSAY RESULTS 

Bioassay Reference Test Conditions Response Coaaants 

Viral enhancement of cell transformation (jn vitro) 

Casto, et al, 1981 

Lewtas ,1981 

Cell transformation (in vitro) Curren, et al, 1981 

Lewtas, I96 T 

U.S. EPA. 1983 

ot 

Ol 

Skin painting initiation studies 

tn SENCAR aice 

Nesnow, et al. 1983 

Slaga, eTaT7 1981 

Nesnow, et al. 1982 

Skin tuaors at injection site Pott and Stober, 1983 

Co-carcinogenic evaluation of 

exhaust and diethylnitrosaaine 

(OEN) treatment 

Pott and Stober, 1983 

Priaary Syrian hamster 

embryo cell explants 

(containing SA7 virus) 

were exposed to organic 

solvent extracts of LO and 

HO diesel and gasoline 

engine emissions. 

A series of tests using the 

Balb/c 313 cell line were 

used to compare a similar 

set of organic extracts with 

those used in the enhancement of viral transformation assay. These studies 

were conducted with and 

without aetaboltc activation. 

Each saaple was initiated 

with 5 doses using 40* & 

40 9 (80 aice) per dose. 

Beginning 1 week after 

treatment, 2.0 ug TPA was 

administered twice weekly. 

Skin tuaors were recorded 

weekly. 

Subcutaneous injection 

of automobile exhaust condensate (AEC) were administered to atce tn a single 

injection. The frequency 

of mice with tuaors at the 

injection site are calculated. 

HaasUrs pretreated with DEN 

at 1.5 and 4.5 ag/kg BW and 

then exposed to whole diesel 

exhaust and to the vapor 

phase only for 120 weeks. 

The animals were scored for 

papillomas of the larynx. 

LO diesel emission extracts 

enhanced viral transformation 

in SHE cells over a dose range 

of 60-250 ug/ml. The HD saaple 

was active at 500 ug/al. The 

results were generally dose 

related. 

Balb/c 3T3 results were positive for L0 diesel and gasoline 

engines without S9. When tested 

with S9, only the gasoline eaission saaple attained statistical 

significance. Independent tests 

using the extract of two different L0 diesel engines were 

negative in this bioassay. One 

HO diesel emission exhaust was 

negative In this test. 

Saaples froa L0 diesel engines 

and LO gasoline engine produced 

dose-related increases in skin 

tuaors. The maximum activities 

were observed at dose levels of 

1-10 mg/g extract per aouse. 

The AEC used in this study was 

not clearly defined as to 

origin. The dose levels and 

tumor incidences were also 

difficult to evaluate. The 

results for AEC were clearly 

positive in this test. 

Exposure to whole exhaust and to 

the vapors Increased the larynx 

papillomas at 4.5 mg/kg OEN 

but not at 1.5 mg/kg. The 

vapor component was clearly 

iaplicated since whole exhaust 

did increase the effect further. 

The aachanita of this 

bioassay ts not 

clearly elucidated. 

The nature of the 

data from a quantitative and dose 

related basis 

suggests that these 

results are not 

easily interpreted. 

The availability of 

PAHs aay Influence 

these results as 

saaples with higher 

BAP content showing 

greater activity. 

The most potent diesel 

saaple was tested as 

a complete carcinogen 

in SENCAR mice and 

was not active. 

This bioassay Is 

dependent to a 

large extent on 

the level of PAH 

in the test saaple. 

The cocarcinogen 

effect aay be the 

result of SO,. NO, 

or oione as well as 

specific chemicals 

in the vapor phase. 

00 
P.82 

SELECTION OF BIOASSAYS TO FORM A MINIMUM EVALUATION MATRIX 

Objective analysis of the current data base for automobile combustion 

emissions and test performance criteria has resulted in the selection of 

three bioassay types which appear to merit further study as screens for 

automotive emission certification. 

The Ames Salmonel1 a/microsome reverse mutation assay appears to be useful 

for several reasons. It responds very sensitively to the mutagens bound 

to emission particulates especially the nitro-PAHs (Rosenkranz, 1981). The 

test is flexible and has been used to assess the mutagenic components of 

the vapor phase of automobile emissions (Stenberg, 1983) and the bioavailability of mutagens in biological fluids (King et al., 1981; Brooks et aL , 

1981). The data generated by the Ames test meet aTT quantitative and dosedependency requirements. Also, the sample size required to perform the test 

is extremely small compared to that required in other types of bioassays. 

Cytogenetic endpoints and especially sister chromatid exchange (SCE) 

showed very good sensitivity to the genotoxic components of automotive 

emissions. The tests can be conducted J_Q vitro with particle extracts as 

well as |n vivo using whole exhaust or only the vapor phase with the 

expectation of detecting significant responses. The SCE endpoint, although 

not directly linked to a toxic phenomenon, is perhaps the most versatile 

and applicable to emission testing among the three tests selected. 

The i_n vitro mammalian cell gene mutation assay is the third test Considered applicable to emission screening. This determination 1s based on 

the assay's quantitative response to organic solvent extracts and, in 

some cases, to whole unextracted particles (Cheschier et aj_., 1980; 

McCormick, 1979). The CHO and Mouse Lymphoma assays generate data from 

automobile emissions which meet all quantitative and dose-dependency 

criteria. The endpoint is relevant, but as noted in the detailed analysis 

of test response, there were a few contradictory responses which should 

be reviewed and, if possible, resolved prior to developing protocols for 

test implementation. 

Some of the information used to support the selection of these three tests 

is shown in Table 34 which identifies the range of susceptibilities of 

various bioassays to the types of samples evaluated. With the exception 

of Tradescantia and the in- vivo cocarcinogenicity studies, both of which 

fail to meet the specifications required for routine application to emission assessment, the three bioassay (endpoint) types selected for emission 

testing are the only bioassays which demonstrate positive responses beyond 

organic solvent extract samples. The ability to respond to unextracted 

particles suggests a higher level of sensitivity of these tests to the 

possibly relevant genotoxic agents bioavailable from emission particles. 

While the use of organic solvent extracts may represent the most feasible 

routine test sample for testing, future methods may rely more upon! examination of unextracted particles. ! 

A detailed analysis of the reliability, reproducibility and sensitivity 

of these bioassays is presented in Part III of this document. This information will provide the basis to interpret test responses from new emission 

sources for their genotoxic potential. 

67 
TABLE 34. DISTRIBUTION OF POSITIVE RESPONSES IN SUSCEPTIBLE BIOASSAYS 

ACROSS SAMPLE CATEGORIES 

Bioassays 

Gene Mutation 

• Microbial 

• Mammalian Cells In Vitro 

• Plants 

• Insects 

• Mammals In Vivo 

Cytogenetic Endpoints 

• Mammalian Cells In Vitro 

• Plants 

• Manuals in Vivo 

Primary DNA Effects 

• In Vitro Hawaiian 

• Tn Vivo/In Vitro Mamalian 

Carcinogenesis Endpoints 

• Mammalian Cells In Vitro 

• In Vivo MaMals (Tcoaplete) 

• Iii Vivo Initiation 

• Tn Vivo Co-carcinogenesis 

Organic Solvent 

Extracts of 

Particles 

i 

-7 

i 

-V 

Biological 

Extracts 

Particl. 

(V) 

Fluid 

of 

ss 

Unextracted 

Particles 

U) 

t V 

Vapor Phase 

Alone 

U) 

V 

Whole 

Exhaust 

i 

-7 

(V) = data consists of limited experimentation which may not apply to all bioassays of this class. 
P.84 

REFERENCES 

1. Barfknecht, T.R., R.A. Hites, E.L. Cavaliers and W.G. Thilly. Human 

Cell Mutagenicity of Polycyclic Aromatic Hydrocarbon Components of 

Diesel Emissions. Elsevier Science Publishing Co., 1982. 

2. Brooks, A.L., R.K. Wolff, R.E. Royer, CR . Clark, A. Sanchez and 

R.O. McClellan. Deposition and Biological Availability of Diesel 

Particles and their Associated Mutagenic Chemicals. Environmental 

International, Vol. 5, 1981. pp. 263-267. 

3. Casto, B.C., G.G. Hatch, S.L. Huang, J. Lewtas, S. Nesnow and M.D. 

Waters. Mutagenic and Carcinogenic Potency of Extracts of Diesel 

and Related Environmental Emissions: I_n Vitro Mutagenesis and Oncogenic Transformation. Environment International, Vol. 5, 1981. 

pp. 403-409. 

4. Chesccheir, G.M., III, N.E. Garrett, J.O. Shelbarne, J.L. Huisingh, 

and M.D. Waters. Mutagenic Effects of Environmental Particulates 

in the CHO/HGPRT System. In: Short-Term Bioassays in the Analysis 

of Complex Mixtures II, M.O. Waters, S.S. Sandhu, J.L. Huisingh, 

L. Claxton and S. Nesnow (eds.) Plenum Press, New York, 1981, ! 

pp.337-350. 

5. Claxton, L.D. Mutagenic and Carcinogenic Potency of Diesel and 

Related Environmental Emissions: Salmonella Bioassay. Environment 

International, Vol. 5, 1981. pp. 389-391. 

6. Claxton, L.D. The Utility of Bacterial Mutagenesis Testing in the 

Characterization of Mobile Source Emissions: A Review. Elsevier 

Science Publishing Co., Inc., 1982. 

7. Curren, R.D., R.E. Kouri, CM . Kim and L.M. Schechtman. Mutagenic 

and Carcinogenic Potency of Extracts from Diesel Related Environmental 

Emissions: Simultaneous Morphological Transformation and Mutagenesis 

1n Balb/c 3T3 Cells. Environmental International, Vol. 5, 1981. 

pp. 411-415. 

8. Oepass, L.R., D'.C Chen and L.G. Petersen. Dermal Carcinogenesis 

Bioassays of Diesel Particulates and Oichloromethane Extract of 

Diesel Particulates in C3H Mice. Elsevier Science Publishing Co., 

Inc., 1982. 

9. Guerrero, R.R. and D.E. Rounds. In Vitro Analysis of Mammalian 

Cells Exposed J_n Vitro and Jji V1vo to Airborne Agents, in press. 

10. Guerrero, R.R., D.E. Rounds, J. Orthoefer. Genotoxicity of Syrian 

Hamster Lung Cells Treated J_n Vivo with Diesel Exhaust Particulates. 

Environmental International, Vol. 5, 1981. pp. 445-454. 

69 
JL85_ 

11. Health Effects of Exposure to Oiesel Exhaust. National Academy 

Press, 1981. 

12. Heinrich, R., L. Peters, W. Funcke, F. Pott, U. Mohr and W. Stober. 

Investigation of Toxic and Carcinogenic Effects of Diesel Exhaust 

in Long-Term Inhalation Exposure of Rodents. Elsevier Science 

Publishing Co., Inc., 1982. 

13. King, L.C, M.J. Kohan, A.C Austin, L.D. Claxton and J.L. Huisingh. 

Evaluation of the Release of Mutagens from Oiesel Particles in the 

Presence of Physiological Fluids. Environ. Mutagen., 3:109-121, 

1981. 

14. Lewtas, J. Mutagenic Activity of Diesel Emissions. Elsevier Science 

Publishing Co., Inc., 1982. 

15. Lewtas, J. Evaluation of the Mutagenicity and Carcinogenicity of 

Motor Vehicle Emissions in Short-Term Bioassays. Environmental 

Health Perspectives, Vol. 47, 1983a. pp. 141-152. 

16. Li, A.P., A.L. Brooks, CR . Clark, R.W. Shimizu, R.L. Hanson and 

J.S. Outcher. Mutagenicity Testing of Complex Environmental Mixtures 

with Chinese Hamster Ovary Cells, in press. 

17. Li, A.P. and R.E. Royer. Diesel Exhaust-Particle Extract Enhancement of Chemical-induced Mutagenesis in Cultured Chinese Hamster 

Ovary Cells: Possible Interaction of Diesel Exhaust with Environmental Carcinogens. Mutat Res. 103:349-355, 1982. 

18. Liber, H.L., B.M. Andon, R.A. Hites and W.G. Thilly. Environment 

International, Vol. 5, 1981. pp. 281-284. 

19. Ma, T.H., V.A. Anderson, and S. Sandhu. A Preliminary Study of the 

Clastogenic Effects of Diesel Exhaust Fumes Using the Tradescantia 

Micronucleus Bioassay. In: Short-Term Bioassays in the Analysis 

of Complex Environmental Mixtures. M.D. Waters, S. Sandhu, J.L. 

Huisingh, L. Claxton and S. Nesnow (eds.) Plenum Press, New York, 

1981, pp.351-358. 

20. McClellan, R.O., A.L. Brooks, R.G. Cuddihy, R.K. Jones, J.L. Mauderly 

and R.K. Wolff. Inhalation Toxicology of Oiesel Exhaust Particles. 

In: J. Lewtas (ed.), Toxicological Effects of Emissions from Diesel 

Engines, Elsevier Biomedical, New York, 1982, pp. 99-120. 

21. McCormick, J.J., R.M. Zator, B.B DaGal, and V.M. Maher. Studies on 

the Effects of Diesel Particulate on Normal and Xeroderma Pigmentosum 

Cells. Proc. EPA Internat. Symposium on Health Effects of Diesel 

Engine Emissions. W.E. Peplko, R.M. Danner and N.A. Clarke (eds.) 

EPA-600/9-80-0576, 1980, pp.413-415. 

70 
P.86 

22. Mitchell, A.D., E.L. Evans, M.M. Jotz, E.S. Riccio, E. Mortelmans 

and V.F. Simmon. Mutagenic and Carcinogenic Potency of Extracts of 

Oiesel and Related Environmental Emissions: In Vitro Mutagenesis 

and ONA Damage. Environment International, Vol. 5, 1981. pp. 

393-401. 

23. Nachtman, J.P., X. Xiao-bai, S.M. Rappaport, R.E. Talcott and E.T. 

Wei. Mutagenic Activity in Diesel Exhaust Particulates, Bull. 

Environm. Contain. Toxicol., 27:463-466, 1981. 

24. Nachtman J.P. and S. Wolff. Activity of Nitro-Polynuclear Aromatic 

Hydrocarbons in the Sister Chromatid Exchange Assay With and Without 

Metabolic Activation. Environ. Mutagen., 4:1-5, 1982. 

25. Nesnow, S. and J. Lewtas. Mutagenic and Carcinogenic Potency of 

Extracts of Diesel and Related Environmental Emissions: Summary of 

Discussion of the Results. Environment International, Vol. 5^ 

1981. pp. 425-429. 

26. Nesnow, S., L.L. Triplett, and T.J. Slaga. Mouse Skin Tumor Initiation-Promotion and Complete Carcinogenesis Bioassays: Mechanisms 

and Biological Activities of Emission Samples. Environmental Health 

Perspectives, Vol. 47, 1983. pp. 255-268. 

27. Pepelko, W.E. EPA Studies on the Toxicological Effects of Inhaled 

Diesel Engine Emissions. Elsevier Science Publishing Co., Inc., 1982. 

28. Pereira, M.A. Genotoxicity of Diesel Exhaust Emissions in Laboratory 

Animals. Elsevier Science Publishing Co., Inc., 1982. 

29. Petersen, B.A. and C C Chuang. Methodology of Fractionation and 

Partition of Diesel Exhaust Particulate Samples. Elsevier Science 

Publishing Co., Inc., 1982. 

30. Pott, F. and W. StSber. Carcinogenicity of Airborne Combustion 

Products Observed in Subcutaneous Tissue and Lungs of Laboratory 

Rodents. Environ. Health Perspect., Vol. 47, 1983. pp. 293-303. 

31. Ramel, C. Advantages of and Problems with Short-Term Mutagenicity 

Tests for the Assessment of Mutagenic and Carcinogenic Risk. Environ. Health Perspect., Vol. 47, 1983. pp. 153-159. 

32. Schairer, L.A., R.C. Sautkulis and N.R. Tempel. Monitoring Ambient 

Air Using the Higher Plant Tradescantia in Genotoxic Effects of 

Airborne Agents. R.R. Tice, D.L. Costa, K.M. Scaich (eds.) Plenum 

Press, 1981, pp. 121-140. 

33. Schiff, L.J., S.F. Elliot, S.J. Moore, M.S. Urcan and J.A. Graham. 

Unscheduled DNA Synthesis in Hamster Tracheal Epithelium Exposed In 

Vitro to Chemical Carcinogens and Environmental Pollutants. U.S. 

Environmental Protection Agency, pp. 114-118, 1981. 

71 
P.87 

34. Shefner, A.M., B.R. Collins, L. Dooley, A. Fiks, J.L. Graf, and 

M.M. Preache. Respiratory Carcinogenicity of Diesel Fuel Emissions 

Interim Results. Elsevier Science Publishing Co., Inc., 1982. 

35. Skopek, T.R., H.L. Liber, J.J. Krolewski, and W.G. Thilly. Quantitative Forward Mutation Assay in Salmonella typhimurium using 

8-Azaguanine Resistance as a Genetic Marker. Proc. Natl. Acad. 

Sci. USA, Vol. 75, No. 1, pp. 410-414, January 1978. 

36. Slaga, T.J., L.L. Triplett, and S. Nesnow. Mutagenic and Carcinogenic Potency of Extracts of Oiesel and Related Environmental Emissions: Two-Stage Carcinogenesis in Skin Tumor Sensitive Mice 

(SENCAR). Environment International, Vol. 5, 1981. pp. 416-423. 

37. Sugimura, T. and S. Takayama. Biological Actions of Nitroarenes in 

Short-Term Tests on Salmonella, Cultured Mammalian Cells, and Cultured 

Human Tracheal Tissue"!": Possible Basis for Regulatory Control. 

Environ. Health Perspect., Vol. 41, 1983. pp. 171-176. 

38. U.S. Environmental Protection Agency. Short-Term Genetic Bioassays 

Used in the Evaluation of Unregulated Automobile Emission: 1981-1982 

Contract Reports. D.J. Brusick, R.R. Young and D.R. Jagannath (eds.) 

EPA 68-02-3382. Technical Directive 008, October 1983. 

39. Wang, Y.Y., R.E. Talcott, D.A. Seid and E.T. Wei. Antimutagenic 

Properties of Liver Homogenates, Proteins and Glutathione on Diesel 

Exhaust Particulates. Cancer Letters, 11:265-275, 1981. 

40. Wei, E.T., Y.Y. Wang and S.M. Rappaport. Diesel Emissions and the 

Ames Test: A Commentary. Reprint from APCA Journal, Vol. 30, No. 3, 

March 1980. 

41. Zweidinger, R.B. Emission Factors from Diesel and Gasoline Power 

Vehicles: Correlation with the Ames Test. Elsevier Science Publishing Co. , Inc., 1982. 

72 
P.88 

PART II 

SECTION 4 

EFFECTS OF ENVIRONMENTAL CONDITIONS ON BIOLOGICAL 

ACTIVITY OF EMISSION SAMPLES 

EFFECT OF SAMPLING CONDITIONS 

The procedure for particulate sampling and measurement involves driving 

a vehicle or engine on a dynamometer in accordance with Federal Test 

Procedure driving cycle to simulate typical vehicle speeds and loads. 

The exhaust is passed into a dilution tunnel connected to a constant 

volume sampler, where it is mixed with filtered ambient air. The net 

flow through the tunnel is kept constant (I.e., the sum of exhaust gas 

and dilution air), to provide proportional sampling. Sampling the diluted • 

exhaust at a constant rate will give an integrated sample representative 

of a particular driving cycle. The average dilution of the exhaust is 

generally from 10:1 to 20:1 depending on the engine size and average 

operating speed. Under highway conditions, the dilution ratio reaches 

1000:1 shortly after the exhaust exits the tailpipe. However, as indicated 

by the investigations of Black and High (1979) and Williams and Chock 

(1980), the composition of the particulate is fixed when it leaves the 

tailpipe and is not significantly influenced by further dilution. The 

data from Williams and Chock (1980) indicate that the B(a)P content tends 

to increase with dilution while the extractable fraction remains constant 

(Table 35). No differences were observed in either the particulate emission 

rates or the molecular weight distribution of the soluble organic fraction 

(S0F). More recently, studies conducted at the Allegheny Mountain tunnel 

found the diesel-produced aerosol at Allegheny to be quite similar 

to the aerosol encountered in dilution-tube experiments with respect to both 

mutagenicity in the Ames test and molecular-weight distribution (Pierson 

et al., 1981). Thus, if one hypothesizes that the particle-bound organics 

a redetermined primarily during the initial cooling and dilution of the 

exhaust, the constant-volume-sampling procedure could result in particles 

chemically and biologically similar to ambient particles. 

AMBIENT CONDITIONS 

None of these above studies, however, addresses the question of what 

happens to the particulate organics during prolonged exposure to sunlight 

and/or to other chemical species present in-the atmosphere such as ozone. 

Ambient conditions can affect the condensation of organic compounds onto 

particles, influence the interaction between organics, alter the organic 

species emitted by a source, and provide for various other interactions. 

73 
P.89 

TABLE 35. THE EFFECT OF DILUTION RATIO 

ON PARTICULATE MASS CONCENTRATION 

ANO COMPOSITION3 

Open Air 

Dilution Tunnel 

Dilution 

Ratio 

<2 

5.1-7.0 

11-17 

21-24 

44-88 

135-150 

220-350 

13.8 

26.5 

No . of 

Samples 

1 

5 

4 

3 

4 

2 

2 

1 

1 

Specific 

Particulate 

Concentration 

(ug/m3 

particulate/ 

ppm of C02) 

..b 

5.0 

4.1 

3.5 

4.5 

6.5 

3.7 

3.9 

3.8 

Extractables 

(%) 

15 

26 

28 

28 

29 

28. 

—

c 

27 

27 

BaP 

(ng/mg) 

5 

19 

22 

28 

25 

32H 

20d "• 

23 

22 

*Data from Williams and Chock (1980). 

.C02 concentration was above instrument capability. 

jExtractable mass was too small for accurate determination. 

Large background correction to mass makes this number uncertain. 

The destruction of polynuclear aromatic hydrocarbons (PAH's) under atmospheric conditions is well known. For example, Falk et aj. (1956) found 

that benzo(a)pyrene absorbed on combustion soot was decomposed 10% on 

exposure to light and air for 48 hours and 18% on exposure to light and 

smog for only 1 hour. Claxton and Barnes (1981) studied ambient-like 

conditions using the Calspan smog chamber. Their results indicate that 

the presence of ozone in the chamber tends to reduce the mutagenic response 

of the organic material collected, the decrease being most pronounced 

without metabolic activation. 

Model experiments in simulated atmospheres suggest that PAH and other 

aromatic compounds are transformed in the atmosphere (Pitts, 1982; Nielson 

et a]_., 1982). The dominant pathways will depend upon seasonal and 

meteorological conditions, e.g., light intensity, humidity, and temperature, as well as the presence of reactive primary and secondary co-pollutants. Reactions with nitrous acid and sulfur oxides may also occur. 

At higher solar intensities, photo oxidations as well as reaction with 

photochemically generated pollutants such as ozone and peroxyacetyl 

74 
P.90 

nitrate, may also be important. Photo oxidation reactions transform at 

least some PAH in model experiments to direct mutagens (McCoy et aL , 

1979). 

EFFECT OF TEMPERATURE 

The effect of temperature on emission has been studied by Braddock (1981). 

In this study, the Federal Test Procedures were conducted at ambient 

temperatures (average of 12°F above crankcase temperature) without heating 

of dilution air. The results indicate that decreasing the temperature 

results in increased particulates and soluble organic fraction (SOF) 

caused by uncombusted diesel fuel. On a revertant per ug SOF basis, the 

mutagenic activity without activation appeared to decrease concomitant 

with decreasing temperature. 

ARTIFACT FORMATION DUE TO COLLECTION METHODS 

The role of N0/N02 during sampling of particles has generated much interest 

among investigators. It has been demonstrated by various means that 

mutagenic nitroaromatic compounds are contained in organic extracts!of 

filterrcollected particles. Pitts et aj. (1978) have shown that nitrated 

species can be formed under certain experimental conditions. It has also 

been concluded that the possible formation of nitro-polycyclics is dependent 

on the filter material (Lee et a_h , 1980). Basically, nitro-aromatics 

may be produced in three processes: (1) combustion, (2) exhaust, as 

organics interact and condense upon the particles, or (3) collection, as 

an artifact. The possibility of nitration during realistic filter sampling 

has been investigated. In one experiment, the N02 concentration was 

increased by altering the dilution ratio, by adding N02 into the dilution 

tunnel. An increase in the mutagenic activity was noted at higher 

concentrations of N02 (Bradow, 1980; Claxton, 1980). Fuel aromatics and 

fuel aliphatics, when exposed to excess N02 at 25°C, generated direct 

acting mutagens for the strain TA-100 (Henderson et aTL , 1981). Another 

experiment involved re-exposure of filters previously used. The results 

obtained with diesel exhausts showed increased levels of 1-nitropyrene 

and 6-nitro-benzo(a)pyrene as well as heightened mutagenic activity 

(Gibson et ah , 1981). Formation of 1-nitropyrene, a strong mutagen, 

was shown to have a decisive influence on mutagenicity (Salmeen et aj., 

1982). 

SOLVENT SYSTEMS ANO STORAGE METHOD 

Hydrocarbons adsorbed on the surface of particulates have been frequently 

extracted from the particulates and concentrated by using organic solvents 

in order to assess their specific biological activity. Investigations 

of solvents representing a wide range of polarities have generally indicated 

dichloromethane (DCM) to be the most efficient single solvent for extracting 

mutagens from diesel particulates (Siak et a_L , 1980, Huisingh et al., 

1978). McGrath et a L (1978) tested whole particles suspended Tn dimethylsulfoxide (DMSO) and obtained negative to moderately positive results 

for those particles tested using the Ames test. Organic compounds can 

75 
P.91 

also be removed by biological fluids, however, the removal process is 

not nearly so efficient as with DCM or DMSO. King et a h (1981) confirmed 

that organic solvents are more efficient than physioTogTcal fluids in 

removing mutagens from diesel particles. 

Since there is usually a time lapse of several days to several weeks 

before generated samples can be tested, storage may be an important factor. 

Huisingh et ah (1978) showed no difference in the mutagenic activity of 

samples afTer storage for up to 8 weeks (Figure 8) either on the filter 

or as an extract. Direct acting components also seem to increase in 

toxicity with storage. Williams and Chock (1980) conducted a filtersample-storage study in which eleven filter samples collected from the 

dilution tunnel were stored in a room at 24° ± 0.5°C and 45%±1X relative 

humidity with normal fluorescent light. The extractable percentage and 

B(a)P concentrations were analyzed on randomly selected filters for up 

to 150 days after collection. Their results show that the total B(a)P 

loss in 150 days was 57%; B(a)P concentration decreased linearly with 

time at a rate of 0.046 ng/mg/day, extractable percentage showed no 

systematic change with storage time (Table 36). 

TABLE 36. EFFECT OF FILTER SAMPLE.STORAGE 

ON DIESEL EXHAUST PARTICULATE* 

Sample 

Number 

1 

3 

3 

4 

5 

6 

7 

8 

9 

10 

11 

Storage 

Period 

(days) 

9 

9 

9 

9 

28 

28 

28 

91 

91 

147 

147 

Extractable 

Percentage 

34.1 

32.9 

32.2 

32.0 

38.8 

39.2 

40.0 

44.6 

40.1 

36.5 

37.8 k 

3O±4.0b 

B(a)P 

Concentration 

(ng/mg) 

12.1 

11.1 

11.1 

10.9 

9.8 

9.0 

10.1 

7.7 

6.3 

5.5 

4.3 

% t a from Williams and Chock (1980). 

One standard deviation 

These results suggest that the storage conditions should be carefully 

controlled to avoid excessive loss of B(a)P by unidentified processes. 

Good Laboratory Practice would dictate minimizing storage time. 

76 
P.92 

1400 

0.2 OJ 

Mg RELATED 'ARTICULATE 

Figure 8. Characterization of diesel particle emissions, Husingh et ah,!1978. 

77 
P.93 . 

REFERENCES 

1. Black F.M. and L.E. High. Methodology for Determining Particulate 

and Gaseous Diesel Hydrocarbon Emissions. SAE Paper 790422, 1979. 

2. Braddock, J.N. Emissions of Diesel Particles and Particulate 

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Raleigh, NC, October 1981. 

3. Bradow, R.L. Diesel particle emissions. Bull. NY Acad Med., 

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4. Claxton, L.D. In: Health Effects of Diesel Engine Emissions, 

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5. Claxton, L.D. and H.M. Barnes. The Mutagenicity of Diesel Exhaust 

Particle Extracts Collected Under Smog-Chamber Conditions Using the 

Salmonella typhimurium Test System. Mutat. Res., 88:255-272, 1981. 

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Their Fate Following Emission into the Atmosphere. AMA Arch. Ind. 

Hlth., 13:13-17, 1956. 

7. Gibson, T.L., A.I. Ricci, and R.L. Williams. Measurements of polynuclear aromatic hydrocarbons, their derivatives and their reactivity 

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M. Cooke and A.J. Dennis, eds. Battelle Press, Columbus, OH, 1981. 

pp. 707-717. 

8. Henderson, T.R., A.P. Li, R.E. Royer, and CR . Clark. Increased 

cytotoxicity and mutagenicity of diesel fuel after reaction with 

N02. Environ. Mutagen., 3:211-220, 1981. 

9. Huisingh, J.L., R. Bradow, R. Jungers, L. Claxton, R. Zweidinger, 

S. Tejada, J. Bumgarner, F. Duffleld, M. Waters, V.F. Inmon, C. Hare, 

C. Rodriguez, and L. Snow. Application of bioassay to the characterization of diesel particle emissions. In: Application of Short-Term 

Bioassays in the Fractionation and Analysis of Complex Environmental 

Mixtures. M.D. Waters, S. Nesnow, J.L. Huisingh, S.S. Sandhu and 

L. Claxton, eds. Plenum Press, New York, 1978. p. 383-418. 

78 
P.94 

10. King, L.C, M.J. Kohan, A.C. Austin, L.D. Claxton, and J.L. Huisingh. 

Evaluation of the release of mutagens from diesel particles in the 

presence of physiological fluids. Environ. Mutagen., 3:109-121, 1981. 

11. Lee, F.S.C, W.R. Pierson, and J. Ezike. The problem of PAH degradation during filter collection of airborne particles--an evaluation 

of several commonly used filter media. In: Polynuclear Aromatic 

Hydrocarbons. A. BjSrseth and A.J. Dennis, eds. Battelle Press, 

Columbus, Ohio, 1980. pp. 543-563. 

12. McCoy, E.C , J. Hyman, and H.S. Rosenkranz. Conversion of environmental pollutant to mutagens by visible light. Biochem. Biophys. 

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13. McGarth, J.J., R.M. Schreck, and J.S. Siak. Mutagenic Screening of 

Diesel Particulate Material. Proceedings of the 71st Annual Meeting 

of APCA, Houston, Texas, 1978. 

14. Nielsen, T., T. Ramdahl, and A. Bjorseth. The fate of airborne 

polycyclic organic matter. Environ. Health Perspect., 47, in press. 

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15. Pierson, W.R., R.A. Gorse, Jr., A.C. Szkarlat, W.W. Bracheczek, 

S.M. Japar, F.S.C. Lee, R.B. Zweidinger, and L.D. Claxton. Mutagenicity and Chemical Characteristics of Carbonaceous Particulate Matter 

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NC, October 1981. , 

16. Pitts, J.N., Jr. Formation and fate of gaseous and particulate 

mutagens and carcinogens in real and simulated atmospheres. Environ. 

Health Perspect., 47, in press. 1982. 

17. Pitts, J.N., Jr., K.A. van Cauwenberghe, D. Grosjean, J.P. Schmid, 

D.R. Fitz, W.L. Belser, G.B. Knudson, and P.M. Hynds. Atmospheric 

reactions of polycyclic aromatic hydrocarbons: facile formation of 

mutagenic nitro derivatives. Science, 202:515-519, 1978. 

18. Salmeen, I., A.M. Duirisin, T.J. Prater, T. Riley, and D. Schuetzle. 

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Engine Emissions, Proceedings of an International Symposium, 

December 3-5, 1979, W.E. Pepelko, R.M. Danner, and N.A. Clarke, 

eds. U.S. Environmental Protection Agency, EPA-600/9-80-057a, 

1980. pp. 245-262. 

20. Williams, R.L., and D.P. Chock. Characterization of Diesel Particulate Exposure. In: Health Effects of Diesel Engine Emissions. 

Proceedings of an International Symposium, December 3-5, 1979, 

W.E. Pepelko, R.M. Danner, and N.A. Clarke, eds. U.S. Environmental 

Protection Agency, EPA-600/9-80-057a, November 1980. 

79 
P.95_ 

PART II 

SECTION 5 

EFFECTS OF ENGINE TYPE, FUEL, AND FUEL ADDITIVES 

ON BIOLOGICAL ACTIVITY OF EMISSION SAMPLES 

INTRODUCTION 

A variety of factors, including engine design, fuels, lubricants, engine 

age and operating conditions, may influence the formation of exhaust particulate compounds in exhaust from gasoline- and diesel-powered vehicles 

(Clark et ah , 1981; Zweidinger, 1982; and Claxton, 1983). Short-term 

ui vitro bToassays have provided a tool for investigating the mutagenic 

properties of automotive exhausts. 

EFFECT OF ENGINE TYPE ON EXHAUST MUTAGENICITY 

Comparison of spark-ignition (gasoline)- and diesel-engine-exhaust particle 

extracts show that gasoline engines produce fewer nitrated PAHs than diesel 

engines (Nishioka et ah , 1982; Gibson, 1982). Diesel engines have been 

reported to produce up to 100 times more respirable particulates per km 

than gasoline engines with catalytic control. The gaseous emissions of 

diesel engines contain more NO as N02, relative to gasoline engines, 

which contributes to the formation of nitroarenes (Holmberg and Ahlborg, 

1983). Diesel vehicles were found to emit about six times as much organic, 

solvent-extractable particle mass as leaded gasoline vehicles and 20 times 

more than in-use catalyst cars. Diesel vehicles were also reported to 

have higher mutagenic emission rates per kilometer than gasoline noncatalyst 

vehicles, which in turn had higher rates than gasoline catalyst vehicles. 

Lofroth, et a h (1980) also found a higher level of mutagenic components 

in diesel than in gasoline exhaust: 100 revertants per liter of diesel 

exhaust compared to 15 revertants per liter of gasoline exhaust in 

Salmonella typhimurium TA98. Zweidinger (1981) reports that the average 

revertants per mile values (TA98 without S9) were 509,000 for six diesel 

vehicles; 152,000 for four in-use leaded gasoline vehicles; and 42,000 

for 15 unleaded gasoline vehicles. A comparison of three gasoline and 

six diesel engine equipped cars (Table 37) showed that dichloromethane 

extracts of particulate exhaust from the gasoline cars were only slightly 

more mutagenic than those observed for the diesel cars, but the mass of 

solvent-extractable material on the particulates was lower. Combined 

with a lower particulate emission rate, this resulted in significantly 

lower values for revertants per mile for the gasoline-engine-equipped 

cars (Clark, 1981). This demonstrates the need for normalizing bioassay 

data for specific needs and comparisons. 

80 
P.96 

TABLE 37. MUTAGENICITY OF SPARK-IGNITION AND DIESEL ENGINE EXHAUST 

PARTICLE EXTRACTS (Clark, 1981) 

Vehicle 

Spark Ignition 

Ford Escort 

Oldsmobiie Cutlass 

Chevrolet Citation 

Oiesel 

Peugeot 504 

Oldsmobiie D-88 

VW Rabbit 

Audi 5000 

Mercedes 3000 

Fiat 131 

Revertants 

per ug 

Extract* 

10 

18 

23 

8 

12 

13 

10 

13 

3 

Extractable 

Fraction 

(%) 

8 

5 

5 

29 

20 

26 

43 

13 

71 

Extractable 

Fraction 

(mg/mi) 

2 

2 

2 

60 

75 

40 

190 

40 

240 

Revertants 

per mile 

(no-3) 

20 

36 

46 

480 

900 

520 

1900 

520 

720 

Slope of linear portion of dose-response curve in TA 100, without the 

baddition of liver S-9. 

Total mass of extractable material from filter r number of miles in test 

cycle (spark-ignition - 26 miles, diesel - 7.6 miles). The spark-ignition 

engine cars were operated on unleaded indolene fuel and the diesel cars 

on a standard No. 2 diesel fuel. 

Variability of repeat samples from one car and of samples collected from 

different identical vehicles is of great concern when evaluating the 

mutagenicity of mobile-source emissions. Both Claxton and Kohan (1981) 

and Clark (1981) have investigated the variability associated with multiple 

sampling of automotive emission. 

Variability of repeat sampling on different days of individual automobiles 

has been shown by Claxton and Kohan and by Clark to vary by less than 

twofold for all variables compared. Oata reported by Clark (Table 38) 

show that revertants per microgram of extract in three samples collected 

on different days with each of three different cars were not significantly 

different (p>0.01). Slightly more variability was noted when mutagenic 

activity was converted to revertants per milligram of particles and 

revertants per mile. The results demonstrate the repeatability Of sample 

collection, preparation and bioassay. 

A comparison of samples from vehicles of the same make, model and operation pattern were shown by Clark (1981) and Claxton and Kohan (1981) to 

have an increased variability over multiple samples taken from one vehicle. 

Clark observed that the fraction of extractable organics was more variable 

with five different diesel equipped Oldsmobiie Delta 88 automobiles, than 

observed in triplicate runs with one car of the same type (Table 39). 

81 
P.97 

TABLE 38. VARIABILITY IN MUTAGENICITY OF REPEATED COLLECTIONS OF DIESEL 

PARTICULATE EXHAUST (Clark et ah , 1981) 

Car 

VW Rabbit 

Oldsmobiie 

(#218) 

Oldsmobiie 

(#214) 

E* 

189 

364 

192 

134 

280 

283 

9896 

9892 

9893 

Revertants 

per ugh 

Extract0 

14 (±1.2) 

15 (±0.6) 

14 (±1.0) 

12 (±0.7) 

14 (±1.2) 

13 (±1.0) 

11 (±0.9) 

10 (±0.4) 

11 (±0.3) 

Extractable 

Fraction 

26 

29 

24 

22 

21 

20 

21 

18 

21 

Revertants 

per mg 

Particles 

3600 

4200 

3200 

2500 

3000 

2600 

2300 

1700 

2300 

Particle 

Emissions 

(g/mi)a 

0.17 

0.16 

0.19 

0.31 

0.36 

0.38 

0.29 

0.38 

0.36 

Revertants 

x IO3 

per Mile 

600 

700 

600 

800 

1100 

1000 

700 

600 

800 

Cars operated according to EPA Federal Test Procedure (hot start) on 

chassis dynamometer, ambient temperature 75°F. The three sampling periods 

hfor each car were on different days. 

Slope (± standard deviation) of linear portion of dose-response curve in 

Salmonella strain TA100 without the addition of a liver enzyme preparation. 

^Percent (w/w) dichloromethane extractable organics associated with particles. 

Grams of particles emitted from exhaust per mile of vehicle operation. 

TABLE 39. MUTAGENICITY OF EXTRACTS OF EXHAUST PARTICLES COLLECTED FROM 

FIVE DIFFERENT OLDSMOBILE OIESEL AUTOMOBILES3 (Clark et ah , 1981) 

Car-Tesj 

No. 

Revertants 

per ug 

Extractc 

Extractable 

Fraction 

Revertants 

per mg 

Particles 

Particle 

Emissions 

(g/mi)e 

Revertants 

x IO3 

per mile 

214-181 

215-9893 

216-9942 

217-284 

218-252 

10 (±0.9) 

11 (±0.3) 

7 (±0.7) 

10 (±0.3) 

7 (±0.6) 

18 

21 

25 

16 

23 

1800 

2300 

1800 

1600 

1600 

0. 

0. 

0. 

0. 

0. 

38 

30 

33 

34 

33 

700 

700 

600 

500 

500 

a1980 model year Oldsmobiie Delta 88 diesel cars tested after a 4,000 

jnile break-in period. 

Operated according to EPA Federal Test Procedure (hot start) on chassis 

dynamometer, ambient temperature 75°F. 

Slope (± standard deviation) of linear portion of dose-response curve 

din Salmonella strain TA-100, without the addition of liver enzyme preparation. 

^Percent (w/w) dichloromethane extractable organics associated with particles. 

Grams of particles emitted from exhaust per mile of vehicle operation. 

82 
P.98 

However, the mutagenicity, when normalized to revertants per milligram 

of particles, was quite similar for each sample, suggesting an inverse 

correlation between mutagenicity of the extracts and extractable fraction. 

Claxton and Kohan observed much greater variability in samples taken from 

five similar gasoline vehicles (Table 40) than seen with either repeat 

sampling of one vehicle (Table 38) or in comparative testing of similar 

diesel vehicles (Table 39). There was nearly a tenfold difference in 

revertants per microgram of organic material among the data from the five 

cars. As seen with the diesel work, the variability for the bioassay was 

partially normalized when results were expressed as revertants per gram 

of particles or revertants per mile. By normalizing the data, variation 

was reduced from a 10-fold to a 5-fold difference. 

Claxton and Kohan also investigated the variation in exhaust particulate 

mutagenicity among six different light-duty diesel vehicles (Table 41). 

The variation among different diesel vehicles is similar to variation 

between the different Ford LTD gasoline automobiles in Table 40 and is 

also similar to a comparison done by Clark, et al. (1981) with six different light-duty diesel vehicles. 

The mutagenicity of exhaust particulates when evaluate in terms of revertants per mile have been seen to vary by two-fold to as much as five-fold 

in a series of comparitive studies with either gasoline or diesel engines. • 

Repeat sampling of one vehicle produced relatively similar samples that 

varied less than two-fold in terms of revertants per mile. More variability, 

up to five-fold difference, was observed when both single samples of identical vehicles and different vehicles within a given class (diesel, gasoline) were compared. This would indicate that collection, processing 

TABLE 40. MUTAGENICITY OF EXTRACTS OF EXHAUST PARTICLES FROM FIVE DIFFERENT FORD LTD GASOLINE AUTOMOBILES (Claxton and Kohan, 1981) 

Test 

No. 

0032 

0033 

0034 

0035 

0036 

Mean 

Car 

Lt. blue 

Mid. blue 

Gray 

Silver 

Brown 

Standard dev. 

Coeff. of var. 

Slope3 

(Rev/plate/ug) 

7.54 

7.51 

9.49 

9.61 

1.02 

7.03 

3.51 

0.50 

* h 

Ext.b 

5.8 

3.4 

3.2 

3.8 

21.4 

7.52 

7.83 

1.04 

Rev x 10s 

/g Partic. 

4.37 

2.55 

3.04 

3.65 

2.18 

3.16 

0.87 

0.28 

PERC 

(g/mile) 

0.0079 

0.0100 

0.0180 

0.0100 

0.0050 

0.0102 

0.0048 

0.47 

Rev/ 

Mile 

3455 

2553 

5466 

3652 

1091 

3243 

1602 

0.49 

bSlope of linear regression line. 

^Percent dichloromethane-extractable mass. 

Particle emission rate. 

83 
P.99 

TABLE 41. MUTAGENICITY OF EXTRACTS OF EXHAUST PARTICLES COLLECTED FROM 

SIX DIFFERENT DIESEL VEHICLES (Claxton and Kohan, 1981) 

Vehicle 

Olds 350 

Olds 260 

Chev. Truck 

350 diesel 

M.B. 3000 

Opel Record E 

VW Dasher 

Mean 

Standard 

deviation 

Coefficient of 

variaion 

Slope3 

(Rev/plate/ug) 

1.85 

1.51 

2.64 

3.23 

1.15 

1.45 

1.97 

0.80 

0.41 

Mean. 

% Ext.D 

21.1 

16.5 

53.4 

23.3 

51.6 

53.7 

36.6 

18.0 

0.49 

Rev x 10s 

/g Partic. 

3.90 

2.49 

14.10 

7.53 

5.93 

7.79 

6.96 

4.06 

0.58 

PERC 

(g/mile) 

0.846 

1.005 

0.623 

0.840 

0.483 

0.322 

0.687 

0.256 

0.37 

Rev x 10s 

/mile 

3.30 

2.50 

8.78 

6.32 

2.87 

2.51 

4.38 

2.59 

0.59 

?Slope of linear regression line. 

^Percent dichloromethane-extractable mass. 

Particle emission rate. 

and bioassay of automotive particulate emission samples can be standardized to produce reproducible results. Results also indicate that sample 

variation may be expected to arise from either operating characteristics 

of a particular test vehicle or from basic differences in combustion 

related to engine design. The most dramatic difference observed of the 

effect of engine type on exhaust mutagenicity was the difference between 

gasoline and diesel engines. When normalized to revertants per mile, 

both Claxton and Kohan (1981) and Clark et ah (1981) observed a 50- to 

100-fold higher level of mutagenicity with dTesel exhaust particulate 

than with gasoline exhaust particulate. 

84 
P.1 

EFFECT OF FUELS ON EXHAUST MUTAGENICITY 

Many variables have been shown to influence particulate exhaust emission 

rates of diesel engines. These include fuel density, aromaticity and 

distillation properties, fuel additives, and make and model of car (Clark 

et a L , 1982a; Hare, et ah , 1978; Springer and Baines, 1977; Lipkea, et 

aT., 1978; Burley andTosebrock, 1979). The correlation between fuel 

composition and mutagenicity of diesel exhaust particulates has not been 

so clearly established. Several studies have investigated the correlation 

between fuel aromatic content and mutagenicity of organic extracts of 

exhaust particulate material (Zweidinger, 1982; Sklarew et al., 1981; 

Clark et ah , 1982a). Generally, no well-defined relationship between 

aromatic content of the fuel and mutagenicity of the exhaust particle 

extract was noted. Clark et aL , suggested that the relative abundance 

of various PAH species may~Ee~7mportant since higher boiling fuels generally produced more mutagenic combustion products than lower boiling fuels. 

However, Zweidinger (1982) investigated several fuel parameters and their 

correlation with mutagenic activity (revertants per microgram of extract). 

The highest correlations noted were associated with nitrogen content of 

the fuel. Subsequent studies of the effect of nitrogen levels (obtained 

by doping a base fuel with isoquinoline) did not show any correlation 

between mutagenicity and fuel nitrogen level. Slight correlation was 

reported between aromaticity and activity, particularly without activation. 

In investigating the effect of aromatic content on exhaust particulate 

mutagenicity, Clark et a L (1982a) burned a variety of fuels (with aromatic 

content ranging from~34 to 13 percent) in two different vehicles. All 

samples were mutagenic in TA-100 without metabolic activation. However, 

the data, summarized in Tables 41 and 42, do not show a clear-cut relationship between mutagenicity of dichloromethane extracts or revertants per 

mile and fuel aromatic content. For the Oldsmobile, higher aromatic fuels 

gave particle extracts with higher mutagenic potency but the mass of 

extractable material was lower (Table 42). In the Peugeot, higher aromatic 

content fuels did not result in the lower particle extract masses as seen 

in the Oldsmobile (Table 43). Also, there was no large difference in 

particle mutagenicity and revertants per mile as noted with some Oldsmobile 

fuel tests. These results suggest an influence of engine design on the 

production of mutagenic particle extracts. 

Clark reported further evidence that mutagenicity is not directly related 

to fuel aromatic content. An Oldsmobile was operated on three fuels, 

ranging from 32 to nearly 0 percent aromatics. As can be seen in Table 44, 

high aromaticity was associated with high particle emission rates but 

lower extractable particulate fraction. The isoparaffln fuel of almost 

0 percent aromatic content had higher mutagenicity than the jet fuel with 

17 percent aromatics. 

85 
pyt^ 

TABLE 42. MUTAGENICITY OF EXTRACTS OF PARTICLES COLLECTED FROM THE EXHAUST 

OF AN OLDSMOBILE DIESEL CAR* 

(Clark et aL , 1982a) 

OPERATEO ON OIFFERENT FUELS 

Fuel' 

% 

Aromatics 

Revertants Extractable Revertants 

per ugh Fraction per mg 

Extract0 (%)c Particles 

Particle 

Emissions 

(g/nri)a 

Revertants 

per Mile 

(x IO3) 

7943 

7926 

7940 

7942 

7939 

7938 

7941 

34 

32 

32 

20 

20 

13 

13 

10 (0.4) 

18 (1.2) 

8 (0.5) 

7 (0.2) 

5 (0.4) 

7 (0.4) 

7 (0.3) 

22 

21 

22 

41 

25 

30 

30 

2200 

3800 

1800 

2900 

1300 

2100 

2100 

0.22 

0.31 

0.25 

0.23 

0.21 

0.22 

0.24 

500 

1200 

400 

700 

300 

500 

500 

1980 model year Oldsmobile Delta 88 (5.7 liter) operated according to EPA 

Federal Test Procedure (hot start) on a chassis dynomometer, ambient 

.temperature 75°F. 

Slope (t standard deviation) of linear portion of dose-response curve in 

Salmonella strain TA-100, without the addition of Liver S-9. 

^Percent (w/w) dichloromethane extractable residue associated with particles. 

Grams of particles emitted from exhaust per mile of vehicle operation. 

TABLE 43. MUTAGENICITY OF EXTRACTS OF PARTICLES COLLECTED FROM THE EXHAUST 

OF A PEUGOT DIESEL CARa OPERATED ON OIFFERENT FUELS 

(Clark et al., 1982a) 

Fuel' 

% 

Aromatics 

Revertants Extractable 

Per ugh 

Extract0 

Fraction (%r 

Revertants 

per mg 

Particles 

Particle 

Emissions 

(g/mi)a 

Revertants 

per Mile 

(x IO3) 

7943 

7926 

7940 

7942 

7939 

7941 

34 

32 

32 

30 

20 

13 

4 (0.3) 

7 (0.2) 

6 (0.2) 

3 (0.3) 

7 (0.2) 

4 (0.3) 

17 

32 

30 

23 

22 

18 

700 

2200 

1800 

700 

1500 

700 

0.18 

0.20 

0.17 

0.18 

0.23 

0.17 

100 

400 

300 

100 

400 

100 

1979 Model year Peugeot 504 (2.3 liter) operated according to EPA Federal 

hTest Procedure (hot start) on a chassis dynomometer, ambient temperature 75°F. 

Slope (± standard deviation) of linear portion of dose-response curve in 

Salmonella strain TA-100, without the addition of Liver S-9. 

^Percent (w/w) dichloromethane extractable residue associated with particles. 

Grams of particles emitted from exhaust per mile of vehicle operation. 

86 
P.3 

TABLE 44. INFLUENCE OF A JET "A" AND ISOPARAFFINIC FUELS ON MUTAGENICITY OF 

EXHAUST PARTICLE EXTRACTS OF AN OLDSMOBILE DIESEL CAR3 (Clark, 

et aL , 1982a) 

Revertants Revertants Particle Revertants 

h per ug Extractable per mg Emissions per Mile 

Fuel" Extract X Fraction0 Particles (g/m1)e (x IO3) 

Reference (32%) 13 23 2900 0.33 950 

Jet "A" (17%) 5 25 1200 0.20 240 

Isoparaffin (0.4%) 8 40 3000 0.16 480 

1980 Model Oldsmobile Delta 88 (5.7 liter) operated according to EPA 

Federal Test Procedure (hot start) on a chassis dynomometer, ambient 

htemperature 75°F. 

Reference Fuel - 7926; Jet "A" fuel - 8017; isoparaffin fuel - 7812; % 

aromatic content in parentheses. 

Slope (± standard deviation) of linear portion of dose-response curve 1n 

dSalmonella strain TA-100, without the addition of liver S-9. 

Percent (w/w) dichloromethane extractable organics associated with particles. 

Grams of particles emitted from exhaust per mile of vehicle operation. 

Chemical analysis of extracts of exhaust particulates indicates that mutagenic direct-acting nitroaromatic compounds may be responsible for much 

of the observed mutagenic activity. It has been suggested that Mtroaromatics, such as nitropyrenes and nitronaphthalenes, are normal components 

of diesel and even some gasoline exhaust (Schuetzle et aL , 1982; Nishioka 

et aL , 1981). These nitroaromatics may be caused partially by reactions 

57 unburned fuel PAH and NO formed in the combustion process (Henderson 

et al., 1981); or they may fie associated with particulate exhaust material 

contributed by the engine oil (Mayer et aL , 1980). 

The'definitive relationship between the mutagenicity of partlculateassociated organic material produced by combustion and aromatic content 

of fuels has not been established. Aromatic content may be influential 

at high aromatic levels (>30%)» but appears to depend upon engine type. 

Because of the variability in diesel fuel composition associated with 

the variety of crude oils and refining techniques, control of fuel 

properties to affect emission mutagenicity may be a futile strategy 

(Seizinger et aL , 1982). 

The addition of fuel extenders has been shown to affect the emission 

characteristics of diesel fuels (Clark et al., 1982b). Addition of 

butanol, ethanol or middle distillate fracUon of SRC-II (a solvent 

refined coal liquid) to diesel fuel decreased the specific mutagenicity 

of dichloromethane extracts of diesel exhaust particles, but increased 

the particle-associated organic material two- to four-fold. This resulted 

87 
P.4 

in higher mutagenicity (revertants per mile) using the three extenders 

than using diesel fuel alone. Rannug (1983) found comparable mutagenicity in acetone extracts of particulates collected from engines powered 

by leaded gasoline, unleaded gasoline, or a lead-free gasoline with 15 

percent methanol. In studies of acetone extracts of particles, he noted 

that three distinct mutagenic groups were formed depending upon the fuel 

used. Using Ames data with TA98 and TA100, light-duty diesel vehicles 

formed a high mutagenicity group giving between 50-250 revertants/L of 

exhaust or 100,000 to 700,000 revertants/kn. The second group contained 

the engines fueled by leaded and lead-free gasoline as well as gasolinealcohol fuels; these gave 10-50 revertants/L of exhaust or 10,000 to 

20,000 revertants/km. The final group of fuels, methanol and propane 

gave less than 10 revertants/L or less than 20,000 revertants/km. 

MUTAGENICITY OF CRUDE OILS AND FUELS 

Mutagenicity of diesel and gasoline engine emissions would indicate that 

the mutagenic components occur in the fuel or are synthesized at some 

point in the combustion/exhaust cycle. A variety of crudes and their 

fractions have been tested for mutagenic activity. Huisingh et al. (1978) 

reported that fractions of a diesel fuel were not found to be mutagenic, 

suggesting that mutagens detected in the exhaust were products of the 

combustion process itself. Lebowitz et a L (1979) reported diesel fuel 

as non-mutagenic in a battery of shorPterm assays. Wang et al. (1978a, 

1978b) noted that a diesel fuel, JP-4, and two types of gasoline were 

not mutagenic with TA-98 in the Salmonella bioassay. 

Some investigators have found some heavier crude oils including petroleum-, 

coal- and shale-derived distillates to be mutagenic. Synthetic (shalederived) oils have been shown to have mutagenic activity in Salmonella 

(Epler et al., 1979; Pelroy and Petersen, 1979; Rao et aL , 1978). 

Guerin et IT. (1978), Epler et al. (1978) and Ellenton et al. (1981) found 

fractions of natural crudes To hive mutagenic activity in Salmonella. 

When fractionated, they found that mutagenic activity was located in the 

PAH-containing (neutral) fraction. Epler et aL (1978) and Guerin et 

a L , (1980) showed that a coal-derived petroleum substitute was ten times 

more mutagenic than a petroleum-derived product. Additionally, they 

reported that activity of natural petroleum crudes was located mainly in 

the neutral fractions, while significant activity was found in both the 

neutral and basic fractions of coal-derived fuels. 

MUTAGENICITY OF LUBRICATING OILS 

Crankcase oils from both diesel and spark-Ignition vehicles have been 

studied for their mutagenic components and for the contribution lubricating 

oils make to mutagenic components in vehicular emissions. Unused crankcase 

oils have been reported to be non-mutagenic in the Salmonella bioassay, but 

used crankcase oils became mutagenic in gasoline engines (Payne et al., 1978; 

Wang et aL , 1978; Hermann et a L , 1980; L8froth et a L , 1980). TofToth 

reported that this mutagenic activity Increased in proportion to the number 

of kilometers driven and, with TA-98 that metabolic activation increased 

the mutagenic activity of used motor oil. Used motor oil from diesel 

engine gave small increases in the reversion frequency in the Salmonella 

assay but the effect was not dose-dependent (Lo'froth et a L , 1980). 

88 
P.5 

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1. Burley, H.A. and T.L. Rosebrock. Automotive diesel engines. Fuel 

composition vs. particulates. Society of Automotive Engineers. 

Paper 790923. 1979. 

2. Clark, CR., R.E. Royer, A.L. Brooks, R.O. McClellan, W.F. Marshal, 

T.M. Naman and D.E. Seizinger. Mutagenicity of Diesel Exhaust Particle 

Extracts: Influence of Car Type. Fundam. Appl. Toxicol. 1:260-265, 

1981. 

3. Clark, CR . Mutagenicity of Diesel Exhaust Particle Extracts.; 

Publication LMF-96, Category: UC-48. Lovelace Biomedical and Environmental Research Institute, Albequerque, NM, April 1982, 32 pp. 

4. Clark, CR., R.E. Royer, A.L. Brooks, T.R. Henderson, R.O. McClellan, . 

W.F. Marshall, T.M. Naman. Mutagenicity of diesel exhaust particle 

extracts: Influence of fuel composition in two diesel engines. 

Fundam. Appl. Toxicol., 2:38-43, 1982a. 

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Seizinger. Mutagenicity of diesel exhaust particle extracts: influence of non-petroleum fuel extenders. Arch. Environm. Contam. Toxicol. 

11:749-752, 1982b. 

6. Claxton, L.D. Bacterial Mutagenesis and Evaluation of Emissions. 

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1983. 

8. Ellenton, J.A. and O.F. Hallett. Mutagenicity and Chemical Analysis 

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i 

10. Epler, J.L., J.A. Young, A.A. Hardigree, T.K. Rao, M.R. Guerin, I.B. 

Rubin, C.-H. Ho, and B.R. Clark. Analytical and Biological Analyses 

of Test Materials from the Synthetic Fuel Technologies. I. Mutagenicity of crude oils determined by the Salmonella typhimuriurn/microsomal activation system. Mutat. Res., 57:265-276, 1978. 

89 
P.6 

11. Gibson, T.L. Nitroderivatives of Polynuclear Aromatic Hydrocarbons 

in Airborne and Source Particulate Matter. Atmos. Environ., 16:2037-

2040, 1982. 

12. Guerin, M.R., J.L. Epler, W.H. Griest, B.R. Clark, and T.K. Rao. 

Polycyclic aromatic hydrocarbons from fossil fuel conversion processes. 

In: Carcinogenesis, Vol. 3, Polynuclear Aromatic Hydrocarbons, P.W. 

Jones and R.J. Freudenthal, eds., New York, Raven, 1978, pp. 21-31. 

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Res., 23:42-53, 1980. 

14. Hare, CT. , K.J. Springer, and R.L. Bradow. Fuel and additive effects 

on diesel particulate development and demonstration of methodology. 

Society of Automotive Engineers. Paper 760130. 1976. 

15. Henderson, T.R., A.P. Li, R.E. Royer and CR . Clark. Increased 

cytotoxicity and mutagenicity of diesel fuel after reaction with 

N02. Environ. Mutagen., 3:211-220, 1981. 

16. Hermann, M., 0. Chaude, N. Weill, H. Bedouelle, and M. Hofnung. 

Mutat. Res., 77:327-339, 1980. 

17. Holmberg, B. and U. Ahlborg, eds. Consenus Report: Mutagenicity 

and Carcinogenicity of Car Exhausts and Coal Combustion Emissions. 

Environ. Health Perspect., 47:1-30, 1983. 

18. 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. In: Application of Short-Term Bioassays 

in the Fractionation and Analysis of Complex Environmental Mixtures, 

Waters, M.D., Nesnow, S., Huisingh, J.L., Sandhu, S.S. and Claxton, 

L., eds., U.S. Environmental Protection Agency, EPA-600/9-78-027, 

1978. pp. 1-32. 

19. Lebowitz H., D. Brusick, D. Matheson, D.R. Jagannath, M. Reed, S. 

Goode, and G. Roy. Commonly used fuels and solvents evaluated in a 

battery of short-term bioassays. Environ. Mutagen., 1:172-173, 1979. 

20. Lipkea, W.H., J.H. Johnson, and CT . Vuk. The physical and chemical 

character of diesel particular emissions measurement techniques and 

fundamental considerations. Society of Automotive Engineers. Paper 

780108. 1978. 

21. LSfroth, G. In: Health Effects of Diesel Engine Emissions, Proceedings of an International Symposium, December 3-5, 1979, Pepelko, 

W.E., Danner, R.M. and Clark, N.A. eds. U.S. Environmental Protection Agency, EPA-600/9-80-057a, 1980. pp. 327-344. 

22. Mayer, W.J., D.C Lechman, and D.L. Hilden. The Contribution of 

Engine 011 to Diesel Exhaust Particulate Emissions. Society Automotive Engineers, Paper 800256, 1980. 

90 
P.7 

23. Nishioka, M.G., B.A. Petersen, and J. Lewtas. Comparison of nitroaromatlc content and direct-acting mutagenicity of diesel emissions. 

Paper presented at 6th International Symposium on Polynuclear Aromatic 

Hydrocarbons. Columbus, OH, Oct. 27-29, 1981. 

24. Payne, J.F., I. Martins, and A. Rahimtula. Crankcase oils: Are 

they a major mutagenic burden in the aquatic environment? Science, 

200:329-330, 1978. 

25. Pelroy, R.A. and M.R. Petersen. Use of Ames test in evaluation of' 

shale oil fractions. Environ. Health Perspect., 30:191-203, 1979. 

26. Rannug, U. Data from short-term tests on motor vehicle exhausts, i 

Environ. Health Perspect., 47:161-169, 1983. 

27. Rao, T.K., J.A. Young, A.A. Hardigree, W. Winton, and J.L. Epler. 

Analytical and Biological Analyses of Test Materials from the Synthetic Fuel Technologies. II. Extended genetic and biochemical studies 

with mutagenic fractions. Mutat. Res., 54:185-191, 1978. 

28. Schuetzle, 0., T. Riley, T.J. Prater, T.M. Harvey, and D.F. Hunt. 

The identification of nitrated derivatives of PAH 1n diesel particulates. Ana. Chem. 54:265-271, 1982. 

29. Seizinger, J.E., T.M. Naman, W.F. Marshall, CR . Clark, R.O. McClellan. 

Diesel Particulates and Bioassay Effect of Fuels, Vehicles, and Ambient 

Temperature. Society of Automotive Engineers. Paper 820813, Passenger 

Car Meeting, Troy, Michigan, June 1982. 9 pp. 

30. Sklarew, D.S., R.A., Pelroy, S.P. Oowney, R.H. Jungers and J. Lewtas. 

Chemical and Mutagenic Characteristics of Diesel Exhaust Particles 

from Different Fuels. EPA Internationl Diesel Emissions Symposium, 

Raleigh, NC (Extended Abstract), 1981. 

31. Springer, K.J. and T.M. Baines. Emissions from diesel engines of 

passenger cars. Society of Automotive Engineers. Paper 770818. 

1977. 

32. Wang, Y.Y., S.M. Rappaport, R.F. Sawyer, R.D. Talcott, and E.J. Wei. 

Cancer Letters, 5:39-47, 1978a. 

33. Wang, Y.Y., R.F. Sawyer, and E.J. Wei. U.S. Environmental Protection Agency, EPA-600/9-78-027, 1978b. 587 pp. 

34. Zweidinger, R.B. Emission Factors from Diesel and Gasoline Powered 

Vehicles: Correlation with the Ames Test. EPA International Diesel 

Emissions Symposium, October 1981, Raleigh, NC, (Extended Abstract), 

1981. 

35. Zweidinger, R.B. Emission Factors from Diesel and Gasoline Powered 

Vehicles: Correlation with the Ames Test. In: Toxicological Effects 

of Emissions from Diesel Engines. J. Lewtas, ed. Elsevier Biomedical, 

New York, 1982. pp. 83-96. 

91 
P.8 

PART III 

SECTION I 

SELECTION OF BIOASSAYS FOR ROUTINE SCREENING 

OF AUTOMOBILE EMISSIONS 

INTRODUCTION 

The first two parts of this document addressed the issues of whether 

short-term tests were amenable to the assessment of auto emission genotoxicity and if so what has been the experience of the specific tests in 

this regard. 

The conclusions indicated that, for several reasons, short-term tests 

would be extremely useful in the assessment of mutagenic and/or carcinogenic potential. Among the numerous tests which have been used to 

evaluate the genotoxicity of automobile emissions, three appear to offer 

the best assessment battery. They are: Ames Salmonella/liver microsome 

assay, Sister Chromatid Exchange (SCE) analysis in Chinese Hamster Ovary 

(CHO) cells, and tests for gene mutation in cultured mammalian cells 

(particularly the Mouse Lymphoma assay). This latter conclusion, of 

course, is based upon an analysis of the current data. As the data base 

expands, it may become evident that other assays provide an improved 

assessment; thus, the utility of the test battery selected should be 

periodically reviewed with this possibility in mind. 

The three tests identified as those currently considered to be most useful 

in emission evaluation have also been identified as predictive tests for 

human health effects, in particular, cancer initiation. The reliability 

of these three tests in combination in predicting animal carcinogenicity 

is very good (Bartsch et. aL , 1983; Clive et aL , 1979; Wolff, 1983). 

The reliability of these tests or animal cancer studies to predict human 

cancer risk 1s limited by the available human epidemiology data base 

(see Table 11). 

PROCEDURES 

The Office of Pesticides and Toxic Substances (OPTS) of the EPA has published guideline protocols for each of the candidate screening bioassays 

identified. Copies of these protocols are available through the National 

Technical Information Service (5285 Port Royal Road, Springfield, 

Virginia 22161). Each protocol has been subjected to peer review and 

meets the minimal criteria for a screening procedure. However, several 

components of these protocols require modification if they are to be 

adapted to emission screening. 

92 
P.9 

1. Emissions exist in multiphase forms (gas, solid, extractable) which 

necessitate that sample collection and preparation information unique 

to the type emission assessment be added to the protocol. At the 

present time, it is recommended that analyses be limited to extracts 

of the particle phase of the emission. 

2. The analysis of test sample results should go beyond qualitative 

evaluations of activity or nonactivity. The primary usefulness ofi 

this approach will rely upon quantitative comparative assessments ; 

among samples. The experimental results from numerous tests of 

engines, running conditions, and fuel types indicate that quantitative variation among positive responses will be involved in decisionmaking. Few, if any, combustion samples are inactive when evaluated 

as particle extracts. Therefore, techniques for quantitative analysis 

and comparison among samples should be included in the study designs. 

Section 2 of Part III of this report will be devoted to these two parameters. 

DATA INTERPRETATION 

Interpretation of test results in a manner that would permit the establishment of minimal criteria for engine or fuel assessment will be the 

most difficult issue to resolve. An approach is one in which bioactivity 

ceilings (limits) are established. These activity limits will not necessarily reflect safety factors because problems associated with adverse 

health consequences of automobile emissions have yet to be resolved; 

however, the limits must be established on the basis of the best available 

information obtained from published reports. 

A secondary parameter of this problem is one of data format (e.g., expression of the data in constant units). Ames test results are often converted 

to revertants per mile (or kilometer) or revertants per unit of fuel 

combusted. While adequate for such a test, these conversions would not 

necessarily work equally well for SCE or quantitative mutation frequencies 

generated in mouse lymphoma L5178Y or other cell lines. 

Problems of data interpretation and standardized criteria for engine and/or 

fuel certification will be addressed in Section 3 of this part of the 

report. 

POTENTIAL PROBLEMS 

While the prospects for using short-term bioassays in the evaluation of 

exhaust emissions look promising based on the published literature, 

other technology related issues must be addressed. Claxton summarized 

these issues in a report published in 1982. A summary of these pertinent 

issues is given in Table 45. 

93 
P.10 

TABLE 45. DIFFICULTIES ENCOUNTERED IN THE INTERPRETATION 

OF MOBILE SOURCE GENOTOXICITY STUDIES 

Stage Type of Difficulty 

Generation 

Collection 

Extraction 

Fractionation 

Bioassay 

Statistical 

Characterization of the source; 

Repeatability of testing procedure; 

Vehicle-to-vehicle variation. 

Artifact formation (NO , organic interaction, 

ozone); 

Control versus ambient conditions. 

Physiological versus organic; 

Selective loss and recovery of compounds; 

Artifact formation. 

Recovery efficiencies; 

Complexity. 

Relevance; 

Reproducibility; 

Endogenous activation; 

Bioassay versus human metabolism. 

Collection/recovery efficiencies; 

Data transformations; 

Data summary methods; 

Stati stical/correlation methods. 

From: Claxton, L.D. The utility of bacterial mutagenesis testing in 

the characterization of mobile source emissions: A review. 

In: 1981 Diesel Emissions Symposium. U.S. Environmental Protection Agency, pp. 81-94, 1982. 

94 
P.11 

PART III 

SECTION 2 

TEST PROCEDURES 

PROTOCOL RECOMMENDATIONS 

Copies of protocols for the Ames test, SCE test using CHO cells, and for 

specific locus gene mutation in Mouse Lymphoma L5178Y (TK+/-) cells* and 

Chinese Hamster Ovary (CHO) (HGPRT+)** cells are attached. The only 

procedural comment regarding the conduct of these tests relates to the 

fact that each should be conducted in such a manner that: 

i 

1. Dose levels will cover toxic as well as nontoxic concentrations of 

the sample. 

2. Sufficient dose levels will be used to enable a multipoint dose 

response curve to be constructed for each test condition employed. 

3. Exposures will be calibrated in a manner that will permit data to 

be expressed as induced events per unit of emission released per 

mile traveled or mass of fuel consumed. 

4. Each assay should be calibrated against a standard sample (i.e., 

standard diesel particulate, standard gasoline particle). The 

standardization would consist of periodic inclusion of an extract 

of the standard sample in the bioassay. A dose-response curve 

following within a specified range would be mandatory before continued testing of routine samples. Also, it would be highly advantageous to include a single dose from the response curve of the standard sample in each bioassay set conducted on a given day or with a 

given cell pool. 

5. It 1s recommended, to fulfill the data requirements for quantitative 

comparisons, that each bioassay be conducted with two replicates 

and repeated once, no earlier than 24 hours following the first trial. 

*Five specific assay acceptance criteria for deciding whether an assay is 

usable have been developed by NTP under their testing program. 

**Use of CHO cells should be limited to the modified exposure method 

reported by Li et aL , 1982. 

95 
P.12 

RECOMMENDATIONS FOR COLLECTION AND PROCESSING OF SAMPLES 

Ideally, one would like to be able to compare engines or fuels using 

whole-exhaust emissions under conditions similar to human exposure. 

Experience to date, however, indicates that the ideal is probably not 

feasible with current state-of-the-art techniques. The best situation 

would be to evaluate the fractionated whole exhaust (i.e., gas phase and 

solid phase separately). This requires that the whole exhaust be trapped 

and specially processed for in vitro bioassay procedures. Part II, 

Section 1 describes many of tHe sampling procedures applied to exhaust 

emissions. The dilution tunnel method has worked well and can be adapted 

to both gas phase and particle trapping. At the present time, either 

the absorbant resin or cryogenic trapping of gas phase materials would 

appear adequate, although both have significant limitations and additional 

research into the best method for gas phase evaluation is needed. Jji 

situ methods may also be of value. 

Part II, Section 2 describes the techniques for preparing collected 

particulates for bioassay. The typical process involves dichloromethane 

soxhlet extraction followed by solvent exchange to DMSO. This procedure 

is applicable to all three of the candidate bioassays. While particle 

size and loading properties might vary slightly as fuels or engines vary, 

for the purposes of comparison, these factors as well as differences in 

bioavailability from particle to particle will not be considered, and 

extracts prepared under uniform conditions will be compared for potency. 

It may be possible at some point to develop a reliable bioassay responsive 

to the toxic properties of whole exhaust but, until that time, the most 

efficient and reproducible method consists of dilution tunnel trapping 

(particulates and/or gases), extraction with organic solvents, and quantification of the total organics collected followed by solvent exchange 

and the selected bioassay(s). 

This process is shown in a general manner in Figure 9 and should be used 

to form the basic steps of a routine testing program. 

RECOMMENDATIONS FOR DATA ANALYSIS 

Each protocol previously recommended contains a series of criteria for 

the determination of a positive response. In all cases, this involves a 

statistical analysis of the data. Because of the use of these tests in 

a program for engine or fuel assessment, the analysis should be more 

extensive and should synthesize the responses of the three candidate 

screening procedures into the final determination of genotoxic activity. 

96 
P.13 

EMISSION 

[Produced under controlled conditions] 

COLLECTED IN STANDARD 

DILUTION TUNNEL OEVICE 

[See Part II, Section 1] 

4. 

4. 

GAS PHASE 

[Resin or Cryogenic Trapping] 

PARTICULATE PHASE 

[Filter Trapping] 

SOLVENT EXTRACTION OR RECOVERY SOLVENT EXTRACTION 

ORGANIC ANALYSIS ORGANIC ANALYSIS 

SOLVENT EXCHANGE SOLVENT EXCHANGE 

BIOASSAY (SCE, Ames, Mutation) 

Figure 9. Flow Chart for Sample Collection and Processing. 

97 
P.14 

Data from the Ames test have been subjected to numerous statistical 

evaluation, potency assessment (Russell and Meselson, 1977; Bartsch, 

1983; Hooper et aL , 1977) and normalization (i.e., revertants/ km; 

revertants per mass of fuel combusted) with varying degrees of success. 

Whether or not these same approaches can be used with the Mouse Lymphoma 

or SCE assays has not been established. 

One must at this time caution against any presumption that potency of a 

sample J_n vitro will be an accurate indication of potency for health 

effects~In vivo. 

Probably the most useful generalization reflecting the relevance of positive responses in the ui vitro bioassays is that they indicate a potential 

for hazard, and that, within a given bioassay, the stronger the response, 

the greater the indication of potential hazard. 

APPROACH TO DATA ANALYSIS USED IN COMPARING GENETIC ACTIVITY OF EMISSION 

SAMPLES 

Analysis of the bioassay data should accomplish several objectives: 

1. Interpretation should be based on test results expressed in a manner 

which will relate biological activity to emissions rate in a useful 

manner. 

2. Responses from all three bioassay types should be used in evaluating 

the genetic toxicity of a specific sample. 

3. Interpretation of the test results should be based on sound statistical methods which capture the maximum amount of information available. 

4. Analysis of a specific sample should clearly demonstrate the degree 

of biological activity relative to a body of similar samples and 

determine whether that biological activity falls outside the range 

of the historical data base. 

The procedures described in this section attempt to satisfy each of the 

objectives. To be useful, a description of biological activity must 

include an estimate of the specific activity of a sample in each test 

employed. For the three tests recommended as the battery, the linear 

slope of the dose-response curve, expressed as revertants/ug, SCE/cells/ 

ug/rnl and mutant frequency/pg/ml, has been selected. The specific activity 

should then be adjusted to an estimate of particle potency by determining 

the level of organic extractabi1ity (percent extractable) from the material 

collected. The final calculation should consider the particle potency vs. 

the rate of particle emission (PER). The following formula gives the 

relationship among these factors in developing a Sample Activity Rate (SAR). 

98 
P.15 

Genetic Activity ug organics pg particles _ Genetic Activity 

pg organics 100 pg particles kilometer (km) " km 

t t t 

Slope t 

(Specific Activity) % Extractables Particle Emission Sample Activity 

Rate (PER) Rate (SAR) 

Particle Potency 

The SAR developed for the three bioassays selected would be expressed in 

the following manner: 

Test SAR Format 

Ames Revertants/km 

Mouse Lymphoma Mutant Frequency/km 

SCE SCE/km 

The proposed approach allows all factors contributing to the genetic 

activity of a sample to be converted to a constant unit of emission release. 

No attempt is implied to associate these values with any quantitative 

estimate of genetic or carcinogenic risk. 

Table 44 identifies a set of samples obtained by the EPA (HERL/RTP) and 

tested in the three candidate bioassays. Different engines and fuels 

are represented in this sampling and, in many respects, this group of 

samples represents a realistic cross-section of data likely to be encountered in routine emission evaluation. 

Under the proposed testing scheme, future test samples will be accompanied 

by more data from independent trials; thus, estimates of variability will 

be more accurate. 

SAR for the 14 samples were calculated for the Ames test (Table 46), 

Mouse Lymphoma assay (Table 47), and SCE assay (Table 48). Values for 

nonactivation and activation trials are shown. Further analysis of the 

data sets resulted in the conclusion that S9 mix did not significantly 

alter the SAR and all subsequent analyses of the data treated nonactivation and activation runs as equivalent. 

Objectives No. 2 through No. 4 necessitate application of statistical 

methods which would identify the samples that produced responses in one 

or more of the selected bioassays which fell outside a "normal" range of 

biological activity. 

99 
P.16 

TABLE 46. DESCRIPTION OF SOURCES FOR 

FOURTEEN EMISSION SAMPLES COMPARED FOR MUTAGENIC ACTIVITY 

Sample 

No. 

2 

3 

4 

5 

6 

10 

11 

Engine (Fuel) 

1970, 6 cylinder Ford Van 

(leaded gas) 

1979, 300D Mercedes Diesel 

(#2 diesel fuel) 

Caterpillar 3208 Diesel 

(#2 diesel fuel, Ave.) 

Caterpillar 3208 Diesel 

(#2 diesel fuel, Min.) 

Caterpillar 3208, Diesel 

(#2 diesel fuel, Prem.) 

GM DO 8V-71 Diesel 

(#2 diesel fuel, Ave.) 

GM DD 8V-71 Diesel 

(#2 diesel fuel, Min.) 

Sample 

No. 

12 

13 

14 

16 

17 

18 

19 

Engine (Fuel) 

GM DD 8V-71 Diesel 

(#2 diesel fuel, Prem.) 

GM DD 8V-71 Diesel 

(#1 diesel fuel) 

GM DD 8V-71 Diesel 

(Jet fuel) 

Oldsmobile 350 

(#2 diesel fuel, Min.) 

Mustang II 

(unleaded gasoline) 

Nissan Diesel 220C 

(#2 diesel fuel, Min.) 

Volkswagon Rabbit 

(#2 diesel fuel, Min.) 

The approach which was selected employed an analysis of variance which 

compared the experimental means at the 0.05 level of significance for 

each sample in each bioassay. The sample means are plotted in tables 

for comparative purposes (Figures 10-12). All data from a given bioassay 

were included in an analysis because there was no evidence which indicated 

a significant S9 effect and no evidence which indicated an interaction 

effect. 

Several methods may be used to ask the question of how to identify the 

outliers among the samples in Figures 10-12. For example, it is possible 

to group samples using various statistical methods and find those tests 

which differ significantly from the remaining tests. Samples 5 and 18, 

for example, are clearly outliers using the Ames test (Figure 10). 

100 
TABLE 47. TIER STUDY - AMES TEST - TA98 

ANALYSIS OF REVERTANTS PER KILOMETER 

"ATF Slope 

7 PER* pg/km % EX I* REV. Per KM2 

2 

3 

3 

3 

3 

4 

4 

4 

4 

5 

5 

5 

5 

6 

6 

6 

6 

10 

10 

10 

10 

11 

11 

11 

11 

12 

12 

12 

12 

13 

13 

13 

13 

14 

14 

14 

14 

16 

16 

17 

17 

18 

18 

19 

19 

2 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

2 

1 

1 

2 

1 

2 

1 

2 

1 

2 

1 

T"630 

5.110 

2.380 

4.380 

5.400 

2.040 

1.020 

1.050 

0.900 

1.040 

1.760 

1.500 

1.340 

1.300 

0.940 

0.800 

1.070 

0.830 

0.045 

0.124 

0.043 

0.081 

0.207 

0.233 

0.184 

0.199 

0.102 

0.120 

0.068 

0.053 

0.081 

0.090 

0.039 

0.037 

0.118 

0.074 

0.074 

0.051 

1.500 

2.200 

3.500 

1.600 

13.000 

11.000 

3.000 

3.800 

0. 

0. 

0. 

0. 

0. 

0.0300 

0.0300 

0.2400 

0.2400 

0.2400 

.2400 

.5600 

.5600 

.5600 

.5600 

0.5900 

0.5900 

0.5900 

0.5900 

0.5500 

0.550 

0.5500 

0.5500 

2.0700 

2.0700 

2.0700 

2.0700 

1.9400 

1.9400 

1.9400 

1.9400 

1.9600 

1.9600 

1.9600 

1.9600 

1.3600 

1.3600 

1.3600 

1.3600 

1.2200 

1.2200 

1.2200 

1.2200 

0.8400 

0.8400 

0.0085 

0.0085 

0.5300 

0.5300 

0.2900 

0.2900 

"18TT" 

18.7 

8.4 

8.4 

8.4 

8.4 

53.9 

53.9 

53.9 

53.9 

58.7 

58.7 

58.7 

58.7 

56.8 

56.8 

56.8 

56.8 

17.5 

17.5 

17.5 

17.5 

17.7 

17.7 

17.7 

17.7 

18.2 

18.2 

18.2 

18.2 

23.1 

23.1 

23.1 

23.1 

29.5 

29.5 

29.5 

29.5 

17.0 

17.0 

43.0 

43.0 

8.0 

8.0 

18.0 

18.0 

37.194 

28.667 

47.981 

88.301 

108.864 

41.126 

307.877 

316.932 

271.656 

313.914 

609.541 

519.495 

464.082 

450.229 

293.656 

249.920 

334.268 

259.292 

16.301 

44.919 

15.577 

29.342 

71.080 

80.008 

63.182 

68.333 

36.385 

42.806 

24.257 

18.906 

25.447 

28.274 

12.252 

11.624 

42.468 

64.062 

26.633 

18.355 

214.200 

314.160 

12.792 

5.848 

551.200 

466.400 

156.600 

198.360 

Activation conditions are: ACT 1 = -S9; ACT 2 » +S9. 2Slope of linear portion of dose response curve. 3Particle Emission Rate. 4Percent of total particle weight extracted. 5SAR (Revertants/KM) Sample Activity Rate. 

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Several conclusions can be made based on analysis of even this small set 

of samples. 

1. Comparisons between nonactivated and S9-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 likely, 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 Figure 13. By performing independent trials, the variability arising from replicating 

the collection and extraction procedures as well as bioassay techniques are important data components. Claxton and Kohan (1981) 

showed that there are small but consistent variances resulting from 

sample preparation differences. 

DATA INTERPRETATION 

If engine or fuel assessments studies can be conducted under conditions 

in which certain variables can be carefully controlled (running cycles, 

collection conditions and sample preparation), comparisons of hazard 

potential can be generated as described previously. These will be best 

estimates with reasonably large limits of variation. Standardized engine 

running conditions and particle collection methods should be defined in 

a separate document. 

As indicated in an earlier section of this report, the ability to quantify 

hazard or risk on the basis of short-term testing will require continued 

research but, if one assumes that the qualitative risks from automobile 

emission will be similar, then comparative differences based on a statistical analysis of the biological activity parameters might be a reasonable 

means to identify high vs_. low polluting engines or fuels. 

CONCLUSIONS 

1. Mutagens exist in vapor and solid phase probably generated by combustion and mono or poly substituted PAH's or nitroaromatics which can 

be activated to genotoxic agents by bacteria or animal cells (liver, 

macrophages, etc.). 

2. 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. 

107 
FIGURE 13. COMPOSITION AND LOGISTICS 

OF AN EMISSION ASSESSMENT 

Stepa 

1 

2 

3 

4 

*> 

Ames Test 

Conducted with 

Triplicate 

Plates/Dose 

Trial Ab 

Particle Collection 

from Filter(s) 

Extraction of Organics 

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*> 

Mouse Lymphoma 

Assay Conducted 

with Duplicate 

Cultures 

4 

Data Analysis 

i 

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Conducted with 

Duplicate 

Cultures 

*> 

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Conducted with 

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Trial Bb 

Particle Collection 

from Filter(s) 

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Bioassay 

4 

4 

Mouse Lymphoma 

Assay Conducted 

with Duplicate 

Cultures 

4 

Data Analysis 

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Step 1 - Independent trials for determination of 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 particulate required per trial is approximately 0.5g. 
P.25 

3. The levels and composition of chemicals found in emissions may be 

influenced by: 

i 

A. Engine types 

B. Fuel types 

C. Operating conditions 

0. Control devices 

E. Environmental conditions of collection. 

4. An efficient screening program to compare quantitative variations 

in one of the above parameters (by holding all other parameters 

constant) can be accomplished with a series of short-term jji vitro! 

tests; however, the assessment cannot be conducted on whole emission 

collections; rather it requires a standard sample collection and 

processing protocol for the preparation of organic compounds recovered from the solid phase. 

5. A uniform data interpretation scheme has been defined in which the 

relative biological activity of each bioassay can be quantitatively 

compared to an existing data base developed from similar engines, 

fuels, or fuel additives. Samples which show statistically significant deviations from the data base can be interpreted as a signal 

that an engine, fuel, or additive might warrant further Investigation 

for possible health impact. 

The proposed approach should be adequate for making quantitative comparisons of the biological activity of emissions generated by different 

engines or fuels under standardized running and collection protocols and 

for identifying responses which fall outside the normal range of engines, 

fuels, or additives. The results will not be suitable to quantify the i 

absolute health risk of emissions because of the many uncertainties 

associated with bioavailability and dosimetry of emissions to heterogenous 

human populations under normal exposure conditions and the lack of formal 

linkages between the responses in short-term tests and human disorders. 

109 
P.26 

REFERENCES 

1. Bartsch, H. Bacterial-mammalian mutagenesis correlations: Mechanistic significance for carcinogenesis. Ann. N.Y. Acad. Sci., 1983 

G.M. Williams, V.C. Ounkel and V.A. Ray (eds.) NY Acad. Sci. New 

York, Vol. 407, pp. 351-361. 

2. Brusick, O.J. and R.R. Young. IERL-RTP Procedures Manual: Level 1 

Environmental Assessment Biological Tests. EPA-600/8-81-024, NTIS 

PB 82-228966, Litton Bionetics, Inc., Kensington, MD, October 1981, 

150 pp. 

3. Clark, CR., R.E. Royer, A.L. Brooks, T.R. Henderson, R.O. McCellan, 

W.F. Marshall, T.M. Naman. Mutagenicity of Diesel Exhaust Particle 

Extracts: Influence of Fuel Compositon in Two Diesel Engines. 

Fundam. Appl. Toxicol., 2:38-43, 1982. 

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.O. Waters, S.S. Sandhu, 

J. Lewtas Huisingh, L. Claxton, S. Nesnow, eds. Plenum Publishing 

Corp., 1981. pp 229-317. 

5. Clive, 0., K.O. Johnson+J.F.S. Spector. Validation and characterization of the L5178Y/TK mouse lymphoma mutagen assay system. 

Mutat. Res., 59:61-108, 1979. 

6. Hooper, N.K., A.D. Friedman, CB . Sawyer and B.N. Ames. Carcinogenic 

potency: Analysis, utility for human risk assessment and relation 

to mutagenic potency in salmonella. Progress Report for IARC/WHO 

meeting, Lyon, France, October 1977. 

7. Li, A.P. Quantification of Mutation at the Na+-K+ATPase and Hypoxanthine-Quanine Phosphoribosyl Transferase (HGPRT) Gene Foci in 

Chinese Hamster Ovary Cells. J. Tissue Culture Methods, 7:1, 27-32, 

1982. 

8. Meselson, M. and K. Russell. Comparisons of carcinogenic and mutagenic potency, In: Origins of Human Cancer, Book C, Human Risk 

Assessment, Vol. 4, H.H. Hiatt, J.D. Watson and J.A. Winsten, eds. 

Cold Spring Harbor Laboratory, 1977. pp. 1473-1481. 

9. U.S. Environmental Protection Agency. Short Term Genetic Bioassays 

Used in the Evaluation of Unregulated Automobile Emissions: 1981-1982 

Contract Reports. D.J. Brusick, R.R. Young and D.R. Jagannath, eds., 

EPA 68-02-3382. Technical Directive 008, 1983. 

110 
P.27 

10. 

as - test for mutagenic carcino 

„ c lister chromatid exchange as a *•«* v.c. 0ynkel and 

*

olff' !• i v Acad. Sci. (1983) G.M.J}1'^? i42-l53. 

gens. Ann. N.Y. * » Mew York Vol. 4U#, 

V.A. Ray, eds. NY Acaa. 

U l