Mutagenicity of diesel exhaust particle extracts: Influence of car type

FUNDAMENTAL AND APPLIEDTOXICOLOGY 1:26o-265 (1981)

Mutagenicity of Diesel Exhaust Particle Extracts: Influence of Car Type C.R. CLARK A, R.E. ROYERA, A.L. BROOKS ^, R.O. McCLELLANA, W.F. MARSHAL B, T.M. NAMAN Band D.E. SEIZINGER B ^Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87115; BBartlesville Energy Technology Center, Bartlesville, Oklahoma 74003

ABSTRACT

Mutagenicity of Diesel Exhaust Particle Extracts: Influence of Car Type. Clark, C.R., Royer, R.E., Brooks, A.L., McClellan, R.O., Marshal, W.F., Naman, T.M. and Seizinger, D.E. (1981). Fundam. Appl. Toxicol. 1:260-265. Mutagenicity of extracts of particles collected from the exhaust of six different European and American diesel cars was evaluated in Salmonella strains TA 1535, TA 100, TA 1537, TA 1538"and TA 98. The extracts demonstrated direct, dose-related mutagenicity in all strains except TA 1535, with the potencies varying from 6-17 revertants/pg in TA 100, the most sensitive strain. Addition of Aroclor 1254 induced rat liver homogenate fractions decreased the direct response in TA 100 and increased the response in four of the six extracts in TA 98. Differences in the extractable organic fraction of the particles and the particulate emission rates for the six cars had a greater influence on the amount of mutagenicity emitted (revertants per mile) than the actual mutagenic potency of the organics. The ranking of the cars by revertants/mile was different than ranking by revertants//zg extractable organics. The response of two of the six extracts in a nitroreductase deficient strain of Salmonella (TA 100 FR1) were significantly lower than the response in TA 100, suggesting that r e d u c t i o n of nitroaromatics by bacterial enzymes may be influential in the direct response. Results of testing triplicate samples collected in three different cars demonstrated good repeatability in sampling and bioassay procedures. INTRODUCTION Automobile manufacturers project a marked increase in future sales of diesel powered automobiles and light duty trucks. Because of the significantly greater (30-100• particulate exhaust emissions produced by diesel engines, studies designed to evaluate the potential environmental effects and health risks of increased diesel vehicle usage have been initiated. Diesel engine exhaust particles (soot) are 0.1- to 0.5-/~mdiameter (mass median) chain aggregates of much smaller primary particles and provide a carbonaceous surface to which organics are easily adsorbed. Studies using 0.1 rcm chain aggregate aerosols have shown that they readily deposit in the deep lung where clearance is more difficult (Wolff et al., in press). Particle associated organics can comprise from 1070% of the weight of the particle and have been shown to be Research performed under U, S. Department of Energy Contract DE-AC0476EV01013 and conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.

mutagenic in in vitro bacterial test systems (Huisingh et al., 1978; Siak eta/., 1979; Clark and Vigil, 1980). Most studies have shown that extracts of the particles contain a mixture of direct and indirect (i.e. require activation by metabolic enzymes) acting mutagens. The greatest activity is found in fractions that do not require activation and most of the mutagenic activity appears to be of the fra me-shift .type (Clark and Vigil, 1980). While the exact identity of the mutagens has not been established, analysis of mutagenic fractions of the extracts indicate the presence of polycyclic aromatic hydrocarbons (PAH) and oxygenated and nitrated PAH (Schuetzle, et al., 1980). A number of factors, including engine design, fuels, driving patterns, engine age and operating temperatures may influence the formation of mutagens in the exhaust. For example, benzo(a)pyrene concentrations have been found to vary as a function of engine type (Springer and Baines, 1977). We report here results of studies designed to determine the influence of various diesel engine types on the mutagenicity of particle associated organics. Extracts of particles collected from the exhaust of six different American and European diesel cars were evaluated in the Salmonella mutagenicitytest. Sampling strategy was designed to provide information on the repeatability of the sampling and testing procedures, and variability in mutagenic activity of exhaust emissions produced by similar and different car types. Mutagenic potencies of the particle extracts were compared to the amount of organics associated with the particles and particle emission rates of the various cars to provide an estimate of the amount of genotoxic materials emitted from the exhaust. The results of these studies suggest that differences in engine type have very little influence on the resultant mutagenicity of the particle associated organics when the cars are operated on the same fuel. METHODS AND MATERIALS Diesel particles were collected from the exhaust of late model American and European cars (Table 1) at the Bartlesville Energy Technology Center, Bartlesville, Oklahoma. Cars were acquired new from dealers or on loan from the manufacturer; a 4000 mile break-in period was allowed before testing for all cars except the Fiat. All maintenance was performed at manufacturer's recommended service intervals.

The cars were operated according to the EPA Federal Test Procedure on a chassis dynamometer while the exhaust was diluted in a tunnel sized to cool the air/exhaust mixture to below 125 ~ (approximately 10:1 dilution of raw exhaust). All tests were done using the same #2 diesel fuel. The entire particulate portion of the exhaust was collected on 40 • 40-

Copyright 19ai, Society of Toxicology 260

Fundam. Appl. Toxicol. (!)

3lay/June, 1981

DIESEL PARTICLE EXTRACT MUTAGENICITY TABLE 1 D i e s e l A u t o m o b i l e s U s e d in S t u d y

Car ^

Model Year

Regulated Engine Emissionsc Displacement B (grams per mile) (liters) CO HC NOx

Peugeot 504

1979

2.3 (4)

0.8

0.3

1.5

Oldsmobile Delta 88

1980

5.7 (8)

1.4

0.4

1.6

Volkswagen Rabbit Audi 5000

1980 1980

1.5 (4) 2.0 (5)

1.1 2.9

0.3 0.9

1.2 1.9

1980

3.0 (5)

1.2

0.3

1.7

Prototype

2.4 (4)

2.5

0.7

1.7

Mercedes 300D Fiat 131

^All cars, except the Fiat, were acquired new from the dealer and a 4000 mile break-in period allowed before testing. BNumber of cylinders indicated in parentheses. CMeasured while cars were operated according to the EPA Federal Test Procedure on a chassis dynamometer, ambient temperature 75 ~ Values represent weighted averages of emissions measured during cold (43%) and hot (57%) start opera tion. CO = carbon monoxide; HC = total hydrocarbons; NOx = nitrogen oxides (NO and NO2).

inch and 47 mm Pallflex T60A20 filters which were weighed before and after sampling to determine mass loading (Fig. 1). Carbon monoxide, total hydrocarbons, and nitrogen oxide emissions were measured during the sampling period for both hot and cold starts and values are reported in Table 1. Particle associated organics were extracted by ultrasonication in dichloromethane (Clark and Vigil, 1980) and reconstituted in spectral quality dimethylsulfoxide for evaluation in the Sa/monella mutagenicity test (Ames et aL, 1975). All extracts were evaluated in strains TA 1535, TA 1537, TA 1538, TA 98, TA 100, and TA 100-FR1 at 4-6 concentrations each (ranging from 1-300/~g extract per plate) using triplicate plates. Liver enzyme fractions (S-9) were prepared from Aroclor-1254induced rats (Fischer-344, male), according to the method of Ames et al. (1975). The S-9 (32 mg protein/mL) comprised 3% of the S-9/cofactor mixture. Sodium azide (TA-100, 1535),

2-nitrofluorene (TA-98, 1538), 9-aminoacridine (TA 1537), benzo(a)pyrene, and 2-aminoanthracene were used to confirm the reversion properties of the tester strains indicated and the activity of the liver S-9. The lack of nitroreductase activity in strain TA 100-FR1 was confirmed by comparing survival in the presence of nitrofurazone to TA 100 (McCalla et aL, 1970). Revertant colonies were counted on an Artek Model 880 automatic colony counter interfaced to an Artek-Compuprint, programmed to correct counts out of the linear range of the instrument. Results are reported as slope :t: standard deviation of the linear portion of the dose-response curve (revertants per ~ug extract) calculated by linear regression analysis. The criteria used for a positive mutagenic response was demonstration of a close-related increase in the number of revertant colonies greater than the 95% confidence limits established for the spontaneous (background) reversion rate for each strain. The

Discharge to Atmosphere

.IDil"tod s t In

[tl

III

Filter

I II D.uted _

I

" Vehicle Exhaust

_

ou=

Critical Flow Venturl

300 r

Inlet

FIG. 1. Schematic of the particulate sampling s y s t e m at the Bartlesville Energy Technology Center. Fundamental and Applied Toxicology

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261

CLARK, ROYER, BROOKS AND McCLELLAN

Student's t test was used to determine significance of the dependence of number of'revertants on dose, and the F test to determine significant differences among various slopes. Raw data is available upon request from the Environmental Mutagen Information Center, Oak Ridge, Tennessee. Since day-today variation in response of the tester strains is common, particle extracts from the six cars were evaluated on the same day for each strain. Since results of mutagenicitytesting reflect onlythe genetic toxicity of organics extracted from the particles, the mutagenic potencies were normalized for the amount of organics associated with the particles (% extractable) and the particle emission rate ( g / m i ) to provide the f o l l o w i n g estimates of mutagenicity: Revertants per mg Farticles

Revertants = per/~g Extract

X 1000 X E x t r a c t a b l e Fraction

Revertants p e r mile

Revertants = per mg Particles

G r a m s p e r mile X 1000 X Particles Emitted

RES UL TS Mutagenicity of samples collected in triplicate test runs using three different cars demonstrate the repeatability achieved in sample collection, preparation and bioassay(Table 2). The mutagenic potencies of the organics extracted from the particles (revertants per/~g extract) in the three samples collected for each car are not significantly different (p > 0.01). Slightly more Variabilitywas observed in specific mutagenicity of the particles (revertants per mg particles) and revertants per mile.

The mutagenicity of extracts of particles collected from five different Oldsmobiles, operated under the same conditions, also did not vary markedly (Table 3). Despite larger differences in the extractable fraction in the five cars than observed in triplicate runs of the same car (Table 2), the mutagenicity per

mg of particles was quite similar suggesting an inverse correlation between mutagenicity of the extracts and extractable fraction. The amount of mutagenicity emitted from the exhaust (revertants/mi) was very similar for the five Oldsmobiles. Table 4 summarizes the mutagenicity of extracts of particles collected from six different American and European cars in four Salmonella tester strains. The extracts were positive in all four strains without the addition of liver S-9; the strains are listed in order of increasing sensitivity with TA 100 being the most sensitive. The addition of S-9 had little effect on the direct response in TA 1537 a nd TA 1538, but increased the response in TA 98 and decreased the response in TA 100. The relative ranking of extract potencies for the six cars was almost identical in the four strains. None of the extracts were capable of reverting strain TA 1535 at concentrations as high as 300 ~ug/plate. Carbon monoxide, total hydrocarbon and nitrogen oxide emissions measured during the sampling periods are summarized in Table 1; no correlation was observed between these emissions and mutagenic potency of the particle extracts. The TA 98 data (without S-9) were used to calculate the mutagenicity associated with the particles (revertants/mg particles) and mutagenicity emitted from the exhaust (revertants per mile) so that differences in extractable fraction and particulate emission rates between the six cars could be accounted for (Table 5). The mutagenicity of the particles differed only by about two-fold between the six cars. The ranking of the cars by revertants/mile was quite different than the ranking by mutagenicity of the extracts emphasizing the importance of including extractable fraction and emission rates when assessing the quantity of potentially hazardous components produced by the various cars. In a separate study, the responses of the particle extracts from the six cars were c.ompared in Salmonella strain TA 100 and its nitroreductase deficient counterpart, TA 100-FRI. The response to TA 100-FR1 was significantly lower (p < 0.01) than TA 100 for extracts of particles collected from the Oldmobile and Audi (Table 6).

TABLE 2 V a r i a b i l i t y i n M u t a g e n i c i t y of R e p e a t e d C o l l e c t i o n s of Diesel Particulate Exhaust Test No. ^

Revertants per/~g Extract B

Extractable Fraction c

Revertants per mg Particles

Particle Emissions (glmi) D

Revertants X 10 s per mile

VW Rabbit

189 364 192

14 (-I- 1.2) 15 (4- 0.6) 14 (-t- 1.0)

26 29 24

3600 4200 3200

0.17 0.16 0.19

600 700 600

Oldsmobile (#218)

134 280 283

12 (-t- 0.7) 14 (-I- 1.2) 13 (-t- 1.0)

22 21 20

2500 3000 2600

0.31 0.36 0.38

800 1100 1000

Oldsmobile (#214)

9896 9892 9893

11 (-t- 0.9) 10 (-t- 0.4) 11 (4- 0.3)

21 18 21

2300 1700 2300

0.29 0.38 0.36

700 600 800

Car

ACars operated according to EPA Federal Test Procedure (hot start) on chassis dynamometer, ambient temperature 75 ~ The three sampling periods for each car'were on different days. BSlope (+ standard deviation) of linear portion of dose-response curve in Salmonella strain TA 100 without the addition of a liver enzyme preparation. Cpercent (w/w) dichloromethane extractable organics associated with particles. ~ of particles emitted from exhaust per mile of vehicle operation. 262

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DIESEL PARTICLE E X T R A C T M U T A G E N I C I T Y TABLE 3 M u t a g e n i c i t y of E x t r a c t s o f E x h a u s t P a r t i c l e s C o l l e c t e d F r o m Five D i f f e r e n t O l d s m o b i l e D i e s e l A u t o m o b i l e s ^ Car-Test No. B

Revertants per/~g Extract c

Extractable Fraction D

Revertants per mg Particles

Particle Emissions (glmi) E

Revertants X 10 s per mile

214-181

1 0 ( • 0.9)

18

1800

0.38

700

215-9893 216-9942

11 (• 0.3) 7 (~0.7)

21 25

2300 1800

0.30 0.33

700 600

217-284

10 (• 0.3)

16

1600

0.34

500

218-252

7 ( ~ 0.6)

23

1600

0.33

500

t1980 model year Oldsmobile Delta 88 diesel cars tested after a 4000 mile break-in period. BOperated according to EPA Federal Test Procedure (hot start) on chassis dynamometer, ambient temperature 75 ~ CSlope (+ standard deviation) of linear portion of dose-response curve in Salmonella strain TA 100, without the addition of a liver enzyme preparation. Dpercent (w/w) dichlorometha ne ex tractable organics associa ted with particles. EGrams of particles emitted from exhaust per mile of vehicle operation.

DISCUSSION

Under the conditions of vehicle operation described in these studies, the mutager~ic potencies of extracts of exhaust particles collected from six different diesel automobiles were remarkably similar. The vehicles tested were represented by 4, 5, and 8 cylinder cars with engine displacements ranging from 2.0 to 5.7 liters (Table 1). As expected, greater variability was observed in the revertants/mi data (Table 5) which is an estimate of the genotoxic material emitted from the exhaust per mile of vehicle operation. Errors inherent in the measurement of m utagenicity, extractable fraction, and particulate emission rates all contribute to the variability in revertants per mile

values. In three separate tests of the extracts in TA 100, the variability in response ranged from 10-30% (two of the tests are reported in Tables 5 and 6). The repeatability of extractable fraction measurements for triplicate test runs ranged from 5 to 10% (VW and Oldsmobile, Table 2). The repeatability of grams/mile particle emissions was 5% in 30 tests each conducted on the Oldsmobile and Peugeot cars (data not reported here). The relative sensitivity of the four tester~strains to the extracts is the same as that reported by Huisingh et aL (1978) and Clark and Vigil (1980). The finding that the particle extracts from the six cars had the same relative potencies in

TABLE 4 M u t a g e n i c i t y o f P a r t i c l e A s s o c i a t e d O r g a n i c s in Six D i f f e r e n t D i e s e l C a r s TA 1537

Revertants//~g Extract a TA 1538 TA 98

TA 100

without S-9

with S-9

without S-9

with S-9

without S-9

with S-9

without S-9

with S-9

Fia t

0.3 (0.05)

0.5 (0.02)

1.1 (0.03)

1.1 (0.05)

4 (0.2)

8c (0.4)

6 (0.2)

2c (0.2)

Peugeot

0.6 (0.04)

0.9 c (0.05)

1.8 (0.10)

2.1 (0.13)

7 (0.Z)

10 c (0.4)

13 (0.4)

7c (0.2)

Audi

0.7 (0.14)

1.3 c (0.05)

2.1 (0.09)

2.4 (0.06)

8 (0.4)

10 (0.3)

13 (0.4)

6c (0.3)

Oldsmobile

1.3 (0.14)

1.1 (0.0S)

3.2 (0.1e)

3.5 (0.16)

10 (0.2)

12 c (0.3)

17 (0.3)

8c (015)

VW Rabbit

2.3 (0.15)

2.2 (0.06)

3.7 (0.14)

5.5 e (0.20)

11 (0.3)

13 c (0.6)

16 (0.8)

7c (0.4)

Mercedes

2.4 (0.06)

2.1 (0.08)

5.1 (0.28)

4.4 (0.16)

12 (0.5)

13 (0.6)

15 (1.0)

7c (0.4)

Car B

^Slope (+ standard deviation) of linear portion of dose-response curve in Sahnonella strains indicated, with and without the addition of Aroclor 12.54 induced liver S-9. BCars operated according to EPA Federal Test Procedure (hot start) on chassis dynamometer, ambient temperature 75 ~ CResponse to addition of S-9 significantly different (p < 0.01) from response without S-9 (F-test). Fundamental and Applied Toxicology

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263

CLARK, ROYER, BROOKS AND McCLELLAN TABLE 5 M u t a g e n i c A c t i v i t y E m i t t e d f r o m t h e E x h a u s t of Six D i e s e l C a r s

Car ^ Fiat Peugeot Audi Oldsmobile VW Rabbit Mercedes

Revertants per/~g Extract s

4 7 8 10 11 12

(0.2) (0.2) (0.4) (0.2) (0.3) (0.5)

Extractable Fraction e

Revertants per mg Particles

Particle Emissions (g/mi) u

Revertants X 103 per mile

71 29 43 20 26 13

2800 2000 3400 2000 2900 1600

0.34 0.21 0.51 0.38 0.17 0.26

1000 400 1700 800 500 400

aAll cars operated according to the EPA Federal Test Procedure (hot start) on a chassis dynamometer, 75 ~ ambient temperature. BSIope (:t: standard deviation) of linear portion of dose-response curve in Salmonella strain TA 98, without the addition of liver S-9. Cpercent (w/w) dichloromethane extractable organics associated with the particles. DGrams of particles emitted from exhaust per mile of vehicle operation.

the four strains (Table 4), suggests that the differences relate to differences in sensitivity of the four strains to the same mutagenic components rather than to different classes of m utagens. The complexity of the extracts make further speculation concerning the nature of the mutagenic components difficult, especially based only on differential responses in the tester strains. Work is currently in progress to further characterize the mutagenicity of the six extracts by testing fractions prepared by high pressure liquid chromatography. Responses of the tester strains to the addition of rat liver S-9 provide little aclditional information on the nature of the mutagens. The addition of liver S-9 reduced the direct response by 2-3-fold in TA 100. This reduction is primarily a result of binding of direct acting mutagens to non-specific proteins in the S-9 since the addition of serum, bovine serum albumin and S-9 w i t h o u t cofactors (required for oxidative capability) produce a similar response (Clark and Vigil, 1990). The response to the addition of S-9 in TA 98 was significantly increased in four of the six cars suggesting the presence of promutagens in the extracts. The complexity of the extracts and of PAH activation/detoxification pathways make it difficult to predict the relative amou nts of promutage ns in the extracts based only on differences in response to S-9. No attempt was made to optimize the S-9 concemration for each of the six extracts. In vitro tests for mutagenesis and DNA damage are widely used to screen materials for carcinogenic potential because of evidence that genetic alterations may be a critical step in cancer induction (Ames, 1979). Their operational flexibility make them useful for evaluating large numbers of samples not amenable to study via conventional animal carcinogenesis tests. However, the tests are only predictive and not definitive for carcinogenesis, and test results should be interpreted with caution when extrapolating risk to higher animals and man. With respect to interpretation of diesel particle extract mutagenicity data in Salmonella, the question of sampling and test result artifacts cannot be ignored. Pitts et al. (1978) showed that benzo(a)pyrene, w h e n deposited on a glass fiber filter and exposed to various gaseous pollutants, was converted from an indirect to a direct-acting mutagen. These findings cast general uncertainties on the interpretation of data obtained from filter collected particulate samples. Subsequent studies in which diesel exhaust particles were collected on 264

three different filter types demonstrated no differences in mutagenicity, but did not rule out the occurrence of artifacts on all three filter types (Clark et al., 1981). Of particular concern would be the occurrence, artifactual or otherwise, of nitro-PAH species in the extracts since Salmonella is known to contain nitroreductases capable of metabolizing nitroarenes to mutagenic and carcinogenic nitroso and n-hydroxy species (Rosenkranz and Speck, 1975; Claxton and Barnes, 1979). Higher nitroreductase levels in bacteria than in mammalian cells has been offered as an explanation for the high mutagenic potency of nitro-containing chemicals in bacteria, yet low mammalian carcinogenicity (Ames eta/., 1975). Mutagenicity of particle extracts from two of the six cars were significantly lower (p < 0.01 ) in a nitroreductase deficient strain of Salmonella (Table 6), suggesting that nitroarenes may be partially responsible for the observed mutagenicity. Also pertinent are the recent findings of Rosenkranz e t a / . (1980) who showed that the activity of TABLE 6 M u t a g e n i c i t y of P a r t i c l e E x t r a c t s i n Nitroreductase Proficient and Deficient Strains Revertantsl/~g Extract (:1: S.D.) a TA 100 TA 100-FR1 c

Car n Peugeot Oldsmobile VW Rabbit Audi Mercedes Fiat

8 14 16 12 16 5

(0.4) (0.3) (i.5) (0.9) (0.8) (0.2)

7 11 13 11 16 4

(0.2) (0.5) D (1.0) n (0.4) (0.5) (0.2)

^Slope (+ standard deviation) of linear portion of doseresponse curve, without the addition of liver S-9. SAil cars operated according to EPA Federal Test Procedure (hot start) on a chassis dynamometer, ambient temperature 75 ~ CNitroreductase deficiency confirmed by survival on nitrofurazone plates. DResponse to TA 100-FR1 significantly lower (p < 0.01) than TA 100. Yundam. Appl. Toxicol. (!)

l$1ay/June, 1981

DIESEL PARTICLE EXTRACT MUTAGENICITY nitropyrenes, among the most potent of bacterial mutagens tested, is not reduced in nitroreductase deficient strains, presumably because activation is accomplished by a bacterial enzyme different from the classical nitroreductases. They have succeeded in isolating a strain resistant to the mutagenic action of dinitropyrenes but capable of being reverted by other nitro-containing chemicals. It is not known if this "'new" enzymatic activity is unique to bacteria. The repeatabiliW of sampling and bioassay procedures demonstrated in these studies, and similarity in relative responses of the four tester strains to the six extracts, justify ranking of the diesel cars by mutagenicityof the dichloromethane extractable organics as shown in Table 4. Since the potency of mutagens detected in the Salmonella assay varies over a range of about 108(McCann and Ames, 1976), the differences observed are minimal. Since the same fuel was used in all tests, the results suggest that engine design has very little influence on the mutagenicity of organics associated with the particles. The differences in extractable organic fraction and particulate emission rates are most likely related to engine design. We are reluctant to rank the cars according to particle mutagenicity and the amount of mutagenic components emitted from the exhaust since factors influencing the extractable fraction and particulate emission rates have not been addressed in these studies. Furthermore, all samples were collected under well defined conditions of engine operation, driving pattern, fuel composition, environmental temperature and engine age. The small differences observed under controlled conditions suggest that considerable overlap may exist in particle mutagenicity in the six cars under actual driving conditions. Subsequent papers will address the influence of fuel type, driving cycle and environmental temperature on the mutagenicity of diesel particulate exhaust. A CKNO WLEDGMENTS

The authors wish to acknowledge the technical assistance of Amy Federman, Nicole Dumont, Denine Buckman, and Ingrid Hansen in the conduct of the mutagenicity assay; Sylvia Crain and Sandy Sinclair for extraction and preparation of filter samples for bioassay; Drs. Bruce Ames and Herbert Rosenkranz for donation of Salmonella tester strains; and the helpful discussion of our colleagues at the Inhalation Toxicology Research Institute and the Bartlesville Energy Technology Center. REFERENCES

Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens as mutagens with the Salmonella/mammalian microsome mutagenicity test. 3[utat. Res. 31:347-364.

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Ames, B.N. (1979). Identifying environmental chemicals causing mutagens and cancer. Science 204:587-593. Clark, C.R. and Vigil, C.L. (1980). Influence of rat lung and liver homogenates on the mutagenicity of diesel exhaust particulate extracts. ToxieoL Appl. PharmaeoL 56:110-115. Clark, C.R., Truex, T.I., Lee, F.S.C. and Salmeen, I.T. (1981). Influence of sampling filter type on the mutagenicity of diesel exhaust particulate extracts. Atmos. Environ. 15:397-402. Claxton, L. and Barnes, H. (1979). The mutagenicity of diesel exhaust exposed to smog chamber conditions as shown by Salmonella ophimurhtm. Proceedings, EPA International Symposium on the Health Effects of Diesel Engine Emissions.

Cincinnati, Ohio. Huisingh, J., Bradow, R., Jungers, R., Claxton, L., Zweidinger, R., Tejada, S., Bumgarner, J., Duffield, F., Waters, M., Simmon, " V.F., Hare, C., Rodriques, C. and Snow, L. (1978). Application of Bioassa)' to the Characterization of Diesel Particle Emissions, EPA-600/9-78-027, pp. 1-32.

McCalla, D.R., Reuvers, A. and Kaiser, C. (1970). Mode of action of nitrofurazone. Am. Sor 3lierobiol. 104:1126-1134. McCann, I. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Prec. NatL Acad. ScL 73:950-954. Pitts, I.N., lr., VanCauwenberghe, K.A., Grosjean, D., Schmid, J.P., Fitz, D.R., Belser, W.L., Jr., Knudson, G.B. and Hynds, P.M. (1978). Atmospheric reactions of polycyclic aromatic hydrocarbons: Facile formation of mutagenic nitro derivatives. Science 202:515-519. Rosenkranz, H.S. and Speck, W.T. (1975). Mutagenicity of Metronidazole: Activation by mammalian liver microsomes. Biochem. Biophys. Res. Comm. 66:520-525. Rosenkranz, H.S., McCoy, E.C., Sanders, D.R., Butler, M., Kiriazides, D.K. and Mermelstein, R. (1980). Nitropyrenes: Isolation, identification, and reduction of mutagenic impurities in carbon black toners. Science 209:1039-1043. Schuetzle, D., Lee, F.S.C. and Teyada (1980). The identification of polynuclear aromatic hydrocarbon derivatives in mutagenic fractions of diesel particulate extracts. Proceedings, lOth Annual Symposium on the Analytical Chemistry o f Pollutants,

Dortmund, Germany. Siak, J., Chan, T. and Lee, P. (1979). Diesel particle extracts in bacterial test systems. Proceedings, EPA International Symposium on the Health Effects of Diesel Engine Emissions,

Cincinnati, Ohio. Springer, K.J. and Baines, T.M. (1977). Emissions from diesel engines of passenger cars. Society of Automotive Engineers, Paper 770818. Wolff, R.K., Kanapilly, G.M., DeNee, P.B. and McClellan, R.O. (1980). Deposition of 0.1 /~m chain aggregate aerosols in Beagle dogs. J. Aerosol Sci., in press.

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