Diesel exhaust particulate material expression of in vitro 

genotoxic activities when dispersed into a phospholipid 

component of lung surfactant 

Shi X C, Keane M J, Ong T M, Harrison, J C, Slaven J E, Bugarski A D, Gautam 

M, Wallace W E. 

US Centers for Disease Control and Prevention, National Institute for Occupational 

Safety and Health, Health Effects Laboratory Division, Morgantown, WV, USA 

Abstract. Bacterial mutagenicity and mammalian cell chromosomal and DNA damage in vitro 

assays were performed on a diesel exhaust particulate material (DPM) standard in two 

preparations: as an organic solvent extract, and as an aqueous dispersion in a simulated 

pulmonary surfactant. U.S. National Institute for Standards and Technology DPM SRM 2975 

expressed mutagenic activity in the Salmonella reversion assay, and for in vitro genotoxicity to 

mammalian cells as micronucleus induction and as DNA damage in both preparations: as an 

acetone extract of the DPM mixed into dimethylsulfoxide, and as a mixture of whole DPM in a 

dispersion of dipalmitoyl phosphatidyl choline. Dispersion in surfactant was used to model the 

conditioning of DPM depositing on the deep respiratory airways of the lung. DPM solid 

residue after acetone extraction was inactive when assayed as a surfactant dispersion in the 

micronucleus induction assay, as was surfactant dispersion of a respirable particulate carbon 

black. In general, a given mass of the DPM in surfactant dispersion expressed greater activity 

than the solvent extract of an equal mass of DPM. 

1. Introduction 

Some organic solvent extracts of filter-collected diesel exhaust particulate material (DPM) express in 

vitro genotoxic activities to bactetria or mammalian cells [1,2]. These activities vary with engine 

design and operating conditions, fuels, and other engineering factors [3]. We have reported that some 

filter-collected DPM also can express bacterial mutagenicity and in vitro mammalian cell 

chromosomal or DNA damage activities when the material is mixed into an aqueous dispersion of 

simulated lung surfactant, e.g., aqueous dispersion of dipalmitoyl phosphatidyl choline (DPPC) [4,5]. 

Genotoxic activity is not expressed by the DPPC extract [6]; instead it resides with the non-dissolved 

but surfactant-dispersed solid ultrafine particles. 

In the study reported here, a U.S. National Institute for Standards and Technology standard DPM 

reference material, SRM 2975, was assayed as both a solvent extract and as a surfactant dispersion in 

three in vitro assays: bacterial mutagenicity as Salmonella reversion to histidine independence, using 

the YG1024 and YG1029 tester strains with and without microsomal enzyme S9 activation; 

mammalian cell clastogenic activity as micronucleus induction in Chinese hamster fibroblast-derived 

V79 cells; and DNA damage as single- or double-strand breaks in V79 cells. SRM 2975 was prepared 

as an acetone extract mixed into dimethylsulfoxide; or as whole DPM mixed into an aqueous 

dispersion of DPPC. To investigate the role of particles per se, that is, particles bearing no solvent1
 

extractable genotoxicants, micronucleus assays also were performed on DPPC surfactant dispersion of 

the residue of previously acetone-extracted SRM 2975, and on a respirable particulate carbon black. 

2. Materials and methods 

2.1. Diesel particle material (DPM)
 

Standard Reference Material, SRM 2975, a forklift-generated diesel exhaust particulate material from
 

the U.S. National Institute of Standards and Technology, was used for the study.
 

2.2. Treatment of samples 

2.2.1. Solvent extraction For preparation of DPM- solvent extract, 500 mg of the SRM 2975 DPM
 

and 100ml of acetone were sonicated together in water bath for 2 h. Then the liquid was evaporated 

under N2 to reduce the volume to <40ml, and then centrifuged at 3000xg for 30 min. The supernatant
 

was removed and syringe-filtered. The filtrate was evaporated to dryness under N2. This acetone 

extraction procedure yielded extract material equal to 4% of the mass of the original SRM2975 DPM
 

when performed for samples for mutagenicity testing; 5.6% for samples for micronucleus and DNA 

damage assays, and 8 % for samples for micronucleus comparisons of DPPC-dispersed carbon black
 

with extracted or dispersed DPM. This evaporated solvent extract residue was mixed into 

dimethylsulfoxide (DMSO) at 36 mg DPM-extract per ml DMSO solution as a stock solution.
 

Samples were diluted serially with complete Eagle’s minimal essential medium ( MEM, Gibco ) to
	

desired concentrations for in vitro assay.
 

2.2.2. Surfactant Dispersion For the preparation of surfactant dispersion, 2.5mg DPPC (Calbiochem, 

La Jolla, CA) per ml was mixed with 0.85% physiological sterile saline (PSS) and sonicated under N2 

at 40 W (Heat system-ultrasonic, Plainview, NY) for 10 min. The probe was wiped with 95% ethanol
 

and was covered with parafilm during sonication. Two mg DPM per ml DPPC stock dispersion was
 

made after agitating the sample in a water bath for 60 min at 37oC. The sample was diluted serially
 

with complete Eagle’s MEM medium to desired concentrations.
 

For surfactant dispersion of acetone-extracted SRM 2975 residue or of carbon black, 2mg per ml 

DPPC dispersions were prepared and diluted serially with complete medium to required 

concentrations. 

2.2.3. Ames Salmonella Assay Mutagenic activity was analyzed by the pre-incubation variant of the 

Ames Salmonella microsuspension assay system [7]. Two bacterial test strains, YG1024 and 

YG1029, were used to identify frame-shift and base- pair substitution mutations. A 10% concentration 

of rat liver microsomal fraction S9 was used for metabolic activation for half of the samples. Coincubation of bacteria, test sample, and S9 was performed by a rotary shaker at 37oC for 30 min before 

plating. Duplicate plates were made for each concentration of test sample and the control. Three 

readings of revertant colonies per plate were scored by an automatic colony counter (Accu Count 

1000, Biologics Inc., Gainville, VA) at 48 h for YG1029 and at 72 h for YG1024 after incubation. The 

average number of revertant colonies and standard deviation per concentration were calculated. A 

sample was considered to be positive for mutagenic activity if the number of revertant colonies per 

plate was twice as high as that of the DMSO solvent control or DPPC surfactant control in at least one 

concentration and if there was a clear concentration-response relationship. 

2.1.4 Micronucleus (MN) Assay Micronucleated cells (MNC) were scored according to specific 

criteria [8,9]. Micronuclei located within the cytoplasm but separated from the main nuclei and with 

diameter between one-twentieth and one-fifth that of the nucleus were scored as a MN. The frequency 

of micronucleated cell (MNC) expressed as the number of MNC per 1000 cells was based on 6000 

cells scored for each treatment. Chinese hamster lung fibroblasts ( V79 cells ) at a concentration of 1 

2
 

×106 were cultured in a 25ml flask containing 5 ml of Eagle’s minimal essential medium ( MEM, 

Gibco) supplemented with 10% inactivated fetal bovine serum and 2% penicillin-streptomycin in a 

humidified incubator at 37oC in an atmosphere of 5% CO2 for 24 h. Cells were treated for 24 h in 

5ml of fresh complete MEM medium containing the test sample which had been prepared as a 

solution in 0.5% DMSO or dispersion in DPPC. Cytotoxicity of V79 cells after treatment was 

determined by cell density measurement and viability was determined by using the trypan blue 

exclusion assay. Cells were rinsed with PBS and re-incubated in 5ml fresh complete MEM medium 

for an additional 24 h. Cells were harvested by trypsinization, collected by centrifugation, and re

suspended in medium at about 4-5× 105/ml. Slides were prepared by a cytocentrifuge ( Shandon 

Cytospin 3, Shandon Inc., Pittsburgh, PA ). An aliquot of 75μl of the cell suspension was added into a 

chamber and the cells were pelleted onto the slide at 500 rpm for 5 min. Slides were dried on a slide 

warmer at 37oC for 10 min, fixed with absolute methanol, and stained with Diff-Quik stain ( Dage 

Diagnostics, Aguada, Puerto Rico ). 

­

2.1.5 Single Cell Gel Electrophoresis ( SCGE ) The procedure for the SCGE assay followed that 

reported by Tice et al. [10] with minor modifications. V79 cell culture and sample treatment for SCGE 

assay was the same as that for micronucleus assay. After challenge, cells were rinsed with phosphatebuffed saline (PBS, Gibco) and harvested by trypsinization. The cell suspensions were centrifuged at 

600 x g at 4oC for 10 min and the cell pellets were resuspended in 0.5 ml of complete medium 

(approximately 1.5 - 2 x 106/ml). Tubes containing the resuspended cells were kept on ice for making 

sandwich gel slides. 

Electrophoresis was carried out at 300mA and approximately 25 volts (0.7 v cm-1 ̄1) for 30 min at 

room temperature. Slides were transferred to Coplin Jars, soaked in Tris buffer for 5 min three times to 

neutralize the gel. The slide was then stained with 50 μl of 2 μg/ml ethidium bromide (Sigma-Aldrich) 

and covered with a cover slip for 5 min, followed by rinsing in distilled water. The finished slides 

were covered with a cover slip and stored in a humidified airtight container at 4oC for analysis. Image 

analysis was performed at 200x magnification using a fluorescence microscope (Zeiss, 7082 

Oberkochen, Germany) equipped with a 100 W Hg vapor lamp, a 515 – 560 nm excitation filter and a 

590 nm emission filter. The tail length of DNA migration was determined with an eyepiece 

micrometer by measuring the distance between the edge of the nucleus to the end of the tail. For each 

treatment, 25 cells per slide were analyzed for 4 replicate slides. 

3. Results 

3.1. Mutagenicity 

Results of the salmonella gene mutation test are shown in Table 1, and data for the assay using the 

YG1024 tester strain without S9 activation are graphed in Figure 1, showing response as revertants per 

plate, while concentration values in the Table and Figure are in terms of the mass of SRM 2975 DPM 

dispersed for the DPM-DPPC dispersed samples and in terms of mass of SRM 2975 DPM subjected to 

extraction for the DPM-Solvent Extract samples. Regression equations and corresponding p-values 

are given in Table 2 for linear fits to the concentration-response data. Both DPM acetone solvent 

extract and DPM dispersion in DPPC showed statistically significant positive mutagenic activity to 

both YG1024 and YG1029 strains both with and without S9 activation; and YG1024 gave a stronger 

mutagenic response than YG1029 to both DPM solvent extract and DPM dispersion in DPPC samples 

The mutagenic activity in general, was stronger without S9 metabolic activation. Overall, activity for 

DPM dispersion in DPPC was stronger than that of DPM solvent extract samples. 

3
 

 

 

 

Concentration (ug/mL)

0 20 40 60 80 100 120 140

Revertants/plate

0

200

400

600

800

1000

1200

Extract

Surfactant

1024-S9 Figure 1: Mutagenic 

Activity of SRM 2975 

Diesel Particulate 

Material as solvent 

extract or as surfactant 

dspersion using 

Salmonella YG1024 

reversion to histidine 

independence, without 

sample activation by S9 

microsomal enzyme. 

4

 

Table 1: Revertant Colonies Induced by Diesel Particle Material (DPM) SRM 2975 in Salmonella 

Typhimurium YG1024 and YG1029 

Test Sample a Concentration

(μg/plate) 

b Average number of revertant colonies/plate

YG1024 YG1029 

DMSO 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

d 2AA

1-NPe 

10c 

13.3 

40 

120 

10c 

13.3 

40 

120 

0.05 

0.05 

w/out S9

18 ±2 

146 ±51 

221± 8 

252± 7 

23 ±1 

295 ±13 

550± 33 

1065± 84 

403±10 

 with S9 

29±5 

24±3 

77±4 

435±32 

41±5 

167±3 

456±65 

1007±81 

w/out S9

27± 4 

63± 1 

152± 23 

351± 11 

62± 3 

173± 3 

357± 25 

750± 60 

 

428±2 

 with S9 

26±6 

51±5 

79±7 

192±2 

79±2 

99±13 

188±16 

560±9 

 794±16 

a 

b 

Concentration based on original DPM subject ed to extraction or dispersion. c d e Average of 6 counts. μl/plate. 2-aminoanthracene. 1-nitropyrine. 
Table 2: Regression Equations for Salmonella mutagenic activity in Revertants/plate versus sample 

concentration in terms of original SRM2975 extracted or dispersed 

Extract Surfactant p-value 

1024+S9 

1024-S9 

1029+S9 

1029-S9 

y = -11.84 + 3.57x 

y = 76.37 + 1.70x 

y = 34.81 + 1.29x 

y = 27.07 + 2.74x 

y = 64.14 + 8.06x 

y = 119.86 + 8.20x 

y = 53.94 + 4.13x 

y = 89.91 + 5.62x 

< 0.0001 

< 0.0001 

< 0.0001 

< 0.0001 

 

 

 

 

 

 

 

 

 

 

3.2. Micronucleus Induction 

Results of the comparative study of micronucleus induction in V79 cells, based on equal concentration 

of SRM 2975 DPM subjected to acetone extraction or DPPC dispersion, are given in Table 3 and 

plotted in Figure 2. DPM solvent extract induced significant micronucleus formation at concentrations 

of 180μg/ml (P<0.05) and 240 μg/ml (P<0.01 ). DPM dispersion in DPPC induced significant 

micronucleus formation with positive concentration-response behavior for all of the concentrations 

tested. Micronucleus formation activity in V79 cells was stronger for surfactant-dispersed whole soot 

particles than for the organics extracted from an equal mass of soot. Tables 3, 4 and 5 and Figure 2 

also show the results of micronucleus assay of DPPC-dispersion of the solid residue of acetone 

solvent-extracted SRM 2975 DPM, and of DPPC-dispersed ultrafine carbon black. The residue of 

acetone-extracted SRM 2975 was inactive for micronucleus induction when assayed as a dispersion in 

DPPC. Carbon black, assayed as an ultrafine particulate material bearing no extractable genotoxicants, 

also was inactive for micronucleus induction as a dispersion in DPPC. 

5

Table 3: Comparative Studies of Micronucleus Induction by Diesel Particle Material (DPM) SRM 

2975 in V79 Cells 

Test sample a Concentration

( μg/ml ) 

b MNC/1000 cells

( Mean±SD ) 

DMSO 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

DPM Solvent Residue-DPPC 

DPM Solvent Residue-DPPC 

DPM Solvent Residue-DPPC 

Carbon Black-DPPC 

Carbon Black-DPPC 

Carbon Black-DPPC 

d MNNG

5c

40 

180 

240 

40 

180 

240 

40 

180 

240 

40 

180 

240 

0.25 

5.6± 2.3 

6.3± 1.6 

7.6± 2.3* 

9.9± 2.2** 

5.8 ±1.9 

7.2± 2.3* 

11.1 ±2.8** 

16.1 ±2.2** 

6.5± 1.7 

6± 3 

5.2± 2 

5.6± 2.6 

6.6± 3.3 

5.6± 1.8 

23.4± 2.8** a 

c 

 

b Based on original DPM extracted or dispersed. Micronucleated cell counts were based on 12000 cells scored. d μl/ml. N-methy-N-nitro-nitrisoquanine. * P < 0.05 compared with negative control. ** P < 0.01 
Additional micronucleus induction assays were performed comparing the activity of a mass of 

SRM 2975 DPM dispersed in DPPC with an equal mass of extract obtained by acetone extraction of a 

(larger) mass of SRM 2975 DPM. Shown in Table 4 and Figure 3, both preparations caused 

significant micronucleated cells at all such concentrations ranging from 13.3 to 180 μg/ml, and both 

exhibited a concentration-dependent increase in activity. 

Table 4: Regression Equations for number of micronucleated cells versus concentration in terms of 

original SRM2075 DPM extracted or dispersed. 

Extract: y = 5.44 + 0.016x 

Surfactant: y = 5.25 + 0.041x 

Carbon Black: y = 5.72 + 0.001x 

Residue: y = 6.83 – 0.006x 

 

Concentration (ug/mL)

0 50 100 150 200 250

# MNCs

0

2

4

6

8

10

12

14

16

18

Surfactant

Extract

Carbon Black

Residue

MNC assay

Figure 2. 

Micronuclei 

Induced by 

SRM2979 DPM 

dispersed in DPPC 

surfactant or 

extracted by 

acetone; by carbon 

black dispersed in 

DPPC; and by 

DPPC dispersion 

of the residue of 

acetone extraction 

of DPM. 

6
Table 5: Induction of Micronucleus by Diesel Particulate Material SRM 2975 in V79 Cells 

Test sample a Concentration

(μ g/ml) 

b MNC/1000 cells

(Mean±SD) 

Multiple Nucleus 

cell/1000 cells 

(Mean±SD) 

DMSO 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

NC 

d MNNG

5c

13.3 

40 

120 

180 

13.3 

40 

120 

180 

0.25 

5.5 ±2.3 

15.7± 2.2** 

17.5 ±2.1** 

21.7± 6.3** 

32.5 ±2.8** 

7.8± 2.5 

9.8 ±3.5 

12.2 ±1.6** 

15.3± 4.1** 

19.3 ±3.6** 

7.5± 2.6 

29.7± 4.2** 

11.8± 3.4 

22.8± 3.1** 

26.7 ±3.8** 

25.0 ±7.1** 

39.8 ±5.7** 

18.6± 3.0 

26.2± 8.2* 

39±.3 4.6** 

34.2± 12.6** 

57.0± 13.7** 

15.5± 1.9 

11.5 ±2.5 

a For solvent extraction the concentration was based on extracted material, for DPPC dispersion the b concentration was based on original DPM dispersed. Micronucleated cell count (MNC) was based c d on 1000 cells. A total of 6000 cells were scored. μl/ml. N-methy-N-nitro-nitrisoquanine * P< 0.05 

compared with negative control. ** P < 0.01 compared with negative control. 

Table 6: DNA damage Induced by Diesel Particle Material (DPM) SR M in V79 Cells with 

SCGE 

Test Sample 

DMSO 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPM Solvent Extract 

DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

DPM-DPPC 

Complete Medium 

d MNNG

a Concentration

( μg/ml ) 

5c 

13.3 

40 

120 

180 

13.3 

40 

120 

180 

0 

0.25 

Damaged 

100 Cells 

14 

20 

22 

33 

54 

13 

10 

29 

38 

41 

9 

89 

Cells/ b Tail Length ( μ m )

(Mean±SE) 

1.1± 0.28 

1.1± 0.25 

1.5 ±0.29 

2.4± 0.37** 

2.6± 0.27** 

1.2 ±0.32 

0.8± 0.25 

2.1 ±0.34* 

2.6 ±0.36** 

2.9± 0.37** 

0.7± 0.21 

8.1± 0.35** 

a For solvent 

dispersion the 

nitrisoquanine. 

extraction the concentration was based on mass of extracted 

concentration was based on mass of original DPM dispersed. 

b c Based on 100 cells. μl/ml * P< 0.05 **p< 0.01 vs. control. 

material, for DPPC

d N-methy-N-nitro

 

­

7
 

3.3. DNA damage 

Results from the SCGE assay of DNA damage in V79 cells were scored as tail length (TL) in μm. 

Assays were performed comparing the activity of a mass of SRM 2975 DPM dispersed in DPPC with 

an equal mass of extract obtained by acetone extraction of a (larger) mass of SRM 2975 DPM. Any 

cell with a nonzero tail was classified as damaged cell. Both exposure of V79 cells to DPM solvent 

extract and to DPM dispersion in DPPC caused significant DNA migration at concentrations ranging 

from 120 to 180 and 40 to 180 μg/ml, respectively and displayed a concentration-dependent increase 

in tail length in Table 6 and Figure 2. The percentage of damaged cells increased with concentrationresponse manner in both DPM solvent and dispersion in DPPC. 

Table 7: Logistic Regression Equations for number of DNA -damaged cells /100cells versus 

concentration in terms of original SRM2075 DPM extracted or dispersed. 

Extract: y = 1.6248 – 0.0092x
 

Surfactant: y = 1.6217 – 0.0079x
 

4. Discussion 

Numerous in vitro genotoxicity studies have been performed upon diesel exhaust particulate materials 

(DPM) collected by filtration and assayed as organic solvent extracts. In many but not all cases such 

extracts have been found to express positive genotoxic activities. However, the physiologically 

anomalous use of organic solvent extracts of DPM questions the biological availability of DPM-borne 

genotoxicants under conditions of deposition in the lung. And such conventional in vitro genotoxicity 

assays applied to DPM organic solvent extracts cannot identify the possible effects of exhaust 

particulate size or structure on genotoxic activity. 

We have reported that some DPM can express in vitro genotoxic activities as mixtures into aqueous 

dispersion of some principal phospholipid pulmonary surfactants, e.g., dipalmitoyl phosphatidyl 

choline, DPPC. Extraction of DPM by DPPC does not result in significant genotoxic activity by the 

extract; however, the non-dissolved particulate phase of direct mixture of DPM into DPPC dispersion 

can express in vitro genotoxic activities. Results suggest that particles adsorb surfactant onto their 

surfaces from aqueous dispersion, making the so-conditioned particle surfaces hydrophilic and 

permitting their dispersion in aqueous media. This retains particle structure and its effects on the 

expression of toxicities, partially modeling particle surface conditioning upon inhalation and 

deposition in the deep lung. This permits the application of in vitro bioassays in a manner which 

avoids anomalous test preparation destruction of particle size and structure, factors which may be 

important in the expression of toxicities by ultrafine or nano-particles. Further, it partially models the 

effects on DPM toxicity of the conditioning of respirable particle surfaces which occurs promptly 

upon respirable particle deposition in the deep lung onto the surfactant-rich aqueous hypophase 

coating of the respiratory bronchioles and alveoli. Surfactant dispersion concentration requirements 

for this have been determined in terms of particulate surface area [11] 

The current study objective was to compare some in vitro genotoxic activities between solventextraction and surfactant- dispersion preparations for a generally available diesel exhaust reference 

material, U.S. National Institute for Standards and Technology DPM standard reference material SRM 

2975. Assays were chosen to represent bacterial mutagenicity and mammalian cell chromosomal or 

DNA damage. 

In Salmonella mutagenicity assays using either of two tester strains, both solvent extract and 

surfactant dispersions of the DPM were active, expressing for at least one sample concentration more 

than twice the solvent or surfactant control activity, and demonstrating a positive concentration – 

mutagenic activity response. Comparing mutagenic activities versus concentrations measured on the 

basis of mass of original DPM dispersed into DPPC or mass of original DPM subjected to solvent 

8
extraction, it was found that the surfactant dispersion preparation was significantly more active for a 

given concentration than the solvent extract. 

Those, similarly, were the results for micronucleus induction in mammalian-derived V79 cells: 

both surfactant dispersion and solvent extract preparations of SRM 2975 DPM were active, with 

surfactant dispersion being significantly more active than the solvent extract for concentration based 

upon mass of DPM extracted or dispersed. Comparison also was made using the micronucleus assay 

to identify a possible contribution to the activity from the presence of non-dissolved particles per se in 

the surfactant dispersion preparations. This was approached by assaying a carbon black particulate 

material in surfactant dispersion, and by assaying in surfactant dispersion the non-dissolved particulate 

residue surviving organic solvent extraction of SRM2975 DPM. Surfactant dispersions of both types 

of insoluble particles carrying no extractable genotoxicants were inactive. Micronucleus induction 

also was compared between the two preparations also on the basis of mass of original SRM 2975 

DPM dispersed in surfactant versus equal mass of solvent extracted DPM material (from a larger 

amount of original SRM 2975 DPM). In that comparison, a mass of extracted material is significantly 

more active than an equal mass of dispersed DPM. It is noted that a given mass of extract requires the 

extraction of a much larger mass of DPM for this SRM2975 DPM with solvent extractables of about 

10% the mass of the DPM extracted. 

DNA damage was compared between the two preparations only on the basis of concentrations of 

surfactant dispersed SRM 2975 DPM versus equal concentrations of material extracted from a (larger) 

amount of SRM 2975 DPM. In this SCGE assay under this manner of concentration comparisons, 

there was no significant difference in activity between the two preparations. Again, with the low 

solvent extractable fraction of this SRM 2975 DPM, this suggests surfactant dispersion of this DPM is 

at least as active and reasonably more active than the solvent extract of an equal mass of original SRM 

2975 DPM. 

The implication of these results is that for this SRM 2975 DPM, a given mass of extractable DPMassociated genotoxic materials express greater in vitro genotoxic activities if they are associated with 

surfactant-dispersed particles than is expressed by the genotoxicants when extracted from the particles. 

Lack of in vitro genotoxic activity of surfactant-dispersed carbon black and of surfactant-dispersed 

SRM2975 which had been stripped of genotoxic compounds by acetone extraction, suggest the greater 

activities seen for this DPM in surfactant dispersion may be a synergistic effect of particle

genotoxicant association rather than an additive effect of activity of the carried genotoxicants plus 

activity of carrier genotoxicant-barren particles. 

That not all DPM solvent extracts express genotoxic activity; and that diesel engine design and 

operational factors affect the amount of emissions and specific genotoxic activities of DPM solvent 

extracts, has suggested that in vitro assays of DPM might be a source of convenient first-tier guidance 

for diesel design or exhaust control technology development with the objective of minimizing 

hazardous diesel exhaust emissions. The results of in vitro assays of surfactant-dispersed DPM suggest 

that such efforts can be extended to include a more physiologically-relevant preparation of DPM for in 

vitro testing which is not blind to considerations of the size and structure of DPM and its conditioning 

upon deposition in the lung. Our current research includes extension of the findings of in vitro 

activities of surfactant-dispersed DPM to the concept of collection of exhaust DPM directly into 

surfactant dispersion for bioassay. 

­

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10