Environ. Sci. Technol.
2007, 41, 2622-2629
Toxicity of Parked Motor Vehicle
Indoor Air
J E R O E N T . M . B U T E R S , * ,†
WOLFGANG SCHOBER,†
JAN GUTERMUTH,† THILO JAKOB,‡
ANTONIO AGUILAR-PIMENTEL,§
JOHANNES HUSS-MARP,†
CLAUDIA TRAIDL-HOFFMANN,†
SABINE MAIR,| STEFAN MAIR,|
FLORIAN MAYER,| KLAUS BREUER,| AND
HEIDRUN BEHRENDT†
Division of Environmental Dermatology and Allergy
GSF/TUM, ZAUM-Center for Allergy and Environment,
Technical University Munich, Munich, Germany, Clinical
Research Group Allergology, Department of Dermatology,
University Freiburg, Germany, Division of Molecular and
Clinical Allergotoxicology, Department of Dermatology and
Allergy, Technical University Munich, Munich, Germany, and
Fraunhofer-Institute for Building Physics IBP, Branch Institute
Holzkirchen, Valley, Germany
The interior of motor vehicles is made of a wide variety
of synthetic materials, which emit volatile organic
compounds (VOC). We tested the health effects of emissions
from vehicles exposed to “parked in sunshine” conditions.
A new and a 3 year old vehicle with identical interior
were exposed to 14 000 W of light. Indoor air was analyzed
by GC-MS. Toxicity of extracts of indoor air was assayed
in human primary keratinocytes, human lung epithelial
A549 cell line, and Chinese hamster V79 lung fibroblasts.
In addition, toxicity after metabolic activation by CYP1A1,
CYP1A2, CYP1B1, CYP2A6, CYP2B6, and CYP2E1 was assayed.
The effect on type I allergic reaction (IgE-mediated
immune response), type IV allergic reaction (T-cell mediated
immune response), and irritative potential was evaluated
also. A total of 10.9 and 1.2 mg/m3 VOC were found in new
and used motor vehicle indoor air, respectively. The
major compounds in the new vehicle were o,m,p-xylenes,
C3 and C4-alkylbenzenes, dodecane, tridecane, and
methylpyrrolidinone. In the used vehicle they were acetone,
methylpyrrolidinone, methylcyclohexane, acetaldehyde,
o,m,p-xylenes, ethylhexanol, and toluene. No toxicity was
observed in any cell line with or without metabolic
activation. Neither did we find an effect on type IV
sensitization or an irritative potential. A slight but statistically
significant aggravating effect on IgE-mediated immune
response of only the new vehicle indoor air was determined
(p < 0.05). The IgE-response modulating effect of indoor
air might be relevant for atopic individuals. Else no direct
* Corresponding author phone: (+) 49-89-41403487; fax: (+) 49-
89-41403453; e-mail: buters@lrz.tum.de. Corresponding author
address: ZAUM-Center for Allergy and Environment, Technical
University Munich, Biedersteiner Strasse 29, 80802 Munich, Germany.
† Division of Environmental Dermatology and Allergy GSF/TUM,
ZAUM-Center for Allergy and Environment, Technical University
Munich.
‡ University Freiburg.
§ Department of Dermatology and Allergy, Technical University
Munich.
| Fraunhofer-Institute for Building Physics IBP.
2622 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007
toxicity, no toxicity after metabolic activation by cytochrome
P450, and no irritative or type IV sensitizing potential of
motor vehicle indoor air were found, neither from the new
nor used vehicle. Our investigations indicated no apparent
health hazard of parked motor vehicle indoor air.
Introduction
Humans are exposed to components found in indoor air of
motor vehicles, sometimes for prolonged periods. Due to
ventilation during driving indoor pollutants are diluted (1,
2), and outside pollutants enter the motor vehicle indoor
environment. Air pollution inside driving motor vehicles was
often studied (3-6). However, little is known about the health
effects of emissions from the interior of cars itself (7, 8). These
emissions can be high, especially in new cars or after
increased temperatures, as are found after “parked in
sunshine” conditions (9, 10).
Indoor air components influence the well being of exposed
individuals at the work place, and maximal work place
concentrations are regulated (11). However, no regulation
limits the concentration of components in motor vehicle
indoor air (7). In the interior of motor vehicles many novel
materials are used which emit volatile organic compounds.
Studies on the quantitation of these emissions are reported
(2, 9, 10), but no study investigated the potential adverse
effects of these emissions found in motor vehicle indoor air.
We therefore sampled the indoor air of a new and a used
motor vehicle under “parked in sunshine” condition as a
“worst case” scenario for high release of volatile organic
compounds from the interior of motor vehicles and assayed
the direct toxicity, toxicity after metabolic activation, and
effects on the immune system of extracts of motor vehicle
indoor air.
Materials and Methods
Extracts of Parked Motor Vehicle Indoor Air. Two motor
vehicles of identical brand and assembly (metallic silver
vehicles with black leather interior) but of different ages were
sampled. The new vehicle was sampled within 1 month after
production and 8 km of usage, and the used vehicle was
sampled about 3 years after production and 107 000 km of
usage.
Both vehicles were parked in a laboratory under 28 halogen
lamps (14 000 W), split in 4 groups, and positioned about 0.5
m away from the front, back, and side windows and roof to
mimic environmental sunshine exposure. Through the
driver’s window an inlet was created by which 12 temperature
sensors, one humidity sensor, a sampling tube, an air inlet,
and a paddle entered the vehicle. The assumed head position
of the driver and navigator served as reference, and the
illumination was variable adjusted to maintain a temperature
of 65 °C throughout the experiment. Cleaned pressured air
was passed through an activated charcoal filter, dehumidified
by a cold-trap, again filtered through an activated charcoal
filter and particle filter, and flow adjusted by mass-flow
regulators, and humidity was adjusted to 50% before entering
the vehicles. Flow of air entering the motor vehicle was
adjusted to 0.75 m3/h. A paddle with charcoal bearings was
used to avoid contaminating indoor air by aliphatic hydro-
carbons of the grease of normal bearings. Four blades of
about 100 cm2 each circulated the air at 72 rpm.
Air sampling was set at 0.75 m3/h, an average respiration
rate for motor vehicle drivers (12), under permanent moni-
toring. Sampled air was passed through 4 cold traps at -80
°C. The first trap was empty and served to dehumidify the
10.1021/es0617901 CCC: $37.00  2007 American Chemical Society
Published on Web 03/02/2007
FIGURE 1. Cytotoxicity of extracts (DMSO transferred) of indoor air of a new and a used motor vehicle with human primary keratinocytes
of a nonatopic individual (graphs A and B) or human epithelial A549 cells (graphs C and D). Cytotoxic endpoints after 48 h incubations
were determined with sulforhodamine B (A and C) and Alamar Blue (B and D). Cell density of cells receiving no compounds was put at
100%. For each point the mean of 8 determinations is given. For clarity the standard deviation of the curve with the highest deviation are
depicted only. Air eq is liter indoor air equivalents per liter cell culture medium. O1 is new, O2 is used vehicle.

air, the second and third traps contained diethyl ether, and
the fourth trap acquired escaped vapors.
After sampling the diethyl ether and aqueous phases (from
freezing of humidity in the sample) of all traps were
combined, shaken, and separated in a separating funnel.
The diethyl ether phase was dried over anhydrous sodium
sulfate and concentrated to 20 mL at 40 °C at ambient
pressure in a Laborota 4000 (Heidolph, Kelheim, Germany)
and stored at -70 °C until use.
Direct Sampling of Parked Motor Vehicle Indoor Air. In
addition to the ether extracts, motor vehicle indoor air was
analyzed directly by drawing 0.5-1.0 L of air from the center
of the car through the sampling tube inserted through the
driver’s window and tightened by emission free aluminum
adhesive foil onto a Carbotrap adsorber tube (9 × 0.6 cm,
Supelco, Taufkirchen, Germany) at 18 °C using a pump at a
velocity of 100 mL/min.
Analysis of Parked Motor Vehicle Indoor Air Extracts.
Motor vehicle indoor air extracts were analyzed by gas
chromatography-mass spectrometry (GC-MS, Agilent 6890
Series, Agilent Technologies, Palo Alto, CA, U.S.A.) equipped
with a carbon dioxide oven cooling system, a HP-5 capillary
column (50 m × 0.32 mm, film thickness 1.05 µm, J&W
Scientific, Folsom, U.S.A.), a flame-ionization-detector (FID),
and a mass selective detector (MSD 5793, Agilent Technolo-
gies, Palo Alto, CA, U.S.A.). The initial temperature of the GC
oven of 20 °C was held for 5 min, then raised at a rate of 5
°C/min to 280 °C, then raised at 20 °C/min to 300 °C, and
held for 10 min. Mass spectra were generated at 70 eV by
electrical (also called electron impact) ionization.
The concentrations of the volatile organic compounds
(VOC) were calculated against the external standard toluene.
Analysis of Direct Samples of Parked Motor Vehicle
Indoor Air. The direct sampled indoor air was analyzed by
installing the loaded Carbotrap into an automatic thermal
desorption system (ATD 400, Perkin-Elmer, Wellesley, U.S.A.)
and analyzing according to ISO 16000-6. Samples were
desorbed from the trap in the ATD by heating up to 360 °C
in a stream of helium (33 mL/min) for 10 min. The volatiles
were cryofocused in a cold trap, which was piezzo-electrically
cooled down to 5 °C. For injection the trap was heated at 350
°C for 3 min, and the volatiles were flushed in a stream of
helium onto the gas chromatography column. Analysis was
performed by GC-FID-MS (see above). Identification of
volatile organic compounds was based on retention time
and mass spectra in accordance with authentic reference
compounds, IBP-own, Wiley, or NIST-MS-library search.
Concentrations were determined by comparing the FID signal
area counts with an external toluene standard calibration
curve.
Analysis of Aldehydes and Ketones in Direct Sampled
Parked Motor Vehicle Indoor Air. For analysis of volatile
aldehydes and ketones 60 L of in-car-air was drawn on silica
cartridges impregnated by dinitrophenylhydrazine (Sep-Pak
DNPH cartridges, Waters, Eschborn, Germany). The dini-
trophenylhydrazone derivatives of the aldehydes and ketones
formed were eluted from the cartridge by 2 mL of acetonitrile.
Analysis of the DNPH-derivatives was performed by high-
performance liquid chromatography with a diode array
detector (HPLC-DAD, 1100 series, Hewlett-Packard/Agilent
Technologies, Palo Alto, CA, U.S.A.) on a C18 HPLC column
(150 × 3.9 mm, Nova-Pak, Waters, Eschborn, Germany)
according to ISO 16000-3. Identification was based on
comparison of retention time and UV-spectra with authentic
reference compounds, and quantification was done by
compound specific calibration using standard solutions of
different concentrations. Detection limit was 0.5-3.0 µg/
m3, depending on the compound.
Toxicity in Cell Culture. Diethyl ether extracts of motor
vehicle indoor air were mixed with an equal volume of DMSO,
and ether was evaporated at room temperature under a gentle
stream of nitrogen within 15 min. Chinese hamster V79 lung
fibroblasts (13), human alveolar epithelial A549 cells (14),
and human primary keratinocytes isolated by suction blister

VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2623

FIGURE 2. Cytotoxicity after metabolic activation by cytochromes P450 CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, and CYP2E1. Cytochromes
were expressed in Chinese hamster V79 lung fibroblasts and incubated for 3 days with dilutions of extracts of motor vehicle indoor air
dissolved in DMSO or DMSO alone. Toxicity was determined with sulforhodamine B (A), Alamar Blue (B), and CellTiter-Glo (C). Air eq.
is liter indoor air equivalents per liter cell culture medium. Each data point represents the mean and standard deviation of 8 replicates.

(15) from an healthy male nonatopic individual were
incubated with dilutions of motor vehicle indoor air extracts.
After incubation toxicity was assessed by sulforhodamine B
(Sigma-Aldrich Inc., St. Louis, MO) (16), alamar blue (Serotec
Ltd., Oxford, U.K.) (17), or CellTiter-Glo (Promega Inc.,
Madison, WI) (18). An identical dilution of the solvent DMSO
was run in parallel with each assay. Keratinocytes and A549
cells were incubated for 48 h, V79 cells for 72 h.
To assess cytotoxicity after metabolic activation a cDNA-
directed expression system of cytochromes P450 in V79 cells
was used, using the same endpoints as with the other cells.
The expressed cytochromes P450 were catalytically active as
tested with substrates specific for the individual cytochromes
and are being published elsewhere (Schober et al. Toxicology
submitted 2006).
β-Hexosaminidase Release from the Murine Mast Cell
Line L138.8A. The bone marrow derived murine mast cell
line L138.8A (19) was grown in the presence of 0.02 U/mL
IL-3. Before an experiment cells were grown overnight in the
presence of purified mouse IgE (5 µg/mL, Pharmingen Inc.,
San Diego, CA). The cells were washed and incubated for 60
min with 0.5 µg/mL anti-IgE (R35-72, Pharmingen) and motor
vehicle indoor air extracts in Tyrode’s buffer at 37 °C.
β-Hexosaminidase release in the supernatant from 200 000
cells was assessed colorimetric by p-nitrophenol liberation
from p-nitrophenol-D-2-acetamido-2-deoxyglucopyranoside
(Sigma-Aldrich) at 405 nm. Mast cells treated with 0.5%
Triton-X revealed maximal β-hexosaminidase release. IgE/
anti-IgE concentrations were adjusted to yield about 20% of
maximal β-hexosaminidase release. This basic activation level
served as control value, depicted as 100% in Figure 3 (20).
Mouse Local Lymph Node (LLNA) and Ear Swelling
Assay. Irritative and sensitizing potential of motor vehicle
indoor air was assessed in a modified mouse LLNA and ear-

2624 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007


FIGURE 3. Enhancement of antigen induced β-hexosaminidase
release by parked motor vehicle indoor air extracts of a new (A)
and a used (B) but otherwise identical vehicles. DMSO was e0.1%
v/v in all experiments. Mean ( SD of at least three experiments
are depicted. Air eq. is liter indoor air equivalents per liter cell
culture medium. β-Hexosaminidase release of incubations with
only IgE/anti-IgE were put at 100%. * p < 0.05
swelling test (21). Groups of five 8 week old female BALB/c
mice were treated for 3 days with 25 µL of DEE-solvent
(dimethylacetamide 40%:diethyl ether 30%:ethanol 30%) on
the dorsal side of both ears (negative controls). Mice serving
as positive controls received croton oil as irritant or oxazolone
(4-ethoxymethylene-2-phenyloxazolin-5-one, Sigma-Aldrich)
as a sensitizing agent, respectively. In verum groups, diethyl
ether in DEE was replaced by motor vehicle indoor air extract
in ether. On day zero, before the first application of the test
substances and on day 3, ear thickness was determined using
a micrometer (B.C. Ames Co., Waltham, MA). Subsequently,
mice were killed, and the ear-draining auricular lymph nodes
were excised. Single cell suspensions of lymph node cells
were stained with a CD45-FITC labeled antibody (Pharm-
ingen Inc.) for 15 min at 4 °C, and the absolute numbers of
CD45+ leukocytes were quantified by flow cytometry (FACS
Calibur, Becton Dickinson, NJ) using TrueCount beads
according to the manufacturers instructions (BD Biosciences).
Lipopolysaccharide (LPS) was determined by the Limulus
amebocyte lysate test according the manufacturers instruc-
tions (QCL-1000, Cambrex, Baltimore, MD).
Statistical Analysis. Differences were analyzed by a paired
Students’ t-test after testing for normal distribution with the
Shapiro-Wilk test (22). p < 0.05 was considered statistically
significant.
Results
Sampling and Analysis of Parked Motor Vehicle Indoor
Air. Of the new and the used motor vehicle 45.25 m3 and
45.44 m3 indoor air could be sampled in one period of 60.25
and 60.5 h, respectively. A total of 10.929 µg/m3 and 1.235
µg/m3 of volatile organic compounds (VOC) in the new and
the used vehicle, respectively, could be determined directly
from the inside of the vehicles. Air entering the car contained
<10 µg/m3 TVOC. The ether extracts represented 99% and
70% of the direct sampled amounts of the new and the used
vehicle, respectively. The identity and relationship of the
compounds between direct versus ether extract are given in
Table 1. About 400 µg/m3 of aldehydes (30%, listed in Table
1) were determined by direct air sampling and HPLC analysis
instead of GC-MS analysis of the extracts.
Motor vehicle indoor air contained many compounds.
The main components in the new motor vehicle were m-
and p-xylene, C3 and C4-alkylbenzenes, dodecane, o-xylene,
tridecane, and methylpyrrolidinone, as determined directly
by GC-MS analysis from indoor air. In the ether extract the
main components were similar: C3 and C4-alkylbenzenes,
dodecane, m- and p- xylene, tridecane, and o-xylene.
In the used motor vehicle the main components after
direct GC-MS analysis were acetone, methylpyrrolidinone,
methylcyclohexane, acetaldehyde, o,m,p-xylene, ethylhex-
anol, and toluene. In the ether extract of the used motor
vehicle these were heptane (likely impurity in the diethyl
ether), BHT-quinonemethide (from the antioxidant BHT in
the diethyl ether), nonanal, m- and p- xylene, toluene, and
methylisobutylketone.
The categories n-alkanes, isoalkanes, aromatic com-
pounds, alkylbenzenes, aldehydes and ketones, phthalates,
amines, and miscellaneous were all low in the used motor
vehicle with alkanes and aromates representing only 4 and
5% of the amounts found in the new vehicle, respectively.
Aldehydes and ketones persisted in the used motor vehicle
with a value 66% in the used compared to the new motor
vehicle.
Cytotoxicity of Parked Motor Vehicle Indoor Air. Com-
pounds extracted from motor vehicle indoor air were
transferred from ether into DMSO to be mixable with cell
culture medium. DMSO was not toxic in any used cell line
or in primary keratinocytes tested up to a concentration of
0.3% (data not shown). As a precaution DMSO was added to
a maximum of 0.1% v/v or less.
No cytotoxicity was observed with organic extracts of the
new or used motor vehicle in human alveolar epithelial A549
cells nor in human primary keratinocytes nor in Chinese
hamster V79 lung fibroblasts (see Figures 1 and 2).
Cytotoxicity after Metabolic Activation. Compounds
from motor vehicle indoor air did not exhibit any toxicity
after metabolic activation by cytochrome CYP1A1, CYP1A2,
CYP1B1, CYP2A6, CYP2B6, and CYP2E1 when exposed for 72
h to the tested concentration of up to 2260 L indoor air per
L cell culture medium, see Figure 2.
β-Hexosaminidase Release from the Murine Mast Cell
Line L138.8A. Murine mast cells were incubated with murine
IgE to load the FcRI-receptor present in this cell line with
IgE. Then anti-IgE in a concentration to obtain a basal level
of activation of about 20-30% was added to the cells with
or without motor vehicle indoor air. β-Hexosaminidase
release of the cells was increased by 30% only with extracts
of indoor air of the new vehicle (Figure 3, p < 0.05).
Local Lymph Node Assay and Ear Swelling Test. As
expected (23) ear painting with the contact sensitizer
oxazolone at concentrations of 0.1 and 0.3% induced a
significant increase of the total leukocyte counts in the
draining auricular lymph node (p < 0.05) (Figure 4 A).
Oxazolone treatment did not induce a significant ear swelling
response when tested at lower concentrations and only a
mild response when tested at the highest concentration
(Figure 4B). In contrast, ear painting with the irritants croton
oil (phorbol ester) at concentrations of 0.1 and 0.3% induced
a significant ear swelling response (p < 0.05) (Figure 4B),
while no or only a minor increase in leukocyte numbers in
VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2625
TABLE 1. Comparison between Direct, GC-FID-MS Determination versus Diethyl Ether Extract GC-FID-MS Analysis of Indoor Air
of a New and a Used Parked Motor Vehicle, Sorted According to Mass Prevalence in the New Vehicle
new vehicle used vehicle
direct ether direct ether
samplinga extractb sampling extract
compound CAS no. [µg/m3] [µg/m3] [µg/m3] [µg/m3]
m-/p-xylenes 106-42-3 1570.7 1341.1 46.3 48.3
C4-alkylbenzenes 1041.1 1653.2
dodecane 112-40-3 982.9 1510.2 33.6 25.6
o-xylene 95-47-6 879.8 829.4 24.4 32.4
C3-alkylbenzenes and not identified 693.5 99.5
tridecane 629-50-5 687.1 1109.1 18.5 27.2
not identified MW g 117c 467.7 148.5
methylpyrrolidinone 872-50-4 425.1 0.0 93.0 0.0
C13-isoalkanes 398.4 743.5
undecane 1120-21-4 352.3 435.8 15.3 15.8
ethylbenzene 100-41-4 306.4 311.6 11.7 20.3
ethanol 64-17-5 299.6 frontd
cycloalkanes 254.6 293.1
acetonee 67-64-1 238.6 front 250.0 front
C14-isoalkanes 218.3 469.1
C3-alkylbenzenes 210.3 180.9 35.1 30.5
decane 124-18-5 168.4 152.1
tetradecane 629-59-4 143.2 326.6 15.6 28.5
not identified MW g 339 131.5 0.0
C12-isoalkanes 130.9 165.1 12.5 12.7
methylcyclohexane 108-87-2 125.8 60.4 91.7 24.4
sesquiterpene MW 204 103.5 113.5
methylhexanone 110-12-3 100.7 116.7
formaldehydee 50-00-0 92.4 0.0 24.3 0.0
acetaldehydee 75-07-0 86.8 0.0 73.8 0.0
toluene 108-88-3 84.3 111.3 47.1 48.0
styrene 100-42-5 83.4 79.7 24.9 10.2
2-butanonee 78-93-3 80.9 f 5.0
BHT-quinonemethide 2607-52-5 75.0 218.3 26.5 72.7
heptane 142-82-5 74.6 207.9 12.3 185.3
siloxane 59.2 0.0
butylated hydroxytoluene (BHT) 128-37-0 54.1 lots 0.0 lots
C11-isoalkanes 53.7 110.2
propionaldehydee 123-38-6 50.5 front 40.8 front
pentane 109-66-0 48.0 front
methylisobutylketone (MIBK) 108-10-1 37.8 38.1 44.7 45.4
hexanale 66-25-1 29.8 40.2 17.6 24.0
crotonaldehydee 4170-30-3 29.4 5.7
benzaldehydee 100-52-7 23.8 4.0
3-/4-methylbenzaldehydee 620-23-5/104-87-0 15.6 4.4
butyraldehydee 123-72-8 12.0 6.2
valeraldehydee 110-62-3 7.2 3.7
C12-isoalkenes 29.9 42.3
heptanal 111-71-7 7.2 10.6
ethylhexanol 104-76-7 56.2 34.6
C5-isoalkanes 24.6 front
C9-isoalkanes 45.9 36.9
caryophyllene 87-44-5 18.4 22.5
nonanal 124-19-6 32.0 54.3
octanal 124-13-0 15.0 18.7
diethyl ether 60-29-7 solvent 17.1 solvent
sum 10928.8 10865.1 1234.9 871.2
a On Carbotrap, Supelco. b Diethyl ether extract. c Molecular weight of the largest fragment. d In solvent front. e Direct sampling determined by
HPLC. f Not detected.

the draining lymph node was observed (Figure 4A). The
solvent (DEE) did not induce significant changes in ear
thickness, nor in leukocyte numbers in draining lymph nodes.
Similarly, repetitive application of new motor vehicle indoor
air extract on mouse ears had no significant effect on
leukocyte counts in the draining lymph nodes and did not
induce significant ear swelling responses, even when tested
at the highest concentration (171 L air equiv) (Figure 4C/D).
Lipopolysaccharide was below detection limit in both
extracts.
Discussion
We determined the toxicity of parked motor vehicle indoor
air sampled from one new and one used vehicle under
“parked in sunshine” conditions at 65 °C. Indoor air was
sampled in ether and transferred into DMSO for testing in
cell culture. No toxicity was observed in human primary
keratinocytes or in the human lung alveolar epithelial A549
cell line or in Chinese hamster V79 lung fibroblasts. Indoor
parked motor vehicle air extract after metabolic activation
by cytochromes P450 was devoid of cytotoxicity also. No
irritative or sensitizing potential (T cell mediated, type IV
sensitization) in a mouse model could be detected.
The parked motor vehicle indoor air did show activation
in a test system for IgE-mediated mast cell activation. These
results indicate that parked motor vehicle indoor air is devoid
of any direct toxicity. In individuals who intrinsically have
high levels of IgE, like atopic individuals, IgE-mediated
responses could be increased.

2626 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 7, 2007

FIGURE 4. Irritative (B, D) and sensitizing (A, C) potential of extracts of new parked motor vehicle indoor air. Up to 171 L air equivalents
were painted once daily on the ears of BALB/c mice for three consecutive days. Ear thickness was recorded before the first application
of test substances and on day 3. The total number of CD45+ leukocytes in the draining auricular lymph nodes was determined on day
4. Croton oil was the positive control for an irritating compound; oxazolone was the positive control for a sensitizing compound. DEE
(dimethylacetamide:diethyl ether:ethanol)4:3:3) was used as solvent control. Mean of 5 mice per group ( SD are depicted.
The main components sampled in the new motor vehicle more volatile organic components during the lifetime of a
were o,m,p-xylenes, alkylbenzenes, and alkanes like dode- vehicle, skewing motor vehicle indoor air toward lesser
cane, which was similar to the compounds found by others volatile compounds. Second, compounds released from
(10). Differences between components found in the extracts deeper compartments of a vehicle could maintain concen-
of indoor air of the new and the used vehicle were especially trations over more prolonged periods of time. Although
quantitative, with the new vehicle showing an about 9-fold vehicles of identical brand and equipment were investigated,
higher concentration of indoor air components than the used differences in the production process of the vehicles not
vehicle (Table 1). known to us could explain these differences too.
The total VOC in the new motor vehicle of 10.9 mg/m3 Ether extracts were made to obtain sufficient quantities
was above the value of 3 mg/m3 proposed by Siefert et al. of indoor air components to be able to perform toxicity testing
(24). This high value of TVOC in parked motor vehicle indoor in cell culture. About 99% of components directly sampled
air might not be so distressing as Seifert et al. deduced this from parked motor vehicle indoor air were accounted for in
value for building indoor air at room temperature, whereas the ether extract from the new vehicle (Table 1). We tested
we sampled a parked motor vehicle heated up to 65 °C. primary human keratinocytes and two lung cell lines for three
Besides, concentrations in motor vehicle indoor air declined toxicologic endpoints in cell culture: sulforhodamine B for
rapidly with forced ventilation or open windows (25), what toxicity evidenced by growth inhibition as a general toxicity
most passengers will use when occupying a heated vehicle. test, Alamar Blue for disturbed mitochondrial function, and
The value for single components like the major component CellTiter-Glo for cellular ATP content. None of the extracts
xylene of the new vehicle (2.45 mg/m3) was below its reported showed any direct toxicity in the used test systems up to a
extrapolated average values to 65 °C encountered in other concentration of 2260 L new vehicle indoor air equivalent
motor vehicles (14.7 mg/m3) (7, 10) and was below its per L cell culture medium. A normal individual breathes about
proposed maximal value of 8.8 mg/m3 (26). For some 500 mL per breath, 12-18 times per minute, which equals
compounds in our extract a maximum work place concen- 9 L air/min, 540 L per hour. As the lung volume is 6 L, and
tration is deduced (11), and toluene, xylenes, trimethylben- assuming sink conditions, the exposure is 90 L per hour
zenes, methylpyrrolidone, methylcyclohexane, formalde- indoor parked motor vehicle air per liter lung volume. We
hyde, 2-butanone, and MIBK were below the allowed exposed cells to 2260 L air equiv for 48 h (keratinocytes and
concentrations. All components declined substantially over A549) or 72 h (V79). Thus our exposure is above the expected
3 years, similar to reports by others (10, 27), except for environmental exposure found in parked motor vehicle
aldehydes and ketones that were found at 66% of the indoor air.
concentrations of the new motor vehicle. Aldehydes and During the transfer into DMSO, a condition essential for
ketones are present in cleaning agents, and it can be assumed dosing the compounds to the cells, we expect to have lost
that some of them might have been introduced during few compounds as DMSO is a solvent freely mixable with
cleaning of the vehicle. An alternative explanation could be ether and has a favorable lipophilicity to maintain all
that aldehydes and ketones, which are also found in air- compounds sampled in the ether phase.
polluted streets, had absorbed to the interior of the used Toxicity is not confined to the parent compounds only
motor vehicle and were released upon sampling (4). but is often mediated by reactive metabolites. Cytochromes
The identity of the analyzed compounds was also different P450 are the dominating enzymes forming reactive metabo-
between the vehicles. This could be due to evaporation of lites (28). Among the cytochromes P450 tested none activated

VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2627

parked motor vehicle indoor air extracts to direct toxic
compounds. We did not test for mutagenic potential of the
reactive species. Also, activation by other cytochromes P450
not included in our test system like CYP3A4 and CYP2C9, the
major hepatic cytochrome P450 (29) cannot be excluded.
However, these cytochromes P450 are not or expressed at
low levels in human skin and lung epithelial cell (28, 30-32).
Metabolic activation by other human enzymes other than
cytochromes P450 cannot be excluded but is generally less
common (29, 33).
Environmental pollutants from roadside emissions aug-
mented IgE-mediated effector cells activation (34). The same
was true for extracts of the indoor air of the new parked
motor vehicle but not for extracts of the used vehicle (see
Figure 3). We used a murine mast cell line as stable FcRI
expressing human mast cells are not easily available. The
FcRI is the high affinity receptor for IgE and is necessary for
IgE-mediated mast cell activationsone facet of immediate-
type (type I) allergic diseases. β-Hexosaminidase release is
an accepted indicator for mast cell activation (20), and mast
cells are important effector cells of the immune system.
In contrast to indoor air in buildings, which showed an
aggravating effect on symptoms in allergic individuals in
controlled human studies (35), only slight effects were noticed
with parked motor vehicle indoor air. The aggravation of
IgE-mediated activation of murine mast cells by indoor air
compounds of the new vehicle was dose dependent and was
significant at 2260 L air equivalent/L medium. The effect of
a more concentrated extract was not statistically different,
probably due to a bell shaped dose response curve generally
found in mast cells (36).
The irritative and sensitizing potential (T cell mediated
type IV immune response) of new motor vehicle indoor air
extract was tested in vivo using a modified mouse local lymph
node assay and mouse ear swelling test. The test systems
were validated using the known contact sensitizer oxazolone
and the irritant croton oil. Even at the highest concentration
tested (171 L air equiv), the extract did not induce a significant
increase in leukocyte numbers in the draining lymph nodes,
nor did it induce a significant ear swelling response.
Therefore, in vivo an irritant potential of new parked motor
vehicle indoor air seems unlikely. Similarly, there was no
indication that the new parked motor vehicle indoor air
extract at the concentrations tested would act as contact
sensitizer.
We studied the toxicity of parked motor vehicle indoor
air. The only effect of motor vehicle indoor air we noticed
was the aggravating effect of indoor air components of only
the new vehicle on the IgE-mediated immune response,
which could be relevant for atopic individuals. Else no direct
toxicity, no toxicity after metabolic activation by cytochrome
P450, and no irritative and no type IV sensitizing potential
of motor vehicle indoor air were found, neither in the new
nor used vehicle at concentrations relevant to humans. Our
investigations gave no indications for an apparent health
hazard of parked motor vehicle indoor air.
Acknowledgments
The excellent help of Katrin Bitterer, Claudia Möbius, and
Antje Wallmuth for performing the cytotoxicity testing,
Simone Knabl for preparing the human keratinocyte culture
and Britta Dorn for LPS determination is greatly acknowl-
edged. Part of this study was funded by a grant from the
Bayerische Forschungsstiftung AZ476/01.
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Received for review July 27, 2006. Revised manuscript re-
ceived October 31, 2006. Accepted January 19, 2007.
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