Acute Toxicity of Marking Pen Emissions

Journal of Toxicology and Environmental Health, Part A, 66:829–845, 2003
Copyright© 2003 Taylor & Francis
1528-7394/03 $12.00 + .00
DOI: 10.1080/15287390390156173
ACUTE TOXICITY OF MARKING PEN EMISSIONS
Rosalind C. Anderson, Julius H. Anderson
Anderson Laboratories, Inc., West Hartford, Vermont, USA
To evaluate complaints of adverse reactions to marking pen emissions, groups of mice were
exposed for 1 h to the emissions of 8 brands of felt-tip markers or white-board cleaner. Pneu
motachographs and a computerized version of ASTM E-981 test method were used to measure
changes in respiration. Sensory irritation (SI), pulmonary irritation (PI), and/or air flow limitation
(AFL) of differing intensities were documented with each of the eight brands tested. At the
peak of the effects, the largest SI was observed with pen F (72% of the breaths); the largest PI
occurred with pen D (13% of the breaths), and the largest AFL was seen with pen F (25% of
the breaths). Pens G and H produced minimal SI, PI, or AFL. A functional observational battery
was used to screen for signs of neurotoxicity. Emissions from all eight of the pens produced
behavioral abnormalities such as altered posture and gait, tremors, falling, and hyperactivity.
The exposure concentrations were similar to the total volatile organic compounds (TVOC)
values near marking pens in actual use. Gas chromatography identified mixtures of alcohols,
acetates, and/or ketones. Exposures to white-board cleaner solution resulted in similar toxicity
(SI, PI, AFL, and neurotoxicity). These results document that some marking pens and white-board
cleaner emit mixtures of chemicals that can produce acute respiratory toxicity and acute
behavioral abnormalities in normal mice. These results provide a toxicological explanation for
some of the human complaints concerning respiratory and neurological reactions to marking
pen emissions.
Several complaints have been received by our laboratory concerning adverse
reactions to the emissions of marking pens or air in the vicinity of white (dry-erase)
boards. After sitting near students using marking pens to highlight lecture notes, one
professional student experienced blurred vision, severe nasal congestion, ringing
in the ears, headache, dizziness, difficulty concentrating, drowsiness, memory
deterioration, decreased fine motor coordination, difficulty breathing, nausea,
and swollen ankles. Other individuals have complained of burning face and
eyes, difficulty breathing, mild mental confusion, and/or lightheadedness when
near white boards or marking pen emissions. Although many marking pens are
labeled “nontoxic,” marking pen emissions are deliberately inhaled by some
drug abusers to induce solvent intoxication (McGarvey et al., 1999). Some of
Received 11 June 2002; sent for revision 9 July and 13 September 2002; accepted 29 October 2002.
Scott Hopkinson provided excellent technical assistance. Gary Cude of Matrix Analytical Labs in Addison,
TX, provided excellent chemical analyses. Dr. Yves Alarie made many helpful suggestions in the preparation
of the manuscript. There was no financial support for this research from outside of Anderson Laboratories
(a private corporation). A preliminary report of this research appeared in Indoor Air 2002, Proceedings of
the 9th International Conference on Indoor Air Quality and Climate, 30 June–5 July 2002, Monterey, CA.
Address correspondence to Rosalind Anderson, Anderson Laboratories, Inc., PO Box 323, West Hartford,
VT 05084, USA. E-mail: JHARCA@hotmail.com
829

830 R. C. ANDERSON AND J. H. ANDERSON
the complaints about marking pens might be due to solvent effects (Cornish,
1980), but other airborne chemicals might also be involved.
The discrepancy between the labels (“nontoxic”) and the human symptoms
(which sound “toxic”) led us to systematically study the acute toxic potential of
marking pen emissions using laboratory mice as objective test subjects. The study
design was to expose mice for 1h to the mixtures of volatile chemicals emitted
by various marking pens. The mice were monitored with pneumotachographs
to determine whether there were any effects on breathing rate, pattern, or air
flow velocity. This is a sensitive way to detect toxic effects on the trigeminal
nerve endings in the face and upper airway (sensory irritation), vagus nerve end
ings in the lower airway (pulmonary irritation), and bronchoconstriction effects
(Vijayaraghavan et al., 1993; Anderson & Anderson, 1997, 1998, 1999a, 1999b,
1999c, 1999d, 2000). After exposures mice were examined and scored using
a functional observational battery (FOB) to screen for evidence of neurotoxicity
as previously done with carpet emissions (Anderson, 1995) and fragrance prod
ucts (Anderson & Anderson, 1997, 1998). The emissions were subjected to gas
chromatography to identify the major chemical components. Analogous studies
were performed with white-board cleaner solution.
MATERIALS AND METHODS
Samples
Eight brands of marking pens and one type of white-board cleaner solution
were purchased in local stores between January 1999 and December 2001. All
the marking pens were labeled “nontoxic” and “conforms to ASTM D 4236”—an
industry standard for labeling potentially toxic art materials (ASTM web site,
www.ASTM.org, 2001). Pens A and B were made for use on white boards; the
label on Pen B recommended use in “a well-ventilated space.” Pens C, D, E, F,
and G were sold for general use, Pen C was permanent, Pen D contained a
fragrance, and Pen E was washable. Pen H was sold for use on transparencies.
The white-board cleaner was made for use as a spray; its label indicated it
contained 2-butoxy ethanol/acetate and isopropanol (proportions not stated)
and that it was irritating to the eyes and skin and should be used in a well-
ventilated space away from children.
Animals
Male Swiss-Webster mice were obtained from Taconic Farms in Germantown,
NY, and housed on corn-cob chips in polypropylene cages with 12h light–
12h dark cycles according to published guidelines (Anonymous, 1996). Purina
lab chow and bottled water were available except during exposures. The
animal weights were between 25 and 28g after a 1-wk acclimation. Extensive
reports of source colony animal health were provided regularly by the animal
supplier. Histological evaluation of lungs from our unexposed colony mice
showed no signs of bacterial, viral, or parasitic infections.

831MARKING PEN TOXICITY
Test Atmospheres
One or more marking pens were uncapped and placed in an all-glass
chamber (40L; 0.25× 0.30× 0.50m), which was sealed, warmed to 26°C, and
allowed to equilibrate for one hr before use. The temperature in the animal
chamber averaged 23°C (Cole Palmer thermister model 8402-00). The relative
humidity in the animal chamber was 35% (range 20 to 50%). To study the
white-board cleaner, 20 or 40g was placed in an open Petri dish inside the glass
chamber and warmed as already described; about 10% of the board cleaner
evaporated during these experiments.
Total Volatile Organic Compounds
The total volatile organic compounds (TVOC) in the exposure chamber was
determined at 1- or 5-min intervals using flame ionization detection (Beckman
Industrial, model 400A) with methane (100 ppm) as calibration gas. Additional
TVOC values were determined after 1 or 4 markers were uncapped and used
to draw 10-cm circles on a piece of tablet paper for 2min. The sampling probe
was positioned 26cm above this drawing or above a sheet of glass being
cleaned with white-board cleaner; 26cm was an arbitrary choice and other
distances were not examined.
Chemical Analysis of the Emissions
A 50-mg piece of felt tip was placed in a 1-L glass vessel. After 12h the
headspace was analyzed on 2 gas chromatography columns of differing
polarities: (a) 30m of 100% dimethyl polysiloxane, and (b) 30m of 95%
dimethyl–5% diphenyl polysiloxane. Peaks were detected by flame ionization
and identified by retention times and referencing the material safety data (MSD)
sheets related to these pens. This work was performed by Matrix Analytical
Laboratories in Addison, TX.
Animal Exposures
For each test four mice were positioned in the glass exposure chamber as
previously described (Vijayaraghavan et al., 1993). The head of each mouse
extended into the central exposure area with the body in a side arm, which
served as a whole-body plethysmograph. During a 15-min “baseline period” the
animal exposure chamber was continuously ventilated with charcoal-filtered air.
Five minutes later the animals began to breathe marking pen emissions carried
by charcoal-filtered air, passed at 6L/min through the sample holding chamber
to the animal exposure chamber and then out of the building. After 60min the
animals again inhaled charcoal-filtered air for a 15-min “recovery period.” The
animals were then removed from the exposure chamber and allowed to rest
for 15min. The mice were then scored over a 20-min period using the FOB.
Over 200 mice were exposed to marking pen emissions, 4 mice per
experiment. As controls, over 200 mice received sham exposures (the same
insertion into the restraining tube for 95min of monitoring while breathing

charcoal-filtered air), 4 mice per experiment. Each laboratory day involved a
control experiment and three experiments with marking pen emissions. Each
mouse served as its own control for statistically diagnosing adverse effects; group
statistics involve pooled data from different mice tested on different days.
American Society for Testing and Materials (ASTM) E-981
ASTM E-981 is a standardized toxicological test method for measuring
acute biological effects of airborne irritant chemicals (ASTM, 1984; Alarie et al.,
2000). Alarie and associates (Vijayaraghavan et al., 1993, 1994; Boylstein et al.,
1995) added pneumotachographs to continuously measure air flow velocity
during each breath from each mouse. Digital computer programs (Alarie, 2000)
sample the analog data 500 times per second and integrate flow rates and
calculate volume changes during each respiratory cycle of each mouse. The
program determines the duration of pause after inspiration (TB, time of break),
the duration of pause after expiration (TP, time of pause), the mid-expiratory air
flow velocity (VD), the respiratory rate, and the tidal volume. The software
determines the mean for each parameter for each 15-s period. During the first
15min of each experiment, approximately 3500 breaths are used to define
the baseline values for each mouse.
In each experimental hour, approximately 60,000 breaths are analyzed
and averaged in 15-s units. To summarize this data, the digital programs provide
two complementary approaches. First, adverse effects can be directly measured
as changes in TB, TP, VD, respiratory rate, and/or tidal volume. These values can
be compared to baseline values or compared to values for sham-exposed (con
trol) mice. Second, one can compare each of these parameters to the baseline
values for that mouse and only report (as diagnostic abnormalities) those that
reflect statistically significant deviations from the baseline mean values. A diagno
sis of sensory irritation (SI) requires an increase of TB by more than 2 times the
standard deviation (SD) from the baseline mean value for that mouse that day.
A diagnosis of pulmonary irritation (PI) requires an increase in the TP to greater
than 2 SD from the baseline mean for that mouse that day. Air flow limitations
(AFL) are diagnosed when VD falls 1.5 SD below the baseline mean value for
that mouse that day. Both forms of data presentation were used: the second
approach yields only those changes that were statistically significant, while the
first approach more directly indicates the magnitude of the adverse effects.
The computer programs allow filtering and smoothing of the data using a
polynomial spline procedure named maximum likelihood estimate–generalized
cross validation (MLEGCV) (Boylstein et al., 1995). To generate tables comparing
brands or doses, plots of the time course for each mouse were prepared,
and the peak value for each parameter or diagnosis was manually extracted
and tabulated.
FOB
Each mouse was individually observed in an open field and scored for 25
parameters related to appearance and behavior (Moser & Padilla, 1998):

posture; gait; overall activity; tremors; balance; climbing ability; reach reflex;
grip strength; righting reflex; response to touch, click, and tail pinch; abdominal
muscle tone; and orientation and foot placement while walking on a vertical
or horizontal grid. We noted (when present) twitching of an eye or ear, gasping,
severe facial swelling, severe lacrimation, piloerection, petechiae in ears, bleeding
from eyes or ears, body tortion, weakness or paralysis of one or more limbs,
repetitive stereotyped movements (such as lip smacking or hand washing),
aggressive behavior, stupor, coma, and convulsions. These evaluations were made
by a single technician who was aware of the previous treatment of the mice
(not blinded). Videotapes were used to document severe reactions.
Data were tabulated as either the percentage of animals showing a specific
abnormality or an FOB score reflecting the mean number of abnormalities
observed per mouse. Individual components of the FOB were assessed used
chi-square analysis of 2× 2 contingency tables with the Yates correction factor
(Zar, 1984). This analysis was applied while the data were in the form of
proportions; the data were later changed to percent values for clarity of
presentation.
Statistics and Graphics
SigmaPlot, Version 3 (Jandel Corporation, San Raphael, CA), was used to
graph the data and perform t-tests on tabulated data. The criterion for signifi
cance was set at p<.05.
RESULTS
Exposure TVOCs
The mouse exposures involved TVOC values (68 to 56,300ppm) that
overlapped the range of TVOC values measured near simulated art projects
(Table 1). While some values appear to be high numbers (e.g., 56,300ppm),
none of these exposure concentrations exceeded four times the TVOC con
centrations found in the simulated art project (Table 1). The TVOC values in
the exposure chamber varied 10% during the 60-min exposures. The control
mice inhaled charcoal-filtered air with a TVOC of 12ppm.
Time Course of a Representative Experiment
Figure 1 shows an experiment with Pen F. Pen F emissions were present
from 20 until 80min. Effects began within seconds after introduction of the
test atmosphere (TVOC= 4900ppm). The value for TB rapidly increased to
170% of baseline and then gradually returned toward baseline and remained
there for the rest of the exposure. The value of TP slowly rose about 10% over
the course of the exposure. VD rapidly declined to about 80% of baseline
value, then fell to about 70% of baseline value toward the end of the exposure
(Figure 1). The results of the statistical diagnostic program are shown in Figure 2.
At the peak of the effects the TB elevations reached diagnostic levels for SI in

TABLE 1. TVOC Values
TVOC values (ppm)
Mouse chamber Room air
1 pen 4 pens 9 pens 1 pen 4 pens
Pen A
Pen B
Pen C
Pen D
Pen E
Pen F
Pen G
Pen H
3800
5700
8900
1800
530
5100
70
68
17,300
26,000
32,000
4000
2400
14,900
380
250
40,700
35,900
56,300
11,800
4120
42,900
610
530
2370
2830
4700
1500
420
4150
48
46
11,400
14,500
24,800
5600
2100
14,700
240
160
Note. Data represent the means of two or four experiments. The mouse
chamber blank (no pens present) was 12 ppm, and the room air blank was
13 ppm.
FIGURE 1. Time course of representative experiment. Emissions of Pen F (TVOC = 4900 ppm) were intro
duced at 20 min; at 80 min the mice again breathed charcoal-filtered air. During the baseline period (0 to
15 min) the average values (SD) were TB = 0.029 (0.006) s, TP = 0.025 (0.003) s, VD = 2.563 (.387) ml/s,
tidal volume = 0.205 (0.020) ml, and respiratory frequency = 266 (35) breaths/min. The thick lines show the
means of 4 mice; the thin lines show the 95% confidence intervals.
47% of the breaths; the fall in VD at the end of the exposure allowed an AFL
diagnosis in 30% of the breaths (Figure 2). The small elevation in TP resulted in
diagnosis of PI in less than 5% of the breaths, so this line has been omitted
from Figure 2.

FIGURE 2. Statistical analysis of respiratory data. The data from Figure 1 were converted to diagnostic
abnormalities using the software described. The thick lines represent the means; the thin lines represent the
95% confidence intervals for the diagnoses. Data for PI were omitted for clarity.
Variability
Swiss Webster mice are outbred and demonstrate genetic variability in
their physiological responses to various stimuli; their responses to felt tip
marker emissions reflected this variability. For example, the TB elevation pro
duced by Pen F peaked at 110, 165, 210, and 240% of baseline in the 4 mice
that simultaneously received this exposure (average shown in Figure 1). These
peaks for TB elevations occurred between 1 and 4min after the onset of the
exposure. Subsequent statements refer in general to the averages for groups of
12 or 16 mice.
Peak Effects
The peak effects for SI were markedly different with the eight brands of
pens (Table 2). Pen F induced the most frequent SI (72% of the breaths), while
Pens G and H produced no statistically significant effect (Table 2). The largest
elevation of TB (TB= 264% of baseline) occurred with 4 units of Pen B. In the
sham exposures 7% of the breaths were classified as SI (due to 12% random
fluctuations in TB values).
All pens except C produced small amounts of PI, but the results were not
markedly dose related (Table 3). The maximum TP value observed was 133%
of baseline with nine units of Pen D.
AFL occurred with all pens except G (Table 3). Pen E was the most potent
when tested as a single unit (AFL= 17% of the breaths), while Pen F produced
the largest AFL (25% of the breaths, peak effect from 9 units of Pen F). The

TABLE 2. Sensory irritation
Percent of breaths
1 pen 4 pens 9 pens
Pen A 4 35a
46a

Pen B 63a
69a
57a

Pen C 59a
63a
33a

Pen D 13a
30a
70a

Pen E 5 — 21a

Pen F 35a
50a
72a

Pen G 4 — 6

Pen H 4 7 12

Note. Summary of peak effects during experiments with one,
four, or nine pens open in the sample chamber. There were 220
shams and 12 or 16 mice in the other groups. Values are means
(SE mean varied from 1 to 10). The sham value for SI was 7.
a
Significant at p < .05 compared to shams.
TABLE 3. Pulmonary irritation and air flow limitation
Percent of breaths
PI AFL

1 pen 4 pens 9 pens 1 pen 4 pens 9 pens

Pen A 9a
3 4 7 8a
5

Pen B 8a
4 3 6 3 9a

Pen C 3 3 2 9a
16a
11a

Pen D 6 11a
13a
9a
1 19a

Pen E 4 n.a. 10a
17a
n.a. 16a

Pen F 3 7a
10a
10a
14a
25a

Pen G 12a
n.a. 4 5 n.a. 4

Pen H 8a
5 12a
4 7 11a

Note. Summary of peak effects during experiments with one,
four, or nine pens open in the sample chamber. There were 220
shams and 12 or 16 mice in the other groups. Values are means
(SE mean varied from 1 to 10). The sham value for PI was 4; the
sham value for AFL was 4; n.a., not assessed.
a
lowest VD recorded was 31% below baseline in experiments with one unit of
Pen D.
Observational Abnormalities
High frequencies of behavioral abnormalities occurred with posture, gait,
tremors, hyperactivity, facial swelling, and gasping (Table 4). Severe lacrimation
was seen with Pens A, B, and E. To preserve readability not all observations are

TABLE 4. Behavioral abnormalities
Percent of mice with abnormality
Sham A B C D E F G H
Posture 5 67a
75a
42a
33a
100a
83a
17 0
Gait 8 42a
75a
58a
75a
56a
42a
0 0
Tremor 17 92a
94a
50a
67a
75a
75a
50a
13
Disorientation 2 8 6 17 33a
12 0 0 0
Falling 0 25a
6 0 33a
31a
8 0 13a
Hyperactivity 3 0 0 0 83a
56a
0 0 0
Swelling 0 67a
31a
17a
33a
19 67a
0 0
Lacrimation 0 17a
37a
17a
0 33a
0 0 0
Gasping 0 83a
88a
58a
42a
44a
92a
67a
0
FOB score 4 12a
12a
10a
9a
9a
11a
7a
4
Note. Data indicate percent of mice with the abnormality in experiments with one pen open in sample
chamber. The last line gives the FOB score, the sum of the number of abnormalities noted. The different
columns represent data from different pens as indicated. There were 64 shams and 12 or 16 mice in the
other groups.
a
shown in Table 4. Avoidance behavior was observed in 50% of the mice
exposed to Pen B. The hyperactivity with Pen D was so severe that 75% of
these mice were recorded as “explosive.” Decreased grip strength was observed
in several mice exposed to Pens D and F, and the righting reflex was slow in
several mice exposed to Pen B. Higher concentrations produced similar types of
abnormalities. The last line in Table 4 shows the FOB score (a simple counting
of all abnormalities) for comparison.
FOB Scores
The FOB score was related to the exposure concentration as shown in
Figure 3. Data are consistent with a linear relationship between FOB and log
TVOC with r2
= .711.
Chemical Evaluation of Pen Emissions
The pens with greatest sensory irritation (B, C, and F) emitted various different
mixtures of methanol, ethanol, propanol, butanol, acetone, butyl acetate, and
methyl isobutyl ketone. The least toxic pens (E, G, and H) emitted only traces
of ethanol and isopropanol. Intermediate toxicity pens (A and D) emitted
larger amounts of ethanol and isopropanol; D also emitted an ester. It was not
possible to attribute all the irritancy to any individual chemical.
White-Board Cleaner
Mice were exposed to white-board cleaner at concentrations (Table 5) that
were slightly higher than the TVOC values developed when using the cleaner
on glass in an open room (4270ppm). The lower concentration tested with
mice (5400ppm) produced SI and AFL, while the higher concentration tested

FIGURE 3. FOB score versus exposure TVOC. Each point represents data from 12 or 16 mice exposed for
1 h to marking pen emissions. Regression line has r2
= .711.
TABLE 5. White-board cleaner
Percent of breaths
Exposure TVOC (ppm) SI PI AFL FOB score
5400 47a
5 24a
12a
14,700 54a
11a
11a
14a
Note. Sixteen mice in each test.

a
Significant at p < .05 compared to 64 shams.

(14,700ppm) affected all three (SI, PI, and AFL) end points (Table 5). TB maximum
was 228% of baseline; TP maximum was 113% baseline, and VD minimum
was 25% below baseline. The FOB scores were significantly elevated (12 to 14
abnormalities/mouse) at both exposure concentrations. The observational
abnormalities with high frequency of occurrence were posture (100%), gait
(83%), tremor (92%), falling (50%), hyperactivity (92%), facial swelling (42%),
and gasping (83% of the mice).
DISCUSSION
Several brands of marking pens emitted mixtures of chemicals that produced
SI, PI, AFL, and behavioral changes in normal mice. These adverse effects
occurred at TVOC concentrations similar to those a human would encounter
near an art project involving one to four marking pens. One can only speculate

whether there might be even higher TVOC concentrations in poorly ventilated
classrooms with 30 students using marking pens continuously over 45 to
60min.
These studies involved only the mixtures of VOCs actually emitted by these
pens; effects of individual components were not evaluated. Interactions between
the effects of various chemical components of the emission mixtures may
account for the fact that there are simple dose-response relationships present
in some parts of the data but not others. For example, lines 1, 4, 6, and 8 in Table
2 show simple relationships, line 2 suggests a saturation phenomenon, and line 3
might represent a biphasic relationship. The presence of simple dose-response
relationships (as in lines 1, 2, 4, 6, and 8) suggests that one or a few compon
ents of the emission mixture is dominant in producing the overall effect. In explain
ing a biphasic or complex dose-response relationship (as in line 3) one must
consider both (a) interference between different physiological reflexes (e.g., SI
reflex vs. PI reflex) and (b) interference between effects of different chemicals
in the emission mixtures. For example, a mixture of a stimulant with a low ED50
and an inhibitor with a high ED50 might give a biphasic dose-response relation
ship. The data in Table 3 concerning PI and AFL involve smaller effects, and one
must avoid overinterpretation of these numbers. Considering the potential
complexities of evaluating multiple neurological effects of mixtures of chemicals,
the relative simplicity of the dose-response relationship in Figure 3 is rather
remarkable.
The pens involved in this study were all labeled “nontoxic.” These certifi
cation labels indicate that a toxicologist has reviewed “the complete formulas,”
“each ingredient and its quantity,” “possible adverse interactions with other
ingredients,” and “potential acute and chronic harm to any part of the human
body” (Art & Creative Materials Institute [ACMI], 2002). These evaluations
considered both children and adults with use and misuse (such as ingestion) by
a small child (ACMI, 2002). The toxicity data presented in this article make
one seriously question the adequacy of the approach outlined above. The
anecdotal evidence reflected by human complaints about marker emissions
suggests that laboratory testing allows more accurate predictions than does the
review process.
These results demonstrate a toxicological basis for some of the complaints
received concerning acute toxicity due to marking pen emissions. It is not known
whether marking pen emissions are causing widespread health problems or
whether the toxic effects are evident in only a few sensitive individuals.
SI Testing in Mice and Humans
SI results from chemical irritation of trigeminal nerves in the conjunctivae,
facial skin, and nasal mucosa (Alarie, 1973, 2000; Neilsen, 1991). The microscopic
details of the nerve terminations in the conjunctivae have been elaborated
(MacIver & Tanelian, 1993). The receptors on the surface of these nerves have
been named vanilloid receptors, because they have been extensively studied
using capsaicin aerosols, and the vanilloid portion of the capsaicin molecule is

important in ligand binding (Carterina et al., 1997). The stereospecificity of
these receptors has been documented (Kasanen et al., 1998; Larsen et al., 2000).
When airborne irritants bind to these tetrameric receptors (Kuzhikandathil
et al., 2001), there is a calcium flux, release of substance P, and local neuro
genic inflammation (Bascom et al., 1997). Trigeminal nerve firing rate is propor
tional to the sum of the individual stimulations by various airborne chemicals
(Carstens et al., 1998). The trigeminal nuclei in the pons (Chamberlin & Saper,
1998) process the afferent information and produce efferent signals in the phrenic
nerves to change the respiratory pattern and rate (Alarie, 1973).
Data concerning SI in mice can be directly extrapolated to predict human
experience and/or threshold limit values for occupational exposure for more
than 80 volatile organic chemicals (VOCs) (Shaper, 1993; Alarie, 1981). Because
Swiss-Webster mice are less sensitive than humans to many airborne irritants
(Y. Alarie, personal communication), the SI test is not likely to give “false positive
results” when used as a screening tool (Tepper & Costa, 1992).
SI testing with mice has proved useful in studies of individual VOCs (Kasanen
et al., 1999; Neilsen et al., 1999; Larsen & Neilsen, 2000) and mixtures of VOCs
derived from mold cultures (Korpi et al., 1999) and consumer products
(Anderson & Anderson, 1997, 1998, 1999a, 1999b, 1999c, 2000). The objec
tivity of the mouse tests has proved useful in evaluating samples of air taken
from schools and offices with complaints of indoor air pollution (Anderson &
Coogan, 1994; Anderson & Anderson, 1999d).
SI in humans has been studied with single VOCs and mixtures of VOCs
(Hudnell et al., 1992; Cometto-Muniz et al., 1997, 1998a; Hemple-Jorgensen
et al., 1999; Millqvist et al., 1999; Molhave et al., 2000). The conjuctivae of
the eyes and the nasal mucosa are approximately equal in chemesthetic
sensitivity (Cometto-Muniz et al., 1998b). Conjunctival hyperemia has been
photographically documented (Hemple-Jorgensen et al., 1998), and mucosal
inflammation (Buckley et al., 1984; Meggs et al., 1996) has been assessed by
analyzing nasal exudate (Koren et al., 1992). The physical properties of volatile
chemicals can be used to develop mathematical equations to predict the effects
of mixtures of chemicals as sensory irritants in humans (Alarie et al., 2000).
The VOCs that produce SI in mice cause humans to experience burning of
eyes, throat, skin, nose, or chest; conjunctivitis, lacrimation; coughing; and/or
gagging (Alarie, 1973), plus fatigue and shortness of breath (Millqvist et al.,
1999). Thus the SI observed in mice exposed to marking pen emissions and
white-board cleaner probably explains the complaints of burning face and eyes
in some humans exposed to marking pen emissions and air near white boards.
PI Reactions in Mice and Humans
Several of the pens and the white-board cleaner produced PI at high
concentrations. Mice and humans react in a similar manner to agents that
induce lower airway irritation and inflammation (Alarie, 1973), but extrapolation
of PI from mice to humans is not as numerically precise as it is with SI
(Schaper, 1993).

AFL in Mice and Humans
The VD measurement used with these mice and the FEV1 measurement
(forced expiratory volume in 1s) employed to evaluate human asthmatics are
both reflections of expiratory air flow velocity. The VD values in Figure 1
decreased by 30% during this exposure, similar to the decreases in FEV1 seen
in human asthmatics during bronchoconstriction episodes (O’Byrne et al., 1997).
The procedure for measuring VD reactions in mice is relatively new, so there is
not yet sufficient experience to allow direct extrapolation to humans. Although
mice have fewer respiratory bronchioles (Mercer & Crapo, 1992) and faster
breathing rates, both mice and humans decrease air flow velocity when chal
lenged with cholinergic agents such as carbamylcholine and methacholine
(Boylstein et al., 1995). Both mice and asthmatics react with air flow decreases
on challenge with fragrance products (Kumar et al., 1995; Anderson & Anderson,
1998) and other mixtures of VOCs (Pappas et al., 2000). Until more informa
tion develops, it is prudent to believe that a chemical or mixture that produces
AFL in mice is probably capable of causing air flow reduction in some humans.
Because AFL occurred during the first hour of encounter between mice and
marking pen emissions, these air flow reductions probably represent acute
toxic effects rather than allergic phenomena. Toxic changes in air flow are proba
bly involved in production and/or exacerbation of some forms of occupational
asthma (Chan-Yeung & Malo, 1995).
Time Course of Respiratory Reactions
The TB response was transient despite a constant TVOC value in the
presented emissions; it is possible that the transience of the SI response reflects
physiological adaptation. Similar transient responses of TB occurred in analogous
experiments with fragrance products and mattresses (Anderson & Anderson,
1998, 1999c), while prolonged responses occur with individual chemicals such
as propanolol and 2-chlorobenzylchloride (Vijayaraghavan et al., 1994) and
with the mixtures of chemicals emitted by air fresheners, fabric softeners, and
vinyl mattress covers (Anderson & Anderson, 1997, 1999a, 2000).
FOB Scores
The FOB scores are based on gross changes in behavior or appearance.
Without more invasive experimentation one can only speculate about the
mechanisms of these adverse effects. Some of the abnormalities (e.g., abnormal
posture, abnormal gait, tremors, and falling) appear to reflect neurological or
neuromuscular effects. Some of the other findings (e.g., facial swelling and
severe lacrimation) might indicate local inflammatory responses or autonomic
nervous system effects (Bascom et al., 1997). The difficulties involved in
interpreting gross behavior effects of solvent toxicity have been extensively
discussed (Warren et al., 2000).
The FOB results in the mice suggest a toxicological basis for some of the
complaints of humans exposed to marker emissions. Data suggest some of the

VOCs entered the central nervous system and caused toxic changes that
resulted in gross abnormalities in appearance or behavior of the mice. Because
of the complexity of the central nervous system, one cannot make a one-to-one
extrapolation of signs in mice to symptoms in humans.
Chemistry of Emissions and Dose-Response Curve for FOB
It is not known which of the chemicals present in the emission mixtures
were most responsible for the adverse effects measured. It was assumed that
the results are probably due to combined effects of several of the moieties in
these mixtures. Individually, propanol, isopropanol, butanol, and methyl isobutyl
ketone produce eye, nose, and throat irritation; central nervous system depres
sion; liver and kidney toxicity; and muscle weakness or tenderness (Wilson,
1993; Anonymous, 2002; Noraberg & Arlien-Soborg, 2000; Neilsen et al., 1988).
Combinations of solvents can act additively or synergistically in human toxicity
studies (Cometto-Muniz et al., 1997; Noraberg & Arlien-Soborg, 2000). The
apparent linearity of the data in Figure 3 would be consistent with nonspecific
solvent effects as the primary mechanism of action for the neurological toxicity
of these mixtures of VOCs. However, data are not sufficient for a critical evalu
ation of this hypothesis.
White-Board Cleaner
At concentrations similar to those that a human would encounter in actual
use, white-board cleaner produced significant adverse effects in mice. The
product label identified 2-butoxy ethanol/acetate and isopropanol as present, but
the relative contributions of the two moieties to the toxic effects is not known.
The label on the white-board cleaner clearly warns of irritant effects and
instructs users to have a well-ventilated space. Unfortunately, many schools are
using marking pens and white-board cleaner in rooms with low ventilation
rates. Marking pens and white-board cleaner may be partially responsible for
symptoms of indoor air pollution (eye irritation, pulmonary irritation, and
neurotoxicity) in certain schools (Anderson & Coogan, 1994; Anderson &
Anderson, 1999d).
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