TOXICOLOGICAL SCIENCES 115(2), 435–443 (2010)
Advance Access publication February 22, 2010
Irritancy and Allergic Responses Induced by Topical Application of
Stacey E. Anderson,1Christina Umbright, Rajendran Sellamuthu, Kara Fluharty, Michael Kashon, Jennifer Franko,
Laurel G. Jackson, Victor J. Johnson, and Pius Joseph
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
1To whom correspondence should be addressed at Health Effects Laboratory Division, National Institute for Occupational Safety and Health,
1095 Willowdale Drive, Morgantown, WV 26505. Fax: (304) 285-6126. E-mail: firstname.lastname@example.org.
Received December 7, 2009; accepted February 13, 2010
Although ortho-phthalaldehyde (OPA) has been suggested as an
alternative to glutaraldehyde for the sterilization and disinfection
of hospital equipment, the toxicity has not been thoroughly inves-
tigated. The purpose of these studies was to evaluate the irritancy
and sensitization potential of OPA. The EpiDerm Skin Irritation
Test was used to evaluate in vitro irritancy potential of OPA and
glutaraldehyde. Treatment with 0.4125 and 0.55% OPA induced
irritation, while glutaraldehyde exposure at these concentrations
did not. Consistent with the in vitro results, OPA induced irri-
tancy, evaluated by ear swelling, when mice were treated with
0.75%. Initial evaluation of the sensitization potential was con-
ducted using the local lymph node assay at concentrations ranging
from 0.005 to 0.75%. A concentration-dependent increase in
lymphocyte proliferation was observed with a calculated EC3
value of 0.051% compared to that of 0.089%, previously deter-
mined for glutaraldehyde. Immunoglobulin (Ig) E-inducing
potential was evaluated by phenotypic analysis of draining lymph
node (DLN) cells and measurement of total and specific serum
IgE levels. The 0.1 and 0.75% exposed groups yielded significant
increases in the IgE1B2201 cell population in the lymph nodes
while the 0.75% treated group demonstrated significant increases
in total IgE, OPA-specific IgE, and OPA-specific IgG1. In
addition, significant increases in interleukin-4 messenger RNA
and protein expression in the DLNs were observed in OPA-treated
groups. The results demonstrate the dermal irritancy and allergic
potential of OPA and raise concern about the proposed/intended
use of OPA as a safe alternative to glutaraldehyde.
Key Words: OPA; hypersensitivity; asthma; IgE.
Ortho-Phthalaldehyde (OPA) is an aromatic dialdehyde used
as a high-level antimicrobial disinfectant for medical equip-
ment which is sensitive to normal heat or steam sterilization
processes. For 40 years, glutaraldehyde has been the primary
choice for disinfecting heat-sensitive medical devices; how-
ever, it has been reported to induce occupational asthma and
other health effects (Gannon et al., 1995). For these reasons,
less offensive and presumably safer alternatives to glutaralde-
hyde have been introduced. OPA, the active ingredient present
in Cidex OPA, has shown superior anti-mycobactericidal
activity as compared to glutaraldehyde (Lerones et al., 2004),
allowing for its use at lower concentrations. In addition, low
volatility and no need for activation have increased the use of
OPA as a more practical alternative to glutaraldehyde.
It is estimated that 3253 workers were potentially exposed to
(NIOSH, 1990). After the selection of OPA as an alternative for
workers could now be exposed. The estimated use of OPA in
2002 was between 10,000 and 500,000 pounds (USEPA, 2006).
Along with being approved for disinfecting medical devices,
OPA has also been approved for use as an indoor antimicrobial
pesticide; an intermediate for the synthesis of pharmaceuticals,
medicines, and other organic compounds (ChemicalLand 21,
and as a diagnostic for urea nitrogen test system(USFDA,2006).
Although the health effects have not been thoroughly tested,
Cidex OPA has been used as a ‘‘safe’’ replacement for
glutaraldehyde for the past 10 years. Currently, there are no
regulations regarding proper use and safe exposure levels of
OPA in spite of the potential of exposure for a large number
of healthcare workers and their patients. Concentrations of
OPA ranging from 1.0 to 13.5 ppb have been detected in air
samples collected from an endoscope cleaning unit of a hospital
that used OPA as its primary disinfectant (Tucker, 2008). On the
other hand, in addition to the required use of hand, eye, and
respiratory protection, stringent occupational exposure ceiling
There are very little data available regarding toxicity for
OPA with the majority of the information from case reports. The
most notable case report describes four patients who experienced
using glutaraldehyde to OPA for cystoscope disinfection (Sokol,
? The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: email@example.com
the immediate and late-phase skin reactions strongly suggest an
immunoglobulin (Ig) E-mediated mechanism for the observed
that lasted for24 h. Two potentialcasesof occupationalasthma in
healthcare workers disinfecting endoscopes and similar devices
with Cidex OPA have also been reported (Franchi and Franco,
2005). More recently, Fujita et al. (2006) investigated a case
involving a female nurse who exhibited slight dyspnea and dry
papules and urticaria after working with OPA. For reasons
described above, OPA was nominated by National Institute for
Occupational Safety and Health (NIOSH) for toxicological
evaluation by the National Toxicology Program.
Animal data investigating the health effects associated with
OPA exposure are limited. To date, the only published study
suggests that OPA injection may act as an adjuvant in a murine
ovalbumin (OVA) model (Hasegawa et al., 2009). The high
reactivity of OPA, suspected dermal and respiratory irritation,
sensitization potential, and structural similarity to glutaralde-
hyde raise concerns about exposure and the need for regulation.
Our laboratory has previously tested the sensitization potential
of glutaraldehyde after dermal exposure in mice (Azadi et al.,
2004). It was identified as a sensitizer in the local lymph node
assay (LLNA) with increases in local and systemic IgE levels,
suggesting as IgE-mediated allergic mechanism. The purpose
of these studies was to determine the irritancy and sensitization
potential of dermal exposure to OPA, provide insight into the
mechanism of sensitization, and then to compare these results
to those previously reported for glutaraldehyde.
MATERIALS AND METHODS
Test articles. OPA (more than 99%) (OPA) (CAS #643-79-8), alpha-
hexylcinnamaldehyde (HCA) (CAS #101-86-0), N, N-dimethylformamide
(DMF) (CAS #68-12-2), 2,4-dinitrofluorobenzene (DNFB) (CAS #70-34-8),
glutaraldehyde solution (25%) (CAS #111-30-8), and potassium hydroxide
(KOH) (CAS #1310-58-3) were purchased from Aldrich Chemical Company,
Inc. (Milwaukee, WI).
Tissue selection. EpiDerm (Standard EpiDerm [EPI-200], MatTek Corpo-
ration) inserts, consisting of highly differentiated epidermis tissue derived from
irritation potential of OPA and to compare the result with that of glutaraldehyde.
The in vitro EpiDerm Skin Irritation Test utilizes a normal, human cell–derived,
metabolically active skin model that closely mimics the human epidermis both
corneum overlaying 8–12 cell layers consisting of basal, spinous, and granular
cells grown at an air-liquid interface situated on 9-mm diameter, chemically
modified, collagen-coated membranes with 0.4 lm pore size. This model is
endorsed by the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) Scientific Advisory Committee for its use to
assess the dermal corrosion potential of chemicals (NIEHS, 2002).
EpiDerm culture and exposure. EpiDerm inserts (tissues) were preincu-
bated in six-well plates containing 900 ll Dulbecco’s Modified Eagle’s Medium
treated in duplicate for 1 h with 100 ll test material. During testing, the apical
surface of each tissuewas exposedto test material while each tissue wasfed with
DMEM culture medium through the basolateral surface. The treatment doses
were dissolved in water at 0.1375, 0.275, 0.4125, 0.55, and 1.1% (wt/vol) OPA;
0.1375, 0.275, 0.4125, 0.55, and 1.1% (vol/vol) glutaraldehyde; 8 N KOH; and
with Dulbeccos Phosphate Buffered Saline and media, and the cell viability was
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability
2,5-diphenyl tetrazolium bromide (MTT) cell viability assays according to
the manufacturer’s procedure (MatTek Corporation). Briefly, the EpiDerm
samples were collected immediately following exposure, rinsed twice with PBS,
and placed in a fresh 24-well plate containing 300 microliters per well MTT
solution (1 mg/ml in MTT diluent). The cultures were then incubated for 3 h
at 37?C and 5% CO2. Next, each insert was removed carefully, the bottom was
blotted with soft tissue paper, and the insert was placed in a fresh 24-well plate
temperature for 2 h in the dark. After extraction, the inserts were discarded and
an additional 1 ml extraction solution per well was added. The contents of
each well were mixed thoroughly, and, in triplicate, 200 ll from each well was
pipetted into a 96-well plate to obtain optical density (OD) values. MTT assay
results were determined by measuring the ODs of each sample at 570 nm and
background corrected with OD values at 650 nm. Because the neat test articles,
OPA and glutaraldehyde, result in a reduction of MTT directly (data not shown),
the assay was also performed on dead EpiDerm treated identically to the
live tissue samples for each chemical and concentration tested. The background-
corrected ODs of the dead tissue were then subtracted from the background-
and their interaction were assessed for these analyses. Based on the manu-
a percent viability less than or equal to 50% of the H2O control.
Species selection. FemaleBALB/cmicewereusedinthisstudy.Thismouse
Taconic (Germantown, NY) at 6–8 weeks of age. Upon arrival, the animals were
allowed to acclimate for a minimum of 5 days. Each shipment of animals was
randomly assigned to treatment group, weighed, and individually identified via
tail markingusinga permanent marker.ApreliminaryANOVAon bodyweights
was performed to insure a homogeneous distribution of animals across treatment
groups. The animals were housed at a maximum of five per cage in ventilated
31 modified 6% irradiated rodent diet (Harlan Teklad), and tap water was
provided from water bottles, ad libitum. The temperature in the animal facility
was maintained between 68 and 72?F and the relative humidity between 36 and
57%. The light/dark cycle was maintained on 12-h intervals. All animal experi-
ments were performed in the Association for Assessment and Accreditation of
Laboratory Animal Care International-accredited NIOSH animal facility in
accordance with an animal protocol approved by the Institutional Animal Care
and Use Committee.
Combined LLNA and irritancy assay. To determine the irritancy and
sensitization potential of OPA, a combined LLNA was conducted. OPA dosing
concentrations (0.005–0.75%) and vehicle (DMF) were selected based on
previously published data on glutaraldehyde (Azadi et al., 2004) for com-
parison purposes. The LLNA was performed according to the method described
in the ICCVAM Peer Review Panel report (NIEHS, 1999) with minor
modifications. Briefly, mice (five per group) were topically treated with DMF
vehicle, increasing concentrations of OPA, or positive control (30% HCA) on
the dorsal surface of each ear (25 microliters per ear) once a day for three
consecutive days. DNFB was used as a positive control for the irritancy portion
of the experiment. Irritancy measurements were performed as previously
described (Woolhiser and Munson, 1999). The thickness of the right and left
ANDERSON ET AL.
ear pinnae of each mouse was measured using a modified engineer’s
micrometer (Mitutoyo Co.) before the first chemical administration and 24 h
following the final exposure. The mean percentage of ear swelling was
calculated based on the following equation: [(mean postchallenge ear
thickness ? mean prechallenge ear thickness)/mean prechallenge thickness] 3
100. Animals were allowed to rest for 2 days following the last exposure. On
day 6, mice were injected, intravenously, via the lateral tail vein with 20 lCi
3H-thymidine (Dupont NEN; specific activity 2 Ci/mmol). Five hours after
3H-thymidine injection, animals were euthanized via CO2inhalation, and the left
and right superficial parotid draining lymph nodes (DLNs) located at the
bifurcation of the jugular vein were excised and pooled for each animal. Single-
cell suspensions were made and incubated overnight in 5% trichloroacetic acid,
and samples were counted using a Packard Tri-Carb 2500TR liquid scintillation
analyzer (Perkin Elmer). Stimulation indices (SI) were calculated by dividing the
mean disintegrations per minute (DPM) per test group by the mean DPM for the
vehicle control group. EC3 values (concentration of chemical required to induce
a threefold increase over the vehicle control) were calculated based on the
equations from Azadi et al. (2004) and Basketter et al. (1999).
Phenotypic analysis of DLN cells. To determine if the chemicals induced
a type I or type IV response, the number of IgEþB220þ cells in the DLNs was
quantitated after dermal exposure to OPA using flow cytometry. For the
cell phenotypes were analyzedusingflow cytometry as described byManetzand
of OPA topically on the dorsal surface of each ear (25 microliters per ear) once
a dayfor four consecutive days. Animalswere allowed to rest for 6 days after the
final treatment and then euthanized on day 10 by CO2inhalation. Animals were
weighed and examined for gross pathology at the end of the experiment. The
spleen, kidneys, and thymus. DLNs were also collected (two nodes/animal/tube)
Cell counts were performed using a Coulter Counter (Z2 model, Beckman
Coulter),and13 106cellspersamplewereaddedto thewells ofa 96-wellplate.
incubated with anti-CD45RA/B220 (phycoerythrin, clone RA3-6B2) and anti-
IgE antibodies (fluorescein isothiocyanate, clone R-35-72) or the appropriate
iodide (PI). All antibodies and isotype controls were purchased from BD
Pharmingen. After a final wash, cells were resuspended in staining buffer and
analyzed with a Becton Dickinson FACSVantage flow cytometer using a PI
Total serum IgE. ForanalysisoftotalIgE,OPAwastestedatconcentrations
up to 0.75%.Mice were treated with DMF,increasing concentrations of OPA, or
0.75% glutaraldehyde topically on the dorsal surface of each ear (25 microliters
per ear) once a day for four consecutive days. Animals were allowed to rest for
Following euthanasia of animals, blood samples were collected via cardiac
puncture. Sera were separated by centrifugation and frozen at ?20?C for
subsequent analysis of IgE by ELISA. The standard colorimetric sandwich
ELISA was performed as previously described (Butler, 2000).All antibodies and
plates (Dynatec Immulon-2) were coated with (2 lg/ml in PBS) purified
monoclonal rat anti-mouse IgE antibody (clone R35-72), sealed with plate
sealers,andincubatedovernightat4?C. The plateswerewashedthree times with
PBS/Tween-20andthenblockedfor 1h with2% newborncalfserum(NCS)and
the serum samples, and IgE control standards were prepared at 500 ng/ml. All
control standard (mouse IgE anti-trinitrophenyl, clone C38-2) were serially
temperature for 1 h. The plates were washed three times with PBS/Tween-20.
Biotin-conjugated rat anti-mouse IgE (clone R35-92) was added in a 100-ll
volume, and plates were incubated at room temperature for 1 h. The plates were
washed three times with PBS/0.05%Tween-20. Streptavidin-alkaline phospha-
tase was added (100 ll of a 1:400 dilution), and plates were incubated for 1 h at
room temperature. P-nitrophenyl phosphate (Sigma) was used as the alkaline
Vmax plate reader (Molecular Devices) at 405–605 nm. Data analysis was
performed using the IBM Softmax Pro 3.1 (Molecular Devices), and the IgE
concentrations for each sample were interpolated from a standard curve using
OPA-specific antibodies. Following euthanasia of animals used in the
were separated by centrifugation and frozen at ?20?C for subsequent analysis of
proteins were detected using a custom indirect ELISA. Briefly, Immulon-4
microtiter plates (Nunc, Thermo Scientific) were coated overnight at 4?C with
0.5% OPA in distilled deionized water for 1 h at room temperature. Plates were
washed three times with PBS/0.05% Tween-20 wash buffer, and nonspecific
(200 microliters per well) for 30 min. A twofold dilution series (1/10 to 1/5120) of
times with PBS/0.05% Tween-20, and then biotin-conjugated antibodies specific
For detection of OPA-specific IgE, total IgG was removed from the sera using
protein-G conjugated to Dynabeads (Invitrogen) that were coated with anti-mouse
incubated with the beads for 30 min followed by bead removal with a magnet.
Finally, plates were washed four times, and avidin-horse radish peroxidase
washes. Tetramethylbenzidine-Turbo substrate (Pierce) was added (50 microliters
per well) for 30 min followed by additionof 2M H2SO4stopsolution. Absorbance
development and at 450 nm following addition of stop solution. To confirm the
parallel with the nonheated sera.
Cytokine messenger RNA analysis. To evaluate if T-helper 1 cells (Th1)
and Th2 cytokines were involved in OPA sensitization, the DLNs were analyzed
for messenger RNA (mRNA) expression after dermal application. The Th1
cytokines assessed were interferon-gamma (IFN-c) and interleukin (IL)-12, and
the Th2 cytokines were IL-4, IL-5, and IL-10. Mice were exposed to DMF or
increasing concentrations of OPA topically on the dorsal surface of each ear (25
microliters per ear) once a day for four consecutive days. Animals were then
and the DLNs for each animal were collected in 1 ml of TRI Reagent (Molecular
Research Center). RNA was isolated using TRI Reagent as specified by the
manufacturer. To further purify the RNA, the RNeasy Mini Kit (QIAGEN) with
optional DNase treatment was used following the manufacturer’s protocol. The
concentration of RNA was determined using an nanodrop-1000 spectrophotom-
eter (Thermo Scientific Nanodrop). Reverse transcription of 1 lg RNA was
performed using the Advantage RT for PCR Kit (Clontech) as directed by the
PCR System (Applied Biosystems) using SYBR Green PCR Master Mix
(Applied Biosystems) as specified in the manufacturer’s protocol. Quantitative
RT-PCR data are collected and expressed as relative fold increase over control,
calculated by the following formula: 2DDCt¼ DCtsample? DCtcontrol3 DCt ¼
Cttarget? CtGAPDH, where Ct ¼ cycle threshold as defined by manufacturer’s
instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as
the endogenous control.
IRRITANCY AND SENSITIZATION POTENTIAL OF OPA
Analysis of IL-4 production by DLN cells. To determine IL-4 protein
production in the DLN, OPA was tested at concentrations up to 0.75%. DLNs
were collected (two nodes/animal/tube) in 2 ml PBS from the mice analyzed for
total IgE and dissociated using the frosted ends of two microscope slides. Cell
counts were performed using a Coulter Counter (Z1 model, Beckman Coulter),
and cells were adjusted to 1 3 106cells per milliliter using sterile Roswell Park
Memorial Institute media containing 10% fetal calf serum. Cells were added to
a 48-wellplateina 500-llvolume,stimulated witha-CD3anda-CD28(2lg/ml
of each; BD Pharmingen), and incubated for 24 h at 37?C and 5% CO2.
Supernatants were analyzed for IL-4 production using an OptEIA ELISA kit
purchased from BD Biosciences according to the manufacturer’s instructions.
Supernatant samples collected from each culture (two stimulated and two
unstimulated for each mouse) were added to the plates in triplicate along with
serial dilutions of the standards. Plates were read at 450 nm (OD values for
standards ranging from 0.77 to 1.93) using a SpectraMax M2 spectrophotometer
(Molecular Devices). IL-4 concentration was extrapolated from the standard
curve. The final data are expressed as the mean value generated when the
concentration identified for the unstimulated cultures was subtracted from the
value generated from the stimulated cultures for each mouse.
Statistical analysis. To determine statistical significance for the MTT cell
assay, a two-way mixed model ANOVA (SAS Proc Mixed) was utilized for the
analysis, and experimental block was incorporated as a random effect. For
analysis of animal studies, the data were first tested for homogeneity using
the Bartlett’s chi-square test. If homogeneous, a one-way ANOVA was
conducted.If the ANOVA showed significance at p < 0.05 or less, the Dunnett’s
Multiple Range t-test was used to compare treatment groups with the control
group. Linear trend analysis was performed to determine if OPA had exposure
concentration-related effects for the specified endpoints. For analysis of OPA-
specific antibodies, data were tested for outliers using Grubb’s Test (p < 0.05),
further analysis. One-way ANOVA was then used to test for overall significance
within antibody isotypes, and following significant ANOVA, post hoc analysis
using protected Student’s T-test was performed to compare treatment groups
(JMP, SAS, Cary, NC). Results were considered significant at p < 0.05.
In Vitro OPA Application Induced Dermal Irritation and
Results of the EpiDerm skin irritation test, comparing the
irritation potential of OPA with that of glutaraldehyde, are
presented in Figure 1. No significant change in viability was
observed at the lower concentrations (0.1375 and 0.275%) for
either chemical when compared to the untreated control.
Treatment with 0.4125% OPA resulted approximately in a 60%
(p < 0.01) decrease in cell viability while 0.55 and 1.1% OPA
resulted in 90–95% decreases (p < 0.01) in cell viability.
Treatment with glutaraldehyde only resulted in 30% (p < 0.01)
decrease in cell viability at the highest (1.1%) concentration
when compared to the vehicle control. OPA was significantly
more cytotoxic than glutaraldehyde (p < 0.01) at all con-
centrations tested. The positive control, 8 N KOH, decreased
cell viability by 98% (data not shown).
In Vivo Treatment with OPA Did Not Induce Local or
There were no OPA-related animal deaths for these studies.
All mice appeared clinically normal with no overt clinical
toxicity (visual monitoring for appearance, ruffled fur, and
discharge from eye, nose, and anus) throughout the course of
these studies, and no significant loss of body weight was
observed (data not shown).
In Vivo Studies Identify OPA to be an Irritant and Allergic
To confirm the results from the in vitro irritancy studies, ear
swelling was evaluated in mice after dermal exposure to OPA.
A dose-dependent (Linear Trend test; p < 0.01) increase in ear
swelling was observed after OPA treatment reaching statistical
24 h after final chemical exposure (Fig. 2). A 0.3% DNFB
an average significant increase of 109% ear swelling after
group and untreated control. ‘‘#’’ Indicates significant difference (p < 0.01)
between OPA and glutaraldehyde at indicated concentration.
irritation after topical application of OPA. Bars represent mean ± SE of five
mice (10 ears) per group. Levels of statistical significance are denoted as ‘‘*’’
(p < 0.01) as compared to DMF vehicle.
Ear swelling as a result of topical application of OPA. Analysis of
ANDERSON ET AL.
application (data not shown). In the LLNA, dose-dependent
(Linear Trend test; p < 0.01) increase in DLN proliferation was
0.75% exposed animals significantly elevated over the vehicle
control animals (Fig. 3). SI values of 2.2, 2.9, 7.5, and 47.6 were
identified for the 0.005, 0.05, 0.1, and 0.75% exposure groups,
respectively. An EC3 value of 0.051% (Fig. 3) was calculated.
HCA (30%) was used as a positive control for these experiments
and resulted in an average SI value of 8.8.
Exposure to OPA Induced an Increase in Local and Systemic
The mechanisms of OPA sensitization were further inves-
tigated using phenotypic analysis of B220þ and IgEþB220þ
expressing cells in the DLNs. Phenotypic analysis of the DLNs
of mice exposed to OPA showed dose-dependent (Linear
Trend test; p < 0.01) increases in both the B220þ and
IgEþB220þ cell populations. Consistent with the LLNA re-
sults, a statistically significant increase in percentage of B220þ
cells was observed for all concentrations tested. Statistically
significant increases in IgEþB220þ expressing cells were
identified in the DLN of mice treated with 0.1% (percent
counts) and 0.75% (percent and absolute counts) OPA (Table 1).
Serum IgE is commonly used as an indicator of type I hyper-
sensitivity to dermal sensitizers. Supporting the phenotyping
results, exposure to OPA produced dose-dependent (Linear
Trend test; p < 0.01) elevations in total serum IgE levels (Fig. 4)
reaching statistical significance at the 0.5 and 0.75% treatment
groups. When tested in the same study, dermal treatment with
0.75% glutaraldehyde produced 206 ± 26 ng/ml of serum IgE
compared to 1081 ± 87 ng/ml for 0.75% OPA (data not shown).
Exposure to OPA-Induced Increases in OPA-Specific
Consistent with the results for total IgE, a significant
elevation in OPA-specific IgE was also observed after treat-
ment with 0.75% OPA (Fig. 5A). Three out of the five mice
exposed to 0.75% OPA had detectable levels of OPA-MSA–
specific IgE antibodies in their serum. The levels were low but
significantly elevated relative to all other treatment groups.
Heat treatment of the serum at 56?C for 4 h abolished the signal
from the ELISA, supporting that heat labile IgE was present.
Mice treated with 0.75% OPA also showed increases in IgG
isotypes specific for OPA-MSA. The incidence as well as level
of IgG1(five out of five mice, Fig. 5B) antibodies were greater
than those for IgG2a(three out of five mice, Fig. 5C). Some of
the mice treated with 0.75% OPA also showed increased serum
levels of anti-MSA IgG1(three out of five mice, Fig. 5B) and
IgE (one out of five mice, Fig. 5A), although the serum levels
were lower than for OPA-MSA–specific antibodies.
Exposure to OPA Increased Expression of IL-4 mRNA and
Protein Expression in the DLN
Cytokine mRNA in the DLNs was analyzed to further
evaluate the effect of OPA exposure on Th1/Th2 balance.
Cytokine mRNA levels analyzed included IL-4, -10, -12, and
Analysis of the allergic sensitization potential of OPA using the LLNA.
3H-thymidine incorporation into DLN cells of BALB/c mice following
exposure to vehicle or concentration of OPA. Numbers in boxes appearing
above the bars represent the SI for each concentration tested. Bars represent
mean ± SE of five mice per group. Levels of statistical significance are denoted
as ‘‘*’’ (p < 0.05) and ‘‘**’’ (p < 0.01) as compared to DMF vehicle.
Allergic sensitization potential after dermal exposure to OPA.
LLNA, Phenotypic, and IgE Analysis after In Vivo OPA Treatment
Dose group LLNA (DPM)
IgEþB220þ (% lymphocyte population)B220þ (% lymphocyte population)
% Cells 3 106
%Cells 3 106
515 ± 88 1.24 ± 0.43 0.16 ± 0.0918.39 ± 0.72 1.6 ± 0.38
476 ± 360
3845 ± 582
24,020 ± 4616**
1.62 ± 0.48
3.51 ± 1.61**
23.42 ± 3.23**
0.27 ± 0.14
0.52 ± 0.14
1.4 ± 0.24**
25.55 ± 1.4*
26.33 ± 2.5**
31.80 ± 1.2**
0.42 ± 0.04
3.8 ± 1.4
15.90 ± 1.3**
Note. Levels of statistical significance are denoted as * (p ? 0.05) and ** (p ? 0.01) as compared to vehicle (DMF). Values present group mean (n ¼ 5) ± SE.
IRRITANCY AND SENSITIZATION POTENTIAL OF OPA
IFN-c. An increase in IL-4 mRNA expression was observed
following OPA treatment reaching statistical significance
(p < 0.01) at 0.75% (Fig. 6A). Levels of IFN-c, IL-10, or
IL-12 mRNA were not modulated following treatment with any
of the OPA concentrations tested (data not shown). Consistent
with the mRNA results, a dose-responsive (Linear Trend test;
p < 0.01) increase in IL-4 protein production by DLN was
observed after dermal treatment with OPA reaching statistical
significance at concentrations of 0.25% and higher (Fig. 7).
When tested in the same study, dermal treatment with 0.75%
glutaraldehyde only generated 217 ± 46 pg/ml of IL-4 in the
DLN compared to 873 ± 129 pg/ml for the 0.75% OPA
treatment group (data not shown).
Work-related asthma has become the most frequently
diagnosed occupational respiratory illness, accounting for
10–25% of adult asthma with occupations in healthcare having
the highest risk (Kogevinas et al., 2007). Of the identified 250
substances suspected to cause occupational asthma, ~90 are
low molecular weight (LMW) organic chemicals (Jarvis et al.,
2005). The mechanisms by which LMW chemicals cause
asthma due to sensitization are believed to be different from
that of high molecular weight substances and remain poorly
defined (Wild and Lopez, 2003). Glutaraldehyde (100.13 MW)
and OPA (134.132 MW) are dialdehydes capable of cross-
linking proteins, thus functioning as effective biocides and
tissue fixatives. Covalent bonding to primary amines and other
protein moieties can result in the formation of hapten-carrier
total IgE levels in mouse sera after dermal exposure to OPA. Bars represent
mean fold change ± SE of five mice per group. Levels of statistical significance
are denoted as ** (p < 0.01) as compared to DMF vehicle. Dermal treatment
with 0.75% glutaraldehyde produced 206 ± 26 ng/ml of IgE (p < 0.05).
Serum total IgE levels after dermal exposure to OPA. Analysis of
conjugates determined using indirect ELISA. Samples were considered to be positive if the OD was 10 times that of the DMF control. For mice treated with 0.75%
OPA, five out of five, three out of five, and three out of five mice were positive for OPA-MSA IgG1, IgG2a, and IgE, respectively. Bars represent the mean fold
change ± SE of five mice per group. The heat treated-0.75 group represents serum from 0.75% OPA group that was heat treated to destroy IgE while leaving IgG
intact. Significant difference at p < 0.05 relative to *DMF control group and ‘‘#’’ respective MSA antibody titer.
Serum levels of antibodies specific for OPA protein adducts. Analysis of serum antibodies (A, IgE; B, IgG1; and C, IgG2a) specific for OPA-MSA
ANDERSON ET AL.
complexes with host proteins and may induce immunological
responses. Defining the mechanism by which these chemicals
induce sensitization is a critical step toward early diagnosis and
prevention of work-related asthma.
While research on OPA is limited, there is extensive litera-
ture available on the adverse health effects associated with
glutaraldehyde exposure (Gannon et al., 1995; Rideout et al.,
2005; Waters et al., 2003). Our laboratory has previously
investigated the allergic sensitization caused by exposure to
glutaraldehyde. It was identified as a moderate contact sensi-
tizer in LLNA with an EC3 value of 0.089% (Azadi et al.,
2004). Although precise comparisons cannot be made because
the experiments were not conducted simultaneously, the OPA
studies attempted to parallel those previously described for
glutaraldehyde; vehicle, mouse strain, and reagents were kept
consistent between the studies. Table 2 summarizes the results
from the two studies. The EC3 value for glutaraldehyde was
extrapolated because the lowest concentration tested yielded an
SI value greater than 3. For comparison purposes, two EC3
values are presented for OPA, one calculated based on the
equation described by Basketter et al. (1999) and the other
calculated based on the equation used to determine the EC3
value for glutaraldehyde as cited by Azadi et al. (2004). The
EC3 values calculated for OPA, using both equations (0.079
and 0.051%), are similar to that described for glutaraldehyde
(0.089%). In addition, the SI values for the 0.1 and 0.75%
exposure groups were 3.5 and 12.7 for glutaraldehyde and 7.5
and 47.6 for OPA, respectively.
however, in vitro studies showed that lower concentrations of
OPA (0.4125%) had increased toxicity compared to glutaralde-
hyde (1.1%) when both chemicals were tested simultaneously.
Skin irritation and the associated inflammatory response may be
immune response and the process of sensitization. In this light,
datafrombothinvitroand invivostudies suggest thatOPA isan
irritant as evidenced by the direct cytotoxicity to primary skin
Quantitative real-time PCR analysis of IL-4 (A) or IFN-c expression (B). Bars
represent mean fold change ± SE of five mice per group. Levels of statistical
significance are denoted as ‘‘*’’ (p < 0.01) as compared to DMF vehicle.
DLN gene expression measured by quantitative real-time PCR.
generated by stimulated DLN after dermal exposure to OPA. Bars represent
mean fold change ± SE of five mice per group. Levels of statistical significance
are denoted as ‘‘*’’ (p < 0.05) and ‘‘**’’ (p < 0.01) as compared to DMF
vehicle. Dermal treatment with 0.75% glutaraldehyde generated 217 ± 46 pg/ml
of IL-4 in the DLN (p < 0.01).
DLN IL-4 protein expression. Analysis of IL-4 protein expression
Comparison of Glutaraldehyde and OPA Sensitization
298 ± 37 21.5 ± 1.6
359 ± 11 39.7 ± 1.3
2.2 ± 0.9
7.2 ± 0.9 3.5
12.7 513 ± 98 47.2 ± 1.1 19.1 ± 2.9
117 ± 13 18.4 ± 0.7
154 ± 20 26.3 ± 2.5
1081 ± 87 31.8 ± 1.2
1.2 ± 0.4
3.5 ± 1.6
23.4 ± 3.2
aData represent results published by Azadi et al. (2004).
bCalculated using equation cited by Azadi et al. (2004).
cCalculated using equation described by Basketter et al. (1999).
IRRITANCY AND SENSITIZATION POTENTIAL OF OPA
cultures and the marked increase in ear swelling observed
following topical exposure in mice. Concomitant with irritation
was significant elevations in total and OPA-specific IgE serum
antibodies and IgEþB220þ cell population in the DLNs of
OPA-exposed mice. Manetz and Meade (1999) have shown that
select chemicals capable of inducing IgE-mediated allergic
IgEþB220þ and B220þ populations and tend to become
significantly elevated at equivalent concentrations. A similar
trend was observed after treatment with 0.75% OPA
(IgEþB220þ population increased to 23.42 ± 3.23% and
B220þ population increased to 31.80 ± 1.2% of total
significant increases in the IgEþB220þ cell population (0.1%)
was lower than the concentration significantly elevating total
DLNs, which may occur before IgE elevations in the serum.
further support the involvement of IgE in the allergic response.
Similar elevations in IL-4 and IgEþB220þ cells in DLN were
higher concentrations were required relative to OPA (Azadi
et al., 2004). IL-4 is crucial for IgE expression because it is
required for increased expression of CD23 on the B cells, B-cell
proliferation, isotype switching, and IgE synthesis, and its
expression often supports polarization to a Th2 hypersensitivity
response. Further supporting the Th2 polarization, a significant
elevation in OPA-specific IgG1, a Th2-driven isotype, was
observed for the 0.75% treatment group compared to vehicle
control (Snapper et al., 1988a). With the exception of a single
mouse, this elevation was approximately five- to eightfold
greater than that observed for OPA-specific IgG2aantibody
levels. Elevations in IgG2atypically represent an inflammatory
response and suggest polarization to a Th1 response with class
switching most often caused by elevations in IFN-c and tumor
necrosis factor-a (Snapper et al., 1988b). However, no increase
in IFN-c mRNA was observed when the DLNs of these mice
were analyzed, further supporting a Th2 response. These results
suggest that OPA acts as a Th2 sensitizer and may have
implications for respiratory allergy. It is also possible that OPA
may exacerbate existing allergy by establishing a Th2-support-
ing immunological milieu. Hasegawa et al. (2009) showed
significant increases in OVA-specific serum IgE, IL-4 mRNA,
to OVA only. Consistent with our results, IL-4 mRNA was also
current LLNA) along with significant elevations in IgE and IL-4
expression at concentrations below or similar to that designated
as the working solution (0.55%) strongly suggests that OPA is
a sensitizing chemical and that this chemical would be expected
to cause significant activation of the immune system following
Thesearethefirst studies todescribe immunotoxicityinduced
developed and validated for the identification of contact
sensitizers, and while LMW chemical respiratory allergens,
such as toluene diisocyanate and trimellitic anhydride (TMA),
induce positive responses in the LLNA, not all LLNA-positive
chemicals are associated with respiratory allergy or asthma.
Although it is often thought that the most common route of
exposure to respiratory allergens is inhalation, published animal
and human data have shown that dermal exposure may result in
respiratory tract sensitization (Fukuyama et al., 2009; Herrick
et al., 2002; Petsonk et al., 2000). Studies have shown that
topical application is effective in sensitizing rats to TMA,
resulting in airway reactivity after inhalational challenge (Zhang
et al., 2004). In addition, other literature has also shown that
specific IgE and airway hyperreactivity upon respiratory
challenge (Howell et al., 2002). Therefore, skin exposure needs
to be addressed in the risk assessment for OPA.
The identification of OPA as an irritant and sensitizing
IgE, OPA-specific IgG1, and published case reports raises
concern that OPA may function as an IgE-mediated sensitizer.
Comparison of these data to that obtained for glutaraldehyde
demonstrates that the sensitizing potential for OPA is compara-
ble to that of glutaraldehyde, suggesting that it may not be a safe
alternative. Similar to glutaraldehyde, in an effort to reduce and
prevent occupational exposure and disease, regulations for the
use of this chemical may need to be established.
Interagency Agreement National Institute of Environmental
Health Sciences (Y1-ES0001-06).
The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the
National Institute for Occupational Safety and Health, Centers
for Disease Control and Prevention.
Azadi, S., Klink, K. J., and Meade, B. J. (2004). Divergent immunological
responses following glutaraldehyde exposure. Toxicol. Appl. Pharmacol.
Basketter, D. A., Lea, L. J., Dickens, A., Briggs, D., Pate, I., Dearman, R. J.,
and Kimber, I. (1999). A comparison of statistical approaches to the
derivation of EC3 values from local lymph node assay dose responses.
J. Appl. Toxicol. 19, 261–266.
ANDERSON ET AL.
Butler, J. E. (2000). Enzyme-linked immunosorbent assay. J. Immunoassay 21, Download full-text
ChemicalLand 21 (Undated). o-Phthaladehyde. Available at: http://www
Accessed August 19, 2009.
Franchi, A., and Franco, G. (2005). Evidence-based decision making in an
endoscopy nurse with respiratory symptoms exposed to the new ortho-
phthalaldehyde (OPA) disinfectant. Occup. Med. (Lond) 55, 575–578.
Fujita, H., Ogawa, M., and Endo, Y. (2006). A case of occupational bronchial
asthma and contact dermatitis caused by ortho-phthalaldehyde exposure in
a medical worker. J. Occup. Health 48, 413–416.
Fukuyama, T., Tajima, Y., Ueda, H., Hayashi, K., Shutoh, Y., Harada, T., and
Kosaka, T. (2009). Allergic reaction induced by dermal and/or respiratory
exposure to low-dose phenoxyacetic acid, organophosphorus, and carbamate
pesticides. Toxicology 261, 152–161.
Gannon, P. F., Bright, P., Campbell, M., O’Hickey, S. P., and Burge, P. S.
(1995). Occupational asthma due to glutaraldehyde and formaldehyde in
endoscopy and x ray departments. Thorax 50, 156–159.
Hasegawa, G., Morinaga, T., and Ishihara, Y. (2009). ortho-Phthalaldehyde
enhances allergen-specific IgE production without allergen-specific IgG in
ovalbumin-sensitized mice. Toxicol. Lett. 185, 45–50.
Herrick, C. A., Xu, L., Wisnewski, A. V., Das, J., Redlich, C. A., and
Bottomly, K. (2002). A novel mouse model of diisocyanate-induced asthma
showing allergic-type inflammation in the lung after inhaled antigen
challenge. J. Allergy Clin. Immunol. 109, 873–878.
Howell, M. D., Weissman, D. N., and Meade, B. J. (2002). Latex sensitization
by dermal exposure can lead to airway hyperreactivity. Int. Arch. Allergy
Immunol. 28, 204–211.
Jarvis, J., Seed, M. J., Elton, R., Sawyer, L., Agius, R., and Ishihara, Y. (2005).
Relationship between chemical structure and the occupational asthma hazard
of low molecular weight organic compounds. Occup. Environ. Med. 62,
Klink, K. J., and Meade, B. J. (2003). Dermal exposure to 3-amino-5-mercapto-
1,2,4-triazole (AMT) induces sensitization and airway hyperreactivity in
BALB/c mice. Toxicol. Sci. 75, 89–98.
Kogevinas, M., Zock, J. P., et al. (2007). Exposure to substances in the
workplace and new-onset asthma: an international prospective population-
based study (ECRHS-II). Lancet 370, 336–341.
Lerones, C., Mariscal, A., Carnero, M., Garcia-Rodriguez, A., and Fernandez-
Crehuet, J. (2004). Assessing the residual antibacterial activity of clinical
materials disinfected with glutaraldehyde, o-phthalaldehyde, hydrogen
peroxide or 2-bromo-2-nitro-1,3-propanediol by means of a bacterial toxicity
assay. Clin. Microbiol. Infect. 10, 984–989.
Manetz, T. S., and Meade, B. J. (1999). Development of a flow cytometry assay
for the identification and differentiation of chemicals with the potential to
elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses.
Toxicol. Sci. 48, 206–217.
National Institute of Environmental Health Sciences (NIEHS). (1999). The
murine local lymph node assay: a test method for assessing the allergic
contact dermatitis potential of chemicals/compounds. Fed. Regist. 64,
National Institute of Environmental Health Sciences (NIEHS). (2002).
ICCVAM Evaluation of EPISKIN?, EpiDerm? (EPI-200), and the Rat
Skin Transcutaneous Electrical Resistance (TER) Assay: In Vitro Test
Methods for Assessing Dermal Corrosivity Potential of Chemicals. NIH
Publication No: 02-4502.
National Institute for Occupational Safety and Health (NIOSH). (1990).
National Occupational Exposure Survey (NOES) 1981-1983. Estimated
Number of Employees Potentially Exposed to Specific Agents by Occupation
with 2-Digit Standard industrial Classification (SIC).
Occupational Health and Safety Administration (OSHA). (2006). Best
Practices for the Safe Use of Glutaraldehyde in Health Care. U.S.
Department of Labor, OSHA 3258-08N.
Petsonk, E. L., Wang, M. L., Lewis, D. M., Siegel, P. D., and Husberg, B. J.
(2000). Asthma-like symptoms in wood product plant workers exposed to
methylene diphenyl diisocyanate. Chest 118, 1183–1193.
Rideout, K., Teschke, K., Dimich-Ward, H., and Kennedy, S. M. (2005).
Considering risks to healthcare workers from glutaraldehyde alternatives in
high-level disinfection. J. Hosp. Infect. 59, 4–11.
Snapper, C. M., Finkelman, F. D., and Paul, W. E. (1988a). Regulation of IgG1
and IgE production by interleukin 4. Immunol. Rev. 102, 51–75.
Snapper, C. M., Peschel, C., and Paul, W. E. (1988b). IFN-gamma stimulates
IgG2asecretion by murine B cells stimulated with bacterial lipopolysaccha-
ride. J. Immunol. 140, 2121–2127.
Sokol, W. N. (2004). Nine episodes of anaphylaxis following cystoscopy
caused by Cidex OPA (ortho-phthalaldehyde) high-level disinfectant in 4
patients after cytoscopy. J. Allergy Clin. Immunol. 114, 392–397.
Suzukawa, M., Komiya, A., Koketsu, R., Kawakami, A., Kimura, M., Nito, T.,
Yamamoto, K., and Yamaguchi, M. (2007). Three cases of ortho-
phthalaldehyde-induced anaphylaxis after laryngoscopy: detection of specific
IgE in serum. Allergol. Int. 56, 313–316.
Suzukawa, M., Yamaguchi, M., Komiya, A., Kimura, M., Nito, T., and
Yomamoto, K. (2006). Ortho-phthalaldehyde-induced anaphylaxis after
laryngoscopy. J. Allergy Clin. Immunol. 117, 1500–1501.
Tucker, S. P. (2008). Determination of ortho-phthalaldehyde in air and on
surfaces. J. Environ. Monit. 10, 1337–1349.
U.S. Environmental Protection Agency (USEPA). (2001). Method 531.2.
Measurement of n-Methylcarbamyoloximes and n-Methylcarbamates in
Water by Direct Aqueous Injection HPLC with Postcolumn Derivatization.
EPA #815-B-01–002. Available at: http://www.epa.gov//safewater/methods
/pdfs/methods/met531_2.pdf. Accessed August 19, 2009.
U.S. Environmental Protection Agency (USEPA). (2006). Inventory Update
Rule. Office of Pollution, Prevention and Toxics, USEPA. [Search by cas no.
for 643798]. Available at: http://www.epa.gov/oppt/iur/tools/data/2002-vol
.htm. Accessed August 19, 2009.
U.S. Food and Drug Administration (USFDA). (2006). Device Evaluation
Information: FDA Cleared Sterilants and High Level Disinfectants with
General Claims for Processing Reusable Medical and Dental Devices:
September 28, 2006. FDA, Center for Devices and Radiological Health
(CDRH). Available at: http://www.fda.gov/cdrh/ode/germlab.html. Accessed
August 19, 2009. Last updated on June 16, 2009.
Waters, A., Beach, J., and Abramson, M. (2003). Symptoms and lung function
in health care personnel exposed to glutaraldehyde. Am. J. Ind. Med. 43,
Wild, L. G., and Lopez, M. (2003). Occupational asthma caused by high-
molecular-weight substances. Immunol. Allergy Clin. North Am. 23,
Woolhiser, M. R., Munson, A. E., and Meade, B. J. (1999). Role of
sensitization routes in the development of type I hypersensitivity to natural
rubber latex in mice. Am. J. Ind. Med. (Suppl. 1), 139–141.
Woolhiser, M. R., Munson, A. E., and Meade, B. J. (2000). Comparison of
mouse strains using the local lymph node assay. Toxicology 146, 221–227.
Zhang, X. D., Fedan, J. S., Lewis, D. M., and Siegel, P. D. (2004). Asthmalike
biphasic airway responses in Brown Norway rats sensitized by dermal
exposure to dry trimellitic anhydride powder. J. Allergy Clin. Immunol. 113,
IRRITANCY AND SENSITIZATION POTENTIAL OF OPA