Preventing false-negatives in the in vitro skin sensitization testing of acid anhydrides using interleukin-8 release assays

Article abstract of DOI:10.1016/j.tiv.2017.04.008

In vitro safety tests may be used as replacements for animal tests owing to their accuracy and high-throughput performance. However, several in vitro skin sensitization tests produce false-negative results such as acid anhydride. Here, we investigated the relationship between false-negative results of acid anhydride and its hydrolysis by aqueous vehicle. Differences in the pattern of hydrolysis for phthalic anhydride (PAH) due to addition of 1 drop of stock solution of PAH in liquid paraffin (LP) dispersion medium and PAH in DMSO were analyzed in a cell-free system. The results showed that use of LP dispersion medium stabilized the concentration of PAH in water over 5 min by sustained-release, although almost all PAH converted to phthalic acid in water within 5 min using DMSO. Additionally, treatment of THP-1 cells with PAH and phthalic acid using LP dispersion medium for 5 min resulted in a 32-fold increase in IL-8 release for PAH as compared with that in the vehicle control. In contrast, for PAH using aqueous vehicle and phthalic acid using LP dispersion medium, there were no significant increases in IL-8 release. Similarly, using LP dispersion medium, trimellitic anhydride significantly increased IL-8 release was observed.

Full text of DOI:10.1016/j.tiv.2017.04.008

Toxicology in Vitro 42 (2017) 69–75
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
Preventing false-negatives in the in vitro skin sensitization testing of acid
anhydrides using interleukin-8 release assays
Kazuto Naritaa,b,⁎, Phuc Thi Hong Voa, Kenta Yamamotoa, Hajime Kojimab, Hiroshi Itagakia
a
Department of Chemical and Energy Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
Division of Risk Assessment, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
In vitro
Alternative
Skin sensitization test
False-negative
Drug exposure
THP-1 cells
In vitro safety tests may be used as replacements for animal tests owing to their accuracy and high-throughput
performance. However, several in vitro skin sensitization tests produce false-negative results such as acid
anhydride. Here, we investigated the relationship between false-negative results of acid anhydride and its
hydrolysis by aqueous vehicle. Differences in the pattern of hydrolysis for phthalic anhydride (PAH) due to
addition of 1 drop of stock solution of PAH in liquid paraffin (LP) dispersion medium and PAH in DMSO were
analyzed in a cell-free system. The results showed that use of LP dispersion medium stabilized the concentration
of PAH in water over 5 min by sustained-release, although almost all PAH converted to phthalic acid in water
within 5 min using DMSO. Additionally, treatment of THP-1 cells with PAH and phthalic acid using LP dispersion
medium for 5 min resulted in a 32-fold increase in IL-8 release for PAH as compared with that in the vehicle
control. In contrast, for PAH using aqueous vehicle and phthalic acid using LP dispersion medium, there were no
significant increases in IL-8 release. Similarly, using LP dispersion medium, trimellitic anhydride significantly
increased IL-8 release was observed.
1. Introduction
particular, augmentation of IL-8 mRNA expression or protein synthesis/
release in THP-1 cells has been suggested as a promising biomarker for
Recently, animal tests have been affected by increasing ethical,
social, and political concerns regarding animal welfare. Indeed, several
countries have banned the testing the safety of cosmetic products and
their ingredients on animals (Adler et al., 2011). Accordingly, it is
necessary to develop alternatives to animal experiments, particularly
for in vitro safety testing. Several in vitro safety tests have been
proposed. Currently, most researchers are focusing on the development
of tests based on not only cytotoxicity but also complex toxic mechan-
isms, such as skin sensitization tests.
Skin sensitization is a phenomenon in which rash occurs on the skin,
i.e., allergic contact dermatitis (ACD), owing to T-cell-mediated com-
plicated immune responses. In the induction phase of skin sensitization,
antigen-presenting cells, such as dendritic cells (DCs), and Langerhans
cells are activated. Sensitizers cause morphological and functional
changes in DCs, altering the expression of CD40, CD80, CD83, CD86,
and HLA-DR (Aiba et al., 1997; Aiba, 1998; Arrighi et al., 2001; De
Smedt et al., 2001) and the production of tumor necrosis factor-α (TNF-
α), interleukin-1β (IL-1β), IL-8/CXCL8, and IL-12p40 (Coutant et al.,
1999; De Smedt et al., 2001; Aiba et al., 2003; Toebak et al., 2006).
Accordingly, various skin sensitization tests have been developed; in
skin sensitization (Miyazawa et al., 2008; Nukada et al., 2008; Mitjans
et al., 2008, 2010; Takahashi et al., 2011; Kimura et al., 2015; Parise
et al., 2015). IL-8 levels are significantly increased by poison ivy/oak in
human patch-tests (Griffiths et al., 1991). Importantly, IL-8 is well
established as a potent chemotactic peptide for neutrophils and T
lymphocytes (Leonard et al., 1990). Thus, analysis of IL-8 in THP-1
cells, based on ACD mechanisms, may provide a promising alternative
skin sensitization test.
However, similar to other in vitro skin sensitization tests, these tests
still produced false-negative results (e.g., for water-insoluble chemicals,
pro/pre-hapten, volatile chemicals, and chemicals which are categor-
ized as weak sensitizers) compared with local lymph node assay (LLNA)
and human clinical data. For example, Nukada et al. (2008) reported
that three skin sensitizers (i.e., phthalic anhydride [PAH], ethylene
diamine, and geraniol) out of 16 did not show the capacity to induce IL-
8 production.
PAH is classified as a strong skin sensitizer (EC3 value = 0.16% w/
v) by LLNA (Dearman et al., 2000). Although the false-negative results
observed for PAH are thought to be related to its water-insolubility,
hydrolysis, and respiratory sensitization, no studies have described the
⁎
Corresponding author at: Department of Chemical and Energy Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
E-mail address: narita-kazuto-vy@ynu.jp (K. Narita).
http://dx.doi.org/10.1016/j.tiv.2017.04.008
Received 26 October 2016; Received in revised form 15 March 2017; Accepted 10 April 2017
Available online 11 April 2017
0887-2333/ © 2017 Elsevier Ltd. All rights reserved.

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
cause of false-negative results for PAH using IL-8 analysis by THP-1
cells. More importantly, several studies have shown that almost all in
vitro skin sensitization tests do not evaluate PAH correctly (e.g., the IL-
8 Luc assay, h-CLAT, the Myeloid U937 Skin Sensitization Test, the test
based on reactive oxygen species production in THP-1 cells, and the
ARE-Nrf2 luciferase test) (Kimura et al., 2015; Ashikaga et al., 2010;
Piroird et al., 2015; Saito et al., 2013; Emter et al., 2013). Therefore, we
hypothesized that false-negative results for PAH may not be associated
with cell lines, tissues, skin sensitization-related endpoints, or criteria
for evaluation, but may instead by attributed to a common factor of in
vitro tests, i.e., hydrolysis by aqueous vehicle. Indeed, further support-
ing this hypothesis, trimellitic anhydride (TAH), which has also been
classified as a strong skin sensitizer (EC3 value = 0.60% w/v) by LLNA
(Dearman et al., 2000), was shown to have false-negative results by IL-8
analysis in THP-1 cells (Mitjans et al., 2008) and the IL-8 Luc assay
(Kimura et al., 2015).
In this study, we aimed to determine whether hydrolysis of acid
anhydride was the underlying cause of the false-negative results from in
vitro skin sensitization tests measuring IL-8 release in a cell-free system
and in THP-1 cells. Furthermore, we investigated whether the prevent-
ing hydrolysis of acid anhydride could be prevented using liquid
paraffin (LP) could be used to accurately evaluate these chemicals. LP
has been used in cosmetics and for medical purposes for many years
(Freund and McDermott, 1942), and LP exhibits chemical stability and
water nonmiscibility. Therefore, we selected LP as a dispersion medium
in this study. Our findings provide important insights into the applica-
tions of these alternative methods for toxicity evaluation.
Abstract Service (CAS) numbers, octanol-water partition coefficient
(LogKow) values calculated with KOWWIN in EPI Suite (Environmental
Protection Agency, Washington, DC, USA), skin sensitization potencies
based on EC3 LLNA values, clinical case reports, patch tests, and human
maximization test results. PAH, maleic anhydride (MAH), TAH, nickel
sulfate hexahydrate (NiSO4), and sodium lauryl sulfate (SLS) were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Phthalic acid,
DMSO, and LP were purchased from Wako Pure Chemical Industries
Ltd. and were of the highest available purity.
2.3. UV spectrophotometric analysis
The absorption measurements of PAH and phthalic acid were made in
a SpectraMax M5e (Molecular Devices, LLC, Sunnyvale, CA, USA) at
room temperature. The absorption spectra of PAH and phthalic acid were
determined immediately and at 1 week after dissolving in distilled water
(Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan) or dimethyl sulfoxide
(DMSO; Japanese Industrial Standards Special Grade; Wako Pure
Chemical Industries Ltd.). A quartz cell (optical path length = 1 cm)
was then filled with 2 mL of these solutions, and the absorbance was
measured at every 4 nm from 250 to 360 nm. The hydrolysis of PAH was
determined by adding a stock solution of PAH in 10 μL DMSO and 200 μL
liquid paraffin (LP; Wako Pure Chemical Industries Ltd.) to a 1-cm quartz
cell containing 2 mL distilled water at room temperature. Optical density
readings at 300 nm were taken every 1 min after mixing well.
2.4. Preparation of chemicals and chemicals exposure of THP-1 cells
2. Materials and methods
Twenty-four-hour aqueous vehicle exposure tests were carried out
as follows. Test chemicals were first solubilized in saline or DMSO and
diluted. These solutions were diluted into culture medium. Fifty
microliters of this diluted solution aqueous solution was then further
mixed with 50 μL of 2 × 106 THP-1 cells/mL prepared in 96-well flat-
bottomed plates. The exposure plates were then incubated for 24 h at
37 °C in an atmosphere containing 5% CO2. The final concentrations of
saline and DMSO were less than 0.2% and 0.8% (v/v), respectively.
For the 5-min LP dispersion medium exposure assays, test chemicals
were first dispersed in LP and diluted. These suspensions (100 μL) were
then mixed with 100 μL THP-1 cell suspension (1 × 107 cells/mL)
prepared in 1.5 mL tubes for 5 min at room temperature. After 5 min
of exposure, cells were washed twice with phosphate-buffered saline
and resuspended in 1 mL culture medium. Next, 100 μL cell suspension
was transferred to individuals wells of a 96-well plate, and plates were
then incubated for 24 h at 37 °C in an atmosphere containing 5% CO2.
2.1. Cells
THP-1 cell, human monocytic leukemia cells, were purchased from
American Type Culture Collection (ATCC; Manassas, VA, USA) and
cultured in RPMI1640 (Wako Pure Chemical Industries Ltd., Osaka,
Japan) supplemented with 10% heated-inactivated fetal bovine serum
(Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 units/
mL penicillin (Wako Pure Chemical Industries Ltd.), 100 μg/mL strep-
tomycin (Wako Pure Chemical Industries Ltd.), and 0.05 mM 2-mer-
captoethanol (Wako Pure Chemical Industries Ltd.) at 37 °C with 5%
CO2. THP-1 cells were routinely passaged and maintained at densities of
1–8 × 105 cells/mL. Cells were used before passage 30 for all experi-
ments.
2.2. Chemicals
The test chemicals are listed in Table 1, along with their Chemical
Table 1
List of chemicals tested.
⁎
Test chemicals
Abbreviation
PAH
CAS No.
85-44-9
Estimated LogKow
EC3 (%) and potency category⁎⁎
Human data⁎⁎⁎⁎
Positived
Phthalic anhydride
Maleic anhydride
2.07
1.62
1.95
2.04
0.16
Stronga
0.16
MAH
108-31-6
552-30-7
10101-97-0
Positivee
Stronga
0.60
Trimellitic anhydride
Nickel sulfate hexahydrate
TAH
Positived
Stronga
4.80
NiSO4
Positivef
Moderateb
nf⁎⁎⁎
Phthalic acid
Sodium lauryl sulfate
88-99-3
151-21-3
1.07
1.69
nf⁎⁎⁎
SLS
1.5–17.0
Negativef
Moderate–weakc
⁎LogKow value calculated with KOWWIN in EPI Suite.
⁎⁎The potency of skin sensitization was estimated using LLNA (aDearman et al., 2000; bRyan et al., 2002; cLoveless et al., 1996).
⁎⁎⁎nf, not found.
⁎⁎⁎⁎The clinical case report was from dMoffitt and Sansom (2002), patch test data were from eMotolese et al. (1993), and human maximization test data were from fKligman, 1966.
70

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
2.5. Analysis of cell viability
decreased each minute, rapidly reaching a minimum value. The
absorbance of distilled water solution containing PAH solute was
comparable to that of phthalic acid solution (final concentration of
phthalic acid in solution: 0.5 mM) after 5 min (Fig. 2a). Moreover,
changes in absorbance were not observed from 5 min onward (data not
shown). To maintain the concentration of PAH in distilled water over a
long period, we dropped an LP suspension of PAH into distilled water.
Because LP and distilled water were not compatible, absorbance
measurements were made at 300 nm using distilled water. For the LP
dispersion-liquid containing PAH, the absorbance at 300 nm increased
from 0 min (absorbance = 0.4) to 5 min, with maximum induction at
2–4 min (absorbance = 0.9; Fig. 2b). When LP dispersion-liquid con-
taining PAH was mixed, the absorbance at 300 nm increased. Dropping
only LP and LP dispersion-liquid containing phthalic acid did not cause
this increase in absorbance at 300 nm.
After incubation and exposure to test chemicals, cytotoxicity was
assessed with water-soluble tetrazolium salt (WST-8) assays using a Cell
Counting Kit-8 (Dojin Laboratory Co. Ltd., Kumamoto, Japan) in 96-
well plates according to the manufacturer's instructions. The half-
maximal inhibitory concentration (IC50) values of PAH and phthalic
acid were calculated by linear regression analysis of the data. Prior to
analysis, the background absorbance values of the test chemicals were
determined.
2.6. Analysis of IL-8 release
After incubation and exposure to test chemicals, cell-free culture
supernatants were collected and centrifuged at 700 ×g for 10 min. IL-8
production was assessed using a specific sandwich enzyme-linked
immunosorbent assay (ELISA) kit (R & D Systems, Inc., Minneapolis,
MN, USA). Procedures were conducted according to the manufacturer's
instructions. Results are expressed in pg/mL.
3.3. Effects of 5-min exposure to PAH or phthalic acid in LP dispersion
medium on IL-8 release
To examine whether PAH was hydrolyzed under cell culture
conditions, we carried out cytotoxicity assays using THP-1 cells. THP-
1 cells were exposed to PAH or phthalic acid with aqueous vehicle
(culture medium containing 0.8% v/v DMSO) for 24 h. This vehicle did
not affect cell viability relative to the DMSO-free vehicle (data not
shown). From this analysis, the IC50 value for PAH was 1315 μg/mL in
aqueous vehicle after a 24-h exposure; that for phthalic acid was
1305 μg/mL. However, in LP dispersion medium after a 5-min expo-
sure, the IC50 value for PAH was 736 μg/mL, which was only half that
of phthalic acid (1421 μg/mL).
Based on the cytotoxicity results observed using LP dispersion
medium, we investigated the effects of LP dispersion medium exposure
on IL-8 release in THP-1 cells. THP-1 cells were exposed to PAH or
phthalic acid with the same aqueous vehicle for 24 h. Alternatively, THP-
1 cells were exposed to PAH or phthalic acid suspended in LP dispersion
medium for 5 min. The release of IL-8 was measured in supernatants 24 h
after exposure and culture. Under our experimental conditions, the
concentration of aqueous vehicle-induced IL-8 released from THP-1 cells
2.7. Statistical analysis
Cell-based assays were performed at least three times, and repre-
sentative results are shown. The data presented are expressed as the
mean
standard deviation (SD). One-way analysis of variance fol-
lowed by Dunnett's post-hoc test was used to evaluate statistical
significance using EZR (Kanda, 2013), which is a graphical user
interface for R (The R Foundation for Statistical Computing, version
3.3.0). For comparisons with data from vehicle-treated cells, differences
with p values of less than 0.05 (asterisks) were considered statistically
significant.
3. Results
3.1. Comparison between the absorption spectra of PAH and phthalic acid
dissolved in vehicle
First, we confirmed the measurement wavelengths that could be
used to distinguish between PAH and phthalic acid. Fig. 1 shows the
molar extinction coefficient of PAH 1 week after dissolving in distilled
water and that of phthalic acid immediately after dissolving in distilled
water; these values were similar, ranging from 250 to 360 nm. In
contrast, the molar extinction coefficient of PAH immediately after
dissolving in DMSO was higher than that of phthalic acid immediately
after dissolving in DMSO. In particular, at 300 nm, the molar extinction
coefficient of PAH immediately after dissolving in DMSO (ε = 2586)
was 8.6 fold higher than that of phthalic acid immediately after
dissolving in DMSO (ε = 302). These results demonstrated that mea-
suring absorbance at 300 nm could distinguish between PAH and
phthalic acid, consistent with a previous study examining the time of
PAH hydrolysis using methanol (Peters, 1959). In contrast, the molar
extinction coefficient of PAH 1 week after dissolving in DMSO at
300 nm decreased by nearly 10% (ε = 273) as compared with that
immediately after dissolving in DMSO (ε = 2586). This molar extinc-
tion coefficient value was similar to that of phthalic acid immediately
after dissolving in DMSO (ε = 302).
was 9.4
6.4 pg/mL (n = 6). In contrast, that observed for LP disper-
25.0 pg/mL (n = 9).
sion medium after a 5-min exposure was 25.6
With aqueous vehicle, PAH failed to stimulate significant IL-8 release
(Fig. 3a). This observation was consistent with a previous analysis by
Nukada et al. (2008). In contrast, with LP dispersion medium, PAH
significantly induced IL-8 release (Fig. 3b; maximum IL-8: 821 pg/mL;
cell viability: 26%; exposure concentration: 1250 μg/mL). Finally, with
aqueous vehicle, phthalic acid stimulated IL-8 release (Fig. 3c; maximum
IL-8: 178 pg/mL; cell viability: 38%; exposure concentration: 1608 μg/
mL). However, with LP dispersion medium, phthalic acid did not
significantly augment IL-8 release.
3.4. Effects of 5-min exposure to other acid anhydride skin sensitizers using
LP dispersion medium on IL-8 release
To investigate whether LP dispersion medium contributes to IL-8
release in response to exposure to other acid anhydrides, we used the
skin sensitizers MAH and TAH. After exposure to MAH with aqueous
vehicle (culture medium containing 0.2% v/v DMSO) for 24 h, THP-1
cells exhibited baseline IL-8 release of 8.3
8.6 pg/mL (n = 12).
3.2. Hydrolysis of PAH by vehicle
Because TAH is a water-insoluble chemical, we selected aqueous vehicle
containing 0.8% v/v DMSO as culture medium.
To demonstrate whether PAH was hydrolyzed during the prepara-
tion of test solutions, we examined the hydrolysis of PAH following
addition of 1 drop of a stock solution of PAH or phthalic acid in DMSO
to a quartz cell containing distilled water by measuring changes in
absorbance at 300 nm. The maximum absorbance (0.9) of distilled
water solution contain PAH solute (final concentration of PAH in
solution: 0.5 mM) was observed at 300 nm. Subsequently, this value
With aqueous vehicle, MAH caused an increase in IL-8 release
(Fig. 4a; maximum IL-8: 1506 pg/mL; cell viability: 41%; exposure
concentration: 400 μg/mL). Thus, MAH increased IL-8 release by over
180-fold compared with that of the vehicle control. Significant aug-
mentation of IL-8 release was also induced within a wide cytotoxicity
range, also affecting cell viability. Similarly, using LP dispersion
medium, MAH also increased IL-8 release (Fig. 4b; maximum IL-8:
71

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
PAH
(DMSO)
PAH
(Water)
Phthalic acid
(DMSO)
PAH
(after 1 week DMSO)
Phthalic acid
(Water)
PAH
(after 1 week water)
Fig. 1. Absorption spectra of 0.5 mM phthalic anhydride (PAH; broken line) and phthalic acid (solid line) determined immediately after dissolving in DMSO or distilled water. The spectra
for 0.5 mM PAH (dotted line) dissolved in DMSO or distilled water after a lapse of 1 week are also shown.
PAH
Phthalic acid
Vehicle
Mitjans et al. (2008). In contrast, using LP dispersion medium, TAH
significantly increased IL-8 release (Fig. 4d; maximum IL-8: 432 pg/mL;
cell viability: 49%; exposure concentration: 1250 μg/mL). Under these
conditions, TAH increased IL-8 release by over 17-fold compared with
that of the vehicle control.
DMSO
a
1.0
0.8
0.6
0.4
0.2
0.0
3.5. Effects of 5-min exposure to other skin sensitizer and irritant using LP
dispersion medium on IL-8 release
0
1
2
3
4
5
Time (min)
To investigate whether LP dispersion medium contributed to IL-8
release following exposure to skin sensitizers other than acid anhy-
drides, we used NiSO4 and the skin irritant SLS. THP-1 cells were
exposed to NiSO4 or SLS with aqueous vehicle (culture medium
containing 0.2% v/v saline) for 24 h. Under these conditions, baseline
PAH
Phthalic acid
Vehicle
Liquid paraffin
b
1.0
0.8
0.6
0.4
0.2
0.0
IL-8 release of 22.2
13.9 pg/mL (n = 6) was observed.
With aqueous vehicle, NiSO4, a moderate skin sensitizer, signifi-
cantly increased IL-8 release (Fig. 5a; maximum IL-8: 57,207 pg/mL;
cell viability: 26%; exposure concentration: 142 μg/mL) by more than
100-fold compared with that of the vehicle control, and significant
augmentation of IL-8 release was induced in the range of cell viability
from 19% to 95%. Similarly, with LP dispersion medium, NiSO4 also
increased IL-8 release (Fig. 5b; maximum IL-8: 3447 pg/mL; cell
viability: 19%; exposure concentration: 5000 μg/mL). However, this
amount of IL-8 was only one-sixteenth that obtained from aqueous
vehicle exposure for 24 h, and significant augmentation of IL-8 release
was induced in the range of cell viability from 21% to 67%.
0
1
2
3
4
5
Time (min)
Fig. 2. The hydrolysis of phthalic anhydride (PAH; solid line) was measured following
addition of 1 drop of stock solution of PAH or phthalic acid (broken line; a: DMSO vehicle,
b: liquid paraffin vehicle) to a 1-cm quartz cell containing 2 mL distilled water. The
absorbance of the distilled water layer was measured at 300 nm with a spectrometer
every 1 min.
The skin irritant SLS, increases IL-8 release (Fig. 5c; maximum IL-8:
162 pg/mL; cell viability: 1.5%; exposure concentration: 71 μg/mL),
and augmentation of IL-8 release was induced in the range of cell
viability from 1.5% to 10%. However, with LP dispersion medium, SLS
did not significantly induce augmentation of IL-8 release (Fig. 5d).
1191 pg/mL; cell viability: 32%; exposure concentration: 313 μg/mL).
Notably, with aqueous vehicle, TAH failed to produce a significant
increase in IL-8 release (Fig. 4c), consistent with a previous study by
72

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
a
b
PAH in aqueous vehicle
PAH in LP
*
1000
100
50
0
1000
500
0
100
50
0
*
500
0
Concentration (µg/mL)
Concentration (µg/mL)
c
d
Phthalic acid in aqueous vehicle
Phthalic acid in LP
1000
500
0
100
50
0
1000
500
0
100
50
0
*
Concentrations (µg/ml)
Concentrations (µg/ml)
Fig. 3. THP-1 cells were treated with various concentrations of phthalic anhydride (PAH; a) or phthalic acid (c) in aqueous vehicle (culture medium with 0.8% v/v DMSO) for 24 h.
Alternatively, THP-1 cells were cultured in medium for an additional 24 h after a 5-min exposure to various concentrations of PAH (b) and phthalic acid (d) in liquid paraffin (LP)
dispersion medium. IL-8 secretion in culture medium supernatants was measured by ELISA (open bars) and expressed as pg/mL, while cell viability was measured by using WST-8 assays
(black squares).
4. Discussion
assays. PAH, MAH, and TAH are highly immunogenic in LLNA,
exhibiting strong sensitization potential (Dearman et al., 2000). There-
fore, we believe that developing improve methods for evaluation of acid
anhydride should be a research priority.
In previous studies, PAH was evaluated as false negative in IL-8
release assays and several in vitro skin sensitization tests (Ashikaga
In this study, we aimed to help prevent false-negative results in in
vitro skin sensitization test using analysis of IL-8 release by THP-1 cells.
In particular, we examined why acid anhydrides, which exhibit strong
skin sensitizing potential, often show false-negative results in these
MAH in aqueous vehicle
MAH in LP
a
b
1506
*
*
*
1000
1000
500
0
100
50
0
100
*
500
0
50
0
*
Concentration (µg/mL)
Concentration (µg/mL)
TAH in aqueous vehicle
TAH in LP
c
d
1000
500
0
100
50
0
1000
500
0
100
50
0
*
Concentration (µg/mL)
Concentration (µg/mL)
Fig. 4. THP-1 cells were treated for 24 h with various concentrations of maleic anhydride (MAH; a) and trimellitic anhydride (TAH; c) in aqueous vehicle (culture medium with 0.2% and
0.8% v/v DMSO). THP-1 cells were cultured in medium for an additional 24 h following a 5-min exposure to various concentrations of MAH (b) or TAH (d) in liquid paraffin (LP)
dispersion medium. IL-8 secretion in culture medium supernatants was measured by ELISA (open bars) and expressed as pg/mL, while cell viability was measured using WST-8 assays
(black squares).
73

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
NiSO4 in aqueous vehicle
NiSO4 in LP
a
b
6885
57207
6021
11460
4958
3447
*
*
*
*
*
*
*
1000
500
0
100
50
0
1000
500
0
100
50
0
Concentration (µg/mL)
Concentration (µg/mL)
SLS in aqueous vehicle
SLS in LP
c
d
1000
500
0
100
50
0
1000
100
500
0
50
0
*
*
Concentration (µg/mL)
Concentration (µg/mL)
Fig. 5. THP-1 cells were treated for 24 h with various concentrations of NiSO4 (a) or SLS (c) in aqueous vehicle (culture medium with 0.2% v/v saline). THP-1 cells were cultured in
medium for an additional 24 h following a 5-min exposure to various concentrations of NiSO4 (b) or SLS (d) in liquid paraffin (LP) dispersion medium. IL-8 secretion in culture medium
supernatants was measured by ELISA (open bars) and expressed as pg/mL, while cell viability was measured using WST-8 assays (black squares).
et al., 2010; Emter et al., 2013; Saito et al., 2013; Takenouchi et al.,
2013; Piroird et al., 2015; Kimura et al., 2015). In LLNA, PAH is
dissolved in acetone/olive oil (4:1 v/v) solution (Dearman et al., 2000).
However, in all of the above in vitro tests, PAH is dissolved in aqueous
vehicle, i.e., culture medium containing DMSO. Generally, in vitro test
results are more strongly affected by water than in vivo test results (e.g.,
water solubility, hydrolysis reactions, hydration reactions). PAH is
known to form the corresponding phthalic acid quickly via hydrolysis.
The rate constant for the forward reaction for PAH in aqueous solution
at 25 °C is 7.9 × 10−3 s−1 (Hawkins, 1975). Therefore, we hypothe-
sized that hydrolysis of PAH may be the underlying cause of the false-
negative results.
In order to clarify these results and validate our hypothesis, we
measured the hydrolysis of PAH in a cell-free system using ultraviolet
(UV) spectrophotometry, which is relative inexpensive, highly sensi-
tive, and quick. In addition, this method has been used to measure the
hydrolysis of PAH in previous studies (Peters, 1959; Hawkins, 1975).
However, quantitative analysis of unknown substances is difficult using
UV spectroscopy. Our preliminary analysis revealed that the greatest
difference between PAH and phthalic acid were observed at 300 nm;
subsequent analyses at this wavelength revealed that PAH was hydro-
lyzed within approximately 5 min in water containing DMSO or within
1 week in DMSO. This could be explained by the hygroscopic properties
and chemical reactivity of DMSO. For example, the amount of water
taken up by DMSO into an open bottle at constant relative humidity of
98% at 22 °C is 40 g per 100 g DMSO (Lebel and Goring, 1962), and the
product is obtained from the reaction of DMSO and acetic anhydride
within 12–24 h (Albright and Goldman, 1965).
phthalic acid. Moreover, PAH in LP dispersion medium induced an
increase in IL-8 release from THP-1 cells, further validating our
hypothesis. Similar results were observed for MAH and TAH, support-
ing that the use of LP dispersion medium prevented the hydrolysis of
acid anhydrides, facilitating THP-1 cell exposure.
In GARD assay, which are in vitro assays based on a genomic
biomarker signature, both PAH and TAH scored as respiratory sensiti-
zers despite the use of aqueous vehicle (Forreryd et al., 2015). The
predictive genomic biomarker signature was identified on the assump-
tion that PAH and TAH were the respiratory sensitizers, regardless of
aqueous environment. In this procedure, whether PAH and TAH were
hydrolyzed was not considered. Moreover, the Ensembl transcript ID,
which was used as a gene identifier, did not include IL-8 as a predictor
gene in the GARD Respiratory Prediction Signature. Therefore, it may
not be possible to compare our data with the results of this assay. In
accordance with the considerations above, we could not determine
whether both PAH and TAH were scored as sensitizers, despite the use
of aqueous vehicle.
Notably, MAH showed a preference for LP dispersion medium,
although it also significantly induced augmentation of IL-8 release in
aqueous vehicle. This phenomenon was considered in terms of the
sensitization potential properties of hydrolyzed MAH. Most impor-
tantly, maleic acid classified clear positive results in LLNA (Kreiling
et al., 2008). Therefore, it is likely that hydrolysis did not suppress IL-8
release induced by MAH. Furthermore, acid anhydrides bind covalently
to free amino groups, particularly the amino group of lysine(Newman
Taylor et al., 1987; Lindh and Jönsson, 1998; Lalko et al., 2012).
However, Lalko et al. (2012) found that MAH also increased cysteine
reactivity when compared with PAH and TAH in chemical analyses.
Thus, we hypothesized that the skin sensitization mechanism of MAH
may differ from those of PAH and TAH.
In this study, we used LP as a dispersion medium for PAH. By mixing
an LP/water binary-phase solvent, we found that PAH was present for
at least 5 min in the water phase because of the sustained release of
PAH from LP. Therefore, we further analyzed the validity and effec-
tiveness of our LP dispersion medium methods for in vitro skin
sensitization tests using THP-1 cells of acid anhydrides. Notably, when
dispersed in LP medium, PAH exhibited an IC50 that was approximately
half that of phthalic acid, despite having similar IC50 values in aqueous
vehicle, confirming that PAH was hydrolyzed by aqueous vehicle to
However, for MAH and NiSO4, the maximum IL-8 release in LP
dispersion medium was significantly decreased compared with that in
aqueous vehicle, which may have been related to exposure time.
Accordingly, additional studies are needed to develop a long-term
exposure method using LP dispersion medium.
In our study, SLS augmented IL-8 release during a 24-h exposure
74

K. Narita et al.
Toxicology in Vitro 42 (2017) 69–75
Biol. Med. 49 (4), 548–553.
using aqueous vehicle. Toebak et al. (2006) reported similar results in
monocyte-derived undifferentiated dendritic cells exposed to subtoxic
concentrations of SLS. For this same reason, phthalic acid caused a
slight augmentation of IL-8 release during a 24-h exposure using
aqueous vehicle.
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5. Conclusion
We found that PAH, MAH, maleic acid, and TAH induced IL-8
release in THP-1 cells, whereas phthalic acid and trimellitic acid did
not. Moreover, the false-negative results of PAH and TAH were caused
by hydrolysis by aqueous vehicle. Furthermore, 5-min exposure to LP
dispersion medium may be a promising method for evaluation of skin
sensitization by acid anhydrides. These findings may have applications
in in vitro skin sensitization tests using THP-1 cells (e.g., h-CLAT).
However, additional studies are needed to develop
exposure method using LP dispersion medium.
a long-term
Transparency document
The http://dx.doi.org/10.1016/j.tiv.2017.04.008 associated with
this article can be found, in online version.
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Acknowledgments
We are thankful to Akira Motoyama, Ph.D. at Quality Assessment
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