The Journal of Toxicological Sciences (J. Toxicol. Sci.

) 163
Vol.39, No.1, 163-171, 2014

Letter

Effect of simultaneous exposure to mixture of two skin

sensitizers on skin sensitization response in guinea pigs
and mice
Takashi Morimoto1,2, Tamaki Higaki1, Mika Ota1, Kunifumi Inawaka1, Satoshi Kawamura1
and Takashi Bungo2

Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98, Kasugadenaka 3-chome,
1

Konohana-ku, Osaka 554-8558, Japan

2Graduate School of Bioresource Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528,

Japan

(Received September 26, 2013; Accepted November 22, 2013)

ABSTRACT — Skin sensitization resulting in allergic contact dermatitis is a common occupational

health issue. In this study, the effect of mixing two skin sensitizers on the skin sensitization response was
investigated. Skin sensitizers are generally classified into T helper type 1 (Th1) or T helper type 2 (Th2),
depending on the induced cytokine profile. Dinitrochlorobenzene (DNCB) and oxazolone (Oxa) are Th1
skin sensitizers and phthalic anhydride (PA) and toluene diisocyanate (TDI) are Th2 skin sensitizers. We
investigated the effect on skin sensitization response to mixtures of three pairs of these sensitizers: DNCB
and Oxa, DNCB and PA, and PA and TDI, using guinea pig maximization test and mouse ear swell-
ing test. In guinea pigs sensitized with the mixture of DNCB and Oxa or PA and TDI, there were chang-
es of skin sensitization response to DNCB and Oxa, and that to PA. On the other hand, there was no mix-
ture effect in guinea pigs sensitized with the mixture of DNCB and PA. The skin sensitization responses
were decreased in mice sensitized with the mixtures of DNCB and Oxa or PA and TDI, whereas the mix-
ture effect was not observed in mice sensitized with the mixture of DNCB and PA. The present findings
revealed that mixture effect on the skin sensitization response was observed after simultaneous exposure
to two skin sensitizers, and the effect was determined by combinations of mixed skin sensitizers.

Key words: Mixture effect, Skin sensitization, T helper type 1, T helper type 2

INTRODUCTION
Skin sensitization resulting in allergic contact derma-
titis (ACD) is by far the most frequent manifestation of
immune toxicity in humans and a common occupation-
al disease. It is a T cell-mediated delayed type hyper-
sensitivity response to a low molecular weight reactive
chemical called hapten. Skin sensitization develops in
two distinct phases: induction and challenge phases. The
induction phase includes several events following the first
contact with hapten and is completed when individual is
sensitized and capable of giving a positive ACD reaction.
The challenge phase begins upon elicitation by hapten
and results in the clinical symptoms of ACD.
To evaluate the skin sensitizing potency of chemicals,
animal experiments have been conducted. Historically,
Correspondence: Takashi Morimoto (E-mail: morimotot2@sc.sumitomo-chem.co.jp)
guinea pigs have been the most common species used for
these studies (Magnusson and Kligman, 1969; Buehler,
1965), and more recently mice have been chosen (Kimber
et al., 1994; Gad et al., 1986).
Since humans are exposed to a wide variety of sub-
stance, there is increasing concern in the general pub-
lic about the mixture effect. Science Committees of the
European Commission have recently published a paper
for toxicity and assessment of chemical mixture (EU,
2011). However, the literature on mixture effect of skin
sensitization was not analyzed in the paper.
On the other hand, many studies have reported the
effect of some chemicals on skin sensitization response in
mice. For example, Wille et al. (1998) demonstrated that
ethacrynic acid inhibited the skin sensitization responses to
2,4-Dinitrochlorobenzene (DNCB) and oxazolone (Oxa).

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T. Morimoto et al.
Di butyl phthalate enhance skin sensitization response
to toluene diisocyanate (TDI), but not Oxa (Matsuda
et al., 2010). However, there have been no reports on the
mixture effect of simultaneous exposure to multiple skin
sensitizers in term of skin sensitization response.
The objective of this study was to investigate the mix-
ture effect on skin sensitization response to each com-
ponent by induction with mixture of skin sensitizers. To
assess the effect, the changes of challenge response were
confirmed by guinea pig maximization test (GPMT) and
mice ear swelling test (MEST). We also selected DNCB,
Oxa, phthalic anhydride (PA) and TDI as skin sensitizers,
and investigated the effect on skin sensitization response
to mixtures of three pairs of these sensitizers: DNCB and
Oxa, DNCB and PA, and PA and TDI. Skin sensitizers are
generally classified as T helper type 1 (Th1) or T helper
type 2 (Th2), depending on the induced cytokine profiles.
DNCB induced high levels of INF-γ, while TDI upregu-
lated the production of Th2-type cytokines such as IL-10
and IL-4 (Dearman et al., 1996, 2002). Hayashi et al.
(2001) demonstrated IL-12p expression was elevated in
lymph node of mouse exposed to Oxa and enhancement
of IL-4 expression was observed in mouse exposed to
phatalic anhydride (PA). Therefore, DNCB and Oxa were
classified as Th1 skin sensitizers, while PA and TDI were
classified as Th2 skin sensitizers.
MATERIALS AND METHODS
Test materials
2,4-Dinitrochlorobenzene (DNCB, purity: min 99%) and
toluene diisocyanate (TDI, purity: 97%) were purchased
from Wako Pure Chemical Industry, Co., Ltd. (Osaka,
Japan). Oxazolone (Oxa, purity: 99%) and phthalic anhy-
dride (PA, purity: 97%) were purchased from Sigma-
Aldrich (St Louis, MO, USA) and Nakalai Tesque, Inc.
(Kyoto, Japan), respectively. Information on the sensitiza-
tion of these skin sensitizers is summarized in Table 1.
Table 1. Skin sensitizers used in this study
CAS No.
Chemicals
2,4-dinitrochlorobenzene (DNCB) 97-00-7
Oxazolone (Oxa) 15646-46-5
Phthalic anhydride (PA) 85-44-9
Toluene diisocyanate (TDI) 584-84-9
van Och FM et al. (2000)
a)
NA: Not applicable
Vol. 39 No. 1
Animals
Female guinea pigs of the albino Hartley strain and
female CBA/J mice were purchased from SLS Japan and
Charles River Laboratories Japan, Inc., respectively. At
the start of the test, guinea pigs were 5 to 7 weeks old
and mice were 9 to 10 weeks old. They were given free
access to filtered tap water and diets. The animal facility
was maintained with a 12-hr light/dark cycle.
Guinea pig maximization test (GPMT)
GPMT was conducted in accordance with OECD
test guideline No. 406, except animal number and chal-
lenge application. Five guinea pigs were assigned for
each group. Corn oil and acetone were selected for intra-
dermal induction and epidermal induction, respectively.
Chemical and FCA were injected in intradermal induction
phase, and chemical was applied in epidermal induction
phase. Two weeks after the epidermal induction, chem-
ical dilution in acetone was applied on shaved back at
0.01 ml/site. The sites were scored separately for ery-
thema and swelling (0-3 scale: 0, No reaction; 1, Slight
reaction; 2, Moderate reaction; 3, Severe reaction) at 24,
48, and 72 hr after the challenge application. The mean
responses (the total scores for erythema and swelling/3,
max 6) and the average of the mean responses were cal-
culated (Nakamura et al., 1998). Preliminary irritation
tests were carried out and mildly to moderately irritating
concentrations were selected for induction and non-irri-
tating concentration for challenge (Table 2).
Mouse ear swelling test (MEST)
MEST was carried out as reported previously (Garrigue
et al., 1994), with minor modifications. Five mice were
assigned for each group. Acetone and olive oil 4:1, v/v
(AOO) was selected as a vehicle for this test. Backs of
mice were shaved at 2 cm × 4 cm/site and 50 μl of test
chemical dilution was applied to the shaved site. A week
after the application, mice were challenged with applica-
tion of 25 μl of chemical dilutions to the dorsum of each
Result of Skin sensitizationa)
Maximization test Local Lymph Node Assay
+ +
+ +
+ +
NA +
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Skin response to sensitizer mixture in guinea pigs and mice

Table 2. Concentrations for inductions and challenge in GPMT

Mixture of DNCB and Oxa
Final concentration in dosing solution
Group
Intradermal induction Typical induction Challenge
Control Vehicle Vehicle
DNCB DNCB: 0.025% DNCB: 0.25% DNCB: 0.00001%~0.1%
Oxa: 0.000001%~0.1%
Oxa Oxa: 0.05% Oxa: 12.5%
DNCB+Oxa DNCB: 0.025% +Oxa:0.05% DNCB: 0.25% +Oxa: 12.5%

Mixture of DNCB and PA

Final concentration in dosing solution
Group
Intradermal induction Typical induction Challenge
Control Vehicle Vehicle
DNCB DNCB: 0.025% DNCB: 0.25% DNCB: 0.00001%~0.1%
PA: 0.0001%~10%
PA PA: 0.1% Oxa: 5%
DNCB+PA DNCB: 0.025% +PA:0.1% DNCB: 0.25% +Oxa: 5%

Mixture of PA and TDI

Final concentration in dosing solution
Group
Intradermal induction Typical induction Challenge
Control Vehicle Vehicle
PA PA: 0.1% PA: 0.25% PA: 0.0001%~10%
TDI: 0.000001%~0.1%
TDI TDI: 0.1% TDI: 5%
PA+TDI PA: 0.1% +TDI: 0.1% PA: 0.25% +TDI: 5%

ear. Ear thickness was measured by Digimatic Micrometer
(Mitutoyo, Japan) before the challenge and at 48 hr after
the application to ear. The increases for ear thickness in
chemical treated groups were compared with that in con-
trol group (AOO). Animal grouping and concentrations
for inductions and challenge are shown in Table 3.
Statistical analysis
Statistical analysis was performed with StatLight#05
version 2 (Yukms Co., Ltd., Tokyo, Japan). The homoge-
neity of variance in ear thickness increase was analyzed
among control group and chemical treated groups by
Bartlett’s test. When the difference between the varianc-
es was not significant, Dunnett’s multiple comparison test
was performed. When the difference between the varianc-
es was significant, Steel’s multiple comparison test was
performed. For the mean response in GPMT and mix-
ture effect in MEST, the data was analyzed using F-test
to compare to the mixture group. When the difference
between the variances was not significant, student’s t-test
was performed. When the difference between the varianc-
es was significant, Welch’s test was performed.
Animal welfare
This study was performed in accordance with the
Guide for Animal Care and Use of Sumitomo Chemical
Co., Ltd.
RESULTS
Guinea pig maximization test
Mixture of DNCB and Oxa (Table 4)
To investigate possible mixture effects on skin sensiti-
zation response in guinea pigs by mixing DNCB and Oxa,
the responses in four groups - DNCB, Oxa, DNCB+Oxa,
and control group - were compared. Animals in DNCB
and Oxa groups showed a positive response to DNCB
and Oxa, respectively. In all animals of DNCB+Oxa
group, both DNCB and Oxa provoked a response. The
response to DNCB in DNCB+Oxa group was com-
pared with that in DNCB group, and it was found that

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T. Morimoto et al.

Table 3. Concentrations for induction and challenge in MEST

Mixture of DNCB and Oxa

Final concentration in dosing solution
Group Induction Challenge
Dorsal Left ear Right ear
Control Vehicle
DNCB DNCB: 1%
DNCB: 1% Oxa: 0.2%
Oxa Oxa: 5%
DNCB+Oxa DNCB: 1% Oxa: 5%

Mixture of DNCB and PA

Final concentration in dosing solution
Group Induction Challenge
Dorsal Left ear Right ear
Control Vehicle
DNCB DNCB: 1%
DNCB: 1% PA: 10%
PA PA: 10%
DNCB+PA DNCB: 1% PA: 10%

Mixture of TDI and PA

Final concentration in dosing solution
Group Induction Challenge
Dorsal Left ear Right ear
Control Vehicle
TDI TDI: 10%
TDI: 0.5% PA: 10%
PA PA: 10%
TDI+PA TDI: 10% PA: 10%

the number of animals showing positive response was
lower in DNCB+Oxa group compared to DNCB group.
Positive responses were observed at the challenge site
with 0.01% DNCB in all 5 animals in DNCB group and
2 out of 5 animals in DNCB+Oxa group. At the challenge
site with 0.001% DNCB, 5 animals showed a positive
response in DNCB group, but no response was observed
in DNCB+Oxa group. In addition, a significant decrease
(p < 0.01) in mean response score was observed at 0.01%
and 0.001% DNCB.
The response to Oxa in DNCB+Oxa group was com-
pared with that in Oxa group. At the challenge site
with 0.00001% Oxa, a positive response was observed
in 3 out of 5 animals of Oxa group, but no response
was observed in any animals of DNCB+Oxa group. A
decrease in number of positive animals was observed at
only 0.00001% Oxa, but there were significant decreas-
es in mean response scores at 0.001% and 0.0001% Oxa.
It was concluded that the skin sensitization responses to
DNCB and Oxa were decreased by mixing DNCB and
Oxa.
Mixture of DNCB and PA (Table 5)
To investigate the effect of mixing DNCB and PA, the
skin sensitization responses in four groups - DNCB, PA,
DNCB+PA, and control group - were compared. Animals
in DNCB and PA groups showed a positive response to
DNCB and PA, respectively. In all animals of DNCB+PA
group, both DNCB and PA provoked a positive response.
The response was compared between DNCB+PA and
DNCB groups, and DNCB+PA and PA groups. At the
challenge site with 0.001% DNCB, positive response to

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Skin response to sensitizer mixture in guinea pigs and mice

Table 4. Skin sensitization response to DNCB and Oxa in GPMT

Number of animals showing positive reaction ／ Number of sensitized animals
Challenge chemical Concentration (%) (average of mean response)
DNCB group Oxa group DNCB+Oxa group Control group
DNCB 0.1 5/5 （4.3） 0/5 （ 0 ） 5/5 （3.9b） 0/5 （ 0 ）
0.01 5/5 （2.9） 0/5 （ 0 ） 2/5 （0.3 ）
a 0/5 （ 0 ）
0.001 5/5 （0.9） 0/5 （ 0 ） 0/5 （ 0 ）
a 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
0.00001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
Oxa 0.1 0/5 （ 0 ） 5/5 （4.0） 5/5 （4.5c） 0/5 （ 0 ）
0.01 0/5 （ 0 ） 5/5 （3.1） 5/5 （3.0e） 0/5 （ 0 ）
0.001 0/5 （ 0 ） 5/5 （2.9） 5/5 （2.3c） 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 5/5 （1.1） 5/5 （0.6 ）
d 0/5 （ 0 ）
0.00001 0/5 （ 0 ） 3/5 （0.3） 0/5 （ 0 e） 0/5 （ 0 ）
0.000001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
a Significantly different (p < 0.01) from DNCB group
b Not Significantly different from DNCB group
c Significantly different (p < 0.05) from Oxa group

d Significantly different (p < 0.01) from Oxa group

e Not significantly different from Oxa group

Table 5. Skin sensitization response to DNCB and PA in GPMT

Number of animals showing positive reaction ／ Number of sensitized animals
Challenged chemical Concentration (%) (average of mean response)
DNCB group PA group DNCB+PA group Control group
DNCB 0.1 5/5 （4.7） 0/5 （ 0 ） 5/5(4.9a） 0/5 （ 0 ）
0.01 5/5 （3.3） 0/5 （ 0 ） 5/5 （2.9 ）
a 0/5 （ 0 ）
0.001 3/5 （0.5） 0/5 （ 0 ） 3/5 （0.5 ）
a 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
0.00001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
PA 10 0/5 （ 0 ） 5/5 （3.7） 5/5 （3.5b） 0/5 （ 0 ）
1 0/5 （ 0 ） 5/5 （2.5） 5/5 （2.5b） 0/5 （ 0 ）
0.1 0/5 （ 0 ） 5/5 （1.1） 5/5 （0.8b） 0/5 （ 0 ）
0.01 0/5 （ 0 ） 1/5 （0.2） 0/5 （ 0 ）
b 0/5 （ 0 ）
0.001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
a Not Significantly different from DNCB group
b Not Significantly different from PA group

DNCB was observed in 3 out of 5 animals of both DNCB
and DNCB+PA groups. One out of 5 animals in PA group
showed a positive response to 0.01% PA and none in
DNCB+PA group. However, this change was small, not
observed at just above challenge concentration, and there
was no significant change in mean response scores. No
clear mixture effect on skin sensitization response was
detected due to mixing of DNCB and PA.
Mixture of PA and TDI (Table 6)
To investigate the effect of mixing PA and TDI, the
skin sensitization responses in four groups - PA, TDI,

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T. Morimoto et al.

Table 6. Skin sensitization response to PA and TDI in GPMT

Number of animals showing positive reaction ／ Number of sensitized animals
Challenged chemical Concentration (%) (average of mean response)
PA group TDI group PA+TDI group Control group
PA 10 5/5 （4.7） 0/5 （ 0 ） 5/5 （3.4a） 0/5 （ 0 ）
1 5/5 （2.2） 0/5 （ 0 ） 5/5 （1.7b） 0/5 （ 0 ）
0.1 5/5 （1.1） 0/5 （ 0 ） 2/5 （0.6b） 0/5 （ 0 ）
0.01 2/5 （0.2） 0/5 （ 0 ） 0/5 （ 0 b） 0/5 （ 0 ）
0.001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
TDI 0.1 0/5 （ 0 ） 5/5 （5.1） 5/5 （4.5c） 0/5 （ 0 ）
0.01 0/5 （ 0 ） 5/5 （2.3） 5/5 （2.0c） 0/5 （ 0 ）
0.001 0/5 （ 0 ） 1/5 （0.4） 3/5 （0.5c） 0/5 （ 0 ）
0.0001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
0.00001 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ） 0/5 （ 0 ）
a Significantly different (p < 0.05) from PA group
b Not significantly different from PA group

c Not significantly different from TDI group

PA+TDI, and control group - were compared. Animals in
PA and TDI groups showed a positive response to PA and
TDI, respectively. In all animals of PA+TDI group, both
PA and TDI provoked a response. The response to PA in
PA+TDI group was compared with that in PA group. Pos-
itive response was observed at the challenge site with
0.1% PA in all 5 animals of PA group and 2 out of 5 ani-
mals of PA+TDI group. At the challenge site with 0.01%
PA, 2 out of 5 animals showed a positive response in PA
group, but no response was observed in PA+TDI group.
The number of animals showing a positive response to PA
was lower at these two challenge concentrations. In addi-
tion, a significant decrease (p < 0.05) in mean response at
10% PA and decreasing trend at 1, 0.1, and 0.01% were
observed.
The response to TDI in PA+TDI group was compared
with that in TDI group. All 5 animals showed a posi-
tive response to 0.01% TDI in both PA+TDI and TDI
groups. At the challenge site with 0.001% TDI, a positive
response was observed in 1 out of 5 animals of TDI group
and 3 out of 5 animals of PA+TDI group. The change in
number of positive animals was observed at only one con-
centration, and there was a no change in mean response.
It was concluded that by mixing PA and TDI the skin sen-
sitization response to PA was decreased, but the response
to TDI was the same.
Mouse Ear Swelling Test
Mixture of DNCB and Oxa (Fig. 1)
To investigate the effect on skin sensitization response
in mice by mixing DNCB and Oxa, the responses in four
groups - DNCB, Oxa, DNCB+Oxa, and control group -
were compared. Mice in DNCB and Oxa groups showed
a significant ear thickness increase by the application of
DNCB and Oxa, respectively.
The response was compared between DNCB+Oxa and
DNCB groups, and DNCB+Oxa and Oxa groups. Ear
thickness increase to DNCB in DNCB+Oxa group was
significantly less (p < 0.01) than that in DNCB group, but
there was no change in ear response to Oxa.
Mixture of DNCB and PA (Fig. 2)
To investigate the effect of mixing DNCB and PA, the
skin sensitization responses in four groups - DNCB, PA,
DNCB+PA, and control group - were compared. Mice in
DNCB and PA groups expressed a significant ear thick-
ness increase (p < 0.01) to DNCB and PA, respectively. In
mice sensitized with the mixture of DNCB and PA, a sig-
nificant ear thickness increase (p < 0.01) to both DNCB
and PA was observed. When the response was compared
between DNCB+PA and DNCB groups, and DNCB+PA
and PA groups, there was no change of response due to
mixing.

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Skin response to sensitizer mixture in guinea pigs and mice
Fig. 1. Ear thickness increase using DNCB and Oxa in mice.
(A) Response to DNCB, (B) Response to Oxa. Data
of individual mice are plotted. The horizontal bars
and error bars show the mean and SD, respectively.
# Significantly different (p < 0.05) from Control group.
*p < 0.01, n.s. = not statistically significant.
Mixture of PA and TDI (Fig. 3)
To investigate the response by mixing PA and TDI,
the skin sensitization responses in four groups - PA, TDI,
PA+TDI, and control group - were compared. Mice in
TDI groups expressed a significant ear thickness increase
(p < 0.05) only by TDI. Mice in PA group showed a sig-
nificant ear thickness increase (p < 0.05) in response to
PA, and to a lesser extent to TDI. In mice sensitized with
the mixture PA and TDI, a significant increase in ear
thickness in response to TDI was observed, but not to PA.
The response was compared between PA+TDI and
PA, and PA+TDI and TDI. Ear thickness increase to PA
in PA+TDI group was significantly less than that in PA
group, and the response to TDI in PA+TDI group was
equivalent to that in TDI group.
DISCUSSION
Many studies have reported the effect of chemi-
cals on skin sensitization response (Wille et al., 1998;
169
Fig. 2. Ear thickness increase using DNCB and PA in mice.
(A) Response to DNCB, (B) Response to PA. Data
of individual mice are plotted. The horizontal bars
and error bars show the mean and SD, respectively.
## Significantly different (p < 0.01) from Control
group. n.s. = not statistically significant.
Matsuda et al., 2010). However, there have been no reports
on the mixture effect on skin sensitization response by
induction with mixture of skin sensitizers. In this study,
we investigated possible mixture effects on skin sensitiza-
tion response in guinea pigs by mixing three pairs of skin
sensitizers: DNCB and Oxa, DNCB and PA, and PA and
TDI. We found that the decreased effects on skin sensiti-
zation response were observed in animals sensitized with
the mixture of DNCB and Oxa, and the mixture of PA and
TDI. On the other hand, skin response of animals sensi-
tized with the mixture of DNCB and PA was similar to
that treated with DNCB or PA alone. Thus, our findings
suggested that certain combination of two skin sensitizers
induce a decrease of skin sensitization response.
By conducting a similar experiment in mice, spe-
cies differences between guinea pigs and mice in effect
on skin sensitization response were examined and it was
concluded that the mixture effect observed in guinea
pig was very similar to that in mice. That is, there was
no effect on skin sensitization response due to mixing of
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T. Morimoto et al.
Fig. 3. Ear thickness increase using PA and TDI in mice.
(A) Response to PA, (B) Response to TDI. Data
of individual mice are plotted. The horizontal bars
and error bars show the mean and SD, respectively.
# Significantly different (p < 0.05) from Control group.
*p < 0.01, n.s. = not statistically significant.
DNCB and PA. And mixture of PA and TDI produced the
decrease effect on response to PA but not to TDI. When
DNCB was mixed with Oxa, the decreased response to
DNCB was observed. These results support the conclu-
sion above. On the other hand, by mixing of DNCB and
Oxa, guinea pigs exhibited the decreased response to Oxa
but mice did not. The difference between guinea pig and
mice can be attributed to the fact that the concentration
of Oxa used for induction was excessive in mice exper-
iment.
Mice in PA group also showed a significant ear thick-
ness increase in response to PA, and to a lesser extent to
TDI. This ear thickness increase by TDI would not be a
skin sensitization response, but an augmented irritating
response by the increase of Th2 cytokine. It was report-
ed that signal transducer and activator of transcription 6
(STAT-6) transgenic mice, in which Th2 cytokine such
as IL-4 increased, showed a greater epidermal thickness
increase against an irritant than WT mice (DaSilva et al.,
2012). Thus, these results may suggest the effect on irri-
tating response by the increase of Th2 cytokine.
Vol. 39 No. 1
Interestingly, our result indicates that the effect on skin
sensitization response may not be observed by mixing
DNCB and PA. Meek et al. (2011) have suggested that
chemicals that act by discrete modes of action or on dif-
ferent target cells, tissues, or organs, may act independ-
ently when animals are exposed to them simultaneously.
Thus, it can be suggested that DNCB (Th1 skin sensi-
tizer) and PA (Th2 skin sensitizer) act through different
mechanism(s) to develop skin reaction in the test sys-
tems employed in this study. In addition, guinea pig and
mouse models used in this study can be applied to dis-
criminate modes of action associated with skin sensitiza-
tion between chemicals.
In the present study, we investigated the mixture effects
by examining dose response relationship during challenge
phase with limited combinations of selected chemicals.
Further study using more variable sets of other skin sensi-
tizers, would be desired in order to reveal the whole pic-
ture of mixture effect on skin sensitizers.
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M.H. and Travers, J.B. (2012): Increased skin barrier disrup-
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