Journal of Oral Rehabilitation 2006 33; 216–220
Quantitative analysis of allergenic ingredients in eluate
extracted from used denture base resin
M. MIKAI, M. KOIKE & H. FUJII Division of Removable Prosthodontics and Management of Oral Function, Department
of Developmental and Reconstructive Medicine, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical
Sciences, Nagasaki, Japan
SUMMARY Allergenic ingredients extracted from
used denture base resin were quantitatively ana-
lysed using a gas chromatograph/mass spectrometer
and high performance liquid chromatography.
Methyl methacrylate (MMA), hydroquinone (HQ),
formaldehyde (FMA), benzoyl peroxide (BPO), ben-
zoic acid (BA) and methyl benzoate (MB) were
detected in a eluate from all of the dentures, while
ethyleneglycol dimethacrylate (DME) was detected
in the eluate from 87 dentures in use for <15 years.
MMA, HQ, FMA, BPO and MB showed a decrease in
correlation to the period of denture use, but con-
tinuing to be evident even after 29 years of use.
Introduction
Acrylic denture base resin (acrylic resin) is a useful
biomaterial in prosthetic dentistry, but clinicians occa-
sionally encounter allergic reactions to chemical ingre-
dients of the acrylic resin. In some cases, moreover, the
patient develops these reactions after wearing the
denture for a number of years (1, 2). Several authors
have already identified certain chemical ingredients of
acrylic resin that induce allergies (3–6), and many
others have confirmed that these chemicals are eluted
from the acrylic resin (7–11). However, most of these
studies examined fresh moulded specimens. Except for
the report by Sadamori et al. (12) on the analysis of
residual monomer, there have been no studies to date
investigating dentures worn for some period of time in
the oral cavity.
The purpose of the present study is to conduct a
quantitative analysis of some of the purported aller-
genic ingredients in used denture base resin, namely,
ª 2006 Blackwell Publishing Ltd
The MMA showed the highest level of elution and a
relatively moderate decrease over time. The elution
of BA, on the contrary, showed an increase with
the period of denture use up to about 10 years and
subsequently reached a plateau. Our results indi-
cate that purported allergens exist in the resin base
and can be eluted into the oral cavity, even in
patients using an old denture for a period of nearly
30 years.
KEYWORDS: acrylic resin, allergenic ingredient,
GCMS, high-performance liquid chromatography
Accepted for publication 10 November 2004
methyl methacrylate (MMA) (4–6, 13), ethyleneglycol
dimethacrylate (DME) (13), formaldehyde (FMA) (14),
hydroquinone (HQ) (3, 4), benzoyl peroxide (BPO)
(3–5, 14) and benzoic acid (BA) (15) and methyl
benzoate (MB) which is a decomposition product of
BPO in methanol (7).
Materials and methods
Dentures
From 140 patients (69 male and 71 female) whose
informed consent was obtained, 152 dentures were
collected. They visited the first author’s clinic to obtain
new dentures from May 1998 to August 2001. Of these
152 dentures, 144 had been made at the first author’s
clinic using heat-curing acrylic resin, and 99 of them
had undergone base repair at some point in the past.
The dentures had been used for a period ranging from
6 months to 29 years. For the purposes of this study,
doi: 10.1111/j.1365-2842.2005.01561.x
 INGREDIENTS FROM USED DENTURE BASE RESIN
Table 1. Number of dentures tested
Full denture Partial denture
Denture groups and duration
of their usage in years Upper Lower Upper Lower
Short-term denture, 26 6 44 20
0Æ0 <
< 5Æ0
Middle-term denture, 13 0 9 10
5Æ0 £
< 10Æ0
Long-term denture, 6 8 8 2
10Æ0 £
< 30Æ0
the dentures were divided into three groups: dentures
used for <5 years (96 dentures; referred to hereafter as
‘short-term dentures’), dentures used for more than
5 years but <10 years (32 dentures; referred to here-
after as ‘middle-term dentures’) and dentures used for
more than 10 years but < 29 years (24 dentures;
referred to hereafter as ‘long-term dentures’) (Table 1).
The length of denture use and the history of repair were
assessed on the basis of clinical records, patient inter-
views and macroscopic observation of the dentures.
The dentures were cleaned mechanically using a
denture brush and denture cleanser*. The dentures
were then placed in beakers containing denture clean-
ing agent† and subjected to chemical cleansing for
10 min using an ultrasonic cleaner (Ultrasonic Cleaner
UT-51N)‡ at an output of 35 W, 32 kHz. After rinsing
with running water, the dentures were allowed to dry
naturally for 3 days. The dried dentures were then
placed in airtight glass containers and kept under dark
conditions at room temperature.
Preparation of specimens for analysis
Specimens were obtained from the base of the dentures
by using nippers to slice fragments of 10 mm in width
and more than 25 mm in length from the resin plate.
Care was taken to exclude the repaired portion, of
which colour differed from intact portion in the most
cases, from specimen. These were then trimmed to a
uniform size for insertion in test tubes, and stains were
removed from the surface of the specimens using 600
grids SiC paper in water. One specimen was obtained
from each denture, or a total of 152 specimens.
*Dento Jyeru, Kobayashi Pharmaceutical Co., Osaka, Japan.
† ††
Pika, Shofu Co., Kyoto, Japan.
‡ ‡‡
Sharp Co., Osaka, Japan.
ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 216–220
217
Table 2. Size of specimens (mean and s.d.)
Weight Length Width Thickness
(g) (mm) (mm) (mm)
0Æ41 (0Æ05) 20Æ90 (3Æ45) 7Æ79 (0Æ70) 2Æ78 (0Æ84)
The length and width of the specimens were meas-
ured with hand-held slide calipers (1/20 mm). The
thickness was calculated as the average value of the
thickness at the four corners and centre of each
specimen, and the weight was measured using an
electronic analytical balance (ER-60A)§. The respective
mean values are shown in Table 2.
Extraction and analysis of resin ingredients
The specimens were completely immersed in borosili-
cate glass low-actinic culture tubes with Teflon cap
lining (17 ml, B-TST-SCR16-150)¶ containing 10 mL of
methanol (99Æ8%)** and shaken 80 times min)1 for a
period of 4 weeks under dark conditions at 37
C. The
extracted eluate was filtered with 0Æ2 lm pore-size
membrane filters (Acrodisc LC13PVDF)††.
Methyl methacrylate (MMA), DME and FMA were
analysed using a gas chromatograph/mass spectro-
meter (GC/MS) (GCMS-QP5050A)‡‡ equipped with a
60 mm · 0Æ32 mm I.D. capillary column (0Æ5 lm mem-
brane thickness; DB-WAX)‡‡. Helium was used as a
carrier gas, and the gas pressure was set at 120 kPa. The
injection temperature was set at 250
C, while the oven
temperature was set at 70
C for 1 min and raised to
230
C at a rate of 15
C min)1. After that, the oven
temperature was maintained for 20 min at 230
C. The
GC/MS interface temperature was set at 230
C. The
split mode was employed for the injection of eluate,
with the split ratio and injection volume set at 50:1 and
1 lL, respectively. The ionization voltage and current of
the MS were set at 70 eV and 60 lA EI, respectively.
The selected ion monitoring method was used for
measurements, and the selected fragment ions were
FMA:M/Z ¼ 29, 30; MMA:M/Z ¼ 41, 69, 100 and
DMA:M/Z ¼ 69, 112, 113.
§
A&D Vo., Tokyo, Japan.
¶
Iwaki Glass, Chiba, Japan.
**Wako Pure Chemical Industries, Ltd., Osaka, Japan.
Gelman Sciences, Ann Arbor, MI, USA.
Shimadzu Co., Kyoto, Japan.
218 M . M I K A I et al.

mg g–1 mg g–1
25·00 0·20
MMA DME

12·50 0·10

0·00 0·00

1·20 1·00
FMA HQ

0·60 0·50

0·00 0·00

4·00 5·00
BPO BA

2·00 2·50

0·00 0·00 Fig. 1. Allergenic ingredients in

0 10 20 30
eluate extracted from used denture
1·00 Year
base resin analysed by high-perfor-
MB mance liquid chromatography and
gas chromatograph/mass spectro-
meter. MMA, methyl methacrylate;
0·50
DME, ethyleneglycol dimethacrylate;
FMA, formaldehyde; HQ, hydroqui-
none; BPO, benzoyl peroxide; BA,
benzoic acid, MB: methyl benzoate;
0·00
0 10 20 30 Abscissa, duration of denture use in
Year years.

The analysis of HQ, BPO, BA and MB was conducted
using high-performance liquid chromatography (HPLC)
(VP Series HPLC System LC-10Avp)‡‡ with a
150 mm · 4Æ6 mm I.D. column (Simpack VP-ODS)‡‡.
For the eluate, 10 mM sodium phosphate buffer solu-
tion (pH 2Æ6) and acetonitrile were used. The acetonit-
rile was increased rectilinearly from 30% to 80% over a
period of 20 min using the gradient elution method.
The total flow rate and column oven temperature were
set at 1Æ0 mL min)1 and 40
C, respectively. The injec-
tion volume was fixed at 10 lL. An absorbance detector
(High Sensitivity UV-Vis Detector SPD-10 AVVP)‡‡ was
‡‡
Shimadzu Co., Kyoto, Japan.
used for detection. The detection wavelength was
254 nm.
The quantitative analysis was conducted using the
two-point absolute calibration method in GC/MS and
HPLC.
Results
The results of the analysis are shown in Fig. 1, and the
minimum, maximum and median values in each
specimen group are presented in Table 3.
The elution of MMA, which showed a median of
3Æ5377 mg g)1 in the short-term group, was the greatest
among the seven chemical ingredients analysed.

ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 216–220

 INGREDIENTS FROM USED DENTURE BASE RESIN 219

Table 3. Allergenic ingredient

Subgroup of denture tested
contents of eluate extracted (mg g)1)
and their number MMA DME FMA HQ BPO BA MB
from the sample of denture base
resin Short-term denture (n ¼ 96)
med. 3Æ5377 0Æ0013 0Æ3060 0Æ1684 1Æ8598 1Æ7609 0Æ5788
min. 1Æ8646 0Æ0000 0Æ1326 0Æ0508 0Æ7973 1Æ1285 0Æ0000
max. 11Æ3627 0Æ0157 1Æ0806 0Æ8420 3Æ3085 2Æ6484 0Æ9144
Middle-term denture (n ¼ 32)
med. 3Æ8278 0Æ0015 0Æ1963 0Æ1174 0Æ6722 2Æ6140 0Æ3690
min. 1Æ9512 0Æ0000 0Æ0109 0Æ0356 0Æ0873 1Æ6393 0Æ0000
max. 22Æ0436 0Æ1960 0Æ5040 0Æ3405 1Æ9214 4Æ6769 0Æ4730
Long-term denture (n ¼ 24)
med. 2Æ0227 0Æ0000 0Æ0604 0Æ0366 0Æ1929 2Æ5320 0Æ3472
min. 1Æ4327 0Æ0000 0Æ0165 0Æ0049 0Æ0103 1Æ6985 0Æ1367
max. 12Æ4234 0Æ0102 0Æ2629 0Æ2807 1Æ0041 4Æ6070 0Æ4635

MMA, methyl methacrylate; DME, ethyleneglycol dimethacrylate; FMA, formaldehyde; HQ,

hydroquinone; BPO, benzoyl peroxide; BA, benzoic acid, MB: methyl benzoate; med., median;
min., minimum; max., maximum.

Although the elution volume decreases in correlation to
the increase in the number of years of denture use, a
median of 2Æ0227 mg g)1 was observed in the long-
term dentures, an elution volume that was still
approximately 60% of that observed in the short-term
dentures (Table 3). Moreover, nine of the dentures that
had been used for between 7 and 10 years showed an
elution volume exceeding 10 mg g)1 (Fig. 1).
The elution of DME was observed in 87 of the 152
dentures, but the period of use was <15 years in all
these 87 dentures (Fig. 1), and the elution volume was
the lowest among the seven chemical ingredients
analysed, the median in the short-term and long-term
dentures being 0Æ0013 mg g)1 and 0Æ0000 mg g)1,
respectively (Table 3).
The elution volume of FMA, HQ and BPO in short-
term dentures was 0Æ3060, 0Æ1684 and 1Æ8598 mg g)1,
respectively (Table 3). Here again, the elution volume
decreases in correlation to the increase in the number
of years of denture use (Fig. 1). But the rate of decrease
was higher than that in MMA, the elution in long-term
dentures being some 10–24% of that in short-term
dentures.
With regard to MB, the decrease in elution over time
reaches a plateau at about 10 years (Fig. 1), the middle-
term and long-term dentures showing very little
difference at 0Æ3690 and 0Æ3472 mg g)1, respectively
(Table 3).
The elution of BA showed changes over time that
differed from those observed in the other chemical
ingredients. That is, the elution of BA increased over
time until reaching a plateau level after about 10 years
of denture use (approximately 2Æ5 mg g)1: Fig. 1,
Table 3). Moreover, the elution volume exceeded
3Æ5 mg g)1 in nine specimens obtained from dentures
used for periods between 7 and 10 years (Fig. 1), and
these nine dentures were the same as the dentures in
which the elution of MMA exceeded 10 mg g)1 (Fig. 1).
Discussion
Methyl methacrylate (MMA) analysed in the present
study is the principal ingredient of the resin monomer
in the acrylic denture base, and DME and HQ are
contained in the monomer as a cross-linking agent and
inhibitor (or stabilizer), respectively (2, 3, 11). BPO,
meanwhile, is contained as an initiator in the polymer
of heat-curing acryl resin (2, 11, 14).
Formaldehyde (FMA) is not an essential ingredient of
acrylic resin, but the elution of FMA from acrylic base
resin has been reported (16). FMA can be produced in
the initial, inhibition phase of the polymerization
process as a primary oxidant product. FMA can also
be formed as a decomposition product of oxygen–MMA
copolymer, or as an oxidation product of residual mono-
meric MMA in the polymerized denture base material
(14). BA and MB are also decomposition products of
BPO, and the former has been detected in water (11)
and methanol (7, 17), and the latter in the methanol
(7) in which dentures had been immersed. Therefore,
MB cannot be excluded from the list of analysed
substances when assessing the content of BPO.
The results of the present study showed that the
elution of MMA, FMA, HQ, BPO and MB decrease in

ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 216–220

220 M . M I K A I et al.
correlation to the number of years of denture use. On
the contrary, however, the elution of BA, which is a
decomposition product of BPO, increased with time.
This disparity may be attributable to the fact that (i) the
resin polymers manufactured over 10 years ago con-
tained more BPO than those manufactured recently, or
(ii) the resin underwent qualitative changes as a result
of long-term use, i.e. changes involving an increase in
the decomposition and elution of BPO. However, it is
beyond the scope of this study to speculate as to which
possibility is supported by the present results.
The nine dentures (used for periods between 7 and
10 years) in which high levels of MMA and BA elution
were observed (Fig. 1) all had a history of repair using
self-curing acrylic resin. The elution of MMA and BA is
known to be higher in self-curing resin than in heat-
curing acrylic resin (11). Thus, the high-level elution of
MMA and BA observed in some of the dentures in the
present study is thought to be due primarily to the
influence of the self-curing acrylic resin used in denture
repair. However, as we could not precisely determine
whether self-curing resin was contained in the im-
mersed specimens used in the present study, we cannot
rule out the influence of factors such as differences
among heat-curing acrylic resins made by different
manufacturers, differing polymerization techniques
(11, 18) and differing methods of denture management
after polymerization. As regards the BA, it is reasonable
to surmise that its elution volume when the denture is
in contact with saliva in the oral cavity will be lower
than that observed in the present study, since the BPO
eluted from resin immersed in methanol solution
decomposes into BA and MB (7).
Within the scope of the present study, it is not
possible to establish an exact standard value for the
minimum amount of the analysed chemical ingredients
necessary to induce an allergic reaction. However, our
results indicate that purported allergens exist in the
resin base and can be eluted into the oral cavity, even in
patients using an old denture for a period of nearly
30 years. The clinician cannot, therefore, assure a
patient that a resin-base denture used for many years
will not induce a resin allergy.
Acknowledgments
This study was partially supported by Grant-in-Aid for
Scientific Research (B) No. 1437063 from The Japanese
Ministry of Education, Science, Sports and Culture.
ª 2006 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 33; 216–220
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Correspondence: Dr Marie Koike, Division of Removable Prosthodon-
tics and Management of Oral Function, Department of Developmental
and Reconstructive Medicine, Course of Medical and Dental Sciences,
Nagasaki University Graduate School of Biomedical Sciences,1-7-1
Sakamoto, Nagasaki, 852-8588 Japan.
E-mail: marie-k@net.nagasaki-u.ac.jp