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
Sciences, Nagasaki, Japan 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

SUMMARY Allergenic ingredients extracted from used denture base resin were quantitatively analysed using a gas chromatograph/mass spectrometer and high performance liquid chromatography. Methyl methacrylate (MMA), hydroquinone (HQ), formaldehyde (FMA), benzoyl peroxide (BPO), benzoic 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 continuing to be evident even after 29 years of use.

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 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. KEYWORDS: acrylic resin, allergenic ingredient, GCMS, high-performance liquid chromatography Accepted for publication 10 November 2004

Introduction
Acrylic denture base resin (acrylic resin) is a useful biomaterial in prosthetic dentistry, but clinicians occasionally encounter allergic reactions to chemical ingredients 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 allergenic ingredients in used denture base resin, namely,
ª 2006 Blackwell Publishing Ltd

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 Denture groups and duration of their usage in years Short-term denture, 0Æ0 <  < 5Æ0 Middle-term denture, 5Æ0 £  < 10Æ0 Long-term denture, 10Æ0 £  < 30Æ0 Upper 26 13 6 Lower 6 0 8 Partial denture Upper 44 9 8 Lower 0Æ41 (0Æ05) 20 10 2 20Æ90 (3Æ45) 7Æ79 (0Æ70) 2Æ78 (0Æ84) Table 2. Size of specimens (mean and s.d.) Weight (g) Length (mm) Width (mm) Thickness (mm)

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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 hereafter 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 interviews 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 cleaning 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.

The length and width of the specimens were measured 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 borosilicate 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 spectrometer (GC/MS) (GCMS-QP5050A)‡‡ equipped with a 60 mm · 0Æ32 mm I.D. capillary column (0Æ5 lm membrane 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.

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&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.

§

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

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mg g–1 25·00 MMA 12·50 0·10 mg g–1 0·20 DME

0·00 1·20 FMA 0·60

0·00 1·00 HQ

0·50

0·00 4·00 BPO 2·00

0·00 5·00 BA

2·50

0·00 1·00 MB 0·50

0·00

0

10

20

30 Year

0·00

0

10

20

30 Year

Fig. 1. Allergenic ingredients in eluate extracted from used denture base resin analysed by high-performance liquid chromatography and gas chromatograph/mass spectrometer. MMA, methyl methacrylate; DME, ethyleneglycol dimethacrylate; FMA, formaldehyde; HQ, hydroquinone; BPO, benzoyl peroxide; BA, benzoic acid, MB: methyl benzoate; Abscissa, duration of denture use in 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 solution (pH 2Æ6) and acetonitrile were used. The acetonitrile 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 injection volume was fixed at 10 lL. An absorbance detector (High Sensitivity UV-Vis Detector SPD-10 AVVP)‡‡ was
‡‡

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.

Shimadzu Co., Kyoto, Japan.

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

INGREDIENTS FROM USED DENTURE BASE RESIN
Table 3. Allergenic ingredient contents of eluate extracted (mg g)1) from the sample of denture base resin

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Subgroup of denture tested and their number

MMA

DME

FMA

HQ

BPO

BA

MB

Short-term denture (n ¼ 96) med. 3Æ5377 min. 1Æ8646 max. 11Æ3627 Middle-term denture (n ¼ 32) med. 3Æ8278 min. 1Æ9512 max. 22Æ0436 Long-term denture (n ¼ 24) med. 2Æ0227 min. 1Æ4327 max. 12Æ4234

0Æ0013 0Æ0000 0Æ0157 0Æ0015 0Æ0000 0Æ1960 0Æ0000 0Æ0000 0Æ0102

0Æ3060 0Æ1326 1Æ0806 0Æ1963 0Æ0109 0Æ5040 0Æ0604 0Æ0165 0Æ2629

0Æ1684 0Æ0508 0Æ8420 0Æ1174 0Æ0356 0Æ3405 0Æ0366 0Æ0049 0Æ2807

1Æ8598 0Æ7973 3Æ3085 0Æ6722 0Æ0873 1Æ9214 0Æ1929 0Æ0103 1Æ0041

1Æ7609 1Æ1285 2Æ6484 2Æ6140 1Æ6393 4Æ6769 2Æ5320 1Æ6985 4Æ6070

0Æ5788 0Æ0000 0Æ9144 0Æ3690 0Æ0000 0Æ4730 0Æ3472 0Æ1367 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 longterm 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 shortterm 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 middleterm 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 monomeric 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

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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 contained 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 heatcuring 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 immersed 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.

References
1. Crissey JT. Stomatitis, dermatitis, and denture materials. Arch Dermatol. 1965;92:45–48. 2. Anusavice KJ. Phillips’ science of dental materials, 10th edn. Philadelphia (PA): W. B. Saunders; 1996:238–239, 265–268. 3. Fisher AA. Contact stomatitis, glossitis, and cheilitis. Otolaryngol Clin North Am. 1974;7:827–843. 4. Gebhardt M, Geier J. Evaluation of patch test results with denture material series. Contact Dermatitis 1996;34:191–195. 5. Danilewicz-Stysiak Z. Allergy as a cause of denture sore mouth. J Prosthet Dent. 1971;25:16–18. 6. Kanzaki T, Kabasawa Y, Jinno T, Isayama K. Contact stomatitis due to methyl methacrylate monomer. Contact Dermatitis 1989;20:146–148. 7. Hongo T, Sato A. Elution patterns of leachable components from a polymeric organic material. J J Dent Mater. 1996;15(Special Issue 28):172–173. 8. Hongo T, Sato K, Sato A. Amounts of residual monomer leached from clinically used acrylic resin denture base. J J Dent Mater. 1994;13(Special Issue 23):252–253. 9. Kalipcilar B, Karaagaclioglu L, Hasanreisoglu U. Evaluation of the level of residual monomer in acrylic denture base materials having different polymerization properties. J Oral Rehabil. 1991;18:399–401. 10. Jerolimov V, Krhen J, Besic J. The role of residual monomer in PMMA powder and methods of polymerization in the finding of residual monomer in poly (methylmethacrylate) denture base. Acta Stomatol Croat. 1991;25:17–23. 11. Koda T, Tsuchiya H, Yamauchi M, Hoshino Y, Takagi N, Kawano J. High-performance liquid chromatographic estimation of eluates from denture base polymers. J Dent. 1989;17:84–89. 12. Sadamori S, Kotani H, Hamada T. The usage period of dentures and their residual monomer contents. J Prosthet Dent. 1992;68:374–376. 13. Kiec-Swierczynska M. Occupational allergic contact dermatitis due to acrylates in Lodz. Contact Dermatitis. 1996;34:419–422. 14. Devlin H, Watts DC. Acrylic ’Allergy’? Br Dent J. 1984;20:272–275. 15. Wray D, Rees SR, Gibson J, Forsyth A. The role of allergy in oral mucosal diseases. Q J Med. 2000;93:507–511. 16. Wennstom A. Formaldehyde content and cytotoxicity of ¨ some denture base materials. Odontol Tidskr. 1966;74:212–216. 17. Fujisawa S, Masuhara E. Analysis of eluate extracted from dental filling resin by high performance liquid chromatography. J Japan Soc Dent Appar Mater. 1979;20:121–131. 18. McCabe JF, Basker RM. Tissue sensitivity to acrylic resin. A method of measuring the residual monomer content and its clinical application. Br Dent J. 1976;140:347–350.

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.

Correspondence: Dr Marie Koike, 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,1-7-1 Sakamoto, Nagasaki, 852-8588 Japan. E-mail: marie-k@net.nagasaki-u.ac.jp

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

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