Environmental Research 151 (2016) 339–343

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Environmental Research

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Synthetic phenolic antioxidants, including (BHT), in resin-based dental sealants

Wei Wang a, Pranav Kannan a, Jingchuan Xue a, Kurunthachalam Kannan a,b,n a Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States b Biochemistry Department, Faculty of Science, Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia article info abstract

Article history: Resin-based dental sealants (also referred to as pit-and-fissure sealants) have been studied for their Received 28 June 2016 contribution to bisphenol A (BPA) exposure in children. Nevertheless, little attention has been paid to the Received in revised form occurrence of other potentially toxic chemicals in dental sealants. In this study, the occurrence of six 29 July 2016 synthetic phenolic antioxidants (SPAs), including 2,6-di-tert-butyl-4-hydroxytoluene (BHT), 2,6-di-tert- Accepted 30 July 2016 butyl-4-(hydroxyethyl)phenol (BHT-OH), 3,5-di-tert-butyl-4-hydroxy-benzaldehyde (BHT-CHO), 2,6-di- tert-butylcyclohexa-2,5-diene-1,4-dione (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) Keywords: and 2-tert-butyl-4-methoxyphenol (BHA), was examined in 63 dental sealant products purchased from Dental sealant the U.S. market. BHT was found in all dental sealants at median and maximum concentrations of 56.8 and Synthetic phenolic antioxidant 1020 mg/g, respectively. The metabolites of BHT and BHA were detected in 39–67% of samples, at con- BHT centration ranges of oLOQ to 242 mg/g. BHT is likely used in sealants to inhibit oxidative reactions, Human exposure Resin remove free radicals, and inhibit potential polymerization, which would eventually prolong the shelf-life of the products. The estimated daily intake (EDI) of BHT, following sealant placement, based on a worst- case scenario (application on eight teeth at 8 mg each tooth), was 930 and 6510 ng/kg bw/d for adults and children, respectively. The EDI of BHT from dental sealants was several orders of magnitude lower than the current acceptable daily intake (ADI) proposed by the European Food Safety Authority (EFSA). & 2016 Elsevier Inc. All rights reserved.

1. Introduction metabolites of BHT have been shown to elicit cellular DNA da- mage, genotoxicity, and carcinogenicity in animal models (Oikawa Synthetic phenolic antioxidants (SPAs) such as 2,6-di-tert-bu- et al., 1998). Some BHT metabolites, such as 3,5-di-tert-butyl-4- tyl-4-hydroxytoluene (BHT) are the most frequently used anti- hydroxy-benzaldehyde (BHT-CHO) and 2,6-di-tert-butylcyclohexa- oxidants in a wide range of consumer products worldwide, in- 2,5-diene-1,4-dione (BHT-Q), were reported to be more toxic than cluding foodstuffs, cosmetics, and plastics (Wang et al., 2016). Al- BHT itself (Nagai et al., 1993). Similarly, other related antioxidants, though considered safe for human health at authorized levels, such as 2-tert-butyl-4-methoxyphenol (BHA), were reported to be their widespread use, multiple sources of exposure, and con- tumor promoters, endocrine disruptors, or carcinogens (Grice, troversial toxicological data are a cause for concern (Nieva-Eche- 1988; Kahl and Kappus, 1993). Nevertheless, debate continues over varria et al., 2014). A number of animal studies have reported toxic the carcinogenic potentials of BHT and BHA (Hirose et al., 1981; effects of BHT, including carcinogenicity and reproductive toxicity Shirai et al., 1982; Williams et al., 1990; Bomhard et al., 1992; (Clapp et al., 1973; Grogan, 1986; Lindenschmidt et al., 1986; Olsen Iverson, 1995; Whysner et al., 1996; Williams et al., 1999; Botter- et al., 1986; Shlian and Goldstone, 1986; Inai et al., 1988; Takahashi, weck et al., 2000). Despite the widespread use of SPAs in various consumer products, little is known about human exposure doses. 1992; Rao et al., 2000; Al-Akid et al., 2001). Further, the Sources other than foodstuffs can contribute to BHT exposures, and, thus, such sources should be taken into account in the as- n Corresponding author at: Wadsworth Center, New York State Department of sessment of risks of BHT. Health, and Department of Environmental Health Sciences, School of Public Health, Dental sealants are thin plastic coatings that are painted on the State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States. chewing surfaces of teeth to provide a barrier for the protection E-mail address: [email protected] (K. Kannan). against bacterial decay (Truman et al., 2002; Ahovuo-Saloranta http://dx.doi.org/10.1016/j.envres.2016.07.042 0013-9351/& 2016 Elsevier Inc. All rights reserved. 340 W. Wang et al. / Environmental Research 151 (2016) 339–343 et al., 2008; Beauchamp et al., 2008; Griffin et al., 2008; Momoi nitrogen stream, and the solvent was exchanged to 150 mL ethyl et al., 2012). Dental sealants are widely used in the U.S., with 50% acetate. The remaining eight SPAs were analyzed by high perfor- of children, aged 9–11 years, have had at least one dental sealant mance liquid chromatograph-tandem mass spectrometer (HPLC- on a permanent tooth as well as 31% of children, aged 6–8 years, MS/MS). and 43% of adolescents, aged 12–19 years, in 2011–2012 (Dye et al., 2015). Human exposure to synthetic and toxic chemicals such as bisphenol A (BPA) present in dental sealants has received con- 2.4. Instrumental analysis siderable attention (Rathee et al., 2012; Kloukos et al., 2013; Van Landuyt et al., 2011). Resin-based dental sealants may release BHT was analyzed using a GC (Agilent Technologies 6890) other chemical additives, including SPAs, in the oral environment. coupled to an MS (Agilent Technologies 5975) in the selected ion Nevertheless, limited information is available on the occurrence of monitoring (SIM) mode. A fused-silica capillary column (DB-5; SPAs and their metabolites in dental sealants. Given the growing 15 m 0.25 mm i.d. 0.25 mm film thickness) was used for se- public concern about the release of toxic components from resin- paration. Other SPAs were determined using an Agilent 1260 HPLC based dental materials (Rathee et al., 2012; McKinney et al., 2014), (Agilent Technologies Inc., Santa Clara, CA, USA) interfaced with an there is a need to investigate the occurrence of SPAs in dental Applied Biosystems QTRAP 4500 mass spectrometer (ESI-MS/MS; sealants. In this study, SPAs, including BHT and its metabolites, Applied Biosystems, Foster City, CA, USA). An analytical column were determined in 21 brands of dental sealants collected in the U. (Betasils C18, 100 2.1 mm column; Thermo Electron Corpora- S. market, for the first time, to elucidate the occurrence and hu- tion, Waltham, MA, USA) connected to a Javelin guard column man exposure through dental material restoration. s (Betasil C18, 20 2.1 mm) was used for chromatographic se- paration. The negative ion multiple reaction monitoring (MRM) mode was used. Nitrogen was used as both a curtain and collision 2. Chemicals and reagents gas. The MS/MS parameters were optimized by infusion of in- fl 2.1. Sample collection dividual compounds into the MS through a ow injection system (Table A2; Appendix. A). The MRM transitions of the target che- Dental sealants (n¼63; pit-and-fissure) were purchased from micals monitored are listed in Table A3 (Appendix. A). June to August 2015 from several vendors and distributors of the products. They were products of the U.S., Korea, Greece, and 2.5. Quality assurance and quality control (QA/QC) Liechtenstein and represented popular brands used in the U.S. The resin-based sealant samples represented 18 brands, featured in With every set of 20 samples analyzed, a procedural blank, a various shades (e.g., opaque, clear, natural, off-white). Approxi- pair of pre-extraction matrix spikes (standards fortified in samples mately 20% of the samples were labeled as containing fluoride, 86% were pit-and-fissure, and all were light-cure-type (100%) prior to extraction), a pair of post-extraction matrix spikes (stan- fi products. dards forti ed in sample extracts after extraction), and duplicate samples were analyzed. Trace levels of BHA (median: 0.67 ng/g) 2.2. Chemicals and reagents were found in procedural blanks, and a background subtraction was performed when reporting concentrations of this compound. The target chemicals and their structures are shown in Table A1 Recoveries of SPAs in spiked dental sealant matrices ranged from (Appendix. A; Supporting information). BHT, BHT-d21, 2,6-di-tert- 93.0712.2% for BHT-OH to 114712.4% for BHT (Table A3). Du- butyl-4-(hydroxyethyl)phenol (BHT-OH), BHT-Q, BHT-CHO, and plicate analysis of randomly selected samples (n¼3) yielded a 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) were ob- coefficient of variation of o20%. Quantification of SPAs was per- tained from Sigma-Aldrich (St. Louis, MO, USA). Isotopically- formed by an isotope-dilution method, based on the responses of 13 13 13 labeled C12-methyl paraben ( C12-MeP; RING- C12, 99%) was 13 BHT-d21 (for BHT) and C12-MeP (for BHT-Q, BHA, BHT-OH, BHT- obtained from Cambridge Isotope Laboratories (Andover, MA, CHO, and BHT-COOH). The limits of quantification (LOQs) were USA). HPLC-grade was supplied by J.T. Baker (Phillips- 1 ng/g for BHA and BHT-Q, 2 ng/g for BHT-CHO, 4 ng/g for BHT-OH, burg, NJ, USA). Ultra-pure water (18.2 Ω) was generated using a 5 ng/g for BHT, and 10 ng/g for BHT-COOH (Table A3). A midpoint Milli-Q system (Millipore, Billerica, MA, USA). calibration standard (in methanol) was injected to check for in- strumental drift in sensitivity after every 20 samples, and a pure 2.3. Sample preparation solvent (methanol) was injected as a check for carry-over of target fi Briefly, 0.1 g of dental sealant resin was weighed and trans- chemicals. Instrumental calibration was veri ed by the injection of ferred into a 15 mL polypropylene (PP) tube. After spiking with standards at concentrations that ranged from 0.02 to 500 ng/g for 13 BHA, BHT-OH, BHT-Q, BHT-CHO, and BHT-COOH and from 0.1 ng/g BHT-d21 (40 mg) and C12-MeP (40 ng) as internal standards (IS), the sealant was extracted with 6 mL of methanol by shaking in an to 50 mg/g for BHT. The regression coefficient of the calibration orbital shaker (Eberbach Corporation, Ann Arbor, MI, USA) for curve (r2)was40.99. 60 min. The mixture was centrifuged at 4500 g for 10 min (Ep- pendorf Centrifuge 5804, Hamburg, Germany), and the super- natant was transferred into a new PP tube. Extraction was re- 2.6. Data analysis peated twice, and the supernatants were combined and passed s through an ENVI-Carb solid phase extraction (SPE) cartridge Statistical analyses (correlation analysis, test for normality, and (Sigma Aldrich, St. Louis, MO, USA), which was preconditioned ANOVA) were performed with SPSS 16.0 software. Normality of with 6 mL of methanol. Analyte residues were eluted with the data was checked by the Shapiro-Wilk test. Differences be- 3 2 mL of methanol. The elutes were combined and concentrated tween groups were compared using a one-way ANOVA, followed under a gentle nitrogen stream to 2 mL. Prior to the analysis of by a Tukey test. Prior to the one-way ANOVA, the data were log- BHT by gas chromatograph-mass spectrometer (GC–MS), 150 mLof transformed to meet the normality assumptions. The probability the methanol extract was evaporated to dryness under a gentle value of pr0.05 was set for statistical significance. W. Wang et al. / Environmental Research 151 (2016) 339–343 341

3. Results and discussion concentrations of metabolites of BHT were two to six orders of magnitude lower than the concentrations of BHT. Transformation 3.1. BHT in dental sealants of BHT occurs not only in living organisms but also in foods and abiotic environments such as air and water (Fries and Püttmann, BHT was the most frequently detected phenolic antioxidant in 2002, 2004; Fernandez-Alvarez et al., 2009; Rodil et al., 2010, resin-based dental sealants (detection rate [DR]: 100%), which 2012). Therefore, the BHT metabolites present in dental sealants suggests its widespread usage in resins (Table 1). The concentra- may have originated from abiotic transformation of BHT (Wang tions of BHT in dental sealants ranged from 0.04 to 1020 mg/g et al., 2016). However, no correlations were found between the (mean: 113, median: 56.8 mg/g). Considerable variations (up to five concentrations of BHT and its derivatives or between BHT and BHA orders of magnitude) in BHT concentrations among different concentrations in dental sealants. The formation of BHT metabo- brands of dental sealants were observed. However, no significant lites from BHT may be influenced by various environmental fac- differences were found between products that originated in dif- tors, including storage conditions. Further studies are needed to ferent countries (Table 1). BHT is likely added in sealants to inhibit elucidate the sources of BHT derivatives in dental sealants. oxidative reactions, remove free radicals, and inhibit potential polymerization, which would enable a longer shelf life of the 3.3. Release and exposure of SPAs from dental sealants products (Hamid and Hume, 1997; Michelsen et al., 2007; Seiss et al., 2009). The European Food Safety Authority (EFSA) has suggested an acceptable daily intake (ADI) of 0.25 mg/kg bw/d for BHT, based on 3.2. Other SPAs in dental sealants effects on reproduction and hepatic enzyme induction in two in- dependent 2-generation studies in rats (Olsen et al., 1986; EFSA, The derivatives/metabolites of BHT also were found in dental 2012). We estimated the release/exposure of BHT following 8 mg sealants, although at lesser frequency in comparison to BHT. BHT- sealant application (assuming that one quadrant is sealed and that OH, BHT-Q, BHT-CHO, and BHT-COOH were detected in 39%, 44%, 8 mg of sealant is used for each tooth) (Gruninger et al., 2015), and 56%, and 67%, respectively, of the samples analyzed. Among BHT the exposure dose ranged between 0.29 and 8140 ng (Table A4). metabolites, BHT-CHO was found at the highest mean concentra- This estimate is based on the concentrations of SPAs found in tion of 3.96 mg/g, followed by BHT-OH (0.95 mg/g) and BHT-Q methanol extracts of sealants, and, thus, this would represent a (0.77 mg/g). BHA was present in 39% of the sealants analyzed, at a worst-case exposure scenario. The release of BHT into saliva fol- concentration range of oLOQ–1.37 mg/g. Overall, the lowing dental sealant placement is expected to be much lower.

Table 1 Concentrations (mg/g) of synthetic phenolic antioxidants (SPAs) in dental sealants collected from the U.S. market and of various geographical origin (Korea, Greece and Liechtenstein).

No. BHT-OH BHT-Q BHT-CHO BHT-COOH BHA BHT

U.S. #1 (N¼1) 1.12 1.31 0.7 0.01 oLOQ 51.4 #2 (N¼1) oLOQ oLOQ 0.05 0.11 oLOQ 0.04 #3 (N¼3) 3.22/5.62a oLOQ 1.26/3.62 0.08/0.20 0.02/0.03 414/614 #4 (N¼15) oLOQ oLOQ 16.1/242 oLOQ 0.49/1.37 55.3/64.4 #5 (N¼4) oLOQ 0.09/0.34 oLOQ 0.02/0.06 0.002/0.008 58.0/76.9 #6 (N¼6) oLOQ oLOQ oLOQ oLOQ oLOQ 33.9/54.2 #7 (N¼2) oLOQ oLOQ oLOQ oLOQ 0.01/0.02 144/188 #8 (N¼3) oLOQ oLOQ oLOQ oLOQ 0.21/0.26 75.6 #9 (N¼3) 3.99/6.38 1.51/4.52 0.02/0.06 0.22/0.63 0.14/0.25 86.3/139 #10 (N¼1) oLOQ oLOQ 0.04 0.04 oLOQ 0.1 #11 (N¼1) oLOQ oLOQ oLOQ 0.06 oLOQ 144 #12 (N¼4) oLOQ oLOQ oLOQ 0.007/0.01 oLOQ 14.5/17.8 #13 (N¼4) 0.18/0.49 0.21/0.42 0.01/0.02 0.02/0.04 oLOQ 21.8/23.3 Median/Mean oLOQ/0.49 oLOQ/0.15 oLOQ/5.14 oLOQ/0.03 oLOQ/0.18 54.0/78.3 Min-Max oLOQ-6.38 oLOQ-4.52 oLOQ-242 oLOQ-0.63 oLOQ-1.37 0.04–614

Korea #1 (N¼4) oLOQ 4.49/10.9 0.03/0.08 0.20/0.28 0.01/0.04 153/213 #2 (N¼4) 3.17/6.08 1.45/2.24 0.23/0.76 0.03/0.05 oLOQ 113/123 #3 (N¼2) 6.13/12.0 5.88/11.8 0.94/1.09 0.04/0.04 oLOQ 88.8/177 Median/Mean 1.07/2.50 2.02/3.55 0.07/0.29 0.05/0.10 oLOQ/0.005 123/125 Min-Max oLOQ-12.0 oLOQ-11.8 oLOQ-1.09 oLOQ-0.28 oLOQ-0.04 0.06–213

Greece #1 (N¼4) 2.69/4.58a 1.16/3.28 oLOQ oLOQ oLOQ 562/1020 Median/Mean 3.09/2.69 0.68/1.16 oLOQ/oLOQ oLOQ/oLOQ oLOQ/oLOQ 554/562 Min-Max oLOQ-4.58 oLOQ-3.28 oLOQ oLOQ oLOQ 116–1020

Liechtenstein #1 (N¼1) 0.89 1.40 oLOQ oLOQ oLOQ 162

Total Median (N¼63) oLOQ oLOQ oLOQ oLOQ oLOQ 56.8 Mean 0.95 0.77 3.96 0.04 0.14 113 Min oLOQ oLOQ oLOQ oLOQ oLOQ 0.04 Max 12.0 11.8 242 0.63 1.37 1020

(# each number within a country of origin represents different brands). a “/” indicates “mean/maximum”. 342 W. Wang et al. / Environmental Research 151 (2016) 339–343

Table 2 In that study, it was shown that, even if 100 mg of dental com- Estimated daily intake of BHT (ng/kg/day) for adults and children after dental posites were applied at each office visit (Seiss et al., 2009) and a a sealant placement . theoretically maximum BHT concentration of 102 mg/L eluted from

Adults Children saliva was assumed (with an average saliva production of 1 L per U.S. 1 Tooth 4 Teeth 8 Teeth 8 Teeth Sealed day) (Graham and Mount, 2005), the EDI of BHT for adults (70 kg), sealed sealed Sealed following dental restoration, was estimated at 1.36 mg/kg bw/day, which was 250 times below the ADI. In this study, the EDI of BHT #1 5.87 23.5 47 329 for children in the worst-case scenario was estimated at 9.52 mg/kg #2 0.004 0.02 0.03 0.23 #3 47.3/70.2b 189/281 378/562 2650/3930 bw/day, 25 times lower than the ADI. #4 6.32/7.36 25.3/29.5 50.5/58.9 354/412 A few studies have reported dietary intakes of BHT. The re- #5 6.63/8.79 25.3/29.5 50.5/58.9 354/412 ported mean dietary intakes of BHT for the general populations in #6 3.88/6.19 15.5/24.8 31.0/49.5 217/347 Korea and the U.S. were 40 and 390 mg/kg bw/day, respectively #7 16.5/21.5 65.9/85.9 132/172 922/1200 #8 8.64 34.6 69.2 484 (Nieva-Echevarria et al., 2014; JECFA, 2000). The EDIs of BHT cal- #9 9.86/15.9 39.4/63.6 78.9/127 552/891 culated from dental sealants during a single clinical visit were one #10 0.01 0.04 0.09 0.61 to two orders of magnitude lower. Nevertheless, in addition to its #11 16.5 65.8 132 922 use as a dental sealant, BHT is used as a plasticizer in medical #12 1.66/2.03 6.65/8.12 13.3/16.2 93.1/114 products (e.g., syringes) (Wahl et al., 1999; Inoue et al., 2002). #13 2.49/2.66 9.97/10.7 19.9/21.3 140/149 Median/Mean 6.17/8.95 24.7/35.8 49.4/71.6 345/501 Therefore, the exposure assessment of BHT from medical products Min-Max 0.004–70.2 0.02–281 0.03–562 0.23–3930 should take into account overall exposure doses and associated Korea risks. #1 17.4/24.4 69.8/97.4 140/195 977/1360 This is the first study to report the concentrations of SPAs in #2 12.9/14.1 51.6/56.4 103/113 723/790 dental sealants that are available in the U.S. market. Measurable #3 10.2/20.3 40.6/91.1 81.2/162 568/1140 concentrations of BHT (mean: 113 mg/g, max: 1020 mg/g) in dental Median/Mean 14.1/14.3 56.4/57.2 113/114 790/801 Min-Max 0.007–24.4 0.03–97.4 0.05–195 0.38–1360 sealants suggest widespread use of this chemical as an antioxidant in these products. The highest amount of BHT that can be released Greece (worst-case scenario) from dental sealants, following the appli- #1 64.3/116 257/465 514/930 3600/6510 fi Median/Mean 63.3/64.3 253/257 506/514 3540/3600 cation in a single clinical visit, was signi cantly below the ADI. Min-Max 13.3–116 53.0–465 106–930 743–6510 However, potential additive effects of BHT with other components

Liechtenstein (e.g. bisphenols) in dental sealants need further investigation. #1 18.6 74.2 148 1040

TOTAL Median 6.49 25.9 51.9 363 Competing interests Mean 12.9 51.8 104 725 Min 0.004 0.02 0.03 0.23 All authors declare that they have no competing interests. Max 116 465 930 6510

a Body weight of 70 kg and 10 kg were used to calculate daily intake of BHT for adults and children, respectively (Gruninger et al., 2015) and with the assumption Acknowledgements that 8 mg of sealant was used for each tooth) (Gruninger et al., 2015). b “ ” “ ” / indicates mean/maximum . We thank Mr. Karl Brosch for help with the collection of samples. The body weight-normalized exposure doses of BHT were es- timated based on an average body weight of 70 kg for adults and 10 kg for children (Gruninger et al., 2015). The highest estimated Appendix A. Supplementary material daily intake (EDI) of BHT for adults was 116, 465 and 930 ng/kg bw/day when one, four, and eight teeth, respectively, were sealed Supplementary data associated with this article can be found in (Table 2). This dose pertains to the highest concentration of BHT the online version at http://dx.doi.org/10.1016/j.envres.2016.07. measured in sealants. Further, under a worst-case scenario of an 042. 8 mg sealant application at the highest concentration for eight teeth, the estimated BHT exposure was 0.93 mg/kg bw/day, which fi m was signi cantly lower than the ADI (250 g/kg bw/day). References To estimate BHT exposure in children following dental sealant application, we assumed that sealants were applied to eight pri- Ahovuo-Saloranta, A., Hiiri, A., Nordblad, A., Makela, M., Worthington, H.V., 2008. mary molars and premolars during a single office visit at an Pit and fissure sealants for preventing dental decay in the permanent teeth of amount of 8 mg to each tooth (Gruninger et al., 2015). The EDI of children and adolescents. Cochrane Database Syst. Rev., 4. BHT, following dental sealant application in children, was sig- Al-Akid, Y.F., El-Rahman, A.E.A., Hussein, H.A., Wassif, G.A., 2001. Nephro- and pneumotoxic response to chronic administration of butylated hydroxytoluene nificantly (po0.05) higher than that of adults, with median and (BHT) in adult albino rats. Al-Azhar. J. Pharm. Sci. 28, 171–195. maximum values of 363 and 6510 ng/kg bw/day, respectively Beauchamp, J., Caufield, P.W., Crall, J.J., Donly, K., Feigal, R., Gooch, B., Ismail, A., (Table 2). However, these values are below the ADI recommended Kohn, W., Siegal, M., Simonsen, R., American Dental Association Council on Scientific Affairs, 2008. Evidence-based clinical recommendations for the use of by the EFSA. The worst-case scenario of EDI for BHT, following pit and fissure sealants: a report of the American Dental Association Council on application of a sealant with the highest concentration to eight Scientific Affairs. J. Am. Dent. Assoc. 139, 257–268. m Bomhard, E.M., Bremmer, J.N., Herbold, B.A., 1992. Review of the mutagenicity/ teeth for children, was 6.51 g/kg bw/day, which was still 40-times – m genotoxicity of butylated hydroxytoluene. Mutat. Res. 277, 187 200. http://dx. below the ADI (250 g/kg bw/day). doi.org/10.1016/0165-1110(92)90043-9. Seiss et al. (2009) reported the worst-case scenario for BHT Botterweck, A.A., Verhagen, H., Goldbohm, R.A., Kleinjans, J., van den Brandt, P.A., release from 100 mg of polymerized resin-based dental composite 2000. Intake of butylated hydroxyanisole and butylated hydroxytoluene and stomach cancer risk: results from analyses in the Netherlands Cohort Study. at 0.11 mg after immersion in methanol for 24 h; this value was Food Chem. Toxicol. 38 (7), 599–605. used as the maximum concentration that could be found in saliva. Clapp, N.K., Tyndall, R.L., Cumming, R.B., 1973. Hyperplasia of hepatic bile ducts in W. Wang et al. / Environmental Research 151 (2016) 339–343 343

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