Mutagenesis, Vol. 16, No. 4, 303-307,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay
Laboratory of Microbiology, Institute of Food Analysis and Research (IIAA), University of Santiago, 15706 Santiago de Compostela, Spain
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Abstract |
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The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was ~10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.
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Introduction |
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The di-epoxide bisphenol A diglycidyl ether (BADGE) (Figure 1
) is a liquid compound obtained by condensation of two molecules of epichlorohydrin with one molecule of bisphenol A (Henry and Neville, 1982; Wicks et al., 1992). This compound is the basic monomer used as a precursor of numerous commercial epoxy resins. Epoxy-based coatings are frequently used as internal lacquer coatings of cans and storage vessels in the food industry (Tice and McGuiness, 1987; Tice, 1988). BADGE is also employed as an additive, functioning as a stabilizer and plasticizer in a variety of other plastic materials (Kroschwitz, 1987).
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In studies performed using official EU aqueous food simulants (European Commission, 1993
) it has been found (Simal Gandara et al., 1993; Paseiro Losada et al., 1997) that residual BADGE from finished epoxy coatings can be hydrolyzed and generates two degradation products, which correspond to its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively; Figure 1
). In addition to the presence of these new compounds, for which no toxicological data exist, increased concern has been expressed by the EU Scientific Committee on Food concerning the identification in the coatings of cans of chlorohydrins of BADGE (i.e. the bis-chlorohydrin of BADGE; Figure 1), due to their structural analogy to the genotoxic mono-chloropropanediol. The latter compounds are mainly formed by reaction of BADGE with chlorine ions during the curing process at high temperatures, and can migrate from the coating, or can also be formed directly in salty foods (Scientific Committee on Food, 1997, 1999).
Epoxy compounds are highly reactive bifunctional alkylating agents, which may attack and bind covalently to DNA and cause mutagenic events (Hemminki et al., 1980a). With regard to the mutagenicity of BADGE, several reports of positive results in bacterial systems have already been published (Andersen et al., 1978; Hemminki et al., 1980b; Ringo et al., 1982; Canter et al., 1986; Environmental Protection Agency, 1981). Also, studies performed with rat liver cells exposed in vitro to BADGE have shown that this compound has the capacity to induce sister chromatid exchanges and increase the percentage of cells with chromatid gaps (Environmental Protection Agency, 1981). However, no induction of unscheduled DNA synthesis in human mononucleated cells treated in vitro with this compound was reported (Environmental Protection Agency, 1977). In the absence of adequate human data the International Agency for Research on Cancer (1999) classified BADGE in Group 3 on the basis of limited carcinogenic evidence in experimental animals. Furthermore, the most recent opinion of the Scientific Committee on Food published on BADGE concluded that no evidence exists for a systematic tumourigenic effect of either pure or technical grade BADGE when topically applied, but there is still concern about its effects by oral exposure (Scientific Committee on Food, 1999).
BADGE has been included in Directive 90/128/EEC (European Commission, 1990), which contains the list of monomers and other starting substances that may be used in the manufacture of plastic materials and articles intended to come into contact with foodstuffs. This Directive establishes restriction limits with respect to the residual concentration of BADGE in plastics and its migration into food. Moreover, the latest amendment to the above Directive on monomers also includes restrictions on the hydrolysis products of BADGE and its chlorohydrins, about which there is toxicological concern (European Commission, 1999).
The present study was designed to re-examine the mutagenic effect of BADGE, as the starting product from which the other compounds derive, and to evaluate the unknown mutagenicity of the diol epoxide, bis-diol and bis-chlorohydrin derivatives of BADGE using the Salmonella mutagenicity test. Assays without and with various concentrations of S9 fraction were performed in each case to investigate possible activation–deactivation effects of the metabolic system on the compounds tested.
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Materials and methods |
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Chemicals
BADGE (2,2-[bis(4-hydroxyphenyl) propane-bis(2,3-epoxypropyl)ether]; CAS no. 1675-54-3; analytical grade), 2-aminofluorene (CAS no. 153-78-6), 2-aminoanthracene (CAS no. 613-13-8), sodium azide (CAS no. 26628-22-8) and 9-aminoacridine (CAS no. 90-45-9) were purchased from Sigma (St Louis, MO). 2,4,7-Trinitro-9-fluorenone (CAS no. 129-79-3) was obtained from Aldrich (Steinheim, Germany). The first hydrolysis product of BADGE (the diol epoxide, 2-[4-(2,3-epoxypropanyloxy)phenyl-2,4-(2,3-dihydroxypropanyloxy)phenyl]propane) and the second hydrolysis product of BADGE (the bis-diol, 2,2-bis[4-(2,3-dihydroxypropanyl)phenyl]propane) were kindly provided by Prof. P.Paseiro (University of Santiago de Compostela, Spain), who purified the compounds as described by Simal Gándara et al. (1993). The chlorohydrin of BADGE [the bis-chlorohydrin, bisphenol A bis(3-chloro-2-hydroxypropyl)ether; CAS no. 4809-35-2] was purchased from Fluka (Buchs, Switzerland). Dimethylsulphoxide (DMSO) was supplied by Merck (Darmstadt, Germany). All other chemical reagents were of the highest commercial quality available. The chemical structures of BADGE and the BADGE derivatives studied are shown in Figure 1
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Bacterial strains
The mutagenicity assay was performed using Salmonella typhimurium strains TA98 (hisD3052, rfa, 
uvrB, pKM101), TA100 (hisG46, rfa, uvrB, pKM101), TA1537 (hisC3076, rfa, uvrB) and TA1535 (hisG46, rfa, uvrB) which were kindly supplied by Prof. B.N.Ames (University of California, Berkeley, CA). All compounds were tested with these four strains except for the bis-chlorohydrin of BADGE, for which TA1537 was not used.
Mutagenicity assays
The standard S.typhimurium plate incorporation assay was carried out in this study (Maron and Ames, 1983). The S9 fraction from the livers of male Sprague–Dawley rats pretreated with a phenobarbital/methylcholanthrene mixture was purchased from Iffa Credo (l'Arbesle, France) and used as the external metabolic activation system at various final concentrations in the S9 mix. Final S9 concentrations of 2, 4, 10 and 20% were used for the hydrolysis products and the chlorohydrin of BADGE with all the strains on which they were tested and with strains TA98 and TA1537 for BADGE. This compound was also tested with strain TA100 using 2, 4 and 10% S9 fraction and with TA1535 using 4, 10 and 20%. The concentrations of cofactors in the S9 mix (before adding them to the overlay) were 4 mM NADP, 5 mM glucose 6-phosphate, 33 mM KCl, 8 mM MgCl2 and 100 mM sodium phosphate buffer (pH 7.4). For the assay a mixture of a bacterial strain culture grown overnight in Oxoid nutrient broth No. 2 (100 µl), test compound in DMSO or DMSO alone (100 µl) and either 0.2 M sodium phosphate buffer (pH 7.4) or freshly prepared S9 mix (500 µl) was added to 2 ml of 0.05 mM histidine/biotin top (soft) agar. This mixture was layered on minimal glucose agar plates that were then incubated at 37°C for 72 h, after which the revertants per plate were counted.
The positive controls without S9 mix were sodium azide for TA100 (1 µg/plate) and TA1535 (0.5 µg/plate), 2,4,7-trinitro-9-fluorenone for TA98 (0.01 µg/plate) and 9-aminoacridine for TA1537 (50 µg/plate). When the assays were carried out with S9 mix the positive controls were 2-aminofluorene for TA98 (1 µg/plate) and TA100 (2.5 µg/plate) with 4% S9 fraction in the S9 mix and 2-aminoanthracene for TA1535 and TA1537 (both at 50 µg/plate) with 10% S9 fraction. The positive controls were dissolved in DMSO except for sodium azide, for which purified water (MilliRo; Millipore, Bedford, MA) was used. Purified water (100 µl) was also used as a negative control in this case.
At least two complete assays were performed for each tested compound. All experiments were carried out in triplicate using a minimum of five doses. When the compound under study showed a negative response at low doses it was then assayed at either the maximum tolerated dose, based upon a background lawn evaluation, or its solubility limit in DMSO. Data from one representative assay for each compound with the test doses evaluated are shown in Results.
Statistical evaluation
The statistical analysis of the data was based on biological mechanistic models proposed by Margolin et al. (1981). The SALM program (Kim and Margolin, 1999) was used to fit Margolin's models to the data and select the model with the greatest likelihood. The goodness of fit test calculated for each data set allowed us to determine whether the data followed the fitted model. The estimated slope of the initial linear region (no. of revertants/µg) was used as a measure of mutagenic potency and to determine mutagenicity via a significance test. In this way a positive response was obtained when the mutagenicity test P value was <0.05. A chemical was deemed to be mutagenic if at least one strain/activation combination yielded a reproducible positive response.
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Results |
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Table I
shows that BADGE has the ability to induce mutations in strains TA100 and TA1535, which revert by base pair substitutions. The dose levels assayed for this starting monomer were between 10 and 2000 µg/plate in these strains. In the absence of added metabolic activation the increase in the number of revertants for TA100 was 2.294 per µg BADGE. The response obtained with TA1535 was much lower but still positive (P < 0.05). Addition of rat liver homogenate for metabolic activation also produced a positive response in both strains. However, the induced revertants were strongly reduced in TA100 (ranging from 0.988 to 0.583 revertants/µg), whereas they were increased in TA1535 (between 0.764 and 1.206 revertants/µg). On the other hand, negative results were obtained for BADGE in strains TA98 and TA1537 at the doses tested (between 500 and 5000 µg/plate), either without or with various concentrations of S9 fraction in the S9 mix (data not shown). These latter data indicate that the compound did not induce frameshift mutations in the test system. No toxicity was evident at the highest dose assayed (5000 µg/plate).
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Table II
summarizes the results obtained with the diol epoxide of BADGE (from 50 to 2000 µg/plate) when strains TA1535 and TA100 were used in the assay. This compound induced mutations in TA100, either in the presence (0.135–0.106 revertants/µg) or absence (0.162 revertants/µg) of S9 fraction. A positive response was only obtained in TA1535 when the external metabolic system was incorporated (between 0.072 and 0.262 revertants/µg). In both strains the number of revertants induced was ~10 times lower than with BADGE. Like BADGE, the diol epoxide of BADGE had no effects in TA98 and TA1537 at the doses tested (between 100 and 2000 µg/plate; data not shown). In this case the highest dose tested with these strains was lower than with BADGE because of the lower solubility of the compound in DMSO.
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Tables III and IV
list data obtained for the bis-diol and bis-chlorohydrin of BADGE, respectively, both in the absence and presence of one concentration of S9 fraction selected from among those previously tested. As shown in Table III, the bis-diol of BADGE was negative in the Ames Salmonella assay at doses from 500 to 5000 µg/plate. Absence of induced revertants was also found for the bis-chlorohydrin of BADGE in strains TA98, TA100 and TA1535 (Table IV). In this case the doses tested with TA98 and TA100 ranged from 500 to 5000 µg/plate. Concentrations of between 50 and 500 µg/plate without S9 fraction and from 50 to 2000 µg/plate with S9 were used to estimate the potential mutagenic activity of the compound with TA1535, due to its toxicity at the highest levels tested (up to 1000 µg/plate without S9 fraction and up to 2000 µg/plate with S9). Strain TA1537 was not used with this compound. A negative response was also obtained for both compounds with the other proportions of S9 fraction employed (data not shown).
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Discussion |
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The present study shows the mutagenic potential of BADGE in the Ames Salmonella assay by virtue of its ability to induce base pair substitutions, both in the presence and absence of added metabolic activation. These results give further support to those previously published using this starting compound in bacterial systems (Andersen et al., 1978
; Hemminki et al., 1980b; Ringo et al., 1982; Canter et al., 1986; Environmental Protection Agency, 1981; International Agency for Research on Cancer, 1999).
This work also provides the first experimental data on the mutagenic potential of the two hydrolysis products of BADGE and one of its chlorohydrin derivatives. As might be expected, the diol epoxide of BADGE was less mutagenic than the di-epoxy monomer BADGE (by a factor of 10) and no mutagenic activity was shown by the bis-diol or bis-chlorohydrin of BADGE, which have no epoxy rings. These data are also consistent with the view that diol compounds are less toxic than their epoxy starting products when the potential genotoxic hazard associated with their presence in canned foodstuffs is assessed. The Ames Salmonella test thus suggests that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE. These findings imply that food cans with BADGE-based linings are a greater potential risk to the consumer when used for oily foods, in which BADGE is protected against hydrolysis, than when used for aqueous foods (Philo et al., 1997; Scientific Committee on Food, 1999).
On the other hand, it was also shown that deactivation of BADGE and its diol epoxide derivative in TA100 was directly associated with an increased proportion of S9 fraction in the S9 mix. A reverse effect was found for both compounds when the same assays were carried out with strain TA1535. These differences could be associated with different metabolic routes by which the compounds are processed in the two strains. This hypothesis, as previously reported by Andersen et al. (1978), is supported by the finding that the mutagenicity of BADGE in the presence of S9 was NADP-dependent in TA1535 but not in TA100 (data not shown). Studies made in vivo with mice by Climie et al. (1981) have shown that BADGE is rapidly metabolized. They proposed that the major route of transformation is by hydrolytic ring opening to form the bis-diol of BADGE, which occurs enzymatically through epoxide hydrolase. This metabolite is then oxidized to carboxylic acid derivatives or undergoes oxidative dealkylation to form a phenol and glyceraldehyde. Given that epoxide hydrolase is active in rat liver microsomal preparations without NADP, this route could explain the reduction in activity observed for both epoxy compounds in strain TA100 with incorporation of S9 mix in the assay. In relation to the activation found for both compounds in strain TA1535, it is suggested that incorporation of S9 fraction in the assay led to formation of metabolites which are more mutagenic than the starting products. In addition, it is not an uncommon observation that S9 fraction can enhance the mutagenicity of alkylating agents (Canter et al., 1986).
Finally, we consider that an important extension of this work would include an examination for genotoxicity of these derived products of BADGE in mammalian assay systems using different end-points. This aspect is currently in progress in our laboratory.
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Acknowledgments |
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We are grateful to Prof. Paseiro for providing us with the two hydrolysis products of BADGE and to María Jose Blanco, Begoña Buceta and Beatriz Freire for their excellent technical assistance. The authors are also grateful to Jose Antonio Veira for his help in editing the manuscript. This work was supported financially by the Xunta de Galicia (XUGA 20316B95).
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Notes |
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1 To whom correspondence should be addressed. Tel: +34 981 563 100; Fax: +34 981 547 171; Email: mprosaan{at}usc.es
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References |
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Received on October 31, 2000; accepted on January 24, 2001.
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