Evidence of Absence: Estrogenicity Assessment of a New Food

Article pubs.acs.org/est

Evidence of Absence: Estrogenicity Assessment of a New FoodContact Coating and the Bisphenol Used in Its Synthesis Ana M. Soto,† Cheryl Schaeberle,† Mark S. Maier,‡ Carlos Sonnenschein,† and Maricel V. Maffini*,§ †

Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, Massachusetts 02111, United States ‡ The Valspar Corporation, Packaging Division, Sewickley, Pennsylvania 15143, United States § Independent Consultant, Germantown, Maryland 20874, United States S Supporting Information *

ABSTRACT: Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using in vitro and in vivo assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the ESCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible.

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INTRODUCTION Polymeric coatings used inside metal food and beverage cans are essential to protect against corrosion, microorganisms, and metallic taste.1 4,4′-Methylenediphenol polymer chain functionality (Figure 1A) imparts unique physical properties to polymeric coatings not available from other materials such as olefins, acrylics, or polyesters.2 In particular, properly cured 4,4′-methylenediphenol polyether polymers have closely packed floccules that result in flexible, high integrity barriers able to protect food for long periods under extreme conditions across an entire spectrum of metal food-contact applications.3 Bisphenols are the only commercially viable starting materials from which to derive this critical 4,4′-methylenediphenol functionality. Bisphenol A (BPA), the most commonly used monomer, has become the subject of intense scrutiny with respect to its estrogenic activity (EA) and growing concern about health effects associated EA, including reproductive and developmental effects.4,5 This has created a substantial demand for alternatives lacking EA. However, other bisphenols either have scant health-effects data or have demonstrated EA.6−8 The challenge, therefore, has been to find a novel bisphenol that lacks EA and that will impart technical performance and integrity into polymers necessary to ensure food safety. To meet this challenge, we used the Safety by Design assessment framework as a guidance principle. This framework identifies testable critical biological effects based on molecular © XXXX American Chemical Society

structure−activity relationship, a priori knowledge, and plausible exposure levels from food-contact materials. Within this context, EA was selected as a testable biological effect based on the structure activity of bisphenols and a priori knowledge of available testing methods. This was complemented by quantitative analytical methods for measuring migration to inform on potential effects at plausible human exposure levels. On the basis of structure−activity, tetramethyl bisphenol F (TMBPF; CASN 5384-21-4) was selected as a potential monomer candidate since predictive modeling indicated TMBPF was unlikely to bind to estrogen receptor (ER). Using ligand-based virtual screening, TMBPF was selected as a potential monomer candidate because it exhibits an unusually low score among bisphenols for ER ligand activity by surface similarity order (LASSO).9 Also, TMBPF can be reacted in a unique new way to separate it from the final polymerization step, effectively eliminating unbound chemical from the final product (Figure 1A), while imparting the needed functionality into the polymer.10 An additional polymer development challenge is to ensure that migrants from the final product, usually a mixture of reaction byproducts and impurities, also Received: September 16, 2016 Revised: December 20, 2016 Accepted: January 3, 2017

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DOI: 10.1021/acs.est.6b04704 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

Figure 1. (A) Route of synthesis for the polymeric coating. TMBPF provides essential 4,4′-methylenediphenol functionality (red). Unlike BPAbased polymers, TMBPF is not used in the final polymerization reaction and, despite its name, is not made from bisphenol F. In BPA-based polymers, BPA monomer (inset) provides 4,4′-methylenediphenol functionality and is also used instead of the aromatic diol. Low levels of unreacted BPA monomer from the final polymerization step may remain in the polymer and be subject to migration. In contrast, due to the inherent reactivity of the substituents with each other and subsequent heat-treated polymerization and curing steps, TMBPF and TMBPF-DGE were not detected in the simulants (LOD = 0.2 ppb). (B) E-SCREEN dose−response curves for TMBPF, positive control 17β-E2, and estrogenic activity (EA) reference substance BPA. TMBPF showed no estrogenic activity. In contrast, both BPA and 17β-E2 displayed typical estrogen-dependent cell proliferation dose−response curves. Cell proliferation is expressed as the average ratio over controls (# cells obtained after exposure to test substance divided by # cells in the untreated control wells) for five experiments. (C) TMBPF induced cytotoxicity at 1 μM and above. Cytotoxicity is evidenced when 100 pM 17β-E2 fails to allow maximal cell proliferation.

bioassay,13 the juvenile rat uterotrophic assay,14 and pubertal developmental assays in both male and female rats.15 Mammary gland development was also assessed in pubertal females.16 The amalgamated migrant from the finished polymeric coating was assessed by E-SCREEN.

lack EA since it has been stated that the biological properties of food contact polymers cannot be inferred by the properties of the starting substances.11 Here, we present the results of a collaborative and integrative approach to test the hypothesis that TMBPF lacks EA and does not persist in the final polymer. We also describe steps taken to assess potential EA of the amalgamated substances that may migrate from the polymeric coating into food and the qualitative analysis of the extracted mixture. Our findings indicate that TMBPF lacks EA when assessed using in vitro and in vivo studies and its migration from the final polymer is negligible. Furthermore, the migrants extracted from the finished polymer also lack EA. TMBPF was studied using estrogen receptor (ER) transactivation assay,12 E-SCREEN

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EXPERIMENTAL SECTION

Test Chemical. Tetramethyl bisphenol F (TMBPF) (lot J14032, 99.63% pure), used in all studies, was purchased from DeePak Chemicals Group (Ahmedabad, India). TMBPF assayed by E-SCREEN was analytically verified by Colorado State University (CSU) who retained an aliquot and transferred the remaining material to Tufts University. TMBPF used for in

B


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flow of 11 L/min, nozzle voltage of 1000 V, skimmer voltage of 65 V, fragmentor voltage of 150 V, and octopole RF voltage of 750 V. The mass range measured was 100−1100 m/z. The raw data files were mined for mass features of interest using the MassHunter Qualitative software (Agilent). Additional details are found in Supporting Information pages S4−S6. Estrogen Receptor Transactivation Assay. Chemicals. 17β-Estradiol (17β-E2), 98% pure, was purchased from SigmaAldrich (St. Louis, Missouri). TMBPF was provided by Valspar. Transactivation Assay. A variant of human breast cancer estrogen-responsive MCF7 cells containing the firefly luciferase gene under transactivational control of the ER (VM7Luc4E2) was used. The assay was conducted per the Organization for Economic Co-Operation and Development (OECD) Test Guideline 455 Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonist.12 Briefly, ER activation induces the expression of luciferase whose activity is measured as relative light units (Supporting Information pages S9−S11). E-SCREEN Bioassay. Chemicals. 17β-E2 (>97% pure) was purchased from Calbiochem and BPA (>99% pure) from Sigma-Aldrich (St. Louis, Missouri). TMBPF (99.63% pure) was provided by Valspar via Colorado State University, and oligomeric extracts were provided by Valspar via FERA. 17βE2, BPA, and TMBPF chemical stock solutions and dilutions were made in DMSO and stored at −20 °C. E-SCREEN. Human breast cancer estrogen-sensitive MCF7 cells were used.21 The assay was described elsewhere.13 Briefly, 24 h after seeding, cells were rinsed and the medium was replaced with either (a) TMBPF or (b) polymeric coating extracts both diluted with 5% charcoal-dextran stripped FBS (CDFBS) in phenol red-free medium. Each experiment included a 15-point 17β-E2 standard dose−response curve (0.05 pM to 10.0 nM) run simultaneously with the samples. BPA was included as EA reference chemical (0.1 nM to 10 μM). Eight concentrations of TMBPF (10 pM−100 μM) and seven concentrations of each of the polymeric coating extracts, 0.5−25 ppm (ppm), were tested in duplicate, plus negative (5% CDFBS) and positive (5% CDFBS + 100 pM 17 β-E2) controls. Assays were repeated three times. The lack of dose− response was further evaluated to assess whether it was due to absence of EA or cell toxicity. This was performed by adding 100 pM 17 β-E2 to each TMBPF and polymeric coating extracts solutions to induce maximal proliferation. Lack of toxicity is demonstrated by maximal proliferating activity in all E2-containing wells. Cell Counting and Data Analysis. Cell number was measured on day six post-treatment after fixing and staining the cells using sulforhodamine B dye; aliquots were transferred to 96-well plates and scanned using a microplate reader.22 The 17β-E2 dose−response curve was used as standard to quantify estrogenic activity of test samples in estradiol equivalents (E2Eq) (Supporting Information page S11). Quantitative Real Time PCR (q-RTPCR). Four genes previously identified as being either up- or down-regulated by ER23 were used to identify whether spikes in cell proliferation were associated with EA. Progesterone receptors A and B (PGRAB), BCL2-Interacting Kill (BIK), apolipoprotein D (APOD), and WNT1 inducible signaling pathway protein 2 (WISP2) were assessed using q-RTPCR. L19 was used as a normalizing housekeeper gene.

vivo assays was analytically verified by each contract laboratory (Figures S1−S4). Polymeric Coating Extracts. Oligomeric migrants were obtained by exposing cured polymer-coated metal panels to 50% aqueous ethanol (v/v) or 3% aqueous acetic acid (w/v) under standardized food simulant conditions per US Food and Drug Administration guidance on food contact substance migration procedures.17 Acetic acid, as 3% aqueous solution, is meant to represent acidic foods, like tomato products and fruits, while 50% aqueous ethanol covers aqueous, fatty, and alcoholic foods. These simulants represent the full range of canned foods and beverage in which the TMBPF-based polymer is expected to be used. After exposure to food simulants, aliquots of these migrant solutions were either set aside or evaporated and redissolved in dimethyl sulfoxide (DMSO). These solutions were submitted for analysis by Valspar to FERA,18 a national reference laboratory for materials and articles in contact with food. TMBPF Migration Quantification and Migrant Composition. TMBPF monomer was determined by liquid chromatography and tandem mass spectrometry (LC-MS/ MS) using electrospray ionization in negative mode. A calibration line was prepared using authentic TMBPF standards at concentrations of 1.3, 3.3, 6.8, 13, and 27 ng/mL and produced a linear line with R2 value of 0.997. The limit of detection (LOD) for TMBPF in migration solutions was 0.2 μg-migrant/6 dm2 of panel surface area or 0.2 ppb (surface to volume ratio assumed to be 6 dm2 to 1 kg food)19 for both 3% acetic acid and 50% ethanol simulants. The LOD was calculated as three times the signal-to-noise of the TMBPF response in an overspiked aliquot of the simulant. Analysis was by LC-MS/MS using an Acquity-TQS system (Waters, Wilmslow, UK). See detailed information in Supporting Information pages S4−S6. TMBPF was ionized using negative electrospray ionization (ESI) with the quantification transition of 255.2 > 132.8 (collision energy, 27 eV) and confirmation transition of 255.2 > 104.7 (collision energy, 25 eV), with both dwell time of 0.08 s and cone voltage of 30 V. Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) was used to determine masses of the oligomeric migrants, a major component of so-called “nonintentionally added substances” (NIAS) that migrate from polymeric coatings.20 The primary aim of this assessment was to verify that the NIAS oligomeric material sent to be tested in the ESCREEN was representative of migrants expected from the finished polymeric coating and, secondarily, to provide mass abundance data for qualitative comparison of migration samples. Samples of the unexposed simulant solutions and DMSO solvent were used as procedural blanks; TMBPF was used as a standard addition overspike for TMBPF determination (Supporting Information pages S4−S6). The reporting limit for the migrant concentrates redissolved in DMSO were 0.06 μg/6 dm2 (0.06 ppb) and 0.01 μg/6 dm2 (0.01 ppb) for 3% acetic acid and 50% ethanol, respectively. Analysis was by LC-TOF-MS (Agilent, Santa Clara, CA, USA) consisting of a 1200 Series LC and a 6230 time-of-flight mass spectrometer. Chromatographic separation was achieved on an Atlantis dC18, 150 × 2.1 mm, 3 μm column (Waters) kept at 30 °C. TOF-MS detection was carried out in positive and negative mode electrospray with nebulizer pressure of 40 psi, capillary voltage of 4000 V (positive ionization) or 3500 V (negative ionization), gas temperature of 350 °C, drying gas flow of 9 L/min, sheath gas temperature of 380 °C, sheath gas C


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examination. Tissues were processed into paraffin blocks, sectioned, and stained with hematoxylin and eosin (H&E). Statistical Analysis. Statistical tests followed EPA testing guidelines. Mean values were presented with standard deviations. The ratio of uterus weight relative to final body weight was subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett’s test or a two-sample t test was used to compare TMBPF-treated and EEtreated groups to the vehicle control. Pubertal Assay. Chemicals. Corn oil (Spectrum Chemicals, New Brunswick, NJ) was used as a vehicle in the preparation of TMBPF. Study Design. The protocol and number of animals followed EPA guidelines for the assessment of pubertal development and thyroid function in juvenile male and female rats.15 Fifteen PND22 females and 15 PND23 males were assigned to each group; one female and one male per litter were allocated to each treatment group. Females were euthanized on PND42 and males on PND53. The study was GLP compliant. Doses and Treatment. Animals were dosed orally by gavage once daily; females’ treatment was from PND22 to PND42, inclusive; males were treated from PND23 to PND53, inclusive. The oral dosage volume was 5 mL/kg bw. The treatment groups were as follows: Group 1, vehicle control; Group 2, TMBPF 200 mg/kg bw/d (TMBPF 200); Group 3, TMBPF 600 mg/kg bw/d (TMBPF 600). The doses were selected on the basis of uterotrophic assay outcomes. Table 1 summarizes the groups and doses. Animals. Animals were housed in Envigo Research facilities (Princeton, NJ); it is fully accredited by AAALAC International, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.25 Timemated F0 female Sprague-Dawley [Crl:CD(SD)] dams were purchased from Charles River Laboratories (Raleigh, NC). F1 pups were weaned on PND21 (Supporting Information page S15). Pubertal Maturation Evaluation. Females were examined for vaginal opening (VO) daily beginning on PND22 and continuing until completion of the VO. Daily vaginal smears to assess estrous cyclicity began on the day VO was achieved. Males were examined for preputial separation (PPS) daily, beginning on PND23. Tissue Collection and Histopathology. Male and female reproductive organs were collected, weighed, and preserved in 10% neutral buffered formalin. Female mammary glands were also collected; the left fourth and fifth glands were prepared for histology while the contralateral glands were whole-mounted, fixed, and stained with carmine-alum. Tissue processing and analysis were described elsewhere.26 Microscopic examination was performed on reproductive organs and mammary glands from all animals in the vehicle control and both TMBPF-dosed groups. A qualitative assessment of the whole-mounted mammary glands was performed using a grading system based on prevalence of terminal end buds (TEB), branching and budding, and ductal extension: minimal, slight, moderate, marked, and severe. Statistical Analysis. For all parameters, significant differences between control and TMBPF-treated groups were expressed as p < 0.05 or p < 0.01. The statistical analysis was performed following the EPA testing guideline.15 Briefly, parameters to be analyzed were identified as continuous, discrete, or binary. TMBPF-treated groups were then compared

After conditioning the cells with 5% CD-FBS medium, estradiol (positive control, final concentration of 0.1 nM) or TMBPF (0.1, 1, and 2.5 μM) was added to the cells and incubated for 48 h (Supporting Information page S12 and Table S2). Uterotrophic Assay. Chemicals. Corn oil (Spectrum Chemical, New Brunswick, NJ) was used as the vehicle in the preparation of TMBPF and positive control 17αethynylestradiol (EE, 99% pure) (Sigma, St. Louis, MO) and for the administration to the control group. Study Design. The test system was the immature female Sprague-Dawley rat. The protocol and number of animals were in accordance with U.S. Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program Test Guideline OPPTS 890.1600.14 Six postnatal day (PND) 18 females were assigned to each group and treatment began at PND19. All females were euthanized by intraperitoneal injection of sodium pentobarbital at PND22, approximately 24 h after the last dose was administered. The study was conducted in compliance with Good Laboratory Practice (GLP) standards. Doses and Treatment. EE was used at a 0.04 mg/mL final concentration. TMBPF formulations were prepared in corn oil at 20, 60, and 200 mg/mL, respectively. Animals were dosed orally by gavage once daily for three consecutive days from PND19 to 21.14 The oral dosage volume was 5 mL/kg body weight (bw). The treatment groups were as follows: Group 1, vehicle control; Group 2, EE 0.2 mg/kg bw/day (d) (EE); Group 3, TMBPF 100 mg/kg bw/d (TMBPF 100); Group 4, TMBPF 300 mg/kg bw/d (TMBPF 300); Group 5, TMBPF 1000 mg/kg bw/d (TMBPF 1000). The doses were selected at intervals that were expected to detect effects associated with EA but not to cause toxicity.24 Table 1 summarizes the groups and doses. Table 1. Animal Studies Treatment Groups group 1 2 3 4 5 1 2 3

substance

dosage level (mg/kg/d)

Uterotrophic Assay (n = 6/group) vehicle control 17α-ethynylestradiol (positive control) TMBPF TMBPF TMBPF Pubertal Assay (n = 15/group/sex) vehicle control TMBPF TMBPF

0 0.2 100 300 1000 0 200 600

Animals. Animals were housed in WIL Research facilities (Ashland, OH) (Supporting Information page S13); it is fully accredited by AAALAC International, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.25 Sprague-Dawley [Crl:CD(SD)] dams with 10 pups/dam were purchased from Charles River Laboratories (Raleigh, NC). All rats were weaned at PND18, weighed, examined for physical abnormalities, and assigned randomly to each of the treatment and controls groups. Tissue Collection and Histopathology. Uteri were dissected and trimmed; each “wet” uterus was weighed intact (with the luminal fluid), then opened longitudinally and blotted with filter paper to remove the luminal fluid, and weighed again. The vagina and mammary glands were also dissected. Tissues were preserved in 10% neutral-buffered formalin for histopathologic D


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Environmental Science & Technology to the control using various statistical tests depending on the parameter classification and whether the dose−response was monotonic or nonmonotonic.

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RESULTS TMBPF Migration Was below the Level of Analytical Detection. TMPBF was not detected either in simulant exposed to polymer-coated panels or in the DMSO migrant concentrates (Table S1). The LOD for TMBPF in each of the neat simulant samples was 0.2 μg/6 dm2 (0.2 ppb), and in the DMSO concentrate, the LOQ was 0.06 and 0.01 μg/6 dm2 (0.06 and 0.01 ppb) for 3% acetic acid and 50% ethanol, respectively. NIAS Composition Was Comparable between Migration Samples and Concentrates Assessed in E-SCREEN. The composition of the individual simulant solutions (50% aqueous ethanol (v/v) and 3% aqueous acetic acid (w/v)) was compared to the composition of the same solutions that were evaporated and redissolved in DMSO for bioassay testing. They were evaluated in two manners, namely, first by measuring and comparing the amount of residual monomer (TMBPF) present and second by comparing the NIAS detected by LC-TOF-MS. The amounts of monomer present were comparable, in that no TMBPF was detected above 0.2 μg/6 dm2 in any samples tested. The NIAS compositions were also similar (Figures S5 and S6) All 650 masses detected by LC-TOF-MS in the 50% ethanol were also detected in the corresponding DMSO sample. Other masses were detected only in the DMSO samples, but these may have originated from procedural contributions. A similar number of masses (651) were detected in both the exposed 3% acetic acid simulant and the corresponding DMSO sample. However, there were additional masses detected only in the 3% acetic acid exposure sample and not in the DMSO, and additional masses were detected only in the DMSO and not in the initially exposed 3% aqueous acetic acid (w/v). No analysis for gravimetric losses was performed. TMBPF Does Not Activate Estrogen Receptor or Increase MCF7 Cell Proliferation. None of the seven TMBPF concentrations induced luciferase activity in the estrogen-sensitive VM7LucE2 cells (Figure S7). TMBPF also did not show estrogen antagonist activity in a similar assay (Figure S8). Treatment with eight different concentrations of TMBPF also did not increase MCF7 cell number (Figure 1B). Both, 17βE2 and BPA, showed dose−response curves compatible with their known EA (Figure 1B). A small increase in cell number at 1 μM TMBPF concentration was observed in some repeats. However, when this region was further analyzed by testing concentrations between 0.1 and 2.5 μM, cell numbers were below quantifiable counts of estrogen equivalents at all points tested (Figure 1B). The absence of EA at the cell proliferation level was further confirmed by lack of activity of estrogen-regulated genes PR, BIK, APOD, and WISP2 in real time RT-PCR (Figure 2). TMBPF was cytotoxic at concentrations of 1 μM and higher (Figure 1C) in the ESCREEN and 3.9 μM and higher in the estrogen receptor transactivation assays (Figures S7 and S8). Migrants from Polymeric Coating Do Not Induce Cell Proliferation. MCF7 cells did not proliferate in the presence of migrant concentrates derived from 3% acetic acid and 50% ethanol simulants. Additionally, both polymeric coating extracts showed cytotoxicity at 2 ppm and higher (Figure 3).

Figure 2. TMBPF does not activate known estrogen-responsive genes. Gene expression, downregulation (A) and upregulation (B), was assessed at 1 μM concentration, the dose showing a small increase over baseline in cell count. A higher and a lower dose were added for completion. 17β-E2 was the positive control, and cell culture medium stripped of steroids was the negative control.

Figure 3. E-SCREEN dose−response curves for extracts of polymeric coating. Coated metal was in contact with 3% acetic acid (A) and 50% ethanol (B) food simulants representing acidic and fatty foods, respectively. The resulting migrants did not stimulate cell proliferation and showed cytotoxicity at concentrations above 2 ppm.

TMBPF Does Not Increase Uterine Weight in the Uterotrophic Assay. Mean wet and blotted uterus weights, both absolute and relative to final body weight, in the TMBPF 100, 300, and 1000 groups were comparable to the vehicle control group (Table 2). The mean wet and blotted absolute uterus weights in the EE group were significantly higher (p < 0.0001) than the vehicle control group (7.9- and 4.0-fold, respectively). Their relative uterus weight was also significantly higher (p < 0.01). No TMBPF-related microscopic findings were noted in the mammary glands. However, five out of six EE-treated rats showed mild hyperplasia in the glandular epithelium. Hyperplasia is an increase in cell number manifested as a thickening of the mammary ductal epithelium. Normal ducts have one to two layers of epithelial cells while hyperplastic ducts may have three to five. It is not uncommon in immature animals receiving an estrogenic stimulus (Figure S9). No clinical findings were noted during the daily examinations related to TMBPF. A few TMBPF-treated rats showed infrequent dried yellow material in the urogenital area; this was detected once in three animals from TMBPF 100, twice in two animals from TMBPF 300, and three times in one animal from TMBPF 1000. When the entire treatment period (PND19−22) was evaluated, a statistically significant (p = 0.0112) lower mean body weight gain was noted at TMBPF 1000 compared to the vehicle control group, likely due to nutritional deficiency (Table S3). On the contrary, mean body weight gain in the TMBPF 100 and 300 groups were like the vehicle control group during the same interval. Daily mean body weights in the E


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Environmental Science & Technology Table 2. Summary of Uterus Weight and Uterus Weight Relative to End of Experiment Body Weighta group vehicle EE TMBPF 100 TMBPF 300 TMBPF 1000 a

uterus blotted 0.034 0.134 0.036 0.038 0.036

± ± ± ± ±

0.007 0.020c 0.006 0.007 0.009

uterus wet 0.041 0.320 0.043 0.045 0.043

± ± ± ± ±

final body weight

0.008 0.047c 0.008 0.009 0.009

50 48 48 48 47

± ± ± ± ±

3.0 1.9 4.2 1.4 3.0

relative uterus blottedb 0.068 0.279 0.074 0.081 0.079

± ± ± ± ±

relative uterus wet

0.017 0.046 0.01 0.014 0.019

0.082 0.667 0.089 0.094 0.092

± ± ± ± ±

0.017 0.113 0.016 0.019 0.018

Number of rats per group = 6. bWeight of uterus (grams)/body weight (grams) × 100. cp < 0.001 using a two sample t test.

TMBPF 300 and TMBPF 1000 were up to 9.0% and 8.3% lower, respectively, than the vehicle control group but without reaching statistical significance (Table S4). In the EE-treated animals, mean body weight gains were slightly lower (p = 0.0455) compared to the vehicle control group throughout the treatment period, yet daily mean body weights were similar between groups. TMBPF Does Not Alter Pubertal Maturation in Female and Male Rats. All animals including controls, TMBPF 200, and TMBPF 600 survived the treatment and did not show any overt effects. In females, the age at VO was not statistically different between the control and both TMBPF-treated groups. The mean age at achievement was 34.5, 33.6, and 33.7 days for control, TMBPF 200, and TMBPF 600, respectively. Additionally, vaginal cytology and estrous cycling were comparable among the three groups (Table S5), and vaginal histopathology was also similar among the vehicle and both TMBPF-treated groups (Table S6). In males, mean PPS age was delayed in the TMBPF 600 rats (48.7 vs 47.3 days, respectively; p = 0.05), but it was not considered statistically different from controls per EPA guidance. Table 3 summarizes the age at achieving sexual

Figure 4. Mean body weight per sex and treatment. Treatment with TMBPF for 20 and 30 days in females (A) and males (B), respectively, did not induce body weight gain.

Table 3. Summary of Sexual Maturation End Points group

age at VO (mean days ± SD)a

age at PPS (mean days ± SD)

vehicle control TMBPF 200 TMBPF 600

34.5 ± 2.2 33.6 ± 2.3 33.7 ± 2.3

47.3 ± 1.2 48.2 ± 2.0 48.7 ± 2.2b

a

Figure 5. Representative whole-mounted mammary glands from pubertal female rats. (A) Vehicle control. (B) TMBPF 200. (C) TMBPF 600. No differences were found in the pattern of development, maturation, and frequency of terminal end buds (arrowheads), branching, and budding. Scale bar: 5000 μm.

SD: standard deviation. bp = 0.05.

structures such as terminal end buds, branching, and budding were present and no qualitative differences were noticed. Ductal extension was also similar between control and TMBPF groups. Table 4 summarizes the frequency of structures observed in the whole-mounted mammary glands.

maturation in males and females, and Figure S10 shows the similarity in age and number of animals achieving sexual maturation between the vehicle and TMBPF-treated groups. TMBPF did not affect female and male body weight at either dose (Figure 4). However, the TMBPF 600 males were heavier than controls when they achieved PPS (mean body weight of 284.8 g for TMBPF 600 and 264 g for vehicle control; p: 0.007). The weight of reproductive organs was unchanged in both females and males of the TMBPF 200 and TMBPF 600 groups (Tables S7 and S8). On a few occasions and in all female groups, there were discrepancies between the estrous phases observed microscopically in the vagina and the vaginal cytology prior to necropsy, likely due to the animals’ young age. The mammary glands were evaluated using traditional histopathology and whole-mount methods. No microscopic changes or alteration in the growth pattern of the glands were observed in the TMBPF-treated animals compared to controls. Figure 5 shows a representative micrograph of whole-mounted mammary glands from control (A), TMBPF 200 (B), and TMBPF 600 (C). In all cases, typical pubertal mammary

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DISCUSSION We have rigorously assessed the potential EA of a new bisphenol that contributes the essential 4,4′-methylenediphenol polymer chain functionality required to produce a high Table 4. Number of Animals Distributed Across a Five-Point Grading System Based on Prevalence of Structures in Whole-Mounted Mammary Glands structure

control

TMBPF 200

TMBPF 600

terminal end buds branching

15 marked 1 slight 14 moderate 5 slight 7 moderate 3 marked

15 marked 15 moderate

15 marked 15 moderate

3 slight 7 moderate 5 marked

3 slight 8 moderate 4 marked

budding

F


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In the present study, we tested an absence hypothesis, namely, that TMBPF does not have estrogenic activity. We are cognizant of the heterodoxy of publishing negative results, yet this is the very nature of safety evidence. We also recognize and emphasize the importance of transparency in chemical safety assessments, especially for new products that could potentially affect consumers. We advocate for independence, data integrity, and transparency. To that end, an independent third-party provided analytical verification of substances (i.e., TMBPF and NIAS) tested in the E-SCREEN using chain-of-custody. Moreover, ESCREEN results were cross-verified under unrestricted academic research arrangements between Tufts University and Colorado State University for interlaboratory comparison. Animal studies were performed using GLP standards with independent analytical verification of TMBPF by the respective contract laboratories. This approach strengthens objectivity and minimizes the potential for interpretive bias to which evidence of absence hypothesis may be especially vulnerable.40 Finally, we also call for public disclosure of data which we accomplish here. In conclusion, our findings provide strong evidence for the absence of EA by TMBPF and the polymeric coating with 4,4′methylenediphenol functionality derived from TMBPF. This conclusion is supported by the integration of evidence derived from in vitro and in vivo studies. On the basis of quantitative migration analysis, the plausibility of human exposure to TMBPF is also exceedingly small. We believe that environmental health, in general, and chemical safety assessment strategies in particular will be enhanced by focusing on hazard elimination and exposure reduction and minimizing health risks in a transparent fashion using the Safety by Design approach as a guiding principle.

performance polymeric coating for food contact. We found consistent evidence that TMBPF and migrants from polymeric coating lack EA across different levels of biological complexity (i.e., receptor binding, gene expression, cell proliferation, and in the whole animal). Further, TMBPF migration into food simulants was below the limit of analytical detection. To our knowledge, this is the first study in which both the monomer and the NIAS migrant from a final polymeric coating have been tested for EA. While screening for contaminants in paper and board, Bengtström et al.27 used a similar approach by testing migrants present in extracts from commercial samples intended for direct contact with food in a broad panel of in vitro assays. The main difference with our study is that their screening was nontargeted while our target was EA. The composition of the individual simulant solutions (50% aqueous ethanol and 3% aqueous acetic acid) exposed to the polymer and the composition of the same solutions after being evaporated and redissolved in DMSO for bioassay testing were comparable. Specifically, no TMBPF could be detected above the LOD/reporting limit, and the NIAS composition determined by LC-TOF-MS was comparable between simulants. No further identification of individual NIAS was needed because the extracts were not estrogenic. This is in contrast with the Vinggaard et al.28 findings where some of the paper extracts that were positive for EA were further analyzed to identify the estrogenic chemicals. TMBPF did not show evidence of EA in any of the assays described here. Unlike TMBPF, BPA, bisphenol S (BPS), and bisphenol F (BPF) have been shown to increase uterine weight.24,29−31 In contrast to TMBPF, BPA, BPF, and BPS induced cell proliferation in the E-SCREEN.32−34 One possible explanation for TMBPF’s lack of EA is the limited flexibility of the methylene bridge between the aromatic rings imparted by the 2,2′,6,6′-tetra-methyl ring substitution (Figure 1A). During development of the polymeric coating, we observed that steric effects that limit flexibility of the methylene bridge, as observed in high temperature gas-phase equilibrium models, were the best predictor of reduced or absent EA in vitro. This inflexibility at the methylene bridge of a bisphenol would likely prevent conformational changes necessary for the molecule to reach binding equilibrium with the ER binding domain. The small increase in cell number observed in the ESCREEN was unlikely due to EA because estrogen-target genes were not activated at or around that dose. Also, the cell number was below the level of quantitation making the calculation of estrogenic equivalency impossible. This event could be a potential response to rapid cytotoxic effect of TMBPF starting at 1 μM. The cytotoxic effect of TMBPF observed in both in vitro assays was like that reported for BPA, BPF, bisphenol M, and dimethylbisphenol A in various cell lines.35−38 These bisphenols also showed cytotoxicity at the micromolar concentration. The TMBPF doses used in animal studies followed EPA’s guidance protocols and published studies on BPA.24,29 It could be argued that these were high doses of TMBPF and further studies should be conducted to identify a potential low-dose effect or the presence of a nonmonotonic dose response. However, the absence of increased cell yields using a wide range of TMBPF concentrations (10 pM to 10 μM) in the ESCREEN and TMBPF’s inability to interact with estrogenresponsive genes23,39 suggests that TMBPF is unlikely to have EA in vivo at lower doses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b04704. Chromatograms of chemical analysis performed before animal testing was conducted; chromatograms of polymeric migrant evaluation and analysis; transactivation assay; estrogen activity calculation; RT PCR and primers; migrant quantification; uterotrophic and pubertal studies (body weight, male and female organ weights, cyclicity); histopathology of the mammary gland (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; telephone: 617-470-3842. ORCID

Maricel V. Maffini: 0000-0002-3853-9461 Notes

The authors declare the following competing financial interest(s): Valspar Corporation has given Tufts University some unrestricted funds for Dr. Soto's laboratory. The funds were used to pay a technician and run bioassays to determine whether or not the materials were estrogenic. The unrestricted fund format, a gift, was chosen so that Drs. Soto and Sonnenschein and Mrs. Schaeberle would remain completely free from Valspar influence. Drs. Maffini and Maier were paid G


Article


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by Valspar to consult on the project; Dr. Maier is also a former Valspar employee. The full reports for migrant evaluation, the transactivation assay, and uterotrophic and pubertal assays can be accessed at http://www.valsparpackaging.com/valpure/item/ourmaterials/#omNBPAE.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by The Valspar Corporation. The authors express their gratitude to Dr. Zhe Wang, Ms. Skylar Klager, and Mr. Michael Sweeney and the Colorado State University Center for Environmental Medicine for their excellent technical contributions.

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Evidence of Absence: Estrogenicity Assessment of a New Food

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