The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Is it time to reassess current safety standards for glyphosate-based herbicides?

Use of glyphosate-based herbicides (GBHs) increased ∼100-fold from 1974 to 2014. Additional increases are expected due to widespread emergence of glyphosate-resistant weeds, increased application of GBHs, and preharvest uses of GBHs as desiccants. Current safety assessments rely heavily on studies conducted over 30 years ago. We have considered information on GBH use, exposures, mechanisms of action, toxicity and epidemiology. Human exposures to glyphosate are rising, and a number of in vitro and in vivo studies challenge the basis for the current safety assessment of glyphosate and GBHs. We conclude that current safety standards for GBHs are outdated and may fail to protect public health or the environment. To improve safety standards, the following are urgently needed: (1) human biomonitoring for glyphosate and its metabolites; (2) prioritisation of glyphosate and GBHs for hazard assessments, including toxicological studies that use state-of-the-art approaches; (3) epidemiological studies, especially of occupationally exposed agricultural workers, pregnant women and their children and (4) evaluations of GBHs in commercially used formulations, recognising that herbicide mixtures likely have effects that are not predicted by studying glyphosate alone.

Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement.

The broad-spectrum herbicide glyphosate (common trade name "Roundup") was first sold to farmers in 1974. Since the late 1970s, the volume of glyphosate-based herbicides (GBHs) applied has increased approximately 100-fold. Further increases in the volume applied are likely due to more and higher rates of application in response to the widespread emergence of glyphosate-resistant weeds and new, pre-harvest, dessicant use patterns. GBHs were developed to replace or reduce reliance on herbicides causing well-documented problems associated with drift and crop damage, slipping efficacy, and human health risks. Initial industry toxicity testing suggested that GBHs posed relatively low risks to non-target species, including mammals, leading regulatory authorities worldwide to set high acceptable exposure limits. To accommodate changes in GBH use patterns associated with genetically engineered, herbicide-tolerant crops, regulators have dramatically increased tolerance levels in maize, oilseed (soybeans and canola), and alfalfa crops and related livestock feeds. Animal and epidemiology studies published in the last decade, however, point to the need for a fresh look at glyphosate toxicity. Furthermore, the World Health Organization's International Agency for Research on Cancer recently concluded that glyphosate is "probably carcinogenic to humans." In response to changing GBH use patterns and advances in scientific understanding of their potential hazards, we have produced a Statement of Concern drawing on emerging science relevant to the safety of GBHs. Our Statement of Concern considers current published literature describing GBH uses, mechanisms of action, toxicity in laboratory animals, and epidemiological studies. It also examines the derivation of current human safety standards. We conclude that: (1) GBHs are the most heavily applied herbicide in the world and usage continues to rise; (2) Worldwide, GBHs often contaminate drinking water sources, precipitation, and air, especially in agricultural regions; (3) The half-life of glyphosate in water and soil is longer than previously recognized; (4) Glyphosate and its metabolites are widely present in the global soybean supply; (5) Human exposures to GBHs are rising; (6) Glyphosate is now authoritatively classified as a probable human carcinogen; (7) Regulatory estimates of tolerable daily intakes for glyphosate in the United States and European Union are based on outdated science. We offer a series of recommendations related to the need for new investments in epidemiological studies, biomonitoring, and toxicology studies that draw on the principles of endocrinology to determine whether the effects of GBHs are due to endocrine disrupting activities. We suggest that common commercial formulations of GBHs should be prioritized for inclusion in government-led toxicology testing programs such as the U.S. National Toxicology Program, as well as for biomonitoring as conducted by the U.S. Centers for Disease Control and Prevention.

Evidence that bisphenol A (BPA) can be accurately measured without contamination in human serum and urine, and that BPA causes numerous hazards from multiple routes of exposure.

There is extensive evidence that bisphenol A (BPA) is related to a wide range of adverse health effects based on both human and experimental animal studies. However, a number of regulatory agencies have ignored all hazard findings. Reports of high levels of unconjugated (bioactive) serum BPA in dozens of human biomonitoring studies have also been rejected based on the prediction that the findings are due to assay contamination and that virtually all ingested BPA is rapidly converted to inactive metabolites. NIH and industry-sponsored round robin studies have demonstrated that serum BPA can be accurately assayed without contamination, while the FDA lab has acknowledged uncontrolled assay contamination. In reviewing the published BPA biomonitoring data, we find that assay contamination is, in fact, well controlled in most labs, and cannot be used as the basis for discounting evidence that significant and virtually continuous exposure to BPA must be occurring from multiple sources.

Holding thermal receipt paper and eating food after using hand sanitizer results in high serum bioactive and urine total levels of bisphenol A (BPA).

Bisphenol A (BPA) is an endocrine disrupting environmental contaminant used in a wide variety of products, and BPA metabolites are found in almost everyone's urine, suggesting widespread exposure from multiple sources. Regulatory agencies estimate that virtually all BPA exposure is from food and beverage packaging. However, free BPA is applied to the outer layer of thermal receipt paper present in very high (∼20 mg BPA/g paper) quantities as a print developer. Not taken into account when considering thermal paper as a source of BPA exposure is that some commonly used hand sanitizers, as well as other skin care products, contain mixtures of dermal penetration enhancing chemicals that can increase by up to 100 fold the dermal absorption of lipophilic compounds such as BPA. We found that when men and women held thermal receipt paper immediately after using a hand sanitizer with penetration enhancing chemicals, significant free BPA was transferred to their hands and then to French fries that were eaten, and the combination of dermal and oral BPA absorption led to a rapid and dramatic average maximum increase (Cmax) in unconjugated (bioactive) BPA of ∼7 ng/mL in serum and ∼20 µg total BPA/g creatinine in urine within 90 min. The default method used by regulatory agencies to test for hazards posed by chemicals is intra-gastric gavage. For BPA this approach results in less than 1% of the administered dose being bioavailable in blood. It also ignores dermal absorption as well as sublingual absorption in the mouth that both bypass first-pass liver metabolism. The elevated levels of BPA that we observed due to holding thermal paper after using a product containing dermal penetration enhancing chemicals have been related to an increased risk for a wide range of developmental abnormalities as well as diseases in adults.

Should oral gavage be abandoned in toxicity testing of endocrine disruptors?

For decades, hazard assessments for environmental chemicals have used intra-gastric gavage to assess the effects of 'oral' exposures. It is now widely used--and in some cases required--by US federal agencies to assess potential toxicity of endocrine disrupting chemicals (EDCs). In this review we enumerate several reasons why gavage is not appropriate for the assessment of EDCs using bisphenol A (BPA) as a main example. First, whereas human dietary exposures interact with the oral mucosa, gavage exposures avoid these interactions, leading to dramatic differences in absorption, bioavailability and metabolism with implications for toxicokinetic assumptions and models. Additionally, there are well acknowledged complications associated with gavage, such as perforation of the esophagus that diminish its value in toxicological experiments. Finally, the gavage protocol itself can induce stress responses by the endocrine system and confound the assessment of EDCs. These serious flaws have not been taken into account in interpreting results of EDC research. We propose the exploration of alternatives to mimic human exposures when there are multiple exposure routes/sources and when exposures are chronic. We conclude that gavage may be preferred over other routes for some environmental chemicals in some circumstances, but it does not appropriately model human dietary exposures for many chemicals. Because it avoids exposure pathways, is stressful, and thus interferes with endocrine responses, gavage should be abandoned as the default route of administration for hazard assessments of EDCs.

Bisphenol A (BPA) pharmacokinetics with daily oral bolus or continuous exposure via silastic capsules in pregnant rhesus monkeys: Relevance for human exposures.

We measured serum dBPA in non-pregnant and pregnant female rhesus monkeys, fetuses and amniotic fluid. dBPA was administered by a daily oral bolus or sc implantation of Silastic capsules; both resulted in daily average serum unconjugated dBPA concentrations of <1ng/ml. We observed lower serum concentrations of unconjugated dBPA in pregnant females relative to pre-pregnancy values, and generally lower concentrations in fetal serum than in maternal serum. Differences in pharmacokinetics of dBPA were evident between pre-pregnancy, early and late pregnancy, likely reflecting changes in maternal, fetal and placental physiology. The serum ratio of conjugated to unconjugated dBPA after continuous sc release of dBPA was similar to values reported in human biomonitoring studies and markedly lower than with oral administration, suggesting oral bolus exposure is not an appropriate human exposure model. We report elsewhere that there were numerous adverse effects on fetuses exposed to very low serum dBPA in these studies.

Metabolic disruption in male mice due to fetal exposure to low but not high doses of bisphenol A (BPA): evidence for effects on body weight, food intake, adipocytes, leptin, adiponectin, insulin and glucose regulation.

Exposure to bisphenol A (BPA) is implicated in many aspects of metabolic disease in humans and experimental animals. We fed pregnant CD-1 mice BPA at doses ranging from 5 to 50,000µg/kg/day, spanning 10-fold below the reference dose to 10-fold above the currently predicted no adverse effect level (NOAEL). At BPA doses below the NOAEL that resulted in average unconjugated BPA between 2 and 200pg/ml in fetal serum (AUC0-24h), we observed significant effects in adult male offspring: an age-related change in food intake, an increase in body weight and liver weight, abdominal adipocyte mass, number and volume, and in serum leptin and insulin, but a decrease in serum adiponectin and in glucose tolerance. For most of these outcomes non-monotonic dose-response relationships were observed; the highest BPA dose did not produce a significant effect for any outcome. A 0.1-µg/kg/day dose of DES resulted in some but not all low-dose BPA outcomes.

Regulatory decisions on endocrine disrupting chemicals should be based on the principles of endocrinology.

For years, scientists from various disciplines have studied the effects of endocrine disrupting chemicals (EDCs) on the health and wellbeing of humans and wildlife. Some studies have specifically focused on the effects of low doses, i.e. those in the range that are thought to be safe for humans and/or animals. Others have focused on the existence of non-monotonic dose-response curves. These concepts challenge the way that chemical risk assessment is performed for EDCs. Continued discussions have clarified exactly what controversies and challenges remain. We address several of these issues, including why the study and regulation of EDCs should incorporate endocrine principles; what level of consensus there is for low dose effects; challenges to our understanding of non-monotonicity; and whether EDCs have been demonstrated to produce adverse effects. This discussion should result in a better understanding of these issues, and allow for additional dialog on their impact on risk assessment.

Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses.

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

Role of p21 and cyclin E in normal and secalonic acid D-inhibited proliferation of human embryonic palatal mesenchymal cells.

Secalonic acid D (SAD), a cleft palate-inducing teratogen, has been shown to inhibit proliferation/cell cycle progression in association with alteration in the levels of cell cycle regulators, p21 and cyclin E. These studies were conducted to test the hypotheses that p21 and cyclin E play an important functional role in normal human embryonic palatal mesenchymal (HEPM) cell cycle and that their up- and down-regulation, respectively, by SAD is functionally significant to its cell cycle block. Using small interfering RNA (siRNA) to silence p21 gene and transient transfection to overexpress cyclin E in control & SAD-treated HEPM cells, cell proliferation was assessed using a combination of cell numbers, thymidine uptake, CDK2 activity and Ki-67 expression. The results showed that silencing of p21 gene, although increased cell proliferation/numbers and CDK2 activity in normal HEPM cells, failed to counteract SAD-induced anti-proliferative effect despite inducing partial recovery of CDK2 activity. Similar effects were apparent with cyclin E overexpression. It is concluded that p21 and cyclin E are important for normal HEPM cell proliferation. However, SAD-induced deregulation of either protein, singly, may not be sufficient to induce anti-proliferative effect. Involvement of other cell cycle proteins such as cyclin D1 or of multiple proteins in SAD-induced cell cycle block needs to be examined.

Similarity of bisphenol A pharmacokinetics in rhesus monkeys and mice: relevance for human exposure.

OBJECTIVE: Daily adult human exposure to bisphenol A (BPA) has been estimated at <1 µg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women. METHODS: Eleven adult female rhesus macaques were fed 400 µg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography-mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 µg/kg. RESULTS: In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice. CONCLUSIONS: BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 µg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.

Bisphenol A data in NHANES suggest longer than expected half-life, substantial nonfood exposure, or both.

BACKGROUND: It is commonly stated in the literature on human exposure to bisphenol A (BPA) that food is the predominant BPA exposure source, and that BPA is rapidly and completely cleared from the body. If this is correct, BPA levels in fasting individuals should decrease with increased fasting time. OBJECTIVES: We set out to investigate the relationship between urine BPA concentration and fasting time in a population-based sample. METHODS: We modeled log BPA urine concentration as a function of fasting time, adjusted for urine creatinine and other confounders, in 1,469 adult participants in the 2003-2004 National Health and Nutrition Examination Survey. We estimated the BPA "population-based half-life" (pop(1/2)) for a fasting time of 0-24 hr, < 4.5 hr, 4.5-8.5 hr, and > 8.5 hr. RESULTS: The overall pop(1/2) for the 0- to 24-hr interval was 43 hr [95% confidence interval (CI), 26-119 hr]. Among those reporting fasting times of 4.5-8.5 hr (n = 441), BPA declined significantly with fasting time, with a pop(1/2) of 4.1 hr (95% CI, 2.6-10.6 hr). However, within the fasting time intervals of 0-4.5 hr (n = 129) and 8.5-24 hr (n = 899), we saw no appreciable decline. Fasting time did not significantly predict highest (> 12 ng/mL) or lowest (below limit of detection) BPA levels. CONCLUSIONS: Overall, BPA levels did not decline rapidly with fasting time in this sample. This suggests substantial nonfood exposure, accumulation in body tissues such as fat, or both. Explaining these findings may require experimental pharmacokinetic studies of chronic BPA exposure, further examination of BPA levels and effects in fat, and a search for important nonfood sources.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

BACKGROUND: In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Genomic responses from the estrogen-responsive element-dependent signaling pathway mediated by estrogen receptor alpha are required to elicit cellular alterations.

Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) alpha and beta. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ERalpha through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ERalpha also modulates gene expression by interacting with transregulators bound to cognate DNA-regulatory elements, and this regulation is referred to as the ERE-independent signaling pathway. The relative importance of the ERE-independent pathway in E2-ERalpha signaling is unclear. To address this issue, we engineered an ERE-binding defective ERalpha mutant (ERalpha(EBD)) by changing residues in an alpha-helix of the protein involved in DNA binding to render the receptor functional only through the ERE-independent signaling pathway. Using recombinant adenovirus-infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that E2-ERalpha(EBD) modulated the expression of a subset of ERalpha-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ERalpha(EBD) did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ERalpha.ERalpha(EBD) in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ERalpha, on the other hand, effectively repressed growth in this cell line. Our findings suggest that genomic responses from the ERE-dependent signaling pathway are required for E2-ERalpha to induce alterations in cellular responses.

Soybeans as a phytochemical reservoir for the production and stabilization of biocompatible gold nanoparticles.

The present study demonstrates an unprecedented green process for the production of gold nanoparticles by simple treatment of gold salts with soybean extracts. Reduction capabilities of antioxidant phytochemicals present in soybean and their ability to reduce gold salts chemically to nanoparticles with subsequent coating of proteins and a host of other phytochemicals present in soybean on the freshly generated gold nanoparticles are discussed. The new genre of green nanoparticles exhibit remarkable in vitro stability in various buffers including saline, histidine, HSA, and cysteine solutions. MTT assays reveal that the green gold nanoparticles are nontoxic and thus provide excellent opportunities for their applications in nanomedicine for molecular imaging and therapy. The overall strategy described herein for the generation of gold nanoparticles meets all 12 principles of green chemistry, as no "man-made" chemicals, other than the gold salts, are used in the green nanotechnological process.