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.

Modeling Mammary Organogenesis from Biological First Principles: A Systems Biology Approach.

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, 3D models are essential. We have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo and that respond to the hormonal cues that regulate mammary gland morphogenesis and function. Due to their simplicity when compared to in vivo studies, and to their accessibility to visualization in real time, these models are well suited to conceptual and mathematical modeling.

European Medicines Agency Conflicts With the European Food Safety Authority (EFSA) on Bisphenol A Regulation.

The European Food Safety Authority (EFSA) has revised their estimate of the toxicity of bisphenol A (BPA) and, as a result, have recommended reducing the tolerable daily intake (TDI) by 20 000-fold. This would essentially ban the use of BPA in food packaging such as can liners, plastic food containers, and in consumer products. To come to this conclusion, EFSA used a systematic approach according to a pre-established protocol and included all guideline and nonguideline studies in their analysis. They found that Th-17 immune cells increased with very low exposure to BPA and used this endpoint to revise the TDI to be human health protective. A number of regulatory agencies including the European Medicines Agency (EMA) have written formal disagreements with several elements of EFSA's proposal. The European Commission will now decide whether to accept EFSA's recommendation over the objections of EMA. If the Commission accepts EFSA's recommendation, it will be a landmark action using knowledge acquired through independent scientific studies focused on biomarkers of chronic disease to protect human health. The goal of this Perspective is to clearly articulate the monumental nature of this debate and decision and to explain what is at stake. Our perspective is that the weight of evidence clearly supports EFSA's proposal to reduce the TDI by 20 000-fold.

A vision for safer food contact materials: Public health concerns as drivers for improved testing.

Food contact materials (FCMs) and food contact articles are ubiquitous in today's globalized food system. Chemicals migrate from FCMs into foodstuffs, so called food contact chemicals (FCCs), but current regulatory requirements do not sufficiently protect public health from hazardous FCCs because only individual substances used to make FCMs are tested and mostly only for genotoxicity while endocrine disruption and other hazard properties are disregarded. Indeed, FCMs are a known source of a wide range of hazardous chemicals, and they likely contribute to highly prevalent non-communicable diseases. FCMs can also include non-intentionally added substances (NIAS), which often are unknown and therefore not subject to risk assessment. To address these important shortcomings, we outline how the safety of FCMs may be improved by (1) testing the overall migrate, including (unknown) NIAS, of finished food contact articles, and (2) expanding toxicological testing beyond genotoxicity to multiple endpoints associated with non-communicable diseases relevant to human health. To identify mechanistic endpoints for testing, we group chronic health outcomes associated with chemical exposure into Six Clusters of Disease (SCOD) and we propose that finished food contact articles should be tested for their impacts on these SCOD. Research should focus on developing robust, relevant, and sensitive in-vitro assays based on mechanistic information linked to the SCOD, e.g., through Adverse Outcome Pathways (AOPs) or Key Characteristics of Toxicants. Implementing this vision will improve prevention of chronic diseases that are associated with hazardous chemical exposures, including from FCMs.

Effects of perinatal exposure to bisphenol A on induction of prostate cancer in Sprague Dawley rats by MNU and testosterone.

Perinatal and neonatal exposure to bisphenol A (BPA) has been linked to enhancement of prostate carcinogenesis in rats induced by combined treatment with estradiol and testosterone, but human data are lacking. This study aimed to determine the effects of perinatal BPA exposure on induction of prostate cancer in rats by sequential treatment with N-methyl-N-nitrosamine (MNU) and continuous low dose administration of testosterone. Pregnant Sprague Dawley rats were exposed to BPA administered by subcutaneous Alzet minipumps at doses of 2.5 or 25 µg/kg body weight/day from gestational day 9 until postnatal day 28 when pups were weaned providing exposure of offspring in utero and via the mother's milk. At 10-12 weeks of age, one male offspring per litter was treated with an intraperitoneal injection of MNU after hormonal stimulation of prostatic cell proliferation followed two weeks later by subcutaneous insertion of Silastic implants containing testosterone until the termination of the study 57-58 weeks after MNU injection. The perinatal BPA exposure did not significantly affect the incidence of prostate carcinomas which was slightly lower in exposed rats (33-23 %) than in control animals (40 %). Carcinomas in all accessory sex glands combined were also insignificantly less frequent in exposed (46-48 %) than in control rats (60 %). The incidence of malignant tumors at any site in the body was significantly lower in exposed rats (81-65 %) than in controls (93 %). In conclusion, perinatal BPA exposure did not significantly modify prostate cancer induction by MNU plus testosterone in rats, unlike the enhancement of prostate carcinogenesis induced by treatments involving estradiol administration. Which of the two models of prostate carcinogenesis is more relevant for the human situation is unclear at present.

Chemical Effects on Breast Development, Function, and Cancer Risk: Existing Knowledge and New Opportunities.

Population studies show worrisome trends towards earlier breast development, difficulty in breastfeeding, and increasing rates of breast cancer in young women. Multiple epidemiological studies have linked these outcomes with chemical exposures, and experimental studies have shown that many of these chemicals generate similar effects in rodents, often by disrupting hormonal regulation. These endocrine-disrupting chemicals (EDCs) can alter the progression of mammary gland (MG) development, impair the ability to nourish offspring via lactation, increase mammary tissue density, and increase the propensity to develop cancer. However, current toxicological approaches to measuring the effects of chemical exposures on the MG are often inadequate to detect these effects, impairing our ability to identify exposures harmful to the breast and limiting opportunities for prevention. This paper describes key adverse outcomes for the MG, including impaired lactation, altered pubertal development, altered morphology (such as increased mammographic density), and cancer. It also summarizes evidence from humans and rodent models for exposures associated with these effects. We also review current toxicological practices for evaluating MG effects, highlight limitations of current methods, summarize debates related to how effects are interpreted in risk assessment, and make recommendations to strengthen assessment approaches. Increasing the rigor of MG assessment would improve our ability to identify chemicals of concern, regulate those chemicals based on their effects, and prevent exposures and associated adverse health effects.

Best practices to quantify the impact of reproductive toxicants on development, function, and diseases of the rodent mammary gland.

Work from numerous fields of study suggests that exposures to hormonally active chemicals during sensitive windows of development can alter mammary gland development, function, and disease risk. Stronger links between many environmental pollutants and disruptions to breast health continue to be documented in human populations, and there remain concerns that the methods utilized to identify, characterize, and prioritize these chemicals for risk assessment and risk management purposes are insufficient. There are also concerns that effects on the mammary gland have been largely ignored by regulatory agencies. Here, we provide technical guidance that is intended to enhance collection and evaluation of the mammary gland in mice and rats. We review several features of studies that should be controlled to properly evaluate the mammary gland, and then describe methods to appropriately collect the mammary gland from rodents. Furthermore, we discuss methods for preparing whole mounted mammary glands and numerous approaches that are available for the analysis of these samples. Finally, we conclude with several examples where analysis of the mammary gland revealed effects of environmental toxicants at low doses. Our work argues that the rodent mammary gland should be considered in chemical safety, hazard and risk assessments. It also suggests that improved measures of mammary gland outcomes, such as those we present in this review, should be included in the standardized methods evaluated by regulatory agencies such as the test guidelines used for identifying reproductive and developmental toxicants.

The cancer puzzle: Welcome to organicism.

During the fifty years since President Nixon declared the "War on Cancer", those inside and outside the cancer community have witnessed the systematic moving of the goalposts attitude to accommodate evidence into an inadequate theory, that is, the Somatic Mutation Theory (SMT). This sorry state promoted a renewable yearly promise that at the end of the next 10-year period the promises uttered in 1971 would become reality. Each failure triggered calls to do more of the same research under the same theory, routinely using more and more sophisticated technology. Meanwhile, in the last few years, an unambiguous general consensus has emerged acknowledging that this overall long, intensive effort has failed, and that it is likely that the solution to the cancer problem resides elsewhere, namely, in alternative theoretical principles of biology. In this essay we concentrate, first, on the big picture, from the philosophical stance (reductionism versus organicism) to the need to adopt rigorous theories. From this novel perspective we conceptualize cancer as a disease of tissue organization akin to development gone awry. Finally, having identified both a promising stance and a useful theory, i.e., the tissue organization field theory (TOFT), we call for abandoning the SMT and for adopting the more promising TOFT.

Matrix Composition Modulates Vitamin D3's Effects on 3D Collagen Fiber Organization by MCF10A Cells.

Vitamin D3 (vitD3) has been implicated in various cellular functions affecting multiple tissue types. Epidemiological and laboratory studies suggest that vitD3 may be effective as a preventive or therapeutic option for breast cancer. However, randomized clinical trials have yet to confirm these suggestions. Breast neoplasias can arise from developmental alterations; based on this evidence, we seek to understand vitD3's role in normal breast development, particularly its role in epithelial morphogenetic processes such as ductal elongation, branching, and alveolar formation. These processes require extensive changes in the extracellular microenvironment, such as collagen fiber organization, and are largely influenced by hormones. Here, we build upon our past work to shed light on calcitriol's effects on collagen fiber organization by breast epithelial cells, and how such effects are modulated by extracellular matrix composition. We embedded MCF10A normal human breast epithelial cells in two different matrices-collagen type I and collagen type I + 10% Matrigel; treatment with calcitriol resulted in flatter epithelial structures. Next, using two-photon microscopy, we examined changes in collagen fiber organization and corresponding changes in epithelial structures. Applying a novel three-dimensional (3D) image analysis method, we show that increasing doses of calcitriol result in denser collagen fiber bundles in the localized area surrounding the epithelial structures, and that these bundles are aligned in a more parallel direction to epithelial structures when exposed to the highest vitD3 dose. Changed patterns in fiber organization may explain the flattening of epithelial structures; in turn, changes in biophysical forces in the matrix abutting these structures may be responsible for changes in the referred patterns. Addition of 10% Matrigel dampened the effects of calcitriol on both epithelial morphogenesis and fiber organization. Overall, we report novel functions of calcitriol in the breast epithelium and add to the growing body of evidence documenting how hormones affect biophysical processes. Impact statement In this study, we report novel functions of calcitriol in the breast epithelium and use a novel quantitative metric to parse the effects of calcitriol on collagen fiber organization that cannot be detected through conventional histological procedures. Despite the large body of literature on vitamin D3 (vitD3) and calcitriol's effects on cellular functions across tissue types, little is known about how they affect collagen fiber organization, an early critical step for breast epithelial development. This work provides further evidence that hormones affect morphogenesis by means of biophysical forces, with implications for a comprehensive view on vitD3's effects in breast development and neoplasia.

From Wingspread to CLARITY: a personal trajectory.

In the three decades since endocrine disruption was conceptualized at the Wingspread Conference, we have witnessed the growth of this multidisciplinary field and the accumulation of evidence showing the deleterious health effects of endocrine-disrupting chemicals. It is only within the past decade that, albeit slowly, some changes regarding regulatory measures have taken place. In this Perspective, we address some historical points regarding the advent of the endocrine disruption field and the conceptual changes that endocrine disruption brought about. We also provide our personal recollection of the events triggered by our serendipitous discovery of oestrogenic activity in plastic, a founder event in the field of endocrine disruption. This recollection ends with the CLARITY study as an example of a discordance between 'science for its own sake' and 'regulatory science' and leads us to offer a perspective that could be summarized by the motto attributed to Ludwig Boltzmann: "Nothing is more practical than a good theory".

Information, programme, signal: dead metaphors that negate the agency of organisms.

The metaphorical adoption of the concepts of information, program and signal introduced into biology the logic and implicit causal structure of the mathematical theories of information; this is inimical to biology. In turn, those metaphors have hindered the development of a theory of organisms by transferring the agency of organisms to natural selection and to DNA. Moreover, those metaphors introduced into biology the dualism software-hardware and a Laplacian causal structure. Instead, we propose to uphold the agency of the living by adopting three foundational principles for a theory of organisms: namely, 1) the principle of biological inertia (i.e., the default state of cells is proliferation and motility), 2) the principle of variation, and 3) the principle of organization.

Data integration, analysis, and interpretation of eight academic CLARITY-BPA studies.

"Consortium Linking Academic and Regulatory Insights on BPA Toxicity" (CLARITY-BPA) was a comprehensive "industry-standard" Good Laboratory Practice (GLP)-compliant 2-year chronic exposure study of bisphenol A (BPA) toxicity that was supplemented by hypothesis-driven independent investigator-initiated studies. The investigator-initiated studies were focused on integrating disease-associated, molecular, and physiological endpoints previously found by academic scientists into an industry standard guideline-compliant toxicity study. Thus, the goal of this collaboration was to provide a more comprehensive dataset upon which to base safety standards and to determine whether industry-standard tests are as sensitive and predictive as molecular and disease-associated endpoints. The goal of this report is to integrate the findings from the investigator-initiated studies into a comprehensive overview of the observed impacts of BPA across the multiple organs and systems analyzed. For each organ system, we provide the rationale for the study, an overview of methodology, and summarize major findings. We then compare the results of the CLARITY-BPA studies across organ systems with the results of previous peer-reviewed studies from independent labs. Finally, we discuss potential influences that contributed to differences between studies. Developmental exposure to BPA can lead to adverse effects in multiple organs systems, including the brain, prostate gland, urinary tract, ovary, mammary gland, and heart. As published previously, many effects were at the lowest dose tested, 2.5µg/kg /day, and many of the responses were non-monotonic. Because the low dose of BPA affected endpoints in the same animals across organs evaluated in different labs, we conclude that these are biologically - and toxicologically - relevant.

Safeguarding Female Reproductive Health against Endocrine Disrupting Chemicals-The FREIA Project.

Currently available test methods are not well-suited for the identification of chemicals that disturb hormonal processes involved in female reproductive development and function. This renders women's reproductive health at increasing risk globally, which, coupled with increasing incidence rates of reproductive disorders, is of great concern. A woman's reproductive health is largely established during embryonic and fetal development and subsequently matures during puberty. The endocrine system influences development, maturation, and function of the female reproductive system, thereby making appropriate hormone levels imperative for correct functioning of reproductive processes. It is concerning that the effects of human-made chemicals on the endocrine system and female reproductive health are poorly addressed in regulatory chemical safety assessment, partly because adequate test methods are lacking. Our EU-funded project FREIA aims to address this need by increasing understanding of how endocrine disrupting chemicals (EDCs) can impact female reproductive health. We will use this information to provide better test methods that enable fit-for-purpose chemical regulation and then share our knowledge, promote a sustainable society, and improve the reproductive health of women globally.

A Combined Morphometric and Statistical Approach to Assess Nonmonotonicity in the Developing Mammary Gland of Rats in the CLARITY-BPA Study.

BACKGROUND: The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA). OBJECTIVES: We aimed to a) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c) compare those methods. METHODS: Sprague-Dawley rats were exposed to BPA, vehicle, or positive control [ethinyl estradiol (EE2)] by oral gavage beginning on gestational day (GD)6 and continuing with direct dosing of the pups after birth. There were two studies: subchronic and chronic. The latter used two exposure regimes, one stopping at postnatal day (PND)21 (stop-dose) the other continuing until tissue harvest (continuous). Glands were harvested at multiple time points; whole mounts and histological specimens were analyzed blinded to treatment. RESULTS: The subchronic study's semiquantitative analysis revealed no significant differences between control and BPA dose groups at PND21, whereas at PND90 there were significant differences between control and the lowest BPA dose and between control and the lowest EE2 dose in animals in estrus. Quantitative, automatized analysis of the chronic PND21 specimens displayed nonmonotonic BPA effects, with a breaking point between the 25 and 250µg/kg body weight (BW) per day doses. This breaking point was confirmed by a global statistical analysis of chronic study animals at PND90 and 6 months analyzed by the quantitative method. The BPA response was different from the EE2 effect for many features. CONCLUSIONS: Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking nonmonotonic dose-response curves. At all time points, lower doses resulted in larger effects, consistent with the core study, which revealed a significant increase of mammary adenocarcinoma incidence in the stop-dose animals at the lowest BPA dose tested. https://doi.org/10.1289/EHP6301.

Over a century of cancer research: Inconvenient truths and promising leads.

Despite over a century of intensive efforts, the great gains promised by the War on Cancer nearly 50 years ago have not materialized. Since 1999, we have analyzed the lack of progress in explaining and "curing" cancer by examining the merits of the premises that determine how cancer is understood and treated. Our ongoing critical analyses have aimed at clarifying the sources of misunderstandings at the root of the cancer puzzle while providing a plausible and comprehensive biomedical perspective as well as a new theory of carcinogenesis that is compatible with evolutionary theory. In this essay, we explain how this new theory, the tissue organization field theory (TOFT), can help chart a path to progress for cancer researchers by explaining features of cancer that remain unexplainable from the perspective of the still hegemonic somatic mutation theory (SMT) and its variants. Of equal significance, the premises underlying the TOFT offer new perspectives on basic biological phenomena.