US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

SCCS scientific opinion on Butylated hydroxytoluene (BHT) - SCCS/1636/21.

OPINION TO BE CITED AS: SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

Lack of genotoxicity of rhubarb (rhizome) in the Ames and micronucleus in vitro tests.

Hydroxyanthracene derivatives are widely distributed in the plant kingdom, mainly in botanicals such as the Hypericum, Rheum, Rhamnus and Aloe genera. For centuries, plants containing hydroxyanthracene derivatives have been used as herbal remedies, mainly as laxatives. The root and underground stem (rhizome) are used to make medicine, primarily for digestive complaints including constipation, diarrhoea, heartburn, stomach pain, gastrointestinal bleeding, and preparation for certain gastrointestinal diagnostic procedures. The use of hydroxyanthracene-containing botanicals has raised the attention of European Food Safety Authority (EFSA) for the potential genotoxicity activity, that in 2018 concluded "[.] and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists". No genotoxic activity has been reported with other constituents such as rhein, physcion and chrysophanol. In the present study, Rhubarb ethanolic extract of ground rhubarb rhizome (hydroxyanthracene total content 1.39 %) was tested in the Ames Assay in Salmonella typhimurium and Escherichia coli, up to 5000 µg/plate and up to 5000 µg/mL in human lymphocytes Micronucleus Test (OECD 471 and 487 respectively) in vitro mutagenic and genotoxic effects. Under the experimental conditions used, the rhubarb rhizome extract showed no genotoxic activity.

The EU chemicals strategy for sustainability: in support of the BfR position.

The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.

Lack of in vivo genotoxic effect of dried whole Aloe ferox juice.

Aloe ferox Mill is widely used as a traditional herbal medicine for the treatment of a broad spectrum of illnesses given its laxative, anti-inflammatory, bitter tonic, anti-oxidant, antimicrobial and anti-cancer properties. Using the in vivo alkaline comet assay in animals (OECD 489), this study investigated the potential in vivo genotoxicity of dried Aloe ferox juice at dose levels of 500, 1000, and 2000 mg/kg/day in mice. Aloe ferox showed no genotoxic activity in preparations of single cells from the colon of the treated Hsd:ICR (CD-1) male mice. No statistically significant increase in DNA migration over the negative control was observed by analysis of variance for both comet parameters, tail moment and tail intensity, apart from the positive control ethyl methanesulphonate that induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The new reported scientific evidence unequivocally demonstrates that dried Aloe ferox juice containing hydroxyanthracene derivatives does not induce DNA damage in preparations of single cells from colon in in vivo comet genotoxicity studies. This suggests that the hyperplastic changes and mucosal hyperplasia observed after long-term administration of Aloe vera non-decolourised whole leaf extract may be attributed to an epigenetic effect of the material under investigation.

Aloe-emodin, a hydroxyanthracene derivative, is not genotoxic in an in vivo comet test.

Aloe-emodin, one of the molecules belonging to the group of hydroxyanthracene derivatives, was recently described as genotoxic in vivo. Indeed, the EFSA judged that aloe-emodin, together with other similar molecules (emodin and danthron) and extracts from the leaf of Aloe species containing hydroxyanthracene derivatives, could represent a risk factor for colorectal cancer mediated by a genotoxic effect. Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion is based, a new in vivo study (in vivo alkaline comet assay in mice - OECD 489) was conducted to test the potential genotoxicity of aloe-emodin at doses of 250, 500, 1000 and 2000 mg/kg bw/day on preparations of single cells from the kidney and colon of treated male mice. Following treatment with the test item, no clinical signs were observed in animals in any treatment group. Slight body-weight loss was randomly observed in all groups treated with the test item and was more evident in the groups dosed at 1000 and 2000 mg/kg bw/day. Under these experimental conditions, aloe-emodin showed no genotoxic activity. Possible oxidative damage to colon tissues could not be excluded based on the results obtained after repair enzyme treatment.

Development of a consensus approach for botanical safety evaluation - A roundtable report.

Botanical safety science continues to evolve as new tools for risk assessment become available alongside continual desire by consumers for "natural" botanical ingredients in consumer products. Focusing on botanical food/dietary supplements a recent international roundtable meeting brought together scientists to discuss the needs, available tools, and ongoing data gaps in the botanical safety risk assessment process. Participants discussed the key elements of botanical safety evaluations. They provided perspective on the use of a decision tree methodology to conduct a robust risk assessment and concluded with alignment on a series of consensus statements. This discussion highlighted the strengths and vulnerabilities in common assumptions, and the participants shared additional perspective to ensure that this end-to-end safety approach is sufficient, actionable and timely. Critical areas and data gaps were identified as opportunities for future focus. These include, better context on history of use, systematic assessment of weight of evidence, use of in silico approaches, inclusion of threshold of toxicological concern considerations, individual substances/matrix interactions of plant constituents, assessing botanical-drug interactions and adaptations needed to apply to in vitro and in vivo pharmacokinetic modelling of botanical constituents.

Effect of plant extracts on the genotoxicity of 1'-hydroxy alkenylbenzenes.

Food-borne alkenylbenzenes are potential risks for human health because they are known to induce liver tumors in rodent bioassays at high dose levels. This carcinogenicity is ascribed to the conversion of their 1'-hydroxymetabolites to the ultimate DNA reactive and carcinogenic 1'-sulfoxymetabolites. The aim of this study was to investigate the in vitro genotoxicity of some botanical extracts used as Plant Food Supplements (PFS) and to compare it with the individual substances, estragole, safrole and their 1'-hydroxy-derivative activity. The genotoxicity of the PFSs was evaluated in HepG2 cell line by comet and micronucleus assays. Unlike the 1'-hydroxy derivatives, PFS extracts and parent alkenylbenzenes did not show genotoxicity at any of the tested concentrations. The sulfotransferase inhibitor pentachlorophenol (PCP) reduced the 1'-hydroxy compound-induced response in the comet and micronucleus assays, thus confirming that the formation of sulfoxy-metabolites is essential for inducing genotoxic effects. When the cells were treated with hydroxylated alkenylbenzenes in the presence of PFSs, a reduction in genotoxic activity of synthetic compounds was observed.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

The chemical composition of ultrafine particles and associated biological effects at an alpine town impacted by wood burning.

This work is part of the TOBICUP (TOxicity of BIomass Combustion generated Ultrafine Particles) project which aimed at providing the composition of ultrafine particles (UFPs, i.e. particles with aerodynamic diameter, dae, lower than 100nm) emitted by wood combustion and elucidating the related toxicity. Results here reported are from two ambient monitoring campaigns carried out at an alpine town in Northern Italy, where wood burning is largely diffused for domestic heating in winter. Wintertime and summertime UFP samples were analyzed to assess their chemical composition (i.e. elements, ions, total carbon, anhydrosugars, and polycyclic aromatic hydrocarbons) and biological activity. The induction of the pro-inflammatory cytokine interleukin-8 (IL-8) by UFPs was investigated in two human cells lines (A549 and THP-1) and in human peripheral blood leukocytes. In addition, UFP-induced oxidative stress and genotoxicity were investigated in A549 cells. Ambient UFP-related effects were compared to those induced by traffic-emitted particles (DEP) taken from the NIES reference material "vehicle exhaust particulates". Ambient air UFPs induced a dose-related IL-8 release in both A549 and THP-1 cells; the effect was more relevant on summer samples and in general THP-1 cells were more sensitive than A549 cells. On a weight basis our data did not support a higher biological activity of ambient UFPs compared to DEP. The production of IL-8 in the whole blood assay indicated that UFPs reached systemic circulation and activated blood leukocytes. Comet assay and γ-H2AX evaluation showed a significant DNA damage especially in winter UFPs samples compared to control samples. Our study showed that ambient UFPs can evoke a pulmonary inflammatory response by inducing a dose-related IL-8 production and DNA damage, with different responses to UFP samples collected in the summer and winter periods.

Insights on wood combustion generated proinflammatory ultrafine particles (UFP).

This study aimed to collect, characterize ultrafine particles (UFP) generated from the combustion of wood pellets and logs (softwood and hardwood) and to evaluate their pro-inflammatory effects in THP-1 and A549 cells. Both cell lines responded to UFP producing interleukin-8 (IL-8), with wood log UFP being more active compared to pellet UFP. With the exception of higher effect observed with beech wood log UFP in THP-1, the ability of soft or hard woods to induce IL-8 release was similar. In addition, on weight mass, IL-8 release was similar or lower compared to diesel exhaust particles (DEP), arguing against higher biological activity of smaller size particles. UFP-induced IL-8 could be reduced by SB203580, indicating a role of p38MAPK activation in IL-8 production. The higher activity of beech wood log UFP in THP-1 was not due to higher uptake or endotoxin contamination. Qualitatively different protein adsorption profiles were observed, with less proteins bound to beech UFP compared to conifer UFP or DEP, which may provide higher intracellular availability of bioactive components, i.e. levoglucosan and galactosan, toward which THP-1 were more responsive compared to A549 cells. These results contribute to our understanding of particles emitted by domestic appliances and their biological effects.

Understanding chemical allergen potency: role of NLRP12 and Blimp-1 in the induction of IL-18 in human keratinocytes.

Keratinocytes (KCs) play a key role in all phases of skin sensitization. We recently identified interleukin-18 (IL-18) production as useful end point for determination of contact sensitization potential of low molecular weight chemicals. The aim of this study was to identify genes involved in skin sensitizer-induced inflammasome activation and to establish their role in IL-18 production. For gene expression analysis, cells were treated for 6 h with p-phenylenediamine (PPD) as reference contact allergen; total RNA was extracted and examined with a commercially available Inflammasome Polymerase Chain Reaction (PCR) array. Among genes induced, NLRP12 (Nod-like receptor P12) was selected for further investigation. NLRP12 promoter region contains Blimp-1 (B-lymphocyte-induced maturation protein-1)/PRDM1 binding site, and from the literature, it is reported that Blimp-1 reduces NLRP12 activity and expression in monocytes/macrophages. Their expression and role in KCs are currently unknown. To confirm NLRP12 expression and to investigate its relationship with Blimp-1, cells were exposed for different times (3, 6 and 24 h) to the extreme sensitizer 2,4-dinitrochlorobenzene (DNCB) and the strong sensitizer PPD. Allergens were able to induce both genes, however, with different kinetic, with DNCB more rapidly upregulating Blimp-1 and inducing IL-18 production, compared to PPD. NLRP12 and Blimp-1 expression appeared to be inversely correlated: Blimp-1 silencing resulted in increased NLRP12 expression and reduced contact allergen-induced IL-18 production. Overall results indicate that contact allergens of different potency differently modulate Blimp-1/NLRP12 expression, with strong allergen more rapidly downregulating NLRP12, thus more rapidly inducing IL-18 production. Data confirm that also in KCs, NLRP12 has an inhibitory effect on inflammasome activation assessed by IL-18 maturation.

Upholding science in health, safety and environmental risk assessments and regulations.

A public appeal has been advanced by a large group of scientists, concerned that science has been misused in attempting to quantify and regulate unmeasurable hazards and risks.1 The appeal recalls that science is unable to evaluate hazards that cannot be measured, and that science in such cases should not be invoked to justify risk assessments in health, safety and environmental regulations. The appeal also notes that most national and international statutes delineating the discretion of regulators are ambiguous about what rules of evidence ought to apply. Those statutes should be revised to ensure that the evidence for regulatory action is grounded on the standards of the scientific method, whenever feasible. When independent scientific evidence is not possible, policies and regulations should be informed by publicly debated trade-offs between socially desirable uses and social perceptions of affordable precaution. This article explores the premises, implications and actions supporting the appeal and its objectives.

The European Registered Toxicologist (ERT): Current status and prospects for advancement.

Following its inception in 1994, the certification of European Registered Toxicologists (ERT) by EUROTOX has been recognized as ensuring professional competence as well as scientific integrity and credibility. Criteria and procedures for registration are contained in the ERT "Guidelines for Registration 2012". The register of ERT currently has over 1900 members. In order to continue the harmonisation of requirements and processes between national registering bodies as a prerequisite for official recognition of the ERT title as a standard, and to take account of recent developments in toxicology, an update of the ERT Guidelines has been prepared in a series of workshops by the EUROTOX subcommittees for education and registration, in consultation with representatives of national toxicology societies and registers. The update includes details of topics and learning outcomes for theoretical training, and how these can be assessed. The importance of continuing professional development as the cornerstone of re-registration is emphasised. To help with the process of harmonisation, it is necessary to collate and share best practices of registration conditions and procedures across Europe. Importantly, this information can also be used to audit compliance with the EUROTOX standards. As recognition of professionals in toxicology, including specialist qualifications, is becoming more important than ever, we believe that this can best be achieved based on the steps for harmonisation outlined here together with the proposed new Guidelines.

Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions.

Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1-2mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-ß and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure-activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions.

Health risk assessment of chemicals--a commentary on requirements for the provision of training.

Today, only a limited number of training courses specifically in human health risk assessment are available in Europe and although some basic training in health risk assessment is part of most toxicology university programmes, the preparation is often not enough. The purpose of this commentary is to present provision of training program in risk assessment based on common European criteria, easily adopted by institutions across Europe and focusing on risk assessment methodology and procedure. It is worth mentioning that the following paper does not deal with microbiological risk assessment and ecotoxicological risk assessment but mainly with chemical risk assessment. The project focuses on understanding the profile and training requirements of risk assessors in order to design a modular training program covering a range of disciplines in risk assessment and providing a model to establish guidelines for the training and recognition of risk assessors in accordance to a well-defined and properly acknowledged training standard.

Metals in cosmetics: an a posteriori safety evaluation.

According to EU Regulation No. 1223/2009/CE cosmetic products for daily use can contain 'technically unavoidable traces' of metals. This definition is too vague. Authorities should set well-defined limits, considering the risks associated with metal contamination of personal care products (PCPs). This paper characterizes the risk arising from a number of metals (antimony, arsenic, cadmium, cobalt, chromium, mercury, nickel, lead) that may occur in 'unavoidable traces" in raw materials and, consequently, in PCPs. A 'worst case scenario' was adopted, based on the following assumptions: (i) the individual ingredients contained the maximum amount in traces allowed for each metal; (ii) the hypothetical PCP was produced exclusively with that single ingredient; (iii) when absorption through the skin was not known, data related to oral absorption were used. Risk characterization was performed calculating the Systemic Exposure Dosage (SED) and the Margin of Safety (MoS=NOAEL or BMDL10/SED). Exposure to the allegedly 'technically unavoidable' maximum amounts of metals in cosmetic ingredients resulted in MoSs exceeding 100 (safety threshold) with one exception. This suggests that the availability of experimental dermal absorption rates could enable significant improvement in MoS, thus increasing safety levels. Although results are reassuring, the authors recommend minimization of contamination, according to the state of the art of manufacturing methods.

Role of ROS and HMGB1 in contact allergen-induced IL-18 production in human keratinocytes.

Keratinocytes have a key role in all phases of allergic contact dermatitis. We have recently identified the possibility to use IL-18 production for the in vitro identification of contact allergens. The purpose of this study was to characterize the molecular mechanisms underlying allergen-induced IL-18 production, in order to identify the cellular source of reactive oxygen species (ROS) and the danger signals involved. The NCTC2544 cell line was exposed to three contact allergens, namely p-phenylenediamine (PPD), 2,4-dinitrochlorobenzene (DNCB), and citral, in the presence or absence of diphenylene iodonium (DPI), allopurinol, and rotenone to identify the source of ROS, and to anti-Toll-like receptor 4 antibody and glycirrizic acid to characterize the danger-associated molecular pattern molecules. In the case of PPD, the induction of IL-18 can be modulated by rotenone, allopurinol, and DPI. In the case of DNCB, rotenone completely prevents the induction of IL-18, whereas for citral, DPI completely prevents the induction of IL-18. We demonstrated the ability of all allergens tested to induce the release of high-mobility group protein B1 (HMGB1). Its sequester by glycirrizic acid significantly modulates PPD-induced IL-18 production and completely prevents DNCB- and citral-induced IL-18. We found that different intracellular sources of ROS are triggered by contact allergens, and an important role for HMGB1 in chemical allergen-induced IL-18 production was demonstrated.