Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Support for Regulatory Assessment of Percutaneous Absorption of Retronecine-type Pyrrolizidine Alkaloids through Human Skin.

1,2-unsaturated pyrrolizidine alkaloids are found naturally in Symphytum officinale, well known as comfrey, which has a longstanding use for the topical treatment of painful muscle and joint complaints. Pyrrolizidine alkaloids (PA) are a relevant concern for the safety assessment due to their liver genotoxicity profile, and close attention is paid during manufacturing to minimizing their levels. Current regulatory risk assessment approaches include setting limits that derive from toxicity data coming from the oral route of exposure. This study investigated to what extent pyrrolizidine alkaloids are bioavailable following topical exposure, assessing penetration of retronecine-type PAs in an in vitro human skin model. A single comfrey root formulation was spiked with 3 different congeners (a 7R-monoester, an open-chained 7R-diester, and a cyclic diester) and percutaneous absorption measured per OECD guidelines and good laboratory practices. The measured penetration for all 3 PAs was low and compared favourably with existing in vitro data. Although consideration of different regulatory guidance influences the determination of dermally absorbed dose, these data facilitate the understanding of absorption differences following topical exposure, which in turn can be taken into account in the risk assessment.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Use of in vitro metabolism and biokinetics assays to refine predicted in vivo and in vitro internal exposure to the cosmetic ingredient, phenoxyethanol, for use in risk assessment.

A novel approach was developed to help characterize the biokinetics of the cosmetic ingredient, phenoxyethanol, to help assess the safety of the parent and its major stable metabolite. In the first step of this non-animal tiered approach, primary human hepatocytes were used to confirm or refute in silico predicted metabolites, and elucidate the intrinsic clearance of phenoxyethanol. A key result was the identification of the major metabolite, phenoxyacetic acid (PAA), the exposure to which in the kidney was subsequently predicted to far exceed that of phenoxyethanol in blood or other tissues. Therefore, a novel aspect of this approach was to measure in the subsequent step the formation of PAA in the cells dosed with phenoxyethanol that were used to provide points of departure (PoDs) and express the intracellular exposure as the Cmax and AUC24. This enabled the calculation of the intracellular concentrations of parent and metabolite at the PoD in the cells used to derive this value. These concentrations can be compared with in vivo tissue levels to conclude on the safety margin. The lessons from this case study will help to inform the design of other non-animal safety assessments.

A 10-step framework for use of read-across (RAX) in next generation risk assessment (NGRA) for cosmetics safety assessment.

This paper presents a 10-step read-across (RAX) framework for use in cases where a threshold of toxicological concern (TTC) approach to cosmetics safety assessment is not possible. RAX builds on established approaches that have existed for more than two decades using chemical properties and in silico toxicology predictions, by further substantiating hypotheses on toxicological similarity of substances, and integrating new approach methodologies (NAM) in the biological and kinetic domains. NAM include new types of data on biological observations from, for example, in vitro assays, toxicogenomics, metabolomics, receptor binding screens and uses physiologically-based kinetic (PBK) modelling to inform about systemic exposure. NAM data can help to substantiate a mode/mechanism of action (MoA), and if similar chemicals can be shown to work by a similar MoA, a next generation risk assessment (NGRA) may be performed with acceptable confidence for a data-poor target substance with no or inadequate safety data, based on RAX approaches using data-rich analogue(s), and taking account of potency or kinetic/dynamic differences.

Use of New Approach Methodologies (NAMs) in regulatory decisions for chemical safety: Report from an EPAA Deep Dive Workshop.

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a 'Blue Sky Workshop' on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. "Disruptive thinking" is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity.

The future trajectory of adverse outcome pathways: a commentary.

The advent of adverse outcome pathways (AOPs) has provided a new lexicon for description of mechanistic toxicology, and a renewed enthusiasm for exploring modes of action resulting in adverse health and environmental effects. In addition, AOPs have been used successfully as a framework for the design and development of non-animal approaches to toxicity testing. Although the value of AOPs is widely recognised, there remain challenges and opportunities associated with their use in practise. The purpose of this article is to consider specifically how the future trajectory of AOPs may provide a basis for addressing some of those challenges and opportunities.

One science-driven approach for the regulatory implementation of alternative methods: A multi-sector perspective.

EU regulations call for the use of alternative methods to animal testing. During the last decade, an increasing number of alternative approaches have been formally adopted. In parallel, new 3Rs-relevant technologies and mechanistic approaches have increasingly contributed to hazard identification and risk assessment evolution. In this changing landscape, an EPAA meeting reviewed the challenges that different industry sectors face in the implementation of alternative methods following a science-driven approach. Although clear progress was acknowledged in animal testing reduction and refinement thanks to an integration of scientifically robust approaches, the following challenges were identified: i) further characterization of toxicity pathways; ii) development of assays covering current scientific gaps, iii) better characterization of links between in vitro readouts and outcome in the target species; iv) better definition of alternative method applicability domains, and v) appropriate implementation of the available approaches. For areas having regulatory adopted alternative methods (e.g., vaccine batch testing), harmonised acceptance across geographical regions was considered critical for broader application. Overall, the main constraints to the application of non-animal alternatives are the still existing gaps in scientific knowledge and technological limitations. The science-driven identification of most appropriate methods is key for furthering a multi-sectorial decrease in animal testing.

SEURAT: Safety Evaluation Ultimately Replacing Animal Testing--recommendations for future research in the field of predictive toxicology.

The development of non-animal methodology to evaluate the potential for a chemical to cause systemic toxicity is one of the grand challenges of modern science. The European research programme SEURAT is active in this field and will conclude its first phase, SEURAT-1, in December 2015. Drawing on the experience gained in SEURAT-1 and appreciating international advancement in both basic and regulatory science, we reflect here on how SEURAT should evolve and propose that further research and development should be directed along two complementary and interconnecting work streams. The first work stream would focus on developing new 'paradigm' approaches for regulatory science. The goal here is the identification of 'critical biological targets' relevant for toxicity and to test their suitability to be used as anchors for predicting toxicity. The second work stream would focus on integration and application of new approach methods for hazard (and risk) assessment within the current regulatory 'paradigm', aiming for acceptance of animal-free testing strategies by regulatory authorities (i.e. translating scientific achievements into regulation). Components for both work streams are discussed and may provide a structure for a future research programme in the field of predictive toxicology.

Identification and semi-quantification of protein allergens in complex mixtures using proteomic and AllerCatPro 2.0 bioinformatic analyses: a proof-of-concept investigation.

The demand for botanicals and natural substances in consumer products has increased in recent years. These substances usually contain proteins and these, in turn, can pose a risk for immunoglobulin E (IgE)-mediated sensitization and allergy. However, no method has yet been accepted or validated for assessment of potential allergenic hazards in such materials. In the studies here, a dual proteomic-bioinformatic approach is proposed to evaluate holistically allergenic hazards in complex mixtures of plants, insects, or animal proteins. Twelve commercial preparations of source materials (plant products, dust mite extract, and preparations of animal dander) known to contain allergenic proteins were analyzed by label-free proteomic analyses to identify and semi-quantify proteins. These were then evaluated by bioinformatics using AllerCatPro 2.0 (https://allercatpro.bii.a-star.edu.sg/) to predict no, weak, or strong evidence for allergenicity and similarity to source-specific allergens. In total, 4,586 protein sequences were identified in the 12 source materials combined. Of these, 1,665 sequences were predicted with weak or strong evidence for allergenic potential. This first-tier approach provided top-level information about the occurrence and abundance of proteins and potential allergens. With regards to source-specific allergens, 129 allergens were identified. The sum of the relative abundance of these allergens ranged from 0.8% (lamb's quarters) to 63% (olive pollen). It is proposed here that this dual proteomic-bioinformatic approach has the potential to provide detailed information on the presence and relative abundance of allergens, and can play an important role in identifying potential allergenic hazards in complex protein mixtures for the purposes of safety assessments.

Evaluation of mixture toxicity literature and chemical space: a data centric approach.

Using the Abstract Sifter tool to analyse PubMed, we reveal published mixture related research most commonly relates to water pollutants, pesticides, environmental pollutants, insecticides, soil pollutants, and chemicals described as persistent, bioaccumulative, and toxic. Furthermore, we discern individual chemicals that also identify as priority chemicals in biomonitoring initiatives and using an ontology-based chemical classification, at the level of the chemical subclass, found these priority chemicals overlap with just 9% of the REACH chemical space.

Application of AllerCatPro 2.0 for protein safety assessments of consumer products.

Foreign proteins are potentially immunogenic, and a proportion of these are able to induce immune responses that result in allergic sensitization. Subsequent exposure of sensitized subjects to the inducing protein can provoke a variety of allergic reactions that may be severe, or even fatal. It has therefore been recognized for some time that it is important to determine a priori whether a given protein has the potential to induce allergic responses in exposed subjects. For example, the need to assess whether transgene products expressed in genetically engineered crop plants have allergenic properties. This is not necessarily a straightforward exercise (as discussed elsewhere in this edition), but the task becomes even more challenging when there is a need to conduct an overall allergenicity safety assessment of complex mixtures of proteins in botanicals or other natural sources that are to be used in consumer products. This paper describes a new paradigm for the allergenicity safety assessment of proteins that is based on the use of AllerCatPro 2.0, a new version of a previously described web application model developed for the characterization of the allergenic potential of proteins. Operational aspects of AllerCatPro 2.0 are described with emphasis on the application of new features that provide improvements in the predictions of allergenic properties such as the identification of proteins with high allergenic concern. Furthermore, the paper provides a description of strategies of how AllerCatPro 2.0 can best be deployed as a screening tool for identifying suitable proteins as ingredients in consumer products as well as a tool, in conjunction with label-free proteomic analysis, for identifying and semiquantifying protein allergens in complex materials. Lastly, the paper discusses the steps that are recommended for formal allergenicity safety assessment of novel consumer products which contain proteins, including consideration and integration of predicted consumer exposure metrics. The article therefore provides a holistic perspective of the processes through which effective protein safety assessments can be made of potential allergenic hazards and risks associated with exposure to proteins in consumer products, with a particular focus on the use of AllerCatPro 2.0 for this purpose.

The in vitro genotoxicity potency of mixtures of pyrrolizidine alkaloids can be explained by dose addition of the individual mixture components.

Plant-based 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Due to 21st century toxicology approaches, risk of PAs can be better discerned though an understanding of differing toxic potencies, but it is often mixtures of PAs that are found as contaminants in foods, for example, herbal teas and honey, food supplements and herbal medicines. Our study investigated whether genotoxicity potency of PAs dosed individually or in mixtures differed when measured using micronuclei formation in vitro in HepaRG human liver cells, which we and others have shown to be suitable for observing genotoxic potency differences across different PA structural classes. When equipotent concentrations of up to six different PAs representing a wide range of potencies in vitro were tested as mixtures, the observed genotoxic potency aligned favorably with results for single PAs. Similarly, when the BMD confidence intervals of these equipotent mixtures were compared with the confidence intervals of the individual PAs, only minimal variation was observed. These data support a conclusion that for this class of plant impurities, all acting via the same DNA-reactive mode of action, genotoxic potency can be regarded as additive when assessing the risk of mixtures of PAs.

Assessing the genotoxicity and carcinogenicity of 2-chloroethanol through structure activity relationships and in vitro testing approaches.

The detection of 2-chloroethanol in foods generally follows an assumption that the pesticide ethylene oxide has been used at some stage in the supply chain. In this situation the Pesticide Residues in Food Regulation (EC) 396/2005 requires 2-chloroethanol to be assessed as if equivalent to ethylene oxide, which has been classified as a genotoxic carcinogen. This review investigated whether this is an appropriate risk assessment approach for 2-chloroethanol. This involved an assessment of existing genotoxicity and carcinogenicity data, application of Structure Activity Based Read Across for carcinogenicity assessment, biological reactivity in the ToxTracker assay and micronuclei formation in HepaRG cells. Although we identified there is an absence of a standard oral bioassay for 2-chloroethanol, carcinogenicity weight-of-evidence assessment along with data on relevant structural analogues do not show evidence for carcinogenicity for 2-chloroethanol. The absence of genotoxicity was demonstrated for 2-chloroethanol and suitable analogues. In contrast, ethylene oxide showed reactivity towards markers indicative of direct DNA damage which is consistent with what is known about its mode-of-action. These data facilitate the understanding of 2-chloroethanol and given that it is not a genotoxic carcinogen suggest it must be assessed relative to non-cancer endpoints and a health protective Reference Dose should be established on that basis.

Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments.

A process for evaluating analogs for use in SAR (Structure-Activity Relationship) assessments was previously published (Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

Incorporation of in vitro techniques for botanicals dietary supplement safety assessment - Towards evaluation of developmental and reproductive toxicity (DART).

As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Here we explore in vitro gene expression as well receptor binding and enzyme activity as alternative assays to inform on developmental and reproductive toxicity (DART) relevant modes of action, since DART data gaps are common for botanicals. Specifically, botanicals suspected to have DART effects, in addition to those with a significant history of use, were tested in these assays. Gene expression changes in a number of different cell types were analysed using the connectivity mapping approach (CMap) to identify modes of action through a functional read across approach. Taken together with ligand affinity data obtained using a set of molecular targets customised towards known DART relevant modes of action, it was possible to inform DART risk using functional analogues, potency comparisons and a margin of internal exposure approach.

Threshold of toxicological concern (TTC) for botanicals - Concentration data analysis of potentially genotoxic constituents to substantiate and extend the TTC approach to botanicals.

This paper evaluates use of the Threshold of Toxicological Concern (TTC) approach to assess safety of botanical preparations that may contain potentially genotoxic constituents, based on estimation of the fraction that may be genotoxic. A database of 107 chemical constituents of botanicals was compiled and their potential for genotoxicity evaluated from published data. Forty-three constituents met the criteria for potential genotoxicity. Concentration data on their occurrence in plants provided 2878 data points; the majority were in the low ppm level (range 0.00001-139,965 ppm, by dry weight). Weibull models of the quantitative distribution data were used to calculate 95th percentile values for chemical concentrations, analysing the dataset according to their presence in botanicals (i) as a single chemical, (ii) as two or more chemicals from the same chemical group, or (iii) as two or more chemicals from different chemical groups. The highest 95th percentile concentration value from these analyses was 1.8%. Using the TTC value of 0.15 µg/person per day for potentially genotoxic substances proposed in 2004, this value of 1.8% was used to derive an adjusted TTC value of 10 µg of plant material on a dry weight basis/person per day for assessment of potentially genotoxic substances in botanicals.