Towards achieving a modern science-based paradigm for agrochemical carcinogenicity assessment.

The rodent cancer bioassay has been the standard approach to fulfill regulatory requirements for assessing human carcinogenic potential of agrochemicals, food additives, industrial chemicals, and pharmaceuticals. Decades of research have described the limitations of the rodent cancer bioassay leading to international initiatives to seek alternatives and establish approaches that modernize carcinogenicity assessment. Biologically relevant approaches can provide mechanistic information and increased efficiency for evaluating hazard and risk of chemical carcinogenicity to humans. The application of human-relevant mechanistic understanding to support new approaches to carcinogenicity assessment will be invaluable for regulatory decision-making. The present work outlines the challenges and opportunities that authorities should consider as they come together to build a roadmap that leads to global acceptance and incorporation of fit-for-purpose, scientifically defensible new approaches for human-relevant carcinogenicity assessment of agrochemicals.

Exposure considerations in human safety assessment: Report from an EPAA Partners' Forum.

Understanding and estimating the exposure to a substance is one of the fundamental requirements for safe manufacture and use. Many approaches are taken to determine exposure to substances, mainly driven by potential use and regulatory need. There are many opportunities to improve and optimise the use of exposure information for chemical safety. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a Partners' Forum (PF) to explore exposure considerations in human safety assessment of industrial products to agree key conclusions for the regulatory acceptance of exposure assessment approaches and priority areas for further research investment. The PF recognised the widescale use of exposure information across industrial sectors with the possibilities of creating synergies between different sectors. Further, the PF acknowledged that the EPAA could make a significant contribution to promote the use of exposure data in human safety assessment, with an aim to address specific regulatory needs. To achieve this, research needs, as well as synergies and areas for potential collaboration across sectors, were identified.

Rethinking agrochemical safety assessment: A perspective.

Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.

Towards a mechanism-based approach for the prediction of nongenotoxic carcinogenic potential of agrochemicals.

Chemical substances are subjected to assessment of genotoxic and carcinogenic effects before being marketed to protect man and the environment from health risks. For agrochemicals, the long-term rodent carcinogenicity study is currently required from a regulatory perspective. Although it is the current mainstay for the detection of nongenotoxic carcinogens, carcinogenicity studies are shown to have prominent weaknesses and are subject to ethical and scientific debate. A transition toward a mechanism-based weight-of-evidence approach is considered a requirement to enhance the prediction of carcinogenic potential for environmental (agro)chemicals. The resulting approach should make optimal use of innovative (computational) tools and be less animal demanding. To identify the various mode of actions (MOAs) underlying the nongenotoxic carcinogenic potential of agrochemicals, we conducted an extensive analysis of 411 unique agrochemicals that have been evaluated for carcinogenicity by the United States Environmental Protection Agency (US EPA) and the European Chemicals Agency (ECHA). About one-third of these substances could be categorized as nongenotoxic carcinogens with an average of approximately two tumor types per substance, observed in a variety of organs. For two-third of the tumor cases, an underlying MOA (network) could be identified. This analysis demonstrates that a limited set of MOA (networks) is underlying nongenotoxic carcinogenicity of agrochemicals, illustrating that the transition toward a MOA-driven approach appears manageable. Ultimately the approach should cover relevant MOAs and its associated key events; this will also facilitate the evaluation of the human relevance. This manuscript describes the results of the analysis while identifying knowledge gaps and necessities to achieve a mechanism-based weight-of-evidence approach.

An analysis of the setting of the acute reference dose (ARfD) for pesticides in Europe.

To protect human health, acute reference values have been established for pesticides which have the potential to cause a toxic effect after acute human exposure. These values are used to identify exposure levels below which there is no appreciable risk. Comprehensive reference documents, including OECD criteria, are available to aid identification of relevant toxicological endpoints. Within Europe, there is a concern that the identification process is inconsistent and unnecessarily conservative such that safe products with no established human health risk are being restricted. For this reason, the basis for the setting of an acute reference dose (ARfD) has been investigated for 130 pesticides to better understand how the toxicological endpoints are selected. The investigation has shown that most ARfDs are derived from repeat dose studies and that there is an over-representation of prenatal developmental toxicity studies. There is clear evidence that ARfDs derived from rabbit developmental toxicity studies are set over conservatively with regard to acute maternal effects and often inappropriately. To facilitate an improved system, refinements to the existing process are recommended, the use of maternal data in the rabbit as the basis for deriving an ARfD is critically evaluated and a new, more pragmatic approach to ARfD derivation is proposed.

A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals.

Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.

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.

An in vitro approach for comparative interspecies metabolism of agrochemicals.

The metabolism and elimination of a xenobiotic has a direct bearing on its potential to cause toxicity in an organism. The confidence with which data from safety studies can be extrapolated to humans depends, among other factors, upon knowing whether humans are systemically exposed to the same chemical entities (i.e. a parent compound and its metabolites) as the laboratory animals used to study toxicity. Ideally, to understand a metabolite in terms of safety, both the chemical structure and the systemic exposure would need to be determined. However, as systemic exposure data (i.e. blood concentration/time data of test material or metabolites) in humans will not be available for agrochemicals, an in vitro approach must be taken. This paper outlines an in vitro experimental approach for evaluating interspecies metabolic comparisons between humans and animal species used in safety studies. The aim is to ensure, where possible, that all potential human metabolites are also present in the species used in the safety studies. If a metabolite is only observed in human in vitro samples and is not present in a metabolic pathway defined in the toxicological species already, the toxicological relevance of this metabolite must be evaluated.

Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

Transforming the evaluation of agrochemicals.

The present agrochemical safety evaluation paradigm is long-standing and anchored in well-established testing and evaluation procedures. However, it does not meet the present-day challenges of rapidly growing populations, food insecurity, and pressures from climate change. To transform the current framework and apply modern evaluation strategies that better support sustainable agriculture, the Health and Environmental Sciences Institute (HESI) assembled a technical committee to reframe the safety evaluation of crop-protection products. The committee is composed of international experts from regulatory agencies, academia, industry and nongovernmental organizations. Their mission is to establish a framework that supports the development of fit-for-purpose agrochemical safety evaluation that is applicable to changing global, as well as local needs and regulatory decisions, and incorporates relevant evolving science. This will be accomplished through the integration of state-of-the-art scientific methods, technologies and data sources, to inform safety and risk decisions, and adapt them to evolving local and global needs. The project team will use a systems-thinking approach to develop the tools that will implement a problem formulation and exposure driven approach to create sustainable, safe and effective crop protection products, and reduce, replace and refine animal studies with fit-for-purpose assays. A new approach necessarily will integrate the most modern tools and latest advances in chemical testing methods to guarantee the robust human and environmental safety and risk assessment of agrochemicals. This article summarizes the challenges associated with the modernization of agrochemical safety evaluation, proposes a potential roadmap, and seeks input and engagement from the broader community to advance this effort. © 2022 Health and Environmental Sciences Institute (HESI). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The mouse carcinogenicity study is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides.

Regulatory tests investigating pesticide carcinogenicity potential routinely comprise a battery of in vitro and in vivo genotoxicity studies and two cancer bioassays, one in rats and one in mice. The genotoxicity testing strategy essentially ensures that genotoxic compounds are eliminated, and any carcinogens identified in subsequent lifetime studies are probably nongenotoxic in character. Assessment of 202 pesticide evaluations from the European Union review programme under Directive 91/414/EEC indicated that the mouse carcinogenicity study contributed little or nothing to either derivation of an acceptable daily intake (ADI) for assessment of chronic risk to humans, or hazard classification for labelling purposes. From a pesticide approval perspective, the mouse study did not influence a single outcome. From a risk assessment perspective, the ADI for just one pesticide was based on tumours in mice and this would have barely changed if the mouse data had not been available. In total, only 10 (5%) pesticide ADIs were based solely on the mouse carcinogenicity study and even in these few cases, a similar value would have been identified from other studies if the mouse study had not been available. For pesticides with treatment-related tumours only in mice, just three, or 1.5%, were classified as carcinogens and all were in the lowest category, Category 3 (R40). For pesticides with treatment-related tumours in mice and rats, the mouse data were probably the main, if not the only, cause for another three cases of R40 classification. Absence of the mouse studies would not have influenced assignment of the higher, Category 2 (R45), cancer classification for any substance with treatment-related tumours in both species as all decisions for these substances were limited to Category 3 or 'unclassified' outcomes. Over 100,000 mice were used to test these pesticides. This review shows that the mouse carcinogenicity studies did not provide significant information over and above that provided by the rat studies, and underpins the opportunity, from both a scientific and an animal welfare perspective, to remove the mouse carcinogenicity study from regulatory data requirements for the testing of pesticides.

A critical Assessment of the Genotoxicity Profile of the Fungicide Tricyclazole.

Tricyclazole (8-methyl-[1,2,4]triazolo[3,4-b][1,3]benzothiazole) is a fungicide used globally on rice for treatment of the seasonal rice blast disease. Human exposure to this fungicide can occur via dietary and nondietary routes. In a battery of in vitro assays, tricyclazole did not induce gene mutations in bacteria (Ames test) or at the Hprt locus of CHO cells. It was also negative for the induction of micronuclei in human lymphocyte cultures and unscheduled DNA synthesis (UDS) in primary rat hepatocyte. Paradoxically, tricyclazole induced a mutagenic response at the Tk locus of the mouse lymphoma L5178Ycells (MLA), which occurred equally among small/large colony phenotypes. Selection of preexisting mutants leading to a false-positive response in the MLA was ruled out in follow-up experiments. In vivo, tricyclazole was negative in the rat liver UDS assay, mouse bone micronucleus test and a transgenic (MutaMouse) gene mutation assay in glandular stomach, liver, and kidney. Other supporting evidence for the lack of genotoxicity for tricyclazole comes from an in vivo study for sister chromatid exchanges in Chinese hamsters, and a dominant lethal test in the male germ cells of mice. The combined evidence from the genotoxicity studies together with the evidence from toxicokinetic, carcinogenicity, developmental, and reproductive toxicity studies confirm that mutagenicity does not occur in relevant in vivo systems. Data were also compared to potential animal and human exposure, mechanistic data on biological targets and data on analogues, confirming adequacy of the available data for hazard identification and risk assessment. Environ. Mol. Mutagen. 61:300-315, 2020. © 2019 Wiley Periodicals, Inc.

Implementing a globally harmonized risk assessment-based approach for regulatory decision-making of crop protection products.

A global, harmonized evaluation system for crop protection chemicals based on exposure and risk will improve the ability to inform risk management decisions and better support innovation. This would be achieved through harmonized risk assessment-based regulatory decision-making realized through the application of the best available science, via integration of new methods and traditional data to create tailored exposure-driven risk assessments. A requirement to achieve success is a structure that encourages direct communication between the regulatory community and the regulated industry, which would enable a more rapid incorporation of new technologies and advancing science. An approach that emulates the International Conference of Harmonization (ICH) for pharmaceuticals would bring together regulatory authorities and the regulated industry along with relevant experts from academia and Non-Governmental Organizations to discuss scientific and technical advances and their implementation. These discussions would also encourage the elimination of outmoded practices that no longer serve a purpose resulting in more uniform testing requirements and best practices for data evaluation to support safe use and scientifically defensible human health and environmental risk assessments. New and developing technologies offer exciting opportunities to improve the current toxicity testing paradigms to provide better solutions and diminish animal testing. Implementation of a harmonized approach will increase the speed, efficiency and accuracy of regulatory decision-making for human health and environmental protection while increasing the efficiency of providing safe and effective innovative products to the agriculture community. © 2020 Society of Chemical Industry.

Interlaboratory validation of 1% pluronic l92 surfactant as a suitable, aqueous vehicle for testing pesticide formulations using the murine local lymph node assay.

The mouse local lymph node assay (LLNA) has become the preferred test for evaluating the dermal sensitization potential of chemicals and requirements are now emerging for its use in the evaluation of their formulated products, especially in the European Union. However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned, which could lead to repeat testing using multiple animal models. As pesticide formulations are typically a specific complex blend of chemicals for use as aqueous-based dilutions, traditional vehicles prescribed for the LLNA may change the properties of these formulations leading to inaccurate test results and hazard identification. The objective of this study was to evaluate the effectiveness of an aqueous solution of Pluronic L92 block copolymer surfactant (L92) as a vehicle in the mouse LLNA across five laboratories. Three chemicals with known sensitization potential and four pesticide formulations for which the sensitization potential in guinea pigs and/or humans had previously been assessed were used. Identical LLNA protocols and test materials were used in the evaluation. Assessment of the positive control chemicals, hexylcinnamaldehyde, formaldehyde, and potassium dichromate revealed positive results when using 1% aqueous L92 as the vehicle. Furthermore, results for these chemicals were reproducible among the five laboratories and demonstrated consistent relative potency determinations. The four pesticide formulations diluted in 1% aqueous L92 also demonstrated reproducible results in the LLNA among the five laboratories. Results for these test materials were also consistent with those generated previously using guinea pigs or from human experience. These data support testing aqueous compatible chemicals or pesticide formulations using the mouse LLNA, and provide additional support for the use of 1% aqueous L92 as a suitable, aqueous-based vehicle.