The Institute for In Vitro Sciences (IIVS) has sponsored a series of workshops with the aim of developing recommendations for scientific and technical approaches to conducting in vitro assays to assess potential toxicity within and across tobacco and nicotine products. As well as providing a background overview of oral tobacco and nicotine products, this report focuses on the methods used to generate extracts from these products for in vitro testing, the recommended best testing practices, and suggested areas for future research. This publication was developed by a working group of workshop members, in conjunction with the ninth IIVS in vitro workshop entitled 'In Vitro Models for Testing Oral Tobacco and Nicotine Products and Continued Discussion of In Vitro Models of Toxicity and Disease', which was held in January 2024.
The use of terminology related to animal-free science has grown rapidly over the past two decades; however, definitions and interpretations of key terms remain inconsistent across global regulatory, scientific, and policy contexts. The term “new approach methodology(ies)” (NAM(s)) exemplifies this issue: though rarely used prior to 2019, its uptake in scientific literature has increased substantially in recent years. Despite this growth, the term, and its acronym, “NAM”, is used with varied meanings, leading to misunderstanding regarding the types of methods described. Similarly, the term “animal” is defined differently across common language, scientific discourse, and legal frameworks, resulting in further ambiguity in what constitutes “animal-free science”. These variations can hinder productive dialogue and collaboration, particularly in international settings. This manuscript maps the current landscape of definitions of key terms such as “NAM”, “animal”, and “animal-free”, drawing on regulatory, academic, and institutional sources to enhance understanding of the range of existing interpretations. By improving transparency and clarity in terminology, this effort seeks to support more coherent and effective global communication in the field of animal-free science. In addition, the definition of NAM, as agreed by the members of the International Collaboration on Cosmetics Safety, is described. Tabulated definitions and sources are provided as a reference tool.
The Ames bacterial mutagenesis assay (Ames test) has been used for more than 50 years, to help assess the safety of a variety of single chemicals and complex mixtures in the form of solids, liquids or aerosols. Because of the diverse range of substances that need to be tested, many modifications of this test, as well as modifications of the methods by which the test substances are prepared and applied to the bacterial test system, have been developed. Currently, there is a need for mutagenicity testing of both traditional tobacco products and next generation products (NGPs) such as e-cigarettes, heated tobacco products, tobacco-free nicotine products, oral pouches, etc. There are currently no standardised guidelines that cover the differing, often complex, procedures used by different laboratories to assess NGPs with the Ames test. To address this problem, the Institute for In Vitro Sciences (IIVS) convened a workshop, where representatives of industry, government, academia, contract research organisations (CROs) and non-government organisations (NGOs) could present bespoke experimental techniques and data from their testing platforms. The further aim of the workshop was to help formulate some general guidelines that could be used in the testing of tobacco and nicotine products, to allow better comparisons to be made between laboratories. The workshop discussions also resulted in the identification of some suggested areas for future research activities.
There is increased interest in developing non-animal test systems for inhalation exposure safety assessments. However, defined methodologies are absent for predicting local respiratory effects from inhalation exposure to irritants. The current study introduces a concept for applying in vitro and in silico methods for inhalation exposure safety assessment. Three in vitro systems, representing the upper (MucilAir™—nasal epithelial tissue) and lower (A549 cells and human precision-cut lung slices) human respiratory regions, were exposed to six respiratory irritants. These irritant exposures were conducted as liquid droplets, aerosol, or vapors, and samples were collected over 24 h. Cytotoxicity, cytokine release, epithelial resistance, oxidative stress, and mitochondrial membrane potential were measured. To determine the human relevance of in vitro exposures, airway surface depositions were predicted by simulating airborne concentrations equivalent to the Cramer class III inhalation threshold of toxicological concern limit of 0.47 mg/person/day using an in silico model. A > 100-fold margin of exposure was calculated comparing lowest concentrations showing in vitro effects to in silico simulated values. While further studies are needed, this manuscript presents a basic requirement for employing non-animal methods to inform inhalation exposure safety assessments by combining in vitro and in silico assays.
INTRODUCTION:Regulations require that agrochemicals be labeled to indicate potential harmful effects caused by exposure. The in vivo Draize rabbit eye test has historically been the standard method used to assess the eye irritation or corrosion potential of chemical substances. However, as scientific confidence has been established for certain in chemico, in vitro, and ex vivo methods developed for this purpose, regulators are increasingly accepting data from such methods in lieu of the in vivo test. Defined approaches (DAs) may also be used to derive hazard and potency predictions by applying fixed data interpretation procedures to results from multiple methods, thereby leveraging strengths of different methods. Currently, the DAs accepted by regulators to predict eye irritation or corrosion potential do not specifically list agrochemical formulations within their applicability domains. METHODS:To address this gap, we conducted testing to confirm the applicability of in vitro methods to agrochemical formulations and to develop DAs to predict eye irritation hazard labeling according to the Globally Harmonized System of Classification and Labeling (GHS) and the U.S. Environmental Protection Agency (EPA) classification system. Twenty-nine formulations were tested in up to four methods: bovine corneal opacity and permeability (BCOP; OECD TG 437) including histopathology, EpiOcular Eye Irritation Test (EO; OECD TG 492), SkinEthic time-to-toxicity for liquids (TTL; OECD TG 492B), and EyeIRR-IS. We propose four DAs comprising BCOP with histopathology alone, and combined with EO, TTL, or EyeIRR-IS. RESULTS AND CONCLUSION:Instead of evaluating direct concordance of the four individual DAs with historical in vivo rabbit eye test data, for each formulation, we assessed orthogonal concordance of GHS and EPA classifications predicted across all five approaches. Predictions were considered orthogonally concordant when they aligned with the prediction of at least two other approaches (i.e. a majority, or at least 3 of the 5 approaches, achieved the same prediction), referred to as the 'majority prediction.' We also evaluated hazard labeling and PPE labeling associated with the GHS and EPA predictions, respectively. Relative to the hazard and PPE labeling associated with the majority predictions, each of the four DAs were as, or more, protective of human health than the rabbit test; hence, we conclude that these DAs can be used to predict the GHS and EPA classifications of agrochemical formulations.
