TOXICOLOGY METHODS: APPROACH AND METHODOLOGY

      Our approach to toxicological assessment of smoke-free products

      In addition to toxicology studies required by regulatory bodies, Philip Morris International (PMI) has emerged as a pioneer in research to substantiate its smoke-free product portfolio. Some of the key drivers that have propelled PMI to the forefront of this field are our dedication to scientific rigor, our multidisciplinary approach, and our investment in cutting-edge technologies and analytical tools. Read on to know more about our toxicology research methods, which includes techniques that are widely employed in biotech and pharmaceutical industries.

      Methods in PMI’s toxicology research

      Part of our smoke-free product assessment includes toxicology studies, and three types of methods support our toxicological assessment: 

      In vitro studies are performed on cells or tissues that are outside of their normal biological context. When required, we will also include in vivo studies which are performed on living animals. We also incorporate computational methods in our toxicology research, which is the use of computers and large databases to study and model biological systems.

      When we have demonstrated through the application of in vitro and in vivo study methods, and at times computational biology, a sufficient understanding of the risks inherent to a specific product, we can then move to clinical studies involving human participants. As the field of toxicological assessment advances, we consistently seek out new technologies and methodologies to enhance the efficiency of our studies.

      Summary of PMI's in vitro methods

      Our toxicology studies involve in vitro methods to investigate the effects of smoke-free products on cultured cells (bacteria or mammalian cells) or tissues grown in the laboratory. Some of the most important types of in vitro toxicology tests we use for regulatory submissions are genotoxicity and cytotoxicity assays.

      A genotoxicity assay is designed to detect genetic damage and gene mutations induced by a range of single chemicals and complex environmental and biological mixtures. The most common assay we use is the Ames Assay (or Bacterial Reverse Mutation Assay) which measures the rate of mutation in Salmonella strains. Other common genotoxicity assays employed are the Mouse Lymphoma Assay (MLA), which detects mutagenic events in the L5178Y mouse lymphoma tk+/- cell line, and the micronucleus assay. We have recently phased out the MLA in favor of the micronucleus assay. 

      A cytotoxicity assay is designed to determine the toxicity of a chemical on living cells. The most common assay we use is the Neutral Red Uptake Assay (a cell viability assay). This assay is based on the ability of viable mouse embryo cells (BALB/c 3T3 cells) to incorporate and bind a neutral red dye in the lysosomes of a cell after exposure to a range of chemicals.

      For our in vitro research, we perform other studies to help us understand the effects of our products on different cell systems. Some of these include studies on lung cells, liver cells, and kidney cells. We usually start with 2D cell cultures before increasing in complexity with 3D cell cultures by using, for example, the Transwell system which enables the exposure of cells to air on one side and the cell culture liquid on the other side. This allows them to form an organotypic culture—one with 3D structure and behaviors more like what is found in the human airway. As such, we are using 3D cultures representing the different airway epithelia (nasal, buccal, gingival, bronchial, and small airway epithelium). With this method, our scientists can create cell cultures that better mimic tissues and organ substructures in the human body, as well as allowing whole aerosol exposures.

      Organs-on-a-chip are also used. These are typically made up of a microfluidic device that contains multiple chambers. Each chamber is designed to hold a tissue, and the chambers are connected by channels that allow fluids to flow between them, enabling the cells in the different chambers to interact with each other in a way that is not possible in traditional 2D cultures.

      Multiple cell cultures can now be combined within the same setting to better understand the effects that smoke-free product extracts and aerosols might have on the human body. For example, we developed a lung-and-liver-on-a-chip, where lung cells and liver cells shared a similar environment and could work together thanks to a common cell culture medium circulating between the two.

      Recent developments also include us partnering with TissUse to develop a highly innovative integrated human aerosol test platform that emulates the entire human respiratory tract with regard to dimension and architecture.

      Such capabilities, and more, allow us to strengthen our competence in understanding the effects of aerosols on biological tissues. By itself, the outcome of in vitro toxicity testing informs us if we need to study the toxicity of a substance in a living animal, also known as in vivo studies.

      Summary of PMI's in vivo methods

      PMI takes to heart public concerns about animal research. Animal testing is still the most reliable method to identify potential hazards without compromising human safety, which makes them a crucial component in regulatory approval. Nevertheless, we replace any animal testing method that is possible today with alternatives, similarly to universities and pharmaceutical companies. When replacement is not possible, the number of rodents in each experiment is reduced to a minimum.

      Our traditional in vivo approaches to inhalation toxicological assessment have followed internationally recognized testing guidelines, like the “Organisation for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals.” These guidelines are important for ensuring consistency in testing methods for regulatory requirements. OECD Test Guidelines 412 and 413 are specifically designed for fully characterizing the toxicity of test articles by inhalation and to provide robust data for toxicological risk assessment. As part of the OECD studies, we have conducted further investigations by using systems biology to gain mechanistic understanding of the changes that occur upon exposing animals to aerosols. For all our studies, we ensure that aerosol groups are compared with smoking and nonsmoking groups for reference.

      Some of the disease models we study in vivo are cardiovascular disease (and in particular the progression of atherosclerotic plaques), chronic obstructive pulmonary disease (COPD), and lung cancer.

      We follow, in our in vivo studies, the principles of the 3Rs—Replacement, Reduction, and Refinement—developed over 50 years ago to provide a framework for performing more humane animal research, as well as follow the national and international laws and rules governing the use of animals in scientific procedures. Our motivation in reducing in vivo testing is evidenced by our investment into research and development, our participation in conferences aimed at sharing knowledge and collaborating on the latest advances within animal testing, and our application of the animal welfare guidelines defined by the OECD.

      An overview of PMI's computational methods 

      In our efforts to adopt a systems biology approach as part of our assessment program of smoke-free products, we have incorporated more holistic analyses using omics technologies, resulting in terabytes of data. As such, we have combined mathematical and computational techniques to analyze the effects of aerosols from smoke-free products on biological systems. To do so, we have developed high performance computing infrastructure to store and analyze the data from an ensemble of factors such as molecular make-up, biological pathways, and disease mechanisms.

      Through modeling and extrapolation, computational biology provides us with insights into the mechanisms leading to the development or progression of smoking-related diseases. 

      Research

      See some examples of our studies that involve computational biology

      To leverage our computational capabilities, we have also initiated collaborative projects in the past. One of these was the crowdsourcing project sbv IMPROVER (set up in partnership with IBM Research) which ran a series of open science challenges, projects, and events from 2012 to 2022, with the aim of verifying and validating biological network models. An example of such science challenges was to identify and test new strategies for the diagnosis of inflammatory bowel disease.

      Another area which has greatly benefited from our capabilities in computational methods is computational fluid dynamics (CFD). To assess the toxicity and biological impact of cigarette smoke and smoke-free product aerosols, it is important to understand where and how much of the aerosol is deposited in the respiratory system. To achieve this, we use CFD simulations which integrate smoke/aerosol transport, evolution, and deposition mechanisms. These simulations require significant data and computing power to increase the quality and accuracy of their predictions. 

      Looking ahead, while our current focus is on machine learning and artificial intelligence for data mining and interpretation, we are always looking for new ways to improve and refine our biological models with the aim of further increasing the certainty of our toxicological assessments.

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      Reduced Risk Products ("RRPs”) is the term we use to refer to products that present, are likely to present, or have the potential to present less risk of harm to smokers who switch to these products versus continuing smoking. PMI has a range of RRPs in various stages of development, scientific assessment and commercialization. All of our RRPs are smoke-free products that deliver nicotine with far lower quantities of harmful and potentially harmful constituents than found in cigarette smoke.