These assays are:
These short-term in vitro assays measure two major endpoints: genotoxicity and cytotoxicity. The gentoxicity assays (Ames assay, in vitro micronucleus assay and in vitro mouse lymphoma assay) measure the ability of the Test Articles to cause changes at different levels of the genetic material and each assay has different sensitivities (as discussed below). Therefore, when all three genotoxicity assays are combined, together with the cytotoxicity assay, they are able to detect most mutagens and cytotoxic compounds. The Committee on Mutagenicity (COM) “Guidance on a strategy for Testing of Chemicals for Mutagenicity” currently recommends the Ames test, the in vitro micronucleus assay and the in vitro mouse lymphoma assay for the in vitro testing of chemicals. The CORESTA in vitro toxicology task force recommends the Ames test, a mammalian in vitro assay for cytogenetics or mutation, and the neutral red uptake assay. Therefore, the battery of in vitro assays we use meet the minimum requirements of both COM and the CORESTA in vitro toxicology task force.
The assays are performed in an independent GLP-certified Contract Research Organisation. Each study starts with a detailed protocol and rigorous acceptance and evaluation criteria, including appropriate statistical analysis.
The Ames test was originally developed by Bruce Ames in 1975 . This test is based on the fact that histidine is usually required for bacteria to grow. In the Ames test, the bacteria are altered in such a way that they are extremely sensitive to mutagens, and the gene that encodes for histidine synthesis is mutated, so the bacteria cannot produce histidine for growth, and therefore cannot survive. If these cells are exposed to a genotoxic Test Article, it can induce changes (point mutations) in the gene at the nucleotide level, that encodes for histidine, so the bacteria can produce histidine endogenously, and therefore grow. These bacteria are referred to as ‘histidine revertants’. The Ames test demonstrates the extent to which the Test Article can induce mutation in bacterial cells. For example, the picture above shows a control Ames plate (left) with spontaneous revertant colonies. After exposure to a Test Article, which induces mutation, the number of revertant colonies increase (right).
We use the standard battery of 5 strains of Salmonella typhimurium (Table 1), and as these bacterial cells are metabolically incompetent, a metabolic activation system is also added, which provides sensitivity to a wide range of mutagens. The methods we use have been standardised by the following guidelines: OECD Guideline 476 (1997 ); UKEMS Guidelines (1990 ); ICH Harmonised Tripartite Guidelines 2011); 2000/32/EC Annex V, B13/44 ; and have been recommended by the CORESTA in vitro toxicology task force (2003).
Table 1: Mutations detected by different strains of Salmonella typhimurium.
The in vitro micronucleus assay is a mammalian chromosome damage assay which complements the Ames test. This assay detects structural aberrations in mitotic Chinese hamster V79 cells, measured by micronuclei. These are produced by whole chromosomes or acentric fragments of chromosomes that are unable to attach to the spindle at mitosis, and appear as small darkly staining bodies adjacent to the main daughter nuclei.
Our in vitro micronucleus assay complies with OECD Guideline 487 (2010) and the recommendations of the CORESTA in vitro toxicology task force (2003).
The in vitro mouse lymphoma assay utilises L5178Y cells and the enzyme thymidine kinase to detect gene mutations and chromosome aberrations. This is a salvage enzyme for nucleic acid breakdown products, and if a toxic base analogue (5-trifluorothymidine (TFT)) is present in the medium, the enzyme will incorporate the analogue into the cell, killing it. However, if this enzyme is rendered inactive, by mutation by a Test Article, the mutated cells are unable to incorporate the toxic analogue, and survive in its presence. This picture depicts mutant colonies caused by exposure to a Test Article. The large colonies represent point mutations and the small colonies represent chromosome aberrations
Our in vitro mouse lymphoma assay complies with OECD Guideline 476 (1997), UKEMS Guidelines (1990), ICH Tripartite Harmonised Guidelines (2011), and the recommendations of the CORESTA in vitro toxicology task force (2003).
The Neutral Red uptake assay is a cellular viability assay, based on the ability of cells to incorporate the supravital dye, Neutral Red. Neutral Red can readily penetrate cell membranes by passive diffusion, and accumulates in lysosomes by an energy dependant process. Therefore, this assay measures the cell membrane integrity and the cellular energy status. If a Test Article disrupts these processes, it can be detected by a decrease in the uptake of Neutral Red. This diagram shows cellular uptake of Neutral Red dye in a 96 well plate. The cells in columns 11 and 12 (vehicle control) show maximum uptake of Neutral Red dye. From column 1 to 10 (as the concentration of the cytotoxic Test Article increases), the cells gradually reduce their Neutral Red uptake, due to cytotoxic damage, as shown by a decrease in staining of the wells
Our Neutral Red assays are performed based on guidance described in “ICCVAM: in vitro Cytotoxicity Test Methods for Estimating Starting Doses for Acute Oral Systemic Toxicity Testing (November 2006), and the recommendations of the CORESTA in vitro toxicology task force.
In the routine in vitro evaluation of combustible tobacco products the particulate fraction of the smoke aerosol, particulate matter (PM) from mainstream cigarette smoke is used as the Test Article. As we move forward, we plan to use fresh tobacco smoke aerosol as the Test Article, rather than the particulate phase, as the PM omits gas (vapour) phase constituents. We are currently investigating the feasibility of exposing in vitro cultured cells to freshly generated whole smoke for each of the assays mentioned above. The smoke is generated on a Vitrocell VC10™ smoking robot. Cells on porous membranes are then exposed to the smoke on the upper surface, while being supplied with cell culture media from below. This system allows the direct in vitro exposure to the whole smoke aerosol.