Identifying and developing safer and more effective food additives are essential for a healthy growing population. Regulators such as the FDA and EFSA are responsible for monitoring the safety of these food additives. Current in vitro approaches for assessing genotoxicity of these additives present a lack of consistency in the literature regarding incubation time and analysis.
At the Society of Toxicology (SOT) conference on March 10-14, 2024, Cyprotex presented a poster titled, ‘Validation of a new genotoxicity pre-screening package for food additives’. The research evaluates a high content screening approach with robust data analysis which would be suitable as an early stage genotoxicity screening package for de-risking food additives.
Genotoxins are chemicals that cause DNA or chromosomal damage. This can be assessed using in vitro assays such as the phosphorylation of histone H2AX (pH2AX) and histone H3 (pH3), and the micronucleus test (MNT; OECD guideline 487). By assessing both pH2AX and pH3, it allows for assessment of clastogens (pH2AX) and aneugens (pH3). Clastogens are substances that result in structural damage to the chromosome through DNA double strand breaks. Aneugens are substances which result in the daughter cell having an abnormal number of chromosomes due to deletion or insertion of a whole chromosome. The in vitro MNT detects micronuclei which are formed from the misincorporation of chromosomal material that might be structurally and/or genetically damaged, due to interactions with clastogens and/or aneugens interactions. It is an approach recommended by the regulatory authorities.
For the pH2AX and pH3 assays, HepG2 cells were dosed with the food additives over 24hr. For the in vitro MNT, CHO-K1 cells were dosed with the food additives over 24hr. All assays used automated high content screening with robust data analysis to identify potential genotoxicity. From the 12 food additives assessed, 83% were correctly identified in at least one of the methods. Both of the false negatives (benzoic acid and tartrazine) have been reported to induce DNA damage under certain conditions but not others. This disparity in the literature may explain our results.
In summary, Cyprotex have developed an early stage high throughput screening approach to assess the genotoxic potential of food additives using a panel of assays to determine clastogenic and aneugenic potential in addition to micronucleus formation. As well as genotoxicity markers, the assays provide valuable additional information on cell survival, membrane integrity and cell cycle information.
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