Assess the toxigenic potential of microbial strains through EFSA-aligned cytotoxicity testing, supporting regulatory safety evaluation.
In vitro cytotoxicity testing is used to evaluate whether microbial strains produce biologically active compounds that may have harmful effects on mammalian cells. In line with EFSA guidance on the characterisation of microorganisms used in the food chain, cytotoxicity testing is applied to assess toxigenic potential, particularly for strains where genomic analysis indicates the presence of biosynthetic gene clusters, virulence factors, or other risk indicators.
Cytotoxicity testing is a key component of microbial safety assessment under EFSA frameworks. It is specifically required in certain cases (e.g. Bacillus species with QPS qualification “absence of toxigenic activity”) and is used to confirm whether predicted toxigenic potential translates into biological activity. This ensures that safety conclusions are supported by both genomic and functional evidence.
Cytotoxicity testing was performed using an LDH release assay on culture supernatants, in accordance with EFSA recommendations for the assessment of toxigenic potential. LDH release reflects loss of cell membrane integrity and is particularly relevant for detecting the activity of Bacillus-associated toxins such as haemolysins and enterotoxins. The assay was conducted with appropriate controls to ensure sensitivity and reliability.
Cytotoxicity results are interpreted in combination with whole genome sequencing (WGS), antimicrobial activity testing, and literature review. This integrated approach supports a weight-of-evidence evaluation of toxigenicity and contributes to robust regulatory conclusions.
Potential toxigenic risk is identified through genome analysis (e.g. BGCs, toxin genes) or literature review.
Appropriate cell models, sample types (supernatant or product), and exposure conditions are defined.
Samples are tested in vitro to assess effects on mammalian cell viability.
Results are evaluated in the context of EFSA requirements and integrated into a weight-of-evidence safety assessment.
Aligned with EFSA guidance on toxigenicity and pathogenicity
Combined with WGS and literature study
Use of established mammalian cell-based assays
Results translated into regulatory conclusions
Cytotoxicity testing evaluates whether microbial strains produce biologically active compounds that can harm mammalian cells. It complements genomic analysis by confirming whether predicted toxigenic potential results in functional effects, supporting EFSA-compliant safety assessment.
Cytotoxicity testing is typically performed when WGS analysis identifies genes or biosynthetic gene clusters associated with toxin production, or when required by regulatory frameworks such as QPS qualifications.
Cytotoxicity testing is primarily conducted using culture supernatants, in line with EFSA guidance, to evaluate secreted metabolites that may include toxigenic compounds. The use of supernatants ensures a conservative assessment, as metabolite concentrations in broth cultures are typically higher than those present in final products.
Commonly used models include mammalian cell lines such as VERO cells or other relevant systems, depending on the test design and regulatory context.
WGS identifies the genetic potential for toxigenicity, while cytotoxicity testing confirms whether this potential translates into functional biological effects.
Identify genes related to toxigenicity and virulence
Assess production of biologically active antimicrobial compounds
Work with our experts to design an EFSA-aligned cytotoxicity testing strategy and strengthen your safety assessment. Your first consultation is free and without obligation.
