Multi‐Endpoint Toxicological Assessment of the Food Additive Triammonium Citrate: Differential Responses in Colorectal Cancer Cells and Non‐Neoplastic Colon Fibroblasts

ABSTRACT

Triammonium citrate (TAC; E380) is used as a food additive, making characterization of its effects in malignant and non‐neoplastic colon‐derived cells relevant to applied toxicology. We conducted a multi‐endpoint assessment of 0.5‐ to 2.0‐mg/mL TAC in KRAS ^G13D ‐mutant, mismatch repair‐deficient HCT116 colorectal carcinoma cells and non‐neoplastic CCD‐18Co colon fibroblasts. Exposure durations were endpoint‐specific (60 min, 24 h, or 48 h), and endpoints included viability, primary DNA damage, micronucleus frequency, nuclear division, clonogenic survival, and wound closure. Microbial assays tested antimicrobial and membrane‐disruptive activity; docking was exploratory. The clearest differential response occurred at 2.0 mg/mL after 48 h, when viability was 8.80% ± 2.14% in HCT116 cells and 49.52% ± 11.50% in CCD‐18Co cells. Primary DNA damage increased in both models, with the largest numerical comet response in HCT116 cells at 2.0 mg/mL. HCT116 clonogenic survival decreased to 29.0% ± 4.8% at 2.0 mg/mL after a 24‐h exposure and 9‐day recovery. Micronucleus frequency did not increase in either cell line, although the nuclear division index decreased in both at 2.0 mg/mL after 48 h, indicating cytostasis. TAC also reduced wound closure in both models. No microbial growth inhibition was detected at 0.5–2.0 mg/mL, and no membrane leakage was detected in Staphylococcus aureus up to 200 mg/mL. Docking generated hypotheses involving PFK‐1, KRAS , and PARP‐1 but did not establish binding or target engagement. Collectively, these findings demonstrate pronounced HCT116 cytotoxicity together with primary DNA damage in both models, without detectable chromosome‐level damage, and provide a basis for mechanistic investigation.