Assessment of in vitro toxicity of chloronitramide anion (CNAM)

In 2024, the chloronitramide anion (CNAM) was identified as a degradation product of inorganic chloramines, more than 40 years since its existence was suggested. However, to our knowledge, no studies have evaluated the toxicity of CNAM. In this manuscript, we evaluate CNAM in vitro toxicity using the Chinese hamster ovary (CHO) cells chronic cytotoxicity assay. CNAM exhibited a LC₅₀ value of 9.99 × 10^-5 M (99.9 µM; 9.45 mg/L), where LC₅₀ denotes the concentration causing 50% cell death. These results indicate that CNAM is more toxic than regulated disinfection by-products (DBPs), including trihalomethanes (THMs) and haloacetic acids (HAAs). Toxicity-weighted DBP concentration analyses further demonstrated that CNAM is the major driver in chloraminated drinking water cytotoxicity, accounting on average for 71% of the calculated cytotoxicity index (CTI). In chlorinated tap water, CTI analysis showed that CNAM contributed, on average, 8.0% of the total DBP-associated toxicity. In waters treated with chloramines, toxicity-weighted concentration index values for CNAM exceeded those of all other evaluated DBP classes, including haloacetonitriles (HANs), haloacetamides (HAMs), haloacetaldehydes (HALs), halonitromethanes (HNMs), THMs, HAAs and haloketones (HKs). In chlorinated waters, CNAM would also be a potential major cytotoxicity contributor under currently available occurrence data. Additional in vitro testing further suggests that CNAM induces substantial oxidative stress in CHO cells, whereas mitochondrial dysfunction and DNA damage were not significant under the tested conditions.