Reporting negative Ames test results for Indian Acorus calamus L., rhizome, extracts, and beta asarone.

Objective: Acorus calamus L. (Sweet Flag), known in India as "Vacha," is widely used in traditional medicine, particularly for cognitive enhancement in infants. While traditionally considered safe, β-asarone - a key constituent - has shown potential genotoxicity in some in vitro studies. This study aimed to evaluate the mutagenic potential of Indian A. calamus rhizome, its extracts, and pure β-asarone using the Ames test in accordance with Organization for Economic Co-operation and Development guidelines.

Materials and methods: Samples tested included pure β-asarone, dried rhizome powder, and extracts prepared in acidic and alkaline aqueous media. Marker compounds (β-asarone, α-asarone, and shyobunone) were quantified. The Ames test was conducted under GLP using Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 (Moltox Inc., USA). Each strain was tested with and without metabolic activation using Aroclor 1254-induced rat liver S9 homogenate. Positive controls included 2-aminoanthracene, sodium azide, mitomycin C, 9-aminoacridine, and 4-nitro-o-phenylenediamine. The vehicle was used as a negative control. Doses tested ranged from 39.06 to 5000 µg/plate.

Results: No mutagenic response was observed in any of the tested samples across all five strains under either condition (±S9). Positive controls showed expected increases in revertant colonies. Revertant counts for test samples did not reach the threshold for a positive response (≥3 × control for TA1535, TA97a; ≥2× for TA98, TA100, and TA102).

Conclusions: Under the test conditions, A. calamus rhizome and β-asarone did not exhibit mutagenicity. Further studies, including chronic toxicity and carcinogenicity evaluations, are recommended to confirm safety across formulations.