Study ID 306	Sequani Limited, Neohesperidine dihydrochalcone: In Vitro mammalian cell micronucleus test, Unpublished report dated 08 February 2018
Funding	Private
Good laboratory practice (GLP) compliance and guideline	Good laboratory practice: yes Guideline studies: OECD TG 487 (2016)
Test system	In vitro micronucleus test in human lymphoblast TK6 cells.
Test material	Neohesperidine dihydrochalcone, batch number 017J017, purity 97.8%
Exposure/treatment conditions	Short treatment: 3 h in the presence and absence of S9 mix and harvest after a recovery period of 41 h (3 cell cycles); extended treatment: 27 h (1.5–2 cell cycles). Doses: 200, 650 and 2,000 μg/mL (short treatment with and without S9 mix); 65, 400, 650 and 750 μg/mL (extended treatment). For each dose and vehicle (DMSO) and positive controls, approx. 20,000 nucleated events were scored by flow cytometry in duplicate cultures.
Results	Equivocal with S9 mix, negative without S9 mix. Short treatments resulted in moderate cytotoxicity, with relative increases in cell counts (RICC) of 88% and 57% at the highest dose, with and without S9mix respectively. After treatment in the presence of S9mix, a statistically significant increase in micronucleus frequency, above the historical control range, was observed at the low and intermediate doses. No statistically significant increase in micronucleus frequency was observed without S9mix and there was no clear dose response. Extended treatment in the absence of S9 resulted in a reduction of RICC of approx. 55% (maximum recommended), and a slight increase in micronucleus frequency, within the historical control range, at one single dose (400 μg/mL).
Reliability/relevance	The study is considered reliable with restriction and its relevance limited. In the short‐term treatment the recovery time was longer (3 cell cycles) than recommended in OECD TG. In case of treatments inducing no cell cycle delay (as shown by RICC for treatment with S9mix) a delayed recovery of treated cells may result in the dilution of damage induced by treatment.

Study ID 789	Bioneeds India Private Limited, In vitro Mammalian Cell Micronucleus Test of Neohesperidine DC in Human Lymphocytes, Unpublished report dated 8 April 2022
Funding	Private
Good laboratory practice (GLP) compliance and guideline	Good laboratory practice: yes Guideline studies: OECD TG 487 (2016)
Test system	In vitro micronucleus test in human lymphocytes.
Test material	Neohesperidine dihydrochalcone, batch number 021F043, purity 98.2%. Solvent DMSO.
Exposure/treatment conditions	Duplicate incubations. Concentrations tested were 0.5, 1.0 and 2.0 mg/mL (mild ppt seen at 2.0 mg/mL) 3–6 h exposures with and without metabolic activation. 20–24 h incubations without metabolic activation. Cytochalasin B used to block cytokinesis.
Results	Treatments induced dose‐related cytotoxicity, up to 40%–42% after short exposure (with and without S9, respectively), and 46% after extended treatment without S9. Under all exposure conditions there was no significant increase in micronuclei formation compared to negative controls. Vehicle (DMSO) and positive controls were within historical control ranges.
Reliability/relevance	Reliable without restriction and of high relevance.