*2.8. Cytotoxicity Study*

Particle dispersion in serum-free cell culture media: Particle stock solution (1 mg/mL) was prepared by dispersing the particle powder in deionized water by probe sonication for 30 s of five intervals. The sonicated particle suspensions in DI water were further diluted in serum-free Dulbecco's modified eagle medium (DMEM) medium.

Cell culture and co-incubation with particles: Human epithelial Caco-2 cells between passages 20–40 were cultured in DMEM (Invitrogen, Grand Island, NY, USA) supplemented with 10% (*v*/*v*) fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA), and 1% antibiotics (penicillin/streptomycin; Invitrogen) 10 cm<sup>2</sup> cell culture plates. These plates were incubated at 37 ◦C in a 5% CO2 incubator to reach 70–80% confluency. The cells were trypsinized, washed and seeded at 10,000 cells in 100 μL per well in a 96-well plate. Seeded cells were cultured overnight at 37 ◦C in a 5% CO2 incubator for complete attachment before particles exposure. The stock solution of particles dispersed in DI water was further diluted in serum-free DMEM medium, and particles exposures were carried out at concentrations of 25, 12.5, 6.25, 3.12, 1.56, and 0 μg/mL for 24 h.

Effect of particles on cell viability: Cytotoxicity of the particles on Caco-2 cells were assessed by the Alamar Blue (resazurin) assay after exposure to the desired concentration of the particles. For this, the exposure media was removed from the wells, and the cells were incubated with 100 μL resazurin solution (50 ppm) and incubated at 37 ◦C in 5% CO2 for 4 h. Absorbance at 570 nm was recorded using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA). The readings were exported to an Excel file for comparison and statistical analysis.

Cytotoxicity was calculated as:

$$\text{Cell viability } (\%) = \frac{\text{Abs test}}{\text{Abs control}} \times 100 \tag{1}$$

### *2.9. Application of Alternatives Assessment Framework*

Developing sustainable alternatives to existing solution demands evaluation not only for performance but also for its cost and safety. The alternatives assessment framework allows us to identify sustainable alternatives to existing chemicals that needs replacement [26]. We conducted a preliminary alternative assessment of CCLDH for its potential application as a food pigment. The assessment conforms to the US National Academy's Framework to Guide Selection of Chemical Alternatives. Accordingly, the Efficacy (% reflectance), Cost (USD/g) (Table S1), and Safety (NOAEL) (Table S2) were used for a multiparametric analysis using MATLAB, as detailed in Supplementary Materials.
