*2.6. Water Activity (aw)*

The *aw* was measured on three biscuits for each sample using an AQUALAB 4TE water activity meter (Decagon Devices Inc., Pullman, WA, USA).

#### *2.7. Extraction Protocol for Acrylamide*

For each one of the eight FDOE points, experimental biscuits (around 35 g) were finely ground with a blender. The powder obtained was separated into its particle size classes using decreasing pore size sieves (Giuliani Tecnologie S.r.l., Torino, Italy) with the following meshes: 850 μm, 500 μm, 212 μm, and 63 μm. The powder fraction recovered on the 63 μm sieve (size within 63–212 μm) was used for the extraction.

An aliquot of 2.50 g from each gross sample was transferred into a 50 mL polypropylene flat-top screw cap tube, acrylamide d3 (500 uL; 0.4 mg/kg) was added, and a ceramic homogenizer for QuEChERS was introduced. Afterwards, 7 mL of water and 10 mL of acetonitrile were introduced, and the tube was manually shaken for 1 min following the addition of each solvent. A ready-to-use mixture of QuEChERS pouch composed of MgSO4 4.0 g + NaCl 0.5 g was added and manually shaken for 1 min and then vortexed for 3 min to facilitate the acrylamide migration into the acetonitrile phase. Each tube was twice centrifuged for 3 min at 3500 rpm to assure the separation of the layers. A portion of the upper layer (measured at exactly 5.0 mL) was withdrawn and evaporated to dryness using a rotational vacuum concentrator (Eppendorf concentrator 5301, Eppendorf, Hamburg, Germany). The sample was re-dissolved in 1 mL of water. Following centrifugation (Mini Spin, Eppendorf Italy, Milan, Italy), the supernatant of each sample was introduced into a 2 mL vial. The same extraction procedure was carried out and analyzed in triplicate using three different aliquots of each ground gross sample.
