ᅟ SUMMARY Box 1 | Recommended considerations for toxicological investigation of ingested engineered nano/materials (iENM) and assessment of manuscripts during the peer-review process

1. Test nano/materials should be food-grade (in vitro and in vivo)

2. They have been comprehensively characterized (in vitro and in vivo).

3. Nanoparticle dispersions should be prepared using standard and reproducible dispersion protocols (in vitro and in vivo).

4. Establish a realistic and physiologically-relevant dose range based on estimated daily intake, exposure variability, dose at the target site, and its estimated surface area (in vitro). Estimated daily intake values should be the basis for selection of relevant dose ranges in vivo studies.

5. Using appropriate co-culture or triculture models of gastrointestinal tract or microbiome that represent the exposure/target site (in vitro).

6. Confirmation of mature (intact/normal), immature (non-intact) or disease-state epithelium depending on the aim of the study (in vitro)

7. Consideration of the dynamics of the in vitro system (in vitro dosimetry).

8. Dissolution kinetics of test nanomaterial in relevant cell culture medium (in vitro).

9. Transformation of test nanomaterials through the gastrointestinal tract (if the target site is in the stomach, intestinal or colon segment) in the presence or absence of food matrix (in vitro).