An extensive safety assessment process exists for novel proteins used as enzymes in food and feed.As part of the mammalian safety assessment, novel proteins are evaluated for their toxicological potential.This is accomplished by employing a holistic approach where the host organism, gene, and the gene product (i.e., protein) are evaluated.Some relevant questions initially asked include: 1) Does the host organism have a history of safe use in the manufacture of enzymes? Or does the lack of pathogenicity or toxicity still need to be established? To help answer this first question, an established principle that the safety of the production organism is paramount to the safety of the enzyme (i.e., safe strain lineage) is employed (Pariza and Foster 1983); 2) Does the expressed protein have a history of safe use? To evaluate a novel protein(s), a weight-of-evidence approach, which takes into account a variety of factors for an overall assessment of toxicological potential, is conducted.This assessment is based on what is known about the novel protein and protein toxins, including similarity to known toxins (in silico amino acid sequence identity comparisons to protein databases); stability to pepsin digestion in vitro; acute toxicity evaluation based on OECD Guidelines, mode of action; and exposure level and dietary intake.Novel proteins for food and feed use are also evaluated for skin and eye irritation, genotoxic effects, and in some cases (e.g., proteases) skin sensitization.Novel proteins for food and feed use are further evaluated for safety by conducting repeat dose 90-day feeding studies according to OECD Guidelines in rodents where general health and food consumption, clinical chemistry, hematology,and histopathology are evaluated.