Multiple substance considerations applied to chemical mixtures in ecological risk assessments can be logically extended to nontarget organism (NTO) risk assessment for pyramided trait crops expressing multiple insect resistance genes. A case instance is developed that considers a two-protein pyramid of Cry1F and Cry1Ac synthetic proteins expressed in cotton (Gossypium hirsutum L.). A mixture toxicity approach was used to arrive at the aggregated multisubstance potentially affected fraction (msPAF) of NTOs that may be at risk from exposure to Cry1F + Cry1Ac cotton in representative-use environments. Development of the msPAF for putative susceptible NTOs considered laboratory toxicity data for Lepidoptera expressed in terms of additive mixture toxicity as well as data on in planta expression of the Cry1F and Cry1Ac proteins and their translation into environmental loads and exposure concentrations. The msPAF based on tier 1 estimated environmental concentrations (EECs) and toxicity to Lepidoptera species—used as surrogate data for adverse effects to a putative susceptible species—provided a highly conservative estimate of effects on beneficial species and therefore is a ready means to conduct screening-level NTO risk assessments for pyramided crops. Environ. Toxicol. Chem. 2011; 30:763–772. © 2011 SETAC
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