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A series of botulism challenge studies were conducted to determine if botulinum toxin would be produced in mussels (Mytilus edulis) inoculated with non-proteolytic Clostridium botulinum spores and held under modified atmosphere (MA) packaging conditions at normal (4 °C) and abusive (12 °C) temperatures. Spore mixtures of six strains of non-proteolytic C. botulinum were introduced into live mussels through immersion in a seawater solution with cultured algae. Mussels were packed in a commercial high-oxygen (60-65% O2) MA-package with a buffer, and also packed under a vacuum. Feeding live mussels cultured algae (10(4) cells/ml) with a C. botulinum spore suspension (10(3) spores/ml) in seawater at 4 °C for 6 h resulted in the uptake of spores into mussel tissue (500/g) and the mussel GI tract (100/g). Under all of the experimental conditions evaluated, none of the fresh mussels became toxic, even after spoilage and in the absence of oxygen. However, control samples using tuna or cooked mussel meats became toxic in the absence of oxygen. Botulinum toxin was not produced in fresh mussels packaged under the MA-packaging conditions evaluated, even at an abusive storage temperature (12 °C) for at least 12 days or at normal storage temperate (4 °C) for at least 21 days, which is beyond their shelf life., (Copyright © 2014 Elsevier Ltd. All rights reserved.)