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Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 7 of 12 Germination stimulation and inhibition. To investigate the effect of initiation of germination on the sporicidal effect of BCTP on Bacillus spores, the germination inhibitor, D-alanine [17, 18], and germination enhancer, L-alanine [19, 20], were incubated with the spores and BCTP for up to 1h. Percentage of killing was calculated at different time points. The sporicidal effect of BCTP was delayed in the presence of 10 mM/D-alanine and accelerated in the presence of 5 m/L-alanine (figure 5). All of the individual time points showed a significant difference in killing between the three treatments (P < 002). Figure 5. Effect of germination inhibition and stimulation on sporicidal activity of BCTP diluted 1: 100 against Bacillus cereus spores. Sporicidal activity of BCTP was delayed in presence of 10 mM D-alanine (germination inhibitor) and accelerated in presence of 5 mM L-alanine (germination enhancer). All time points show significant difference between 3 treatments (P < .002). In vivo toxicity testing. CD-1 mice injected with BCTP diluted 1 : 10 in saline did not exhibit signs of distress or inflammatory reaction, either grossly or histologically (figure 64, 6B). Identical results were obtained when the toxicity of BCTP 401 was tested in mice subcutaneously. Intramuscular injection of the BCTP or BCTP 401 diluted 1 : 10 did not have any toxic effects in the form of inflammatory reaction, edema, or necrosis in mice. Open wound irrigation with 2 mL of the emulsions did not result in any pathologic damage. Intranasal instillation of the emulsion was less tolerable because of its viscosity; however, there was no injury from BCTP diluted 1 : 50 and BCTP 401 diluted 1 : 25. Oral administration of 10% BCTP (4 mL/kg of body weight daily) in rats for 1 week did not result in any gross or pathologic changes, and the rats maintained normal weight gain during this period (data not shown). In these tests, pathologic’examination of local tissues and internal organs was done, and no abnormalities were detected. Figure 6. Gross and histologic photographs of animals injected subcutaneously with different combinations of BCTP and Bacillus cereus spores. A, B, animals injected with BCTP alone at dilution of 1 : 10. There was no gross tissue damage, and histology showed no inflammation. C, D, animals injected with 4 x 107 B. cereus spores alone subcutaneously. Large necrotic area resulted, with average area of 1.68 +0.35 cm”. Histologic examination of this area showed essentially complete tissue necrosis of epidermis and dermis, including subcutaneous fat and muscle. £, F, mice injected with 4 x 107 Bacillus spores that had been immediately premixed hitp://www.joumals.uchicago.edu/JID/journal/issues/v180n6/990281/990281 text.html 2/18/2005