49 and five different strains of S. typhimurium (TA97A, TA98, TA100, TA102, and TA1535). This is an incorporation assay in the presence and absence of an exogenous mammalian activation system (S9). Frozen working stocks were used to create working cultures of each bacterial strain. Cultures were grown overnight at 37 �� 2 ��C until a density of 0.6�C1.6 at 650 nm was reached. ntSPONGE? selleck extract or vehicle control plus bacteria culture plus either PBS or metabolic activation solution (S9) was added to a molten top agar of 0.6% Difco agar in 0.5% NaCl supplemented with an l-histidine/0.5 mM biotin solution. The solution was vortexed and allowed to harden and incubated for 48 -72 h at 37 �� 2 ��C. All plates were scored using an automatic image analysis (Domino Image Analyzer) system for colony counting.

Negatives controls were plated with normal saline (NS) or DMSO extraction blanks, with and without S9. Mouse lymphoma assay The assay procedures used were based on those developed by Clive et al., The test materials were extracted in normal saline or DMSO for 72 h at 37 ��C. The saline test article and saline negative control were dosed in 1.0 mL volumes while the DMSO test article and control were dosed at 0.1 mL volumes. A total of 6 �� 106 cells in a final volume of 10 mL (cells plus test article) were incubated at 37 ��C on a shaking incubator (80 rpm) for 4 h. The cells were then washed once and resuspended in 20 mL media and incubated for 48 h. Prior to cloning, cells were adjusted to a final density of 2 �� 105 cells/mL in 20 mL of media.

Mutagenicity was determined by suspending 5 mL from each dose tube (1 �� 106 total cells) in the cloning media. Three plates were prepared from each tube. After 11 d of incubation at 37 ��C, the colonies were counted using a Domino Image Analyzer including software for colony size discrimination. Systemic toxicity Systemic toxicity of ntSPONGE? was evaluated with the test protocol ISO Acute Systemic Injection Test. Twenty mice were injected systemically with two extracts of ntSPONGE? (normal saline [NS] or cottonseed oil [CSO]) or the appropriate vehicle. Animals were observed for fatality/signs of toxicity immediately after injection and at 4, 24, 48, and 72 h post-injection. Animals were also monitored for weight loss. Subchronic intravenous toxicity was evaluated using 20 mice.

Animals were injected intravenously daily for 14 d at a dose of 10 ml/kg. Observations for mortality and clinical signs of pharmacologic and/or toxicologic effects were made immediately post-injection and once a day for 14 d. Blood samples were drawn on day 14 via cardiac puncture. Gross necropsy was also performed. Subacute intraperitoneal toxicity was evaluated using 20 mice. Animals were injected intraperitoneally daily for 14 d at a dose of 1ml/kg in cottonseed oil. Animals were observed immediately post-injection and daily for 14 d for mortality and clinical signs of Brefeldin_A toxicologic and pharmacologic effects.