Safety evaluation of Protaminobacter rubrum: Intravenous pathogenicity and toxigenicity study in rabbits and mice

The iv pathogenicity and toxigenicity of Protaminobacter rubrum was studied in New Zealand White rabbits and CF1 BR mice. Following a probe study, nine groups of six rabbits each were injected iv with: 1 ml of viable-cell suspension (VCS) at concentrations of 2.23 x 108, 106, 104 and 102 organisms/ml; the cell-free supernatants (CFS; in which the test organism had been cultured to a concentration of approximately 3 x 1010 cells/ml) at dilutions of 1:100, 1:10,000 and 1:1,000,000 in phosphate buffered saline (PBS); uninoculated culture medium; or uninoculated PBS. Rabbits were observed daily for 14 days and body weights were recorded on days 0, 7 and 14. Body temperatures were recorded in two rabbits per group and until 18 hr after dosing. On day 14, each rabbit was killed. Blood samples, sections of liver and spleen, and any tissues presenting lesions possibly indicating an infection were excised and cultured for P. rubrum. Deaths occurred in the probe study following 1 ml iv injection of VCS at a concentration of 2.5 x 1010 organisms/ml or of undiluted CFS in which P. rubrum had been grown to a concentration of approximately 2.5 x 1010 organisms/ml. Blood and tissue samples obtained less than 24 hr after treatment from rabbits in the VCS group tested positive for P. rubrum, which would be expected after iv administration of such a high concentration of cells. No deaths occurred, no adverse effects on body-weight gain were seen, and in no instance was P. rubrum recovered from blood or tissue cultures in the definitive study. Overt signs of infection or toxinosis were limited to transient reduced activity in the highest two VCS concentration groups, and the highest CFS group. Modestly elevated body temperatures were also recorded for rabbits that received the highest two concentrations of either VCS or CFS. A similar 14-day study was carried out in mice. Four groups of 20 Crl:COBS CF1 BR mice received a single iv injection of 0.1 ml of VCS (2.5 x 1010 organisms/ml), CFS in which the test organism was cultured to approximately 2.5 x 1010 cells/ml, uninoculated culture medium or uninoculated PBS. The mice were observed daily and body weights were recorded on days 0, 7 and 14. On day 14, each mouse was killed and blood samples as well as liver and spleen sections were obtained and cultured. In this study no deaths occurred, and signs of infection or toxinosis were limited to reduced activity and ptosis for all mice in the VCS and CFS groups. All mice appeared normal by 3-5 days after treatment. The tails (injection site) of several mice in the VCS group exhibited dark discoloration, oedema and occasional eschar. Sections of the tail were cultured for P. rubrum. In no instance was the test organism recovered from blood or tissue cultures. The results of these studies indicate that P. rubrum is not pathogenic and has only a low order of toxigenicity. Intravenous administration of large numbers of organisms and media derived from high-concentration cultures to a rodent and a non-rodent species provides a most rigorous test for potential pathogenicity/toxigenicity of P. rubrum. These findings provide a high degree of confidence that, if P. rubrum or its by-products should accidentally enter the final product, they will not present a hazard.