Report of Sub–chronic toxicity (safety) evaluation of Prosopis juliflora – Wholepod in Sprague Dawley rats Volume-IV

Abstract

Methodology: The investigation was conducted in sixty Sprague Dawley rats (30M+30F), aged 6-8 weeks, weighing 180-200 gm obtained from the National Center for Laboratory Animal Science (NCLAS) after approval of IAEC(Appendix-lV) (No.P8/1–2011/BDK). Test materials: The powdered test material supplied from sponsor has been subjected for compositional analysis. The wheat in the standard pellet diet was replaced with 40% and 80% of test material (Mesocarp) (SOP-Appendix-VIII). The pellet diet with and without test material has been provided to the animals in respective groups (SOP Appendix-VIII). S.No Group Details Test material administration 1. Control Standard Diet (NCLAS Pellet diet) 2. PJWP-50 wheat flour substituted with 50% of wholepod 3. PJWP-100 wheat flour substituted with 100% of wholepod Test Material preparation: The test material supplied by the sponsor was Prosopis juliflora – (wholepod) based on the exposure level (50% and 100%), the above test material has been substituted to wheat flour in NIN standard diet and prepared a pellet for oral feeding ad libitum as per SOP (No.17/PHARM/NIN/CO1/ATI/2007/OL). Diet was autoclaved at 121oC at 15 lb pressure and air dried and canned properly and labeled as Control diet, PJWP-50 and PJWP-100 respectively, and stored at room temperature. Test diet was prepared for every 10 days. Entire procedure was repeated for every 10 days for 3 months. The prepared test material pellet diet was fed daily for 90 days to the respective group animals. Test Material Administration (a) Control Group: The standard NIN pellet Diet was given 15-30 g to each rat daily for 90 days. (b) PJWP-50 Group: The PJWP-50 substituted NIN pellet was given 15-30 g to each rat daily for 90 days. (c) PJWP-100 Group: The PJWP-100 substituted NIN pellet was given 15-30 g to each rat daily for 90 days. Experimental phase: All rats were conditioned for 11 days. The conditioned animals were randomly divided into three groups viz., i. Control (c) ii. PJWP-50 (wholepod-50%) iii. PJWP-50 (wholepod-100%). The dietary profile was recorded daily for nine days after conditioning period. The test material containing 50% and 100% wholepod were fed for 90 days to respective groups. Study Parameters: The body weight was recorded twice in a week, whereas feed intake was monitored daily till end of experiment. The live phase, cage side, physical and neurological activity were examined bi-weekly. The clinical chemistry, hematology, profile was assessed in serum and blood samples after 48 hrs of last exposure. Serum IgG2a, & IgE were determined to assess allergenicity potential if any. The animals were euthanized 48 hours of last exposure followed gross necropsy and recording of weights of vital organs. All organs were subjected for histopathological evaluation. The data was compiled and analyzed for significant differences between individual groups. Results:  No pre-terminal deaths were recorded in control and PJWP-50 groups. One male animal died pre-terminally in PJWP-100 group on 85th day of experimental period.  Significant difference was observed in experimental groups in feed intake during the first 11 days due to the adaptation of animal to the diet regime.  The gain in body weight was significantly lower in PJWP-50 between 4thto 15th day of experiment and in PJWP-100 between 4th -32nd days of experiment as compared to Control group. This can be attributed to adaptation to test material.  There were no abnormal changes in live phase, physical and neurological activity between the control and test groups throughout the study period.  The clinical chemistry profiles were normal in all groups except decreased glucose levels and increased total bilirubin in PJWP-100 . All these changes were in normal historical range.  The hematology profile was normal except for significant difference in eosinophils count in PJWP-50 group as compared to control group. These chages are also in normal range.  The serum total IgG2a and IgE levels in rats fed on PJWP-50 and PJWP-100 were found to be comparable with the control. However, the serum total IgE levels of PJWP100 group were lesser than the control, though this difference was not statistically significant.  A significant difference was observed in weights of liver and kidneys in animals fed with wholepod-50%, where as in wholepod-100% significant difference was observed only in weights of liver when compared to control group.  It was observed that the various changes seen in experimental group were also seen in control group and hence not considered significant and may not be due to administration of test item. Conclusion: All animals were active except one male animal which died in PJWP-100 group on 85th day of experimental period. Though there was significant observations in the initial phase of experiment but found to be normal with the duration of experiment in feed intake and body weight gain. Clinical chemistry and hematology profile indicate no abnormal changes in rats expose to test material. Histopathological changes were comparable between the groups and not considered significant. Study impression: The replacement of Prosopis juliflora mesocarp 50% - 100% with wheat in the standard NIN diet did not induce any morbidity till 90 days of study period in experimental animals.