ACUTE ORAL TOXICITY TEST OF PYKURE CAPSULES 750mg IN MICE
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1 ACUTE ORAL TOXICITY TEST OF PYKURE CAPSULES 750mg IN MICE Address for Correspondence: Nirav Patel a, R. Balaraman b Prof. R. Balaraman Pharmacy Department Faculty of Tech. & Engg., The M. S. University of Baroda, Kalabhavan, Vadodara a nirs_patel@yahoo.com b rbalaraman2000@yahoo.com
2 ACUTE ORAL TOXICITY TEST OF PYKURE CAPSULES 750mg IN MICE: FIXED DOSE PROCEDURE ACCORDING TO OECD 420 GUIDELINES. 1. AIM AND OBJECTIVE OF THE TEST The aim of this test is to observe toxicity using a minimum no of animals, sufficient information on the acute toxicity after single administration, by oral route in mice of a test substance (PYKURE CAPSULES 750mg) for its classification. NO Botanical Name Sanskrit Name Formula /100 mg Formula/750mg 1 Mesua Ferrea Nagpushpa Teminalia Chebularets Haritaki Cuminum Cyminum Jeeraka Azardirachta Indica Nimbi Saraca Indica Kankelli Abutilan Indicum Kangi Red Ochre Kutuki Alowas Geru Picroohiza Kurroa Ela 8 60 Test substance is administered to a group of experimental animals by oral route at one defined dose (5 mg/kg, 50 mg/kg, 300 mg/kg and 2000 mg/kg) according to the available information on test substance. Animals were observed for 7 consecutive days at least, after administration of test substance to detect the signs of toxicity. 2. TEST SUBSTANCE The powder dissolved in distilled water was used as test substance. 3. TEST ANIMALS Species: Swiss albino mice weighing in the range of 20 to 30 g. Strain: Wistar Age: 8 to 10 weeks. Number and sex: 5 nulliparous and non pregnant females Diet: Standard feed prepared in house (PRANAV AGRO)
3 Water: Plain tap water ad libtium. Housing: The animals were housed in 37cm x 23cm x 16 cm polypropylene cages with maximum of 5 animals per cage. The cages were placed in limited access premises of animal house with controlled temperature and humidity. The artificial lighting ensured a sequence of 12 hours light and 12 hours dark. 4. TEST PROCEDURE Administration of the test substance: All animals were weighed again on day 1 before administration. The animals were fasted overnight prior to the test substance administration. As per the OECD 420 guidelines starting dose taken was 2000 mg/kg. The test substance was administered in a single dose p.o. After administration animals were fasted for 4 hrs. Main Study: According to the methodology described in the Annex 3 of the OECD guideline 420, the test was performed with 5 animals receiving the test substance at the dose level of 2000 mg/kg. 5. OBSERVATIONS Animals were observed individually after dosing during the first 30 minutes, periodically during the first 24 hours and daily thereafter for 7 consecutive days. Animal were examined outside the home cage. The different parameters observed were change in skin & fur, eyes & mucous membranes, respiratory function, tremors, convulsions, diarrhea, lethargy, sleep and coma. Body weight: The animals were weighed on day 1 before administration, then 7 days after administration of the test substance. Interpretations of the results: The assessment criteria of the toxicity of the test substance taken into account were Body weight change
4 Behavioral signs The mortality expressed in % of compound related deaths These results enabled to classify the test substance to one of a series of toxicity classes defined by fixed oral LD 50 cut off values, in compliance with Globally Harmonized System (GHS). Category 1 = 0 < LD 50 < 5mg/kg Category 2 = 5 mg/kg < LD 50 < 50 mg/kg Category 3 = 50mg/kg < LD 50 < 300 mg/kg Category 4 = 300 mg/kg < LD 50 < 2000mg/kg Category 5 = 2000mg/kg < LD 50 Category 5 or non classified. 6. Results Body weight: All animals exhibited normal and comparable body weight gain throughout the period of study. The individual weights of the animals assessed during the test period are given in Table1. Observations: All animals were free of intoxication signs throughout the study. The results of the observations are summarized in Table 2 Statistical Analysis: All the values are expressed as mean S.E.M. Statistical significance between more than two groups was tested using one-way ANOVA followed by the Bonferroni multiple comparisons test or unpaired two-tailed student's t-test as appropriate using computer based fitting program (Prism, Graphpad.). Differences were considered to be statistically significant when p < Table1: Weights of the animals assessed during the test Weights of animals in gm Compound Day 1 Day 7 Test substance All the values are expressed as MEAN + S.E, (n=5)
5 Table 2: Results of observations during the test Observation Time Comments Observation time Comments Day 1 (after treatment) Day 1 (after 30 minutes) NTR Day 1 (after 10 hr) NTR NTR Day 1 (after 24 hr) NTR Day 1 (after 1 hr) NTR Day 2 NTR Day 1 (after 2 hr) NTR Day 3 NTR Day 1 (after 3 hr) NTR Day 4 NTR Day 1 (after 4 hr) NTR Day 5 NTR Day 1 (after 5hr) NTR Day 6 NTR Day 1 (after 6 hr) NTR Day 7 NTR NTR Nothing to report Table 3: Results of % MORTALITY OBSERVED during the test Observation Time % Mortality Observation time % Mortality Day 1 (after treatment) Day 1 (after 30 minutes) NIL Day 1 (after 10 hr) NIL NIL Day 1 (after 24 hr) NIL Day 1 (after 1 hr) NIL Day 2 NIL Day 1 (after 2 hr) NIL Day 3 NIL Day 1 (after 3 hr) NIL Day 4 NIL Day 1 (after 4 hr) NIL Day 5 NIL Day 1 (after 5hr) NIL Day 6 NIL Day 1 (after 6 hr) NIL Day 7 NIL NIL No Mortality.
6 7. Conclusion According to the Globally Harmonized system (GHS) for the classification of substances which cause acute toxicity, the powder of PYKURE CAPSULES (750 mg) may be classified in the Category 5 or non classified with a LD 50 higher than 2000 mg/kg. Flow chart showing the main study
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