Preparation and Evaluation of Veterinary 0.1 % Injectable Solution of Atropine Sulphate

This study introduces the know-how of preparing a multiple injection form atropine sulphate solution. An injectable aqueous solution of atropine sulphate at a concentration of 0.1%. was prepared under aseptic conditions in dark glass bottles each containing 50 ml. The preparation was intended for animal use only. It contained 1g atropine sulphate, 9 g sodium chloride as a normal saline, benzyl alcohol 15 ml as a preservative and water for injection up to 1000 ml. The pH of the solution was adjusted to 4.2 (range 3.0-6.5). The preparation of 0.1% atropine sulphate solution was clear colorless solution free from undesired particles. It complied with the requirements for injectable solutions. Further, the preparation was safe when used under laboratory conditions in chicks, rats and donkeys. It was also effective in preventing dichlorvos (an organophosphate insecticide)-induced poisoning in chicks in a manner comparable to a commercial preparation of 0.1% atropine sulphate. In conclusion, the know-how of a preparation of 0.1% atropine sulphate solution is presented for veterinary use.


Introduction
poisoning following ingestion of certain fungi (Plumb, 2002;Bishop, 2005).Furthermore, the Atropine is a naturally occurring tertiary drug has some ophthalmic uses (Bishop, 2005; amine isolated from the Atropa belladonna plant Brunton et al., 2008).(deadly nightshade); it is a competitive acetylcholine The recommended therapeutic dose of atropine antagonist at the muscarinic receptors (Brunton in animals is usually between 0.15-0.3mg/kg, et al., 2008).Atropine sulphate is odorless, intramuscularly (i.m.) as a preanesthetic agent colorless or white crystalline bitter powder, and it and in case of organophosphate poisoning it is 0.5 is highly soluble in water (Parfitt, 1999; mg/kg, (1/4 intravenously (i.v.), rest subcutan- McKeown, 2002).It is mostly used in veterinary eously (s.c.) or intramuscularly), which could be practice as a preanesthetic agent to achieve repeated at 3-4 hours intervals for 1-2 days balanced anesthesia and as a specific antidote (Plumb, 2002;Bishop, 2005).Higher doses of the against organophosphate and carbamate insecticides drug can be used when needed in cases of (Plumb, 2002) which cause parasympathetic effects organophosphate poisoning (Osweiler, 1996).as a result of acetylcholinesterase inhibition Many commercial preparations of the leading to acetylcholine accumulation at the injectable forms of atropine sulphate are available nerve endings (Marrs and Vale, 2006) For safety evaluation of the product, the successful approach to prepare an injectable preparation of 0.1% atropine sulphate was multiple dosage form of atropine sulphate injected in chicks at 2 mg/kg, s.c.(n=10), suitable for use in animals.

Materials and Methods
(n=5), and rats at 2 mg/kg, intraperitoneally (i.p.) (n=10).The animals were monitored for any The chemicals used were atropine sulphate, physical unexpected side effects or lethality.To sodium chloride, benzyl alcohol and water for further document the safety and effectiveness of injection.They were according to specifications the prepared atropine sulphate solution (0.1%) in of BP Vet (2000).The ingredients and their comparison with a commercial preparation of amounts of the injectable multiple dosage form of 0.1% atropine sulphate solution, an experiment 0.1% atropine sulphate are shown in table 1.
was conducted to antagonize poisoning induced were used.They were maintained at a temperature Water for injection to make 1000 ml O of 30-34 C with constant lighting and floor litter Atropine sulphate and sodium chloride were consisted of wood shavings; water and feed were dissolved in about 900 ml water for injection with given ad libitum.Commercial insecticidal continuous stirring.Then, benzyl alcohol was concentrate solution of dichlorvos (55%, SAFA added to the solution with continuous stirring DDVP55EC, Kalite Yonetim, Turkey) was until a clear solution was obtained.When further diluted in distilled water to obtain the necessary, we adjusted the pH to 4.2 (range 3.0desired concentration for oral dosing by a gavage needle in a volume of 5 ml/kg body weight.6.5) with sulfuric acid or sodium hydroxide solutions and thereafter the volume was Chicks were treated i.p. with the physiological saline solution at 5 ml/kg (the control, n=8) or completed to 1000 ml with water for injection.
with the prepared 0.1% atropine sulphate at 2 sulphate complied with the requirements for parenteral solutions.The content of atropine mg/kg (n=9) or with a commercial preparation of sulphate complied with the requirements of this 0.1% atropine sulphate at 2 mg/kg (n=7).This dosage of atropine is supposed to be effective in preparation with a range of 93-100%.The antagonizing organophosphate poisoning prepared dosage form of 0.1% atropine sulphate (Osweiler, 1996;McKeown, 2002).Fifteen was clear colorless solution with a pH value of 4.2 minutes after the injections, all the chicks were (range 3-6.5); it was sterile, free from undesired dosed orally with dichlorvos (active ingredient) particles.It appeared to be safe in chicks, rats and at 25 mg/kg of body weight to induce organophodonkeys and none of the treated animals showed sphate poisoning (Mohammad et al., 2008).The undesirable side effects or death.dose of dichlorvos was determined in a preliminary Dichlorvos at 25 mg/kg, orally produced experiment on chicks, and this dose was found to signs of cholinergic toxicosis in the chicks, and induce death in 80 to 100% of the dosed chicks these included salivation, lacrimation, gasping within 24 h.After the dichlorvos dosing, the chicks and convulsions within 2 h.The 2-h as and 24-h were observed for the occurrence of the signs of lethalities were 100% in the saline treated control organophosphate poisoning that are characteristic group (Table 2).Atropine sulphate at 2 mg/kg, i.p. of cholinergic toxicity (Mohammad et al., 2008).as the prepared or the commercial 0.1% solutions The signs included salivation, lacrimation, gasping given 15 minutes before dichlorvos dosing variably and convulsion.The 2-h and 24-h lethalities were decreased the occurrence of toxic manifestations also recorded.The latencies to onset of any sign of in the chicks and both of the preparations poisoning and death were recorded within 2 h.significantly decreased the latency to onset of Data as multiple means were statistically signs of organophosphate poisoning and analyzed by one way analysis of variance prevented lethality in chicks by 100% as judged followed by the least significant difference test 24 h after the dichlorvos dosing (Table 2).(Petrie and Watson, 1999).The frequency data were subjected to the Fisher's exact probability Discussion test (Runyon, 1977).The accepted level of The prepared injectable dosage form of statistical significance was at p < 0.05.0.1% atropine sulphate was in compliance with The Scientific and Ethics Committee of the the requirements of parenteral solutions intended College of Veterinary Medicine at the University for injection (Ansel et al., 1999).The content of of Mosul (Iraq) has approved the present study, atropine sulphate in the prepared samples was including the toxicity experiment in chicks.All more than 93%; the solution was clear, colorless, experiments complied with our institutional sterile and free from particles.The specifications regulations and ethics addressing animal use, and of the prepared solution were also well within proper attention and care have been given to the those required by BP Vet ( 2000) and USP (2002).animals used in the study.
Atropine sulphate is highly soluble in water, and

Results
sodium chloride is added to help keep the tonicity of the preparation without causing tissue damage The injectable dosage form of 0.1% atropine after injections (Parfitt, 1999;McKeown, 2002).0.1% atropine solution on a commercial basis Benzyl alcohol is used in the preparation as a when needed, as we introduce the know-how of the product intended for veterinary use only.preservative since it is required in preparations intended for use as multiple dosage forms Acknowledgments (Blodingers, 1983;Ansel et al., 1999).Others This study was supported by the Ministry of have used parabens and sulfites in injectable Higher Education and Scientific Research, Iraq products of atropine sulphate (McEvoy, 2002).
(Research grant No. 202/6).The authors thank Further, the 0.1% preparation of atropine sulphate Syphco Co. for Drugs and Chemicals, Damascus, in the present study was tried experimentally at Syria for the drug analysis.therapeutic doses in three animal species: chicks, rats and donkeys, and it was found to be safe Conflict of interest without producing adverse effects in these animals.
Authors declare that they have no conflict of Even at a high dose (2 mg/kg), the prepared interest.atropine sulphate solution was therapeutically similar to a commercial preparation of the drug in References antagonizing organophosphate poisoning caused  countries are usually imported.The present findings 11.Mohammad, F. K., Al-Badrany, Y. M. and Al-Jobory, M. M. (2008).Acute toxicity and would be a contribution for the manufacture of as the commercial preparation and conform to the 4. BP Vet.(2000).British Pharmacopoeia (Vet.).expected efficacy of such a preparation (McEvoy, The Stationary Office, UK. 2002; McKeown, 2002). 5. Brooks, G. F., Butel, J. S. and Morse, S. A. The prepared atropine sulphate solution (2001).Medical Microbiology.New York: should be protected from light and stored in Lange Medical Books.multiple-dose air tight containers, preferably 6. Brunton, L., Parker, K., Blumenthal, D. and amber-colored glass, at a temperature of less than Buxton, L. (2008).Goodman and Gilman's 40 °C (preferably between 15 to 30 ° C).Freezing Manual of Pharmacology and Therapeutics.should be avoided (McEvoy , 2002; Bishop, New York: McGraw-Hill Co., Inc. pp. 114-134.2005).The shelf-life is 2 years from the date of 7. Entriken, T. L. (2001).Veterinary Pharma- th manufacturing if kept under the above conditions ceuticals and Biologicals.12 ed.Edwardsville, (as determined by Syphco Co., Syria).Kansas, USA: Veterinary Medicine Publishing Co.The label of the present preparation of 8. Marrs, T. C. and Vale, J. A. (2006).Management atropine sulphate 0.1% should state the it is a of organophosphorus pesticide poisoning.In: veterinary preparation that can be given s.c., i.m.Gupta, R. C. (ed.).Toxicology of Organophosor i.v. at the dose rates of 0.02-0.045mg/kg body phate and Carbamate Compounds.London: weight.It should be protect from light and kept Elsevier Academic Press.pp.715-733.o between 15-30 C. The expiry date of the product 9. McEvoy, G. (2002).Anticholinergic agents.is usually 24 months from the date of manufacture.Bethesda, MD: AHFS drug information American Hospital Formulary Service.pp.Conclusion 1222-1228.Atropine sulphate injection solutions which 10. McKeown, R. (2002).Monograph on atropine.are used in veterinary practice in Iraq and neighboring Geneva: WHO.pp.1-41.
. Atropine is for use in veterinary practice (Entriken, 2001; also useful in treating diarrhea and muscarine Bishop, 2005).These are sterile solutions in The final solution of atropine sulphate was sterilized by filtration with pore size of 0.2 µm normal saline or water for injection from several (Ansel et al., 1999; BP Vet 2000; USP, 2002), and manufacturers (Entriken, 2001; Bishop, 2005).

Table - 1. The ingredients and their amounts of
(Osweiler, 1996;sphate insecticide dichlorvos in the veterinary injectable solution of 0.1% chicks.Atropine is the standard antidote against atropine sulphate organophosphate poisoning in animals includingIngredientsAmount/1000 ml the avian species(Osweiler, 1996; McKeown,