Test animals— Select male guinea pigs,each weighing at least 500g,but restrict selection so that no guinea pig is more than 30%heavier than the lightest.Withdraw food,but not water,from each animal.

Sodium chloride solution— Use Sodium Chloride Injection containing 0.9%of NaCl.

Standard preparations— Dissolve an accurately weighed quantity of USP Sincalide RSin Sodium chloride solutionto obtain a stock solution having a known concentration of about 10µg of sincalide per mL.Dilute an accurately measured volume of this solution quantitatively with Sodium chloride solutionto obtain a solution containing 0.0624µg of sincalide per kg of the animal's body weight in each 0.1mL.Prepare a series of 1in 2dilutions of this solution with Sodium chloride solutionto contain 0.0312,0.0156,and 0.0078µg of sincalide per kg of body weight.[NOTE—Other dose levels may be used if so indicated by the responses obtained in the Procedure.]

Assay preparations— Constitute 1vial of Sincalide for Injection in a sufficient volume of Water for Injection,accurately measured,to obtain a solution having a concentration of about 1µg of sincalide per mL.Proceed as directed for Standard preparations,beginning with “Dilute an accurately measured volume of this solution.”

Procedure— Anesthetize each guinea pig by injecting it,subcutaneously,with 2.25g of urethane per kg of body weight,administered as a 25%solution.Perform a tracheotomy,then expose a jugular vein,and cannulate with a polyethylene catheter.Tie a thin silk line to the free pole or fundus of the gallbladder,or attach a thin hook with connecting silk line to the wall of the fundus.Gallbladder contractile responses,transmitted through the silk line,cause a change in the line tension.Connect the free end of the silk line to a force transducer,and impose on the system an initial tension of about 2g.Connect the force transducer to a polygraph,which records the contractile responses.Determine the sensitivity or the responsiveness of the guinea pig's gallbladder by making a few trial injections through the jugular vein catheter,then select two nonconsecutive dose levels (e.g.,0.0624and 0.0156)for the assay.Use the same dose levels for the Assay preparationsas for the Standard preparations.Administer the selected dose levels of the Standard preparationsand the Assay preparationsas 0.1-mLdose volumes in random order,taking 2to 3seconds to inject each dose volume and flushing each through the catheter with about 0.5mLof Sodium chloride solution.Make injections at about 10-minute intervals or when the gallbladder has returned to approximately the initial 2g of tension.[NOTE—Three injections of each dose level may be made.As many as 3different samples can be tested on the same animal prior to retiring the animal.]

Calculation— Calculate the potency of each vial as directed under Potencies Interpolated from a Standard Curve(see Design and Analysis of Biological Assays á111ñ),using a log transformation,straight-line method with a least-squares fitting procedure,and a test for linearity.