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MICROBIAL LIMIT TEST VALIDATION PROTOCOL Protocol No.

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TABLE O% CONTENTS &.' PRE APPROVAL ((((((((((((((((((((((. Pa)e numbers *.' OB+ECTIVE(((((((((((((((((((((((((.

,.' SCOPE(((((((((((((((((((((((((((... -.' /.' 1.' 4.' 5.' 6.' &' && &* &, &RE%ERENCE DOC.MENT((((((((((((((((((... RESPONSIBILIT0((((((((((((((((((((((... VALIDATION MET2OLO30(((((((((((((((((... ACCEPTANCE CRITERIA ((((((((((((((((((( REVALIDATION CRITERIA(((((((((((((((((( RES.LT REPORTIN3((((((((((((((((((((( CONCL.SION((((((((((((((((((((((((... 7E0 CONSIDERATIONS((((((((((((((((((.. ABBERIVATIONS(((((((((((((((((((((((. %LO8 C2ART (((((((((((((((((...

POST APPROVAL(((((((((((((((((((((((.
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MICROBIAL LIMIT TEST VALIDATION PROTOCOL

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OB+ECTIVE9 )o establish the documentary evidence that the method for *icrobial limit test for Non+sterile materials is capable of correctly estimating the microbial counts in the *aterials. )he validity of the test results largely upon the ade,uacy of a Demonstration that the test specimens to which they are applied do not- of them +selves- inhibit the multiplication- under the test condition- of microorganisms that may be present. )he validation e.ercise shall demonstrate that the method employed is capable for correct enumeration of microorganisms without adversely effecting the /rowth even in case of materials- which have antimicrobial activity.

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SCOPE9 )his validation protocol is applicable for validating the *icrobial limit test of on+ sterile products and raw materials. )he protocol shall be used for validation of the methods applicable for all Dosage forms and materials- which have re,uirement for *icrobial limit test. 0henever the method is used for *icrobial limit test for scale up$ scale down 1ormulation- the validation shall be done on only one strength of product.

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RE%ERENCE DOC.MENT9 1ollowing documents are referred during preparation of the protocol Do$umen Name *icrobial limit )est "2P Do$umen Number ................

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RESPONSIBILIT09 Tra#ned M#$rob#o"o)#s or a su# ab"e ra#ned au :or#;ed person

1.' VALIDATION MET2ODOLO30 1.& TOTAL VIABLE AEROBIC CO.NT < o= Tween *' #n Bu==ered Sod#um $:"or#de Pep one So"u #on 4.4&5 6 4.4546 4.4756 4.' 6 <Re$o!ery o= M#$roor)an#sms ecovery not obtained ecovery not obtained ecovery less than 746 ecovery up to 746

8.'.' '4 gm$ml of sample 9 :4 ml of Buffered "odium ;hloride Peptone 9 4.'6 )ween &4 $ <4. ("olution !) '#'4. '5 ml solution ! 9 84 ml of Buffered "odium ;hloride Peptone 9 4.' 6 )ween &4 '#54. '4 ml of solution ! 9 :4 ml of Buffered "odium chloride Peptone 9 4.'6 )ween &4 '#'44. 8.'.& Prepared si. test tube containing '4 ml each from "olution ! ('#'4) and follow the same for the other preparation i.e. "olution B ('#54) and "olution ; ('#'44) respectively. 8.'.( /row the bacterial cultures separately in "oyabean casein digest medium at (44; to (54; for &3 hours and 1ungal cultures separately in "oyabean casein

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MICROBIAL LIMIT TEST VALIDATION PROTOCOL

digest medium at &44; to &54; for 3< hours. )he organisms to be used are as mentioned below. + E.coli + ". aures + "almonella + Pseudomonas aeruginosa + ;. albicans + !. niger +Bile tolerant gram+negative bacteria (replaced the EP procedure "Test for Enterobacteria and Certain Other GramNegative Bacteria" 8.'.3 Prepare reference suspension separately of the above organisms by diluting the broth cultures to get containing about not less than '4( viable microorganisms per ml. 8.'.5 !dd separately ' ml of the culture dilutions of ". aureus- E. coli- "almonellaPseudomonas aeruginosa- ;. albicans- !. niger and other bacterial species prepared above in all the three sets of si. test tubes prepared from each solution !-B-;. 8.'.8 1rom each set pipette out ' ml separately in two pre+ sterili=ed Petri plate .Pour &4+&& ml of li,uefied "oyabean casein digest agar for the cultivation of bacteria and &4+&& ml of li,uefied "abouraud de.trose agar for the cultivation of fungi. 8.'.7 >ncubate plates of soyabean casein digest agar at (4 4; to (54; for 5 days and plates of "abouraud de.trose agar at &44; to &54; for 5 days. 8.'.< "ample controls#

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>noculate '.4 ml of solution !- B- and ; in duplicate and add &4+&& ml soyabean casein digest agar for bacterial count and "abouraud de.trose agar for total combined mold and yeasts count respectively. >ncubate at (4 4; to (54; for bacterial count for 5 days and &4 4; to &54; for total combined mold and yeasts count for 5 days. 8.'.: Positive control >noculate' ml of each of the diluted bacterial and fungal culture into two plates Each and add &4+&& ml of soyabean casein digest agar and "abouraud de.trose !gar respectively. >ncubate at (4 4; to (54; for bacterial culture for 5 days !nd at &44; to &54; for total combined mold and yeasts count for 5 days. 8.'.'4 Negative control for media# >noculate '.4 ml of Buffered "odium chloride Peptone "olution in four plates !nd add &4+&& ml of soyabean casein digest agar into & plates and "abouraud de.trose agar into other & plates respectively. >ncubate at (4 4; to (54; for bacterial culture for 5 days and at &4 4; to &54; for total combined mold and yeasts count for 5 day. 1.* TESTS %OR PAT2O3ENS9 8.&.' Prepara #on of sample9 '.4 gm sample 9 '44 ml soyabean casein digest medium 9 36 )ween &4 '.4 gm sample 9 '44 ml 1luid lactose medium 9 36 )ween &4 '44 ml 1luid lactose medium 9 36 )ween &4 (Positive control) '44 ml soyabean casein digest medium 9 36 )ween &4 ( Positive control) 8.&.& *i. well to obtain uniform solution or suspension#
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8.&.( /row the bacterial culture separately in soyabean casein digest medium at (44; to (54; for &3 hours. )he organisms to be used are as mentioned below. + E.coli + ". aures + "almonella + Pseudomonas aeruginosa 8.&.3 Prepare reference suspension separately of above organisms by diluting the )he broth cultures to get not less than '4( viable organisms per ml. *i. e,ual volume of each suspension. 8.&5 Pipette '.4 ml mi. suspension of the microorganism separately in tube of "oyabean casein digest medium and 1luid lactose medium containing the sample to be e.amined. 8.&.5 >ncubate the tube of soyabean casein digest medium and 1luid lactose medium at (44; to (54; for &3 to 3< hours and proceed for >solation and >dentification of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Escherichia coli from this broth as per specified "2P. 4.' ACCEPTANCE CRITERIA 7.' 7.& 7.( ecovery of the test organisms should not be less than 74 6 of the calculated value of the inoculum suspension is to be obtained. )here shall not be failure in isolation and identification of organisms inoculated in the medium along with material. )he negative control should show no growth.

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REVALIDATION CRITERIA> DEMONSTRATION O% MET2OD S.ITABILIT0 ;hange in formulation where in new component has been added. 0hen concentration of preservative has been changed. *a?or change in method for *icrobial limit test like method for deactivation of !ntimicrobial activity.

)hree batches of each product $ materials shall be validated for *icrobial limit )est. )he @absence of specified organisms@ tests to provide procedures for demonstration of the absence of "taphylococcus aureus- Pseudomonas aeruginosa- "almonella species A Escherichia coli. 6.' RES.LT REPORTIN3 > REVIE8IN3 RE?.IREMENTS

eport all results on a method validation report form. >f results are unacceptablethe method accordingly to rule out the affecting factor. &'.' CONCL.SION %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%

&&.' 7E0 CONSIDERATIONS9

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Beeping in view regarding latest international harmoni=ation rules some key considerations should be discussed both by the working C! group and ;CD. &. T:e =undamen a" s:or $om#n)s o= :ese es s #n re)ards o :e $urren )ood manu=a$ ur#n) pra$ #$e @C3MPA reBu#remen s =or Cabsen$e o= obDe$ #onab"e or)an#smsC s:ou"d be d#s$ussed by s$#en #=#$ eams. *. Produ$ r#sk ana"ys#s #n$"ud#n) produ$ use and rou e o= adm#n#s ra #onE )row : po en #a"E preser!a #onE and o :er $ons#dera #ons w:#$: are re$ommended #n P:arma$opoe#a eF s mus be proper"y aken #n o a$$oun . T:e Bua"# y )roup mus ake a proper and reasonab"e s$#en #=#$ approa$: :ow o :and"eE !a"#da e and es #n spe$#a" $ases o= produ$ re$a""s due o presen$e o= obDe$ #onab"e or)an#sms. ,. ReG!a"#da #on o= eF#s #n) es s o a"#)n w# : $urren :armon#;ed s andards and "e!e" o= de a#" s:ou"d proper"y d#s$ussed and made by us#n) a ma r#F approa$:. -. A proper repor #n) =orma s:ou"d be de!e"oped by bo : Bua"# y and do$umen a #on eams. /. Tra#n#n) m#$rob#o"o)#s s =or :e re!#sed es s s:ou"d be $ons#dered as a pr#or# y by bo : !a"#da #on and Bua"# y eam dur#n) rans=er o= pro$edures. &*.' ABBREVIATIONS C! "2P /*P
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