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VALIDATION PROGRAMME OF THE EFFICACY OF DISINFECTANTS

4e Rencontres A3P de Microbiologie – TOURS, les 8 et 9 Juin 2004 – JP. de FARCY

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Qualification of a facility compliant disinfectant efficacy programme

! c GMP’s require the development of an effective programme of microbiological controls in production areas

" see USP 27 / NF 22, 2004, <1116>, Microbiological evaluation of clean rooms and other areas

! This microbiology monitoring programme is a top concernfor FDA, especially the validation of disinfectants

" see FDA 483s, FDA Warning Letters

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Qualification of a facility compliant disinfectant efficacy programme

! These microbiology programmes are highly challenged during FDA facilities inspections for compliance

" see Journal of Validation Technology, November 2003,10(1),15-19, S. Richter, J. McLaughlin, A practical approach to disinfectant qualification for FDA-Regulated industry

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Disinfectants concern position

! Our disinfectants used for rasburicaseproduction are they in adequation with the existing contamination baseline ?

! These disinfectants are they validated for efficacy in conditions of use on our own surfaces?

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Disinfectants concern position before FDA inspection

! Products and conditions of use were established and procedures in place

! Technical dossiers of validation were available for each product from each supplier, except for bleach

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Disinfectants concern position during the FDA inspection

! Presentation of the list of products used and their use

! Presentation of the existing SOP’s! Presentation of the suppliers technical dossiers

of validation for each product concerned , butinsufficient and limited information

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FDA observations on disinfectants concern

! All observations are focused :there must be in house validation studies available demonstrating an efficacy of the disinfectants used on the different facility surfaces

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EP and USP regulations and methods on disinfectants

! No EP / USP direct information on the validation of disinfectants

! Indirect information by description of microbiological evaluation programmes" see, USP 27 / NF 22, 2004, <1116>," see, Guide BPF 98/5 bis, 1998, page 74

! Microbiological methods for efficacy validation on surfaces " AOAC Hard Surface Carrier Method" French standard NF T 72-190, August 1988 :

Water-miscible contact disinfectants used in liquid state.Germ carrier method. Determination of the bactericidal, fungicidal and sporicidal activity.

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Disinfectants validation programme: choice of the method

! The germ carrier method selected must be able to measure the effectiveness of different products on different materials : AOAC method, or AFNOR standard method.

! The AOAC method , qualitative in-vitro test method for qualifying disinfectants,is described for glass carriers and is difficult to perform" not chosen.

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Disinfectants validation programme: choice of the method

! The NF T 72-190 standard, quantitative method forqualifying disinfectants, is described for different carriersand can measure a biocidal activity by logarithmic reduction

" chosen.Specifications for logarithmic reduction :- not less than 5 for sensitive strains, - not less than 4 for resistant strains, - not less than 3 for sporulated.

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Disinfectants validation programme: main elements

DisinfectantDisinfectant

StrainStrainCarrierCarrier

ConcentrationConcentration Contact timeContact time

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Disinfectants validation programme: method NF T 72-190

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Disinfectants validation programme: products to validate

! Bacteranios D : surface routine cleaning / aldehydes / 0.25%

! Amphosurf : surface routine cleaning /substituted tertiary amine / 0.25%

! Bleach solution : floor routine cleaning / 300 ppm! IPA 70% : process routine cleaning / sterile IsoPropylic

Alcohol / pure! Phagospray 45 : process routine cleaning / isopropanol

and formaldehyde / pure

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Disinfectants validation programme: products / carriers to validate

! Bleach solution at 300 ppm / mipolam (floor facility)! Bacteranios D ready to use at 0.25% / lacquered steel (wallfacility)! Bacteranios D ready to use at 0.25% / PVC (wall facility)! Amphosurf ready to use at 0.25% / lacquered steel (wall facility)! Amphosurf ready to use at 0.25% / PVC (wall facility)! Sterile IsoPropylic Alcohol at 70% / stainless steel (processfacility)! Sterile IsoPropylic Alcohol at 70% / glass (process facility)! Phagospray 45 / stainless steel (process facility)! Phagospray 45 / glass (process facility)

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Disinfectants validation program: strains choice and rationale

! Reference strains described in standard NF T 72-190 : bacteria

Pseudomonas aeruginosa " chosen : strictly aerobic, Gram -Escherichia coli " chosen : facultative anaerobic, Gram -Staphylococcus aureus " chosen : potentially pathogenic, Gram +Enterococcus faecium " chosen : presumed resistant, Gram +Mycobacterium smegmatis " not chosen : exotic

Bacillus subtilis, var. niger " not chosen

Bacillus cereus " chosen : in house sporulated strainClostridium sporogenes " not chosen : aerobic process

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Disinfectants validation programme: strains choice and rationale

! Rationale for 7 bacterial strains tested :- 4 reference strains chosen including Enterococcus, the morepresumed resistant one for a bactericidal 4 validation (5 log reduction).- Introduction of 2 in house contaminants isolated from maping (annual contamination baseline): Stenotrophomonas maltophilia : Gram -Staphylococcus epidermidis : Gram +- Choice of a sporulated bacterium (Bacillus cereus), in house contaminant isolated from maping for the sporicidal validation(3 log reduction).

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Disinfectants validation programme: strains choice and rationale

! Reference strains described in standard NF T 72-190 : yeast and fungi Absidia corymbifera " not chosen: exotic

Cladosporium cladosporioides " not chosen: exotic

Candida albicans " chosen : yeastPenicillium verrucosum, var. cyclopium " chosen : fungus

! Rationale for 2 strains tested :1 reference yeast Candida and 1 reference mold Penicillium, presumed resistant fungus the commonly observed one for the fungicidal validation (4 log reduction).

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Disinfectants validation programme: contact time

! 1 contact time tested for all the sensible strains (bacteria and the yeast Candida albicans) : 5 minutes (bleach, IPA, Phagospray), or 15 minutes (Bacterianos, Amphosurf).

! 2 contact times tested for the presumed resistant strains (including the sporulated bacterium Bacillus cereus and fungus Penicillium verrucosum): 5 or 15 minutes as previously, and 30 minutes.

! Only 2 products (bleach / Bacteranios D) described as having a sporicidal activity were tested with Bacillus cereus.

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Disinfectants validation programme: bleach results

! The bleach solution used at the supplier’s concentration on mipolam carrier, eg 300 ppm, shows no bactericidal, fungicidal nor sporicidal activity.

! The bleach solution used at the concentration of 1800 ppm on mipolam carrier has a bactericidal, fungicidal and sporicidal activity :- after 5 minutes for sensible bacteria and yeast, - after 30 minutes for the presumed resistant strains, sporulated and fungus.

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Disinfectants validation programme: Bacteranios D results

! Bacteranios D used at the supplier’s concentration, eg. 0.25% on lacquered steel and PVC carriers has nobactericidal, fungicidal nor sporicidal activity.

! The same product at the concentration 2.0% has :- a bactericidal activity after 15 minutes of contact time (except for Enterococcus faecium on PVC),- a moderate fungicidal activity that may need 30 minutes of contact time, depending on the couple strain / carrier, - and no sporicidal activity, even after 30 minutes of contact time.

! These results are not in accordance with the active concentration indicated by the supplier, nor with the potential / expected sporicidal activity.

! Note that this product is used in alternance with Amphosurf during the weekly cleaning programme.

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Disinfectants validation programme: Amphosurf results

! Amphosurf used at the supplier’s concentration, eg. 0.25% on lacquered steel and PVC carriers has no bactericidal nor fungicidal activity.

! The same product at the concentration 2.0% has :- a bactericidal activity after 15 minutes of contact time (except for Pseudomonas aeruginosa on PVC),- a fungicidal activity against Penicillium (after 15 or 30 minutes) , - no fungicidal activity against Candida after 15 minutes (not tested after 30 minutes).

! These results are not in accordance with the active concentration indicated by the supplier

! Note that this product is used in alternance with Bacterianos D during the weekly cleaning programme.

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Disinfectants validation programme: Amphosurf / Bacterianos alternance results

! Bacterianos D is moderately active against Candida albicans (logarithmic reduction near 4), whereas Amphosurf is inactive.

! Amphosurf is active against Penicillium verrucosum , whereas Bacterianos is inactive.

! Amphosurf is active against Pseudomonas aeruginosa on PVC, whereas Bacterianos is inactive.

! Bacterianos is active against Enterococcus faecium on PVC, whereas Amphosurf is inactive.

! The alternance of Amphosurf and Bacteranios D is successfull to fight against a bacterial and fungicidal contamination.

! For sporulated contaminants, apply an intensive corrective cleaning programme using bleach during 30 minutes.

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Disinfectants validation programme: IPA 70%, Phagospray 45 results

! The two products are used pure in the manufacturing areas and were validated on glass and steel carriers.

! IPA 70% has a bactericidal and fungicidal activity after5 minutes of contact time on glass and steel.

! Phagospray 45 has a bactericidal and fungicidal activity against Candida after 5 minutes of contact time on glass and steel, but not against Penicillium.

! In case of a sporulated contamination, apply a bleachcorrective cleaning programme.

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DISINFECTANTS VALIDATION PROGRAMME CONCLUSIONS

! The bleach use concentration was modified to 1800 ppm (instead of 300 ppm previously).

! The Bacterianos D and the Amphosurf use concentrations were modified to 2 % (instead of 0.25% previously like it was prescribed by the supplier).

! The efficiency of alternating the two products Bacterianos D and the Amphosurf is demonstrated.

! The lack of efficacy for sporicidal activity of Bacteranios D is demonstrated, and the bleach sporicidal activity is confirmed.

! Phagospray and IPA are validated with no change.

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Thanks :

Delphine BEYNET ACM PharmaSandrine MARIE ACM PharmaSylvia MARTIN ACM PharmaEric PETAT ACM PharmaVéronique ROBIN ACM PharmaPhilippe TAILLIEZ ACM Pharma

Alain BAYOL Sanofi∼SynthélaboHervé DROUILLARD Sanofi∼SynthélaboMarie-Noël MAUMUS Sanofi∼SynthélaboCatherine MINTY Sanofi∼SynthélaboAlain PREVOST Sanofi∼SynthélaboGérard RIMLINGER Sanofi∼Synthélabo

Sanofi∼Synthélabo Recherche Centre de LabègeJP. de FARCY / 09.06.04