VALIDATION PROGRAMME OF THE EFFICACY OF DISINFECTANTS

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

Qualification of a facility compliant disinfectant efficacy programme

! c GMPs 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 concern for FDA, especially the validation of disinfectants " see FDA 483s, FDA Warning Letters

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

Disinfectants concern position

! Our disinfectants used for rasburicase production are they in adequation with the existing contamination baseline ? ! These disinfectants are they validated for efficacy in conditions of use on our own surfaces?

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

Disinfectants concern position during the FDA inspection

! Presentation of the list of products used and their use ! Presentation of the existing SOPs ! Presentation of the suppliers technical dossiers of validation for each product concerned , but insufficient and limited information

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

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.

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.

Disinfectants validation programme: choice of the method

! The NF T 72-190 standard, quantitative method for qualifying disinfectants, is described for different carriers and 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.

Disinfectants validation programme: main elements

Carrier Carrier

Strain Strain

Disinfectant Disinfectant

Concentration Concentration

Contact time Contact time

Disinfectants validation programme: method NF T 72-190

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

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 (wall facility) ! 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 (process facility) ! Sterile IsoPropylic Alcohol at 70% / glass (process facility) ! Phagospray 45 / stainless steel (process facility) ! Phagospray 45 / glass (process facility)

Disinfectants validation program: strains choice and rationale

! Reference strains described in standard NF T 72-190 : bacteria Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Enterococcus faecium
Mycobacterium smegmatis Bacillus subtilis, var. niger

" chosen : strictly aerobic, Gram " chosen : facultative anaerobic, Gram " chosen : potentially pathogenic, Gram + " chosen : presumed resistant, Gram +
" not chosen : exotic " not chosen

Bacillus cereus
Clostridium sporogenes

" chosen : in house sporulated strain

" not chosen : aerobic process

Disinfectants validation programme: strains choice and rationale

! Rationale for 7 bacterial strains tested : - 4 reference strains chosen including Enterococcus, the more presumed 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).

Disinfectants validation programme: strains choice and rationale

Reference strains described in standard NF T 72-190 : yeast and fungi

Absidia corymbifera Cladosporium cladosporioides " not chosen: exotic " not chosen: exotic

Candida albicans Penicillium verrucosum, var. cyclopium

" chosen : yeast " 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).

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.

Disinfectants validation programme: bleach results

! The bleach solution used at the suppliers 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.

Disinfectants validation programme: Bacteranios D results ! Bacteranios D used at the suppliers concentration, eg. 0.25% on lacquered steel and PVC carriers has no bactericidal, 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.

! !

Disinfectants validation programme: Amphosurf results ! Amphosurf used at the suppliers 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.

! !

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.

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 after 5 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 bleach corrective cleaning programme.

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.

Thanks : Delphine BEYNET ACM Pharma Sandrine MARIE ACM Pharma Sylvia MARTIN ACM Pharma Eric PETAT ACM Pharma Vronique ROBIN ACM Pharma Philippe TAILLIEZ ACM Pharma Alain BAYOL SanofiSynthlabo Herv DROUILLARD SanofiSynthlabo Marie-Nol MAUMUS SanofiSynthlabo Catherine MINTY SanofiSynthlabo Alain PREVOST SanofiSynthlabo Grard RIMLINGER SanofiSynthlabo

SanofiSynthlabo Recherche Centre de Labge

JP. de FARCY / 09.06.04