Method Activity Aspect EPA Suggestion/Recommendation CSPA Comments Priority

SOP MB-09-04 Carrier Innoculation Size of Petri dishes with and without filter paper EPA’s SOP calls for standard Petri dishes without filter paper. Industry is using filter paper and some labs are using shallow Petri dishes to allow for easier wiping. Action is required on standardizing the use of filter paper and shallow dishes. The use of a shallow petri dish would allow the testing to be performed in the dish and cut down on the handling of the slide. Most labs use filter paper already and this might cut down on the movement of the slide during wiping. H-3

SOP MB-09-04 Carrier Wiping Position of the glass slide carrier during w EPA holds carrier on edge of Petri dish while industry wipes carrier inside Petri dish. Action item – standardization of the position of the carrier during wiping is encouraged. EPA’s method requires two people while the industry approach can be achieved by a single analyst. Methods requiring a single analyst are encouraged. The use of a shallow dish would be better so the slide does not need to be held. This will also allow one person to run the test. H - 1

SOP MB-09-04 Control Carrier Counts Timing of the carrier counts EPA performs counts during the initial phase of the study; however, industry performs counts at the end. Vortexed carriers may be refrigerated prior to dilution and plating (EPA). Parties considered an approach where counts could be split, beginning and end, and then averaged. Also, the group agreed that if a single carrier fell below the established minimum, it was the mean value across all carriers analyzed that would be used to qualify the study. Action item – seek consensus on timing of processing control carrier counts. It is not clear that all aspects of averaging carriers have been reviewed as well as how the average would be used. If the carriers plated last were below the required carrier count number they should not be averaged with carriers from the beginning to bring the results into compliance. It seems that the carriers should be plated at the end of the testing or carriers should be done with each set and used for the set, not averaged. This will be a good item to discuss H - 1

Draft Quantitative Petri Plate Method Wiping Wiping and folding EPA demonstrated the use of 4 folds and the 6 total revolutions/corkscrew process. The group discussed the advantages of folding one time (inverted) when reaching the center to provide a fresh surface. The group generally agreed with this process. Action item – EPA will incorporate this step in the draft procedure. This method was demonstrated at the ASTM meeting showing that the cloth is turned for the second set of 3 swirls. The change should be confirmed. H – 1

Draft Quantitative Petri Plate Method Wiping Performance standard Is it appropriate to put the performance criteria (regulatory language) in the method? How was the Disinfectant level decided and determined to be appropriate? CSPA has gone on record with AOAC that regulatory language does not belong in the method. CSPA would like to discuss the options for performance criteria being in the method or noted in a place which is easily accessible. At the least CSPA would like to have a discussion on EPA’s thoughts on performance standards for NFC sanitizers and disinfectants H - 1

Use-Dilution Method (SOP MB-05) Bioscreening and efficacy testing Efficacy testing (primary and secondary subcultures) EPA consistently uses both primary and secondary subculture tubes, industry typically uses primary tubes only. Some concerns were expressed regarding the need to do both and the increased potential for contamination. EPA does not perform a neutralization assay with every test, thus use of both primary and secondary tubes are deemed necessary. The issue of increased resources and time was also expressed. EPA has not observed increased contamination rate with use of secondary tubes. No action items. This is a big topic of discussion. There is more chance for contamination with the use of secondary sub-culture tubes. If the jacket for each product is available the neutralization media used for registration should be used by EPA. H – 1

SOP MB-09-04 And for SOP MB (GST) Carrier Inoculation Glass slide carriers Use of frosted glass microscope slides (inverted) common by industry – EPA will likely use them in the future as well. No action required. CSPA highly recommends the use a frosted end microscope slide to avoid making any marks on the slide to assure no ink is transferred to the product during testing. M-H – 2

Use-dilution method (SOP MB-05) Culture harvest and carr Carrier exposure EPA uses Teflon-coated (Pyrex) tubes to reduce breakage of tubes during carrier autoclaving. EPA does not remove broth prior to placing carrier on filter paper for drying. Industry suggests removing broth first to mitigate the wetness of carriers. Action item – seek revision to the official method and EPA’s SOP to include this step. Test organism should be removed (poured off or suctioned off) from the tube with the carriers. 2 reasons – the carriers will have less liquid retained on them ensuring more consistent and even drying - soak time will be the same for all carriers.

OECD Method (SOP MB-25) Carriers Source of carriers Several sources of carriers discussed – this may be an issue that impacts the outcome of tests. Action item - CSPA will look into sources of carriers

. Dr. Syed Sattar states that if the same material is used he doesn’t see that there would be an issue. This is something that hasn’t been tested. CSPA participants discussed that the Test Guidelines should indicate that the beveled (smooth edged) side should be the consistent side to use for testing. This will be discussed with the OECD VMG by Diane Falbo and Mary Bennett. OECD doesn’t allow sources for equipment to be listed in guidelines. It is recommended that a single source for the carriers be listed for consistency. M-H - 3

SOP MB-09-04 Container of Towelettes Canister/Container of Towelettes The container of the towelettes is shook prior to removing any towelettes. If there are not directions on the label then the practice shouldn’t be done. M - 3

SOP MB-09-04 Carrier Wiping Timing of exposure General agreement that the contact time begins following the wiping process; however, it would be advisable to indicate this in EPA’s SOP. As long as the carrier has the allotted time for exposure there is no issue. SOP should be updated immediately to reflect this information M – 1, theirSOP MB-09-04 Control Carrier Counts Carrier processing Some disagreement in methodology exists, i.e., EPA vortexes carriers, some in industry sonicate then vortex. Action item – seek consensus on inoculum removal/disaggregation process. NOTE: need some discussion on this point to get input. Method states vortex or sonicating. How will BEAD get consensus? M – 3

OECD Method (SOP MB-25) Carrier treatment Product application This needs further discussion to see whether the use of a positive displacement pipette for product application is equivalent. M - 3

SOP MB-09-04 Carrier Wiping Number of wipes (passes) EPA is currently using 6 passes; other labs are using 4 due to CA “requirements”. The number of passes must be standardized. Action item – follow-up is necessary to seek consensus on the number of passes across all labs. No issue with the use of 6 passes. Who does BEAD refer to as all labs? M – 2

SOP MB-09-04 (Modified AOAC GSPT for Towelettes) Location of the test EPA conducts the method inside the BSC while industry conducts the assay on the open bench. No action required; however, industry indicates that ease of towelette manipulation is greater on the open bench. It may be easier to perform some tests on the lab bench. The canister is shook prior to removing 3-4 wipes that are discarded before the one for testing is removed. http://www.cdc.gov/biosafety/publications/bmbl5/BM BL.pdfC L

SOP MB-09-04 Carrier Inoculation Use of positive displacement pipettes for carrier inoculation EPA uses calibrated positive displacementpipettes and recommends their use for spotinoculation purposes whenever possible. Industry currently does not use them Industry could discuss the use of positive displacement pipettes. There may be some labs that use them for inoculation. Discussion can be added to the method. L

SOP MB-09-04 Carrier Inoculation Spreading the inoculum EPA uses flamed 4 mm loops, others use bent wire needles. No action required. Achieving the carrier count is what is important. L

SOP MB-09-04 Carrier Wiping Discarding towelettes The number of towelettes discarded prior to beginning the official test is not described in the SOP. Action item – indicate the removal of 3-4 towelettes prior to beginning study in the SOP The removal of towelettes prior to testing does not take into account small count sizes or single packs. These smaller size packaging options need to be discussed. L

SOP MB-09-04 Carrier Wiping Towelette folding – 5 folds Slight variations noted; however, as long as fresh surface is exposed per slide there is no issue. No action required The folding technique was familiar to a few – may be some variation but the important part is to have a fresh area for each slide and wipe 10 slides/towelette L

SOP MB-09-04 Testing Testing Industry performs the method in two 30-carrier sets, while EPA conducts a single 60 carrier test. No action required; however, advantages and disadvantages should be explored for each approach. One difference is that the carriers do not sit at room temperature as long for a 30 slide test. Also BEAD does tests in sets of 20. Sixty carriers are inoculated and dried. How long do the carriers set before the last test is run? L

SOP MB-09-04 Overall EPA recommends the preparation of an AOAC method specifically designed for towelettes using the AOAC spray test method (AOAC method 961.02) as the foundation with appropriate consensus-based modifications. Action item – is there agreement to pursue this activity? This is the basis for the towelette method for industry. L

Draft Quantitative Petri Plate Method Carrier inoculation and inoculum recovery Carrier inoculation Use of template showing inoculation sites and positive displacement pipette is encouraged. No action required. This is a good practice. L

Draft Quantitative Petri Plate Method Carrier inoculation and inoculum recovery Dislodging the inoculum EPA demonstrated the process using a small scraper; however, the use of a larger scraper was encouraged by the group. Action item - EPA will explore the use of other devices. Larger scraper option is Thermo Scientific/Nunc #179693 L

Draft Quantitative Petri Plate Method Carrier inoculation and inoculum recovery 40 mL suspension The group recommends rinsing the tube containing the 40 mL suspension with saline or PBS and processing the rinsate. Action item – revise the draft method to include a rinsing step. This is a good recommendation. L

Draft Quantitative Petri Plate Method Carrier inoculation and inoculum recovery Control counts Ensure use of the neutralizer as the solution applied to control plates; no action required, the draft procedure currently states use of the neutralizer. L

Use-dilution method (SOP MB-05) Culture harvest and carrier inoculation Harvest of Pseudomonasusing 20 mL test cultures Careful manipulation of culture is required to avoid disruption of the pellicle. This can be achieved through aspiration of the broth and/or removal of the pellicle itself. Easier manipulation in 20 mL tubes. No action items.CSPA members thought that the growth looked light and the pellicle didn’t look as thick as they see in their labs. L

Use-dilution method (SOP MB-05) Bioscreening and efficacy testing Bioscreening Visual physical screening performed in advance of bioscreening. Failures in the UDM are always separated and rescreened prior to use. Control carrier counts are not considered failed carriers. No action items. This follows industry practice. There wasn’t any discussion. L

Use-dilution method (SOP MB-05) Bioscreening and efficacy testing Efficacy test (disinfectant tubes) Use of Bellco 25 x 100mm tubes encouraged by EPA to reduce potential for contamination of inside of disinfectant tubes – catalog part number provided to participants. Carrier repeated (new carrier) if analyst inadvertently touches inside of the tube. No action items. Belco 2010-25100 25x125mm - $45/144 tubes CSPA would like to confirm this information from BEAD.

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OECD Method (SOP MB-25) Screening Perform screening (reduce carrier numbers) in advance to devise dilution series. No action items. This is screening of the product L

OECD Method (SOP MB-25) Inoculum preparation Preparation and standardization of the inoculum The group discussed the possibility of concentrating both organisms the same way through centrifugation and resuspension. Action item – further discussion required. The Test Guidelines have been changed to have all organisms prepared the same way. L

OECD Method (SOP MB-25) Collaborative study EPA expressed interest in conducting a collaborative study to verify the performance of the OECD method. Several participants expressed interest in this proposal. Action item – EPA will consult with CSPA as the collaborative study is developed. CSPA companies indicated that some would be interested in taking part in the collaborative. L