1 

### UDM/OECD Technical Workshop

– Highlights from Laboratory Sessions ( November 15 th



### , 2011 : Further Standardization of the AOAC UDM )

Method/ Activity Aspect Comments /recommendation s

UDM

Test Cultures/Growth Media Nutrient broth (NB) vs. synthetic broth (SB) Both media are specified in the U se Dilution Methods (U DMs ) ; EPA h as historically used NB for daily and

final test cultures

. MLB stresses the need to identify a single medium.

C lumping of S. aureus has been frequently observed by the group in NB and the medium is difficult to standardize (e.g., beef extract powder vs. paste, boiling of media, color of prepared media).

A h andout w as provided by EPA showing optical density and carrier counts of S. aure us grown in nutrient broth and synthetic broth. O ptical density (O D ) and

counts were similar, regardless of the growth media.

EPA stated that the type of broth culture medium is

identified in approximately 50% of the regulatory jackets. A p articipant spe culated that using different growth media may result in different efficacy testing outcomes.

ACTION ITEM: Consensus on the issue:

EPA will seek a n editorial revision to remove

nutrient broth from the AOAC UDM s ; however, for

ATP efficacy testing, EPA will utilize the medium

identified in the registration data for currently

registered products. For future registrations, synthetic broth will be used. AD will communicate this point following acceptance by AOAC

. Regulatory guidance will not be included in the official

method.

20 mL vs. 10 mL for test EPA briefly explained the history of the 10 and 20 mL2 cultures ; r emoval of pellicle during harvest issue

– ambiguous language was in the 1990 version of

the method. EPA SOP MB

- 05 specifies 20 mL final test cultures. Some labs have converted over to 20 mL, but

most have retained the 10 mL volume. EPA demonstration: Removal of pellicle of P. aeruginosa from 10 mL and 20 mL test culture tubes using vacuum and pipette aspiration.

A h andout was provided showing titer, optical density,

and carrier counts for S. aureus and P. aeruginosa cultures grown in both 10 mL and 20 mL tubes of

nutrient broth

. Titer, optical density, and carrier counts

were similar , regardless of the volume of gr owth media. The g roup concurred on a recommendation to use vacuum aspiration as the principal method to remove the pellicle from 10 mL tubes

. When harvesting either organism, avoid sediment l which has settled to the bottom of the tube; add a

statement to the official UDM. The g roup stressed the need to carefully observe the final test culture tubes for pellicle fragments prior to culture harvest and after pooling of culture.

ACTION ITEM: A recommendation was made to

include a statement in the official me thod regarding inspection of the Pseudomonas broth cultures for

evidence of pellicle.

10 mL test culture tubes are more convenient for efficient pellicle removal; pellicle in 20 mL cultures was less intact, more fragile.

ACTION ITEM: EPA interpretation of method is to use 20 mL test culture volumes; however, EPA is willing to pursue revision to the official UDMs to change the 20 mL specified in the method to a 10 mL

volume.3 Carrier inoculation EPA stated that the AOAC UDM is specific

– 20

carriers added to 20 mL of inoculum One participant stated that there are two ways of inoculating carriers: add carriers to a tube of inoculum

or add inoculum to a tube of carriers.

Some labs remove inoculum at the end of the 15

- m in

inoculation period using vacuum aspiration rather than removing carriers from the tube. Complete coverage of the carriers is necessary. Ratio of carrier to volume of inoculum is important Appropriate sized vessel must be used

– some labs are not using t he prescribed tube size, rather, are using larger vessels while maintaining the 1:1 ratio. Use of a larger vessel is a deviation from the method. Group agreed that removal of the inoculum may aid in transferring less liquid on carriers, thus enhancing the drying process

. ACTION ITEM: EPA will submit a recommendation

t o include rem oval ( aspiration ) of inoculum from the tube as an option.

ACTION ITEM: EPA to consider submitting a

recommendation to include alternate method for use of a larger vessel to inoculate carriers as long as 1:1

carrier to inoculum volume is maintained. This proposal may require data generation by industry.

Carrier transfers The method currently directs to “briefly swirl” carrier upon transferring carrier into the disinfectant. EPA stated that no swirling of the tube is preferred , i.e., why

swirl at all? There is a risk of increasing wash

- off into

the disinfectant . However, some members thought that

s wirling was necessary, especial ly for viscous products that may trap air bubbles.4 Draining the carrier by tapping following the contact time is specified. Some labs do not tap at all while others drain (drip) outside the tube. Important to t ap away from neck of tube

. Avoid upper part of tube while tapping.

ACTION ITEM S : Clarify step for swirling of the

carrier in the disinfectant and tapping . Submit

written recommendation and video clip to AOAC. Use same technique for neutralization as well. Include language to avoid tapping against the upper part of the tube to mitigate cross contamination.

UDM Primary and secondary tubes EPA stated that n eutralization is not conducted for each test, but seconda r y tubes are used. Secondary tube s can be used on an as

- need basis ; a neutralization assay will need to be conducted for each product sample. If EPA has issues with neutralization even after using secondary tubes, EPA will c ontact the company to
discuss.

ACTION ITEM: EPA to revise SOP MB

- 05 to incorporate use of secondary tubes on an “as

- need

basis”.

Turbidity Growth/no growth Use of DE broth : Positive results in DE broth are a

c olor change (Staph) and p resence of p ellicle (Pseudo) can be problematic. It was suggested to incubate the DE tubes for an additional day so t he pellicle will be more visible. Streak all tubes to confirm growth ( can be cumbersome)

Several labs streak the negative tubes on to agar media to verify negative tubes. EPA to change strategy for conducting neutralization confirmation

– this will be performed prior to each UDM with primary only and primary/secondary combinations.5 T here was a question on increasing the volume of the neutralizer to 20 mL, several people said they did it successfully. EPA does not increase the volume of the neutralizer as this is a method deviation. Will require data generation by industry

. Action Item: EPA will propose the addition of new

text to AOAC method describing the different

neutralization/subculture scenarios.

Carrier Counts Syn chronize carrier counts with the product test. Action Item: 3 carriers will be assayed at the beginning of the test and 3 at the end

.

Carrier screening Visual inspection of carriers in necessary; however, the importance of bioscreening was raised by EPA. The group did not concur with the elimination of bioscreening. 

### UDM/OECD Technical Workshop

– Highlights from Laboratory Sessions (November 16 th 

### , 2011: Further Standardization of the OECD Method)

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 assist with sources of carriers

. MLB will continue to work with the OECD VMG to

make a final decision on source(s) and specs prior to

la unch of the collaborative study

. Action Item : Specs will be discussed at the next VMG meeting.

OECD Method (SOP MB

- 25) Carrier s creening Visual screening without the aid of magnification seems

suitable.

Action items : Examples of failed carriers would be helpful

– EPA to provide.

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 cen trifugation and resuspension.6 Action item

– further discussion with the VMG will be required.

OECD Method (SOP MB

- 25)

Carrier treatment Product application Proposal to add “ inoculate extra carriers ” due to possible runoff

– the group concurred. Use of a positive displacement pipette for dispensing the product onto carriers was discussed; there was g eneral agreement to

this idea. Vortex time is not specified in the method

– agreed to add “vortex briefly” immediately following

addition of the neutralizer.

Action Item : Items will be discussed at the next VMG meeting.

OECD Method (SOP MB

- 25) Collaborative study EPA led a collaborative study to verify the performance of the OECD method. Several participants expressed interest in this proposal.

Action ite m

– EPA will consult with CSPA for

volunteers as the collaborative study is developed.

OECD Filtration Group agreed to filte r all samples using 0.22 μm filters

and to eliminate option s for plating controls and use of

0

. 45 micron filter s

. Action Item : I ssues will b e discussed at the next VMG meeting

. Carrier

- based neutralization

assay A d emonstration was conducted and the issue of %

difference (75%) for treatment comparison was discussed

.

Research topics with industry

– pre

- collaborative

studies The group discussed the research priorities associated with

OECD method

– impact on the L og R eduction with quat products. EPA continues to work with the ADBAC group to better understand the impact of growth media, hard water

and the carrier surface on the low efficacy of quat

- based products. Minor changes to the OECD method must be made before the collaborative study early in 2012. ADBAC will provide a set of product samples, EPA has provided product samples

. Management of Pseudomonas pellicle will be an issue if growth medium other than 1:1000 diluted7 TSB is used.

OECD S treamlining label claims The group discussed EPA’s goal to test all disinfectants

with soil, hard water, and a 5 minute contact time.