2011 UDM/OECD Technical Workshop

Collaborative Evaluation of the Draft OECD 

Test Guidelines for Bactericidal Activity 

Stephen Tomasino, Ph.D.

U.S. EPA Office of Pesticide Programs 

Biological and Economic Analysis Division

Microbiology Laboratory

Fort Meade, MD 
2011 UDM/OECD Technical Workshop

Presentation Overview

• Purpose of the 

collaborative 

• Pre-collaborative studies

• Key measurements

• General study plan 

• Timeline

• Potential outcomes and 

use of the data

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2011 UDM/OECD Technical Workshop

Purpose of the collaborative

• Addresses technical issues raised to the Working Group 

of National Coordinators of the TGP (WNT)

• Method/protocol optimization

• Further identify and control the sources of variability

• Support the EPA implementation strategy of the OECD 

TGs

• Evaluate several EPA registered disinfectant products against 

new performance standards 

– Use the method to test commercial products rather than 

active ingredients

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2011 UDM/OECD Technical Workshop

• Demonstrate feasibility of the carrier-based 

neutralization protocol

• Initiate evaluation of a reference standard

• Involve several representative stakeholder labs 

with trained analysts

• Test design consistent with method validation 

– Oversight provided by AOAC International

• Not designed to address performance standards 

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Purpose of the collaborative
2011 UDM/OECD Technical Workshop

• The OECD TG for Bactericidal Activity has been 

converted into a Standard Operating Procedure 

(SOP).

– MLB SOP MB-25 (OECD Quantitative Method for 

Evaluating Bactericidal Activity); see 

http://www.epa.gov/pesticides/methods/atmpa2z.htm

– The SOP is a strict interpretation of the TG and further 

reduces procedural options and increases clarity.

– Contains test forms and data sheets

– Encourage use by stakeholders

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Purpose of the collaborative
2011 UDM/OECD Technical Workshop

• EPA initiated product testing using the OECD 

method in late 2010 with the following goals: 

– Use/optimization of the SOP prior to widespread use 

– Training of EPA staff

– Develop product performance data

– Assist in the implementation process

• Overall observations 

– Training is essential

– Consistent control counts

– Wide range of product efficacy 

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Purpose of the collaborative
2011 UDM/OECD Technical Workshop

* *

† † †

†

†

* *

*

*

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

A B C D E F G H I J K L M

Log10 Reduction

Hydrogen 

peroxide

Phenol Sodium 

hypochlorite

Quaternary ammonium 

compounds

Quat + 

HCl

Citric 

acid

Test Chemicals

* = Value listed represents complete kill. 

† = Value listed represents a value of 1,000 that was substituted at the least concentrated dilution because growth on the filter was a lawn. 7

LR data for 13 EPA registered products against P. aeruginosa

Purpose of the collaborative
2011 UDM/OECD Technical Workshop

Pre-collaborative studies

• Neutralization confirmation

– Successfully demonstrated by EPA 

• Reference standard

– Reagent grade sodium hypochlorite as first candidate

– High efficacy and low efficacy 

– Target concentrations have been identified by EPA 

• Series of studies to address low LR for quaternary 

ammonium compounds – to begin in October 2011

– Impact of growth media

– Impact of carrier surface

– Impact of inoculum distribution on carriers 

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2011 UDM/OECD Technical Workshop

Key Measurements

• Control carrier counts (log density) per prescribed 

performance standard (+0.5 – 1.5 logs above the PS)

• Method performance indicators 

– Repeatability (within-lab variability)

– Reproducibility (between-lab variability)

– Responsiveness 

• Product performance (log reduction) with new default 

test conditions

– Contact time – 5 min

– Hard water - 375 ppm

– Organic burden – 3 part mixture 

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2011 UDM/OECD Technical Workshop

General Study Plan

• Follow the EPA SOP

• 5-8 laboratories with previous training/use of the 

OECD method

• The laboratories taking part in this collaborative 

study are assumed to be statistically 

representative of all laboratories that will be 

conducting this type of test in the future. 

• Two microbes – S. aureus and P. aeruginosa

• Target control carrier counts of 4.5 to 5.5 

• Use of pre-screened carriers – critical step 

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2011 UDM/OECD Technical Workshop

• Commercially available disinfectant products will be tested – 5 

classes of active ingredients

– A single lot of each product will be used.

– Formulation chemistry will be conducted.

– The products will be distributed to the testing laboratories in containers 

that are marked only with a treatment code.

• 1 Quat.

• 1 Alcohol

• 1 Phenolic

• 1 Hydrogen peroxide

• 1 Sodium hypochlorite (2 levels) 

• Random order for treatments

• Three independent replications per product

• Assuming no repeat testing, a total of 12 test days to complete 

the entire study

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General Study Plan
2011 UDM/OECD Technical Workshop

Organism Replicate Test Day Test 

Chemical

S. aureus

1

1 E, A, B

2 D, F, C

2

3 B, E, C

4 A, D, F

3

5 B, E, A

6 F, C, D

P. aeruginosa

1

7 F, E, D

8 C, A, B

2

9 A, F, D

10 B, C, E

3

11 E, F, D

12 B, C, A

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General Study Plan
2011 UDM/OECD Technical Workshop

• A teleconference with the participating laboratories and/or a 

training session provided by EPA will be held in advance of 

testing to discuss the details of the protocol. 

• Maintain documents to ensure that all studies are supported 

by complete, accurate, consistent, and chronological records 

from initial collection of raw data to final analysis 

interpretation and reporting of results.

• The expected level of quality assurance will be consistent

with EPA Good Laboratory Practices. 

• The statistical analysis will produce estimates of the 

repeatability standard deviation, the reproducibility standard 

deviation, for each treatment (log reduction value and the 

control carrier log CFU per carrier), and method 

responsiveness.

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General Study Plan
2011 UDM/OECD Technical Workshop

• Each control carrier count will be log10 transformed to create the log 

density (LD). The statistical properties of the control carrier LDs will 

be calculated using analysis of variance techniques. 

• Log reduction (LR) value – LR is the primary quantitative response and 

most of the statistical work will focus on the LR data. 

• Mean LR – For each test chemical, the mean LR will be calculated 

along with the associated standard error and confidence interval. 

• For the chemical designed to measure responsiveness, a statistical trend 

test will determine whether the mean LR values increase significantly 

with increasing efficacy level (presumed).

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General Study Plan
2011 UDM/OECD Technical Workshop

Timeline

• Pre-collaborative studies – October through 

December 2011

• Collaborative launch date of February 2012

• Data collection complete by April 2012

• Data analysis by Montana State University 

• Report out to the OECD Validation 

Management Group by June 2012 

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2011 UDM/OECD Technical Workshop

Potential Outcomes and Use of the Data

• Report to the OECD Validation Management 

Group and WNT on findings

• Finalize the carrier-based neutralization 

protocol

• Verify the reference standard and pave way for 

the development of additional standards 

• Publish findings in the Journal of AOAC 

International or other methods journal

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2011 UDM/OECD Technical Workshop

• Verify or improve the performance of the method 

• Recommend additional procedural information 

for items such as:

– Carrier screening

– Culture harvest

– Target carrier counts

– Neutralization

• Show progress on the development of reference 

standards

• Use experience for the development of additional 

studies such as for mycobacterial activity 

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Potential Outcomes and Use of the Data
2011 UDM/OECD Technical Workshop

QUESTIONS AND 

COMMENTS?

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