Collaborative Evaluation of 

the Draft OECD Test 

Guidelines for Bactericidal 

Activity 

Stephen Tomasino, Ph.D.

U.S. EPA Office of Pesticide Programs Microbiology Laboratory

Fort Meade, MD 

(Presented at the Test Guidelines Programme – Biocides Programme, Meeting of Experts and 

Regulators, September 13, 2011)
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DISCLAIMER

This information is distributed solely for 

the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally 

disseminated by EPA. It does not represent 

and should not be construed to represent 

any Agency Determination or Policy.

The information provided should not be 

cited or referenced. 
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 Test Guidelines Programme
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 Test Guidelines Programme
Purpose of the collaborative

• 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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2011 Test Guidelines Programme
Purpose of the collaborative

• The OECD TG for Bactericidal Activity have 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.ht

m

▫ 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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2011 Test Guidelines Programme
Purpose of the collaborative

• 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 

2011 Test Guidelines Programme

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Purpose of the collaborative

* *

† † † † †

* * * *

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.

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2011 Test Guidelines Programme

LR data for 13 EPA registered products against P. aeruginosa
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 Test Guidelines Programme
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 Test Guidelines Programme
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 5.5 to 6.5 

• Use of pre-screened carriers – critical step 

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2011 Test Guidelines Programme
General Study Plan

• Up to 6 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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2011 Test Guidelines Programme
General Study Plan

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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2011 Test Guidelines Programme
General Study Plan

• 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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2011 Test Guidelines Programme
General Study Plan

• 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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2011 Test Guidelines Programme
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 Test Guidelines Programme
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 Test Guidelines Programme
Potential Outcomes and Use of the Data

• 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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2011 Test Guidelines Programme
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2011 Test Guidelines Programme