# 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)2 

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# Presentation Overview • Purpose of the collaborative • Pre- collaborative studies • Key measurements • General study plan • Timeline • Potential outcomes and use of the data 3 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 4 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 5 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 6 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 7

## 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 MLog10Reduction

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. 8 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 9 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 10 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 11 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 12 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 13 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. 14 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). 15 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 16 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 17 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 18 2011 Test Guidelines Programme19 2011 Test Guidelines Programme