Study ID: NG1065 Client: Microgen, Inc. Protocol Number ... Expa… · Study ID: NG1065 Client: Microgen, Inc. Protocol Number: ATL SOP 005 Page 2 of 16

Study ID: NG1065 Client: Microgen, Inc. Protocol Number: ATL SOP 005

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Antimicrobial Test Laboratories • 3000 Joe DiMaggio Blvd, Suite 32 • Round Rock, Texas 78665 • (512) 310-8378

FINAL STUDY REPORT

Study Title

Determination of the Antimicrobial Effectiveness of D-125 Disinfectant Against Citrobacter braakii (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445)1, 2

Product Identity D-125 Disinfectant, Lot #6298-2537

Data Requirement This study serves to document the antimicrobial activity level of diluted D-125

Author Benjamin D. Tanner, Ph.D.

Scientific Director

Study Completion Date 05/29/2008

Testing Facility Antimicrobial Test Laboratories

3000 Joe DiMaggio Blvd., Suite #32 Round Rock, Texas 78665

Study ID NG1065

Conclusion in Brief The D-125 Disinfectant Product, Diluted 1:64 in hard water, was completely effective in eliminating contamination

from 10 of 10 contaminated surfaces that were tested for this study.


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STUDY PERSONNEL

SUBMITTER: Microgen, Inc DATE: 05/29/2008 Robert G. Prince President SPONSOR: Microgen, Inc. DATE: 05/29/2008 Robert G. Prince President STUDY ADMINISTRATIVE MANAGER:

DATE: 05/29/2008 Gina Tanner Business Manager

STUDY DIRECTOR: DATE: 05/29/2008 Benjamin D. Tanner, Ph.D. Scientific Director

ADDITIONAL STUDY PERSONNEL: DATE: 05/29/2008 Landra Sweeney Laboratory Technician I


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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study.

Company: Microgen, Inc.

Agent: Robert G. Prince

Title: President

Date: 05/29/2008 Signature:


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LABORATORY QUALITY ASSURANCE STATEMENT

This study was performed in accordance with Antimicrobial Test Laboratories Standard Operating Procedure (SOP) #050, Methods and Procedures for Laboratory Quality Control. Antimicrobial Test Laboratories SOP 050 specifies the following:

- Laboratory equipment and devices are verified to function properly and calibrated internally or externally as appropriate to ensure experimental quality.

- Each experiment is subject to rigorous experimental control measures

o Media sterility controls (negative control) o Vehicle and carrier sterility controls o Media growth controls (positive control) o Neutralization of active ingredient control o Verification of positive cultures as target organism

- Review by Antimicrobial Test Laboratories’ Scientific Director, Benjamin Tanner, Ph.D. for scientific clarity,

accuracy, and completeness.

This study is exempt from 40 CFR Part 160; data not generated to support United States product registration or marketing. The AOAC tests reported here, as well as Microgen’s American-based dossier, are based on "carrier" methods, which require complete kill of microorganisms on dry, contaminated surfaces. In contrast, CEN suspension methods require only partial kill in liquid suspension (e.g., 5-log kill of target bacteria within 5 minutes) to demonstrate the needed efficacy to satisfy European authorities.


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TABLE OF CONTENTS

Title/Cover Page...............................................................................................………........…….....… 1 Study Personnel and Involvement Statement..................................................................................…….. 2 Statement of Data Confidentiality Claims…………….….......................................................……............ 3 Laboratory Quality Assurance Statement............................................................................………......... 4 Table of Contents.........................................……........……....…......................................................... 5 Final Study Report............................................................................................................…….………. 6 Protocol Changes, Deviations, and Amendments.......................………………………………….........……... 11 Study Acceptance Criteria…………………………………………………………………………………………… 12 Calculations and Statistical Analysis………………………………………………………………………………… 13 Study Record and Specimen Retention…………………………………………………………………………….. 14 Study Conclusion……………………………………………………………………………………………………… 15 References……………………………………………………………………………………………………………… 16


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FINAL STUDY REPORT

Study Title

Determination of the Antimicrobial Effectiveness of D-125 Disinfectant Against Citrobacter braakii (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445)

Study Number

NG1065

Study Sponsor Microgen, Inc.

33 Clinton Road, Suite 102 Clinton Square Executive Center West Caldwell, NJ 07006 USA

Test Facility

Antimicrobial Test Laboratories 3000 Joe DiMaggio Blvd., Suite 32

Round Rock, TX 78665 USA

Study Objective

To Determine the Antimicrobial Effectiveness of diluted D-125 Disinfectant by Using the AOAC Use-Dilution Test Method

Importance of the Test Microorganism(s)

Citrobacter braakii – Citrobacter braakii is gram-negative, rod shaped bacterium that is a member of the coliform group of bacteria. As a coliform, it is able to ferment lactose at elevated temperatures. From a clinical perspective, Citrobacter braakii is notable as an opportunistic pathogen. It has been known to cause septicemia in transplant patients and other immunocompromised persons. It can be found in urine, stool, wound infections, fish and animals.3


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Summary of the Test Method

AOAC Use Dilution Test Method 4

Reference: Official Methods of Analysis (2006) 18th ED., AOAC INTERNATIONAL, Methods 955.15 and 964.02, Gaithersburg, MD, Chapter 6. Corresponding Antimicrobial Test Laboratories SOP: ATL SOP 005, Version 1.0.5., Last Modified on September 7, 2007.

Summary in Brief:

� A 48 hour culture of the appropriate microorganism is grown in AOAC nutrient broth. � The culture is vortexed briefly, except in the case of Pseudomonas aeruginosa, which is subject to special

handling procedures. � The culture is allowed to settle. � The culture is applied to a number of stainless steel penicylinders for a predetermined contact time. � The culture is removed from the penicylinders. � Penicylinders are placed individually, on end, into sterile glass Petri dishes containing sterile filter paper. � The contaminated penicylinders are then incubated at 35C for 40 minutes until dry. � The dried, contaminated penicylinders are transferred, individually, using a sterile wire hook into 10 mL of

the disinfectant solution. � After the contact time has elapsed, the treated penicylinders is removed from the product and placed in a

separate test tube containing 10 mL of appropriate neutralization/growth broth. � A control is performed to assure media sterility. � A control is performed to assure media growth promotion. � A control is performed to assure that the test substance is quickly and adequately neutralized by the

neutralization broth. � A carrier sterility control is performed. � A culture purity control is performed. � The broth tubes containing the treated penicylinders are then incubated for 48 hours. � Results are read as the number of test tubes demonstrating growth of the organism as evidenced by turbidity,

color change, etc. � Any positive tubes are tested to ensure that the organism responsible for growth was the test’s target

organism. � Success criteria – all controls must be normal and the product is permitted 0/10 failures for a 10 carrier test

or <2/60 failures for a 60 carrier test.


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Test Facts

Key to Abbreviations Used in the Report NV = Neutralization Verification Control CFU = Colony Forming Units SOP = Standard Operating Procedure AOAC = Association of Official Analytical Chemists 1X = Normal Strength

Client Information Company Name: Microgen, Inc. Sponsor: Robert G. Prince Sponsor’s Phone: (973) 575-9025 Sponsor’s Email: [email protected] General Test Information Test Performed: One 10 Carrier Confirmatory AOAC Use-Dilution Test ATL Study ID: NG1065 SOP Followed: ATL SOP 005 Performed By: B. Tanner and L. Sweeney Date Complete: 05/29/2008 Sample Test Substance: D-125 Disinfectant (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration

Certificate License No. FF-424-10445, Lot 6298-2357) Number Received: One 250 mL bottle Date Received: 04/25/2008 Test Parameters Microorganisms: Citrobacter braakii ATCC # 43162 Subculture Number: 4 Growth Medium: AOAC Nutrient Broth Dilution Tested: 1:64 Diluent: 300 parts per million AOAC hard water Contact Time: 10 minutes Organic Soil Load: Sterile Horse Serum, 5.0% Neutralizer: Difco Letheen Broth (1X) Exposure Temperature: 21C Type of Carrier: Stainless steel penicylinder Number of Test Carriers: One test, 10 test carriers plus controls Carrier Dry Time: 40 minutes at 35 C Incubation Temperature: 35.0 (+/- 1) C Tube Incubation Time: 48 hours


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Test Facts (Continued)

Controls Neutralized: Passed, NV tube positive Media Sterility: Passed, all standard media controls negative Growth Control: Passed, Citrobacter braakii positive Vehicle Control: Passed, carrier negative Test Results Carriers Positive: Zero Confirmation: N/A, all carriers negative Test Valid: Yes Notes Recorded By Study Personnel During the Study D-125 (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445) passed the 10 carrier test with zero positive carriers for Citrobacter braakii.


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Test Facts (Continued)

Table 1.

Test SubstanceDate

TestedDilution Microorganism

Contact

Time

CFU /

Carrier

Carriers

Positive /

10 Tested

D-125 Disinfectact 5/27/2008 1:64 Citrobacter braakii ATCC # 43162 10:00 3.30E+06 0 (PASS)

Chart 1.

Microgen D-125 Disinfectant (Study ID NG1065) Results

for Citrobacter braakii ATCC # 43162

00

1

2

3

4

5

6

7

8

9

10

D-125 Disinfectact

Carr

iers

Po

sit

ive o

f 10 T

este

d

1.E+04

1.E+05

1.E+06

1.E+07

CF

U/C

arr

ier

carriers positive CFU/Carrier


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PROTOCOL CHANGES, DEVIATIONS, AND AMENDMENTS

Changes None Recorded for This Study Deviations None Recorded for This Study Amendments None Recorded for This Study


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STUDY ACCEPTANCE CRITERIA

The following controls must be normal in order for the study to be deemed acceptable by the Scientific Director:

- Media Sterility Control - Media Growth Promotion Control - Carrier Sterility Control - Organism Identity Control (Concurrent w/Media Growth Promotion Control)

- Test Substance Neutralization Control Study Control Results

- Media Sterility Control – Pass - Media Growth Promotion Control – Pass - Carrier Sterility Control – Pass - Organism Identity Control – Pass - Test Substance Neutralization Control – Pass


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CALCULATIONS AND STATISTICAL ANALYSIS

Microbial concentrations were calculated using standard means for this study (dilution and duplicate plating). Dilutions of 1:10,000,000 and 1:1,000 – 1:100,000 were used for the neutralization control and dry carrier enumeration, respectively. Plates having between 25 and 250 colonies were counted, counts were averaged, and then reciprocal dilutions were applied to determine microbial concentrations. No other calculations or statistical analyses were performed for this study.


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STUDY RECORD AND SPECIMEN RETENTION

Study Record Retention This study report and corresponding data sheets will be held in the archives of Antimicrobial Test Laboratories at the following address for at least 2 years after the date of this report: Antimicrobial Test Laboratories 3000 Joe DiMaggio Blvd, Suite #32 Round Rock, Texas, 78665 USA

Specimen Retention

The test specimen will be held for 30 days after study completion at the following facility: Antimicrobial Test Laboratories 3000 Joe DiMaggio Blvd, Suite #32 Round Rock, Texas, 78665 USA


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STUDY CONCLUSION

This study demonstrates that D-125 Disinfectant, diluted 1:64 (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445) is effective, completely eliminating Citrobacter braakii ATCC # 43162 from 10 contaminated surfaces within 10 minutes. The product was challenged with 3.30 x 10^6 bacteria per test carrier, as shown in Table 1. This test method has a limit of detection of 1 CFU/carrier. Thus, the reduction in microbial concentrations was at least 6 log10. Chart 1 illustrates the number of test carriers that showed growth for each test following the 10 minute exposure time, along with the corresponding number of dried microorganisms per carrier, as determined by analysis of three dried carriers, chosen at random from the lot. Neutralization of the test substance was rapid and complete, per the results of the neutralization verification control. This study also affirms the efficacy of D-125 in the presence of organic soil (5% horse serum) and >300 PPM hard water. Based on all the circumstances of the study, including protocol deviations (if any were noted above), the opinion of the study director is that the study produced fair and accurate results.

Report Submitted By Scientific Director: Benjamin D. Tanner, Ph.D. Study Completion Date: 05/29/2008


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REFERENCES

1. 3rd Generation Quaternary Ammonium (D-125 MSDS). Microgen Inc. Revision 17.03.2005. Downloaded 6/22/08 (http://www.microgeninc.com/milestones/PDF/D-125%20MSDS%20EN.pdf).

2. Microgen D-125 (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445)™ USEPA Master Label. May 17, 2007. Downloaded 2/6/08. (http://www.microgeninc.com/milestones/PDF/D-125 (US REG. 61178-1; DCGI REG. 64/FF/09/07-DC, Registration Certificate License No. FF-424-10445)%20Master%20Label.pdf).

3. Manual of Clinical Microbiology (2003) 8th Ed., Chapter 44. ASM Press. Washington, D.C. 4. Official Methods of Analysis (2006) 18th ED., Chapter 6. AOAC INTERNATIONAL, Methods 955.15 and

964.02, Gaithersburg, MD.

Documents

Study ID: NG1065 Client: Microgen, Inc. Protocol Number ... Expa… · Study ID: NG1065 Client: Microgen, Inc. Protocol Number: ATL SOP 005 Page 2 of 16