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FINAL REPORT

USP ANTIMICROBIAL PRESERVATIVE EFFECTIVENESS TEST

PROCEDURE NO. STP0131 REV 02PROTOCOL DETAIL SHEET NO. 200801838 REV 01

LABORATORY NO. 459306

PREPARED FOR:

KEITH MOELLERAMERICAN BIOTECH LABS80 W. CANYON CREST RD.

ALPINE UT 84004

SUBMITTED BY:

NELSON LABORATORIES, INC.6280 S. REDWOOD RD.

SALT LAKE CITY UT 84123-6600801-290-7500 of 10

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NELSON LABORATORIES, INC.

QAU AUDIT STATEMENT

[X] USFDA (21 CFR PART 58) [ ] USEPA (40 CFR PART 160)


LABORATORY NO. 459306

1. The test was conducted in accordance with the USFDA or USEPA Regulations as notedabove.

2. In accordance with the Good Laboratory Practice Regulations, the Plating phase(s) ofthis study was inspected by the Quality Assurance Unit on: 28 Jan 2009. The findings ofthe inspection(s) were reported to the Study Director and to Management on: 02 Mar2009.

3. The Quality Assurance Unit has reviewed this report and has determined that themethods and standard testing procedures are accurately described, and that thereported results accurately reflect the raw data.

4. The name of the study director, the names of other scientists or professionals, and thenames of all supervisory personnel, involved in the study:

Michael NeilsonPeter Croci

Dr. Jerry NelsonJeff Hills

QUALITY ASSURANCE: -'..L,t~~~:r--gz~.i::;c'--.!~~,--·---DATE: o:~ AaI' <dd2

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LABORATORY NUMBER:PROCEDURE NUMBER:PROTOCOL DETAIL SHEET NUMBER:SAMPLE SOURCE:SAMPLE IDENTIFICATION:DEVIATIONS:PROTOCOL APPROVAL DATE:SAMPLE RECEIVED DATE:LAB PHASE START DATE:LAB PHASE COMPLETION DATE:REPORT ISSUE DATE:

459306STP0131 REV 02200801838 REV 01American Biotech LabsRefer to Tables 2-3None21 Jan 200921 Jan 200921 Jan 200902 Mar 200903 Mar 2009

INTRODUCTION:

This study was performed to determine the survival rate of various organisms in the testproduct. The test employed methods designed to determine antimicrobial effectivenessdescribed in the United States Pharmacopeia (USP).

The samples of the product were inoculated, with five standard test organisms. The inoculatedsamples were then incubated for a total of 28 days at 20-25°C. Aliquots from the samples wereimmediately removed and assayed for surviving organisms at 0 hour, 7 day, 14 day, and 28 daytime intervals. The log reduction in the level of the test organisms was calculated for each timeinterval.

ACCEPTANCE CRITERIA:

According to USP, products are placed into categories according to product type. The productcategory will determine the acceptance criteria for the test. The following table provides adescription of the current USP categories.

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American Biotech LabsLab Number 459306

USP Antimicrobial Preservative Effectiveness Test

CATEGORY PRODUCT DESCRIPTION

Injections, other parenterals including emulsions, otic products,1 sterile nasal products, and ophthalmic products made with aqueous

bases or vehicles.Topically used products made with aqueous bases or vehicles, non-

2 sterile nasal products, and emulsions, including those applied tomucous membranes.

3 Oral products other than antacids, made with aqueous bases orvehicles.

4 Antacids made with an aqueous base.

The acceptance criteria for the USP Antimicrobial Effectiveness Test is located in Table 1. Noincrease is defined as not more than a 0.5 10glOunit higher than the previous value measured.

1) Staphylococcus aureus ATCC #6538[Bacteria, Gram (+) cocci]

2) Pseudomonas aeruginosa ATCC #9027[Bacteria, Gram (-) bacillus]

3) Escherichia coli ATCC #8739[Bacteria, Gram (-) bacillus]

4) Candida albicans ATCC #10231[Yeast]

5) Aspergillus niger ATCC #16404[Mold]

PROCEDURE:

The following organisms were tested:

The bacteria were transferred to soybean casein digest agar (SCDA) and incubated at fO-35°Cfor 18-24 hours. The C. albicans and A. niger were transferred to Sabouraud dextr9se agar(SDEX). The C. albicans was incubated at 20-25°C for 44-52 hours and the A. niger wasincubated at 20-25°C for 6-10 days.

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The bacteria and C. albicans were harvested using 0.9% saline (PHSS) and A. niger washarvested with 0.9% saline containing 0.05% polysorbate 80 (Tween® 80). The A. niger wasfiltered through sterile gauze and all cultures were vortexed to break up clumps. Afterharvesting, the organisms were centrifuged and re-suspended in purified water (PURW). Eachsuspension was adjusted to approximately 108 colony forming units (CFU)/mL using visualturbidity. The actual concentration of each culture was determined using the positive controlvalues.

These results represent duplicate analysis plated in triplicate. Individual samples of the testproduct were prepared for each challenge organism. The tubes containing the sample wereinoculated with the test organisms using a calibrated micropipettor. The volume of the inoculumwas between 0.5% and 1.0% of the volume of the product. The final concentration of the testpreparation was approximately 105 - 106 CFU/mL of product. The samples were well mixed.

Positive control tubes were prepared for each organism using sterile water. The volume usedfor the positive control was equivalent to that used in the samples. Negative controls were alsoprepared. The positive controls were then inoculated in the same manner as the test samples.All test samples were stored at 20-25°C for a total of 28 days.

Control tubes and test vials were assayed immediately to determine the initial concentration oforganisms in each tube. The test suspensions were assayed at the following intervals: 0 hour,7, 14, and 28 days.

Sample aliquots at each interval were diluted in Letheen broth (LETH) and plated on SCDA forbacteria and SDEX for C. albicans and A. niger. The bacteria plates were incubated at 30-35°Cfor 3-5 days. The C. albicans plates were incubated at 20-25°C for 3-5 days and the A. nigerplates were incubated at 20-25°C for 3-7 days.

RESULTS:

The results are reported in Tables 2-3. The greater than (» values represent the detectablelimits of the test where zero CFU were observed on the plates. The approximate (-) symbol isapplied to results where plate counts fell outside of the statistically accurate range of 25-250CFU for bacteria and yeast and 8-80 CFU for mold.

Qualification of neutralization was not performed. Sponsor is responsible for provingneutralization of the product.

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American Biotech LabsLab Number459306


All negative controls showed no growth. Testing met the acceptance criteria stated in thisreport.

CONCLUSION:

Interpretation of the data is the responsibility of the sponsor and no conclusion can be made byNelson Laboratories, Inc. (NLI).

DATA DISPOSITION:

The raw data and final report from this study are archived at NLI or an approved off-sitelocation.

STATEMENT OF UNCERTAINTY:

If applicable, the statement of uncertainty is available to sponsors upon request.

Peter/Graci, B:A.Study Director./

Study Completion Date

es

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TABLE 1. USP Antimicrobial Effectiveness Acceptance Criteria

Category 1 Products

Bacteria: Not less than 1.0 log reduction from the initial calculated count at7 days, not less than 3.0 log reduction from the initial count at 14days, and no increase from the 14 days' count at 28 days.

Yeast & Molds: No increase from the initial calculated count at 7, 14, and 28 days.

Category 2 Products

Bacteria: Not less than 2.0 log reduction from the initial count at 14 days,and no increase from the 14 days' count at 28 days.

Yeast & Molds: No increase from the initial calculated count at 14 and 28 days.

Category 3 Products

Bacteria: Not less than 1.0 log reduction from the initial count at 14 days,and no increase from the 14 days' count at 28 days.

Yeast & Molds: No increase from the initial calculated count at 14 and 28 days.

Category 4 Products

Bacteria, Yeast,& No increase from the initial calculated count at 14 and 28 days.Molds:

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TABLE 2. Inoculum Control ResultsSample Identification: ASAP Ultimate Skin and Body Care

Lot #08295

ORGANISM CHALLENGE TITERAT 0 HOUR(CFU / mL)

S. aureus 3.0 x 105

P. aeruginosa 2.1 x 10°E. coli 2.4 x 105

C. albicans 4.2x105A. niger 6.2 x 10°

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TABLE 3. Summary of Log Reduction ResultsSample Identification: ASAP Ultimate Skin and Body Care

Lot #08295

ORGANISM TIME INTERVALo HOUR 7 DAY 14 DAY 28 DAY

S. aureus -0.10 >4.18 >4.18 >4.18P. aeruginosa -2.94 >4.01 >4.01 >4.01

E. coli -2.82 >4.09 >4.09 >4.09C. albicans -3.32 >4.32 >4.32 >4.32

A. niger 1.23 -4.36 >4.49 >4.49

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All reports and letters issued by Nelson Laboratories, Inc. are for the exclusive use of thesponsor to whom they are addressed. These results relate only to the samples tested. Reportsmay not be reproduced except in their entirety. No quotations from reports or use of thecorporate name is permitted except as expressly authorized by Nelson Laboratories, Inc. inwriting. The significance of any data is subject to the adequacy and representative character ofthe samples tendered for testing. Nelson Laboratories, Inc. warrants that all tests are performedin accordance with established laboratory procedures and standards. Nelson Laboratories, Inc.makes no other warranties of any kind, express or implied. Nelson Laboratories, Inc. expresslystates that it makes no representation or warranty regarding the adequacy of the samplestendered for testing for any specific use of application, that determination being the soleresponsibility of the sponsor. Nelson Laboratories' liability for any loss or damage resulting fromits actions or failure to act shall not exceed the cost of tests performed, and it shall not be liablefor any incidental or consequential damages.

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